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The normal steps of spermiogenesis in relation to testis tubule stages in an XY mouse. This figure presents a schematic depiction of the 16 steps of mouse spermiogenesis based on the stage of the spermatogenic cycle (stages I–XII). Photos of step 1–12 spermatid nuclei are shown with PAS staining at the foot of the diagram. The transition from round to elongating spermatids takes place during stage VIII; this marks the beginning of sperm morphogenesis. The shaping of the sperm head and formation of the sperm tail are essentially complete by step 12. However, the sperm are not shed until the following stages VIII-IX; consequently two generations of spermatids are present in stages I-VIII.
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A previous study indicated that genetic information encoded on the mouse Y chromosome short arm (Yp) is required for efficient completion of the second meiotic division (that generates haploid round spermatids), restructuring of the sperm head, and development of the sperm tail. Using mouse models lacking a Y chromosome but with varying Yp gene com...
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... By comparison of X E,Z2 Y* X Sry with XY* X Sxr a (and XY controls) it is clear that Sxr a is more effective than the Zfy2 transgene in supporting the sperm morphogenesis. In agreement with the illustration in Fig 2, in XY males at stage XI sperm head morphogenesis has progressed to the early 'hooked tip' stage and nuclear condensation is evident. As previously reported, in XY* X Sxr a at this stage some of the sperm from the previous cycle have not yet been shed (stained dark blue); the spermatids from the new cycle are retarded with respect to elongation and nuclear condensation is not evident. ...
Citations
... The three models with varying NPYp content (XSxr a O, X E Sxr b O, and X E OSry; Fig. 1) were used to demonstrate that NPYp (MSYp) encode genes necessary for spermiogenesis (Vernet et al., , 2016b. ...
... The NPYp contribution to spermiogenesis was also assessed using males with the same Y chromosome gene content but carrying the meiotic pairing partner Y* X (Fig. 1) and therefore not subjected to the meiotic arrest due to the X chromosome univalency (Yamauchi et al., 2015;Vernet et al., 2016b). The addition of Y* X did not enhance spermatid elongation or sperm morphogenesis. ...
... The addition of Y* X did not enhance spermatid elongation or sperm morphogenesis. X E Y* X Sry and X E OSry males displayed similar arrest at the round spermatid stage, with no sperm head or tail development observed, while X E Sxr b Y* X had similar delayed spermatid elongation, nuclear condensation and acrosome relocation as that observed with X E Sxr b O (Vernet et al., 2016b). When compared to XY, X E Sxr b Y* X males had 2.5 times fewer round spermatids but almost 14 times fewer elongating/elongated spermatids, indicating that not all round spermatids undergo elongation (Yamauchi et al., 2015). ...
The Y-linked zinc finger gene ZFY is conserved across eutherians and known to be a critical fertility factor in some species. The initial studies of the mouse homologues, Zfy1 and Zfy2, were performed using mice with spontaneous Y chromosome mutations and Zfy transgenes. These studies revealed that Zfy is involved in multiple processes during spermatogenesis, including removal of germ cells with unpaired chromosomes and control of meiotic sex chromosome inactivation during meiosis I, facilitating the progress of meiosis II, promoting spermiogenesis, and improving assisted reproduction outcomes. Zfy was also identified as a key gene in Y chromosome evolution, protecting this chromosome from extinction by serving as the executioner responsible for meiosis surveillance. Studies with targeted Zfy knockouts revealed that mice lacking both homologues have severe spermatogenic defects and are infertile. Based on protein structure and in vitro assays, Zfy is expected to drive spermatogenesis as a transcriptional regulator. The combined evidence documents that the presence of at least one Zfy homologue is required for male fertility, and that Zfy2 plays a more prominent role. This knowledge reinforces the importance of these factors for mouse spermatogenesis and informs our understanding of the human ZFY variants, which are homologous to the mouse Zfy1 and Zfy2.
... Prssly (Protease, serine-like, Chr Y) and Teyorf1 (Testis expressed, chromosome Y open reading frame 1) are both acquired single-copy genes located on the distal tip of the short arm of the Y chromosome (Yp) adjacent to telomeric sequence ( Figure 1), with no X chromosome-linked homologues [1]. Both genes are primarily expressed in testicular germ cells [1], have been mapped to the Y short arm derivatives Sxr a and Sxr b [2] (Figure 1), and have been proposed to be candidates to improve sperm morphology in mice with Y chromosome deficiencies and sperm head defects [2,3]. ...
... Prssly (Protease, serine-like, Chr Y) and Teyorf1 (Testis expressed, chromosome Y open reading frame 1) are both acquired single-copy genes located on the distal tip of the short arm of the Y chromosome (Yp) adjacent to telomeric sequence ( Figure 1), with no X chromosome-linked homologues [1]. Both genes are primarily expressed in testicular germ cells [1], have been mapped to the Y short arm derivatives Sxr a and Sxr b [2] (Figure 1), and have been proposed to be candidates to improve sperm morphology in mice with Y chromosome deficiencies and sperm head defects [2,3]. ...
... Most Y chromosome genes do not yet have clearly ascribed functions, and many are predicted to be important for spermatogenesis [42]. The recently discovered Prssly and Teyorf1 were previously suggested as candidates for "fertility" genes [2,3]. Here, we describe generation and characterization of Prssly KO and Teyorf1 KO mice to determine the importance of Prssly and Teyorf1 for male fertility, along with sequence and transcript expression analyses of these genes. ...
Prssly (Protease, serine-like, Chr Y) and Teyorf1 (Testis expressed, chromosome Y open reading frame 1) are two acquired single-copy genes located on the distal tip of the non-pairing short arm of the mouse Y chromosome (NPYp) adjacent to telomeric sequence. Both genes lack X chromosome-linked homologues and are expressed in testicular germ cells. We first performed analysis of Prssly and Teyorf1 genomic sequences and demonstrated that previously reported Prssly sequence is erroneous and the true Prssly sequence is longer and encodes a larger protein than previously estimated. We also confirmed that both genes encode pseudogenes that are not expressed in testes. Next, using CRISPR/Cas9 genome targeting we generated Prssly and Teyorf1 knockout (KO) mice and characterized their phenotype. To create Prssly KO mice we targeted the conserved exon 5 encoding a trypsin domain typical for serine proteases. The targeting was successful and resulted in a frame shift mutation that introduced a premature stop codon, with the Prssly KO males retaining only residual transcript expression in testes. The Teyorf1 targeting removed the entire open reading frame (ORF) of the gene, which resulted in no transcript expression in KO males. Both Prssly KO and Teyorf1 KO males were fertile and had normal testis size and normal sperm number, motility, and morphology. Our findings show that Prssly and Teyorf1 transcripts with potential to encode proteins are dispensable for male fertility.
... This was associated with de novo transcription of these genes during an interphase between the meiotic divisions suggesting that the male-specific meiotic interphase serves to allow for meiosis II (MII) critical X and Y gene reactivation following sex chromosome silencing in meiotic prophase. In our earlier work, we have shown that mouse Zfy2 promotes spermatid elongation [14,15] and enhances the efficiency of round spermatid injection (ROSI) [15]. We also demonstrated that mouse Zfy2 is the sole Y chromosome gene responsible for enabling round spermatids to transform into sperm in the context of limited Y chromosome content. ...
... Several types of males, with Y chromosome deficiencies and variable Zfy contribution, were previously investigated to pinpoint the functional differences between Zfy1 and Zfy2 [10][11][12][13][14][15][16]38]. The findings acquired with these mice have shown that early during meiosis, Zfy1 and Zfy2 play to some extend overlapping but nevertheless distinct roles [10][11][12]38]. ...
... The findings acquired with these mice have shown that early during meiosis, Zfy1 and Zfy2 play to some extend overlapping but nevertheless distinct roles [10][11][12]38]. However, in later meiotic stages, and postmeiotically, Zfy2 becomes more functionally significant, with roles in facilitating the 2nd meiotic division [13] and sperm morphogenesis [14,15]. Based on expression differences, and analyses of prior mouse models, it has been suggested that Zfy1 plays a dual role in spermatogenesis to produce both activator and repressive proteins while Zfy2 serves as a super active transcription factor [13]. ...
Using mice with Y chromosome deficiencies and supplementing Zfy transgenes we, and others, have previously shown that loss of Y chromosome Zfy1 and Zfy2 genes is associated with infertility and spermiogenic defects, and that addition of Zfy transgenes rescues these defects. In these past studies, the absence of Zfy was linked to the loss of other Y chromosome genes, which might have contributed to spermiogenic phenotypes. Here, we used CRISPR/Cas9 to specifically remove open reading frame of Zfy1, Zfy2, or both Zfy1 and Zfy2, and generated Zfy knockout (KO) and double knockout (DKO) mice. Zfy1 KO and Zfy2 KO mice were both fertile, but the latter had decreased litters size and sperm number, and sperm headshape abnormalities. Zfy DKO males were infertile and displayed severe spermatogenesis defects. Post-meiotic arrest largely prevented production of sperm and the few sperm that were produced all displayed gross headshape abnormalities and structural defects within head and tail. Infertility of Zfy DKO mice could be overcome by injection of spermatids or sperm directly to oocytes and the resulting male offspring had the same spermiogenic phenotype as their fathers. The study is the first describing detailed phenotypic characterization of mice with the complete Zfy gene loss. It provides evidence supporting that presence of at least one Zfy homologue is essential for male fertility and development of normal sperm functional in unassisted fertilization. The data also show that while the loss of Zfy1 is benign, the loss of Zfy2 is mildly detrimental for spermatogenesis.
... The downregulation of these two genes was confirmed by qRT-PCR (Fig. 4e). Downregulated genes also included Zfy1/2, two genes located in the Y chromosome and involved in spermatogenesis [38][39][40][41][42][43] ; Uba1y, a gene also localized in the Y chromosome and involved in the survival and proliferation of differentiating spermatogonia [44][45][46] ; Sohlh2, an essential germ cell-specific gene [47][48][49][50] ; Nxf3, a gene localized in the X chromosome and specifically expressed in Sertoli cells 51,52 ; Fkbp6, a meiosis-specific gene 53 ; and the upstream regulator of the EIF2S3Y gene, which, together with SRY, orchestrates the male gametogenesis program [54][55][56] . Finally, among the genes that were downregulated in OE cells were Zfp264 and Usp26. ...
The kinase haspin phosphorylates histone H3 at threonine-3 (H3T3ph) during mitosis. H3T3ph provides a docking site for the Chromosomal Passenger Complex at the centromere, enabling correction of erratic microtubule-chromosome contacts. Although this mechanism is operational in all dividing cells, haspin-null mice do not exhibit developmental anomalies, apart from aberrant testis architecture. Investigating this problem, we show here that mouse embryonic stem cells that lack or overexpress haspin, albeit prone to chromosome misalignment during metaphase, can still divide, expand and differentiate. RNA sequencing reveals that haspin dosage affects severely the expression levels of several genes that are involved in male gametogenesis. Consistent with a role in testis-specific expression, H3T3ph is detected not only in mitotic spermatogonia and meiotic spermatocytes, but also in non-dividing cells, such as haploid spermatids. Similarly to somatic cells, the mark is erased in the end of meiotic divisions, but re-installed during spermatid maturation, subsequent to methylation of histone H3 at lysine-4 (H3K4me3) and arginine-8 (H3R8me2). These serial modifications are particularly enriched in chromatin domains containing histone H3 trimethylated at lysine-27 (H3K27me3), but devoid of histone H3 trimethylated at lysine-9 (H3K9me3). The unique spatio-temporal pattern of histone H3 modifications implicates haspin in the epigenetic control of spermiogenesis.
... Zfy2 is also involved in spermatid morphogenesis, as transgenic integration of Zfy2 in Y-deletants improves spermatid morphology and increases the success rate of assisted reproduction (Figure 4; Yamauchi et al., 2015). Indeed, addition of Zfy2 contributes to the restructuring of the sperm head and development of the sperm tail (Figure 4; Vernet et al., 2016b). Specifically, these morphogenic changes are thought to be regulated by Zfy2 through strong expression of the ZFY2 transactivation domain from an alternative, spermatid-specific Cypt-derived promoter. ...
... These caveats can complicate interpretation of transgene rescue experiments. Even if transgenes are integrated at defined genomic locations, their expression levels can differ from wild-type (Vernet et al., 2016b;Vernet et al., 2014). This is limiting because dosage of ancestral Y genes is thought to be crucial and sensitive for sex-chromosome function (see: Evolutionary forces and functional specialisation of the Y chromosome). ...
The mammalian Y chromosome is critical for male sex determination and spermatogenesis. However, linking each Y gene to specific aspects of male reproduction has been challenging. As the Y chromosome is notoriously hard to sequence and target, functional studies have mostly relied on transgene-rescue approaches using mouse models with large multi-gene deletions. These experimental limitations have oriented the field toward the search for a minimum set of Y genes necessary for male reproduction. Here, considering Y-chromosome evolutionary history and decades of discoveries, we review the current state of research on its function in spermatogenesis and reassess the view that many Y genes are disposable for male reproduction.
... Some studies have shown that reducing the level of Zfx/Zfy mRNA expression will make the physiological function of X/Y sperm worse, which can influence the offspring gender [19,[29][30][31]34]. Zfy controls sperm head and tail formation and neck development [35], which is highly expressed between meiosis I and meiosis II. It can regulate sperm morphology and ROSI efficiency and promotes second meiosis [36,37]. ...
Zinc finger protein X-linked (Zfx) was regarded to be a sex determination factor and plays a critical role in spermatogenesis. RNAi is an effective method of silencing Zfx mRNA expression. However, there has been little research on the use of RNAi technology to control the sex of the offspring of red deer (Cervus elaphus). The objective of this study was first to explore an efficient method to alter the red deer offspring sex-ratio by silencing the gene Zfx during spermatogenesis. Three recombinant expression vectors pLL3.7/A, pLL3.7/B, and pLL3.7/C were constructed to interrupt the Zfx gene. The results showed that the expression of Zfx mRNA was significantly silenced by pLL3.7/A (P
... The most important morphological changes that occur during spermiogenesis are formation of acrosome, condensation of core chromatin, growth of a moving tail and loss of unnecessary parts for spermatozoa which will occur later in spermatid material. Spermiogenesis consists of Golgi, cap, acrosomal and maturation stages (Vernet et al., 2016). ...
... After this concentration, the DNA gains resistance. Protamine must be replaced by histones provided by oocytes before the condensation of the chromatin during fertilization (Vernet et al., 2016). ...
... Spermatozoa: spermatozoa with lengths of approximately 60-75 spm under a light microscope have two parts; a head and tail. These comprise parts such as neck, middle piece, main piece and end piece (Vernet et al., 2016). ...
INTRODUCTION Animals used in research and biological studies designed according to scientific rules are called experimental animals or laboratory animals. The reasons why these animals are preferred as experimental animals are; ease of cultivation, ease of performing complex genetic applications, short pregnancy duration and sexual cycles, easy feeding and care, no need for large cultivation areas and sufficient knowledge gathered over many years. According to the International Laboratory Animals Committee; 40% to 80% of the preferred laboratory animals were reported to be mice (Akman, 2007).
Laboratory animals can be listed as follows according to the frequency of experiments. 1. Mice 2. Rats 3. Rabbit 4. Guinea pigs The male reproductive system and production, nutrition and storage of haploid male sex cells is responsible for the production and secretion of male sex hormones. Male reproductive system organs include effluent channels that transmit these cells from testes that express gonad cells and secrete androgens, of the tubuli recti, rete testis, ductuli efferentes, ductus epididymis, ductus deferens, ductus ejaculatorius and urethra; and the prostate gland, seminal vesicle and bulbourethral glands and external genital penis (Bernal, Aya, De Jesus-Ayson, & Garcia, 2015).
... One of the most generally accepted hypotheses is that the cytoplasmic continuity they provide equalises the genomic contents of postmeiotic cells [7], thus rendering postmeiotic cells phenotypically diploid [8]. Support for this hypothesis comes from the observation that, in postmeiotic cells, X-encoded transcripts pass through intercellular bridges in the form of protein-messenger RNA (mRNA) complexes [9] and that, in general, both the X and Y chromosomes encode postmeiotically expressed genes that are essential for sperm cytodifferentiation and function, e.g., [10][11][12]. Consequently, it is undeniable that postmeiotic equalisation of X-and Y-encoded gene products across the syncytium must be essential for fertility in mammals. Nevertheless, this cannot be the only or even the primary reason for the presence of these bridges, for three reasons: Firstly, cytoplasmic bridges are present in all species studied to date, including species without differentiated sex chromosomes [13], species with female rather than male heterogamety [14], and species with haploid males [15]. ...
During in vitro fertilisation (IVF), pharmacological activation of the murine X chromosome–encoded receptor proteins Toll-like receptor (TLR) 7 and TLR8 reportedly results in male-biased litters by selectively disrupting the motility of X-bearing sperm cells. Thus—in the context of agonist treatment during IVF—these receptors act as ‘suicidal’ segregation distorters that impair their own transmission to the next generation. Such behaviour would, from an evolutionary perspective, be strongly selected against if present during natural fertilisation. Consequently, TLR7/8 biology in vivo must differ significantly from this in vitro situation to allow these genes to persist in the genome. Here, we use our current understanding of male germ cell biology and TLR function as a starting point to explore the mechanistic and evolutionary aspects of this apparent paradox.
... Some studies have shown that reducing the level of Zfx/Zfy mRNA expression will make the physiological function of X/Y sperm worse, which can influence offspring gender [14,17,[20][21][22]. Zfy is closely related to sperm head and tail formation and neck development [23], which is highly expressed between meiosis I and II. It can regulate sperm morphology and ROSI efficiency, and promotes second meiosis [24,25]. ...
Background: Zinc finger protein X-linked (Zfx) was regarded to be a sex determination factor, and plays a critical role in spermatogenesis. RNAi is an effective method of silencing Zfx/Zfy mRNA expression. However, there has been little research on the use of RNAi technology to control the sex of the offspring of Cervus elaphus. The objective of this study was first to explore an efficient method to alter the Cervus elaphus offspring sex-ratio by silencing the genes Zfx during spermatogenesis.
Results: Three recombinant expression vectors pLL3.7/A, pLL3.7/B and pLL3.7/C were constructed to interrupt the Zfx gene. The results showed that the expression of Zfx mRNA was significantly silenced by pLL3.7/A (P < 0.01), compared with the control group. The group injected with pLL3.7/A produced 94 Cervus elaphus, including 68 males and 26 females. The male rates (72.34%) were significantly higher than the control groups (P < 0.01).
Conclusions: Our experiment suggest that the Zfx gene plays a significant role in the process of X-sperm formation. Zfx siRNA may be a useful approach to control offspring sex in Cervus elaphus.
... Another gene on the p arm of the Y chromosome is ZFY which encodes a zinc finger-containing protein and functions as a transcription factor. Expressed in almost all somatic tissues [28,29], it is proposed to play a role in spermatogenesis, particularly in promoting meiotic division and sperm formation [38][39][40][41] Refer Additional file 1: Table S2. While mice knockout for Zfy genes are infertile [42], despite its expression in multiple tissues, a rare deletion of ZFY and SRY in a woman was not associated with Turner syndrome stigmata. ...
The human Y chromosome harbors genes that are responsible for testis development and also for initiation and maintenance of spermatogenesis in adulthood. The long arm of the Y chromosome (Yq) contains many ampliconic and palindromic sequences making it predisposed to self-recombination during spermatogenesis and hence susceptible to intra-chromosomal deletions. Such deletions lead to copy number variation in genes of the Y chromosome resulting in male infertility. Three common Yq deletions that recur in infertile males are termed as AZF (Azoospermia Factor) microdeletions viz. AZFa, AZFb and AZFc. As estimated from data of nearly 40,000 Y chromosomes, the global prevalence of Yq microdeletions is 7.5% in infertile males; however the European infertile men are less susceptible to Yq microdeletions, the highest prevalence is in Americans and East Asian infertile men. In addition, partial deletions of the AZFc locus have been associated with infertility but the effect seems to be ethnicity dependent. Analysis of > 17,000 Y chromosomes from fertile and infertile men has revealed an association of gr/gr deletion with male infertility in Caucasians and Mongolian men, while the b2/b3 deletion is associated with male infertility in African and Dravidian men. Clinically, the screening for Yq microdeletions would aid the clinician in determining the cause of male infertility and decide a rational management strategy for the patient. As these deletions are transmitted to 100% of male offspring born through assisted reproduction, testing of Yq deletions will allow the couples to make an informed choice regarding the perpetuation of male infertility in future generations. With the emerging data on association of Yq deletions with testicular cancers and neuropsychiatric conditions long term follow-up data is urgently needed for infertile men harboring Yq deletions. If found so, the information will change the current the perspective of androgenetics from infertility and might have broad implication in men health.
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