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cDNA clones encoding poly (A) RNAs which first appear at detectable levels in haploid phases of spermatogenesis in the mouse
Abstract
We have isolated several cDNA clones encoding cytoplasmic poly(A)+ RNAs which are enriched in postmeiotic (haploid) spermatogenic cells in the mouse. Seventeen of 750 clones from a testis cDNA library hybridized more strongly to 32P-labeled cDNA copied from cytoplasmic poly(A) RNA of round spermatids than pachytene spermatocytes. Northern gel blots demonstrated that these 17 plasmids hybridized to RNA(s) approximately 0.5 kb (1 clone), 0.7 kb (13 clones), 0.8 kb (1 clone), and 0.9 kb (2 clones). Four plasmids hybridizing to RNAs 0.7 and 0.9 kb were further characterized by Northern blots. The levels of hybridization were about 10-fold greater with RNA from round spermatids, elongating spermatids and residual bodies than from pachytene spermatocytes from adult testis. These plasmids did not hybridize with cytoplasmic poly(A)+ RNA from sexually immature testis, adult liver, or brain, larger precursors in adult testis nuclear RNA, total RNA from cultured Sertoli cells, poly(A)- RNA from adult testis or the mouse mitochondrial genome. These results demonstrate that certain poly(A)+ RNAs are abundant in haploid cells but barely or not detectable in meiotic cells suggesting the accumulation of these RNAs in round spermatids requires transcription in haploid cells.
... Their major function is to neutralize the charge of DNA and to aid in its compaction (Bellv6, 1979;Hecht, , 1988aPoccia, 1986). The mRNAs of two variants of mouse protamine have been found to be synthesized during the haploid phase in round nucleated spermarids, and to be translationally regulated (Kleene et al., 1983(Kleene et al., , 1984Hecht and Penschow, 1987), i.e., translation in elongating spermatids occurs ,x,1 w after mRNA synthesis starts (Kieene et al., 1983(Kieene et al., , 1984Balhorn et al., 1984). The exact stages of spermiogenesis where the transcription and translation of protamine mRNA occurs, are not known. ...
... The mouse protamine cDNA clone pMP1, isolated from a mouse testis cDNA library (Kleene et al., 1983(Kleene et al., , 1985Yelick et al., 1987), was used as the hybridization probe. For in situ hybridization, the 43-/-bp insert was released from its vector by digestion with the restriction enzyme Sal I, and isolated after electrophoresis on 1% agarose gel by binding to a DEAE membrane (Schleicher & Schuell, Inc., Keene, NH). ...
A mouse protamine 1 cDNA probe was used to study P1 protamine gene expression during the cycle of the seminiferous epithelium in the rat. In situ hybridization experiments showed that transcription of the P1 protamine mRNA starts in the middle of step 7 of spermiogenesis during substage VIIc. The mRNA levels stay high in steps 7-14 spermatids but decrease during steps 15-16 and are virtually undetectable in steps 17-19 spermatids. Northern blot analyses of RNAs isolated from microdissected pools of seminiferous tubules show high P1 protamine mRNA concentrations during stages VIIc-XIV-III of the cycle and lower levels during stages IV-VIIb. Owing to a post-transcriptional shortening of the poly(A) tail by 130 bases, a decrease in the size of protamine 1 mRNA from approximately 580 to 450 nucleotides was observed in stages XIII-XIV suggesting an initiation of protamine 1 synthesis in step 13-14 spermatids. In stages II-VI (steps 16-18 spermatids), only the smaller size protamine 1 mRNA was detectable. The expression of protamine 1 mRNAs has been localized in the very last phase of the haploid gene activity. Although the in situ hybridization suggests a disappearance of protamine 1 mRNA after step 16 of spermiogenesis, Northern blot analysis shows that low levels of mRNA are present during the period of final condensation of the chromatin, reflecting the association of protamine with DNA.
... Analysis of the 5' flanking region of the testis-specific gene mP2 has revealed an element that matches the FRG Y2-binding Y box element at 9 of 12 nucleotide positions [13]. Interestingly, several additional mammalian testis-specific genes, including protamine 1 [24,25], transition protein 2 [24,25], phosphoglycerate kinase 2 [26], an alternatively transcribed cytochrome c [27], and the testis-specific H2B histone gene promoter [28] also contain 5' flanking sequences that match the consensus Y box at 9-10 of 12 positions. This level of identity is sufficient for activation of the HSV tsk promoter by FRG Y2 [3]. ...
... Analysis of the 5' flanking region of the testis-specific gene mP2 has revealed an element that matches the FRG Y2-binding Y box element at 9 of 12 nucleotide positions [13]. Interestingly, several additional mammalian testis-specific genes, including protamine 1 [24,25], transition protein 2 [24,25], phosphoglycerate kinase 2 [26], an alternatively transcribed cytochrome c [27], and the testis-specific H2B histone gene promoter [28] also contain 5' flanking sequences that match the consensus Y box at 9-10 of 12 positions. This level of identity is sufficient for activation of the HSV tsk promoter by FRG Y2 [3]. ...
Recent evidence indicates that a member of the Y box-binding family of transcriptional regulators is identical to p56, a predominant protein of messenger ribonucleoprotein complexes. The p56 protein is highly enriched in oocytes and testis, and a functional RNA binding mouse cytoplasmic homologue has been cloned and partially characterized. Because few potential testis-specific transcriptional regulators have been identified, the testis-enriched Y box-binding proteins represent transacting elements of a unique model system for the study of haploid gene expression. The 5' flanking region of the testis-specific, haploid-expressed mouse protamine 2 gene contains an element with a 9-of-12 nucleotide identity with the previously defined Y box consensus sequence. We have investigated the possible role of Y box-binding proteins in transcriptional regulation of protamine 2 using specific antibodies and DNA-protein binding assays. Western blot analyses with two different anti-p54/p56 antibodies demonstrate that a mouse homologue of Xenopus p54/p56 is present in transcriptionally active mouse testis nuclear extracts. Our results further indicate that the Xenopus Y box-binding proteins bind to an element 5' to the mouse protamine 2 gene. Similarly, binding of the mouse testis homologue to the protamine 2 Y box element is demonstrated by gel mobility shift and antibody supershift analyses. The demonstrated interactions between testis-enriched Y box-binding proteins and prqtamine 2 transcriptional control elements therefore represent a unique system for functional studies to determine the mechanism of regulation of haploid gene expression.
... Unlike histones, protamines and sperm specific basic nuclear proteins vary greatly in different among species and their times of gene expression also differ. In the mouse, there are two different protamine variants, whose mRNAs are expressed in haploid cells (5, 7,8,13,16,17). In the rat (3) and rooster (20), protamine and transition protein-1 (TP-1) mRNAs are also expressed only in post-41 9 meiotic cells. ...
... In situ hybridization for the antisense protamine riboprobe showed that medaka protamine mRNA is detectable in spermatids and a fraction of secondary spermatocytes, but not in spermatogonia or primary spermatocytes judged from their size and characteristic nuclei. This is a unique characteristic in medaka, differing from protamine and transition protein 1 mRNAs in mammals (3,5,7,8,13,16,17) and the rooster (20) based on northern hybridization and in situ hybridization analyses. In the rainbow trout, protamine mRNA was found only in spermatid nets, not in spermatogonia or spermatocytes by in situ hybridization analyses (25). ...
Protamines or sperm specific basic proteins are highly basic low molecular weight proteins that substitute histones in the chromatin of sperm during spermatogenesis. They condense sperm DNA into a highly compact, stable and inactive complex. In this study, cDNA of protamine of the medaka, Oryzias latipes, was cloned to elucidate the molecular mechanisms involved in spermatogenesis. A medaka testis cDNA library constructed in lambda gt11 showed 2.78X106 independent recombinants. Several positive clones were obtained by immunoscreening with polyclonal antiserum against medaka protamine. Sequencing showed that one of these positive clones, named MP-1, encoded arginine clusters characteristic of protamine. The putative amino acid sequence of MP-1 revealed a remarkable extent of homology with other fish protamines, such as 71% identity with thynnin Y, a sperm specific basic protein isolated from the bluefin tuna, Thunnus thynnus. Northern hybridization using a MP-1 cDNA probe showed that MP-1 mRNA is present exclusively in the testes and that it gave three detectable bands: a major band of 280 b, and two others of 400 b and 500 b. In situ hybridization of a complementary RNA probe (digoxigenine-UTP-labeled MP-1 RNA) revealed that MP-1 mRNA is localized in some secondary spermatocytes and spermatids, but not in primary spermatocytes or spermatogonia. These results differ from those obtained in studies on the rainbow trout by solution hybridization and in situ hybridization.
... Studies that have focused on the stages of spermatogenesis at which the genes for these SBPs are expressed indicate that, in the rainbow trout, protamine genes are transcribed in the primary spermatocytes (12). In contrast, other studies have shown that the protamine genes in mammals (3,7,15,18) and in the rooster (22) are expressed on a haploid genome in spermatids. ...
... The present study also shows that the genes for protamines in Bufo testis are transcribed for the first time postmeiotically in early spermatids. The timing of the onset of the transcription of Xenopus SP4 is therefore similar to that of protamine in the rainbow trout (12), whereas the situation in Bufo agrees with the well-known haploid expression of genes for protamines in mammals (3,7,15,18) and the Pm. rooster (22), as well as TPl (spermatid nuclear and Xenopus (Fig. 6), hybridized in siru with 35S-labeled antisense RNA probes for protamines (Bufo) and SP4 (Xenopus). ...
The expression of the genes for sperm-specific basic nuclear proteins was examined, using the cDNA clones encoding protamine (P2) of Bufo japonicus and SP4 of Xenopus laevis as probes. Northern analyses showed that the mRNAs for these proteins were present only in the testes. Analyses with total RNA extracted from testicular cells at various spermatogenic stages revealed that in Bufo the transcripts of protamine genes are present in the spermatids, while in Xenopus the mRNAs for SP4 are present in both primary spermatocytes and spermatids. In situ hybridization studies with radiolabeled antisense RNA probes generated from cDNAs indicated that the Bufo protamine mRNAs accumulated first in round spermatids, while the Xenopus SP4 mRNAs did so in the pachytene stage of primary spermatocytes and thereafter.
... I began by screening a genomic library with [ 32 P]labeled DNA reverse transcribed from poly(A) + -RNA extracted from purified round spermatids and pachytene spermatocytes, and wound up many experiments later with 17 recombinant DNAs copied from mRNAs that hybridized to small mRNAs, which were enriched in haploid spermatogenic cells (Kleene et al. 1983). This meandering path involved working through a series of glitches in constructing and screening recombinant DNA libraries without the aid of kits supplied by biotechnology companies. ...
Gordon Dixon’s pioneering work on the replacement of histones by protamines during spermatogenesis inspired research as recombinant DNA became widely used to analyze gene expression in mammalian spermatogenic cells. The impact of recombinant DNA began immediately with the identification of mouse protamine 1 as a haploid-expressed mRNA, resolving a decades-long controversy whether gene expression in haploid spermatogenic cells distorts transmission of alleles to progeny. Numerous insights into the biology of spermatogenesis followed as the sequences of many mRNAs revealed that the patterns of gene expression in spermatogenic cells are astonishingly different from those in other cells in the mammalian body. Studies of these phenomena have generated fundamental insights across reproductive, molecular and evolutionary biology.
Abbreviations: PRM1: protamine 1; PRM2: protamine 2; TCE: translation control element
... Posttranscriptional control plays a major role in the successful development of male gametes (Schafer, Nayernia et al. 1995, Laiho, Kotaja et al. 2013). In the case of chromatin compaction, the mRNAs encoding Prm1 and Prm2 are synthesized in round spermatids and stored as mRNPs for up to 7 days before being recruited for translation in elongating spermatids (Kleene, Distel et al. 1983, Kleene, Distel et al. 1984. Translational repression of Prm1 is mediated by sequences in its 3'UTR (Fajardo, Haugen et al. 1997) and temporal translational delay is essential for completion of spermatid differentiation (Lee, Haugen et al. 1995). ...
Micro RNAs (miRNAs), which are ~22 nucleotide (nt) long RNA molecules and several RNA binding proteins (RBPs) engage in an RNA dependent post-transcriptional gene silencing process known as RNA interference (RNAi). In the canonical miRNA biogenesis pathway, an enzyme known as DICER cleaves the ~70nt pre-miRNA to a ~22nt long miRNA that is loaded into the RNAi effector mechanism, the RNA induced silencing complex (RISC). Several in vitro studies provide suggestive evidence that mammalian double stranded RNA binding proteins (dsRBPs), such as TARBP2, act as DICER cofactors in miRNA processing and RISC loading to promote RNAi activity. A screen attempting to identify translational regulators of the murine Protamine1 gene identified TARBP2 as a potential translation regulator. It is unknown if TARBP2 has a role in miRNA biogenesis in vivo, or if the translation regulation of Prm1 during murine spermatogenesis is dependent on TARBP2 mediated miRNA biogenesis. Murine embryos with a constitutive null allele of Tarbp2 and adult mice with a germ cell-specific loss of TARBP2 were generated to lead to understanding of the role of TARBP2 in miRNA biogenesis and TARBP2 mediated post-transcriptional gene regulation during spermatogenesis. Here, I describe that TARBP2 regulate biogenesis of a sub-set of miRNAs during murine embryonic development and spermatogenesis. Also, the role of TARBP2 dependent miRNAs in post-transcriptional regulation of gene expression during murine spermatogenesis will be discussed.
... A particularly surprising outcome concerned the PRM and TNP. It has been long stated that their mRNAs in mouse, rat and human start to be transcribed in RS [81][82][83][84][85] and remain repressed until the stages of elongating spermatids, when they are translated [86,87]. This means that in mouse, for example, this repression mechanism would be operative for up to a week. ...
Background
Spermatogenesis is a complex differentiation process that involves the successive and simultaneous execution of three different gene expression programs: mitotic proliferation of spermatogonia, meiosis, and spermiogenesis. Testicular cell heterogeneity has hindered its molecular analyses. Moreover, the characterization of short, poorly represented cell stages such as initial meiotic prophase ones (leptotene and zygotene) has remained elusive, despite their crucial importance for understanding the fundamentals of meiosis.
Results
We have developed a flow cytometry-based approach for obtaining highly pure stage-specific spermatogenic cell populations, including early meiotic prophase. Here we combined this methodology with next generation sequencing, which enabled the analysis of meiotic and postmeiotic gene expression signatures in mouse with unprecedented reliability. Interestingly, we found that a considerable number of genes involved in early as well as late meiotic processes are already on at early meiotic prophase, with a high proportion of them being expressed only for the short time lapse of lepto-zygotene stages. Besides, we observed a massive change in gene expression patterns during medium meiotic prophase (pachytene) when mostly genes related to spermiogenesis and sperm function are already turned on. This indicates that the transcriptional switch from meiosis to post-meiosis takes place very early, during meiotic prophase, thus disclosing a higher incidence of post-transcriptional regulation in spermatogenesis than previously reported. Moreover, we found that a good proportion of the differential gene expression in spermiogenesis corresponds to up-regulation of genes whose expression starts earlier, at pachytene stage; this includes transition protein-and protamine-coding genes, which have long been claimed to switch on during spermiogenesis. In addition, our results afford new insights concerning X chromosome meiotic inactivation and reactivation.
Conclusions
This work provides for the first time an overview of the time course for the massive onset and turning off of the meiotic and spermiogenic genetic programs. Importantly, our data represent a highly reliable information set about gene expression in pure testicular cell populations including early meiotic prophase, for further data mining towards the elucidation of the molecular bases of male reproduction in mammals.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-016-2618-1) contains supplementary material, which is available to authorized users.
... The use of recombinant cDNA probes has made it possible t o identify specific mRNAs in various testicular populations (Kleene et al., 1983;Distel et al., 1984;Huggenvik et al., 1984;Ponzetto and Wolgemuth, 1985) and therefore to examine the regulation of gene expression of testis-specific and stage-specific transcripts (Gizang-Ginsberg and Wolgemuth, 1 98 5 ). ...
Localization and stage-dependent levels of transferrin and sulfated glycoprotein-2 (SGP-2) mRNAs were examined in rat testes by in situ and soluble hybridization of mRNA with a single-stranded RNA probe prepared with the SP65 vector. Biotinylated RNA probes were identified in testicular tissue by using a biotinylated glucose oxidase-avidin system followed by a treatment with an appropriate electron carrier and a tetrazolium salt. This procedure demonstrated that the anatomical site of transferrin and SGP-2 gene expression was the Sertoli cells. Tritium-labeled RNA probes were visualized by radioautography. Negative and positive controls as well as in situ hybridization in Sertoli and myoid cells in culture indicated again that the cytoplasm of Sertoli cells was the anatomical site of transferrin and SGP-2 expression. Quantitative radioautography revealed cyclic variations in the level of both transferrin and SGP-2 mRNAs. The level of transferrin mRNA was relatively high from Stage I to Stage VIII. At Stage IX, the level decreased acutely and remained low in Stage X. The level of transcripts increased dramatically at Stage XIII, remaining high until Stage XIV. In the case of SGP-2 mRNAs, levels of transcripts were similar in most stages except at Stages VII and VIII, where higher levels were observed. These data were substantiated by similar results obtained by solution hybridization of both recombinant cRNAs with mRNAs from selected seminiferous tubules staged by transillumination. Thus, our results demonstrated a stage-specific regulation of transferrin and SGP-2 mRNA levels in Sertoli cells.
... In the literature, approximately 14 genes have been shown to be expressed exclusively in mouse male germ cells [22,23]. Many of them were originally identified in differential screening projects [42][43][44]. In these studies, male germ cellspecific cDNA molecules were isolated by screening with labeled cDNA prepared from male germ cells, selecting mainly for highly abundant cDNA molecules. ...
A large number of cDNA clones were isolated from an adult mouse testis cDNA library and partially sequenced. Sequence comparisons revealed that many of them displayed similarities to genes previously identified only in invertebrates and lower eukaryotes, including, e.g., one cDNA clone related to the Drosophila melanogaster suppressor of forked gene. Other cDNA clones were found to be related, but not identical, to rodent genes involved in a variety of cellular activities, e.g., signal transduction and tumor development. The RNA expression patterns of 258 cDNA clones were analyzed through use of Northern blot methodology. Forty-two novel cDNA clones were found to be expressed only in male germ cells, the majority of them during spermiogenesis. One male germ cell-specific mouse cDNA clone was found to be similar to tektin A1, a protein known to interact with the flagellar microtubules of sea urchin sperm.
... Although the sequence context surrounding of testicular genes. Identification and isolation of haploid-specific cDNAs has the third ATG codon is unfavourable for initiation of translation (Kozak, 1987;Grunert and Jackson, 1994), the next inframe been successfully attempted previously by differential screening (Kleene et al., 1983). The advantage of the differential downstream ATG codon (the fifth ATG) possesses a favourable sequence context. ...
Spermatogenesis is a complex differentiation process in which diverse stage-specific proteins are co-ordinately expressed. Previously, subtractive hybridization and differential hybridization have been used in the identification of differentially expressed mRNAs. Although these techniques have been successfully used they require large amounts of RNA and are time consuming. To overcome these problems we have made use of the recently described mRNA differential display technique. The technique is an effective method which can identify and separate cDNAs that are differentially expressed between various cell-types. By comparing RNA from testes of mature (.60 days old) and prepubertal (15‐16 days old) mice we have identified nine differential cDNA bands expressed in mature testes. The differential display cDNA band DDC8 was used to screen a testis cDNA library and the full length cDNA was isolated and sequenced. DDC8 cDNA is 1965 bp with an open reading frame of 533 amino acids which codes for a predicted hydrophilic protein with a calculated molecular weight of 62.04 kDa. RNase protection assays indicate DDC8 to be expressed during the postmeiotic stages of spermatogenesis and database searches using both nucleotide and amino acid sequences show DDC8 to have similarities to structural, cytoskeletal and associated proteins.
... This argues against a major role of H3K79 methylation in transcription during spermiogenesis in Drosophila. In mammals, transcription ceases in midspermiogenesis, with high levels of RNA found in haploid round spermatids (Hecht et al., 1986;Kleene et al., 1983). Concordantly, we detected active RNA polymerase II in the nuclei of spermatocytes and early round spermatids in rat testes, whereas H3K79 methylation and H4 hyperacetylation were only detectable in elongating spermatids, which are devoid of actively transcribing RNA polymerase II. ...
During spermiogenesis, haploid spermatids undergo extensive chromatin remodeling events in which histones are successively replaced by more basic protamines to generate highly compacted chromatin. Here we show for the first time that H3K79 methylation is a conserved feature preceding the histone-to-protamine transition in Drosophila melanogaster and rat. During Drosophila spermatogenesis, the Dot1-like methyltransferase Grappa (Gpp) is primarily expressed in canoe stage nuclei. The corresponding H3K79 methylation is a histone modification that precedes the histone-to-protamine transition and correlates with histone H4 hyperacetylation. When acetylation was inhibited in cultured Drosophila testes, nuclei were smaller and chromatin was compact, Gpp was little synthesized, H3K79 methylation was strongly reduced, and protamines were not synthesized. The Gpp isoform Gpp-D has a unique C-terminus, and Gpp is essential for full fertility. In rat, H3K79 methylation also correlates with H4 hyperacetylation but not with active RNA polymerase II, which might point towards a conserved function in chromatin remodeling during the histone-to-protamine transition in both Drosophila and rat.
The development of gametes was of much interest as the meiotic events are quite different between the two sexes. Female gametogenesis occurred during embryonic development whereas male gametogenesis continued into adulthood and was more easily studied. A practical concern, of much importance for domestic animal breeding, was the separation of X- and Y-bearing sperm and much effort was expended trying to do so. Also, it had generally been assumed that there was no gene expression after meiosis such that the evolutionary selection would only occur on the zygote and not on the gametes. This turned out not to be true and other mechanisms, intracellular bridges for the sharing of postmeiotically synthesized gene products, were found to mostly maintain gamete neutrality. However, the much studied t-alleles provided a major exception to this neutrality and the elucidation of the transmission ratio distortion they produced was a major project for mouse geneticists.
Using several actin isotype-specific cDNA probes, we found actin mRNA of two size classes, 2.1 and 1.5 kilobases (kb), in extracts of polyadenylated and nonpolyadenylated RNA from sexually mature CD-1 mouse testes. Although the 2.1-kb sequence was present in both meiotic and postmeiotic testicular cell types, it decreased manyfold in late haploid cells. The 1.5-kb actin sequence was not detectable in meiotic pachytene spermatocytes (or in liver or kidney cells), but was present in round and elongating spermatids and residual bodies. To differentiate between the beta- and gamma-actin mRNAs, we isolated a cDNA, pMGA, containing the 3' untranslated region of a mouse cytoplasmic actin that has homology to the 3' untranslated region of a human gamma-actin cDNA but not to the 3' untranslated regions of human alpha-, beta-, or cardiac actins. Dot blot hybridizations with pMGA detected high levels of presumptive gamma-actin mRNA in pachytene spermatocytes and round spermatids, with lower amounts found in elongating spermatids. Hybridization with the 3' untranslated region of a rat beta-actin probe revealed that round spermatids contained higher levels of beta-actin mRNA than did pachytene spermatocytes or residual bodies. Both probes hybridized to the 2.1-kb actin mRNA but failed to hybridize to the 1.5-kb mRNA.
RNA from immature mouse testes was shown to lack a low-molecular-weight c-abl transcript previously noted to be the predominant species in adult testes. The developmental pattern of appearance of this c-abl variant was determined by analyzing RNA obtained from purified populations of testicular cells in different stages of spermatogenesis. The appearance of the c-abl testicular variant was coincident with the entry of the germ cells into their haploid state and suggested that the regulated expression of this proto-oncogene may be important in the normal differentiation of the male germ line.
The nuclei of mouse spermatozoa contain two protamine variants, mouse protamine 1 (mP1) and mouse protamine 2 (mP2). The amino acid sequence predicted from mP1 cDNAs demonstrates that mP1 is a 50-amino-acid protein with strong homology to other mammalian P1 protamines. Nucleotide sequence analysis of independently isolated, overlapping cDNA clones indicated that mP2 is initially synthesized as a precursor protein which is subsequently processed into the spermatozoan form of mP2. The existence of the mP2 precursor was confirmed by amino acid composition and sequence analysis of the largest of a set of four basic proteins isolated from late-step spermatids whose synthesis is coincident with that of mP1. The sequence of the first 10 amino acids of this protein, mP2 precursor 1, exactly matches that predicted from the nucleotide sequence of cDNA and genomic mP2 clones. The amino acid composition of isolated mP2 precursor 1 very closely matches that predicted from the mP2 cDNA nucleotide sequence. Sequence analysis of the amino terminus of isolated mature mP2 identified the final processing point within the mP2 precursor. These studies demonstrated that mP2 is synthesized as a precursor containing 106 amino acids which is processed into the mature, 63-amino-acid form found in spermatozoa.
c-mos RNA transcripts have been previously detected in mouse gonadal tissue and in late-term embryos. Here, we show that they are also present at low levels in placenta and in adult mouse brain, kidney, mammary gland, and epididymis. Marked differences are observed in the size of the mos RNA transcripts detected in different tissues. All transcripts appear to end at the same 3' position, and the tissue-specific size variations appear to be due to the use of different promoters. For example, the testicular and ovarian RNA transcripts initiate approximately 280 and approximately 70 base pairs, respectively, upstream from the first initiation codon, but both end at a common site downstream from the mos open reading frame. The expression of mos is developmentally regulated in gonadal tissue. Thus, the level of mos transcripts in testes is low for the first 3 weeks after birth, increases at least 10-fold around day 25, and reaches adult levels by day 30. In contrast, ovaries from preweaning mice contain a higher level of mos mRNA compared to ovaries from adult mice. In cell fractionation experiments we show that mos transcripts are present in haploid germ cells. We find that these transcripts are associated with monosomes and polysomes. The peculiar pattern of mos expression in mouse gonadal tissue suggests a role for the c-mos proto-oncogene in germ cell differentiation.
The protamines are small, basic, arginine-rich proteins synthesized postmeiotically in the testes. Analysis of the regulation of synthesis of the protamine mRNA and protein is restricted by the difficulty in culturing and manipulating the cells in which transcription and translation occur. To avoid these problems, we have produced transgenic mice carrying fusion genes in which sequences 5' to the mouse protamine-2 gene have been linked to exons 2 and 3 of the mouse c-myc gene and, separately, to the simian virus 40 (SV40) early region. We show here that the prot.myc gene is correctly regulated; transcription is detected only in the round spermatids. In one family of transgenic mice carrying the 5' protamine-SV40 T-antigen fusion gene, SV40 early-region mRNA accumulated to the highest level in the testes but was also detected in the thymuses, brains, hearts, and preputial glands of the animals. Although we have demonstrated specific transcription of these fusion genes in the round spermatids, we were not able to detect the SV40 T-antigen protein.
Mouse testis contains two size classes of actin mRNAs of 2.1 and 1.5 kilobases (kb). The 2.1-kb actin mRNA codes for cytoplasmic beta- and gamma-actin and is found throughout spermatogenesis, while the 1.5-kb actin mRNA is first detected in postmeiotic cells. Here we identify the testicular postmeiotic actin encoded by the 1.5-kb mRNA as a smooth-muscle gamma-actin (SMGA) and present its cDNA sequence. The amino acid sequence deduced from the postmeiotic actin cDNA sequence was nearly identical to that of a chicken gizzard SMGA, with one amino acid replacement at amino acid 359, where glutamine was substituted for proline. The nucleotide sequence of the untranslated region of the SMGA differed substantially from those of other isotypes of mammalian actins. By using the 3' untranslated region of the testicular SMGA, a highly specific probe was obtained. The 1.5-kb mRNA was detected in RNA from mouse aorta, small intestine, and uterus, but not in RNA isolated from mouse brain, heart, and spleen. Testicular SMGA mRNA was first detected and increased substantially in amount during spermiogenesis in the germ cells, in contrast to the decrease of the cytoplasmic beta- and gamma-actin mRNAs towards the end of spermatogenesis. Testicular SMGA mRNA was present in the polysome fractions, indicating that it was translated. These studies demonstrate the existence of an SMGA in male haploid germ cells. The implications of the existence of an SMGA in male germ cells are discussed.
Gastrulation occurs between 6.5 and 8.0 days post coitum in the mouse and is associated with a dramatic increase in cell proliferation, being initiated when the embryo consists of some 500-600 cells and resulting in a neural plate stage embryo containing 50,000 cells or more. There is evidence to suggest that some of these areas may be determined with respect to their future development quite early in the process of gastrulation. During this period of development it is clear that the growth rate of the whole embryo, and of its constituent parts, is under intrinsic control and capable of controlled variation aimed presumably at generating an organogenesis stage embryo of a notional target size in which the parts are of appropriate relative size. Investigations in the mouse have shown that growth of the embryo parts and their co-ordination can be profoundly disturbed in early embryonic stages by insult with the cytotoxic agent, Mitomycin C. MMC is an alkylating agent that inhibits DNA synthesis. It causes cell death and reduces pre-organogenesis stage embryos to 10% of normal size as measured by cell number. During the progression of embryogenesis the normal co-ordination of development of different parts of the embryo is abnormal. Considerable and localized growth control is obviously operating in these stages, thus the proto-oncogenes with known growth regulatory functions were looked at as likely candidates controlling the dramatic growth patterns observed. During the gastrulation movements the primordial germ cells are located at the base of the allantois and appear to 'escape' the movements and rearrangements of gastrulation by migrating into the extraembryonic membranes. The primordial germ cells then appear to undergo their own differentiation, proliferate and migrate into the genital ridges. The germ cells multiply as they migrate until some 5000 cells ultimately invade the gonads. The proto-oncogene c-kit expression was analysed, as it has been recently identified with the dominant white spotting (W) locus of the mouse. The W mutations have pleiotropic effects on mouse development, one of which is to cause an impairment of proliferation and/or migration of primordial germ cells. It appears that the c-kit product is necessary for the maintenance of the primordial germ cells during their migratory/mitotically active phase. This finding holds true for overall expression of c-kit in structures of the developing embryo. As the testes develop and become functional, the germ cells undergo further differentiation. There is evidence from Y chromosome deletion mapping that there is a multiple copy gene on the long arm of the mouse Y which is needed for the normal development of the sperm head. A Y-specific genomic DNA sequence (Y353/B) has previously been described which is present in multiple copies on the long arm of the mouse Y and is specifically transcribed in the testis. Using quantitative Northern analysis it was shown that the RNA transcripts are from the multiple copies. In situ hybridization was used to demonstrate that the transcripts are In the germ line and not In the soma, and that the expression is predominantly in round spermatids. This was the first evidence for a Y chromosome gene being transcribed in the testis (and probably anywhere). Y353/B is thus a candidate for being the multiple copy 'sperm morphology gene' on the mouse Y chromosome.
To determine whether male germ cells contain specific storage sites for poly(A)+ RNAs, in situ hybridizations were performed with sections of rat testis and a [3H]polyuridylic acid probe. The highest levels of poly(A)+ RNA were found in spermatocytes and round spermatids, while lower levels of poly(A)+ RNA were detected in spermatogonia, elongated spermatids, Sertoli cells, myoid cells, fibroblasts, macrophages, and Leydig cells. No poly(A)+ RNA was detected in residual bodies of elongated spermatids. At stages IX-XI of the seminiferous cycle, the nuclei and cytoplasm of pachytene spermatocytes contained approximately equal amounts of poly(A)+ RNA, suggesting nuclear RNA storage and/or a reduced processing rate of mRNA precursors at this stage of germ cell differentiation. To examine the distribution of poly(A)+ RNAs in subcellular components of testicular cells, electron microscope radioautography was used. In germ cells and Sertoli cells, poly(A)+ RNA was often seen free in the cytoplasm or associated with the endoplasmic reticulum and was only occasionally found associated with mitochondria, lysosomes, lipid inclusions, and axonemes. As previously reported for the mRNAs of transition protein 1 and protamine 1 [Morales et al., J Cell Sci 1991; 100:119-131], no compartmentalization of poly(A)+ RNAs was detected in the cytoplasm of round and elongated spermatids. No poly(A)+ RNA was detected in association with the radial body and in most sections, the chromatoid body did not contain any significant amounts of poly(A)+ RNA.
DNA-associated proteins of spermatozoa are different from somatic cell histones. In mammals, arginine-rich protamines replace histones and transition proteins in spermatids during compaction of the chromatin (Poccia, 1986). The mouse protamine mRNAs are expressed in early spermatids by a haploid genome (Kleene et al., 1983). To find the exact stages of differentiation where the expression of spermatidal nucleoprotein mRNAs occur, we used three different cDNA-hybridization techniques to measure protamine 1, protamine 2 and transition protein (TP1) mRNA levels.
Spermatogenesis is a complex differentiative process, highly conserved in the course of evolution, represented by the ordered sequence of mitotic, meiotic and differentiative stages. The pioneering work of Oakberg (1956), Monesi (1965) and Clermont (1972) have clearly shown that these stages are characterized by a complex series of biosynthetic processes which make spermatogenesis an excellent model system for the study of molecular events related to cell differentiation. DNA, RNA and protein synthesis were initially studied by autoradiographic and histochemical techniques, and only later with biochemical analysis after the introduction of cell fractionation techniques (Lam et al., 1970; Meistrich, 1973) which made available germ cells at defined stage of differentiation. Further insight into the genetic events and the mechanisms which control germ cell differentiation seems now possible with the use of powerful tools like molecular biology and transgenic techniques.
In eukaryotic cells the expression of a gene is regulated at many different levels. In the nucleus, changes in chromatin structure and DNA methylation control the temporal and spatial transcription of genes. Although the time of transcription is frequently the primary regulator of gene expression in many tissues, including the testis, the actual time of synthesis of many proteins in male germ cells is often dependent upon posttranscriptional mRNA processing events. Posttranscriptional regulation is especially important towards the end of spermatogenesis because, in contrast to virtually every other eukaryotic cell type, nuclear transcription ceases during mid-spermiogenesis in male germ cells. This necessitates that mRNA storage and translational activation play prominent roles in the expression of a large number of spermatid and sperma-tozoal proteins that are synthesized in late stages of germ cell maturation. Many of the structural and isoprotein molecules of the spermatozoon are under this type of posttranscriptional control (reviewed in Eddy and O’Brien 1994; Erickson 1993; Handel 1987; Hecht 1993, 1995; Willison and Ashworth 1987; Wolgemuth et al. 1993).
The testis in mammals has to perform three principal functions. Firstly, it is the place where the male gametes are generated from undifferentiated stem cells (spermatogonia), through regulated processes of proliferation and reduction division. There is a continuous production of haploid nuclei enclosed within highly specialized transport systems (spermatozoa) capable of conveying the haploid nucleus through a relatively inimical environment (the female tract) to undergo specific nuclear fusion with the female gamete. Secondly, the testis is the organ producing the male sex steroid hormone, testosterone (and other hormones) in a regulated fashion (e.g. negative feedback through the pituitary-gonadal axis) to provide an appropriate gender-specific environment for the correct development and management of other organ systems. Thirdly, the testis is the principal organ of evolution, where small changes in the inherited genome are tolerated (encouraged) during spermatogenesis in order to provide individual variation and hence potential species adaptation. This function requires an organ-specific regulation of DNA replication, recombination and repair.
The term spermatogenesis describes the complex series of events in which a terminally differentiated cell, the spermatozoon, is produced from a stem cell [1-3]. The events leading to the formation of the species-specific-shaped spermatozoon are dependent on the products from a large number of temporally expressed genes, many unique to the testis [4]. Prominent among the organ-specific proteins expressed during spermatogenesis are a group of structural DNA-binding proteins.
To understand the transcriptional regulation of the mouse protamine 2 gene, the interactions between nuclear proteins and promoter DNA of the regulatory region was investigated. Within the promoter region, -170 to +8, two protein binding sites were identified. Site 1, -64 to -48, contained a cAMP responsive element, CRE, and site 2, -87 to -67, contained a CAAT-like element. In vitro phosphorylation of testis nuclear proteins increased the binding affinities for these two sites and also transcriptional activity of the mouse protamine 2 promoter.
During the development of pseudopodial spermatozoa of the nematode, Caenorhabditis elegans, protein synthesis stops before differentiation is completed. Colloidal gold conjugates of monoclonal antibody SP56, which binds to the surface of spermatozoa, and TR20, which recognizes the major sperm cytoplasmic protein (MSP), were used to label thin sections of testes embedded in Lowicryl K4M in order to follow polypeptides from their synthesis early in spermatogenesis to their segregation to specific compartments of the mature cell. Both antigens are synthesized in primary spermatocytes and are assembled into a unique double organelle, the fibrous body-membranous organelle (FB-MO) complex. However, the antigens are localized in different regions of this FB-MO complex. As described in detail, the assembly of proteins into the FB-MO complex allows both membrane and cytoplasmic components to be concentrated in the spermatids after meiosis. Then, the stepwise disassembly of this transient structure ensures delivery of each component to its final destination in the mature spermatozoan: MSP filaments in the fibrous body depolymerize, releasing MSP into the cytoplasm and the membranous organelles fuse with the plasma membrane, delivering SP56 antigen to the surface.
In a molecular screen for cDNAs that encode protamine RNA-binding proteins, we obtained seven independent clones that encode Tenr, a testis nuclear RNA-binding protein. Tenr is a 72-kDa protein that has one copy of a previously described RNA-binding domain. Northwestern blotting experiments showed that a maltose-binding protein-Tenr fusion binds to a variety of RNAs in vitro and that it does not bind to single-stranded or double-stranded DNA. The Tenr gene is transcribed exclusively in the testis, and its mRNA is restricted to cells from the pachytene spermatocyte stage through the round spermatid stage. Immunolocalization of the Tenr protein within the testis showed that it is first detected postmeiotically, demonstrating that the Tenr mRNA is under translational control. The Tenr protein is localized to round and early elongating spermatid cells, and confocal microscopy revealed a lattice-like nuclear distribution suggesting association with the nuclear scaffold. We suggest that the Tenr protein may be involved in testis-specific nuclear posttranscriptional processes such as heterogeneous nuclear RNA (hnRNA) packaging, alternative splicing, or nuclear/cytoplasmic transport of mRNAs.
The acrosome of the mammalian spermatozoon contains proacrosin that is autocatalytically activated to become the proteolytic enzyme acrosin during the process of fertilization. Tschesche et al [1982] isolated specific acrosin inhibitors and suggested that they block prematurely activated acrosin. Antibodies against acrosin inhibitors purified from boar spermatozoa were used to demonstrate the evolutionaary relationship and the developmental pattern of the inhibitors in mammals. Using immunofluorescent techniques the following results were obtained: (1) The spermatozoa of man, boar, bull, ram, rat, rabbit, beaver, and mole stained positive in the acrosomal portion. (2) The round-headed spermatozoa of patients with globozoospermia and those in the ejaculates of fertile men lacked immunostaining for the inhibitors. (3) During spermatogenesis in all species, immunofluorescence for the acrosin inhibitors was first demonstrable in haploid spermatids and increased thereafter during spermatid differentiation. The stained area was found adjacent to the nuclear membrane, the position of the acrosome. (4) During teratogenesis of round-headed spermatozoa, the immunofluorescent staining for the inhibitors becomes separated from the nuclear membrane of the spermatids and is lost in late spermatids. Since identical results have been described for acrosin and acrosomal membrane proteins both in spermatozoa and spermatids of mammalian species and during spermiogenesis of patients with globozoospermia, our results are consistent with the localisation of the inhibitors in the acrosome. Immunostaining of spermatozoa of species belonging to five different mammalian orders with the antibody against boar acrosin-inhibitors is indicative of an evolutionary conservation of the inhibitors and their underlying genes.
RNAfromsexually matureCD-1mouse testes. Although the2.1-kb sequence waspresent inbothmeiotic andpostmeiotic testicular cell types, it decreased manyfold inlate haploid cells. The1.5-kb actin sequence wasnotdetectable inmeiotic pachytene spermatocytes (orinliver orkidney cells), butwaspresent inroundandelongating spermatids andresidual bodies. Todifferentiate between theI8- andy-actin mRNAs,weisolated acDNA,pMGA,containing the3' untranslated region ofamousecytoplasmic actin thathashomology tothe3'untranslated region ofahuman -y-actin cDNAbutnottothe3'untranslated regions ofhumana-,i-, orcardiac actins. Dotblot hybridizations withpMGA detected highlevels ofpresumptive -y-actin mRNA inpachytene spermatocytes andround spermatids, withlower amounts found inelongating spermatids. Hybridization withthe3'untranslated region ofarat0-actin proberevealed thatroundspermatids contained higher levels of3-actin mRNA thandid pachytene spermatocytes orresidual bodies. Bothprobes hybridized tothe2.1-kb actin mRNA butfailed to hybridize tothe1.5-kb mRNA. Theactins arebelieved toplay major roles incell division, cell shape changes, secretory processes, phagocytosis, cell andorganelle motility, andmuscle contraction (1,8,11). At least sixdifferent vertebrate actins havebeenidentified, eachtheproduct ofadifferent gene(26, 34). Aminoacid sequencing studies haverevealed thatthemultiple actin isotypes haveevolved fromtwomajor classes, thecytoplas- micandthemuscle actins (15). Inmammals, twoforms of cytoplasmic actin, called ,B- and-y-actin, aregenerally found (26). During spermatogenesis, marked morphological andstruc- tural changes occur asthemalegamete differentiates (3). In this well-ordered process, stemcells, theprimitive typeA spermatogonia, undergo a series ofchanges toproduce highly differentiated spermatozoa. After mitotic spermato- gonial proliferation, theresulting preleptotene spermato- cytesdifferentiate: after duplicating their chromosomes, these functionally tetraploid cells enter meiosis withapro- longed prophase inwhich, atthepachytene stage, homolo- gouschromosomes pair andgenetic recombination occurs. Pachytene spermatocytes arethefirst developmental cell typeexamined inthis paper. Meiotic prophase Iendswith twosuccessive divisions without chromosome duplication, producing thehaploid spermatids, cells which overa2-week period inthemousebecomeelongated anddevelop atail, an elongated nucleus withhighly condensed chromatin, andan acrosome attheanterior end.Thehaploid roundspermatids and,later, elongating spermatids areexamined inthis study, asareresidual bodies, thecytoplasmic vesicles discarded at theendofspermiogenesis whentheelongating spermatids becomespermatozoa. Ofthesixvertebrate actins, onlythetwocytoplasmic actins havebeendetected inmousespermatogenic cells (16). Two-dimensional gelelectrophoresis ofinvivo-labeled mousetesticular proteins hasdemonstrated ahighlevel of radiolabeled -y-actin inmeiotic pachytene spermatocytes whichdecreases inlater haploid cell types, i.e., roundand
Mature sperm of an anuran amphibian Xenopus laevis contain six sperm-specific basic nuclear proteins (SP 1–6). In an attempt to understand how the amounts of SP produced are regulated during transcription and translation, the relative amounts of proteins and messenger RNA (mRNA), and the gene number in nuclei were compared between SP4 and SP5. Measurements of the peak areas of proteins separated by reversed-phase high performance liquid chromatography (HPLC) indicated that SP4 is present in about a five times greater amount than SP5. Nuclease S1 protection assays showed the existence of SP4 mRNA in about a five times greater amount than SP5 mRNA. Southern hybridization analyses of restriction enzyme-digested genomic DNA indicated a single copy of SP5 gene in the haploid genome, as contrasted with the five SP4 genes found in our previous study. Thus the deposition of different amounts of SP4 and SP5 in sperm nuclei reflects the relative number of these genes in the genome.
The purpose of this review is to evaluate the available information concerning nuclear genes specifying sperm structures and functions important for fertilization, to indicate gaps in our understanding, and to suggest experiments that might increase our knowledge of this area. Most of this review is based on work with mice, since, except for humans, their genetics are the best known of the mammalian species. Genetic analysis of sperm fertilizing ability can be approached in several ways. First, one can examine the genetic basis for heritable defects in fertility. As yet most attention has been directed to mutations that cause sterility. Unfortunately, these are usually pleiotropic with effects not only on sperm attributes and sperm production but also on other body functions. Furthermore, only a few of these mutations have been investigated systematically [Chubb and Nolan, 1985; Sotomayor and Handel, 1986; Handel, 1987]. Less severe effects on sperm fertilizing ability have frequently been noted for rodents of various strains or carrying different Y chromosomes, although few attempts have been made to identify the genes responsible. An extension of this approach is to study hereditary abnormalities in sperm characteristics thought to be important for fertilization, such as morphology, motility, and enzyme levels. Another approach is to identify any genes active during sperm differentiation. Because the mature sperm has a condensed nucleus and very few ribosomes, gene expression (transcription and translation) important for fertilizing ability must occur in the diploid precursor of the sperm or in the spermatid prior to nuclear compaction. Genetic regulation of spermatogenesis in mice has recently been reviewed by Handel [1987]; however, genes active in haploid germ cells are specifically discussed here because of their potential for differentially affecting the fertilizing ability of sperm produced by a single individual. Finally, extensive studies of the t complex in the mouse provide strong evidence that in mammals the sperm's own genes can influence its fertilizing ability.
cDNA libraries have played a prominent role in developing the extensive database of gene expression in germ cells and somatic
cells of the mammalian testis. Differential screening of cDNA libraries has allowed investigators to determine the temporal
up-and downregulation of many genes. This chapter discusses how suppressive subtraction hybridization and cDNA sequencing
have been used to define populations of messenger RNAs (mRNAs) that selectively bind, or do not bind, to the germ cell-specific
Y-box protein, MSY2. MSY2 is an abundant DNA/RNA-binding protein that in vitro binds to all mRNAs, but shows selective binding
to a subset of male germ cell mRNAs in cells. This specificity is regulated by MSY2 binding to a conserved sequence in gene
promoters, which facilitates MSY2 binding to the transcripts from these promoters in the nucleus and coordinates the transport,
storage, and translational suppression of these mRNAs in the cytoplasm.
Interspecific matings between Heliothis virescens males and H. subflexa females result in the production of progeny normal in all respects except that the males are sterile. Recurrent backcrossing of fertile females to H. virescens males perpetuates testis dysfunction in these lines. Although the basis of this phenomenon is unknown, characteristic degenerative abnormalities in sperm mitochondria from backcross males suggest that the normal function of this organelle is disrupted. In order to test this notion, mitochondrial gene sequences have been isolated from a cDNA library constructed from H. virescens testis poly (A)+ RNA, aligned on the mitochondrial DNA physical map, and assigned genetic designations based upon their homology with sequenced mouse mitochondrial DNA restriction fragments. Hybridizations of these DNA sequences to RNA molecules from H. virescens and backcross testes revealed two major differences between the two lines: (1) the steadystate levels of mitochondrial transcripts from backcross testes were reduced approximately 3-fold relative to H. virescens; and (2) transcripts encoding mitochondrial proteins were not polyadenylated in backcross testes. Neither of these abnormalities appeared in mitochondrial transcripts from other tissues in backcross moths. These findings suggest that abnormal mitochondrial RNA metabolism is either a direct cause, or a manifestation of, the mechanism of sterility in backcross males.
During animal development and gametogenesis two DNA ligases are found and successively expressed. In this study the two DNA
ligases present in the axolotl egg and the two ligases present during ram sperm cell maturation were distinguished by biochemical
and immunological methods. The expression of the genes for the heavy and light ram DNA ligases has been studied using transplantation
of spermatid and sperm nuclei in axolotl eggs. We found that ram DNA ligases were expressed in axolotl egg cytoplasm. The
exclusion phenomenon between the heavy and light form of DNA ligase is species-specific and involves a cytoplasmic mediator.
In the transplanted ram germ cell nuclei the heavy ram DNA ligase expression was found to be sensitive to inhibitors of transcription
while the light one was not. When mRNA was used, no exclusion process was observed and both the heavy and light enzyme expression
were sensitive to cycloheximide and not to aamanitin. These results are discussed in terms of the possible stability of the
gene-regulated state following nuclear transfer.
The human Y chromosome contains a number of genes and gene families that are necessary for spermatogenesis. Many of these genes are embedded in repetitive elements that are subject to deletion events. Deletions of azoospermia factor (AZF) regions AZFa, AZFb, and AZFc are found in approx 10–15% of men with either unexplained severe oligozoospermia or azoospermia. These deletions fall on different Y chromosome backgrounds and there is no evidence for a link between a Y-chromosome lineage and the presence or absence of an AZF deletion. Several partial AZFc deletions have been described. One of these, which removes around half of all the genes within the AZFc region, appears to be present as in inconsequential polymorphism in populations of northern Eurasia. A second deletion, termed gr/gr, results in the absence of several AZFc genes and has been suggested to be a genetic risk factor for spermatogenic failure. However, the link between the gr/gr deletion and infertility is more complex. First, the gr/gr deletion is actually not a single type of deletion but a combination of deletions that vary in size and complexity and result in the absence of different members of the deleted azoospermia (DAZ) gene family as well as other AZFc genes, such as CDY1. Second, there are regional or ethnic differences in the frequency of gr/gr deletions. In some Y-chromosome lineages, these deletion appear to be fixed and may have little influence on spermatogenesis. Third, these observations have influenced a number of association studies aimed to determine the relationship between the gr/gr deletion and male infertility. Consequently, some studies suggest that the gr/gr deletion confers a strong genetic susceptibility to reduced sperm counts, whereas others suggest that the genetic susceptibility may not exist or be limited to specific Y-chromosome haplotypes.
Sperm dysfunction is the single most common defined cause of infertility. Approximately 1 in 15 men are subfertile and the
condition is increasing in frequency. However, the diagnosis is poor and, excluding assisted conception, there is no treatment
because of our limited understanding of the cellular, biochemical, and molecular functioning of the spermatozoon. The underlying
premise of our research program is to establish a rudimentary understanding of the processes necessary for successful fertilization.
We detail advances in our understanding of calcium signaling in the cell and outline genetic and proteomic technologies that
are being used to improve the diagnosis of the condition.
Nearly 7% of men suffer from male factor infertility. In one-fourth of infertile males, the etiology remains unexplained.
Unlike other multifactorial disorders, gene-gene and gene-environment interactions in the regulation of male fertility have
been poorly characterized. A candidate-gene approach that incorporates biological information from model organisms is likely
to be critical in deciphering the genetic basis of idiopathic male fertility. Genes that fulfill essential roles in spermatogenesis
often have orthologs in several species wherein they serve similar functions. By using a comparative cross-species approach,
major susceptibility genes underlying male infertility can be identified in association studies. With a better understanding
of the molecular regulation of spermatogenesis, proper diagnosis and treatment of male infertility should be realized in the
foreseeable future.
The maintenance of genomic integrity is of key importance for gametogenesis. Nevertheless, the processes of DNA replication,
mitosis, and meiosis are surprisingly error-prone and subject to damage. Accordingly, a series of DNA repair mechanisms have
evolved that recognize and repair DNA damage and DNA replication errors to maintain the fidelity of the DNA sequence. Gradually
translating findings from targeted gene deletion and mutant mouse models to human male infertility, we have learned that the
processes of mitosis and meiosis require proper functioning of the entire DNA repair mechanism in the cell for normal fertility
to be present in the male. This chapter focuses on our current understanding of the processes required for the maintenance
of DNA integrity during spermatogensis.
The intracellular location of the mRNA for the testis-specific isozyme of phosphoglycerate kinase-2 (PGK-2) has been determined for two spermatogenic cell types. The mRNA activity for PGK-2 from the polysomal and nonpolysomal fractions of pachytene primary spermatocytes or round spermatids has been assayed by cell-free translation with the polypeptide products monitored by immunoprecipitation, followed by one-dimensional or two-dimensional electrophoresis and fluorography. The results reveal that the majority of PGK-2 mRNA activity of round spermatids was present in the polysomal fraction while the relatively less abundant PGK-2 mRNA of pachytene primary spermatocytes was present in the nonpolysomal fraction. No PGK-2 mRNA activity was observed in the cytoplasmic RNA from primitive type A spermatogonia or prepubertal Sertoli cells. These data indicate that mature PGK-2 mRNA first appears in the cytoplasm of spermatogenic cells during the prophase of meiosis and increases in amount after meiosis. Although mature PGK-2 mRNA is present in meiotic cells it is not actively translated until after meiosis has been completed. Thus, mRNA accumulation and translational mechanisms are involved in the control of phosphoglycerate kinase-2 synthesis during spermatogenesis.
Following intratesticular injection of [35S]methionine, the multiple isoforms of actin and tubulin from highly purified mouse testicular meiotic and post-meiotic cells have been analysed by high resolution two-dimensional gel electrophoresis. In pachytene spermatocytes both β and γ actin are synthesized, γ actin being made in a significantly greater amount. The relative proportion of synthesis of β and γ actin changes during spermiogenesis, β actin increasing and γ actin decreasing in round spermatids, elongating spermatids, and residual bodies. Both α and β tubulin are synthesized in approximately equal proportion in pachytene spermatocytes. In addition to the tubulin isoforms synthesized during meiosis, at least one new form of both α and β tubulin first appears in post-meiotic (haploid) cells. In elongating spermatids and residual bodies, the synthesis of α tubulin is drastically reduced.
We have used a human phosphoglycerate kinase-1 (PGK-1) cDNA clone to study expression of PGK-2 during mouse spermatogenesis. Hybrid selection, in vitro translation with product identification by 2-D gel electrophoresis demon-strated that the PGK-1 cDNA clone hybridized to PGK-2 mRNA in mouse testes. Northern analyses of RNA purified from separated spermatogenic cells demonstrated a large increase in abundance of PGK-2 mRNA in post-meiotic cells. Thus, post-meiotic transcription of PGK-2 mRNA is demonstrable with cloned DNA probes. Peer Reviewed http://deepblue.lib.umich.edu/bitstream/2027.42/44192/1/10540_2005_Article_BF01119630.pdf
Transgenic mice harboring a chimeric gene linking mouse protamine 1 5'-flanking sequence to the coding sequence of the simian virus 40 T-antigen develop spontaneous rhabdomyosarcomas of the right atria. The presence of the tumors is accompanied by dramatic elevations in plasma atrial natriuretic peptide (ANP) immunoreactivity (1,698 +/- 993 vs. 60 +/- 18 fmol/ml for controls) and hematocrit (56 +/- 8 vs. 51 +/- 2 for controls). The immunoreactive ANP (irANP) present in the tumors is similar in size to irANP found in normal mouse atria. ANP mRNA transcripts present in the tumors also appear to be very similar in overall size and 5'-termini to those produced in normal cardiac tissue. Microscopically, the tumors are composed of a disorganized array of densely packed abnormal-appearing cells. Immunocytochemistry and in situ hybridization analysis reveal considerable heterogeneity in ANP gene expression. ANP peptide and mRNA are detectable throughout the parenchyma of the tumors, but absolute levels of expression vary widely among different cells in the population. These tumors represent a potentially valuable model for the study of inappropriate ANP secretion and may provide a tissue source for the development of an ANP-producing atrial cell line.
Sequential reverse transcriptase, DNA polymerase, and S1 nuclease reactions can be employed to synthesize double-stranded DNA representing messenger RNA. Using reverse transcriptase products made from partially purified lysozyme, ovomucoid, and ovalbumin messengers from hen oviduct, we have characterized the Escherichia coli DNA polymerase I reaction. We have optimized for a high yield of full length second strands under conditions which require only a small amount of mRNA. The effects of several parameters (time, enzyme levels, salt concentration, monovalent cation, and temperature) on the length of products synthesized by DNA polymerase I have been investigated. Each has a significant influence on the proportion of products which are full length. Under our conditions the three reactions are efficient in synthesizing full length duplex DNA from partially purified mRNA fractions or from total poly(A)-containing RNA.
We have cloned double-stranded cDNA copies of a rat preproinsulin messenger RNA in Escherichia coli chi1776, using the unique Pst endonuclease site of plasmid pBR322 that lies in the region encoding amino acids 181-182 of penicillinase. This site was reconstructed by inserting the cDNA with an oligo(dG)-oligo(dC) joining procedure. One of the clones expresses a fused protein bearing both insulin and penicillinase antigenic determinants. The DNA sequence of this plasmid shows that the insulin region is read in phase; a stretch of six glycine residues connects the alanine at position 182 of penicillinase to the fourth amino acid, glutamine, of rat proinsulin.
A procedure is described which permits the isolation from the prepuberal mouse testis of highly purified populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, leptotene and zygotene primary spermatocytes, pachytene primary spermatocytes and Sertoli cells. The successful isolation of these prepuberal cell types was accomplished by: (a) defining distinctive morphological characteristics of the cells, (b) determining the temporal appearance of spermatogenic cells during prepuberal development, (c) isolating purified seminiferous cords, after dissociation of the testis with collagenase, (d) separating the trypsin-dispersed seminiferous cells by sedimentation velocity at unit gravity, and (e) assessing the identity and purity of the isolated cell types by microscopy. The seminiferous epithelium from day 6 animals contains only primitive type A spermatogonia and Sertoli cells. Type A and type B spermatogonia are present by day 8. At day 10, meiotic prophase is initiated, with the germ cells reaching the early and late pachytene stages by 14 and 18, respectively. Secondary spermatocytes and haploid spermatids appear throughout this developmental period. The purity and optimum day for the recovery of specific cell types are as follows: day 6, Sertoli cells (purity>99 percent) and primitive type A spermatogonia (90 percent); day 8, type A spermatogonia (91 percent) and type B spermatogonia (76 percent); day 18, preleptotene spermatocytes (93 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent).
A whole-mount electron microscope technique has allowed direct visualization of the transcription process in mouse spermatids. Thes observations have been supported by light and electron microscope autoradiographic techniques that employ [3H]uridine and [3H]arginine in attempts to clarify mechanisms of RNA synthesis and their relationship to nuclear histone changes throughout spermiogenesis. Early spermatid genomes are dispersed almost completely, whereas in later spermiogenic steps the posterior or flagellar nuclear region is readily dispersed and the anterior or subacrosomal nuclear region remains compact. Display of genome segments permits identification of regions where transcription complexes, presumably heterogeneous nuclear RNA species, are seen related to chromatin. These complexes appear as ribonucleoprotein chains, some of them of considerable length, decreasing progressively in number in late spermiogenic steps. This decrease coincides with diminishing rates of [3H]uridine incorporation. Two distinct patterns of chromatin have been identified: a beaded chromatin type associated with transcription complexes encounterd in early spermatids; and a smooth chromatin type not involved in transcriptive activity observed in advanced spermiogenic genomes. Protein particles staining densely with phosphotungstic acid become apparent in nuclei of spermatids after [3H]arginine incorporation becomes significant. There is no structural or autoradiographic evidence for the presence of nucleoli during spermiogenesis. From these data and from previous experimental findings, we conclude that: (a) spermatogonia, spermatocytes and Sertoli cells are transcriptionally expressed into heterogeneous nuclear RNA and preribosomal RNA species whereas transcription in spermatids is predominantly heterogeneous nuclear RNA; and (b) the modification of the chromatin patterns in late spermiogenic steps indicates a stabilized genome that restricts transcriptive functions.
Following intratesticular injection of [35S]methionine or [3H]leucine, four testicular cell types (pachytene spermatocytes, round spermatids, elongating spermatids and residual bodies) were purified from mouse testicular cell suspensions by unit gravity sedimentation and equilibrium density gradient centrifugation through Percoll. Measurement of the amount of radiolabeled amino acid incorporated into protein in the testicular cells revealed that for a constant number of cells, pachytene spermatocytes incorporated 5.4 times more isotope than round spermatids, which incorporated 2.4 times more isotope than elongating spermatids. Analysis by two-dimensional gel electrophoresis of the polypeptides synthesized in vivo in the four testicular cell types demonstrated qualitative and quantitative changes in protein synthesis during spermatogenesis. At the level of detection provided by the electrophoretic methods used, pachytene spermatocytes and round spermatids synthesized approximately equivalent numbers of polypeptides while the number of polypeptides synthesized in elongating spermatids and residual bodies was decreased. Quantitative changes for polypeptides ranging in molecular weight from 16,500 to 82,000 were detected during spermatogenesis. For each cell type examined, a minimum of 5% of the polypeptides appear to be either unique or greatly enriched. These studies indicate that the expression of a sizable number of polypeptides is specific to certain stages of spermatogenesis.
To study the role of low-abundance, embryonic muscle-specific gene transcripts, we have developed a method to screen cDNA clones from embryonic muscle for such sequences. The protocol involves two stages: first, partial enrichment for cDNA clones carrying possible embryo-specific sequences by selecting clones of low-abundance sequences; and second, determination, by hybridization to RNA attached to diazobenzyloxymethyl-paper, which sequences from this category are regulated in an embryonic muscle-specific manner during development. At least three different clones were obtained which hybridized to sequences present in early muscle development but absent, or present at relatively low levels, at late embryonic and adult muscle stages. Two of these clones were not muscle-specific because they hybridized to poly(A)+RNA from liver or brain or both. The third clone, 106A4, did not detectably hybridize to total poly(A)+RNA at any stage of brain or liver development tested. This sequence also was not detectable in poly(A)+RNA from embryonic muscle progenitor cells. Thus, the 106A4 sequence is a likely candidate for an embryonic muscle-specific sequence. We have demonstrated that the 106A4 sequence is a mRNA, although the specific identity and function of the translated product is unknown. The method used to identify embryonic muscle-specific cDNA clones should be generally applicable for obtaining clones for low abundance transcripts regulated in a tissue-specific or developmental stage-specific manner.
Cells were labelled by intratesticular injection of [35S]methionine. After 14-16 h the relative rates of incorporation of label in spermatocytes, early spermatids and late spermatids were 10:2:1 respectively. Approximately 15% of the soluble (100 000 g supernatant) and 20% of the particulate proteins solubilized by NP-40 (from the 100 000 g pellet) that were detectable on two-dimensional gels showed stage-specific synthesis. A large number of proteins were detectable only in post-meiotic cells and may be valuable for studying the control of gene expression in haploid cells.
The procedure for the determination of nucleic acids described in this chapter is based on the finding that nucleic acids can be separated from other tissue compounds by their preferential solubility in hot trichloroacetie acid. The isolated nucleic acids are then quantitated by means of colorimetric reactions involving the pentose components of the nucleic acids. The determination of nucleic acids in tissues is largely a problem in identification. By means of the extraction procedures described in the chapter and the colorimetric reactions of peptide nucleic acid and DNA, a considerable degree of specificity is placed on the determination of these compounds. Occasionally however, false results will be obtained, owing to the presence of materials in the nucleic acid extracts that interfere with the pentose reactions. It is emphasized that the extraction methods described were developed for nucleic acid determinations by spectrophotometric methods. Although it was at first thought that these procedures might be directly applicable to isotopic work, it has become quite clear that the separations are not sufficiently refined for such studies. The methods have served, however, as starting points for other separation procedures more suitable for isotopic work.
Following intratesticular injection of [35S]methionine or [3H]leucine, four testicular cell types (pachytene spermatocytes, round spermatids, elongating spermatids and residual bodies) were purified from mouse testicular cell suspensions by unit gravity sedimentation and equilibrium density gradient centrifugation through Percoll. Measurement of the amount of radiolabeled amino acid incorporated into protein in the testicular cells revealed that for a constant number of cells, pachytene spermatocytes incorporated 5.4 times more isotope than round spermatids, which incorporated 2.4 times more isotope than elongating spermatids. Analysis by two-dimensional gel electrophoresis of the polypeptides synthesized in vivo in the four testicular cell types demonstrated qualitative and quantitative changes in protein synthesis during spermatogenesis. At the level of detection provided by the electrophoretic methods used, pachytene spermatocytes and round spermatids synthesized approximately equivalent numbers of polypeptides while the number of polypeptides synthesized in elongating spermatids and residual bodies was decreased. Quantitative changes for polypeptides ranging in molecular weight from 16,500 to 82,000 were detected during spermatogenesis. For each cell type examined, a minimum of 5% of the polypeptides appear to be either unique or greatly enriched. These studies indicate that the expression of a sizable number of polypeptides is specific to certain stages of spermatogenesis.
A method for obtaining highly purified fractions of rat testicular cells is described. Single cell suspensions from adult rat testes were separated by centrifugal elutriation. Fractions enriched in pachytene primary spermatocytes, early spermatids, and cytoplasts detached from late spermatids were obtained. These fractions were further separated by equilibrium density centrifugation on gradients of Percoll. In this manner fractions of 3 x 10⁷ pachytene spermatocytes (98% purity), 1.1 x 10⁸ early spermatids (93% purity), and 1.1 x 10⁸ cytoplasts (98% purity) were obtained within 6 h after sacrificing the rats. The cells appeared to be morphologically intact and to have retained their biochemical integrity.
Analysis of acid-soluble nuclear proteins by polyacrylamide gel electrophoresis showed that histone 4 is synthesized during the pachytene stage, and confirmed that testis-specific histones are also synthesized during this stage. Analysis of a microsomal RNA preparation from purified pachytene spermatocytes and purified early spermatids by sucrose gradients indicated that intact ribosomal RNA (rRNA) can be obtained from purified cells. Both cell types are active in synthesizing presumptive messenger RNA (mRNA) with a wide range of sedimentation values, but no appreciable rRNA synthesis was detected.
The quantity of poly(A)-containing RNA is measured in Xenopus laevis oocytes as a function of developmental stage. The amount of poly(A)-containing RNA per oocyte, 0.7 to 1.0% of the total RNA, remains relatively constant from early vitellogenesis until ovulation. It is largely present in the cytoplasm of the oocyte in the form of a ribonucleoprotein complex. The poly(A) sequence is approximately 100 bases in length and is attached to molecules of heterogeneous sedimentation coefficients.
Poly(A)+RNA transcripts were prepared from Lilium microsporocytes at stages spanning premeiotic interphase through pachytene. Transcripts were also prepared from somatic tissues of the anthers. The transcripts were reverse transcribed and cDNA clones prepared using pBR322 as a vehicle. Thirteen clones derived from the most abundant meiotic transcripts which were unique to the meiocytes were selected for study as meiotic-specific clones. The cloned inserts contained repeated sequences shared by at least eleven of the cDNAs. When hybridized to restriction digests of total DNA, the hybrids showed a complexity of sequence associations. The cDNAs were found to be homologous with DNA sequences from wheat, rye, and maize. Despite the complexity of sequence associations, melting curves of the hybrids formed between the cloned inserts of lily and wheat DNA indicated a high degree of sequence conservation.
Round spermatids and elongating spermatids were purified from a suspension of mouse testicular cells by sedimentation at unit gravity coupled with density gradient centrifugation through Percoll. Following separation, the two cell types were fractionated into polysomal and non-polysomal compartments. By comparison with round spermatids, elongating spermatids contain about one-half as much cytoplasmic RNA per cell, one sixth as much poly(A)+ RNA per cell and one-half the concentration of poly (A)+ mRNA in their cytoplasm. About two-thirds of the poly(A)+ messenger RNA (mRNA) was in the non-polysomal fraction in both cell types. Polypeptides whose synthesis was directed by cell-free translation of purified mRNA from each cell fraction were analyzed by two-dimensional gel electrophoresis. At the level of detection provided by the electrophoretic methods used, the majority of peptides from the polysomal and non-polysomal compartments for each cell type were similar. However, between the two cell types, approx. 5–10% of the polypeptides in the polysomal and non-polysomal fractions differed markedly in abundance. When the polypeptides encoded by the polysomal and non-polysomal mRNA from round spermatids were compared to the polypeptides encoded in the equivalent fractions from elongating spermatids, a significant reduction in number of polypeptides from elongating spermatids was seen. The presence of specific mRNAs in the non-polysomal fraction of round spermatids and in the polysomal fraction of elongating spermatids suggests that storage of mRNA in the cytoplasm and subsequent utilization provides a source of mRNA for proteins expressed at a time during spermiogenesis when transcription has terminated.
The intracellular location of the mRNA for the testis-specific isozyme of phosphoglycerate kinase-2 (PGK-2) has been determined for two spermatogenic cell types. The mRNA activity for PGK-2 from the polysomal and nonpolysomal fractions of pachytene primary spermatocytes or round spermatids has been assayed by cell-free translation with the polypeptide products monitored by immunoprecipitation, followed by one-dimensional or two-dimensional electrophoresis and fluorography. The results reveal that the majority of PGK-2 mRNA activity of round spermatids was present in the polysomal fraction while the relatively less abundant PGK-2 mRNA of pachytene primary spermatocytes was present in the nonpolysomal fraction. No PGK-2 mRNA activity was observed in the cytoplasmic RNA from primitive type A spermatogonia or prepubertal Sertoli cells. These data indicate that mature PGK-2 mRNA first appears in the cytoplasm of spermatogenic cells during the prophase of meiosis and increases in amount after meiosis. Although mature PGK-2 mRNA is present in meiotic cells it is not actively translated until after meiosis has been completed. Thus, mRNA accumulation and translational mechanisms are involved in the control of phosphoglycerate kinase-2 synthesis during spermatogenesis.
A procedure is described for screening bacterial colonies containing recombinant plasmids by nucleic acid hybridization at high density, i.e., at 100 000 colonies per 150 mm diameter plate. Small colonies are established on nitrocellulose filters from which they can be faithfully replicated to additional filters. Chloramphenicol amplification may be carried out in situ before screening. The filters may be kept frozen for long-term storage of colonies which may be further replicated after thawing.
A method utilizing sequential enzymatic incubation in collagenase (1 mg/ml) and trypsin (2.5 mg/ml) has been developed for the dissociation of the seminiferous epithelium. A significant advantage of this method is that, following collagenase incubation and washings in an enriched Krebs-Ringer bicarbonate buffer solution, isolated seminiferous tubules are obtained which are free of interstitial cells. The “purified” seminiferous epithelium is then dissociated with trypsin. A further advantage of this dissociation technique has been a reduction in the number of symplasts (multinucleate cells) which form by the opening up of the intercellular bridges that occur between synchronously differentiating clusters of germ cells. Both the elimination of the interstitial cells and the reduction in the number of symplasts have made possible the recovery of more highly enriched germ cell fractions. The homogeneity of the cell fractions was determined by light and electron microscopy. Integrity of the isolated cells was verified by Trypan blue exclusion and measurement of oxygen consumption.
This chapter discusses the cloning of hormone genes from a mixture of complementary deoxyribonucleic acid (cDNA) molecules. The molecular cloning of cDNA synthesized from a purified messenger ribonucleic acid (mRNA) is a well-established method for obtaining purified DNA for sequence analysis and use as a hybridization probe. Only a limited number of purified mRNAs are currently available that include those from specialized cells producing predominantly a single protein, such as globin, immunoglubin, or ovalbumin. The chapter describes the procedures used to isolate and clone specific hormone cDNAs from an impure mRNA population. The method employs the restriction endonuclease cleavage of double-stranded cDNA transcribed from a complex mixture of mRNA. The method does not require any extensive purification of RNA but instead makes use of the transcription of RNA into cDNA, the sequence-specific fragmentation of this cDNA, with one or two restriction endonucleases, and the fractionation of the cDNA restriction fragments on the basis of their lengths. The use of restriction endonucleases eliminates size heterogeneity and produces homogeneous-length DNA fragments from any cDNA species that contains at least two restriction sites. From the initially heterogeneous population of cDNA transcripts, uniform-sized fragments of desired sequence are produced. The chapter discusses a complete scheme for cDNA cloning.
This chapter describes and evaluates separation methods employing two physical parameters— density and sedimentation rate. The chapter also discusses the use of ultrasound or enzymes to destroy cells and nuclei selectively. The techniques, as currently being used in the laboratory are outlined. Velocity sedimentation, equilibrium density centrifugation, and ultrasonic disruption are used to obtain enrichment of all classes of cells and nuclei from adult rodent testes. Several types of nuclei, including pachytene spermatocytes, round spermatids, early elongated spermatids, and late elongated spermatids can be obtained in purities by using the appropriate combinations of methods. The important factors and principles involved in the development of the separation methods are emphasized. The chapter reviews the methods developed in other laboratories and compares the results obtained with the different procedures. Using the cell separation methods, spermatogenesis as a model of cell differentiation can be exploited and the sequence of macromolecular events involved in sperm development can be mapped. The availability of purified populations of specific testicular cell types will permit the elucidation of other important events of spermatogenesis, such as genetic recombination, meiosis, spermatid morphogenesis, and nuclear inactivation and condensation.
It appears that a major control mechanism for the regulation of the expression of protamine genes in differentiating trout testis is at the translational level. The group of protamine mRNAs are transcribed in cells at an early stage of development, the primary spermatocyte, processed and stored in mature 16-18S mRNP particles in the cell sap of succeeding cell stages, secondary spermatocytes and early spermatids. The period of storage in the rainbow trout testis is of the order of 15-30 days. These 'stored' mRNP particles must then undergo an as yet unknown 'activation process' in middle spermatid cells which induces subsequent binding to ribosomal subunits to form di-ribosome complexes actively translating protamine polypeptides.
Intact ribonucleic acid (RNA) has been prepared from tissues rich in ribonuclease such as the rat pancreas by efficient homogenization in a 4 M solution of the potent protein denaturant guanidinium thiocyanate plus 0.1 M 2-mercaptoethanol to break protein disulfide bonds. The RNA was isolated free of protein by ethanol precipitation or by sedimentation through cesium chloride. Rat pancreas RNA obtained by these means has been used as a source for the purification of alpha-amylase messenger ribonucleic acid.
An improved one-step method for the extraction of RNA from rat brain is described. Fresh or frozen tissue is disrupted in the powerful protein denaturant guanidine thiocyanate, and RNA isolated by ultracentrifugation through CsCl. The procedure is advantageous in that it is relatively simple, is rapid and does not expose the sample to enzyme treatments or repeated organic extractions.
Mouse male germ cells at middle-late pachytene and early spermatid stages obtained by velocity sedimentation at unit gravity in albumin gradients were labelled in culture with [3H]uridine. The newly synthesized RNAs extracted from polysomes of the 2 cell types were studied by sucrose gradient fractionation and poly(U) Sepharose chromatography. The results showed that round spermatids, as well as pachytene spermatocytes, synthesize both ribosomal and polyadenylated RNA molecules. Since these latter are engaged in polysomes they are presumably active messenger RNA molecules.
Terminal deoxynucleotidyl transferase, which requires a single-stranded DNA primer under the usual assay conditions, can be made to accept double-stranded DNA as primer for the addition of either rNMP or dNMP, if Mg+2 ion is replaced by Co+2 ion. The priming efficiency in the presence of Co+2 ion with respect to initial rate tested with 2 single-stranded primer, is 5-6 fold higher than that observed with Mg+2 ion. In the presence of Co+2 ion, the primer specificity is altered so that all forms of duplex DNA molecules can be labeled at their unique 3'-ends regardless of whether such ends are staggered or even. Thus, using ribonucleotide incorporation, we have for the first time employed this reaction for sequence analysis of duplex DNA fragments generated by restriction endonuclease cleavages. Furthermore, by using Co+2 ion, it is possible to add a long homopolymer tract of deoxyribonucleotides to the 3'-terminus of double-stranded DNA. Therefore, without prior treatment with lambda exonuclease to expose the 3' terminus as single-stranded primer, this reaction now permits insertion of homopolymer tails at the 3'-ends of all types of DNA molecules for the purpose of in vitro construction of recombinant DNA.
The rate of RNA synthesis at different stages of spermatogenesis in the mouse, and the preservation of RNA from the diploid to the haploid phase of spermatogenesis were studied in homogeneous germ cell fractions separated by velocity sedimentation at unit gravity. The uridine pool expansion method was used for determining the rate of RNA synthesis: seminiferous tubules were labelled in culture with increasing concentrations of [3H]-uridine and the incorporated radioactivity was estimated in cell fractions separated by velocity sedimentation. The results indicate that before nuclear elongation, round spermatids (steps 1 to 8 of spermiogenesis) synthesize RNA at the same rate per DNA content as middle-late pachytene spermatocytes. The preservation of RNA molecules synthesized in meiosis was investigated by labelling pachytene spermatocytes with T3H]uridine in vivo and collecting samples of germ cells at definite stages of spermatogenesis at various time intervals thereafter. The results show that a considerable proportion the RNA synthesized during the pachytene stage is preserved through spermatid development until late spermiogenesis.
Dramatic transitions occur in the nuclear proteins during spermiogenesis in rats. In order to determine more precisely when these transitions occur, we have employed centrifugal elutriation, velocity sedimentation at unit gravity, centrifugation in metrizamide gradients, and sonication to obtain relatively homogeneous populations of testis cells and nuclei. The results indicate that histones are present in step 1–8 spermatid nuclei but are not detectable after step 12. Nuclear proteins designated TP and TP2 are not detectable in step 1–8 spermatids but are present and actively synthesized in step 13–15 spermatids. These two proteins are turned over within 5 days after synthesis. A spermatidal basic nuclear protein, designated TP3, and the sperm basic nuclear protein, S1, are present in step 16–19 spermatids. Biochemical characterization of TP2 and TP3 are presented.
A convenient technique for the partial purification of large quantities of functional, poly(adenylic acid)-rich mRNA is described. The method depends upon annealing poly(adenylic acid)-rich mRNA to oligothymidylic acid-cellulose columns and its elution with buffers of low ionic strength. Biologically active rabbit globin mRNA has been purified by this procedure and assayed for its ability to direct the synthesis of rabbit globin in a cell-free extract of ascites tumor. Inasmuch as various mammalian mRNAs appear to be rich in poly(adenylic acid) and can likely be translated in the ascites cell-free extract, this approach should prove generally useful as an initial step in the isolation of specific mRNAs.
Identification of cells has been made in stained smears of cell suspensions prepared from mouse testes and separated by velocity sedimentation at unit gravity. Comparison of various methods of producing suspensions demonstrated that the best cell separations were achieved using suspensions prepared with trypsin. Various fractions obtained following separation contained 29% Sertoli cells sedimenting at about 14 mm/h, 17% Leydig cells at 11 mm/h, 73% pachytene spermatocytes at 9.5 mm/h, 54% binucleate spermatids and 14% secondary spermatocytes at 6.7 mm/h, 77% round spermatids at 4.5 mm/h, 21% elongating spermatids and 74% cytoplasmic fragments detached from these spermatids at 2.1 mm/h and 37% late spermatids at 0.75 mm/h. The resolution of different size classes of cells was essentially complete, but separation of different types of cells was limited by the occurrence of multinucleate forms of the cells and by fragments of damaged elongated spermatids. Most cells, however, appeared to be intact on light microscopical examination.
A quick and simple procedure based upon CsCl ultracentrifugation for the isolation of RNA from disrupted cells or subcellular fractions is described. rRNA is obtained intact and can be recovered in yields close to 100%. It is shown that the translation of rabbit globin mRNA is not impaired by exposure to this treatment. The yield of poly(A) containing globin mRNA extracted by this method and partially enriched on a poly(U)-Sepharose column was assayed in wheat germ S-30 extracts along with that extracted with phenol undergoing identical treatment. RNA from the CsCl extraction yields RNA six times more active in globin synthesis. It is observed that specific aggregation takes place during CsCl centrifugation at high gravitational fields diminishing the amount of the 16S or 18S rRNA component but not 26S or 28S component. The aggregation can be avoided by slow-speed centrifugation or reversed by heating at 60° for 30 sec. The CsCl centrifugation appears to be superior to phenol extraction in terms of simplicity, processing time, yield, intactness, and biological activity of resulting RNA. The suitability of the method for small amounts of starting material and one step separation of macromolecules from each other seem to recommend the CsCl centrifugation as a general method of isolating RNA over other methods currently in use.
When annealed with synthetic polynucleotides and treated with ribonuclease under appropriate conditions, poly(U) forms the ribonuclease-resistant complexes poly(rA) · poly(U) (1:1), poly(dA) · 2poly(U) (1:2) and poly · (dA)poly(dT) · poly(U) (1:1:1). This forms the basis of a quantitative assay of poly(rA), poly(dA) and poly(dA) · poly(dT) sequences in unlabelled nucleic acids. Using this assay, duck haemoglobin messenger RNA is shown to contain a poly(rA) sequence approximately 100 nucleotides long.Eukaryotic DNAs contain small amounts of sequences that react with poly(U). In the case of duck DNA, these sequences are considerably shorter than the mRNA-associated sequences and are interspersed widely with other sequences. It is concluded that if duck DNA does contain poly(dA) sequences corresponding to mRNA-associated poly(rA) sequences, there are fewer than 8000 of these per haploid genome.
Escherichia coli cells of strain K12 and C can be made competent to take up temperate phage DNA without the use of “helper phage”. This competence is dependent on the presence of calcium ions and is effective for both linear and circular DNA molecules.
Poly(A)-containing protamine messenger ribonucleoprotein particles [poly(A+) pmRNP particles] have been isolated from the polysomal and free cytoplasmic subcellular fractions of trout testis cells by a two-step isolation procedure. Ethylenediaminetetraacetic acid (EDTA) treated particles from both cytoplasmic fractions were first fractionated by sucrose gradient centrifugation and the putative pmRNP particles localized by utilizing 3H-labeled protamine complementary DNA (pcDNA) probes. In addition, particles present in these fractions were characterized by their translational activity in the heterologous, rabbit reticulocyte cell-free system and the protein components of crude mRNP complexes analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoesis. The final purification step involved affinity chromatography of pooled gradient fractions on oligo(dT)-cellulose from which intact pmRNP could be eluted with distilled water at 40 degrees C. Highly purified particles from both polysomal and free cytoplasmic fractions prepared by this procedure had buoyant densities of 1.35-1.37 g/cm3 in CsCl or a protein content of approximately 82%. Particles isolated from EDTA-dissociated polysomes were actively translated in vitro, while their free cytoplasmic counterparts were not. High salt washed pmRNP particles or the RNA extracted from pmRNP preparations, however, directed the synthesis of trout protamines in this system. A model of the activation of stored pmRNP particles in vitro and in vivo is presented.
We have investigated the changes occurring in the pattern of translatable mRNA species in germ cells during spermatogenesis of mice. Highly homogeneous cell populations of spermatocytes or spermatids were purified. Poly(A)+ mRNA was isolated from each cell population by oligo(dT)-cellulose chromatography. A comparison of meiotic and post-meiotic mRNAs was made by two-dimensional gel analyses of their in vitro synthesized translation products. Among spots identified in the fluorograms of two-dimensional gels, a number of qualitatively new proteins appeared after meiosis. The results suggest that some of poly(A)+ mRNAs are transcribed post-meiotically in haploid germ cells.
A simple and rapid method for transferring RNA from agarose gels to nitrocellulose paper for blot hybridization has been developed. Poly(A)+ and ribosomal RNAs transfer efficiently to nitrocellulose paper in high salt (3 M NaCl/0.3 M trisodium citrate) after denaturation with glyoxal and 50% (vol/vol) dimethyl sulfoxide. RNA also binds to nitrocellulose after treatment with methylmercuric hydroxide. The method is sensitive: about 50 pg of specific mRNA per band is readily detectable after hybridization with high specific activity probes (10(8) cpm/microgram). The RNA is stably bound to the nitrocellulose paper by this procedure, allowing removal of the hybridized probes and rehybridization of the RNA blots without loss of sensitivity. The use of nitrocellulose paper for the analysis of RNA by blot hybridization has several advantages over the use of activated paper (diazobenzyloxymethyl-paper). The method is simple, inexpensive, reproducible, and sensitive. In addition, denaturation of DNA with glyoxal and dimethyl sulfoxide promotes transfer and retention of small DNAs (100 nucleotides and larger) to nitrocellulose paper. A related method is also described for dotting RNA and DNA directly onto nitrocellulose paper treated with a high concentration of salt; under these conditions denatured DNA of less than 200 nucleotides is retained and hybridizes efficiently.
Mouse testicular cells labelled in vivo with 3H-uridine for 1 hour were separated into enriched cell populations representing different stages of spermatogenesis by centrifugation in an Elutriator rotor. RNA extracted with phenol and chloroform was sized by electrophoresis on 2.4% acrylamide gels. The percentage of newly synthesized RNA which was not ribosomal RNA (or its precursors) and was not transfer RNA, was higher in early postmeiotic, as compared to late premeiotic, stages. RNA was also extracted from fractionated cells in the presence of guanidinium chloride and the proportion of total 3H-RNA-containing poly(A) sequences was determined by binding to an oligo-(dT)-cellulose column. RNA that bound in 0.5 NaCl was eluted with low salt buffer and reapplied twice more, after heating each time in DMSO to disaggregate any non-poly(A)-containing RNA. The percentage of newly synthesized RNA which contained poly(A) did not decrease in early postmeiotic, as compared to late premeiotic, stages. We suggest that at least some part of the 6-15S 3H-RNA and 3H-poly(A)-containing RNA represents mRNA(s) transcribed postmeiotically in haploid germ cells.
The development of spermatozoon (sperm) from a spermatid involves a complex process of differentiation during which a variety of new gene products appear. It has been generally assumed that no genetic transcription occurs after meiosis and, if this were so, that all the new sperm proteins would have to to be transcribed from stored messenger RNA. However, the biochemical evidence suggests that there is no abrupt change in the rate of RNA synthesis during meiosis and that qualitative changes in RNA synthesis, to the extent that they are known, favor the likelihood of continuing messenger RNA synthesis. Experimental analyses of distorted transmission ratios of t-alleles and unbalanced chromosomal states in makes also suggest that genes are expressed in haploid nuclei after meiosis. It is probable that spermatozoa are functionally equivalent in most respects because of intercellular bridges that create a continuous cytoplasm between developing spermatozoa, facilitating an exchange of most postmeiotic gene products. Plasma membrane proteins which are potential antigens might not be shared across the intercellular bridges but the evidence to date for haploid expression of sperm antigens is poor.
Messenger ribonucleic acid (mRNA) from fractionated mouse testis has been used as a template in the wheat germ and reticulocyte lysate cell-free translation systems. Cell-free translation products of deproteinized RNA from testis polysomes and from a nonpolysomal fraction (< 80 S) have been compared by one- and two-dimensional polyacrylamide gel electrophoresis, followed by autoradiography. Wheat germ and reticulocyte ribosomes translate both polysomal and nonpolysomal RNA from testis with high efficiency. Analysis of the polypeptide products of these cell-free translation systems indicates a compartmentalization between polysome-bound and nonpolysomal mRNA in testis. With the assumption of equal template efficiency for those RNAs tested, three classes of radiolabeled polypeptide products have been distinguished: (1) polypeptide bands which represent an equal abundance of mRNA in each cell compartment; (2) polypeptide bands which represent a higher abundance of mRNA in the polysomal than in the nonpolysomal compartment; (3) polypeptide bands which represent a higher abundance of mRNA in the nonpolysomal than in the polysomal compartment. Compared with liver, the testis contained a larger proportion of ribosomes present as monosomes. Further, more poly(A)+ RNA and an equal or greater template activity were found to be associated with the nonpolysomal portion of testis cytoplasm than the polysomal fraction, suggesting that testis does not have as large a proportion of its messenger RNAs actively involved in protein synthesis as does liver. A comparison of cell-free translation products of deproteinized and nondeproteinized RNAs obtained from total testis cytoplasm had revealed similar polypeptide profiles with a few minor differences. These data suggest that some form of selective mRNA masking or sequestration in a subcellular compartment may be regulating the loading of specific nonpolysomal mRNAs onto polysomes.
Gene expression during murine spermatogenesis has been studied using highly enriched populations of cells obtained by velocity sedimentation at unit gravity and further purified by density gradient centrifugation through Percoll. Polypeptides whose synthesis was directed by total cytoplasmic RNA from round spermatids, pachytene spermatocytes, primitive type A spermatogonia, and Sertoli cells in cell-free translation systems have been compared by two-dimensional polyacrylamide gel electrophoresis, followed by fluorography. At the level of detection provided by the electrophoretic methods used, each population of cells contained mRNAs encoding over 200 polypeptides, many of which were present in high abundance in all four cell types. However, for each cell type examined, a minimum of 5-10% of these polypeptides appear to be either specific to or greatly enriched within a particular cell type. Analysis of the polysomal and nonpolysomal cell fractions from pachytene spermatocytes and round spermatids revealed that the two compartments share many identical mRNAs but specific mRNAs are selectively compartmentalized between the cell fractions and between the two cell types. Movement between compartments was seen; e.g., some polypeptides encoded by mRNA found primarily in the nonpolysomal fraction of pachytene cells were later seen in the polysomal fraction from round spermatids. Virtually every other combination was also observed. These results suggest that the control of gene expression at the level of selective production of mRNA and selective utilization of mRNA are among the mechanisms involved in regulation of spermatogenic cell differentiation.
Mouse sarcoma ascites cells contain several abundant mRNA species that occur to a large extent in an untranslated state. RNA preparations enriched in these species were used as starting material to construct recombinant plasmids. Cloned plasmids bearing sequences homologous to four of the untranslated mRNA species were identified by translation of hybrid-selected material. These plasmids, as well as a recombinant plasmid derived from chick alpha-actin mRNA, were used as probes for the estimation of mRNA levels in polyribosomes and in small ribonucleoprotein (RNP) particles of the ascites cells. Considerable amounts of the mRNA molecules belonging to the untranslated species were present in polyribosomes as well as in mRNPs. The actin mRNA, on the other hand, was present almost exclusively in polyribosomes. The distributions obtained by the hybridization assay resembled those estimated by translation of the same RNA preparations in cell-free systems. This indicates that the mRNA molecules of a given species engaged in translation in the cells and those present as untranslated RNP particles are equally effective in cell-free translation systems.
Total RNA, prepared from immature or mature mouse testes or from spermatogenic cells separated on the basis of sedimentation velocity, was translated in vitro. Mouse protamine-like histone could be identified as an in vitro translational product when [3H]arginine was used as the label. The mRNA for protamine-like histone was detected only after meiosis; the appearance of a peak of radioactivity comigrating with protamine-like histone occurred only when RNA from mature testes or late spermatid cell fractions was translated. Phosphoglycerate kinase-2 (ATP:3-phospho-D-glycerate I-phosphotransferase, EC 2.7.2.3) was identified as an in vitro translational product by affinity chromatography followed by two-dimensional gel electrophoresis or by specific immunoprecipitation when [35S]methionine was the label. The mRNA for phosphoglycerate kinase-2 was detected only in mature testes or late spermatid cell fractions. These translational assays for protamine-like histone and phosphoglycerate kinase-2 mRNAs suggest that these messages are transcribed after meiosis.
The complete DNA sequence of the rRNA genes of mouse L cell mtDNA provides a basis for the examination of the nucleotide sequence of this region in a mutant mouse cell line that is resistant to chloramphenicol, a known inhibitor of mitochondrial protein synthesis. Resistance to chloramphenicol (CAPr) is conferred by a cytoplasmic determinant that is linked to mtDNA restriction endonuclease site polymorphisms. We have determined the sequence of a 212-nucleotide region of mtDNA from a CAPr mouse cell line that encodes a portion of the 1582-nucleotide large rRNA. This sequence is located 107-318 nucleotides from the 5' end of the heavy strand coding sequence, which corresponds to the 3' end of the rRNA. There is a single nucleotide difference in the large rRNA gene from CAPr cells, an A-to-G transition 243 nucleotides from the 5' end of the coding sequence. This single transition is located within a region of 10 nucleotides tht is otherwise completely homologous to human and yeast mitochondrial large rRNAs and Escherichia coli 23S rRNA and is positioned immediately adjacent to a single nucleotide transversion known to occur in a yeast CAPr mutant. This characterization of a mammalian mitochondrial mutant at the nucleotide level directly demonstrates that a mutant phenotype may result from a single mtDNA nucleotide change in an animal cell.
cDNA clones complementary to mRNA of cells from patients having chronic lymphocytic leukemia (CLL) were used to examine quantitative changes in the mRNA levels of specific genes in human leukemia leukocytes. Fourteen (of 400) CLL-positive clones that did not hybridize with placental mRNA were studied. Three of the 14 clones were highly represented in mRNA from CEM, a T-cell line. One clone was highly represented in mRNA from CLL and two were highly represented in mRNA from patients with chronic myelocytic leukemia. Ten clones were not significantly represented in normal leukocytes and spleen mRNA. We have identified several genes that are differentially expressed in human leukemia leukocytes.
A number of parameters were explored to increase the transformation efficiency of E. coli with pBR322/eukaryotic DNA chimera, formed via d(A) . d(T) and d(G) . d(C) homopolymer tails. Of the E. coli strains analyzed, E. coli strain RR1 was the most efficient bacterial host. A clear optimum of nucleotide tail length existed for both types of homopolymer. The optimum hybridization temperature for chimera formation was found to be approx. 57 degrees C. In the case of d(A) . d(T)-linked chimeras, 30 min was sufficient for optimum chimera formation. In contrast, d(C) . d(G)-linked chimeras required up to 2 h to give the best yields (as measured by transformation efficiency). Other minor factors affecting the transformation process are also explored and discussed.
The specific activities and synthesis of the ubiquitous isozyme, PGK-1, and the testis-specific isozyme, PGK-2, have been quantitated and localized in spermatogenic cells of the mouse. There is a fivefold increase in total PGK specific activity between immature and adult testes which begins at approximately 30 days of age, coincident with the appearance of late-middle stage spermatids. The increase in total PGK is entirely due to the appearance and increase of the PGK-2 isozyme. Rates of PGK synthesis were measured by labeling testicular cells in vitro with [3H]leucine and purifying the PGK isozymes. When total testicular cells were examined, PGK-2 synthesis was detectable after 22 days of age at very low levels and increased in older testes to a level of 0.5% of total protein synthesis. PGK-1 synthesis remained relatively constant at all ages at a level 100-fold lower (0.005%). Testicular cells were separated into highly enriched fractions of particular spermatogenic stages by centrifugal elutriation. The PGK-1 synthesis rates were, again, very low and not significantly different between the various spermatogenic stages. PGK-2 synthesis was low to nondetectable in pachytene spermatocytes, increased to 0.07% in early spermatids and represented 0.7% of total protein synthesis in late spermatids. This increased rate of PGK-2 synthesis appears to require an increase in the amount of PGK-2 mRNA in late spermatids, cells in which no active RNA synthesis is detectable.
The pattern of protein synthesis in different stages of spermatogenesis was examined using two-dimensional polyacrylamide gel electrophoresis followed fluorography. The [3H]leucine labelling was performed by incubating in culture either seminiferous tubules before cell separation or isolated germ cells after fractionation by velocity sedimentation at unit gravity in an albumin gradient. The patterns of soluble polypeptides synthesised in middle-late pachytene spermatocytes, round spermatides (steps 1--8 of spermiogenesis) and intermediate spermatids (steps 9--13) have been compared with each other. Approximately 250 fluorographic spots were detected in middle-late pachytene spermatocytes and round spermatids, but only 100 in intermediate spermatids. From the analysis of fluorograms in the three cell stages examined, 5 categories of labelled polypeptides can be identified: (1) those specific of each cell stage; (2) those present in pachytene spermatocytes and early spermatids but absent in intermediate spermatids; (3) polypeptides labelled during spermiogenesis but unlabelled in meiotic cells; (4) polypeptides showing quantitative differences among the three cell types; and finally (5) polypeptides common to the three cell stages. These results are discussed in relation to differential gene expression during spermatogenesis.