Pachytene Spermatocytes Research Articles

The male germline-specific protein MAPS is indispensable for pachynema progression and fertility

Meiosis is a specialized cell division that creates haploid germ cells from diploid progenitors. Through differential RNA expression analyses, we previously identified a number of mouse genes that were dramatically elevated in spermatocytes, relative to their very low expression in spermatogonia and somatic organs. Here, we investigated in detail 1700102P08Rik, one of these genes, and independently conclude that it encodes a male germline-specific protein, in agreement with a recent report. We demonstrated that it is essential for pachynema progression in spermatocytes and named it male pachynema-specific (MAPS) protein. Mice lacking Maps (Maps-/- ) suffered from pachytene arrest and spermatocyte death, leading to male infertility, whereas female fertility was not affected. Interestingly, pubertal Maps-/- spermatocytes were arrested at early pachytene stage, accompanied by defects in DNA double-strand break (DSB) repair, crossover formation, and XY body formation. In contrast, adult Maps-/- spermatocytes only exhibited partially defective crossover but nonetheless were delayed or failed in progression from early to mid- and late pachytene stage, resulting in cell death. Furthermore, we report a significant transcriptional dysregulation in autosomes and XY chromosomes in both pubertal and adult Maps-/- pachytene spermatocytes, including failed meiotic sex chromosome inactivation (MSCI). Further experiments revealed that MAPS overexpression in vitro dramatically decreased the ubiquitination levels of cellular proteins. Conversely, in Maps-/- pachytene cells, protein ubiquitination was dramatically increased, likely contributing to the large-scale disruption in gene expression in pachytene cells. Thus, MAPS is a protein essential for pachynema progression in male mice, possibly in mammals in general.

Genome-wide chromatin occupancy of BRDT and gene expression analysis suggest transcriptional partners and specific epigenetic landscapes that regulate gene expression during spermatogenesis.

BRDT, a member of the BET family of double bromodomain-containing proteins, is essential for spermatogenesis in the mouse and has been postulated to be a key regulator of transcription in meiotic and post-meiotic cells. To understand the function of BRDT in these processes, we first characterized the genome-wide distribution of the BRDT binding sites, in particular within gene units, by ChIP-Seq analysis of enriched fractions of pachytene spermatocytes and round spermatids. In both cell types, BRDT binding sites were mainly located in promoters, first exons, and introns of genes. BRDT binding sites in promoters overlapped with several histone modifications and histone variants associated with active transcription, and were enriched for consensus sequences for specific transcription factors, including MYB, RFX, ETS, and ELF1 in pachytene spermatocytes, and JunD, c-Jun, CRE, and RFX in round spermatids. Subsequent integration of the ChIP-seq data with available transcriptome data revealed that stage-specific gene expression programs are associated with BRDT binding to their gene promoters, with most of the BDRT-bound genes being upregulated. Gene Ontology analysis further identified unique sets of genes enriched in diverse biological processes essential for meiosis and spermiogenesis between the two cell types, suggesting distinct developmentally stage-specific functions for BRDT. Taken together, our data suggest that BRDT cooperates with different transcription factors at distinctive chromatin regions within gene units to regulate diverse downstream target genes that function in male meiosis and spermiogenesis.

BET bromodomain inhibitor JQ1 regulates spermatid development by changing chromatin conformation in mouse spermatogenesis

As a BET bromodomain inhibitor, JQ1 has been proven have efficacy against a number of different cancers. In terms of male reproduction, JQ1 may be used as a new type of contraceptive, since JQ1 treatment in male mice could lead to germ cell defects and a decrease of sperm motility, moreover, this effect is reversible. However, the mechanism of JQ1 acting on gene regulation in spermatogenesis remains unclear. Here, we performed single-cell RNA sequencing (scRNA-seq) on mouse testes treated with JQ1 or vehicle control to determine the transcriptional regulatory function of JQ1 in spermatogenesis at the single cell resolution. We confirmed that JQ1 treatment could increase the numbers of somatic cells and spermatocytes and decrease the numbers of spermatid cells. Gene Ontology (GO) analysis demonstrated that differentially expressed genes which were down-regulated after JQ1 injection were mainly enriched in “DNA conformation change” biological process in early developmental germ cells and “spermatid development” biological process in spermatid cells. ATAC-seq data further confirmed that JQ1 injection could change the open state of chromatin. In addition, JQ1 could change the numbers of accessible meiotic DNA double-stranded break sites and the types of transcription factor motif that functioned in pachytene spermatocytes and round spermatids. The multi-omics analysis revealed that JQ1 had the ability to regulate gene transcription by changing chromatin conformation in mouse spermatogenesis, which would potentiate the availability of JQ1 in male contraceptive.

Immunodistribution of RFamide-related peptide-3 (RFRP-3) during the seminiferous epithelium cycle in a desert rodent Psammomys obesus

The Sand rat, Psammomys obesus, living northwest of the Algerian Sahara, presents a seasonal reproductive cycle. The purposes of this study were firstly to determine the stages of seminiferous epithelium cycle (SEC) by histological and morphometric analysis and secondly to investigate, for the first time, the testicular expression of RFamide-related peptide-3 (RFRP-3) during the SEC by immunohistochemistry. The results showed that the SEC consists of 14 stages according to the tubular morphology method. RFRP-3 was observed in both testicular compartments: the tubular and the interstitial. Leydig cells exhibited the highest RFRP-3 signal (30.73 % ± 4.80) compared to Sertoli cells (13–15 %). In the germline, RFRP-3 was detected during the late prophase I of meiosis in late pachytene, diplotene and metaphasic spermatocytes I. In addition, only round and triangular spermatids were positive during spermiogenesis. Referring to the SEC, it was found that the increased staining of RFRP-3 in spermatocytes I coincided with late pachytene of XI and XII stages (16.90 % ± 0.69 and 16.61 % ± 0.28, respectively). In spermatids, the labeling decreased in the triangular ones at stage IX (8.04 % ± 0.42). These results suggest the involvement of RFRP-3 in the control of SEC in P. obesus.

Comparison of germ cell differentiation of rat testis fragments cultured in knockout serum replacement versus Albumax™ I.

Spermatogenesis complexity makes reliable in vitro testis model development challenging. Previously, we evaluated an in vitro mouse testis organ culture system for assessing testicular toxicity. However, rat models are commonly used for drug/chemical toxicity testing; therefore, we assessed the effects of media on germ cell differentiation in cultured rat testis fragments. Testes from postnatal day 5 Sprague-Dawley (Hsd:SD) rats were cultured in knockout serum replacement (KSR) or Albumax™ I (Albumax) medium. For testis morphology and germ cell differentiation, rat testis fragments were collected on days 20, 27, 35, 42, 49, and 63 of culture for histology/immunohistochemistry using antibodies to spermatogenesis-specific markers. The fragments collected on days 20, 27, 42, 49, and 63 were used for qPCR. Pachytene spermatocyte (PS) differentiation was observed in rat testis fragments cultured in KSR and Albumax. However, there were more seminiferous tubules (STs) with PS in rat testis fragments cultured in Albumax than in KSR. Over 60% of STs with germ cell differentiation were observed in rat testis fragments when cultured in Albumax on days 20, 27, and 35, whereas this figure showed only on day 20 when cultured in KSR. This study found only PS differentiation in rat testis fragments. Compared to KSR, Albumax appears to contribute to increased PS production. This in vitro rat testis organ culture system may be useful for assessing testicular toxicity. However, PS differentiation per ST is lower in rat testis fragments; further studies are required to improve this rat testis organ culture system.

Parp1-Dependent DNA Double-Strand Break Repair in Irradiated Late Pachytene Spermatocytes.

Poly (ADP-ribose) polymerase-1 (Parp1) is a member of nuclear enzymes family involved in to the response to genotoxic stresses, DNA repair, and is critical for the maintenance of genome stability. During gametogenesis, genome stability is essential for inheritance and formation of healthy gametes. The latter involves DNA double-strand break (DSB)-driven pairing of homologous chromosomes in first meiotic prophase. By analysis of DSB repair kinetics in male meiotic prophase cells of homologous recombination (HR) and nonhomologous end joining (NHEJ)-deficient mouse models, we previously demonstrated an interplay between HR and the conventional NHEJ repair pathway. In the current work, we evaluate the relative contribution of Parp1-dependent NHEJ to the repair of ectopic ionizing radiation (IR)-induced DSBs in control and Parp1-inhibited mouse pachytene spermatocytes before and after the completion of meiotic recombination in stages VI-XI. The disappearance of large, exogenous DSB-related γ-H2AX foci was quantified 1 and 8 h after 1 Gy γ-irradiation of control and 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)quinolinone (DPQ) Parp1-inhibited mice. Late pachytene control spermatocytes obtained 8 h after IR had repaired >80% of DSBs observed at 1 h after IR. However, only 64% of DSBs were repaired in late spermatocytes of DPQ-treated (Parp1-inhibited) mice. Thus, it appears that Parp1 contributes to the repair of a fraction of DSBs in late prophase I, providing further insights in DNA repair pathway choreography during spermatogenic differentiation.

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.

Seasonal evaluation of spermatogenesis of the hematophagous bat Desmodus rotundus in the Caatinga biome.

This study was aimed to characterize the spermatogenic process and its seasonal variation in Desmodus rotundus, in the Caatinga biome, a water-limited ecosystem, with marked water restriction during most of the year. Collections of adult animals were performed during the dry and rainy seasons, and after euthanasia, their testes were processed histologically to perform morphological, morphometric, ultrastructural and immunohistochemical analyzes. The percentage of seminiferous epithelium, number of Leydig cells per gram of testis, and population of Sertoli cells and A-type spermatogonia presented by D. rotundus were significantly higher in the rainy season, while the percentage of lumen, mitotic index, support capacity performed by Sertoli cells, and overall yield of spermatogenesis were higher in the dry season. The ultrastructure of spermatogenesis was similar to that described in other mammals, and the immunohistochemical analysis revealed activity of the aromatase enzyme in Sertoli cells, Leydig cells, spermatocytes and spermatids, as well as the presence of androgen receptors in Sertoli cells and Leydig cells. FGF2 activity was detected in primary spermatocytes in zygotene and pachytene, as well as secondary spermatocytes and rounded and elongated spermatids, while the BCL-2 protein was expressed in primary spermatocytes in zygotene and pachytene, secondary spermatocytes, and rounded spermatids. The activity of these molecules was similar in both seasons, and associated with the morphometric findings, indicates maintenance in the integrity of the seminiferous epithelium throughout the year. The seasonal study of D. rotundus spermatogenesis indicates a continuous spermatogenesis pattern and suggests a greater production of spermatozoa in the rainy season in the Caatinga biome.

Sodium vitamin C transporter 2 orchestrates lactate metabolism in mouse Sertoli cells.

Transportation of vitamin C (also called ascorbic acid (AA)), an important water-soluble antioxidant and cofactor in testis, requires glucose transporter family (GLUTs) and sodium/vitamin C cotransporter family (SVCT1 and SVCT2). There is so far scant information vis-à-vis the functional roles of SVCTs in testis, although they possess higher affinity for transportation of AA compared to GLUTs. To analyze the biological effects of SVCT2 in testis, we assessed testicular expression of SVCT2 in different experimental settings and the effect of SVCT2 ablation on spermatogenesis. Persistent expression of SVCT2 was shown in the mouse testis at different stages of postnatal development, demonstrated on day 14 of testicular development in mice consistent with the appearance of pachytene spermatocytes during the first wave of spermatogenesis. Testicular expression of SVCT2 was enriched in the cytoplasm of murine Sertoli cells (SCs). We then showed that in vivo inhibition of SVCT2 in mouse testis significantly impaired male fertility by causing oligozoospermia and asthenospermia, which mainly stemmed from a deficiency in lactate production. By generating the TM4SVCT2-/- cells and by profiling TM4SVCT2-/- cells with a constitutively activated HIF-1α mutant, we demonstrated that SVCT2 deficiency led to impaired lactate synthesis and reduced expression of Ldha mRNA in SCs. Mechanistically, ablation of SVCT2 resulted in ubiquitination and subsequent degradation of HIF-1α protein in the FSH-stimulated SCs. Collectively, our data document a novel testicular site of action of SVCT2 in the control of lactate synthesis by SCs, probably via ubiquitination-dependent regulation of HIF-1α stability.

Unusual Behavior of the XY Body in Spermatogenesis in Mutagenized Mice

For the first time, it is shown that in hybrid F1 CBA × C57BL/6 mice exposed to the chemical mutagen dipin pachytene spermatocytes are formed with the sex XY chromosomes (XY bodies) lying outside the main nuclei. XY bodies isolated from the main nuclei were also found in the spermatid population. Our quantifications show that in the control and the experiment groups the frequency of occurrence of such genetically abnormal pachytene spermatocytes, the nuclei of which have lost the XY bodies, is 0.22 and 2.9%, respectively, and that of aberrant round spermatids is 0.0 and 2.84%. In spermatogenesis of mutant 129 mouse strain, a similar pattern of XY bodies unusual behavior was revealed, both in the control and in the experiment. In the control and in mutagenized 129 mice, the number of genetically abnormal pachytene spermatocytes was 0.67 and 0.25%, respectively, while the number of aberrant round spermatids was 0.1 and 0.17%. The differences established between the two strains of mice regarding the reaction of XY bodies to the action of dipin might be related to the fact that, in mutagenized 129 mice, the death of genetically damaged developing male germ cells at stages prior to pachytene spermatocytes and round spermatids is higher than in hybrid F1 CBA × C57BL/6 mice.

Spermatogonial asynchrony in Tex14 mutant mice lacking intercellular bridges

The existence of cytoplasmic passages between germ cells and their potential function in the control of the spermatogenic process has long been an intriguing question. Evidence of the important role of such structures, known as intercellular bridges (ICB), in spermatogenesis has been implicated by the failure of spermatogenesis in testis-expressed gene 14 (Tex14) mutant mice, which lack the ICBs, to progress past the pachytene spermatocyte stage. Using these Tex14 mutants, the present study evaluated, for the first time, the behavior and synchrony of the spermatogonial lineage in the absence of ICBs. Our data suggest that the absence of these cytoplasmic connections between cells affects the expansion of the undifferentiated type A (Aundiff) spermatogonia compartment and their transition to A1, resulting in a significant numerical reduction of differentiating A1 spermatogonia, but did not interfere with cell amplification during subsequent mitotic steps of differentiating spermatogonia from A1 through intermediate (In). However, beginning at the type B spermatogonia, the synchrony of differentiation was impaired as some cells showed delayed differentiation compared to their behavior in a normal seminiferous epithelium cycle. Thus although spermatogonial development is able to proceed, in the absence of ICBs in Tex14-/- mutants, the yield of cells, specific steps of differentiation, the synchrony of the cell kinetics, and the subsequent progression in meiosis are quantitatively lower than normal.

Cdc14a has a role in spermatogenesis, sperm maturation and male fertility

Cdc14a is an evolutionarily conserved dual-specific protein phosphatase, and it plays different roles in different organisms. Cdc14a mutations in human have been reported to cause male infertility, while the specific role of Cdc14a in regulation of the male reproductive system remains elusive. In the present study, we established a knockout mouse model to study the function of Cdc14a in male reproductive system. Cdc14a−/− male mice were subfertile and they could only produce very few offspring. The number of sperm was decreased, the sperm motility was impaired, and the proportion of sperm with abnormal morphology was elevated in Cdc14a−/− mice. When we mated Cdc14a−/− male mice with wild-type (WT) female mice, fertilized eggs could be found in female fallopian tubes, however, the majority of these embryos died during development. Some empty spaces were observed in seminiferous tubule of Cdc14a−/− testes. Compared with WT male mice, the proportions of pachytene spermatocytes were increased and germ cells stained with γH2ax were decreased in Cdc14a−/− male mice, indicating that knockout of Cdc14a inhibited meiotic initiation. Subsequently, we analyzed the expression levels of some substrate proteins of Cdc14a, including Cdc25a, Wee1, and PR-Set7, and compared those with WT testes, in which the expression levels of these proteins were significantly increased in Cdc14a−/− testes. Our results revealed that Cdc14a−/− male mice are highly subfertile, and Cdc14a is essential for normal spermatogenesis and sperm function.

Whole-body heat exposure causes developmental stage-specific apoptosis of male germ cells.

Humans are occasionally exposed to extreme environmental heat for a prolonged period of time. Here, we investigated testicular responses to whole-body heat exposure by placing mice in a warm chamber. Among the examined tissues, the testis was found to be most susceptible to heat stress. Heat stress induces direct responses within germ cells, such as eukaryotic initiation factor 2αphosphorylation and stress granule (SG) formation. Prolonged heat stress (42°C for 6 hr) also disturbed tissue organization, such as through blood-testis barrier (BTB) leakage. Germ cell apoptosis was induced by heat stress for 6 hr in a cell type- and developmental stage-specific manner. We previously showed that spermatocytes in the early tubular stages (I-VI) form SGs for protection against heat stress. In the mid-tubular stages (VII-VIII), BTB leakage synergistically enhances the adverse effects of heat stress on pachytene spermatocyte apoptosis. In the late tubular stages (IX-XII), SGs are not formed and severe leakage of the BTB does not occur, resulting in mild apoptosis of late-pachytene spermatocytes near meiosis. Our results revealed that multiple stress responses are involved in germ cell damage resulting from prolonged heat stress (42°C for 6 hr).

Improving Freezing Protocols and Organotypic Culture: A Histological Study on Rat Prepubertal Testicular Tissue.

Testicular tissue freezing before gonadotoxic treatments allows the preservation of fertility for children suffering from cancer. Recently, the testis organ culture method was presented as a relevant method to restore the fertility of these patients. However, the yield of spermatozoa production is low in the mouse model and no gamete has been obtained in vitro in the rat model. Here, we assess different cryopreservation protocols and culture conditions to improve the efficiency of in vitro maturation of rat prepubertal testes. Testes from male rats aged 5 or 8 days post-partum were cultured onto agarose gels of different percentages. After determining the best culture conditions, different cryopreservation protocols were assessed. Finally, testicular tissues were cultured with media of various compositions and analyzed at different time points. Our results show that the cryopreservation protocols allow the preservation of tissue architecture, cell proliferation, with no or moderate increase of cell death. In vitro spermatogenesis did not proceed beyond the pachytene spermatocyte stage. Only 2 of the 6 tested media allowed the survival of differentiated germ cells over the 45-day culture period. In conclusion, this study highlights the necessity to further improve the organ culture method before applying it into the clinics.

Hsa-miR-1908-3p Mediates the Self-Renewal and Apoptosis of Human Spermatogonial Stem Cells via Targeting KLF2

Spermatogenesis depends on precise epigenetic and genetic regulation of spermatogonial stem cells (SSCs). However, it remains largely unknown about the roles and mechanisms of small noncoding RNA in regulating the self-renewal and apoptosis of human SSCs. Notably, we have found that Homo sapiens-microRNA (hsa-miR)-1908-3p is expressed at a higher level in human spermatogonia than pachytene spermatocytes. MiR-1908-3p stimulated cell proliferation and DNA synthesis of the human SSC line. Allophycocyanin (APC) Annexin V and propidium iodide staining, determined by flow cytometric analysis and TUNEL assays, showed that miR-1908-3p inhibited early and late apoptosis of the human SSC line. Furthermore, Kruppel-like factor 2 (KLF2) was predicted and verified as the target of miR-1908-3p, and, significantly, KLF2 silencing resulted in the increase of proliferation and DNA synthesis, as well as reduction of apoptosis of the human SSC line. Moreover, KLF2 silencing ameliorated the decrease in the proliferation and DNA synthesis and the enhancement in the apoptosis of the human SSC line caused by miR-1908-3p inhibition. Collectively, these results implicate that miR-1908-3p stimulates the self-renewal and suppresses the apoptosis of human SSCs by targeting KLF2. This study thus provides a novel epigenetic regulatory mechanism underlying the fate determinations of human SSCs, and it offers new endogenous targets for treating male infertility.

BRUCE preserves genomic stability in the male germline of mice.

BRUCE is a DNA damage response protein that promotes the activation of ATM and ATR for homologous recombination (HR) repair in somatic cells, making BRUCE a key protector of genomic stability. Preservation of genomic stability in the germline is essential for the maintenance of species. Here, we show that BRUCE is required for the preservation of genomic stability in the male germline of mice, specifically in spermatogonia and spermatocytes. Conditional knockout of Bruce in the male germline leads to profound defects in spermatogenesis, including impaired maintenance of spermatogonia and increased chromosomal anomalies during meiosis. Bruce-deficient pachytene spermatocytes frequently displayed persistent DNA breaks. Homologous synapsis was impaired, and nonhomologous associations and rearrangements were apparent in up to 10% of Bruce-deficient spermatocytes. Genomic instability was apparent in the form of chromosomal fragmentation, translocations, and synapsed quadrivalents and hexavalents. In addition, unsynapsed regions of rearranged autosomes were devoid of ATM and ATR signaling, suggesting an impairment in the ATM- and ATR-dependent DNA damage response of meiotic HR. Taken together, our study unveils crucial functions for BRUCE in the maintenance of spermatogonia and in the regulation of meiotic HR-functions that preserve the genomic stability of the male germline.

Linker histone variant H1t is closely associated with repressed repeat-element chromatin domains in pachytene spermatocytes

BackgroundH1t is the major linker histone variant in pachytene spermatocytes, where it constitutes 50–60% of total H1. This linker histone variant was previously reported to localize in the nucleolar rDNA element in mouse spermatocytes. Our main aim was to determine the extra-nucleolar localization of this linker histone variant in pachytene spermatocytes.ResultsWe generated H1t-specific antibodies in rabbits and validated its specificity by multiple assays like ELISA, western blot, etc. Genome-wide occupancy studies, as determined by ChIP-sequencing in P20 mouse testicular cells revealed that H1t did not closely associate with active gene promoters and open chromatin regions. Annotation of H1t-bound genomic regions revealed that H1t is depleted from DSB hotspots and TSS, but are predominantly associated with retrotransposable repeat elements like LINE and LTR in pachytene spermatocytes. These chromatin domains are repressed based on co-association of H1t observed with methylated CpGs and repressive histone marks like H3K9me3 and H4K20me3 in vivo. Mass spectrometric analysis of proteins associated with H1t-containing oligonucleosomes identified piRNA–PIWI pathway proteins, repeat repression-associated proteins and heterochromatin proteins confirming the association with repressed repeat-element genomic regions. We validated the interaction of key proteins with H1t-containing oligonucleosomes by use of ChIP-western blot assays. On the other hand, we observe majority of H1t peaks to be associated with the intergenic spacer of the rDNA element, also in association with SINE elements of the rDNA element. Thus, we have identified the genomic and chromatin features of both nucleolar and extranucleolar localization patterns of linker histone H1t in the context of pachytene spermatocytes.ConclusionsH1t-containing repeat-element LINE and LTR chromatin domains are associated with repressive marks like methylated CpGs, histone modifications H3K9me3 and H4K20me3, and heterochromatin proteins like HP1β, Trim28, PIWIL1, etc. Apart from localization of H1t at the rDNA element, we demonstrate the extranucleolar association of this linker histone variant at repeat-associated chromatin domains in pachytene spermatocytes. We hypothesize that H1t might induce local chromatin relaxation to recruit heterochromatin and repeat repression-associated protein factors necessary for TE (transposable element) repression, the final biological effect being formation of closed chromatin repressed structures.

Pluripotency factor NANOG promotes germ cell maintenance in vitro without triggering dedifferentiation of spermatogonial stem cells

Spermatogonial stem cells (SSCs) are defined as the tissue-specific stem cells in the testes that produce sperm and support life-long spermatogenesis. A culture microenvironment could convert mouse SSCs from unipotent to pluripotent states; however, the underlying mechanism is unclear. NANOG has been considered the decisive transcriptional factor for pluripotency transition of stem cells, but NANOG is not expressed in SSCs. Here, we investigated whether NANOG overexpression could result in SSCs being converted into a pluripotent state. We found that rare NANOG-positive cells could be detected in spermatogonia, pachytene spermatocytes, and even round spermatids in mouse testes, and that the induction of NANOG could promote the proliferation of cultured SSCs in vitro and partially compensate for the role of the growth factor GDNF. In vivo allogeneic transplantation of NANOG-overexpressing germ cells did not yield any teratoma-like tissues, but regenerated normal colonies of spermatogenesis in the testes of recipient mice. Collectively, our data showed that overexpression of the pluripotency factor NANOG along did not dedifferentiate testis germline stem cells into a pluripotent state, suggesting that other genetic or epigenetic factors are involved in SSC reprogramming.

M6A-dependent biogenesis of circular RNAs in male germ cells.

The majority of circular RNAs (circRNAs) spliced from coding genes contain open reading frames (ORFs) and thus, have protein coding potential. However, it remains unknown what regulates the biogenesis of these ORF-containing circRNAs, whether they are actually translated into proteins and what functions they play in specific physiological contexts. Here, we report that a large number of circRNAs are synthesized with increasing abundance when late pachytene spermatocytes develop into round and then elongating spermatids during murine spermatogenesis. For a subset of circRNAs, the back splicing appears to occur mostly at m6A-enriched sites, which are usually located around the start and stop codons in linear mRNAs. Consequently, approximately a half of these male germ cell circRNAs contain large ORFs with m6A-modified start codons in their junctions, features that have been recently shown to be associated with protein-coding potential. Hundreds of peptides encoded by the junction sequences of these circRNAs were detected using liquid chromatography coupled with mass spectrometry, suggesting that these circRNAs can indeed be translated into proteins in both developing (spermatocytes and spermatids) and mature (spermatozoa) male germ cells. The present study discovered not only a novel role of m6A in the biogenesis of coding circRNAs, but also a potential mechanism to ensure stable and long-lasting protein production in the absence of linear mRNAs, i.e., through production of circRNAs containing large ORFs and m6A-modified start codons in junction sequences.

Foxn1 and Prkdc genes are important for testis function: evidence from nude and scid adult mice.

Mutations in Foxn1 and Prkdc genes lead to nude and severe combined immunodeficiency (scid) phenotypes, respectively. Besides being immunodeficient, previous reports have shown that nude mice have lower gonadotropins and testosterone levels, while scid mice present increased pachytene spermatocyte (PS) apoptosis. Therefore, these specific features make them important experimental models for understanding Foxn1 and Prkdc roles in reproduction. Hence, we conducted an investigation of the testicular function in nude and scid BALB/c adult male mice and significant differences were observed, especially in Leydig cell (LC) parameters. Although the differences were more pronounced in nude mice, both immunodeficient strains presented a larger number of LC, whereas its cellular volume was smaller in comparison to the wild type. Besides these alterations in LC, we also observed differences in androgen receptor and steroidogenic enzyme expression in nude and scid mice, suggesting the importance of Foxn1 and Prkdc genes in androgen synthesis. Specifically in scid mice, we found a smaller meiotic index, which represents the number of round spermatids per PS, indicating a greater cell loss during meiosis, as previously described in the literature. In addition and for the first time, Foxn1 was identified in the testis, being expressed in LC, whereas DNA-PKc (the protein produced by Prkdc) was observed in LC and Sertoli cells. Taken together, our results show that the changes in LC composition added to the higher expression of steroidogenesis-related genes in nude mice and imply that Foxn1 transcription factor may be associated to androgen production regulation, while Prkdc expression is also important for the meiotic process.