Post-meiotic Spermatogenesis Research Articles

Impact of chronic vitamin A excess on sperm morphogenesis in mice.

The increasing availability of fortified foods and supplements has caused an overconsumption of vitamin A (VA), above the recommended level. To date, the effects of chronic VA excess (VAE) on spermatogenesis remain unclear. This study aims to investigate the long-term excessive intake of VA effects on spermatogenesis in mice. Dams were initially fed a control diet (4IU/g) or a VAE diet (250IU/g), 4weeks prior to mating and during pregnancy. Dams and their male pups continued this diet regimen until the offspring reached 12weeks of age. At 12weeks of age, epididymis caudal spermatozoa and testes were collected. For histological analysis, sections were stained with periodic acid-Schiff-hematoxylin, and quantitative PCR was used to detect changes in gene expression in the testes of the VAE mice. Sperm motility and morphology were evaluated to detect the endpoint of VAE toxicity. Body weights were not significantly different between the control and VAE groups. Testicular cross-sections from the control and VAE mice contained a normal array of germ cells, and the daily sperm production was similar between the two groups. However, the percentage of seminiferous tubules in stages VII and VIII was significantly lower in the VAE mice than in the control. In addition, significant changes in the expression of genes involved in retinoid metabolism, spermatogenesis, and spermiogenesis were detected in the testes of the VAE mice. Consistently, sperm motility and head morphology were significantly impaired in the VAE mice. Our findings suggest that long-term dietary intake of VAE was able to influence both pre- and post-meiotic spermatogenesis. As a result of testicular toxicity, we demonstrated, to the best of our knowledge, for the first time that long-term VAE caused sperm-head abnormalities.

Male mice with large inversions or deletions of X-chromosome palindrome arms are fertile and express their associated genes during post-meiosis

Large (>10 kb) palindromic sequences are enriched on mammalian sex chromosomes. In mice, these palindromes harbor gene families (≥2 gene copies) expressed exclusively in post-meiotic testicular germ cells, a time when most single-copy sex-linked genes are transcriptionally repressed. This observation led to the hypothesis that palindromic structures or having ≥2 gene copies enable post-meiotic gene expression. We tested these hypotheses by using CRISPR to precisely engineer large (10’s of kb) inversions and deletions of X-chromosome palindrome arms for two regions that carry the mouse 4930567H17Rik and Mageb5 palindrome gene families. We found that 4930567H17Rik and Mageb5 gene expression is unaffected in mice carrying palindrome arm inversions and halved in mice carrying palindrome arm deletions. We assessed whether palindrome-associated genes were sensitive to reduced expression in mice carrying palindrome arm deletions. Male mice carrying palindrome arm deletions are fertile and show no defects in post-meiotic spermatogenesis. Together, these findings suggest palindromic structures on the sex chromosomes are not necessary for their associated genes to evade post-meiotic transcriptional repression and that these genes are not sensitive to reduced expression levels. Large sex chromosome palindromes may be important for other reasons, such as promoting gene conversion between palindrome arms.

Dynamin 2 is essential for mammalian spermatogenesis.

The dynamin family of proteins play important regulatory roles in membrane remodelling and endocytosis, especially within brain and neuronal tissues. In the context of reproduction, dynamin 1 (DNM1) and dynamin 2 (DNM2) have recently been shown to act as key mediators of sperm acrosome formation and function. However, little is known about the roles that these proteins play in the developing testicular germ cells. In this study, we employed a DNM2 germ cell-specific knockout model to investigate the role of DNM2 in spermatogenesis. We demonstrate that ablation of DNM2 in early spermatogenesis results in germ cell arrest during prophase I of meiosis, subsequent loss of all post-meiotic germ cells and concomitant sterility. These effects become exacerbated with age, and ultimately result in the demise of the spermatogonial stem cells and a Sertoli cell only phenotype. We also demonstrate that DNM2 activity may be temporally regulated by phosphorylation of DNM2 via the kinase CDK1 in spermatogonia, and dephosphorylation by phosphatase PPP3CA during meiotic and post-meiotic spermatogenesis.

Multimerization of Drosophila sperm protein Mst77F causes a unique condensed chromatin structure.

Despite insights on the cellular level, the molecular details of chromatin reorganization in sperm development, which involves replacement of histone proteins by specialized factors to allow ultra most condensation of the genome, are not well understood. Protamines are dispensable for DNA condensation during Drosophila post-meiotic spermatogenesis. Therefore, we analyzed the interaction of Mst77F, another very basic testis-specific protein with chromatin and DNA as well as studied the molecular consequences of such binding. We show that Mst77F on its own causes severe chromatin and DNA aggregation. An intrinsically unstructured domain in the C-terminus of Mst77F binds DNA via electrostatic interaction. This binding results in structural reorganization of the domain, which induces interaction with an N-terminal region of the protein. Via putative cooperative effects Mst77F is induced to multimerize in this state causing DNA aggregation. In agreement, overexpression of Mst77F results in chromatin aggregation in fly sperm. Based on these findings we postulate that Mst77F is crucial for sperm development by giving rise to a unique condensed chromatin structure.

Ex vivo culture of Drosophila pupal testis and single male germ-line cysts: dissection, imaging, and pharmacological treatment.

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin—the histone-to-protamine switch.Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.

<em>Ex vivo</em> Culture of <em>Drosophila</em> Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin—the histone-to-protamine switch. Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages. The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.

Rapid Male-Specific Regulatory Divergence and Down Regulation of Spermatogenesis Genes in Drosophila Species Hybrids

In most crosses between closely related species of Drosophila, the male hybrids are sterile and show postmeiotic abnormalities. A series of gene expression studies using genomic approaches have found significant down regulation of postmeiotic spermatogenesis genes in sterile male hybrids. These results have led some to suggest a direct relationship between down regulation in gene expression and hybrid sterility. An alternative explanation to a cause-and-effect relationship between misregulation of gene expression and male sterility is rapid divergence of male sex regulatory elements leading to incompatible interactions in an interspecies hybrid genome. To test the effect of regulatory divergence in spermatogenesis gene expression, we isolated 35 fertile D. simulans strains with D. mauritiana introgressions in either the X, second or third chromosome. We analyzed gene expression in these fertile hybrid strains for a subset of spermatogenesis genes previously reported as significantly under expressed in sterile hybrids relative to D. simulans. We found that fertile autosomal introgressions can cause levels of gene down regulation similar to that of sterile hybrids. We also found that X chromosome heterospecific introgressions cause significantly less gene down regulation than autosomal introgressions. Our results provide evidence that rapid male sex gene regulatory divergence can explain misexpression of spermatogenesis genes in hybrids.

Complete deletion of the AZFb interval from the Y chromosome in an oligozoospermic man

Deletion of the entire AZFb interval from the Y chromosome is strictly associated with azoospermia arising from maturation arrest during meiosis. Here, we describe the exceptional case of an oligozoospermic man, 13-1217, with an AZFb + c (P5/distal-P1) deletion. Through the characterization of this patient, and two AZFb (P5/proximal-P1) patients with maturation arrest, we have explored three possible explanations for his exceptionally progressive spermatogenesis. We have determined the precise breakpoints of the deletion in 13-1217, and shown that 13-1217 is deleted for more AZFb material than one of the AZFb-deleted men (13-5349). Immunocytochemical analysis of spermatocytes with an antibody against a synaptonemal complex component indicates synapsis to be largely unaffected in 13-1217, in contrast to 13-5349 where extended asynapsis is frequent. Using PCR-based analyses of RNA and DNA from the same testicular biopsy, we show that 13-1217 expresses post-meiotic germ cell markers in the absence of genomic DNA and transcripts from the AZFb and AZFc intervals. We have determined the Y chromosome haplogroup of 13-1217 to be HgL-M185. Our results indicate that the post-meiotic spermatogenesis in 13-1217 is not a consequence of mosaicism or retention of a key AZFb gene. On the contrary, since the Hg-L Y chromosome carried by 13-1217 is uncommon in Western Europe, a Y-linked modifier locus remains a possible explanation for the oligozoospermia observed in patient 13-1217. Further cases must now be studied to understand how germ cells complete spermatogenesis in the absence of the AZFb interval.

Haspin: a newly discovered regulator of mitotic chromosome behavior

The haspins are divergent members of the eukaryotic protein kinase family that are conserved in many eukaryotic lineages including animals, fungi, and plants. Recently-solved crystal structures confirm that the kinase domain of human haspin has unusual structural features that stabilize a catalytically active conformation and create a distinctive substrate binding site. Haspin localizes predominantly to chromosomes and phosphorylates histone H3 at threonine-3 during mitosis, particularly at inner centromeres. This suggests that haspin directly regulates chromosome behavior by modifying histones, although it is likely that additional substrates will be identified in the future. Depletion of haspin by RNA interference in human cell lines causes premature loss of centromeric cohesin from chromosomes in mitosis and failure of metaphase chromosome alignment, leading to activation of the spindle assembly checkpoint and mitotic arrest. Haspin overexpression stabilizes chromosome arm cohesion. Haspin, therefore, appears to be required for protection of cohesion at mitotic centromeres. Saccharomyces cerevisiae homologues of haspin, Alk1 and Alk2, are also implicated in regulation of mitosis. In mammals, haspin is expressed at high levels in the testis, particularly in round spermatids, so it seems likely that haspin has an additional role in post-meiotic spermatogenesis. Haspin is currently the subject of a number of drug discovery efforts, and the future use of haspin inhibitors should provide new insight into the cellular functions of these kinases and help determine the utility of, for example, targeting haspin for cancer therapy.

Sertoli Cell Androgen Receptor DNA Binding Domain Is Essential for the Completion of Spermatogenesis

We examined the biological importance of Sertoli cell androgen receptor (AR) genomic interaction, using a Cre-loxP approach to selectively disrupt the AR DNA-binding domain (AR-DBD). Sertoli cell (SC)-specific transgenic Abpa or AMH promoters targeted Cre-mediated inframe excision of mouse Ar exon-3, encoding the AR-DBD second zinc-finger (ZF2), generating SC-specific mutant AR(DeltaZF2) lines designated Abp.SCAR(DeltaZF2) and AMH.SCAR(DeltaZF2), respectively. Both SCAR(DeltaZF2) lines produced infertile males exhibiting spermatogenic arrest, despite normal SC numbers and immunolocalized SC nuclear AR. Adult homozygous TgCre((+/+)) SCAR(DeltaZF2) or double-TgCre((+/-)) Abp/AMH.SCAR(DeltaZF2) males displayed equivalent small testes 30% of normal size, representing maximal Cre-loxP-disruption of Sertoli AR function. Hemizygous TgCre((+/-)) vs. homozygous TgCre((+/+)) Abp.SCAR(DeltaZF2) testes were larger (47% normal size) with more postmeiotic development, indicating dose-dependent Cre-mediated disruption of SC-specific AR-DBD activity. SCAR(DeltaZF2) males exhibited adult Leydig cell hypertrophy but normal serum testosterone levels. Sertoli cell-specific Rhox5 and Spinlw1 transcription, regulated by divergent or classical androgen-response elements, respectively, were both decreased in postnatal SCAR(DeltaZF2) vs. control testes, demonstrating SC-specific AR-DBD function as early as postnatal d 5. However, Rhox5 expression declined dose-dependently, whereas Spinlw1 expression increased, in adult TgCre((+/-)) and TgCre((+/+)) SCAR(DeltaZF2) testes, revealing differential temporal control for distinct AR-regulated transcripts. Androgen-repressed Ngfr was not up-regulated in SCAR(DeltaZF2) testes, suggesting maintenance of a nonclassical mechanism independent of AR-DBD. Thus, our unique SCAR(DeltaZF2) paradigm provided dose-dependent Cre-mediated disruption of testicular development and gene expression revealing that the AR-DBD is essential for SC function and postmeiotic spermatogenesis. Nongenomic or AR-DBD-independent pathways appear secondary or play no major independent role in SC function.

Expression of choline acetyltransferase mRNA in spermatogenic cells results in an accumulation of the enzyme in the postacrosomal region of mature spermatozoa.

The gene encoding choline acetyltransferase (ChAT; EC 2.3.1.6), the key enzyme in the synthesis of the neurotransmitter acetylcholine (ACh), is shown to be expressed in rat and human testes. High levels of two ChAT transcripts of 3.5 and 1.3 kilobases were detected by Northern blot analysis of adult rat testis RNA. A single ChAT mRNA species of 3.2 kilobases was detected in human testis. Cells responsible for the synthesis of ChAT mRNA in rat testis were localized by in situ hybridization in the middle part of the seminiferous epithelium, where the labeling was mostly found over spermatocytes and spermatids. Studies on the ontogeny of ChAT mRNA expression showed low levels in prepubertal rats with increasing levels as sexual maturation is reached. A peak of expression was seen at postnatal day 32, correlating with the onset of postmeiotic spermatogenesis. Results from surgical and pharmacological treatments suggest that androgens, as well as pituitary factors, could influence the relative levels of the two ChAT mRNAs detected in rat testis. Evidence for translation of the mRNA detected in the testis was obtained from the demonstration of ChAT-like immunoreactivity in ejaculated human spermatozoa. The staining was restricted to the postacrosomal region of the head, where the membrane of the sperm first fuses with that of the egg during fertilization, and to the annulus, a ring of dense material in the caudal end of the midpiece. Combined, these findings support the hypothesis that the neurotransmitter ACh is involved in reproductive function.

Heritable translocation test and dominant-lethal assay in male mice with methyl methanesulfonate

A dominant-lethal test and a heritable translocation test were performed with methyl methanesulphonate (MMS) at 40 mg/kg by treating the sensitive periods of post-meiotic spermatogenesis i.e. spermatozoa and spermatids. In the dominant-lethal test 25 to 60% dominant-lethal mutations were obtained depending on the mating intervals. In the heritable translocation test 11% sterile and partially sterile F 1 males were observed in 250 offspring of the MMS group. All of the 14 partially sterile and 6 of the 14 sterile F 1 males were demonstrated to be translocation carriers. Fertility of the partial steriles was about 40% of normal fertility. The translocation frequencies in the primary spermatocytes of the partially sterile F 1 males varied between 2 and 99%. Transmission of partially sterility and translocations was confirmed in the F 2 generation. There were no partially sterile or sterile males among the 245 controls.