In vitro reconstitution of epigenetic reprogramming in the human germ line.

Epigenetic reprogramming resets parental epigenetic memories and differentiates primordial germ cells (PGCs) into mitotic pro-spermatogonia or oogonia. This process ensures sexually dimorphic germ cell development for totipotency1. In vitro reconstitution of epigenetic reprogramming in humans remains a fundamental challenge. Here we establish a strategy for inducing epigenetic reprogramming and differentiation of pluripotent stem-cell-derived human PGC-like cells (hPGCLCs) into mitotic pro-spermatogonia or oogonia, coupled with their extensive amplification (about >1010-fold). Bone morphogenetic protein (BMP) signalling is a key driver of these processes. BMP-driven hPGCLC differentiation involves attenuation of the MAPK (ERK) pathway and both de novo and maintenance DNA methyltransferase activities, which probably promote replication-coupled, passive DNA demethylation. hPGCLCs deficient in TET1, an active DNA demethylase abundant in human germ cells2,3, differentiate into extraembryonic cells, including amnion, with de-repression of key genes that bear bivalent promoters. These cells fail to fully activate genes vital for spermatogenesis and oogenesis, and their promoters remain methylated. Our study provides a framework for epigenetic reprogramming in humans and an important advance in human biology. Through the generation of abundant mitotic pro-spermatogonia and oogonia-like cells, our results also represent a milestone for human in vitro gametogenesis research and its potential translation into reproductive medicine.

Msl3 promotes germline stem cell differentiation in female Drosophila.

Gamete formation from germline stem cells (GSCs) is essential for sexual reproduction. However, the regulation of GSC differentiation is incompletely understood. Set2, which deposits H3K36me3 modifications, is required for GSC differentiation during Drosophila oogenesis. We discovered that the H3K36me3 reader Male-specific lethal 3 (Msl3) and histone acetyltransferase complex Ada2a-containing (ATAC) cooperate with Set2 to regulate GSC differentiation in female Drosophila. Msl3, acting independently of the rest of the male-specific lethal complex, promotes transcription of genes, including a germline-enriched ribosomal protein S19 paralog RpS19b. RpS19b upregulation is required for translation of RNA-binding Fox protein 1 (Rbfox1), a known meiotic cell cycle entry factor. Thus, Msl3 regulates GSC differentiation by modulating translation of a key factor that promotes transition to an oocyte fate.

The Drosophila ribosome protein S5 paralog RpS5b promotes germ cell and follicle cell differentiation during oogenesis.

Emerging evidence suggests that ribosome heterogeneity may have important functional consequences in the translation of specific mRNAs within different cell types and under various conditions. Ribosome heterogeneity comes in many forms, including post-translational modification of ribosome proteins (RPs), absence of specific RPs and inclusion of different RP paralogs. The Drosophila genome encodes two RpS5 paralogs: RpS5a and RpS5b. While RpS5a is ubiquitously expressed, RpS5b exhibits enriched expression in the reproductive system. Deletion of RpS5b results in female sterility marked by developmental arrest of egg chambers at stages 7-8, disruption of vitellogenesis and posterior follicle cell (PFC) hyperplasia. While transgenic rescue experiments suggest functional redundancy between RpS5a and RpS5b, molecular, biochemical and ribo-seq experiments indicate that RpS5b mutants display increased rRNA transcription and RP production, accompanied by increased protein synthesis. Loss of RpS5b results in microtubule-based defects and in mislocalization of Delta and Mindbomb1, leading to failure of Notch pathway activation in PFCs. Together, our results indicate that germ cell-specific expression of RpS5b promotes proper egg chamber development by ensuring the homeostasis of functional ribosomes.

TGFB1 stimulates the proliferation of quiescent oogonial stem cells in chicken.

In brief: Oogonial stem cells in the adult ovary can generate oocytes, but they are usually quiescent. TGFB1 is key in stimulating the proliferation of OSC, thereby ensuring the sustained reproductive potential in poultry species. Abstract: Oogonial stem cells (OSCs) are a type of germ stem cell present in the adult ovary. They have the ability to self-renew through mitosis and differentiate into oocytes through meiosis. We have previously identified a population of OSCs in the chicken ovary, but the underlying mechanisms controlling their activation and proliferation were unclear. In this study, we observed that OSCs showed robust proliferation when cultured on a layer of chicken embryo fibroblasts (CEF), suggesting that CEF may secrete certain crucial factors that activate OSC proliferation. We further detected TGFB1 as a potent signaling molecule to promote OSC proliferation. Additionally, we revealed the signaling pathways that play important roles downstream of TGFB1-induced OSC proliferation. These findings provide insights into the mechanisms underlying OSC proliferation in chickens and offer a foundation for future research on in situ activation of OSC proliferation in ovary and improvement of egg-laying performance in chickens.

The DNA damage response is required for oocyte cyst breakdown and follicle formation in mice.

Mammalian oogonia proliferate without completing cytokinesis, forming cysts. Within these, oocytes differentiate and initiate meiosis, promoting double-strand break (DSBs) formation, which are repaired by homologous recombination (HR) causing the pairing and synapsis of the homologs. Errors in these processes activate checkpoint mechanisms, leading to apoptosis. At the end of prophase I, in contrast with what is observed in spermatocytes, oocytes accumulate unrepaired DSBs. Simultaneously to the cyst breakdown, there is a massive oocyte death, which has been proposed to be necessary to enable the individualization of the oocytes to form follicles. Based upon all the above-mentioned information, we hypothesize that the apparently inefficient HR occurring in the oocytes may be a requirement to first eliminate most of the oocytes and enable cyst breakdown and follicle formation. To test this idea, we compared perinatal ovaries from control and mutant mice for the effector kinase of the DNA Damage Response (DDR), CHK2. We found that CHK2 is required to eliminate ~50% of the fetal oocyte population. Nevertheless, the number of oocytes and follicles found in Chk2-mutant ovaries three days after birth was equivalent to that of the controls. These data revealed the existence of another mechanism capable of eliminating oocytes. In vitro inhibition of CHK1 rescued the oocyte number in Chk2-/- mice, implying that CHK1 regulates postnatal oocyte death. Moreover, we found that CHK1 and CHK2 functions are required for the timely breakdown of the cyst and to form follicles. Thus, we uncovered a novel CHK1 function in regulating the oocyte population in mice. Based upon these data, we propose that the CHK1- and CHK2-dependent DDR controls the number of oocytes and is required to properly break down oocyte cysts and form follicles in mammals.

Environmentally-relevant exposure to diethylhexyl phthalate (DEHP) alters regulation of double-strand break formation and crossover designation leading to germline dysfunction in Caenorhabditis elegans.

Exposure to diethylhexyl phthalate (DEHP), the most abundant plasticizer used in the production of polyvinyl-containing plastics, has been associated to adverse reproductive health outcomes in both males and females. While the effects of DEHP on reproductive health have been widely investigated, the molecular mechanisms by which exposure to environmentally-relevant levels of DEHP and its metabolites impact the female germline in the context of a multicellular organism have remained elusive. Using the Caenorhabditis elegans germline as a model for studying reprotoxicity, we show that exposure to environmentally-relevant levels of DEHP and its metabolites results in increased meiotic double-strand breaks (DSBs), altered DSB repair progression, activation of p53/CEP-1-dependent germ cell apoptosis, defects in chromosome remodeling at late prophase I, aberrant chromosome morphology in diakinesis oocytes, increased chromosome non-disjunction and defects during early embryogenesis. Exposure to DEHP results in a subset of nuclei held in a DSB permissive state in mid to late pachytene that exhibit defects in crossover (CO) designation/formation. In addition, these nuclei show reduced Polo-like kinase-1/2 (PLK-1/2)-dependent phosphorylation of SYP-4, a synaptonemal complex (SC) protein. Moreover, DEHP exposure leads to germline-specific change in the expression of prmt-5, which encodes for an arginine methyltransferase, and both increased SC length and altered CO designation levels on the X chromosome. Taken together, our data suggest a model by which impairment of a PLK-1/2-dependent negative feedback loop set in place to shut down meiotic DSBs, together with alterations in chromosome structure, contribute to the formation of an excess number of DSBs and altered CO designation levels, leading to genomic instability.

Production of juvenile masu salmon (Oncorhynchus masou) from spermatogonia-derived sperm and oogonia-derived eggs via intraperitoneal transplantation of immature germ cells.

No effective cryopreservation technique exists for fish eggs and embryos; thus, the cryopreservation of germ cells (spermatogonia or oogonia) and subsequent generation of eggs and sperm would be an alternative solution for the long-term preservation of piscine genetic resources. Nevertheless, in our previous study using rainbow trout, we showed that recipients transplanted with XY spermatogonia or XX oogonia produced unnatural sex-biased F1 offspring. To overcome these obstacles, we transplanted immature germ cells (XX oogonia or XY spermatogonia; frozen for 33 days) into the body cavities of triploid hatchlings, and the transplanted germ cells possessed a high capacity for differentiating into eggs and sperm in the ovaries and testes of recipients. Approximately 30% of triploid recipients receiving frozen germ cells generated normal salmon that displayed the donor-derived black body color phenotype, although all triploid salmon not receiving transplants were functionally sterile. Furthermore, F1 offspring obtained from insemination of the oogonia-derived eggs and spermatogonia-derived sperm show a normal sex ratio of 1:1 (female:male). Thus, this method presented a critical technique for practical conservation projects for other teleost fish species and masu salmon.

Specification and spatial arrangement of cells in the germline stem cell niche of the Drosophila ovary depend on the Maf transcription factor Traffic jam.

Germline stem cells in the Drosophila ovary are maintained by a somatic niche. The niche is structurally and functionally complex and contains four cell types, the escort, cap, and terminal filament cells and the newly identified transition cell. We find that the large Maf transcription factor Traffic jam (Tj) is essential for determining niche cell fates and architecture, enabling each niche in the ovary to support a normal complement of 2-3 germline stem cells. In particular, we focused on the question of how cap cells form. Cap cells express Tj and are considered the key component of a mature germline stem cell niche. We conclude that Tj controls the specification of cap cells, as the complete loss of Tj function caused the development of additional terminal filament cells at the expense of cap cells, and terminal filament cells developed cap cell characteristics when induced to express Tj. Further, we propose that Tj controls the morphogenetic behavior of cap cells as they adopted the shape and spatial organization of terminal filament cells but otherwise appeared to retain their fate when Tj expression was only partially reduced. Our data indicate that Tj contributes to the establishment of germline stem cells by promoting the cap cell fate, and controls the stem cell-carrying capacity of the niche by regulating niche architecture. Analysis of the interactions between Tj and the Notch (N) pathway indicates that Tj and N have distinct functions in the cap cell specification program. We propose that formation of cap cells depends on the combined activities of Tj and the N pathway, with Tj promoting the cap cell fate by blocking the terminal filament cell fate, and N supporting cap cells by preventing the escort cell fate and/or controlling the number of cap cell precursors.

c-Fos Repression by Piwi Regulates Drosophila Ovarian Germline Formation and Tissue Morphogenesis.

Drosophila melanogaster Piwi functions within the germline stem cells (GSCs) and the somatic niche to regulate GSC self-renewal and differentiation. How Piwi influences GSCs is largely unknown. We uncovered a genetic interaction between Piwi and c-Fos in the somatic niche that influences GSCs. c-Fos is a proto-oncogene that influences many cell and developmental processes. In wild-type ovarian cells, c-Fos is post-transcriptionally repressed by Piwi, which destabilized the c-Fos mRNA by promoting the processing of its 3' untranslated region (UTR) into Piwi-interacting RNAs (piRNAs). The c-Fos 3' UTR was sufficient to trigger Piwi-dependent destabilization of a GFP reporter. Piwi represses c-Fos in the somatic niche to regulate GSC maintenance and differentiation and in the somatic follicle cells to affect somatic cell disorganization, tissue dysmorphogenesis, oocyte maturation arrest, and infertility.

Lhx8 ablation leads to massive autophagy of mouse oocytes associated with DNA damage.

Following proliferation of oogonia in mammals, great numbers of germ cells are discarded, primarily by apoptosis, while the remainder form primordial follicles (the ovarian reserve) that determine fertility and reproductive lifespan. More massive, rapid, and essentially total loss of oocytes, however, occurs when the transcription factor Lhx8 is ablated-though the cause and mechanism of germ cell loss from the Lhx8-/- ovaries has been unknown. We found that Lhx8-/- ovaries maintain the same number of germ cells throughout embryonic development; rapid decrease in the pool of oocytes starts shortly before birth. The loss results from activation of autophagy, which becomes overwhelming within the first postnatal week, with extracellular matrix proteins filling the space previously occupied by follicles to produce a fibrotic ovary. Associated with this process, as early as a few days before birth, Lhx8-/- oocytes failed to repair DNA damage-which normally occurs when meiosis is initiated during embryonic development; and DNA damage repair genes were downregulated throughout the oocyte short lifespan. Based on gene expression analyses and morphological changes, we propose a model in which lineage-restricted failure of DNA repair triggers germ cell autophagy, causing premature depletion of the ovarian reserve in Lhx8-/- mice.

Functions of the MRE11 complex in the development and maintenance of oocytes.

The MRE11 complex (MRE11, RAD50, and NBS1) is a central component of the DNA damage response, governing both double-strand break repair and DNA damage response signaling. To determine the functions of the MRE11 complex in the development and maintenance of oocytes, we analyzed ovarian phenotypes of mice harboring the hypomorphic Mre11 (ATLD1) allele. Mre11 (ATLD1/ATLD1) females exhibited premature oocyte elimination attributable to defects in homologous chromosome pairing and double-strand break repair during meiotic prophase. Other aspects of meiotic progression, including attachment of telomeres to the nuclear envelope and recruitment of RAD21L, a component of the meiotic cohesin complex to the synaptonemal complex, were normal. Unlike Dmc1 (-/-) and Trp13 (Gt/Gt) mice which exhibit comparable defects in double-strand break repair and oocyte depletion by 5 days post-partum, we found that oocyte attrition occurred by 12 weeks in Mre11 (ATLD1/ATLD1) . Disruption of the oocyte checkpoint pathway governed by Chk2 gene further enhanced the survival of Mre11 (ATLD1/ATLD1) follicles. Together our data suggest that the MRE11 complex influences the elimination of oocytes with unrepaired meiotic double-strand breaks post-natally, in addition to its previously described role in double-strand break repair and homologous synapsis during female meiosis.

Human foetal ovary shares meiotic preventing factors with the developing testis.

STUDY QUESTION: How can pre-meiotic germ cells persist in the human foetal ovary? SUMMARY ANSWER: Numerous oogonia escaping meiotic entry were retrieved throughout human ovarian development simultaneously with the expression of signalling pathways preventing meiosis, typically described in the rodent embryonic testis. WHAT IS KNOWN ALREADY: The transition from mitosis to meiosis is a key event in female germ cells that remains poorly documented in research on the human ovary. Previous reports described a strikingly asynchronous differentiation in the human female germ line during development, with the persistence of oogonia among oocytes and follicles during the second and third trimesters. The possible mechanisms allowing some cells to escape meiosis remain elusive. STUDY DESIGN SIZE, DURATION: In order to document the extent of this phenomenon, we detailed the expression profile of germ cell differentiation markers using 73 ovaries ranging from 6.4 to 35 weeks post-fertilization. PARTICIPANTS/MATERIALS SETTING, METHODS: Pre-meiotic markers were detected by immunohistochemistry or qRT-PCR. The expression of the main meiosis-preventing factors identified in mice was analysed, and their functionality assessed using organ cultures. MAIN RESULTS AND THE ROLE OF CHANCE: Oogonia stained for AP2γ could be traced from the first trimester until the end of the third trimester. Female germ cell differentiation is organized both in time and space in a centripetal manner in the foetal human ovary. Unexpectedly, some features usually ascribed to rodent pre-spermatogonia could be observed in human foetal ovaries, such as NANOS2 expression and quiescence in some germ cells. The two main somatic signals known to inhibit meiosis in the mouse embryonic testis, CYP26B1 and FGF9, were detected in the human ovary and act simultaneously to repress STRA8 and meiosis in human foetal female germ cells. LARGE SCALE DATA: N/A. LIMITATIONS REASON FOR CAUTION: Our conclusions relied partly on in vitro experiments. Germ cells were not systematically identified with immunostaining and some may have thus escaped analysis. WIDER IMPLICATIONS OF THE FINDINGS: We found evidence that a robust repression of meiotic entry is taking place in the human foetal ovary, possibly explaining the exceptional long-lasting presence of pre-meiotic germ cells until late gestational age. This result calls for a redefinition of the markers known as classical male markers, which may in fact characterize mammalian developing gonads irrespectively of their sex. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the Université Paris Diderot-Paris 7 and Université Paris-Sud, CEA, INSERM, and Agence de la Biomédecine. The authors declare no conflict of interest.

OOgenesis_Pred: A sequence-based method for predicting oogenesis proteins by six different modes of Chou's pseudo amino acid composition.

Regarding to critical roles of oogenesis in formation of ova or unfertilized eggs from the oogonia by mitotic division and subsequent differentiation, the identification of oogenesis-related proteins is of great interest. However, the experimental determination of proteins involved in oogenesis is expensive, time consuming and labor-intensive. Therefore, a new powerful discriminating model is indispensable for classifying oogenesis/non-oogenesis-related proteins with high accuracy and precision. Hereby, for the first time we developed a support vector machine based oogenesis protein prediction method which differentiates oogenesis from non-oogenesis proteins. By means of informative protein physicochemical properties and in addition parameter optimization scheme, our method yields a robust and consistent performance. Our model achieved 87.68% and 84.82% prediction accuracy by five-fold cross validation test for datasets with 90% and 50% identity, respectively. The prediction model was also assessed using the independent dataset and yielded 91.62% and 85.38% prediction accuracy for datasets with 90% and 50% identity, respectively, which further demonstrates the effectiveness of our method. Moreover, by applying 10 different feature weighting methods, the more important protein features for oogenesis/non-oogenesis-related proteins discrimination, including serine and glycine frequency, quasi-sequence-order, pseudo-amino acid composition, distribution and conjoint triad, were determined. The success rates revealed that our model can be considered as a new encouraging and strong model for predicting proteins involved in oogenesis with appropriate performance. To enhance the value of the practical applications of the proposed method, we developed a standalone software for predicting oogenesis candidate proteins called OOgenesis_Pred. This software is the first predictor ever established for identifying oogenesis proteins. We also showed the capability of OOgenesis_Pred by making oogenesis-related proteins prediction for some of the oogenesis candidate proteins. It is anticipated that OOgenesis_Pred will become a powerful tool for future proteomic studies related to oogenesis.

Persistent Requirement and Alteration of the Key Targets of PRDM1 During Primordial Germ Cell Development in Mice.

Primordial germ cells (PGCs) are the foundation of totipotency and vital for reproduction and heredity. PGCs in mice arise from the epiblast around Embryonic Day (E) 7.0, migrate through the hindgut endoderm, and colonize and proliferate in the embryonic gonads until around E13.5 prior to their differentiation either into prospermatogonia or oogonia. PRDM1, a transcriptional repressor, plays an essential role in PGC specification that includes robustly repressing a somatic mesodermal program. Using an inducible conditional knockout system, we show here that PRDM1 is critically required throughout PGC development. When Prdm1 was deleted in migrating PGCs at E9.5 or E10.5, or in male gonadal PGCs at E11.5, PGCs were eliminated by apoptosis from around E10.5, E11.5, or E13.5, respectively. When Prdm1 was deleted in female gonadal PGCs at E11.5, PGCs progressed into the first meiotic prophase in an apparently normal fashion, but the oogonia exhibited an aberrant pachytene phenotype, undergoing abrupt apoptosis from around E16.5. The escape of a fraction of PGCs (∼10%) from the Prdm1 deletion was sufficient to recover fairly normal germ cell pools, both in male and female adults. The key targets of PRDM1 in migrating and/or gonadal PGCs, including genes for development, apoptosis, and prospermatogonial differentiation, showed only a modest overlap with those upon PGC specification, and were enriched with histone H3 lysine 27 trimethylation (H3K27me3). Our findings provide critical insight into the mechanism for maintaining the transcriptional integrity of PGCs.

Scp3 expression in relation to the ovarian differentiation in the protogynous hermaphroditic ricefield eel Monopterus albus.

Synaptonemal complex protein 3 (Scp3), which is encoded by scp3, is a meiotic marker commonly used to trace the timing of gonadal differentiation in vertebrates. In the present study, the ricefield eel scp3 cDNA was cloned, and a fragment encoding amino acids 49 to 244 was overexpressed. The recombinant Scp3 polypeptide was purified and used to generate a rabbit anti-Scp3 polyclonal antiserum. In adult ricefield eels, scp3 mRNA was predominantly detected in the gonads and faintly detected in discrete brain areas. In the gonads, Scp3 immunoreactivities were shown to be localized to the germ cells, including meiotic primary growth oocytes, spermatocytes, and pre-meiotic spermatogonia. During early ovarian differentiation, immunoreactive Scp3 was not detected in the gonads of ricefield eels at 6 days post-hatching (dph) but was found to be abundantly localized in the cytoplasm of some oogonia at 7 dph, coinciding with the appearance of the ovarian cavity and ovarian differentiation. At 14 dph, strong Scp3 immunostaining was detected on one side of the nucleus with a distinct polarity in some germ cells, presumably at the leptotene stage. Consistent with these results, the expression of scp3 mRNA was faintly detected in the gonads of ricefield eels at 6 dph, increased at 8 dph, and then remained relatively high thereafter. Taken together, these results suggest that the appearance of immunoreactive Scp3 in oogonia could be a marker for early ovarian differentiation in ricefield eels. The translocation of the Scp3 protein from the cytoplasm to the nucleus in the oogonium of ricefield eels appears to be a controlled process that warrants further study.

Changes in ultrastructure and expression of steroidogenic factor-1 in ovaries of zebrafish Danio rerio exposed to glyphosate.

Glyphosate is a broad-spectrum organophosphate (OP) herbicide, highly soluble in water, and when applied in terrestrial systems it penetrates into soil, eventually reaching the aquatic community and affecting nontarget organisms. The aim of this study was to evaluate the toxicity of glyphosate on ovaries of zebrafish (Danio rerio). Ovaries (n = 18 per triplicate) were exposed to 65 µg/L of glyphosate [N-(phosphonomethyl) glycine] for 15 d. This concentration was determined according to Resolution 357/2005/CONAMA/Brazil, which establishes the permissible concentration of glyphosate in Brazilian inland waters. Nonexposed ovaries (n = 18 per triplicate) were used as control. Subsequently, morphology and expression of steroidogenic factor-1 (SF-1) of exposed and nonexposed ovaries was determined. No apparent changes were noted in general morphology of exposed and nonexposed ovaries. However, a significant increase in diameter of oocytes was observed after exposure to glyphosate. When ovarian ultrastructure was examined the presence of concentric membranes, appearing as myelin-like structures, associated with the external membranes of mitochondria and with yolk granules was found. After glyphosate exposure, immunohistochemistry and immunoblotting revealed greater expression of SF-1 in the oocytes, which suggests a relationship between oocyte growth and SF-1 expression. These subtle adverse effects of glyphosate on oocytes raised a potential concern for fish reproduction. These results contribute to understanding glyphosate-induced toxicity to nontarget organisms, showing subcellular and molecular impairments that may affect reproduction in +female fish.

The dynamic expression of SOX17 in germ cells from human female foetus and adult ovaries after specification.

Background: SOX17 has been identified as a critical factor in specification of human primordial germ cells, but whether SOX17 regulates development of germ cells after sex differentiation is poorly understood. Methods: We collected specimens of gonadal ridge from an embryo (n=1), and ovaries of foetuses (n=23) and adults (n=3). Germ cells were labelled with SOX17, VASA (classic germ cells marker), phosphohistone H3 (PHH3, mitosis marker) and synaptonemal complex protein 3 (SCP3, meiosis marker). Results: SOX17 was detected in both cytoplasm and nucleus of oogonia and oocytes of primordial and primary follicles from 15 to 28 gestational weeks (GW). However, it was exclusively expressed in cytoplasm of oogonia at 7 GW, and in nucleus of oocytes in secondary follicles. Co-expression rates of SOX17 in VASA+ germ cells ranged from 81.29% to 97.81% in foetuses. Co-staining rates of SOX17 and PHH3 or SCP3 were 0%-34% and 0%-57%, respectively. Interestingly, we distinguished a subpopulation of SOX17+VASA- germ cells in fetal ovaries. These cells clustered in the cortex and could be co-stained with the mitosis marker PHH3 but not the meiosis marker SCP3. Conclusions: The dynamic expression of SOX17 was detected in human female germ cells. We discovered a population of SOX17+ VASA- germ cells clustering at the cortex of ovaries. We could not find a relationship between mitosis or meiosis and SOX17 or VASA staining in germ cells. Our findings provide insight into the potential role of SOX17 involving germ cells maturation after specification, although the mechanism is unclear and needs further investigation.

Analysis of meiosis regulators in human gonads: a sexually dimorphic spatio-temporal expression pattern suggests involvement of DMRT1 in meiotic entry.

The mitosis-meiosis switch is a key event in the differentiation of germ cells. In humans, meiosis is initiated in fetal ovaries, whereas in testes meiotic entry is inhibited until puberty. The purpose of this study was to examine the expression pattern of meiosis regulators in human gonads and to investigate a possible role of DMRT1 in the regulation of meiotic entry. The expression pattern of DMRT1, STRA8, SCP3, DMC1, NANOS3, CYP26B1 and NANOS2 was investigated by RT-PCR and immunohistochemistry in a series of human testis samples from fetal life to adulthood, and in fetal ovaries. DMRT1 was expressed in testes throughout development but with marked spatio-temporal changes. At the early fetal period of 8-20 gestational weeks (GW) and at infantile mini-puberty, DMRT1 was predominantly expressed in Sertoli cells, whereas at later stages of gestation (22-40 GW), during childhood and in post-pubertal testes, DMRT1 was most abundant in spermatogonia, except in the A-dark type. In fetal ovaries, DMRT1 was detected in oogonia and oocytes until 20 GW, but was completely down-regulated following meiotic entry. STRA8, SCP3 and DMC1 were expressed mainly in oocytes and spermatogonia in accordance with their role in initiation and progression of meiosis. The putative meiosis inhibitors, CYP26B1 and NANOS2, were primarily expressed in Leydig cells and spermatocytes, respectively. In conclusion, the expression pattern of the investigated meiotic regulators is largely conserved in the human gonads compared with rodents, but with some minor differences, such as a stable expression of CYP26B1 in human fetal ovaries. The sexually dimorphic expression pattern of DMRT1 indicates a similar role in the mitosis-meiosis switch in human gonads as previously demonstrated in mice. The biological importance of the changes in expression of DMRT1 in Sertoli cells remains to be established, but it is consistent with DMRT1 reinforcing the inhibition of meiosis in the testis.

Co-localization of DrPiwi-1 and DrPiwi-2 in the oogonial cytoplasm is essential for oocyte differentiation in sexualized planarians.

P-Element-induced wimpy testis (Piwi) subfamily proteins form complexes that bind to Piwi-interacting RNA. This interaction is crucial for stem cell regulation and formation, maintenance of germline stem cells, and gametogenesis in several metazoans. Planarians are effective models for studying stem cells. In the planarian Dugesia ryukyuensis, DrPiwi-1 is essential for the development of germ cells, but not somatic cells and sexual organs. DrPiwi-2 is indispensable for regeneration. In this study, we aimed to investigate the effects of Piwi on the differentiation of germ cells using monoclonal antibodies against DrPiwi-1 and DrPiwi-2. DrPiwi-1 and DrPiwi-2 co-localized more in immature germ cells than in mature germ cells in the ovary. DrPiwi-1 was found in the cytoplasm of early oogonia as undifferentiated germ cells, whereas DrPiwi-2 was found to localize not only in the nuclei but also in the cytoplasm of early oogonia. In descendant germ cells (oocytes), DrPiwi-2 was not present in the cytoplasm, but was strongly detected in the nucleolus. Moreover, we found that DrPiwi-1 forms a complex with DrPiwi-2. The cause of DrPiwi-1 depletion may be the severe reduction in the DrPiwi-2 level in the cytoplasm of oogonia. These results suggest that the formation of the DrPiwi-1 and DrPiwi-2 complex in the cytoplasm of oogonia is essential for oocyte differentiation. Our findings support the conclusion that DrPiwi-1 forms a complex with DrPiwi-2 in the cytoplasm of undifferentiated germ cells, and it signifies the start of gametogenesis. In contrast, in the testes, Drpiwi-1 was found in undifferentiated germ cells (spermatogonia), whereas DrPiwi-2 was found in descendant germ cells (spermatocytes). The process of germ cell differentiation from adult stem cells in planarians may be regulated in different ways in female and male germ lines by the Piwi family.

Translation and codon usage regulate Argonaute slicer activity to trigger small RNA biogenesis.

In the Caenorhabditis elegans germline, thousands of mRNAs are concomitantly expressed with antisense 22G-RNAs, which are loaded into the Argonaute CSR-1. Despite their essential functions for animal fertility and embryonic development, how CSR-1 22G-RNAs are produced remains unknown. Here, we show that CSR-1 slicer activity is primarily involved in triggering the synthesis of small RNAs on the coding sequences of germline mRNAs and post-transcriptionally regulates a fraction of targets. CSR-1-cleaved mRNAs prime the RNA-dependent RNA polymerase, EGO-1, to synthesize 22G-RNAs in phase with translating ribosomes, in contrast to other 22G-RNAs mostly synthesized in germ granules. Moreover, codon optimality and efficient translation antagonize CSR-1 slicing and 22G-RNAs biogenesis. We propose that codon usage differences encoded into mRNA sequences might be a conserved strategy in eukaryotes to regulate small RNA biogenesis and Argonaute targeting.