Age-Dependent Alterations in Meiotic Recombination Cause Chromosome Segregation Errors in Spermatocytes.

Faithful chromosome segregation in meiosis requires crossover (CO) recombination, which is regulated to ensure at least one CO per homolog pair. We investigate the failure to ensure COs in juvenile male mice. By monitoring recombination genome-wide using cytological assays and at hotspots using molecular assays, we show that juvenile mouse spermatocytes have fewer COs relative to adults. Analysis of recombination in the absence of MLH3 provides evidence for greater utilization in juveniles of pathways involving structure-selective nucleases and alternative complexes, which can act upon precursors to generate noncrossovers (NCOs) at the expense of COs. We propose that some designated CO sites fail to mature efficiently in juveniles owing to inappropriate activity of these alternative repair pathways, leading to chromosome mis-segregation. We also find lower MutLγ focus density in juvenile human spermatocytes, suggesting that weaker CO maturation efficiency may explain why younger men have a higher risk of fathering children with Down syndrome.

YTHDC2 serves a distinct late role in spermatocytes during germ cell differentiation.

Posttranscriptional regulation of gene expression by RNA-binding proteins can enhance the speed and robustness of cell state transitions by controlling RNA stability, localization, or if, when, or where mRNAs are translated. The RNA helicase YTHDC2 is required to shut down components of the mitotic program to facilitate a proper switch from mitosis to meiosis in mouse germ cells. Here, we show that YTHDC2 has a second essential role in promoting meiotic progression in late spermatocytes. Inducing conditional knockout of Ythdc2 during the first wave of spermatogenesis, after initiation of meiotic prophase, allowed YTHDC2-deficient germ cells to advance to the pachytene stage and properly express many meiotic markers. However, the YTHDC2-deficient spermatocytes mis-expressed a number of genes, some up-regulated and some down-regulated, failed to transition to the diplotene stage, and then quickly died. Coimmunoprecipitation experiments revealed that YTHDC2 interacts with several RNA-binding proteins in early or late spermatocytes, with many of the interacting proteins, including MEIOC, localizing to granules, similar to YTHDC2. Our findings suggest that YTHDC2 collaborates with other RNA granule components to facilitate proper progression of germ cells through multiple steps of meiosis via mechanisms influencing posttranscriptional regulation of RNAs.

Unpacking chromatin remodelling in germ cells: implications for development and evolution.

Germ cells reflect the evolutionary history and future potential of a species. Understanding how the genome is organised in gametocytes is fundamental to understanding fertility and its impact on genetic diversity and evolution of species. Here, we explore principles of chromatin remodelling during the formation of germ cells and how these are affected by genome reshuffling.

Modeling mammalian spermatogonial differentiation and meiotic initiation in vitro.

In mammalian testes, premeiotic spermatogonia respond to retinoic acid by completing an essential lengthy differentiation program before initiating meiosis. The molecular and cellular changes directing these developmental processes remain largely undefined. This wide gap in knowledge is due to two unresolved technical challenges: (1) lack of robust and reliable in vitro models to study differentiation and meiotic initiation; and (2) lack of methods to isolate large and pure populations of male germ cells at each stage of differentiation and at meiotic initiation. Here, we report a facile in vitro differentiation and meiotic initiation system that can be readily manipulated, including the use of chemical agents that cannot be safely administered to live animals. In addition, we present a transgenic mouse model enabling fluorescence-activated cell sorting-based isolation of millions of spermatogonia at specific developmental stages as well as meiotic spermatocytes.

Heat stress induced piRNA alterations in pachytene spermatocytes and round spermatids.

BACKGROUND: Spermatogenesis is a temperature-sensitive process, and elevation in temperature hampers this process quickly and significantly. We studied the molecular effects of testicular heating on piRNAs and gene expression in rat testicular germ cells. METHODS: We generated a cryptorchid rat model by displacing the testis from the scrotal sac (34 °C) to the abdominal area (37 °C) and sacrificed animals after 1 day, 3 days, and 5 days. Pachytene spermatocytes and round spermatids were purified using elutriation centrifugation and percoll gradient methods. We performed transcriptome sequencing in pachytene spermatocytes and round spermatids to identify differentially expressed piRNAs and their probable targets, i.e., TE transcripts and mRNAs. RESULTS: As a result of heat stress, we observed significant upregulation of piRNAs and TE transcripts in testicular germ cells. In addition to this, piRNA biogenesis machinery and heat shock proteins (Hsp70 and Hsp90 family members) were upregulated. mRNAs have also been proposed as targets for piRNAs; therefore, we shortlisted certain piRNA-mRNA pairs with an inverse relationship of expression. We observed that in testicular heat stress, the heat shock proteins go hand-in-hand with the upregulation of piRNA biogenesis machinery. The dysregulation of piRNAs in heat-stressed germ cells, increased ping-pong activity, and disturbed expression of piRNA target transcripts suggest a connection between piRNAs, mRNAs, and TE transcripts. CONCLUSIONS: In heat stress, piRNAs, piRNA machinery, and heat shock proteins are activated to deal with low levels of stress, which is followed by a rescue approach in prolonged stressaccompained by high TE activity to allow genetic mutations, perhaps for survival and adaptability.

Study of spermatogenic and Sertoli cells in the Hu sheep testes at different developmental stages.

Spermatogenesis is a highly organized process by which undifferentiated spermatogonia self-renew and differentiate into spermatocytes and spermatids. The entire developmental process from spermatogonia to sperm occurs within the seminiferous tubules. Spermatogenesis is supported by the close interaction of germ cells with Sertoli cells. In this study, testicular tissues were collected from Hu sheep at 8 timepoints after birth: 0, 30, 90, 180, 270, 360, 540, and 720 days. Immunofluorescence staining and histological analysis were used to explore the development of male germ cells and Sertoli cells in the Hu sheep testes at these timepoints. The changes in seminiferous tubule diameter and male germ cells in the Hu sheep testes at these different developmental stages were analyzed. Then, specific molecular markers were used to study the proliferation and differentiation of spermatogonia, the timepoint of spermatocyte appearance, and the maturation and proliferation of Sertoli cells in the seminiferous tubules. Finally, the formation of the blood-testes barrier was studied using antibodies against the main components of the blood-testes barrier, ß-catenin, and ZO-1. These findings not only increased the understanding of the development of the Hu sheep testes, but also laid a solid theoretical foundation for Hu sheep breeding.

Comparative effects of chemical and green zinc oxide nanoparticles in caprine testis: ultrastructural and steroidogenic enzyme analysis.

Recent advancements in nanotechnology has opened up enormous possibilities in diverse sectors such as industries, agriculture, environmental remediation, electronics, medicine and varied industries. Among metal oxide nanoparticles zinc oxide nanoparticles has gained considerable attention due to their fascinating physiochemical properties. Rapid growth in the use of zinc oxide nanoparticles (ZnONPs) in daily household products, food and feed additives, biological products, medicine, as antimicrobial agents, electronics and agriculture, creates serious toxic potential risks of these engineered nanoparticles on living organisms. The aim of present study was to assess the effects of synthesized chemical ZnONPs and green ZnONPs on testicular tissue of Capra hircus (goat) in vitro. The reproductive stress was analyzed by ultrastructural damage, change in frequency of apoptotic cells and alteration in steroidogenic enzyme activity. The transmission electron micrographs of testicular cells after treatment with chemical and green ZnONPs at three doses (10 µg/ml, 20 µg/ml and 30 µg/ml) for exposure duration 4 h and 8 h illustrated that chemical nanoparticles induced more alterations, identified as ruptured nuclear membrane, condensation and margination of chromatin material in somatic cells and germ cells in the seminiferous tubules, presence of apoptotic bodies in nucleus of spermatocytes and spermatids, reduction in number of cell organelles, vacuolization and hyalinization of cytoplasm. Maximum damage was observed after treatment of testicular tissues with 30 µg/ml of chemical ZnONPs for 8 h exposure duration. However, the green ZnONPs were found to be less toxic as evidenced by few apoptotic characteristics in testicular cells. The results of fluorescence assay by acridine orange staining showed significant increase in the percentage of apoptotic cells in chemical treated groups as compared to green and control groups. Decreased enzyme activity of 3ß-Hydroxysteroid dehydrogenase and 17ß-Hydroxysteroid dehydrogenase was assayed in chemical ZnONPs than green ZnONPs treated groups. Our results confirm that chemical ZnONPs are significantly more toxic in comparison to green ZnONPs and adversely affects the male fertility.

Reactive oxygen species and their role in the andrological factor of couple fertility.

Reactive oxygen species play a significant role in male fertility and infertility. They are essential for physiological processes, but when their concentration becomes excessive, they can be a cause of various sperm pathologies. Seminal leukocytes and pathologically abnormal sperm are the primary sources of oxygen radicals in ejaculate. They negatively affect sperm quality, including DNA fragmentation and sperm motility impairment. Addressing increased concentrations of reactive oxygen species involves various appropriate lifestyle modifications and measures, including the use of antioxidants, treatment of urogenital infections, management of varicocele, weight reduction, and others. In many cases, these interventions can lead to adjustments in the condition and improvement in sperm quality. Such improvements can subsequently lead to enhanced outcomes in assisted reproduction or even an increased likelihood of natural conception. In some instances, the need for donor sperm may be eliminated. However, a key factor is adhering to a sufficiently prolonged treatment, which requires patience on the part of both, the physician and the patient.

Comparative proteomic analysis identifies differentially expressed proteins associated with meiotic arrest in cattle-yak hybrids.

Cattle-yak, the interspecific hybrid between yak and taurine cattle, exhibits male-specific sterility. Massive loss of spermatogenic cells, especially spermatocytes, results in azoospermia in these animals. Currently, the mechanisms underlying meiosis block and defects in spermatocyte development remain elusive. The present study was designed to investigate the differences in the protein composition of spermatocytes isolated from 12-month-old yak and cattle-yak testes. Histological analysis confirmed that spermatocytes were the most advanced germ cells in the testes of yak and cattle-yak at this developmental stage. Comparative proteomic analysis identified a total of 452 differentially abundant proteins (DAPs) in the fluorescence-activated cell sorting (FACS) isolated spermatocytes from cattle-yak and yak. A total of 291 proteins were only present in yak spermatocytes. Gene Ontology analysis revealed that the downregulated DAPs were mostly enriched in the cellular response to DNA damage stimulus and double-strand breaks (DSBs) repair via break-induced replication, while the proteins specific for yak were related to cell division and cycle, spermatogenesis, and negative regulation of the extrinsic apoptotic signaling pathway. Ultimately, these DAPs were related to the critical process for spermatocyte meiotic events, including DSBs, homologous recombination, synapsis, crossover formation, and germ cell apoptosis. The database composed of proteins associated with spermatogenesis, including KPNA2, HTATSF1, TRIP12, STIP1, LZTFL1, LARP7, MTCH2, STK31, ROMO1, CDK5AP2, DNMT1, RBM44, and CHRAC1, is the focus of further research on male hybrid sterility. In total, these results provide insight into the molecular mechanisms underlying failed meiotic processes and male infertility in cattle-yak.

Kinesin-5 Eg5 mediates centrosome separation to control spindle assembly in spermatocytes.

Timely and accurate centrosome separation is critical for bipolar spindle organization and faithful chromosome segregation during cell division. Kinesin-5 Eg5 is essential for centrosome separation and spindle organization in somatic cells; however, the detailed functions and mechanisms of Eg5 in spermatocytes remain unclear. In this study, we show that Eg5 proteins are located at spindle microtubules and centrosomes in spermatocytes both in vivo and in vitro. We reveal that the spermatocytes are arrested at metaphase I in seminiferous tubules after Eg5 inhibition. Eg5 ablation results in cell cycle arrest, the formation of monopolar spindle, and chromosome misalignment in cultured GC-2 spd cells. Importantly, we find that the long-term inhibition of Eg5 results in an increased number of centrosomes and chromosomal instability in spermatocytes. Our findings indicate that Eg5 mediates centrosome separation to control spindle assembly and chromosome alignment in spermatocytes, which finally contribute to chromosome stability and faithful cell division of the spermatocytes.

Unraveling the effect of the inflammatory microenvironment in spermatogenesis progression.

Experimental autoimmune orchitis (EAO) is a chronic inflammatory disorder that causes progressive spermatogenic impairment. EAO is characterized by high intratesticular levels of nitric oxide (NO) and tumor necrosis factor alpha (TNFα) causing germ cell apoptosis and Sertoli cell dysfunction. However, the impact of this inflammatory milieu on the spermatogenic wave is unknown. Therefore, we studied the effect of inflammation on spermatogonia and preleptotene spermatocyte cell cycle progression in an EAO context and through the intratesticular DETA-NO and TNFα injection in the normal rat testes. In EAO, premeiotic germ cell proliferation is limited as a consequence of the undifferentiated spermatogonia (CD9+) cell cycle arrest in G2/M and the reduced number of differentiated spermatogonia (c-kit+) and preleptotene spermatocytes that enter in the meiotic S-phase. Although inflammation disrupts spermatogenesis in EAO, it is maintained in some seminiferous tubules at XIV and VII-VIII stages of the epithelial cell cycle, thereby guaranteeing sperm production. We found that DETA-NO (2 mM) injected in normal testes arrests spermatogonia and preleptotene spermatocyte cell cycle; this effect reduces the number of proliferative spermatogonia and the number of preleptotene spermatocytes in meiosis S-phase (36 h after). The temporal inhibition of spermatogonia clonal amplification delayed progression of the spermatogenic wave (5 days after) finally altering spermatogenesis. TNFα (0.5 and 1 µg) exposure did not affect premeiotic germ cell cycle or spermatogenic wave. Our results show that in EAO the inflammatory microenvironment altered spermatogenesis kinetics through premeiotic germ cell cycle arrest and that NO is a sufficient factor contributing to this phenomenon.

Genotoxicity, DNA damage and sperm defects induced by vinblastine.

BACKGROUND: The treatment with chemotherapy may develop secondary tumors as a result of chemo genotoxicity. Sperm defects is another complication associated with chemo treatment. In this study the genotoxicity of vinblastine (VB) was estimated in both somatic and germ cells. MATERIALS: 85 mice were taken. Four single doses of VB at 3, 4.5, 6 and 10 mg/kg and three successive doses at 3, 4.5 and 6 mg/kg were taken for estimation of chromosomal aberrations (CAs). Four single doses of VB were involved in estimating the DNA fragmentation, and comet assay. For sperm abnormalities mice were injected with three successive doses of VB at 3, 4.5, and 6 mg/kg. RESULTS: The results demonstrated a significant frequency of DNA fragmentation in spleen cells and in the percentage of CAs in bone marrow. Numerical and structural aberrations were recorded with a pronounced number of polyploidy metaphases which reached (11.60%) after treatment with 6 mg/kg for three successive days vs zero for control. VB also induced a significant percentage of CAs in spermatocytes in the form of univalent. Sperm defects in the form of coiled tail, absence of acrosome and shapeless head and a significant DNA damage in the testes were recorded. The frequency of sperm abnormalities reached 11.06 ± 0.14 after treatment with highest tested dose (6 mg/kg) vs 3.04 ± 0.19 for control. CONCLUSION: VB is genotoxic in somatic and germ cells. Sperm defects induced by VB are of serious concern to future generations and may affect the fertility of cancer survivors.

Telomere Length in Human Spermatogenic Cells as a New Potential Predictor of Clinical Outcomes in ART Treatment with Intracytoplasmic Injection of Testicular Spermatozoa.

Predicting the clinical outcomes of intracytoplasmic sperm injection (ICSI) cycles that use the testicular spermatozoa of azoospermic patients presents a challenge. Thus, the development of additional approaches to assessing the competence of a testicular-sperm-derived embryo without causing damage to gametes or the embryo is necessary. One of the key parameters in determining such developmental competence is telomere length (TL). We aimed to analyze TLs in spermatogenic cells from the testicular biopsy samples of azoospermic patients and determine how this parameter influences embryo competence for pre- and post-implantation development. Using Q-FISH, we studied the TL of the chromosomes in spermatogonia and spermatocytes I from the TESE biopsy samples of 30 azoospermic patients. An increase in TL was detected during the differentiation from spermatogonia to spermatocytes I. The patients' testicular spermatozoa were used in 37 ICSI cycles that resulted in 22 embryo transfers. Nine pregnancies resulted, of which, one was ectopic and eight ended in birth. The analysis of embryological outcomes revealed a dependence between embryo competence for development to the blastocyst stage and the TL in spermatogenic cells. The TLs in spermatogonia and spermatocytes I in the testicular biopsy samples were found to be higher in patients whose testicular sperm ICSI cycles resulted in a birth. Therefore, the length of telomeres in spermatogenic cells can be considered as a potential prognostic criterion in assessing the competence of testicular-sperm-derived embryos for pre- and post-implantation development. The results of this study provide the basis for the development of a laboratory test for the prediction of testicular sperm ICSI cycle outcomes.

[Effect of autophagy in cadmium chloride induced apoptosis of mouse spermatogenic cells].

OBJECTIVE: To study the effect of autophagy in cadmium chloride(CdCl_2)-induced apoptosis of mouse spermatocytes(GC-2 spd) cells and explore the underlying molecular mechanisms. METHODS: The cells were treated with different concentrations of CdCl_2(0, 5 and 10 µmol/L) for 24 h. Hoechst33342 staining and monodansylcadaverine(MDC) were performed to explore the formation of autophagosomes and apoptotic bodies. The apoptosis of cadmium-treated cells was examined by TUNEL staining. Autophagy inhibitor 3-methyladenine(3-MA)(60 µmol/L), apoptotic inhibitorCaspase inhibitor Z-VAD-FMK( zVAD-FMK)(50 nmol/L), autophagy inducer rapamycin(RAPA)(50 nmol/L) and lysosomal inhibitor chloroquine(CQ)(10 µmol/L) were added to cell culture in the presence/absence of CdCl_2(10 µmol/L) to treat GC-2 spd cells for 24 h. The expression levels of autophagy-related proteins LC3, P62, and pro-apoptotic proteins cleaved Caspase-3 and cleaved Caspase-9 were examined by Western blot. RESULTS: Autophagosomes aggregated and the number of apoptotic cells increased after exposure to CdCl_2 for 24 h. Western blot result showed that in the 5 and 10 µmol/L CdCl_2 exposure groups, the protein expression levels of LC3II/LC3I increased to 9.23±0.81 and 12.15±0.80 compared with the control group(5.50±0.56)(P<0.05), LC3II protein expression level increased to 3.35±0.14 and 3.47±0.32 compared with the control group(2.35±0.34)(P<0.05), P62 protein expression level increased to 1.48±0.12 and 1.80±0.22 compared with the control group(0.83±0.09)(P<0.05). Compared with the CdCl_2-treated group, the protein expression levels of LC3II/LC3I, LC3II, P62, cleaved Caspase-9 and cleaved Caspase-3 after 3-MA treatment decreased to 0.90±0.07(CdCl_2 group: 1.47±0.06), 1.57±0.14(CdCl_2 group: 2.45±0.29), 0.82±0.05(CdCl_2 group: 1.44±0.18), 0.18±0.01(CdCl_2 group: 0.28±0.01) and 0.61±0.84(CdCl_2 group: 1.15±0.04)(P<0.05). Compared with the CdCl_2-treated group, the protein expression levels of cleaved Caspase-9 and cleaved Caspase-3 after zVAD-FMK treatment decreased to 0.12±0.01(CdCl_2 group: 0.28±0.01) and 0.34±0.01(CdCl_2 group: 1.15±0.04)(P<0.05), while those of LC3II/LC3I, LC3II and P62 had no significant change(P>0.05). Compared with the CdCl_2-treated group, RAPA enhanced cadmium-induced LC3II/LC3I, LC3II and P62 protein expressions to 2.22±0.21(CdCl_2 group: 1.56±0.06), 3.72±0.21(CdCl_2 group: 2.97±0.15) and 2.41±0.19(CdCl_2 group: 1.52±0.35)(P<0.05). Western blot result showed that compared with the CdCl_2 group, the protein expressions of LC3II/LC3I, LC3II, P62 and cleaved Caspase-3 in the CdCl_2 and CQ treatment groups increased to 3.21±0.31(CdCl_2 group: 2.09±0.25), 4.49±0.43(CdCl_2 group: 2.72±0.26), 2.59±0.19(CdCl_2 group: 1.84±0.19) and 2.43±0.23(CdCl_2 group: 1.50±0.27)(P<0.05). CONCLUSION: Cadmium chloride induces apoptosis of mouse spermatocyte cells by inhibiting autophagosome-lysosomal fusion and prompting abnormal aggregation of autophagosomes.

Origanum majorana essential oil improves the rat's sexual behavior and testicular oxidative damage induced by imidacloprid via modulating the steroidogenesis pathways.

The neonicotinoid insecticide imidacloprid has been linked to significant reproductive damage in mammals. Origanum majorana essential oil (OME) is a natural herbal product used in the management of many diseases due to its strong antioxidant effects. The oil was hydrodistilled from O. Majorana and analyzed using GC/MS then its possible protective mechanisms against IMI-induced reprotoxicity in male rats were investigated. 28-adult male Wistar rats were divided into 4 groups as follows: group (1) control group, group (2) OME, group (3) IMI, and group (4) IMI + OME. The treatments were applied daily via oral gavage for 60 days. Remarkable abnormalities in both territorial aggressive and sexual behaviors were observed in IMI-treated rats with a significant elevation of serum FSH and LH as well as altered testicular redox status. Along with inhibition of the testicular expression of StAR and aromatase genes and serum total testosterone in addition to abnormal sperm count, viability, motility, and morphology. Histopathological examination showed severe degeneration and necrosis in both germ cells and Leydig cells with atrophy in most of the seminiferous tubules. Co-administration of OME with IMI notably improved all the above-mentioned studied parameters, and restored rats' spermatogenesis, sexual behavior, and favorably modulates the levels of both testosterone and gonadotropic hormones via its potent antioxidant effect. These findings support the use of OME as a fertility enhancer and suggest that it could be used to manage pesticide-induced male infertility.

[Cadmium induces apoptosis of mouse spermatocytes (GC-2 spd) by promoting mitochondrial fission].

Objective: To study the underlying mechanism of cadmium-induced apoptosis of mouse spermatocytes (GC-2 spd) . Methods: In March 2021, GC-2 spd cells were exposed to different concentrations of CdCl(2) for 24 hours, namely 5 µmol/L CdCl(2) (low-dose) group and 10 µmol/L CdCl(2) (high-dose) group, and unexposed GC-2 spd cells were used as control group. Mitochondrial morphology was observed in the cells stained with Mito-Track Red CMXRos fluorescent probes by confocal microscopy and the mitochrondrial membrane potential was measured by flow cytometry with JC-1 fluorescent probes. Mitochrondrial proteins, cytosolic proteins and total cellular proteins of GC-2 spd cells were extracted using cell mitochondria isolation kit and RIPA buffer, respectively. The expression of mitochondrial homeostasis regulatory proteins (FIS1 and OPA1), and apoptosis-related proteins (Cytochrome c and cleaved Caspase-3) were examined by Western blot. Results: Compared with the cells in the control group, the relative ratio of JC-1 red/green fluorescence signal in the cells of the low-dose and high-dose CdCl(2) groups decreased significantly (0.740±0.071, 0.570±0.028), with a statistically significant difference (P=0.017, 0.004) ; The morphology of mitochondria changed from long tube to point, and the proportion of cells containing point mitochondria increased significantly (45.1%±3.7% and 25.7%±4.9%), the difference was statistically significant (P=0.005, 0.001) ; The relative expression level of mitochondrial FIS1 in cells of low and high dose CdCl(2) groups was significantly higher (1.271±0.120, 1.693±0.155), the difference was statistically significant (P=0.046, 0.000) ; The relative expression level of OPA1 decreased significantly (0.838±0.050, 0.682±0.040), and the difference was statistically significant (P=0.049, 0.001). Compared with the control group, the relative expression level of cytochrome c protein in the cytoplasm of cells in the low dose group of CdCl(2) was not significantly increased (1.249±0.151), and the difference was not statistically significant (P=0.075). However, the relative expression level in the cytoplasm of cells in the high dose group of CdCl(2) was significantly increased (2.355±0.110), and the difference was statistically significant (P=0.000) ; The relative expression level of Cytochrome c in mitochondria of low and high dose CdCl(2) groups decreased significantly (0.681±0.043, 0.619±0.114), with a statistically significant difference (P=0.004, 0.001) ; Moreover, the level of cleaved Caspase-3 protein in cells gradually increased (5.486±0.544, 11.493±1.739), the difference was statistically significant (P=0.004, 0.000) . Conclusion: Cadmium induced cleaved Caspase-3 mediated apoptosis of GC-2 spd cells via promoting mitochrondrial fission and the release of Cytochrome c from the mitochrondria to the cytosol.

Transferrin receptor (TFRC) is essential for meiotic progression during mouse spermatogenesis.

Meiosis is a highly conserved process, and is responsible for the production of haploid gametes and generation of genetic diversity. We previously identified the transferrin receptor (TFRC) in the proteome profile of mice neonatal testes, indicating the involvement of the TFRC in meiosis. However, the exact molecular role of the TFRC in meiosis remains unclear. In this study, we aimed to determine the function of the TFRC in neonatal testicular development by TFRC knockdown using the testis culture platform. Our results showed high TFRC expression in 2-week testes, corresponding to the first meiotic division. Targeting TFRC using morpholino oligonucleotides resulted in clear spermatocyte apoptosis. In addition, we used the chromosomal spread technique to show that a deficiency of TFRC caused the accumulation of leptotene and zygotene spermatocytes, and a decrease of pachytene spermatocytes, indicating early meiotic arrest. Moreover, the chromosomes of TFRC-deficient pachytene spermatocytes displayed sustained γH2AX association, as well as SYCP1/SYCP3 dissociation beyond the sex body. Therefore, our results demonstrated that the TFRC is essential for the progression of spermatocyte meiosis, particularly for DNA double-stranded break repair and chromosomal synapsis.

Meiotic spindle assembly checkpoint and aneuploidy in males versus females.

The production of gametes (sperm and eggs in mammals) involves two sequential cell divisions, meiosis I and meiosis II. In meiosis I, homologous chromosomes segregate to different daughter cells, and meiosis II resembles mitotic divisions in that sister chromatids separate. While in principle the process is identical in males and females, the time frame and susceptibility to chromosomal defects, including achiasmy and cohesion weakening, and the response to mis-segregating chromosomes are not. In this review, we compare and contrast meiotic spindle assembly checkpoint function and aneuploidy in the two sexes.

Drosophila dany is essential for transcriptional control and nuclear architecture in spermatocytes.

The terminal differentiation of adult stem cell progeny depends on transcriptional control. A dramatic change in gene expression programs accompanies the transition from proliferating spermatogonia to postmitotic spermatocytes, which prepare for meiosis and subsequent spermiogenesis. More than a thousand spermatocyte-specific genes are transcriptionally activated in early Drosophila spermatocytes. Here we describe the identification and initial characterization of dany, a gene required in spermatocytes for the large-scale change in gene expression. Similar to tMAC and tTAFs, the known major activators of spermatocyte-specific genes, dany has a recent evolutionary origin, but it functions independently. Like dan and danr, its primordial relatives with functions in somatic tissues, dany encodes a nuclear Psq domain protein. Dany associates preferentially with euchromatic genome regions. In dany mutant spermatocytes, activation of spermatocyte-specific genes and silencing of non-spermatocyte-specific genes are severely compromised and the chromatin no longer associates intimately with the nuclear envelope. Therefore, as suggested recently for Dan/Danr, we propose that Dany is essential for the coordination of change in cell type-specific expression programs and large-scale spatial chromatin reorganization.

Selective advantage of euploid spermatocytes I in an azoospermic 47,XYY man with gonadal mosaicism.

Although most XYY men have normal sperm counts and are fertile (supposedly due to the loss of the extra Y before meiosis), there is a minority who are infertile. In these cases, the XYY spermatocytes are able to enter meiosis and form different synaptic configurations. With regard to mosaics, there is scarce well-defined information on the presence of the second Y and its meiotic behaviour. In this study, the chromosome constitution and the synaptic behaviour of pachytene spermatocytes from an azoospermic man with testicular hypotrophy and non-mosaic 47,XYY karyotype were analysed. Furthermore, we determined the chromosome constitution of the somatic Sertoli cells. Five karyotypically normal men with obstructive azoospermia, but having complete spermatogenesis, were included as controls. Immuno-FISH using specific protein markers of synapsis and recombination (SYCP3, SYCP1, BRCA1, MLH1, CREST) and a specific Yq12 DNA probe were used. In addition, we used the newly developed Super-Resolution Structured Illumination Microscopy (SR-SIM) to clearly define the synaptic configurations. FISH analysis was also performed on Sertoli cells. The histopathological analysis showed variable degrees of spermatogenesis development in the testicular tissue of the propositus. Immuno-FISH analysis showed that most of the primary spermatocytes were euploid 46, XY. The use of SR-SIM confirmed the existence of this euploidy. Only a few pachytene spermatocytes showed an aneuploid X + YY constitution. Sertoli cells showed two different populations with one or two Y chromosomes, in similar proportions. Thus an abnormal niche of sex-trisomic Sertoli cells should be also considered when searching for the origin of spermatogenesis failure in XYY men.