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Haploid Expression of a Mouse Testis α-Tubulin Gene
Abstract
A complementary DNA clone for an alpha-tubulin has been isolated from a mouse testis complementary DNA library. The untranslated
3' end of this complementary DNA is homologous to two RNA transcripts present in postmeiotic cells of the testis but absent
from meiotic cells and from several tissues including brain. The temporal expression of this alpha-tubulin complementary DNA
provides evidence for the haploid expression of a mammalian structural gene.
... Microtubules, formed from heterodimers of a and ,3 tubulin, constitute the primary structural components of mitotic and meiotic spindles, eucaryotic cilia and flagella, and elongated neuronal processes (3). The differential expression of specific tubulins during development in higher eucaryotes (3), the identification of a testis-specific i tubulin in Drosophila melanogaster (6), testis-specific a tubulins in mouse (4,16) and chicken (13) testes, and a male-specific Drosophila a tubulin (14) suggest that tissueor cell-type-specific microtubules could exist in organs such as the testis. ...
... Identification of a divergent testicular a tubulin. A 1,650nucleotide cDNA insert that contains coding and 3'-untranslated sequences of the rat brain a tubulin, pILaT1 (10), was used to isolate a novel a-tubulin cDNA from a mouse testis cDNA library (4). Approximately 20,000 colonies were screened by colony hybridization. ...
... The 1.5-kb actin mRNA in testes of 6-and 8-day-old mice did not hybridize to the 3'-UT of the testicular SMGA. Thus, the days 6 and 8 1.5-kb actin mRNAs are 6,8,12,16,18,20,22,30, and 45 days of age were denatured with formaldehyde, electrophoresed into a 1.1% agarose gel, and transferred to nitrocellulose. The blot was hybridized with (A) the pAL41 probe (14-h exposure) and (B) the 3'-UT of the testicular SMGA (24-h exposure). ...
... In Dictyostelium discoideum, different actin genes coding for the same actin are expressed differentially (46). From the results of the Northern blot analysis with the 3'-UT testicular SMGA probe of RNAs from smooth-muscle tissues (Fig. 3 (8,50), and mouse, rat, and human transition proteins (5,19,22,23,26,31; P. C. Yelick, Y. K. Kwon, J. F. Flynn, K. C. Kleene, and N. B. Hecht, Mol. Reprod. ...
... However, although zebrafish and mammalian isotypes do not strongly cluster phylogenetically, as isotypes do when comparing mammals only, zebrafish also possess neuronal-specific tubulin isotypes (Gulati-Leekha and Goldman, 2006;Oehlmann et al., 2004;Veldman et al., 2010). Similarly, testis-or oocyte-specific isotypes exist in the fly (Kemphues et al., 1982), nematode (Nishida et al., 2021), frog (Wu and Morgan, 1994), chicken (Pratt et al., 1987) and mouse (Distel et al., 1984;Feng et al., 2016). Although grouping isotypes from distant clades into functionally similar groups based on sequence alone is not trivial, these observations suggest that certain isotypes fulfill neuronal-and gamete-specific roles in diverse organisms. ...
The microtubule cytoskeleton is assembled from the α- and β-tubulin subunits of the canonical tubulin heterodimer, which polymerizes into microtubules, and a small number of other family members, such as γ-tubulin, with specialized functions. Overall, microtubule function involves the collective action of multiple α- and β-tubulin isotypes. However, despite 40 years of awareness that most eukaryotes harbor multiple tubulin isotypes, their role in the microtubule cytoskeleton has remained relatively unclear. Various model organisms offer specific advantages for gaining insight into the role of tubulin isotypes. Whereas simple unicellular organisms such as yeast provide experimental tractability that can facilitate deeper access to mechanistic details, more complex organisms, such as the fruit fly, nematode and mouse, can be used to discern potential specialized functions of tissue- and structure-specific isotypes. Here, we review the role of α- and β-tubulin isotypes in microtubule function and in associated tubulinopathies with an emphasis on the advances gained using model organisms. Overall, we argue that studying tubulin isotypes in a range of organisms can reveal the fundamental mechanisms by which they mediate microtubule function. It will also provide valuable perspectives on how these mechanisms underlie the functional and biological diversity of the cytoskeleton.
... 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.
... Thus, it seems likely that none of the five genes described in this paper plays a dominant role in spermatogenesis, and that one or more different testis-specific or testisabundant a-and/3-tubulins exist. In this context, it is noteworthy that a mouse a-tubulin mRNA expressed in spermatids has been reported (8). In addition, a testis-abundant/3tubulin isotype has been described in chicken (16) and a multifunctional, testis-specific a-tubulin has been characterized in Drosophila (18,19). ...
We describe five mouse tubulin cloned cDNAs, two (M alpha 1 and M alpha 2) that encode alpha-tubulin and three (M beta 2, M beta 4, and M beta 5) that encode beta-tubulin. The sequence of these clones reveals that each represents a distinct gene product. Within the sequence common to the two alpha-tubulin cDNAs, the encoded amino acids are identical, though the 3' noncoding regions are wholly dissimilar. In contrast, the three beta-tubulin cDNAs show considerable carboxy-terminal heterogeneity. Two of the beta-tubulin isotypes defined by the cloned sequences are absolutely conserved between mouse and human, and all three beta-tubulin isotypes are conserved between mouse and rat. This result implies the existence of selective constraints that have maintained sequence identity after species divergence. This conclusion is reinforced by the near identity between a third mouse beta-tubulin isotype and a chicken beta-tubulin isotype. The significance of the interspecies conservation of tubulin isotypes is discussed in relationship to microtubule function. We have used non-cross-hybridizing 3' noncoding region probes from the five cloned mouse tubulin cDNAs to study the developmental expression of each isotype in various mouse tissues. M alpha 1 and M beta 2 are expressed in an approximately coordinate fashion, and their transcripts are most abundant in brain and lung. M alpha 2 and M beta 5 are ubiquitously expressed and to a similar extent in each tissue, with the greatest abundance in spleen, thymus, and immature brain. In contrast, M beta 4 is expressed exclusively in brain. Whereas the expression of the latter isotype increases dramatically during postnatal development, transcripts from all four other tubulin genes decline from maximum levels at or before birth. Tissue-specific development changes in the abundance of tubulin isotype-specific mRNAs are discussed in relationship to organogenesis in the mouse.
... In mouse, however, the mRNA is first detectable immediately postmeiosis, continues to accumulate in spermatids, and is translationally regulated, whereas in rainbow trout, mRNA is synthesized in diploid cells and subject to translational control. Finally, mouse testis alfa tubulin mRNA [38] is found exclusively in postmeiotic cells of the testis and is absent from meiotic cells. It remains unclear which of the above mentioned regulatory mechanisms may apply to the expression of HLA molecules found on sperm cells. ...
A diploid expression of class I and class II human leukocyte antigens (HLA) has been found in purified spermatozoa by using double fluorescence labeling cytofluorometry and relevant monoclonal antibodies; this expression has been confirmed for the first time by the analysis of specific HLA mRNA and metabolic 35 S labeling followed by immunoprecipitation, which demonstrates an active ongoing translation of HLA proteins in ger-minal cells. Long-living mRNA coming from diploid germinal cells may be translated to HLA molecules in spermatozoa. This translation is controlled (or at least inversely correlated) by a testicular hormone (inhibin) in a cyclic fashion. Remarkably, serum levels of inhibin, synthesized by Leydig and Sertoli cells, follow a 12-to 13-day cycle, with a peak level at Day 6; this is probably controlled by FSH (not cyclic in males) and other tes-ticular and/or unknown hormones. Peak levels of inhibin concur with the lower density and percentage of spermatozoa expressing both HLA class I and II molecules (close to 3% by cytofluo-rometry); lowest levels of inhibin coincide with the highest numbers (35-40%) of spermatozoa positive for both HLA molecules and a higher surface density. These observations could put to an end a disconcerting and long-lasting controversy on the expres-sion/non-expression of HLA antigens on spermatozoa. The possibility that HLA-bearing spermatozoa are more capacitated for fertilization than those that do not bear HLA, and the implications of our results on male fertility control are also discussed.
... 267 YV I YV 827 -rf--tions: UbI by heat-shock and chemical stress, and UblI during spermatogenesis. Other genes, including 'housekeeping' genes, that express a particular form during spermatogenesis have been described [38,39]. ...
Northern analysis demonstrated that levels of ubiquitin transcript increased during the chicken testis maturation process, in agreement with the previously published increase of ubiquitin during this differentiation process. Specific probes for four different ubiquitin genes (two polyubiquitins, UbI and UbII, and two ubiquitin-fusion genes, UbCep52 and UbCep80) allowed us to analyse the expression of each individual gene. UbI polyubiquitin gene was expressed in all the tissues tested, and its transcript was the most abundant ubiquitin RNA in all of them. Unspliced UbI transcript, already detected in stressed chicken-embryo fibroblast, was also present in immature testis and reticulocytes. UbII, a chicken polyubiquitin gene not previously found expressed and not heat-shock-inducible, was specifically stimulated during the testis maturation process. Two minor ubiquitin fusion transcripts of 0.6 and 0.7 kb, corresponding to UbCep52 and UbCep80 respectively, were also found in chicken testis. Although differentially expressed, it was found that UbI and UbII chicken polyubiquitin genes had an HTF ('HpaII tiny fragments') island (CpG-rich and constitutively unmethylated region) in their 5' proximal non-coding region. In addition, we demonstrated the coexistence of 3' and/or 5' relatively distal methylated sites together with these 5' proximal HTF islands in both chicken polyubiquitin genes. 3' and 5' distal UbI CCGG sites were specifically hypermethylated in mature testis, whereas a 3' distal UbII CCGG site was found to be about 50% methylated in all DNAs tested.
... The germ-cell-specific 1700-nt form of proenkephalin RNA is detected in both meiotic as well as postmeiotic cells-the highest concentrations occurring in round spermatids and pachytene spermatocytes. Other examples of germ-cell-specific mRNAs that undergo developmental regulation have been identified, including those for a-tubulin (26), the protooncogene c-abl (27), /-and y-actin (28), protamines (29), and the presumptive gene product from the mouse homebox gene MH-3 (30,31). ...
Spermatogenic cells have been previously shown to be a major site of testicular proenkephalin gene expression. Using RNA gel-blot analysis of purified mouse and hamster germ cells and of testes from prepuberal and germ cell-deficient mutant mice, we now have demonstrated that, in addition to its previously described expression by somatic (Leydig) cells, the gene for a second opioid peptide precursor, pro-opiomelanocortin (POMC), is also expressed by spermatogenic cells. Of particular significance is the finding that the RNAs for proenkephalin and POMC are differentially regulated during spermatogenesis. Two forms of POMC RNA were detected in mouse testis, a larger component 675- to 750-nucleotides (nt) in size common to somatic and spermatogenic cells and a smaller 625-nt RNA found only in pachytene spermatocytes. Two distinct, cell-specific proenkephalin RNAs were also shown to be present in mouse testis: a 1700-nt transcript previously shown to be expressed by spermatogenic cells and a 1450-nt form associated with somatic cells. These data suggest that proenkephalin- and POMC-derived peptides are produced by both somatic cells and germ cells in the testis and in germ cells these two families of opioid peptides may function at different stages of spermatogenesis.
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.
Five mouse alpha-tubulin isotypes are described, each distinguished by the presence of unique amino acid substitutions within the coding region. Most, though not all of these isotype-specific amino acids, are clustered at the carboxy terminus. One of the alpha-tubulin isotypes described is expressed exclusively in testis and is encoded by two closely related genes (M alpha 3 and M alpha 7) which have homologous 3' untranslated regions but which differ at multiple third codon positions and in their 5' untranslated regions. We show that a subfamily of alpha-tubulin genes encoding the same testis-specific isotype also exists in humans. Thus, we conclude that the duplication event leading to a pair of genes encoding a testis-specific alpha-tubulin isotype predated the mammalian radiation, and both members of the duplicated sequence have been maintained since species divergence. A second alpha-tubulin gene, M alpha 6, is expressed ubiquitously at a low level, whereas a third gene, M alpha 4, is unique in that it does not encode a carboxy-terminal tyrosine residue. This gene yields two transcripts: a 1.8-kilobase (kb) mRNA that is abundant in muscle and a 2.4-kb mRNA that is abundant in testis. Whereas the 1.8-kb mRNA encodes a distinct alpha-tubulin isotype, the 2.4-kb mRNA is defective in that the methionine residue required for translational initiation is missing. Patterns of developmental expression of the various alpha-tubulin isotypes are presented. Our data support the view that individual tubulin isotypes are capable of conferring functional specificity on different kinds of microtubules.
Mouse somatic tissues contain low levels of transcripts homologous to the heat shock-inducible and cognate members of the heat shock protein 70 (hsp70) gene family. An abundant, unique sized hsp70 mRNA of 2.7 kilobases (kb) is present in testes in the absence of exogenous stress. Its expression is restricted to germ cells and is developmentally regulated. The 2.7-kb transcript first appears during the haploid phase of spermatogenesis and is stable throughout the morphogenic stages of spermiogenesis. A 2.7-kb hsp70 mRNA is present in rat and human testes. These observations suggest that a member of the hsp70 gene family plays a role in the development of the mammalian male germ cell lineage.
Transcripts encoding CAPL-B, an apparent member of the cyclic-nucleotide-regulated kinase subfamily in Aplysia californica, are found exclusively in the ovotestis and are concentrated in meiotic and postmeiotic spermatogenic cells. The CAPL-B polypeptide is present in mature spermatozoa, suggesting that the kinase plays a part in regulating events associated with fertilization.
We isolated and sequenced a cDNA clone of the human gene encoded by the 5' half of the ret transforming gene. The nucleotide sequence indicates that it encodes a protein with "finger" structures which represent putative metal- and nucleic acid-binding domains. Transcription of this gene was detected at high levels in a variety of human and rodent tumor cell lines, mouse testis, and embryos. In addition, a unique transcript was observed in testis RNA. When the expression of the unique transcript was examined at different stages of spermatogenesis, a striking increase in mRNA levels accompanied progression from meiotic prophase pachytene spermatocytes to postmeiotic round spermatids. This finger-containing gene may thus function in male germ cell development.
Sequence analysis of a mouse testicular alpha-tubulin partial cDNA, pRD alpha TT1, reveals an isotype that differs from both the somatic and the predominant testicular alpha tubulins at approximately 30% of the 212 amino acid residues determined. Although this mouse testicular cDNA retains the highly conserved sequence, Glu-Gly-Glu-Glu, found in the carboxyl termini of many alpha tubulins, the protein extends substantially beyond this sequence and does not terminate with a C-terminal tyrosine. Using rabbit antiserum prepared to a novel synthetic peptide predicted from this mouse testis alpha-tubulin cDNA, we have have detected by immunoblot and indirect immunofluorescence an antigenic epitope present in testicular alpha tubulin that is not detectable in brain alpha tubulins. We find that the antiserum specifically binds to the manchettes and meiotic spindles of the mouse testis but not with neural fibers or tubulin extracts of the adult mouse brain. These results demonstrate that at least one of the multiple alpha-tubulin isotypes of the mammalian testis is expressed and used in male germ cells but not in the brain.
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.
Spermatogenesis in human males is the process by which spermatogonia develop into spermatozoa, and occurs in the seminiferous tubules of the testis. If this process is disrupted at any stage, the male may become sub- or infertile. Both environmental and genetic factors have been implicated in these cases, and both causes are poorly understood. The aim of this project has been to identify novel genes involved in spermatogenesis by isolating sequences that are expressed either solely or highly in the testis. I have analysed cDNAs using the techniques of Differential Display RT-PCR and cDNA selection. Differential Display is a relatively new technique which has wide applications for identifying tissue or stage-specific transcripts. A subset of mRNAs present in a selected tissue are converted into short cDNA fragments. A comparison of the cDNAs produced from several different tissues (in this case testis, liver, lung, white blood cells, placenta, muscle and brain) allows products specific to one tissue to be identified. Whilst Differential Display will identify testis-specific genes expressed from anywhere in the genome, cDNA selection is basically a hybridization technique which, in this case, was set up to identify sequences in common between those expressed in the testis and those present on the Y chromosome. Using these experimental techniques I have identified previously characterized testis-specific sequences as well as 22 novel sequences and 11 sequences which matched to uncharacterized cDNAs present in the genome database. Several of the latter have been characterized further in terms of expression patterns and chromosomal location.
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.
Most of the integral components of the mature mammalian spermatozoon are made up of cytoskeletal proteins that are synthesized and assembled during the haploid phase of spermatogenesis. With the exception of various isoforms of tubulin composing the microtubules of the sperm tail (1,2) and filamentous (3–5) and nonfilamentous (6, 7) forms of actin localized in diverse regions of the sperm head and tail of various species, the majority of sperm cytoskeletal proteins appear to have no protein or structural counterparts in somatic cells. Specialized cytoskeletal elements found in the sperm tail are the outer dense fibers (ODF), the fibrous sheath (FS), the submitochondrial reticulum (8), the annulus, and the striated collar and capitulum of the neck piece [reviewed by Oko and Clermont (9)]. In the sperm head are found the perinuclear theca (PT), the outer periacrosomal layer (OPL), and the basal plate [reviewed by Oko (10)]. The isolation or extraction of many of these sperm elements is made possible by their differential resistance to protein solubilizing agents (11–21). Compositional studies have revealed that most of these elements are made up of a heterogeneous mixture of proteins of various concentrations (11–21).
There is increasing evidence that the use of sperm structure can be of great help in phylogenetic reconstruction at any taxonomic level [see Baccetti and Afzelius’ (1976) and Jamieson’s (1987) extensive reviews]. Quoting the latter author, ‘opposition to its (sperm structure) use is usually dispelled when its effectiveness for resolving hitherto intractable problems of taxonomic placement and relationship is demonstrated’. The range of sperm structural diversity is so large that it is often possible to ascribe quite reliably any single sperm cell to a hierarchy of taxa (i.e. phylum, order, family, genus and even species). A primitive sperm architecture can be recognized throughout the metazoans in unrelated species which have retained the primitive mode of fertilization (Franzén 1977). As a consequence, animals with a primitive spermatozoon are very unlikely to have arisen from animals with a derived one. Implicit in this, apomorphies and plesiomorphies between related taxa can be clearly determined and hence questionable phylogenetic relationships solved.
This chapter examines the novel structural features of the mammalian spermatozoon, its composition and assembly, and the mechanisms regulating its function. The spermatozoon is a highly specialized cell with unique structural features and functional characteristics required to deliver the male genome to the egg. The head of a spermatozoon contains a highly condensed nucleus, an enzyme-filled acrosome, and a small amount of cytoplasm. The flagellum is a highly novel feature that provides the spermatozoon with the ability to propel itself through liquid medium. New findings about genes that are expressed only in spermatids and encode novel proteins unique to spermatozoa are reviewed in this chapter. The gene knockout approach has identified many proteins required for normal sperm functions in mice that are likely to be required in humans as well. However, many critical questions remain to be answered about the composition, organization, and function of spermatozoa.
During spermatogenesis, the population of stem cells (diploid spermatogonia) divides and differentiates into tetraploid spermatocytes. Spermatocytes undergo meiosis, in which genetic recombination occurs, producing haploid spermatids. Spermatids, through an extraordinary process of metamorphosis called spermiogenesis, develop into a highly specialized motile vector for transportation of genetic information: the spermatozoa (Figure 1).
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.
The aim of the workshop was to present and discuss recent findings and developments in the fields of molecular dissection of chromosome segments, expression and regulation of gene activity, genome evolution, organization of genes and gene complexes, molecules of immunological interest, mutagenesis, and embryonal and teratocarcinoma cell lines, with an effort to combine molecular aspects with certain new aspects of cytogenetics and developmental genetics of the mouse.
A cDNA library has been constructed from Heliothis virescens testes poly(A)+ RNA, and a Heliothis cDNA clone isolated using a testis-specific β-tubulin sequence from Drosophila. The Heliothis cDNA was identified as corresponding to a β-tubulin sequence by hybrid-selected translation. A transcript from the Heliothis β-tubulin gene was abundant in testes, but undetectable in 0–48 h embryos, larval fat body, adult ovaries or ovarian derived cultured cells. The testis β-tubulin gene was expressed throughout all stages of spermatogenesis examined. The data suggest that the encoded testis-specific polypeptide may be involved in the assembly of multiple rather than single microtubular structures formed during sperm cell differentiation. Although the β-tubulin transcript was present throughout all stages of spermatogenesis, the steady-state level of the transcript varied during different stages of spermatogenesis, indicating some degree of temporal regulation of either rate of transcription or RNA stability.
Weisolated andsequenced acDNAclone ofthehumangeneencoded bythe5'half oftherettransforming gene. Thenucleotide sequence indicates thatitencodes aprotein with"finger" structures whichrepresent putative metal- andnucleic acid-binding domains. Transcription ofthis genewasdetected athighlevels ina variety ofhumanandrodent tumorcell lines, mousetestis, andembryos. Inaddition, aunique transcript was observed intestis RNA.Whentheexpression oftheunique transcript wasexamined atdifferent stages of spermatogenesis, a striking increase inmRNA levels accompanied progression frommeiotic prophase pachytene spermatocytes topostmeiotic roundspermatids. Thisfinger-containing genemaythusfunction in malegermcell development.
The nucleotide sequence of a human α-tubulin gene (bαl) is described. This gene is extensively homologous to a rat α-tubulln
gene in its coding regions, 3′-untranslated region and, indeed, in segments of its largest intron. However, with the exception
of three short conserved blocks of homology, the 5′ flanking regions of the rat and human genes are unrelated. Hence, these
genes each encoding an identical protein are transcribed under the influence of divergent promoters.
Blot analyses using RNA from a variety of transformed cells derived from different tissues indicate that expression of the
human α-tubulin gene is restricted to cells of neurological origin. Among neurological cell types bαl expression is further
restricted to adherent cells that are morphologically differentiated. The data presented suggest that the bαl gene encodes
a prominent neuronal and glial α-tubulin and that bαl expression is a function of the differentiated state of these cells.
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
Comparative studies show that variation in sperm morphometry across taxa is associated with the environment in which sperm function, and the species' mating pattern dictating the risk of sperm competition. Accordingly, sperm have evolved to function in a non-self environment (in contrast to somatic cells) and sperm morphometry is predicted to be optimized independently of the individual male producing them, but is the result of selective forces arising directly from the fertilization and competitive environment in which sperm will operate. Males within a population are therefore under stabilizing selection to produce an optimal distribution of sperm sizes. The nature of this distribution was explored using consistent techniques to measure detailed sperm morphometry for 10 species in a range of taxa from insects to humans. Although we expected variance in sperm morphometry to be optimized by every individual male through stabilizing selection at a population or species level, we found the exact opposite; for every species examined there was significant variation between individual males in the total lengths of the sperm they produced. A significant variation is reported between individual males for every species in the sizes of each sperm head, mid-piece and flagellum component. The between-male variation exists consistently in wild, domestic and human populations, subject to a wide range of levels of inbreeding. In gryllid crickets sperm length is shown to be male-specific and is repeatable between successive ejaculates. Between-female variation in ova size (data are presented for trout) is explainable by individual female fecundity optimization strategies; however, the adaptive significance of widespread between-individual variance in male gamete size is counter-intuitive and difficult to interpret, particularly as the limited evidence available shows that sperm morphometry is not condition-dependent or resource-constrained. The differences, however, do suggest negligible influences from haploid expression in the development of sperm morphometry – if haplotypic expression were manifested we would expect more profound variation within a male's sperm population (to reflect the inherent within-male variance in haplotypes derived from recombination) rather than the significant between-male differences we found that suggests the diploid control of spermatozoal phenotype
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.
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.
Eight alpha and twelve beta isoforms of tubulin were isolated from discrete regions of the mouse brain using high-resolution isoelectric focusing and identified by two-dimensional gel electrophoresis and immunoreactivity. In the different regions, the number of isoforms was identical, but their relative proportion varied except for alpha 2, 3, 4, 5, 6 and beta 1 bands. The patterns of the inferior and superior colliculi were nearly similar. The cerebellum, compared with the inferior and superior colliculi is characterized by a decrease of alpha band 7 and beta bands 3 and 7 and an increase of alpha band 8 and beta bands 2, 4, 10, 11, 12. The forebrain displays an intermediate pattern between the cerebellum and the colliculi. These results suggest that in functionally different regions of the brain the number of isotypes of tubulin is identical but their relative proportion differs with an apparent correlation between the function of the neuronal subpopulations and the pattern of isotubulins.
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
We have identified a male meiotic germ cell-specific antigen (Meg 1) with monoclonal antibody (mAb) TRA 369 in mice. The Meg 1 antigen was strongly expressed in specific steps of meiotic germ cells from pachytene spermatocyte to early spermatid, and not in other germ cells or somatic cells. Immunohistochemical examination revealed that the antigen was localized to the cytoplasm and was not distributed in the nucleus or on the cell surface. This antigen was demonstrated to have a molecular weight of 93 kDa and an isoelectric point of 5.2 by Western blotting. This molecule was first detected in the testis of 13-day-old mouse when pachytene spermatocytes first appeared. Thus this is a differentiation-specific antigen in male meiotic germ cells, and mAb TRA 369 is a useful tool to study the regulation of germ cell differentiation and to define germ cell development in a molecular level.
In situ hybridization in immature and mature testis sections shows that the rooster protamine mRNA is transcribed in the post-meiotic stages of spermatogenesis. Two distinct populations of rooster protamine mRNA are expressed as determined by Northern blot analysis. Since there are two copies of the chicken protamine gene per haploid genome, the question was raised of whether the two mRNA populations correspond to the two different genes or was a result of differential mRNA processing. The fact that the two genes differ only in one nucleotide (one extra A in the polyadenylation signal in the second locus) and that random sequencing of several cDNA clones has revealed only one poly-A tail addition site favors the hypothesis that the differences are due to mRNA processing. This is supported by 3' S1 mapping which shows a single poly-A tail addition site, which in turn suggests that the heterogeneity is due to differences in the length of the poly-A tail. The latter is confirmed by RNAse H digestion of mRNA-Oligo-dT hybrids which shows that the two mRNA populations (470 +/- 20 nucleotide (nt) and 430 +/- 20 nt respectively) are converted to a single population of 345 +/- 15 nt in good accordance with the poly-A tail site determined by S1 mapping (347 nt). Thus one species has a poly-A tail of 145 nt appearing in round spermatids and the second, a shorter tail of 105 nt present at the later stages of elongated spermatids.
Transcripts encoding CAPL-B, an apparent member of the cyclic-nucleotide-regulated kinase subfamily in Aplysia californica,
are found exclusively in the ovotestis and are concentrated in meiotic and postmeiotic spermatogenic cells. The CAPL-B polypeptide
is present in mature spermatozoa, suggesting that the kinase plays a part in regulating events associated with fertilization.
cDNAs encoding human and mouse microtubule-associated protein 4 (MAP 4) were isolated. MAP 4 is encoded by a single gene. Multiple MAP 4 mRNAs are transcribed that are differentially expressed among mouse tissues. Open reading frames for the human and mouse MAP 4 clones indicate three distinct regions consisting of related sequences with different motifs. Approximately 30% of the protein is tandem related repeats of approximately 14 amino acids. Another region contains clusters of serine and proline. Four 18-mer repeats characteristic of the microtubule-binding domains of MAP 2 and tau are located at the carboxyl-terminal portion of MAP 4. Amino acid sequence analysis revealed that human and mouse MAP 4 are homologs of the bovine 190-kDa MAP/MAP U (Aizawa, H., Emori, Y., Murofushi, H., Kawasakai, H., Sakai, H., and Suzuki, K. (1990) J. Biol. Chem. 265, 13849-13855). Mouse and human MAP 4 and the bovine 190-kDa MAP are approximately 75% similar, indicating that these proteins are all members of the same class. Domains with extremely high conservation (greater than or equal to 88%) are: 1) the extreme amino terminus; 2) a proline-rich region between the KDM and S,P domains; 3) the microtubule-binding domain; and 4) the extreme carboxyl terminus.
Thymosin beta 10 is one of a small family of proteins closely related in sequence to thymosin beta 4, recently identified as an actin-sequestering protein. A single molecular weight species of thymosin beta 10 mRNA is present in a number of rat tissues. In adult rat testis, an additional thymosin beta 10 mRNA of higher molecular weight was identified. Nucleotide sequencing of cDNA clones complementary to the testis-specific thymosin mRNA indicated that this mRNA differed from the ubiquitous thymosin beta 10 mRNA only in its 5'-untranslated region, beginning 14 nucleotides upstream of the translation initiation codon. These results, together with primer extension experiments, suggest that the two thymosin beta 10 mRNAs are transcribed from the same gene through a combination of differential promoter utilization and alternative splicing. The novel thymosin beta 10 mRNA could be detected only in RNA isolated from sexually mature rat testis. Both mRNAs were present in pachytene spermatocytes; only the testis-specific mRNA was detected in postmeiotic haploid spermatids. Immunoblot analysis using specific antibodies showed that the thymosin beta 10 protein synthesized in adult testis was identical in size to that synthesized in brain. Immunohistochemical analysis showed that the protein was present in differentiating spermatids, suggesting that the testis-specific thymosin beta 10 mRNA is translated in haploid male germ cells.
In the present study we have examined the cellular localization and developmental changes of mRNAs for retinoid-binding proteins in rat testis. We demonstrate that mRNA (0.7 kb) for cellular retinol-binding protein (CRBP) is expressed only in Sertoli cells and peritubular cells. The mRNA for CRBP could not be detected in other testicular cells. In contrast, mRNA for cellular retinoic acid-binding protein (CRABP) was detected primarily in germ cells and to a small extent in tumor Leydig cells. The mRNA for CRABP in germ cells revealed distinct size heterogeneity and three distinct mRNA species were observed (1.0, 1.8, and 1.9 kb), in contrast to previous data for somatic cells where only the 1.0-kb mRNA has been reported. Messenger RNAs for retinoic acid receptor-alpha (RAR alpha) were detected in both somatic and haploid germ cells. The highest level of RAR alpha was seen in Sertoli cells, round spermatids, and tumor Leydig cells. Lower, but distinct, levels were observed in peritubular cells. Furthermore, we observed germ cell-specific species of RAR alpha mRNA (4 kb and approximately 7 kb). The smallest mRNA for RAR alpha (2.7 kb) in somatic cells was absent in germ cells. The levels of mRNAs for the various retinoid-binding proteins in whole testis obtained from rats of various ages confirmed this cellular localization. The mRNAs for CRBP, the small molecular size (2.7 kb) mRNA for RAR alpha (localized to somatic cells), and the 1-kb mRNA for CRABP showed an age-dependent decrease.(ABSTRACT TRUNCATED AT 250 WORDS)
Evolutionary arguments and well-designed experiments (based on false premises, however) had suggested that post-meiotic gene expression did not occur in animals. The techniques of molecular genetics have now clearly demonstrated such genetic activity in mammalian testes. The current problem is to understand why some classes of genes, such as Zfy and many oncogenes, are expressed in this manner.
The cells of the seminiferous epithelium of the rat testis are a rich source of microtubules and contain distinct microtubular structures such as the meiotic spindle and manchette. Microtubule diversity can be maintained by differential genetic expression of the multiple α‐ and β‐tubulin polypeptides or by tubulin monomer acetylation and detyrosination, post‐translational modifications of α‐tubulin. In the present analysis, antibodies that specifically recognize acetylated (antiacetylated), tyrosinated (anti‐Tyr) and detyrosinated (anti‐Glu) α‐tubulins were employed to examine the distribution of post‐translationally modified microtubules in the cells of the seminiferous epithelium.
In the light microscope, a distinct pattern of staining for each antibody was detected using immunoperoxidase techniques on paraffin‐embedded testicular sections. In the case of the anti‐Glu antibody, a dense immunoperoxidase staining was detected in the cytoplasm of steps 4–7 spermatids. Thereafter, staining was noted over the area corresponding to the manchette of steps 8–15 spermatids, but not over their cytoplasm. The tails of spermatids were also reactive with this antibody. The anti‐Tyr antibody was observed to be localized over the cytoplasm of Sertoli cells in their basal, supranuclear, and apical regions. A dense immunoperoxidase staining was also noted in the cytoplasm of pachytene spermatocytes, but it was negligible in the cytoplasm of spermatocytes undergoing their meiotic division; in these cells the centrioles and meiotic spindle were reactive. The spermatid's tails were also reactive. The antiacetylated antibody showed reactivity only over the tails of spermatids.
With the electron microscope, a similar pattern of labeling was noted using immunogold labeling on Lowicryl K4M embedded testicular sections. The anti‐Glu antibody heavily labeled microtubules of the manchette and the axoneme of tails of spermatids as well as microtubules of the proximal and distal centrioles and centriolar adjunct. The anti‐Tyr antibody strongly labeled microtubules of Sertoli cells and the meiotic spindle and midbody of dividing spermatocytes. The anti‐Tyr antibody also labeled the microtubules of the axoneme, centrioles, and centriolar adjunct of spermatids, but to a lesser degree than the anti‐Glu antibodies; the manchette was faintly labeled. Of the three antibodies, the antiacetylated antibody showed the weakest labeling of microtubules of the centrioles, centriolar adjunct, and midbody, whereas those of the manchette and Sertoli cells were unreactive; the axoneme was moderately labeled. All three antibodies, however, labeled the microtubules of the tall columnar epithelial cells, referred to as transitional cells , lining the terminal part (tubulus rectus) of the seminiferous tubule. These results thus suggest that post‐translational modifications of α‐tubulin have an essential role in the functional microtubular diversification of the seminiferous epithelium.
Excerpt
Department of Biology, Tufts University, Medford, MA 02155, USA
Keywords: spermatogenesis; haploid gene expression; gene regulation; testis
Introduction Spermatogenesis offers an experimental system whereby the gene expression of eukaryotic cells with tetraploid, diploid, and haploid chromosome complements can be compared. Starting from a population of stem cells, the diploid spermatogonia follow one of two lineages. One subpopulation of cells initiates a differentiation process ultimately leading to the spermatozoon while a second, presumably distinct, subpopulation of spermatogonia enters a pathway that maintains and repopulates the stem cells of the testis. The cells destined to become spermatozoa undergo several spermatogonial divisions. The last complete replication of DNA during spermatogenesis, in the preleptotene primary spermatocyte, heralds the start of meiosis. During the lengthy interval of meiotic prophase, homologous chromosomes synapse and genetic recombination occurs, producing the genetic diversity required for survival of a species. Following meiotic recombination, the 4N spermatocytes divide twice
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).
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The strategy of shotgun cloning with M13 is based on obtaining random fragments used for the rapid accumulation of sequence data. A strategy, however, is sometimes needed for obtaining subcloned sequences preferentially out of a mixture of fragments. Shotgun sequencing experiments have shown that not all DNA fragments are obtained with the same frequency and that the redundant information increases during the last third of a sequencing project. In addition, experiments have shown that particular fragments are obtained more frequently in one orientation, allowing the use of only one of the two DNA strands as a template for M13 shotgun sequencing. Two new M13 vectors, M13mp8 and M13mp9, have been constructed that permit the cloning of the same restriction fragment in both possible orientations. Consequently, each of the two strands becomes a (+) strand in a pair of vectors. The fragments to be cloned are cleaved with two restriction enzymes to produce a fragment with two different ends. The insertion of such a fragment into the vector can occur only in one orientation. Since M13mp8 and M13mp9 have their array of cloning sites in an antiparallel order, either orientation for inserting a double-digest fragment can be selected by the choice of the vector.
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.
This chapter presents recent studies on in vitro assembly, nucleation, and growth of microtubules along with the properties that shed light on their biological role. The biochemical study of structural proteins such as tubulin and actin is a new area. It has received a considerable amount of attention recently which, it is hoped, reflects progress. Despite the fact that actin is in general the most common cellular protein and that so much was known about it from studies of muscle, not until recently has its role in nonmuscle cells been appreciated. Tubulin, perhaps the second most common intracellular protein, was not described until the 1960s. Functional studies of tubulin assembly were not performed until 1972. The means of regulation and the principles of self-assembly are also presented in the chapter. It discusses the experiments that (1) emanated from the discovery of conditions for assembly of microtubules from brain extracts, (2) relate to the biochemical properties of tubulin, and (3) that utilize in vitro systems for studying nucleation and growth of cytoplasmic microtubules. Certain topics in microtubule assembly have been well studied in the past few years
This paper describes a method of transferring fragments of DNA from agarose gels to cellulose nitrate filters. The fragments can then be hybridized to radioactive RNA and hybrids detected by radioautography or fluorography. The method is illustrated by analyses of restriction fragments complementary to ribosomal RNAs from Escherichia coli and Xenopus laevis, and from several mammals.
Transcripts from the four different Drosophila melanogaster alpha-tubulin genes were detected by filter hybridization experiments that used subcloned fragments from each gene as hybridization probes. These hybridization experiments demonstrated that each gene is transcribed. All of the transcripts are found on polysomes and are long enough to encode an alpha-tubulin protein. The hybridization studies were extended to examine the developmental pattern of RNA concentrations. The concentration of RNAs from the alpha 2 and alpha 4 genes vary independently and dramatically, while those from alpha 1 and alpha 3 have parallel variations. We conclude that at the RNA level of expression, two alpha-tubulin genes are regulated in parallel and two genes are not. We hypothesize that the different concentration patterns reflect different functions for the protein products of each gene.
Screening of two human genomic libraries with a chicken α-tubulin complementary DNA probe has resulted in the isolation of several clones containing a common hybridizing region. Restriction mapping of the cloned fragments reveals that, while the majority of sites are retained in each clone, there are a number of differences, including several within the gene-containing region. These differences cannot be ascribed solely to restriction-site polymorphism, and therefore reflect the existence of a family of closely related α-tubulin genes.
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.
We have isolated four recessive male sterile mutations in the structural gene for the testis-specific Drosophila beta 2-tubulin. Each of these mutations encodes a variant beta 2-tubulin subunit synthesized at normal levels, but which is subsequently unstable and rapidly degraded within the testis. In such testes, the normal alpha tubulins are also synthesized at normal levels and then degraded. Thus in mutant males the testis tubulin pool is drastically reduced relative to wild-type. In males homozygous for any of the recessive beta 2-tubulin mutations, the early mitotic divisions, which are completed before the time of synthesis of beta 2-tubulin, are normal. Thereafter, however, all microtubule-mediated events subsequent to the expression of the altered subunit are defective: meiosis, nuclear shaping and assembly of the axoneme all fail to occur. We thus conclude that the beta 2-tubulin subunit that forms the Drosophila sperm axoneme is not functionally restricted but serves multiple functions in spermatogenesis, including the assembly of both singlet and doublet tubules.
Bacterial clones containing inserted DNA sequences specific for α-tubulin, β-tubulin, β-actin and γ-actin have been constructed from mRNA of embryonic chick brain. Plasmids containing approximately 75, 90 and >90%, respectively, of the sequences present in α-tubulin, β-tubulin and β-actin mRNAs have been isolated as well as clones containing parts of the extensive 3′ untranslated regions of the β- and γ-actin mRNAs. The sequences for the two tubulins do not cross hybridize. Hybridization of labeled, cloned probes for each of the tubulins with chicken DNA digested with several restriction endonucleases reveals about four fragments for α- and four for β-tubulin. This seems to be the number of genes, since both the 5′ and 3′ ends of either cloned tubulin cDNAs hybridize to at least four common fragments in genomic DNA which has been digested with restriction endonucleases. The tubulin probes are able to hybridize under stringent conditions to DNA of all vertebrate genomes tested, as well as to sea urchin DNA, but not to yeast DNA. In digested sea urchin sperm DNA there are more than 20 different fragments which hybridize to both the 5′ and 3′ ends of the tubulin cDNAs. A full-length β-actin cDNA clone hybridizes to 4–7 bands in restricted chicken DNA and cross hybridizes to DNA from every other species tested, including sea urchin and yeast. Hybridization to chicken DNA of cloned probes specific for the 3′ untranslated regions of β- and γ-actin mRNA indicates that the β sequence is present only once in the genome and the γ is present in at most three copies. Neither 3′ untranslated sequence is conserved evolutionarily.
The rat genome contains two segments closely related to a rat alpha-tubulin mRNA. Both have been cloned and complete nucleotide sequences are presented. Analysis of the structure and sequence of one of these establishes it as a functional alpha-tubulin gene. The second segment is a processed alpha-tubulin pseudogene. Comparison of this pseudogene to the mRNA and gene coding for alpha-tubulin strongly suggests that a mature mRNA was involved in its origin. Features of the pseudogene and a dispersed repetitive element inserted within it possibly reflect a common RNA-mediated process of insertion.
Bacterial clones containing complementary DNA sequences specific for rat brain α-tubulin messenger RNA were constructed. One plasmid, pILαTl, contains >95% of the sequences found in the mRNA: the entire coding sequence as well as extensive 5′ and 3′ untranslated sequences. Comparison of the rat amino acid sequence with the known chicken α-tubulin sequence (Valenzuela et al., 1981) reveals the extraordinary evolutionary stability of α-tubulin protein. The presence of only two interspecies amino acid differences within analogous 411 amino acid sequences predicts that amino acid substitutions in this protein are fixed with a unit evolutionary period (Wilson et al., 1977) of 550 million years (i.e. the time required for a 1% difference to arise within a specific protein in two diverging evolutionary lineages). An analysis of the silent nucleotide differences, permissible because of the degeneracy of the genetic code, demonstrates that these might not occur in a random fashion. The high guanine-cytosine bias in silent codon positions within the chicken α-tubulin sequence, previously noted by Valenzuela et al. (1981), is not conserved within the rat sequence. This decrease in guanine-cytosine bias is accompanied by a selective loss of CpG dinucleotides in the rat sequence.