Meiotic Germ Cells Research Articles

Normal male fertility in a mouse model of KPNA2 deficiency.

The nuclear transport of proteins is mediated by karyopherins and has been implicated to be crucial for germ cell and embryonic development. Deletion of distinct members of the karyopherin alpha family has been shown to cause male and female infertility in mice. Using a genetrap approach, we established mice deficient for KPNA2 (KPNA2 KO) and investigated the role of this protein in male germ cell development and fertility. Breeding of male KPNA2 KO mice leads to healthy offsprings in all cases albeit the absence of KPNA2 resulted in a reduction in sperm number by 60%. Analyses of the KPNA2 expression in wild-type mice revealed a strong KPNA2 presence in meiotic germ cells of all stages while a rapid decline is found in round spermatids. The high KPNA2 expression throughout all meiotic stages of sperm development suggests a possible function of KPNA2 during this phase, hence in its absence the spermatogenesis is not completely blocked. In KPNA2 KO mice, a higher portion of sperms presented with morphological abnormalities in the head and neck region, but a severe spermiogenesis defect was not found. Thus, we conclude that the function of KPNA2 in round spermatids is dispensable, as our mice do not show any signs of infertility. Our data provide evidence that KPNA2 is not crucial for male germ cell development and fertility.

Phenotypic continuum and poor intracytoplasmic sperm injection intracytoplasmic sperm injection prognosis in patients harboring HENMT1 variants.

Small RNAs interacting with PIWI (piRNAs) play a crucial role in regulating transposable elements and translation during spermatogenesis and are essential in male germ cell development. Disruptions in the piRNA pathway typically lead to severe spermatogenic defects and thus male infertility. The HENMT1 gene is a key player in piRNAs primary biogenesis and dysfunction of HENMT1 protein in meiotic and haploid germ cells resulted in the loss of piRNA methylation, piRNA instability, and TE de-repression. Henmt1-knockout mice exhibit a severe oligo-astheno-teratozoospermia (OAT) phenotype, whereas patients with HENMT1 variants display more severe azoospermia phenotypes, ranging from meiotic arrest to hypospermatogenesis. Through whole-exome sequencing (WES) of infertile patient cohorts, we identified two new patients with variants in the HENMT1 gene presenting spermatozoa in their ejcaulate, providing us the opportunity to study spermatozoa from these patients. Investigate the spermatozoa of two patients harboring an HENMT1 variant to determine whether or not these scarce spermatozoa could be used with assisted reproductive technologies. HENMT1 variants identified by WES were validated through Sanger sequencing. Comprehensive semen analysis was conducted, and sperm cells were subjected to transmission electron microscopy for structural examination, in situ hybridization for aneuploidy assessment, and aniline blue staining for DNA compaction status. Subsequently, we assessed their suitability for in vitro fertilization using intracytoplasmic sperm injection (IVF-ICSI). Our investigations revealed a severe OAT phenotype similar to knockout mice, revealing altered sperm concentration, mobility, morphology, aneuploidy and nuclear compaction defects. Multiple IVF-ICSI attempts were also performed, but no live births were achieved. We confirm the crucial role of HENMT1 in spermatogenesis and highlight a phenotypic continuum associated with HENMT1 variants. Unfortunately, the clinical outcome of these genetic predispositions remains unfavorable, regardless of the patient's phenotype. The presence of spermatozoa is insufficient to anticipate ICSI pregnancy success in HENMT1 patients.

The chromatin-associated 53BP1 ortholog, HSR-9, regulates recombinational repair and X chromosome segregation in the Caenorhabditis elegans germ line.

53BP1 plays a crucial role in regulating DNA damage repair pathway choice and checkpoint signaling in somatic cells; however, its role in meiosis has remained enigmatic. In this study, we demonstrate that the Caenorhabditis elegans ortholog of 53BP1, HSR-9, associates with chromatin in both proliferating and meiotic germ cells. Notably, HSR-9 is enriched on the X chromosome pair in pachytene oogenic germ cells. HSR-9 is also present at kinetochores during both mitotic and meiotic divisions but does not appear to be essential for monitoring microtubule-kinetochore attachments or tension. Using cytological markers of different steps in recombinational repair, we found that HSR-9 influences the processing of a subset of meiotic double-stranded breaks into COSA-1-marked crossovers. Additionally, HSR-9 plays a role in meiotic X chromosome segregation under conditions where X chromosomes fail to pair, synapse, and recombine. Together, these results highlight that chromatin-associated HSR-9 has both conserved and unique functions in the regulation of meiotic chromosome behavior.

Impaired Spermatogenesis in Infertile Patients with Orchitis and Experimental Autoimmune Orchitis in Rats.

Experimental autoimmune orchitis (EAO) is a well-established rodent model of organ-specific autoimmunity associated with infertility in which the testis immunohistopathology has been extensively studied. In contrast, analysis of testis biopsies from infertile patients associated with inflammation has been more limited. In this work, testicular biopsies from patients with idiopathic non-obstructive azoospermia diagnosed with hypospermatogenesis (HypoSp) [mild: n = 9, and severe: n = 11], with obstructive azoospermia and complete Sp (spermatogenesis) (control group, C, n = 9), and from Sertoli cell-only syndrome (SCOS, n = 9) were analyzed for the presence of immune cells, spermatogonia and Sertoli cell (SCs) alterations, and reproductive hormones levels. These parameters were compared with those obtained in rats with EAO. The presence of increased CD45+ cells in the seminiferous tubules (STs) wall and lumen in severe HypoSp is associated with increased numbers of apoptotic meiotic germ cells and decreased populations of undifferentiated and differentiated spermatogonia. The SCs showed an immature profile with the highest expression of AMH in patients with SCOS and severe HypoSp. In SCOS patients, the amount of SCs/ST and Ki67+ SCs/ST increased and correlated with high serum FSH levels and CD45+ cells. In the severe phase of EAO, immune cell infiltration and apoptosis of meiotic germ cells increased and the number of undifferentiated and differentiated spermatogonia was lowest, as previously reported. Here, we found that orchitis leads to reduced sperm number, viability, and motility. SCs were mature (AMH-) but increased in number, with Ki67+ observed in severely damaged STs and associated with the highest levels of FSH and inflammatory cells. Our findings demonstrate that in a scenario where a chronic inflammatory process is underway, FSH levels, immune cell infiltration, and immature phenotypes of SCs are associated with severe changes in spermatogenesis, leading to azoospermia. Furthermore, AMH and Ki67 expression in SCs is a distinctive marker of severe alterations of STs in human orchitis.

Tryptophanyl-tRNA synthetase-1 (WARS-1) depletion and high tryptophan concentration lead to genomic instability in Caenorhabditis elegans

The fidelity of translation is ensured by a family of proteins named aminoacyl-tRNA synthetases (ARSs), making them crucial for development and survival. More recently, mutations in the tryptophanyl-tRNA synthetase 1 (WARS1) have been linked to various human diseases, from intellectual disability to various types of cancer. To understand the function of WARS1, we investigated the effect of WARS-1 depletion during the mitotic and meiotic cell cycle in the developing germline of Caenorhabditis elegans (C. elegans) and demonstrated the role of WARS-1 in genome integrity. wars-1 knockdown results in cell cycle arrest of the mitotically active germ cells. Such mitotic arrest is also associated with canonical DNA damage-induced checkpoint signaling in mitotic and meiotic germ cells. Significantly, such DNA checkpoint activation is associated with the morphological anomalies in chromatin structures that are the hallmarks of genome instability, such as the formation of chromatin bridges, micronuclei, and chromatin buds. We demonstrated that knocking down wars-1 results in an elevation of the intracellular concentration of tryptophan and its catabolites, a surprising finding emphasizing the impact of cellular amino acid availability and organismal/individual dietary uptake on genome integrity. Our result demonstrates that exposing C. elegans to a high tryptophan dosage leads to DNA damage checkpoint activation and a significant increase in the tryptophan metabolites. Targeting tryptophan catabolism, the least utilized amino acid in nature, can be important in developing new cancer therapeutic approaches. All in all, we have strong evidence that knocking down wars-1 results in defects in genomic integrity.

Regulation of meiotic telomere dynamics through membrane fluidity promoted by AdipoR2-ELOVL2

The cellular membrane in male meiotic germ cells contains a unique class of phospholipids and sphingolipids that is required for male reproduction. Here, we show that a conserved membrane fluidity sensor, AdipoR2, regulates the meiosis-specific lipidome in mouse testes by promoting the synthesis of sphingolipids containing very-long-chain polyunsaturated fatty acids (VLC-PUFAs). AdipoR2 upregulates the expression of a fatty acid elongase, ELOVL2, both transcriptionally and post-transcriptionally, to synthesize VLC-PUFA. The depletion of VLC-PUFAs and subsequent accumulation of palmitic acid in AdipoR2 knockout testes stiffens the cellular membrane and causes the invagination of the nuclear envelope. This condition impairs the nuclear peripheral distribution of meiotic telomeres, leading to errors in homologous synapsis and recombination. Further, the stiffened membrane impairs the formation of intercellular bridges and the germ cell syncytium, which disrupts the orderly arrangement of cell types within the seminiferous tubules. According to our findings we propose a framework in which the highly-fluid membrane microenvironment shaped by AdipoR2-ELOVL2 underpins meiosis-specific chromosome dynamics in testes.

HDAC3 promotes Sertoli cell maturation and maintains the blood-testis barrier dynamics.

Germ cell development depends on the capacity of somatic Sertoli cells to undergo differentiation into a mature state and establish a germ cell-specific blood-testis barrier (BTB). The BTB structure confers an immunological barrier for meiotic and postmeiotic germ cells, and its dynamic permeability facilitates a transient movement of preleptotene spermatocytes through BTB to enter meiosis. However, the regulatory factors involved in Sertoli cell maturation and how BTB dynamics coordinate germ cell development remain unclear. Here, we found a histone deacetylase HDAC3 abundantly expresses in Sertoli cells and localizes in both cytoplasm and nucleus. Sertoli cell-specific Hdac3 knockout in mice causes infertility with compromised integrity of blood-testis barrier, leading to germ cells unable to traverse through BTB and an accumulation of preleptotene spermatocytes in juvenile testis. Mechanistically, nuclear HDAC3 regulates the expression program of Sertoli cell maturation genes, and cytoplasmic HDAC3 forms a complex with the gap junction protein Connexin 43 to modulate the BTB integrity and dynamics through regulating the distribution of tight junction proteins. Our findings identify HDAC3 as a critical regulator in promoting Sertoli cell maturation and maintaining the homeostasis of the blood-testis barrier.

Purification by STA-PUT Technique of Male Germ Cells from Single Mouse and RNA-Extraction for Transcriptomic Analysis.

Transcriptomic analyses of germ cells at different stages of differentiation have shed light on the transcriptional and post-transcriptional mechanisms regulating gene expression that ensure the correct progression of spermatogenesis and male fertility. In this chapter, we describe a method to isolate meiotic and post-meiotic germ cells, based on gravimetric sedimentation, starting from a testicular germ cell suspension isolated from a single adult mouse. We also describe how to assess the purity and quality of the collected fractions of germ cells and how to optimize the extraction from these samples of RNA for subsequent RNA-sequencing experiment. In our experience, this protocol is suitable for germ cell isolation and transcriptomic analysis for mouse models with spermatogenic defects, overcoming the limits that reduced fertility poses to the obtaining of experimental animals.

Disparate roles for C. elegans DNA translocase paralogs RAD-54.L and RAD-54.B in meiotic prophase germ cells.

RAD54 family DNA translocases partner with RAD51 recombinases to ensure stable genome inheritance, exhibiting biochemical activities both in promoting recombinase removal and in stabilizing recombinase association with DNA. Understanding how such disparate activities of RAD54 paralogs align with their biological roles is an ongoing challenge. Here we investigate the in vivo functions of Caenorhabditis elegans RAD54 paralogs RAD-54.L and RAD-54.B during meiotic prophase, revealing distinct contributions to the dynamics of RAD-51 association with DNA and to the progression of meiotic double-strand break repair (DSBR). While RAD-54.L is essential for RAD-51 removal from meiotic DSBR sites to enable recombination progression, RAD-54.B is largely dispensable for meiotic DSBR. However, RAD-54.B is required to prevent hyperaccumulation of RAD-51 on unbroken DNA during the meiotic sub-stage when DSBs and early recombination intermediates form. Moreover, DSB-independent hyperaccumulation of RAD-51 foci in the absence of RAD-54.B is RAD-54.L-dependent, revealing a hidden activity of RAD-54.L in promoting promiscuous RAD-51 association that is antagonized by RAD-54.B. We propose a model wherein a division of labor among RAD-54 paralogs allows germ cells to ramp up their capacity for efficient homologous recombination that is crucial to successful meiosis while counteracting potentially deleterious effects of unproductive RAD-51 association with unbroken DNA.

Sertoli Hücresi ve Kan-Testis Bariyeri

The Sertoli cell is a critical somatic cell that initiates the development of testicular morphology and determines important parameters for spermatogenic function. The blood-testis barrier, also known as the Sertoli cell barrier and one of the tightest tissue barriers in the mammalian body, is an immunological barrier to separate post meiotic germ cell antigens from the systemic circulation. Additionally, creating a unique microenvironment for the development of spermatocytes that exceed into the adluminal compartment from the leptotene stage. It restricts the passage of substances such as paracrine factors, electrolytes, hormones, water, and biological molecules to the apical part of the seminiferous tubule. It separates spermatogenic cells from toxic and drug-containing environmentally harmful substances, hormones, and biomolecules in the systemic circulation. This nearly impenetrable barrier prevents proteins, including antibodies, from reaching the spermatogenic cells. It also prevents protein leakage from developing spermatogenic cells and forming an immune response. This review explains Sertoli's functional properties, the testis barrier's molecular structure, the substances involved in the barrier dynamics, and their importance in realizing spermatogenesis.

Two RNA binding proteins, ADAD2 and RNF17, interact to form a heterogeneous population of novel meiotic germ cell granules with developmentally dependent organelle association.

Mammalian male germ cell differentiation relies on complex RNA biogenesis events, many of which occur in non-membrane bound organelles termed RNA germ cell granules that are rich in RNA binding proteins (RBPs). Though known to be required for male germ cell differentiation, we understand little of the relationships between the numerous granule subtypes. ADAD2, a testis specific RBP, is required for normal male fertility and forms a poorly characterized granule in meiotic germ cells. This work aimed to understand the role of ADAD2 granules in male germ cell differentiation by clearly defining their molecular composition and relationship to other granules. Biochemical analyses identified RNF17, a testis specific RBP that forms meiotic male germ cell granules, as an ADAD2-interacting protein. Phenotypic analysis of Adad2 and Rnf17 mutants identified a rare post-meiotic chromatin defect, suggesting shared biological roles. ADAD2 and RNF17 were found to be dependent on one another for granularization and together form a previously unstudied set of germ cell granules. Based on co-localization studies with well-characterized granule RBPs and organelle-specific markers, a subset of the ADAD2-RNF17 granules are found to be associated with the intermitochondrial cement and piRNA biogenesis. In contrast, a second, morphologically distinct population of ADAD2-RNF17 granules co-localized with the translation regulators NANOS1 and PUM1, along with the molecular chaperone PDI. These large granules form a unique funnel-shaped structure that displays distinct protein subdomains and is tightly associated with the endoplasmic reticulum. Developmental studies suggest the different granule populations represent different phases of a granule maturation process. Lastly, a double Adad2-Rnf17 mutant model suggests the interaction between ADAD2 and RNF17, as opposed to loss of either, is the likely driver of the Adad2 and Rnf17 mutant phenotypes. These findings shed light on the relationship between germ cell granule pools and define new genetic approaches to their study.

Fancm has dual roles in the limiting of meiotic crossovers and germ cell maintenance in mammals

Meiotic crossovers are required for accurate chromosome segregation and producing new allelic combinations. Meiotic crossover numbers are tightly regulated within a narrow range, despite an excess of initiating DNA double-strand breaks. Here, we reveal the tumor suppressor FANCM as a meiotic anti-crossover factor in mammals. We use unique large-scale crossover analyses with both single-gamete sequencing and pedigree-based bulk-sequencing datasets to identify a genome-wide increase in crossover frequencies in Fancm-deficient mice. Gametogenesis is heavily perturbed in Fancm loss-of-function mice, which is consistent with the reproductive defects reported in humans with biallelic FANCM mutations. A portion of the gametogenesis defects can be attributed to the cGAS-STING pathway after birth. Despite the gametogenesis phenotypes in Fancm mutants, both sexes are capable of producing offspring. We propose that the anti-crossoverfunction and role in gametogenesis of Fancm are separable and will inform diagnostic pathways for human genomic instability disorders.

P-001 ClpX is required in maintaining mitochondrial functions during meiosis and spermatogenesis

Abstract Study question What is the role of ClpX in spermatogonia differentiation and spermatogenesis? Summary answer ClpX is required to maintain normal mitochondrial functions in spermatocytes, deficiency of ClpX resulted in meiosisarrest and diruption of spermatogenesis in mice. What is known already ClpX is mitochondria-specific quality control protease, it maintains proteostasis via degrading misfolded or damagedproteins in mitochondrial matrix. ClpX is a AAA+ protease that uses the energy of ATP binding and hydrolysis to degradeunfolded or misfolded proteins. ClpX is reported working as a complex with ClpP, the complex consists of a hexamer ofClpX and a tetradecamer of ClpP. ClpX recognizes the protein substrates via binding with their unstructured peptidesequences, termed as degradation tags or recognition signals. The fragments of the cleaved polypeptides can then exitthe chamber and be further degraded. Study design, size, duration Study design: we generated a germ cell specific ClpX knockout mice line and evaluated size, weight, inner structure oftestis tissue. We also applied biochemical techniques and high-throughput sequencing techniques to investigate themechanism of ClpX in regulating spermatogenesis. Size: more than 50 mice were used in this study, including the control mice. Duration: 2 years Participants/materials, setting, methods Participants/materials: we generated a ClpX conditional knockout (cKO) mice line by CRISPR-gene editing and Cre-LoxP system, which specifically knockout ClpX in the germ cells of male mice. Setting: the siblings of ClpX WT mice and ClpX cKO mice were compared in various of experiments. Mehods: we performed morphology comparement to check the weight and size of tetes in the control and ClpX cKOmice, and applied immunofluorescence, western blot, histological study, high-throughput sequencing andpharmocological treatment. Main results and the role of chance We found ClpX deficiency reduced mitochondrial functions and quantity in spermatocytes, affected energy supply duringmeiosis and attenuated zygotene-pachytene transformation of the male germ cells. The affected spermatocytesexhibited disorder of chromosome synapsis and cross-over events. Their telomeres failed to attach to the nuclearenvelop, which was associated with failure of α-tubulin formation in the ClpX deficient spermatocytes. The dysregulatedspermatocytes finally underwent apoptosis resulting in decreased testicular size and vacuolar structures within theseminiferous tubules. Both transcriptome analysis and m6A-methylation sequencing analysis highlighted dysregulationof metabolic pathways including the mTOR signaling in the isolated spermatocytes. We confirmed over-activation of themTORC1 pathway with increased expression of phosphorylated S6 and 4EBP after deletion of ClpX in spermatocytes.Long-term inhibition of the mTORC1 signaling via rapamycin treatment in vivo partially rescue spermatogenesis in theseminiferous tubules with much less apoptotic germ cells and formation of late stage of meiotic germ cells, such asround spermatids and elongated spermatozoon. The data reveal the novel roles of ClpX in regulating meiosis andspermatogenesis. Limitations, reasons for caution This study is based on animal experiments, thus, these results only indicated the important roles of ClpX in micespermatogenesis rather than human beings. Clinical data has not yet found a Clpx gene mutation in reproductivedisease. Wider implications of the findings Clinical report has already found the mutation in ClpP can induce autosomal recessive Perrault syndrome, while themutation of ClpX has not been reported yet from clinical data. Our resutls demonstrated severe effect onspermatogenesis after knockingout of ClpX, this might give some insights to the clinic. Trial registration number not applicable

The DOT1L-MLLT10 complex regulates male fertility and promotes histone removal during spermiogenesis.

Histone modifications regulate chromatin remodeling and gene expression in development and diseases. DOT1L, the sole histone H3K79 methyltransferase, is essential for embryonic development. Here, we report that DOT1L regulates male fertility in mouse. DOT1L associates with MLLT10 in testis. DOT1L and MLLT10 localize to the sex chromatin in meiotic and post-meiotic germ cells in an inter-dependent manner. Loss of either DOT1L or MLLT10 leads to reduced testis weight, decreased sperm count and male subfertility. H3K79me2 is abundant in elongating spermatids, which undergo the dramatic histone-to-protamine transition. Both DOT1L and MLLT10 are essential for H3K79me2 modification in germ cells. Strikingly, histones are substantially retained in epididymal sperm from either DOT1L- or MLLT10-deficient mice. These results demonstrate that H3K79 methylation promotes histone replacement during spermiogenesis.

DOT1L promotes spermatid differentiation by regulating expression of genes required for histone-to-protamine replacement.

Unique chromatin remodeling factors orchestrate dramatic changes in nuclear morphology during differentiation of the mature sperm head. A crucial step in this process is histone-to-protamine exchange, which must be executed correctly to avoid sperm DNA damage, embryonic lethality and male sterility. Here, we define an essential role for the histone methyltransferase DOT1L in the histone-to-protamine transition. We show that DOT1L is abundantly expressed in mouse meiotic and postmeiotic germ cells, and that methylation of histone H3 lysine 79 (H3K79), the modification catalyzed by DOT1L, is enriched in developing spermatids in the initial stages of histone replacement. Elongating spermatids lacking DOT1L fail to fully replace histones and exhibit aberrant protamine recruitment, resulting in deformed sperm heads and male sterility. Loss of DOT1L results in transcriptional dysregulation coinciding with the onset of histone replacement and affecting genes required for histone-to-protamine exchange. DOT1L also deposits H3K79me2 and promotes accumulation of elongating RNA Polymerase II at the testis-specific bromodomain gene Brdt. Together, our results indicate that DOT1L is an important mediator of transcription during spermatid differentiation and an indispensable regulator of male fertility.

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 break (DSB) 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. This article is protected by copyright. All rights reserved.

Chromatin remodelers HELLS, WDHD1 and BAZ1A are dynamically expressed during mouse spermatogenesis.

Proper regulation of heterochromatin is critical for spermatogenesis. This study reveals the dynamic localization patterns of distinct chromatin regulators during spermatogenesis and disrupted sex chromatin status in spermatocytes in the absence of DICER. Heterochromatin is dynamically formed and organized in differentiating male germ cells, and its proper regulation is a prerequisite for normal spermatogenesis. While heterochromatin is generally transcriptionally silent, we have previously shown that major satellite repeat (MSR) DNA in the pericentric heterochromatin (PCH) is transcribed during spermatogenesis. We have also shown that DICER associates with PCH and is involved in the regulation of MSR-derived transcripts. To shed light on the heterochromatin regulation in the male germline, we studied the expression, localization and heterochromatin association of selected testis-enriched chromatin regulators in the mouse testis. Our results show that HELLS, WDHD1 and BAZ1A are dynamically expressed during spermatogenesis. They display limited overlap in expression, suggesting involvement in distinct heterochromatin-associated processes at different steps of differentiation. We also show that HELLS and BAZ1A interact with DICER and MSR chromatin. Interestingly, deletion of Dicer1 affects the sex chromosome heterochromatin status in late pachytene spermatocytes, as demonstrated by mislocalization of Polycomb protein family member SCML1 to the sex body. These data substantiate the importance of dynamic heterochromatin regulation during spermatogenesis and emphasize the key role of DICER in the maintenance of chromatin status in meiotic male germ cells.

Role of glyoxalase I and II in somatic and spermatogenic testicular cells during the postnatal development of the domestic cat

Like humans, many felid species suffer from teratozoospermia and frequently produce low numbers of normal spermatozoa. Male fertility can be affected by oxidative and dicarbonyl stress. Because of the high level of glycolytic activity in testes, reactive dicarbonyl metabolites may arise as side-products of glycolysis; their generation is further promoted by oxidative stress. Alpha-oxoaldehydes, including methylglyoxal (MG), are reactive dicarbonyl metabolites and substrates for the formation of advanced glycation end products. Elevated levels of both may lead to dicarbonyl stress and cause cellular dysfunction. However, MG and other α-oxoaldehydes can be converted to less dangerous molecules via the glyoxalase pathway. In this pathway, α-oxoaldehydes react with glutathione (GSH), forming a thioacetal, which becomes metabolized by glyoxalase I (GLO I) to S-D-lactoyl-glutathione (SLG). Glyoxalase II (GLO II) converts SLG to d-lactate upon the release of GSH. Nothing is known about the glyoxalase system in the feline testis and its capacity to mitigate an excess of dicarbonyl metabolites. To study whether GLO I and GLO II are present and have a specific function in the testis of the domestic cat, the gene expression of both enzymes were analyzed in testis samples of different developmental stages (prepubertal, pubertal, postpubertal). Furthermore, the presence of GLO I and GLO II proteins was investigated via immunohistochemistry. The GLO I gene expression does not change between developmental stages. Immunohistochemistry revealed strong signals for GLO I in the cytoplasm and nuclei of Sertoli and Leydig cells during all developmental stages. GLO I was described as catalyzing the rate-limiting step in the glyoxalase pathway. This implies a function on the part of this enzyme of sustaining the homeostasis of somatic testicular cells. For GLO II, we observed stage-dependent mRNA expression, which was significantly increased after puberty. In accordance with this observation, clear immunohistochemical GLO II signals were observed in nuclei of individual germ cells. The most intense signals were visible in spermatocytes. The different localizations of the strong GLO I and GLO II signals indicate that GLO II, in addition to the classical glyoxalase pathway, may have additional functions in meiotic germ cells, for example, providing lactate as an energy substrate and/or GSH as an antioxidant. Moreover, protein functions may be modulated via S-glutathionylation.

Absence of Testicular Estrogen Leads to Defects in Spermatogenesis and Increased Semen Abnormalities in Male Rabbits.

Estrogens are steroid hormones produced by the aromatization of androgens by the aromatase enzyme, encoded by the CYP19A1 gene. Although generally referred to as "female sex hormones", estrogen is also produced in the adult testes of many mammals, including humans. To better understand the function of estrogens in the male, we used the rabbit model which is an important biomedical model. First, the expression of CYP19A1 transcripts was localized mainly in meiotic germ cells. Thus, testicular estrogen appears to be produced inside the seminiferous tubules. Next, the cells expressing ESR1 and ESR2 were identified, showing that estrogens could exert their function on post-meiotic germ cells in the tubules and play a role during sperm maturation, since ESR1 and ESR2 were detected in the cauda epididymis. Then, CRISPR/Cas9 CYP19A1-/- genetically modified rabbits were analyzed. CYP19A1-/- males showed decreased fertility with lower sperm count associated with hypo-spermatogenesis and lower spermatid number. Germ/sperm cell DNA methylation was unchanged, while sperm parameters were affected as CYP19A1-/- males exhibited reduced sperm motility associated with increased flagellar defects. In conclusion, testicular estrogens could be involved in the spermatocyte-spermatid transition in the testis, and in the acquisition of sperm motility in the epididymis.

Sub-chronic restraint stress exposure in adult rats: An insight into possible inhibitory mechanism on testicular function in relation to germ cell dynamics.

Psychological stress is now widely recognized as one of the major risk factors for male fertility. Its impact on the dynamics of testicular germ cells, however, has yet to be fully investigated. Therefore, we used the rat restraint stress (RS) model as a psychological stressor to assess the impact of psychological stress on testicular germ cell dynamics. Adult male SD rats were exposed to sub-chronic RS for 1.5 and 3h per day for 30 days. The quality of cauda epididymis spermatozoa was adversely affected by RS exposure, and the frequency of spermatozoa with tail abnormalities was higher than that of spermatozoa with head abnormalities. RS exposure adversely affected testicular daily sperm production by disturbing the meiotic and post meiotic germ cell kinetics in the testis. The histomorphology of the testis was altered by loosening and vacuolization in the seminiferous epithelium, germ cell exfoliation and the presence of giant cells. Seminiferous tubules of stage I-VI and VII-VIII were severely affected in rats exposed to RS for 3h. By interfering with steroidogenic enzymes, RS exposure disrupts testosterone biosynthesis. The testicular oxidative balance was also disturbed by RS exposure, which disrupted the levels/activities of lipid peroxidation, Nrf-2, superoxide dismutase and catalase. There was also an increase in caspase-3 activity and a decrease in the Bax-Bcl2 ratio. In conclusion, our findings suggest that psychological stressors like RS impair testicular functions in rats by disrupting germ cell dynamics, downregulating testicular androgenesis and increasing oxidative stress and apoptosis.