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PIWI family member piwil1, which associates with Piwi-interacting RNA (piRNA), is responsible in regulation of germ cell differentiation and maintenance of reproductive stem cells. In this study, we analyzed the piwil1 gene in Paralichthys olivaceus. Bioinformatics analysis and structure prediction showed that piwil1 had the conserved domains: PAZ domain and PIWI domain. Expression analysis during embryonic development implied that piwil1 gene was maternally inherited. The tissue distribution showed a sexually dimorphic gene expression pattern, with higher expression level in testis than ovary. In situ hybridization results demonstrated that piwil1 was predominantly distributed in oogonia, oocytes, sertoli cells and spermatocytes. A CpG island was predicted in the 5'-flanking region of piwil1 gene, and its methylation levels showed significant disparity between males and females, indicating that the sexually dimorphic expression of piwil1 gene might be regulated by methylation. Furthermore, we explored the distinct roles of human chorionic gonadotropin and 17α-methyltestosterone in regulating the expression of piwil1, and found that piwil1 was interacting with the HPG axis hormones. These results indicated that piwil1 might play a crucial role in gonadal development and gametogenesis in Paralichthys olivaceus.

Teleost sex differentiation largely depends on the number of undifferentiated germ cells. Here, we describe the generation and characterization of a novel transgenic zebrafish line, Tg(piwil1:egfp-UTRnanos3)ihb327Tg, which specifically labels the whole lifetime of germ cells, i.e., from primordial germ cells (PGCs) at shield stage to the oogonia and early stage of oocytes in the ovary and to the early stage of spermatogonia, spermatocyte, and spermatid in the testis. By using this transgenic line, we carefully observed the numbers of PGCs from early embryonic stage to juvenile stage and the differentiation process of ovary and testis. The numbers of PGCs became variable at as early as 1 day post-fertilization (dpf). Interestingly, the embryos with a high amount of PGCs mainly developed into females and the ones with a low amount of PGCs mainly developed into males. By using transient overexpression and transgenic induction of PGC-specific bucky ball (buc), we further proved that induction of abundant PGCs at embryonic stage promoted later ovary differentiation and female development. Taken together, we generate an ideal transgenic line Tg(piwil1:egfp-UTRnanos3)ihb327Tg which can visualize zebrafish germline for a lifetime, and we have utilized this line to study germ cell development and gonad differentiation of teleost and to demonstrate that the increase of PGC number at embryonic stage promotes female differentiation.

335–330 million-year-old cherts from the Massif Central, France, contain exceptionally well-preserved remains of an early forest ecosystem, including plants, fungi and other microorganisms. Here we reinvestigate the original material prepared by Renault and Roche from collections of the Muséum National d’Histoire Naturelle, Paris, and present a re-evaluation of Oochytrium lepidodendri (Renault 1894), originally described as a zoosporic fungus. Confocal laser scanning microscopy (CLSM) was used to study the microfossils, enabling us in software to digitally reconstruct them in three-dimensional detail. We reinterpret O. lepidodendri as a pseudofungus and favour placement within the oomycetes, a diverse clade of saprotrophs and both animal and plant parasites. Phylogenetically, O. lepidodendri appears to belong to a group of oomycetes distinct from those previously described from Paleozoic rocks and most likely related to the Peronosporales s.l. This study adds to our knowledge of Paleozoic eukaryotic diversity and reinforces the view that oomycetes were early and diverse constituents of terrestrial biotas, playing similar ecological roles to those they perform in modern ecosystems.

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.

STUDY QUESTION How are germ cell numbers and initiation of folliculogenesis affected in fetal Turner syndrome (TS) ovaries? SUMMARY ANSWER Germ cell development was severely affected already in early second trimester pregnancies, including accelerated oogonia loss and impaired initiation of primordial follicle formation in TS ovaries, while the phenotype in TS mosaic ovaries was less severe. WHAT IS KNOWN ALREADY Females with TS are characterized by premature ovarian insufficiency (POI). This phenotype is proposed to be a consequence of germ cell loss during development, but the timing and mechanisms behind this are not characterized in detail. Only few studies have evaluated germ cell development in fetal TS and TS mosaic ovaries, and with a sparse number of specimens included per study. STUDY DESIGN, SIZE, DURATION This study included a total of 102 formalin-fixed and paraffin-embedded fetal ovarian tissue specimens. Specimens included were from fetuses with 45,X (N = 42 aged gestational week (GW) 12–20, except one GW 40 sample), 45,X/46,XX (N = 7, aged GW 12–20), and from controls (N = 53, aged GW 12–42) from a biobank (ethics approval # H-2-2014-103). PARTICIPANTS/MATERIALS, SETTING, METHODS The number of OCT4 positive germ cells/mm2, follicles (primordial and primary)/mm2 and cPARP positive cells/mm2 were quantified in fetal ovarian tissue from TS, TS mosaic and controls following morphological and immunohistochemical analysis. MAIN RESULTS AND THE ROLE OF CHANCE After adjusting for gestational age, the number of OCT4+ oogonia was significantly higher in control ovaries (N = 53) versus 45,X ovaries (N = 40, P LIMITATIONS, REASONS FOR CAUTION The analysis of OCT4+ cells/mm2, cPARP+ cells/mm2 and follicles (primordial and primary)/mm2 should be considered semi-quantitative as it was not possible to use quantification by stereology. The heterogeneous distribution of follicles in the ovarian cortex warrants a cautious interpretation of the exact quantitative numbers reported. Moreover, only one 45,X specimen and no 45,X/46,XX specimens aged above GW 20 were available for this study, which unfortunately made it impossible to assess whether the ovarian folliculogenesis was delayed or absent in the TS and TS mosaic specimens. WIDER IMPLICATIONS OF THE FINDINGS This human study provides insights about the timing of accelerated fetal germ cell loss in TS. Knowledge about the biological mechanism of POI in girls with TS is clinically useful when counseling patients about expected ovarian function and fertility preservation strategies. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the International Center for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC). TRIAL REGISTRATION NUMBER N/A.

Meiosis initiation is a crucial step for the production of haploid gametes, which occurs from anterior to posterior in fetal ovaries. The asynchrony of the transition from mitosis to meiosis results in heterogeneity in the female germ cell populations, which limits the studies of meiosis initiation and progression at a higher resolution level. To dissect the process of meiosis initiation, we investigated the transcriptional profiles of 19 363 single germ cells collected from E12.5, E14.5, and E16.5 mouse fetal ovaries. Clustering analysis identified seven groups and defined dozens of corresponding transcription factors, providing a global view of cellular differentiation from primordial germ cells toward meiocytes. Furthermore, we explored the dynamics of gene expression within the developmental trajectory with special focus on the critical state of meiosis. We found that meiosis initiation occurs as early as E12.5 and the cluster of oogonia_4 is the critical state between mitosis and meiosis. Our data provide key insights into the transcriptome features of peri-meiotic female germ cells, which offers new information not only on meiosis initiation and progression but also on screening pathogenic mutations in meiosis-associated diseases.

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., Author summary In mammals, female fertility is limited by the number of germ cells, i.e., the oocytes, females are born with. These oocytes associate with somatic cells to produce follicles that guarantee the production of a fertilizable egg and the synthesis of sex hormones. During fetal development, oocytes initiate meiosis to halve their ploidy. As part of the meiotic program, oocytes self-induce hundreds of DSBs into their genome, which have to be repaired. However, oocytes are not very efficient at repairing DSBs. Also, during fetal development, a great part of the oocytes are eliminated, in a process that has been linked to the formation of follicles. It has been described that the effector kinase of the DDR (CHK2) is responsible for eliminating oocytes with persistent DSBs. Thus, we hypothesized that the DDR may be responsible for eliminating oocytes. In agreement with our hypothesis, we found that CHK2 was responsible for eliminating most fetal oocytes and regulating follicle formation in the mouse. Unexpectedly, we found that there is a CHK1-dependent perinatal oocyte death occurring in Chk2-/- mice that alters follicle formation. These data suggest that the CHK2- and CHK1-dependent DDR control the oocyte pool and follicle formation in mammals.

Reconstituting a human ovary Human pluripotent stem cells (hPSCs) have been induced into human primordial germ cell–like cells (hPGCLCs) in vitro, the first step toward human in vitro gametogenesis. Yamashiro et al. went a step closer to generating mature gametes by culturing hPSCs with mouse embryonic ovarian somatic cells in xenogeneic reconstituted ovaries (see the Perspective by Gill and Peters). Over a period of 4 months, hPGCLCs underwent hallmark epigenetic reprogramming and differentiated progressively into cells closely resembling human oogonia, an immediate embryonic precursor for human oocytes. This study creates opportunities for human germ cell research and provides a foundation for human in vitro gametogenesis. Science , this issue p. 356 ; see also p. 291

This contribution describes the growth of oocytes, addressing the formation of structures that compose the follicular complex, as well as the remodeling of the extracellular matrix, specifically laminin, fibronectin and type IV collagen during gonadal maturation. Thirty-seven females of the Acari zebra fish, Hypancistrus zebra were captured and the ovaries were submitted to histological processing for light and electron microscopy and immunohistochemistry techniques. Oogonia and four stages (I – IV) of oocytes were distinguished, and structures such as the postovulatory follicle and atretic oocytes (initial and advanced atresia) were observed. The follicular complex consists of the mature oocyte, zona radiata (Zr1, Zr2 and Zr3), follicular cells, basement membrane and theca. During oocyte growth, proteins of the extracellular matrix showed different intensities of staining. Based on these observations, a model of oocyte growth is proposed to define specific characteristics of the oocyte and the remodeling of the extracellular matrix in the ovary of H. zebra. This model of oocyte growth can be extended to other species of ornamental fishes. This study contributes data for induced fertilization and eventual conservation of this species.

Information on temporal variations in stock reproductive potential (SRP) is essential in fisheries management. Despite this relevance, fundamental understanding of egg production variability remains largely unclear due to difficulties in tracking the underlying complex fluctuations in early oocyte recruitment that determines fecundity. We applied advanced oocyte packing density theory to get in-depth, quantitative insights across oocyte stages and seasons, selecting the commercially valuable European hake (Merluccius merluccius) as a case study. Our work evidenced sophisticated seasonal oocyte recruitment dynamics and patterns, mostly driven by a low-cost predefinition of fecundity as a function of fish body size, likely influenced also by environmental cues. Fecundity seems to be defined at a much earlier stage of oocyte development than previously thought, implying a quasi determinate – rather than indeterminate – fecundity type in hake. These results imply a major change in the conceptual approach to reproductive strategies in teleosts. These findings not only question the current binary classification of fecundity as either determinate or indeterminate, but also suggest that current practices regarding potential fecundity estimation in fishes should be complemented with studies on primary oocyte dynamics. Accordingly, the methodology and approach adopted in this study may be profitably applied for unravelling some of the complexities associated with oocyte recruitment and thereby SRP variability Te authors thank Ignasi Vallès Casanova for providing temperature data, Dr. Tassya C. dos Santos Schmidt for graphical and statistical advice and S. Rábade, M. Fabeiro and P. R. Tamargo for their assistance in the sampling and histological work. Tis work was funded by the Spanish research grant DREAMER, Recruitment Dynamic of European Hake (CTM2015-66676-C2-1-R). A.S. would like to thank the Annenberg Foundation, CA, USA (Grant No. 16-249) and CSIC for supporting a research stay at IMR Bergen, and the University of Girona for the PhD funding program (BR-UdG 2015). Likewise, C.G.-F. would like to thank the Fundación Tatiana Pérez de Guzmán el Bueno for fnancial support towards her PhD

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.

Macrobrachium nipponense has the characteristics of fast ovarian development cycle, which leads to the coexistence of multiple generations, the reduction of commodity specifications and the low economic benefit. Therefore, the study on the mechanism of ovarian development is of great significance to the development of industry. Cyclin A (CycA)is a key gene regulating ovarian development in vertebrates, but little information was available for its function in crustaceans. In this study, the full-length cDNA of Mn-CycA was obtained from the ovary. The full-length cDNA (2033 bp) with an open reading frame of 1368 bp, encoded a 456-amino acid protein. qRT-PCR revealed tissue-specific expression pattern of Mn-CycA, with abundant expression in the ovary. Results in different developmental stages of ovary indicated that Mn-CycA expression is positively correlated with ovarian maturation. qRT-PCR In different developmental stages, the expression of Mn-CycA mRNA gradually increased during the embryonic stage and decreased significantly on the first day of the hatching stage. At the 25th day of the metamorphosis stage, the expression level of Mn-CycAmRNA in female shrimp was 3.5 times higher than that in male shrimp, which may be related to the proliferation of oogonia and the formation of oocytes. In situ hybridization (ISH) of ovary showed Mn-CycA was examined in all stages and was mainly located in oogonia and oocytes. Compared with the control group, the obvious change of gonad somatic index (GSI) proved that injection of Mn-CycA dsRNA could delay the ovarian development cycle, which provided strong evidence for the involvement of Mn-CycA in ovarian maturation and oogenesis, and expanded a new perspective for studying the fast ovarian development cycle in M. nipponense.

The study of gametogenesis is useful for phylogenetic analysis and can also provide insight into the physiology and biology of species. This report describes oogenesis in the Phoronis embryolabi, a newly described species, which has an unusual type of development, that is, a viviparity of larvae. Phoronid oogonia are described here for the first time. Yolk formation is autoheterosynthetic. Heterosynthesis occurs in the peripheral cytoplasm via fusion of endocytosic vesicles. Simultaneously, the yolk is formed autosynthetically by rough endoplasmic reticulum in the central cytoplasm. Each developing oocyte is surrounded by the follicle of vasoperitoneal cells, whose cytoplasm is filled with glycogen particles and various inclusions. Cytoplasmic bridges connect developing oocytes and vasoperitoneal cells. These bridges and the presence of the numerous glycogen particles in the vasoperitoneal cells suggest that nutrients are transported from the follicle to oocytes. Phoronis embryolabi is just the second phoronid species in which the ultrastructure of oogenesis has been studied, and I discuss the data obtained comparing them with those in Phoronopsis harmeri. Finally, I discuss the distribution of reproductive patterns across both, molecular and morphological phylogenetic trees in Phoronida proving that parental care has evolved independently several times in this phylum.

We analysed the ovarian dynamics of the anadromous semelparous allis shad Alosa alosa for which our working hypothesis was that mature pre-spawning females would have very low or even exhausted primary growth (PG) oocyte reserves; semelparity has been linked with the depletion of the pool of PG oocytes. To test this hypothesis, the PG oocytes were enumerated, their recruitment pattern to the secondary growth (SG) phase was analysed and their potential replenishment from the pool of oogonia was examined in females caught very close to the Mondego River mouth, in central Portugal and along the river. The development of the SG oocytes was also analysed, the fecundity (batch, total and annual) values were estimated and the intensity of atresia was quantified. Ovarian samples and histological sections were investigated in parallel. A dynamic recruitment pattern of PG oocytes to the SG phase was revealed, where all PG oocytes were recruited and were not replenished by oogonia. Annual fecundity was subject to down-regulation due to atresia prior to spawning and its size was multiple times higher than the size of batch fecundity. Lack of population synchronicity in ovarian development and spawning migration was also observed. This multifaceted analysis of the ovarian dynamics of this species will contribute to management efforts for this critically endangered and economically important fish throughout its geographical distribution. The results reported in this study will also assist in unravelling the complexity of the early processes of oogenesis in fish.

The aim of the present article was to investigate the oogenic cycle of Mytilus galloprovincialis sampled in the Bay of Naples, and to immunolocalize 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD), and P450 aromatase, enzymes involved in the synthesis of two sex hormones: testosterone and 17β-estradiol. We demonstrate that the oogenic cycle starts in late summer-early fall and continues in early winter when the first event of spawning occurs; other spawning events take place until June, when the ovary is spent and contains a few empty ovarian follicles and numerous somatic cells, that is, adipogranular cells and vesicular connective tissue cells. During the oogenic cycle, apoptotic events occur at the level of oogonia, previtellogenic oocytes, as well as follicle cells; by contrast, necrosis events probably take place in vitellogenic oocytes, which, once degenerated, transfer their content to healthy oocytes. Finally, the present data demonstrate that 3β-HSD, 17β-HSD, and P450 aromatase are present in the ovary both during the reproductive and nonreproductive phases. The possible role of these enzymes during the Mytilus galloprovincialis reproductive cycle is discussed. Anat Rec, 302:1039-1049, 2019. © 2019 Wiley Periodicals, Inc.

The objective of this study was to understand germ cell renewal and differentiation in the Yesso scallop Mizuhopecten yessoensis throughout its reproductive cycle. First, we identified a Yesso scallop vasa-like gene (my-vlg) as a useful marker for immature germ cells. Both in situ hybridization and immunohistochemistry confirmed the germ-cell-specific expression of my-vlg and protein, especially in spermatogonia, oogonia, and primary oocytes. An immunohistochemistry with anti my-Vasa antibody further revealed that immature germ cells (i.e., gonial cells) are located on the basal membrane of germinal acini and are present during all maturational stages in both the ovary and testis. Quantitative analysis revealed a shift in germ cell number and change in germ cell composition associated with the gonadal maturation process, indicating a persistence of my-Vasa-positive immature germ cells throughout the reproductive cycle. In the testis, a gradual increase in my-Vasa-positive immature germ cells was seen from November to March. In the ovary, an increase in my-Vasa-positive immature germ cells was observed from September until November. We also used gonad index (GI) to summarize and evaluate the maturational changes of scallops in the 2018 generation. This generation had a background of relatively high mortality and shell deformity in the previous year, and we noted a small irregular peak of GI in the summer (July–August). We confirmed that the germ cells proliferating in the summer were my-Vasa-positive, implying that germ cell developmental progression occurred in the 2018 generation during the non-reproductive period. In conclusion, this study establishes an immuno-identification method for my-Vasa-positive immature germ cells and reveals germ cell composition in a bivalve species throughout its reproductive cycle. These results will support monitoring techniques for gonad development and the reproductive status of brood stock in shellfish seed production hatcheries.

In many animals, primordial germ cells (PGCs) migrate into developing gonads. There, they proliferate and differentiate into female and male germ stem cells (GSCs), oogonia and spermatogonia, respectively. Few studies have focused on the molecular mechanisms underlying the development of GSC sex determination. Here, we investigated the expression of the transcription factor Dmrt1 and a phosphorylated form of the histone variant H2AX (γH2AX) during gonadal development in Xenopus laevis. During early sexual differentiation, Dmrt1 was expressed in the GSCs of the ZW (female) and ZZ (male) gonads as well as somatic cells of the ZZ gonads. Notably, the PGCs and primary GSCs contained large, unstructured nuclei, whereas condensed, rounder nuclei appeared only in primary oogonia during tadpole development. After metamorphosis, Dmrt1 showed its expression in secondary spermatogonia, but not in secondary oogonia. Like Dmrt1, γH2AX was expressed in the nuclei of primary GSCs in early developing gonads. However, after metamorphosis, γH2AX expression continued in primary and secondary spermatogonia, but was barely detected in the condensed nuclei of primary oogonia. Taken together, these observations indicate that spermatogonia tend to retain PGC characteristics, compared to oogonia, which undergo substantial changes during gonadal differentiation in X. laevis. Our findings suggest that Dmrt1 and γH2AX may contribute to the maintenance of stem cell identity by controlling gene expression and epigenetic changes, respectively.

Made available in DSpace on 2020-12-12T02:14:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-10-01 The ovarian development of Callinectes ornatus and Arenaeus cribrarius was described using histochemistry and ultrastructure. Both species shows the same ovarian stages, which are the juvenile (JUV), adult rudimentary (RUD), developing (DEV), intermediary (INT), mature (MAT), and spent (OV) stages. The JUV and RUD stages showed similar characteristics, and previtellogenesis is characterized by meiotic prophase chromosomes. In the primary vitellogenesis, the oocyte cytoplasm shows many small and large cytoplasmic glycoprotein vesicles. These vesicles correspond to the dilated cisternae of the rough endoplasmic reticulum (RER), which produces the immature (endogenous) yolk. Secondary vitellogenesis (exogenous phase) begins at the DEV stage with the fusion of pinocytic vesicles and vesicles with immature yolks to form mature yolk granules. At the INT stage, the formation of the chorion begins, and the mature yolks increase in size and number, while the RER diminishes. In the MAT stage, the oocytes are completely formed, and the cytoplasm is filled with mature yolk, lipid droplets, and glycogen. There are no significant variations between the gonadosomatic and hepatosomatic indices, which allows us to infer that the transfer of reserves from the hepatopancreas is nearly constant during ovarian development, since we observed primiparous and multiparous females in the same sampled population. Universidade Estadual Paulista (UNESP) Faculdade de Ciências Agrárias e Veterinárias (FCAV) Departamento de Biologia Aplicada à Agropecuária Laboratório de Morfologia de Invertebrados (LMI), Via de Acesso Prof. Paulo Donato Castellane, s/n Universidade Estadual Paulista (UNESP) Instituto de Biociências de Rio Claro, Rio Claro Laboratório de Bioecologia e Sistemática de Crustáceos (LBSC) Departamento de Biologia Faculdade de Filosofia Ciências e Letras de Ribeirão Preto (FFCLRP) Universidade de São Paulo (USP), Av. Bandeirantes, 3900, Ribeirão Preto Universidade Estadual Paulista (UNESP) Faculdade de Ciências Agrárias e Veterinárias (FCAV) Departamento de Biologia Aplicada à Agropecuária Laboratório de Morfologia de Invertebrados (LMI), Via de Acesso Prof. Paulo Donato Castellane, s/n Universidade Estadual Paulista (UNESP) Instituto de Biociências de Rio Claro, Rio Claro

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., Author summary Faithful chromosome segregation during meiosis, the specialized cell division program that produces haploid gametes (i.e. eggs and sperm) from a diploid organism, is key for successful sexual reproduction. Diethylhexyl phthalate (DEHP), a commonly used plasticizer found in personal care and household products, has emerged as an endocrine disruptor that exerts reprotoxicity in mammals. In this study, we provide mechanistic insight into the modes of action by which environmentally-relevant levels of DEHP and its metabolites impair female meiosis in the C. elegans germline. Exposure to DEHP leads to defects in late prophase I chromosome remodeling, altered chromosome morphology in oocytes at diakinesis, errors in chromosome segregation, and impaired embryogenesis. Underlying these defects are higher levels of DSBs, altered DSB repair, defects in crossover (CO) designation/formation, germline-specific change in prmt-5 gene expression and altered chromosome structure. We propose that DEHP exposure induces an excess number of DSBs by interfering with mechanisms set in place to turn off DSBs once CO designation is accomplished and by altering chromosome structure resulting in increased chromatin accessibility to the DSB machinery.

Identification of germ cell markers is important for investigating reproduction biology in fish. Vasa is one of the most studied germ cell markers in mammals and lower vertebrates including fish. Here, we characterized a vasa homologue from the fish marbled goby (Oxyeleotris marmorata), termed omvasa. The full length of omvasa cDNA is 2344 bp and encodes 658 amino acids, sharing high identities with Vasa homologues of other vertebrates. OmVasa protein contains 15 RG/RGG repeats at N-terminus, 2 ATPase motifs, as well as RNA unwinding and RNA binding domains at C-terminus. Phylogenetic tree showed that omVasa had the closest relationship with the Vasa homologue from the fish Boleophthalmus pectinirostris, the great blue-spotted mudskipper. qRT-PCR analysis indicated that omvasa was specifically transcribed in gonads, and the transcription level was gradually increased during oocyte development. The germ cell-specific distribution of omvasa mRNA was revealed by fluorescent in situ hybridization. In ovary, the signal of omvasa RNA displayed strong-weak-strong dynamics from oogonia over pre-vitellogenic oocytes to vitellogenic oocytes. In testis, omvasa signal was strong in spermatogonia, modest in spermatocytes but undetectable in spermatids and somatic cells. During embryogenesis, the transcription of omvasa mRNA was high at blastula stage, gradually decreased from gastrula stage and maintained at a low level in later developmental stages. Whole mount in situ hybridization indicated that omvasa mRNA was specific to primordial germ cells (PGCs). In summary, marbled goby vasa is a germ cell-specific transcript during gametogenesis, and can be used as an ideal marker for tracing PGC formation and migration, which is pivotal to germ cell manipulation in this species.

International audience; Oocytes, including from mammals, lack centrioles, but neither the mechanism by which mature eggs lose their centrioles nor the exact stage at which centrioles are destroyed during oogenesis is known. To answer questions raised by centriole disappearance during oogenesis, using a transgenic mouse expressing GFP-centrin-2 (GFP CETN2), we traced their presence from e11.5 primordial germ cells (PGCs) through oogenesis and their ultimate dissolution in mature oocytes. We show tightly coupled CETN2 doublets in PGCs, oogonia, and pre-pubertal oocytes. Beginning with follicular recruitment of incompetent germinal vesicle (GV) oocytes, through full oocyte maturation, the CETN2 doublets separate within the pericentriolar material (PCM) and a rise in single CETN2 pairs is identified, mostly at meiotic metaphase-I and-II spindle poles. Partial CETN2 foci dissolution occurs even as other centriole markers, like Cep135, a protein necessary for centriole duplication, are maintained at the PCM. Furthermore, live imaging demonstrates that the link between the two centrioles breaks as meiosis resumes and that centriole association with the PCM is progressively lost. Microtubule inhibition shows that centriole dissolution is uncoupled from microtubule dynamics. Thus, centriole doublets, present in early G2-arrested meiotic prophase oocytes, begin partial reduction during follicular recruitment and meiotic resumption, later than previously thought. Centrioles, found at the poles of mitotic spindles, are vital for reproduction and development. Long thought to be contributed by the sperm during fertilization and lost during fetal oogenesis, they are essential in innumerable processes 1. Indeed, centriole defects appear as the root causes of a broad set of diseases, ranging from blindness and cancers through microcephaly and ciliopathies 2,3. Centrioles are often surrounded by the pericentriolar material (PCM), and together, the two structures define the canonical centrosome, the cell's major microtubule organizing center (MTOC) 3. In most mammals, haploid female gametes produced during oogenesis lose their centrosomes, although the mechanism of when and how remains elusive 4-6. Most studies on centrosome reduction in gametes involve ultrastructural observations 4,7,8. In humans, centrioles have been detected in fetal oogonia at 13-15 weeks post-gestation and within early growing oocytes 9. However, centrioles have not been found in fully grown ger-minal vesicle (GV)-stage oocytes, and the metaphase-I and-II spindles formed after meiotic resumption are anastral, barrel-shaped structures with spindle poles devoid of centrioles or PCM 8. In mice, ultrastructural and marker tracing have identified intact centriole pairs in fetal oogonia and early post-natal stage (P4) mouse pri-mordial oocytes 10-12. In later, preovulatory stages, growing mouse oocytes apparently lose centrioles 13 while maintaining dispersed acentriolar PCM throughout the cytoplasm. As the oocyte reaches maturity and competency to enter meiosis, a perinuclear MTOC, composed of PCM constituents such as γ-tubulin and pericentrin, gradually enlarges near the GV nucleus 14-16. Upon meiotic resumption, the acentriolar PCM fragments along the GV nucleus, mediated by PLK1, which releases the centri-ole adhesion protein cNAP1 (centrosomal Nek2-associated protein-1) 17,18 and then is stretched and fragmented by BicD2-anchored dynein in a microtubule-dependent manner 18. Finally, KIF11 mediates further MTOC

International audience; The extracellular matrix plays an essential role for stem cell differentiation and niche homeostasis. Yet, the origin and mechanism of assembly of the stem cell niche microenvironment remain poorly characterized. Here, we uncover an association between the niche and blood cells, leading to the formation of the Drosophila ovarian germline stem cell niche basement membrane. We identify a distinct pool of plasmatocytes tightly associated with the developing ovaries from larval stages onward. Expressing tagged collagen IV tissue specifically, we show that the germline stem cell niche basement membrane is produced by these "companion plasmatocytes'' in the larval gonad and persists throughout adulthood, including the reproductive period. Eliminating companion plasmatocytes or specifically blocking their collagen IV expression during larval stages results in abnormal adult niches with excess stem cells, a phenotype due to aberrant BMP signaling. Thus, local interactions between the niche and blood cells during gonad development are essential for adult germline stem cell niche microenvironment assembly and homeostasis.

One of the biggest mysteries of ovarian physiology is what controls the emergence of adult primordial follicles from the resting stage, and their steady depletion over the woman's lifetime. A related mystery is why do early oogonia begin meiosis in the fetus and then suddenly arrest for most of fetal and adult life. If fetal oocyte arrest did not occur after meiotic activation, there would be no oocytes left in the female baby by the time she is born. Similarly, without a steady controlled release in the adult ovary of resting follicles, the adult woman would run out of her eggs prematurely and have an early menopause. Could there be a similarity between what causes fetal oocyte arrest and what causes adult oocyte recruitment? The answer begins with the observation of a sudden massive recruitment of primordial follicles after human ovarian transplantation, and the embryologic discoveries about oocyte activation and the time of differentiation of cortex and medulla. The unifying theory is that ovarian cortical tissue pressure controls both fetal oocyte arrest and adult oocyte recruitment.

SummaryWe aimed to analyze the oogenesis of adult females of the cichlid fish Laetacara araguaiae. The specimens’ gonads were removed and processed for light and transmission electron microscopy. Oogenesis in L. araguaiae showed the following characteristics: a germinal epithelium with three types of oogonia (A-undifferentiated, A-differentiated and B-oogonia), oocytes at meiotic prophase stage and ovarian follicle formation. Oocytes showing primary growth with pre-vitellogenic and cortical alveolus were observed. Similar to data for other cichlids, oocytes in secondary growth or vitellogenesis were characterized by the initial deposition of yolk microgranules. The event that characterizes the maturation stage is nucleolus migration, also called the germinal vesicle, to the oocyte periphery in the direction of the micropyle. The follicular complex undergoes several changes throughout the oocyte stages. To the best of our knowledge this study is the first to describe L. araguaiae oogenesis. Moreover, this study is the first step to better understand the reproductive biology of this species, which shows great potential for use as an ornamental fish.

What are the effects of experimentally manipulating meiosis signalling by addition of retinoic acid (RA) in cultured human fetal gonads?RA-treatment accelerated meiotic entry in cultured fetal ovary samples, while addition of RA resulted in a dysgenetic gonadal phenotype in fetal testis cultures.One of the first manifestations of sex differentiation is the initiation of meiosis in fetal ovaries. In contrast, meiotic entry is actively prevented in the fetal testis at this developmental time-point. It has previously been shown that RA-treatment mediates initiation of meiosis in human fetal ovary ex vivo.This was a controlled ex vivo study of human fetal gonads treated with RA in 'hanging-drop' tissue cultures. The applied experimental set-up preserves germ cell-somatic niche interactions and the investigated outcomes included tissue integrity and morphology, cell proliferation and survival and the expression of markers of meiosis and sex differentiation.Tissue from 24 first trimester human fetuses was included in this study, all from elective terminations at gestational week (GW) 7-12. Gonads were cultured for 2 weeks with and without addition of 1 µM RA. Samples were subsequently formalin-fixed and investigated by immunohistochemistry and cell counting. Proteins investigated and quantified included; octamer-binding transcription factor 4 (OCT4), transcription factor AP-2 gamma (AP2γ) (embryonic germ cell markers), SRY (sex determining region Y)-box 9 (SOX9), anti-Müllerian hormone (AMH) (immature Sertoli cell markers), COUP transcription factor 2 (COUP-TFII) (marker of interstitial cells), forkhead box L2 (FOXL2) (granulosa cell marker), H2A histone family, member X (γH2AX) (meiosis marker), doublesex and mab-3 related transcription factor 1 (DMRT1) (meiosis regulator), cleaved poly ADP ribose polymerase (PARP), cleaved Caspase 3 (apoptosis markers) and Ki-67 antigen (Ki-67) (proliferation marker). Also, proliferation was determined using a 5'-bromo-2'-deoxyuridine (BrdU) incorporation assay.A novel ex vivo 'hanging-drop' culture model for human fetal gonads was successfully established. Continued proliferation of cells without signs of increased apoptosis was observed after 2 weeks of culture. In cultured fetal ovaries treated with RA, an increased number of meiotic germ cells (P0.05) and DMRT1-positive oogonia initiating meiosis (P0.05) was observed, which is in agreement with a previous study. In fetal testes, RA-treatment resulted in a decreased number of gonocytes (P0.05), a reduced percentage of proliferating gonocytes (P0.05), altered expression pattern of the somatic cell markers AMH and COUP-TFII, as well as disrupted seminiferous cord structure and testis morphology.The number of samples included in this study was relatively small due to the limited availability of human fetal tissue.The hanging-drop culture, similarly to other organ culture approaches, allows studies of germ cell-somatic niche interactions and determination of effects after manipulating specific signalling pathways. Our novel finding of disrupted fetal testis development after treatment with RA indicates that abnormal meiosis regulation can potentially cause gonadal dysgenesis. Further studies will elucidate the exact mechanisms and timing of observed effects.This work was supported in part by an ESPE Research Fellowship, sponsored by Novo Nordisk A/S to A.Jø. Additional funding for this project was obtained from The Research Council of the Capital Region of Denmark (E.R.-D.M.), The Research Fund at Rigshospitalet (A.Ju. and J.E.N.), Familien Erichssens Fund (A.Jø.), Dagmar Marshalls Fund (A.Jø.) and AaseEjnar Danielsens Fund (A.Jø.). The authors have no conflicts of interest.

Cytological responses in different organs of sentinel organisms have proven to be useful tools for characterizing the health status of those organisms and assessing the impact of environmental contaminants. Our study shows that nickel (II) accumulated in both germ cells (oogonia and developing oocytes) and somatic cells (muscle cells, follicle cells) in the Astacus leptodactylus ovary. Muscle cells from ovarian wall show disorganization and the disruption of cytoplasmic microtubules and pyknosis of the cell nucleus. Follicle cells, both those that surround the developing oocytes and also those that are not associated with the oocytes contained within the cytoplasm vacuoles of different sizes, degenerated mitochondria, myelin bodies, disorganized microtubules, and pyknotic nuclei. The most evident pathological phenomenon was the alteration and disorganization of the basal matrix, which separates the ovarian interstitium from ovarian follicles compartment. Exposure to nickel induces cytoplasmic vacuolation in oogonia and developing oocytes, structural alteration of the developing yolk granules and condensation of the nucleoli. Ultrastructural autometallography has shown grains of silver-enhanced nickel inside the cytoplasm of the muscle cells with altered morphology, including the cytoplasm, nucleus, and basal matrix of the follicle cells, and in intracisternal granules and developing yolk granules of the oocytes.

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.

Oogonia are characterized by diploidy and mitotic proliferation. Human and mouse oogonia express several factors such as OCT4, which are characteristic of pluripotent cells. In human, almost all oogonia enter meiosis between weeks 9 and 22 of prenatal development or undergo mitotic arrest and subsequent elimination from the ovary. As a consequence, neonatal human ovaries generally lack oogonia. The same was found in neonatal ovaries of the rhesus monkey, a representative of the old world monkeys (Catarrhini). By contrast, proliferating oogonia were found in adult prosimians (now called Strepsirrhini), which is a group of ‘lower’ primates. The common marmoset monkey (Callithrix jacchus) belongs to the new world monkeys (Platyrrhini) and is increasingly used in reproductive biology and stem cell research. However, ovarian development in the marmoset monkey has not been widely investigated. Herein, we show that the neonatal marmoset ovary has an extremely immature histological appearance compared with the human ovary. It contains numerous oogonia expressing the pluripotency factors OCT4A, SALL4, and LIN28A (LIN28). The pluripotency factor-positive germ cells also express the proliferation marker MKI67 (Ki-67), which has previously been shown in the human ovary to be restricted to premeiotic germ cells. Together, the data demonstrate the primitiveness of the neonatal marmoset ovary compared with human. This study may introduce the marmoset monkey as a non-human primate model to experimentally study the aspects of primate primitive gonad development, follicle assembly, and germ cell biologyin vivo.

Oogenesis involves a sequence of cellular divisions and developmental changes leading to the formation of oocytes, whose role in development is to transfer genomic information to the next generation. During this process, the gene expression pattern changes considerably concomitant with genome remodeling, while genomic information is maintained. The development of the gonad in zebrafish is unique in that it goes through an initial ovarian phase and subsequently into either ovarian or testicular phases. How the germ cells choose to commit to an oogenic fate and enter meiosis or alternatively not to enter meiosis and commit to a spermatogenetic fate remains a key question in development. Lack of suitable markers has hampered the understanding of the principles controlling sex differentiation in zebrafish. The current study was aimed at finding substantive cytochemical markers to identify specific oocyte stages primarily focusing on the DNA and RNA component of cells, using fluorescent dyes: acridine orange and propidium iodide. The pattern of synthesis and appearance of nucleoli was stage specific and may be used to identify stages of oogenesis. A distinguishing and possibly diagnostic feature of the staining pattern observed was the low level of chromatin staining compared to other cellular structures. This may be related to the more diffuse state of chromatin that occurs prior to thickening of chromosomes from the pachytene stage onwards. Although the fluorescent dyes may be useful in determining the localization of nucleic acids in tissue sections, it was not possible to quantify the relative contribution of the DNA and RNA components of specific stages of oocyte growth.

Bucrates lanista, the most southerly distributed species in the genus Bucrates Burmeister, was originally described from Brazil based on a female collected in the state of Rio Grande do Sul, but the species has not been recorded since 1918. In this work, we report that B. lanista inhabits the Pantanal Wetland in the state of Mato Grosso do Sul and, for the first time, describe the male. Individuals of B. lanista are gregarious and present a brown/green color dimorphism; this behavior and color variation are also observed in species of closely related genera. Individuals from the Pantanal vary slightly from those of Rio Grande do Sul. The karyotype was determined to be 2n♂ = 21 = 20 + X0 and 2n♀ = 22 = 20 + XX. The X chromosome is metacentric and the largest of the complement, and all of the autosomes are submetacentrics. All chromosomes solely present telomeric (TTAGG)n repeats at their ends, and some chromosomes present positive and negative DAPI bands.

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.

Context:The 21-hydroxylase deficiency is the most common cause of congenital adrenal hyperplasia. Pregnant women presenting a risk of genetic transmission may be treated with synthetic glucocorticoids such as dexamethasone (DEX) to prevent female fetus virilization.Objective:The aim of this study was to assess the potential deleterious effects of DEX exposure on fetal ovarian development.Settings:Human fetal ovaries, ranging from 8–11 weeks after fertilization, were harvested from material available after legally induced abortions. They were cultured in the absence or presence of DEX (2, 10, or 50 μm) over 14 d, and histological analyses were performed.Results:The glucocorticoid receptor NR3C1 was present and the signaling pathway active in the fetal ovary as demonstrated by the expression of NR3C1 target genes, such as PLZF and FKBP5, in response to DEX exposure. DEX decreased germ cell density at the 10 and 50 μm doses. Exposure to DEX, even at the highest dose, did not change oogonial proliferation as monitored by 5-bromo-2′-deoxyuridine incorporation and significantly increased the apoptotic rate, detected with cleaved caspase 3 staining. Interestingly, the expression of the prosurvival gene KIT was significantly decreased in the presence of DEX during the course of the culture.Conclusion:We have demonstrated for the first time that in vitro exposure to high doses of DEX impairs human fetal oogenesis through an increase in apoptosis. These data are of high importance, and additional epidemiological studies are required to investigate the female fertility of those women who have been exposed to DEX during fetal life.

Distinct types of oogonia are found in the germinal epithelium that borders the ovarian lamellae of Pimelodus maculatus: A-undifferentiated, A-differentiated and B-oogonia. This is similar to the situation observed for spermatogonia in the vertebrate testis. The single A-undifferentiated oogonia divide by mitosis giving rise to A-groups of single differentiated oogonia, each enclosed by epithelial cells that are prefollicle cells. Subsequently, the single A-differentiated oogonia proliferate to generate B-oogonia that are interconnected by cytoplasmic bridges, hence, forming germline cysts. The prefollicle cells associated with them also divide. Within the germline cysts, B-oogonia enter meiosis becoming oocytes. Meiotic prophase and early folliculogenesis occur within the germline cysts. During folliculogenesis, prefollicle cells grow between the oocytes, encompassing and individualizing each of them. The intercellular bridges disappear, and the germline cysts are broken down. Next, a basement membrane begins to form around the nascent follicle, separating an oocyte and its associated prefollicle cells from the cell nest. Folliculogenesis is completed when the oocyte and the now follicle cells are totally encompassed by a basement membrane. Cells derived from the ovarian stroma encompass the newly-formed ovarian follicle, and become the theca, thereby completing the formation of the follicle complex. Follicle complexes remain attached to the germinal epithelium as they share a portion of basement membrane. This attachment site is where the oocyte is released during ovulation. The postovulatory follicle complex is continuous with the germinal epithelium as both are supported by a continuous basement membrane. The findings in P. maculatus reinforce the hypothesis that ovarian follicle formation represents a conserved process throughout vertebrate evolution. J. Morphol. 2011. © 2011 Wiley-Liss, Inc.

To understand the mechanism of premature ovarian failure (POF). The ultrastructural (electron microscopy) analysis of primordial ovarian follicles in Nobox deficient mice. We studied, for the first time, the fate of oogonia in embryonic (prenatal) mouse ovaries and showed that the abolishment of the transition from germ cell cysts to primordial follicles in the ovaries of Nobox deficient mice is caused by defects in germ cell cyst breakdown, leading to the formation of syncytial follicles instead of primordial follicles. These results indicate that POF syndrome in Nobox deficient mice results from the faulty signaling between somatic and germ line components during embryonic development. In addition, the extremely unusual and abnormal presence of adherens junctions between unseparated oocytes within syncytial follicles indicates that faulty communication between somatic and germ cells is involved in, or leads to, abnormalities in the cell adhesion program.

International audience; The production of fertilizable ova is the consequence of multiple events that start as soon as ovarian development and culminate at the time of ovulation. Throughout their development, germ cells are associated with companion somatic cells, which ensure germ cell survival, growth and maturation. Data obtained in vitro and in vivo on several animal models of germ cell depletion have led to uncover the many roles of germ cells on both ovarian development and folliculogenesis. During ovarian development, germ cells become progressively enclosed within epithelial structures called "ovigerous cords" constituted by pregranulosa cells, lined by a basement membrane. At the end of ovarian development, ovigerous cords fragment into primordial follicles, which are epithelial units constituted by an oocyte surrounded by a single layer of granulosa cells. Germ cells are necessary for the fragmentation of ovigerous cords into follicles, since in their absence, no follicle will form. Germ cells also ensure the differentiation of the ovarian somatic lineage, and they may inhibit the testis-differentiating pathway by preventing the conversion of pregranulosa cells into Sertoli cells, their counterpart in the testis. Regularly, primordial follicles are recruited into the growing follicle pool and initiate their growth. They develop through primary, preantral, antral and preovulatory stages before being ovulated. Interestingly, the action of the oocyte on companion somatic cells tightly depends on the follicular stage. In primordial follicles, the oocyte prevents the transdifferentiation of granulosa cells into cells resembling Sertoli cells. By contrast, as soon as the follicle enters growth, the oocyte regulates the functional differentiation of granulosa cells and at the latest stages, it prevents their premature maturation into luteal cells. Overall, these data demonstrate that the female germ cell act on companion somatic cells to regulate ovarian development and folliculogenesis, thereby actively supporting its own maturation.; Le stock de follicules primordiaux, chacun formé d’un ovocyte entouré par une assise de cellules somatiques folliculaires ou cellules de la granulosa, est définitivement constitué pendant la vie fœtale ou très peu de temps après la naissance chez la majorité des mammifères. Dès la constitution de cette réserve et jusqu'à son épuisement chez la femelle âgée, l’ovaire est le siège de la folliculogenèse qui est l’ensemble des processus de croissance et de maturation des follicules ovariens depuis le stade de follicule primordial jusqu’au stade préovulatoire. Sa finalité biologique est la production, lors de chaque cycle menstruel ou estrien, d’ovocytes aptes à la fécondation et au développement. L’obtention de tels ovocytes implique des régulations paracrines et endocrines complexes. Au cours de ces dernières décennies, de nombreuses données ont clairement établi l’importance des cellules somatiques ovariennes dans la survie, la croissance et la maturation ovocytaires. Mais ce n’est que plus récemment que des travaux ont mis en lumière le rôle clé joué par la cellule germinale dans la folliculogenèse et dans la différenciation ovarienne. Cette revue présente une synthèse des connaissances, basées principalement sur l’interprétation de travaux sur différents modèles animaux, ayant permis de reconsidérer le rôle des cellules germinales dans la différenciation et la fonction de l’ovaire.

Ovarian reserve is determined by the number of primordial follicles in the ovary. Quiescent primordial follicles are activated for growth and pass through stages of development before they reach the antral stage. Then a cohort of antral follicles is recruited for further growth, dominance and ovulation under the cyclic stimulation of gonadotrophins. What triggers the initiation of growth in primordial follicles has remained a mystery for decades. However, recent studies on mutant mouse models have shown that primordial follicles are maintained in a dormant state by the actions of various inhibitory molecules to preserve the follicle pool, such as the transcription factor Foxo3a, PTEN (phosphotase and tensin homolog deleted on chromosome 10) and Tsc-1 (tumour suppressor tuberous schlerosis complex). Mice with deletions of these oocyte-specific genes exhibit premature activation of dormant primordial follicles, and all primordial follicles become depleted in early adulthood, causing premature ovarian failure. Other oocyte and somatic cell-derived growth factors are also involved in the early, gonadotrophin-independent phase of follicle growth via autocrine and paracrine interactions. Interestingly, some of these factors also play critical roles at later stages of follicle growth, such as the process of selecting the dominant follicle, by modifying the response of the follicles to gonadotrophins and inhibiting premature luteinization. Therefore, a thorough understanding of the molecular aspects of folliculogenesis is of paramount importance in the context of translational medicine and future clinical applications in human reproduction.

This review focuses on those mouse mutations that cause an effect on the morphology, viability, and/or behavior of primordial germ cells (PGCs) and gonocytes at specific steps of their fetal development up to the start of spermatogenesis, a few days after birth. To restrict the area covered, mice with mutations that cause abnormal hormone levels or mutations of genes not expressed in germ cells that secondarily cause spermatogenic problems are not discussed. To make our literature search as comprehensive as possible, Pubmed was searched for "(primordial germ cells OR prospermatogonia OR prespermatogonia OR gonocytes OR spermatogonia ormeiosis or spermiogenesis or spermatogenesis) AND mouse AND (knockout or mutant or transgenic)." This search started at 2003 as mutants created earlier were already retrieved for a previous review. The resulting citations were then further selected for complete or partial arrests at the level of PGCs and/or gonocytes. Fifty-nine protein coding genes and two miRNA coding genes were found that arrest the development of PGCs and gonocytes at specific steps providing a better insight into the regulation of the development of these cells. As to be expected, often problems in fetal germ cell development have an effect on the fertility of the mice at adulthood. Summary Sentence: Many gene mutations cause an arrest at specific developmental steps of mouse primordial germ cells and gonocytes, also called prospermatogonia providing valuable clues for the regulation of their development.

In this work we studied the structures of 51 foetal and 14 adult ovaries obtained from slaughtered Kano brown does in Nsukka abattoir. The ages of the adult does were determined by dentition and foetuses by crown rump length method. The foetal and adult ovaries were divided into five different groups using specific age intervals as Gestation day (GD) 50-65, 66-95, 96-125 and 126-145 and adults. For histological studies the ovaries were fixed, processed and routinely stained with H&E. The ovarian follicles were classified into 5 types according to granulosa cell layers surrounding the oocytes. The number of ovarian follicles per microscopic field, number of granulosa cells surrounding type 1 and 1A follicles and diameter of the ovarian follicles were determined for each group at 400x magnification. Grossly the foetal ovaries were like pin head, oval in shape, uniformly smooth and creamy in colour. The adult ovaries had follicles with different sizes. The adult mean ovarian weights were significantly higher (P

The primordial follicles present in neonatal ovary represent the fecundity of a female throughout her reproductive life. Germ cell meiosis and apoptosis are two important events during primordial folliculogenesis. In this study, through focusing on the cytochrome P450 lanosterol 14 alphademethylase (CYP51) and its lanosterol metabolic product(s), we explored the possible regulatory mechanism of the initiation of germ cell meiosis and primordial follicle formation. The expression of CYP51 could be detected in both oocytes and granulosa cells during primordial folliculogenesis by immunochemistry. RS21745, which leads to the reduction of lanosterol metabolic product(s) level, inhibited the primordial follicle formation in a dose-dependent manner, and thus postpone the establishment of the primordial follicle pool when the mouse fetal ovaries were cultured in serum-free medium. In contrast, the number of primordial follicle increased significantly with the accumulation of the lanosterol metabolic products caused by 0.025, 0.0625, and 0.125 µM AY9944-A-7 supplements. AY9944-A-7 also up-regulated the expression of meiotic diplotene stage marker gene msy2 and primordial follicle formation regulatory gene fig-alpha. Furthermore, AY9944-A-7 decreased the expression of apoptosis gene bax and significantly prevented oocyte apoptosis from 15.37 ± 1.97% to 3.68 ± 0.27% (P

SummaryRecent interest in the initial phases of ovarian follicular formation and development has lead to a number of publications in this area, most of which address the autocrine and paracrine factors involved in primordial follicle activation to primary follicle. Primordial follicle assembly (first step in follicle formation) determines the lifetime supply of primordial follicles and remains a poorly understood phenomenon. Despite a number of recent articles that are concentrating on immuno-histochemistry, basic steps in the process are not clear. Hence, we feel it is time to take a step back and see what is available in the literature and identify the gaps in which future research about primordial follicle assembly in humans needs to be directed.

BACKGROUND: Mouse fetal germ cells have been successfully purified from fetal gonads. However, there are no published reports describing a procedure for deriving mature oocytes from isolated fetal germ cells. The purpose of this present study is to explore whether purified fetal germ cells are able to differentiate into mature oocytes through an in vivo grafting procedure. METHODS AND RESULTS: First, intact 11.5 and 12.5 days post-coitum (dpc) female gonads with or without the attached mesonephros and the reaggregated female gonad cells were transplanted into the recipient mice. The results demonstrate both the gonad accompanied by mesonephroi and the innate gonad structure are not absolutely required for 11.5 dpc and 12.5 dpc oogonia to generate mature oocytes. Next, oogonia were purified from female gonads, aggregated with different ovarian somatic cells and transplanted into the recipient mice. Purified 12.5 dpc oogonia were able to generate mature oocytes by aggregating with 12.5 dpc ovarian somatic cells, but not with 16.5 dpc or 0 days postpartum ovarian somatic cells. We also tested 12.5 dpc male germ cells but they were unable to undergo oogenesis. CONCLUSIONS: Our study demonstrates that mature oocytes can be derived from purified fetal germ cells through an aggregation and transplantation procedure. It also suggests that the synchronized interactions between oogonia and gonadal somatic cells are important to ensure normal folliculogenesis.

The effect of the luteinizing hormone (LH) on the oogenesis of ovaries from newly-hatched chicks treated in vivo on days 13, 15, and 17 of embryonic development was analyzed. Changes in oogonial proliferation, meiotic prophase, degeneration of germ cells, and primordial follicular organization were determined. Results indicate that the total number of germ cells was not affected by the LH treatment, but significant differences existed in the number of oogonia and oocytes between the ovaries of control and LH-treated chicks. LH treatment increased the percentage of oocytes and diminished the percentage of oogonia. The mitotic activity of oogonia and degeneration of germ cells decreased, but the number of follicles during development increased in LH-treated ovaries. These findings suggest that LH treatment might trigger a cascade of endocrine events, resulting in inhibition of oogonial proliferation and induction of the meiotic prophase and follicle formation.

We have described the architecture of Bidder's organ, defined its compartmented structure, and affirmed the presence of basal laminae. We did not find morphological differences between sexes in Bidder's organ. All specimens initially developed gonads with a peripheral fertile layer surrounding a thin primary cavity. The first oogenetic wave was observed early, showing all phases of meiosis, including leptotene, zygotene, and pachytene, which had been previously thought to be lacking. The peculiar presence of an asynchronous germ cell nest was discussed. Diplotene oocytes issued from the peripheral layer and migrated inside the primary cavity. They were surrounded by a single layer of follicular cells, which originated from the peripheral layer somatic cells and were delimited by a basal lamina. There were few medulla or central layer cells. At the end of metamorphosis, while the oocytes of the first oogenetic wave came into close contact with blood vessels, a second oogenetic wave took place just as the first, except for the presence of synchronous germ cell nests. The central layer was not visible and we did not observe the formation of an ovarian pocket. Stocks of stem germ cells remained in the peripheral layer during both the first and second oogenetic waves. The asymmetric model, in which there is a tendency toward a primary female differentiation, was confirmed. The female differentiation becomes stable in the Bidder's organ because of the absence of further interaction between germ and medullary somatic cells, which would have led toward a male differentiation.

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 Universite Paris Diderot-Paris 7 and Universite Paris-Sud, CEA, INSERM, and Agence de la Biomedecine. The authors declare no conflict of interest.

In a number of mammalian species, the main events of development of the primordial germ cells (PGCs), the embryonic precursors of the oocytes and spermatozoa, were described during the early twentieth century. Actually, the concept of the origin of germ cells in extragonadal sites before the formation of the gonadal anlagen, was put forward for the human embryo around the first decade of the 1900s (for a review, see De Felici, 2013). PGC development is characterized by two major cellular processes, a movement from the wall of the yolk sac, where the germline is determined, to the gonadal anlagen and an increase in number due to active proliferation. As far as we know, the notion that programmed cell death (PCD) might physiologically occur in mammalian PGCs was for the first time put forward by us in 1993 in the case of the mouse. How we arrived to such a concept and the progress made up to now in the characterization of this process in our and other laboratories mainly in the mouse are the topics of the present review.

Mammalian females enter puberty with follicular reserves that exceed the number needed for ovulation during a single lifetime. Follicular depletion occurs throughout reproductive life and ends in menopause, or reproductive senescence, when the follicle pool is exhausted. The mechanisms regulating the production of a species-specific initial follicle pool are not well understood. However, the establishment of a follicular reserve is critical to defining the length of reproductive cyclicity. Here we show that activin A (rh-ActA), a known regulator of follicle formation and growth in vitro, increased the number of postnatal mouse primordial follicles by 30% when administered to neonatal animals during the time of germline cyst breakdown and follicle assembly. This expansion in the initial follicle pool was characterized by a significant increase in both germ cell and granulosa cell proliferation. However, the excess follicles formed shortly after birth did not persist into puberty and both adult rh-ActA- and vehicle-treated animals demonstrated normal fertility. A follicle atresia kinetic constant (kA) was modeled for the two groups of animals, and consistent with the empirical data, the kA for rh-ActA-treated was twice that of vehicle-treated animals. Kinetic constants for follicle formation, follicle loss and follicle expansion from birth to postnatal day 19 were also derived for vehicle and rh-ActA treatment conditions. Importantly, introduction of exogenous rh-ActA revealed an intrinsic ovarian quorum sensing mechanism that controls the number of follicles available at puberty. We propose that there is an optimal number of oocytes present at puberty, and when the follicle number is exceeded, it occurs at the expense of oocyte quality. The proposed mechanism provides a means by which the ovary eliminates excess follicles containing oocytes of poor quality prior to puberty, thus maintaining fertility in the face of abnormal hormonal stimuli in the prepubertal period.

The mechanism regulating primordial follicle formation remains largely unexplored because of the developmental particularity of female germ cells and their ultimate functional structure as follicles. Using an in vitro follicle reconstitution culture model, we explored, in the present study, the possibility of producing transgenetic follicles in vitro. We found that mouse fetal ovarian germ cells progressively lose the flexibility for gene manipulation with their oogonia-oocyte transformation upon entering meiosis, the borderline of which was at around embryonic age of 13.5 days post coitus (dpc). Interestingly, we further observed that fetal ovarian cells, only at this age or beyond achieve the capacity to reform the follicles in culture. Screening of well-known marker gene (Zp1-3, Figalpha, and Cx43) expression in cultured fetal ovarian cells of various developmental ages revealed that Figalpha is one of the determining factors for normal primordial follicle formation. By conducting reciprocal follicle reconstitution experiments, we provided further evidence that a synchronized germ-somatic cell interaction determines the normal duration of primordial folliculogenesis. Besides uncovering a potentially important regulatory mechanism for normal oocyte differentiation and follicle formation, this observation offers an alternative approach to produce transgenic oocytes/follicles, and thus animal models.

Ovarian follicle development is a complex process that begins with the establishment of what is thought to be a finite pool of primordial follicles and culminates in either the atretic degradation of the follicle or the release of a mature oocyte for fertilization. This review highlights the many advances made in understanding these events using transgenic mouse models. Specifically, this review describes the ovarian phenotypes of mice with genetic mutations that affect ovarian differentiation, primordial follicle formation, follicular growth, atresia, ovulation and corpus luteum (CL) formation. In addition, this review describes the phenotypes of mice with mutations in a variety of genes, which affect the hormones that regulate folliculogenesis. Because studies using transgenic animals have revealed a variety of reproductive abnormalities that resemble many reproductive disorders in women, it is likely that studies using transgenic mouse models will impact our understanding of ovarian function and fertility in women.

The fine structure of human oogonia and growing oocytes has been reviewed in fetal and adult ovaries. Preovulatory maturation and the ultrastructure of stimulated oocytes from the germinal vesicle (GV) stage to metaphase II (MII) stage are also documented. Oogonia have large nuclei, scanty cytoplasm with complex mitochondria. During folliculogenesis, follicle cell processes establish desmosomes and deep gap junctions at the surface of growing oocytes, which are retracted during the final stages of maturation. The zona pellucida is secreted in secondary follicles. Growing oocytes have mitochondria, Golgi, rough endoplasmic reticulum (RER), ribosomes, lysosomes, and lipofuscin bodies, often associated with Balbiani bodies and have nuclei with reticulated nucleoli. Oocytes from antral follicles show numerous surface microvilli and cortical granules (CGs) separated from the oolemma by a band of microfilaments. The CGs are evidently secreted by Golgi membranes. The GV oocytes have peripheral Golgi complexes associated with a single layer of CGs close to the oolemma. They have many lysosomes, and nuclei with dense compact nucleoli. GV breakdown occurs by disorganization of the nuclear envelope and the oocyte enters a transient metaphase I followed by MII, when it is arrested and ovulated. Maturation of oocytes in vitro follows the same pattern of meiosis seen in preovulatory oocytes. The general organization of the human oocyte conforms to that of most other mammals but has some unique features. The MII oocyte has the basic cellular organelles such as mitochondria, smooth endoplasmic reticulum, microfilaments, and microtubules, while Golgi, RER, lysosomes, multivesicular, residual and lipofuscin bodies are very rare. It neither has yolk nor lipid inclusions. Its surface has few microvilli, and 1-3 layers of CGs, aligned beneath the oolemma. Special reference has been made to the reduction and inactivation of the maternal centrosome during oogenesis. The MII spindle, often oriented perpendicular to the oocyte surface, is barrel-shaped, anastral and lacks centrioles. Osmiophilic centrosomes are not demonstrable in human eggs, since the maternal centrosome is nonfunctional. However, oogonia and growing oocytes have typical centrioles, similar to those of somatic cells. The sperm centrosome activates the egg and organizes the sperm aster and mitotic spindles of the embryo, after fertilization.

Oogenesis in the lizard Mabuya brachypoda is seasonal, with oogenesis initiated during May-June and ovulation occurring during July-August. This species ovulates an egg that is microlecithal, having very small yolk stores. The preovulatory oocyte attains a maximum diameter of 0.9-1.3 mm. Two elongated germinal beds, formed by germinal epithelia containing oogonia, early oocytes, and somatic cells, are found on the dorsal surface of each ovary. Although microlecithal eggs are ovulated in this species, oogenesis is characterized by both previtellogenic and vitellogenic stages. During early previtellogenesis, the nucleus of the oocyte contains lampbrush chromosomes, whereas the ooplasm stains lightly with a perinuclear yolk nucleus. During late previtellogenesis the ooplasm displays basophilic staining with fine granular material composed of irregularly distributed bundles of thin fibers. A well-defined zona pellucida is also observed. The granulosa, initially composed of a single layer of squamous cells during early previtellogenesis, becomes multilayered and polymorphic. As with other squamate reptiles, the granulosa at this stage is formed by three cell types: small, intermediate, and large or pyriform cells. As vitellogenesis progresses the oocyte displays abundant vacuoles and small, but scarce, yolk platelets at the periphery of the oocyte. The zona pellucida attains its maximum thickness during late oogenesis, a period when the granulosa is again reduced to a single layer of squamous cells. The vitellogenic process observed in M. brachypoda corresponds with the earliest vitellogenic stages seen in other viviparous lizard species with larger oocytes. The various species of the genus Mabuya provided us with important models to understand a major transition in the evolution of viviparity, the development of a microlecithal egg.