Genital Ridge Research Articles

Primary retroperitoneal extraovarian granulosa cell tumor.

ABSTRACTExtraovarian granulosa cell tumors (GCTs) develop from ectopic gonadal tissue situated along the embryonal route of the genital ridge. Primary retroperitoneal tumors are extremely rare, with an incidence of 02% -06% and 80-85% probability of malignancy. Only eight such case reports have been published previously. We herein, report a rare case of extraovarian retroperitoneal GCT in a 55-year-old woman who presented with intermittent left lumbar region pain of one-year duration. She had a history of hysterectomy and bilateral salpingo-oophorectomy 8 years ago for uterine leiomyoma. Laparotomy revealed a retroperitoneal mass measuring 8cm x 10cm x 20cm in size, solid cystic with areas of necrosis and hemorrhage. The gross features, classical histopathology, and positive immunostaining of the retroperitoneal mass with inhibin, calretinin, PR, WT1 and immunonegativity for EMA were characteristic of adult-type GCT. Excluding any previous history of primary ovarian GCT in this patient, a de-novo retroperitoneal diagnosis was established.

Human SRY Expression at the Sex-determining Period is Insufficient to Drive Testis Development in Mice.

The sex-determining region of the Y chromosome, Sry/SRY, is an initiation factor for testis development in both humans and mice. Although the functional compatibility between murine SRY and human SRY was previously examined in transgenic mice, their equivalency remains inconclusive. Because molecular interaction and timeline of mammalian sex determination were mostly described in murine experiments, we generated a mouse model in which Sry was substituted with human SRY to verify the compatibility. The mouse model had the human SRY open reading frame at the locus of murine Sry exon 1-Sry(SRY) mice-and was generated using the CRISPR/Cas9 system. The reproductive system of the mice was analyzed. The expression of human SRY in the fetal gonadal ridge of Sry(SRY) mice was detected. The external and internal genitalia of adult Sry(SRY) mice were similar to those of wild-type females, without any significant difference in anogenital distance. Sry(SRY) mice obtained gonads, which were morphologically considered as ovaries. Histological analysis revealed that the cortical regions of gonads from adult Sry(SRY) mice contained few follicles. We successfully replaced genes on the Y chromosome with targeted genome editing using the CRISPR/Cas9 system. Because the Sry(SRY) XY mice did not develop testis, we concluded that human SRY was insufficient to drive testis development in mouse embryos. The difference in response elements and lack of glutamine-rich domains may have invalidated human SRY function in mice. Signal transduction between Sry/SRY expression and Sox9/SOX9 activation is possibly organized in a species-specific manner.

Germplasm and the origin of the primordial germ cells in the oriental river prawn Macrobrachium nipponense.

Germplasm is a special cytoplasmic component containing special RNAs and proteins, and is located in specific regions of oocytes and embryos. Only the blastomeres inheriting the germplasm can develop into primordial germ cells (PGCs). By using Vasa mRNA as a germline marker, we previously demonstrated that germline specification followed the preformation mode in the prawn Macrobrachium nipponense. In this study, we raised the Vasa antibody to identify germplasm in the oocyte and trace the origin and migration of PGCs. In previtellogenic oocytes, Vasa protein was detected in the perinuclear region, in which electron-dense granules associated with numerous mitochondria were mostly visualized under a transmission electron microscope. In mature oocytes, immunosignal was localized to a large granule under the plasma membrane. During early embryogenesis, the granule was inherited by a single blastomere from 1-cell to 16-cell stages, and thereafter was segregated into two daughter blastomeres at the 32-cell stage. In gastrula, the Vasa-positive cells were large with typical PGC characteristics, containing a big round nucleus and a prominent nucleolus. The immunosignal was localized to the perinuclear region again. In the zoea stage, the Vasa-positive cells migrated toward the genital ridge and clustered in the dorsomedial region close to the yolk portion. Accordingly, we concluded that the prawn PGCs could be specified from the 16-cell stage by inheriting the germplasm. To our knowledge, this is the first report on the identification of the prawn germplasm and PGCs. The continuous expression of Vasa protein throughout oogenesis and embryogenesis suggests that Vasa protein could be an important factor in germplasm that functions in early germ cell specification.

Histological description of the early gonadal development of Arapaima gigas, paiche

AbstractArapaima gigas, paiche, is the largest freshwater fish species in the South American Amazon. Despite the importance of an accurate understanding of the species' gonadal sexual differentiation mechanisms, the gonadogenesis process is still not well studied. The aim of this study was to determine the histological characteristics of the early gonadal development of paiche fry, belonging to the same “levante” (“levante” is the word used to describe the removal of fry from the male paiche). The fry was sampled during the month of September 2016; from the 5‐day post “levante” (dpl) to the 51 dpl with a minimum size of 2.7 cm and a maximum size of 16.0 cm in total length. The histology showed a large concentration of cells in the dorsal part of the abdominal cavity in 5 dpl fry, being greater in the left side. In addition, the gonadal ridges appeared at 6 dpl and the gonadal primordia appeared at 7 dpl. The two gonadal primordia were observed with different sizes, where the left primordia were larger and more basophilic than the right primordia. Finally, gonad differentiation between presumable females and males were observed with some structural differentiation between presumable females and presumable males since 43 post hatching day.

Ovarian cysts

Ovarian cysts

PAX2 + Mesenchymal Origin of Gonadal Supporting Cells Is Conserved in Birds.

During embryonic gonadal development, the supporting cell lineage is the first cell type to differentiate, giving rise to Sertoli cells in the testis and pre-granulosa cells in the ovary. These cells are thought to direct other gonadal cell lineages down the testis or ovarian pathways, including the germline. Recent research has shown that, in contrast to mouse, chicken gonadal supporting cells derive from a PAX2/OSR1/DMRT1/WNT4 positive mesenchymal cell population. These cells colonize the undifferentiated genital ridge during early gonadogenesis, around the time that germ cells migrate into the gonad. During the process of somatic gonadal sex differentiation, PAX2 expression is down-regulated in embryonic chicken gonads just prior to up-regulation of testis- and ovary-specific markers and prior to germ cell differentiation. Most research on avian gonadal development has focused on the chicken model, and related species from the Galloanserae clade. There is a lack of knowledge on gonadal sex differentiation in other avian lineages. Comparative analysis in birds is required to fully understand the mechanisms of avian sex determination and gonadal differentiation. Here we report the first comparative molecular characterization of gonadal supporting cell differentiation in birds from each of the three main clades, Galloanserae (chicken and quail), Neoaves (zebra finch) and Palaeognathe (emu). Our analysis reveals conservation of PAX2+ expression and a mesenchymal origin of supporting cells in each clade. Moreover, down-regulation of PAX2 expression precisely defines the onset of gonadal sex differentiation in each species. Altogether, these results indicate that gonadal morphogenesis is conserved among the major bird clades.

Abnormal migration behavior linked to Rac1 signaling contributes to primordial germ cell exhaustion in Fanconi anemia pathway-deficient Fancg-/- embryos.

Fanconi anemia (FA) is a rare human genetic disorder characterized by bone marrow failure, predisposition to cancer and developmental defects including hypogonadism. Reproductive defects leading to germ cell aplasia are the most consistent phenotypes seen in FA mouse models. We examined the role of the nuclear FA core complex gene Fancg in the development of primordial germ cells (PGCs), the embryonic precursors of adult gametes, during fetal development. PGC maintenance was severely impaired in Fancg−/− embryos. We observed a defect in the number of PGCs starting at E9.5 and a strong attrition at E11.5 and E13.5. Remarkably, we observed a mosaic pattern reflecting a portion of testicular cords devoid of PGCs in E13.5 fetal gonads. Our in vitro and in vivo data highlight a potential role of Fancg in the proliferation and in the intrinsic cell motility abilities of PGCs. The random migratory process is abnormally activated in Fancg−/− PGCs, altering the migration of cells. Increased cell death and PGC attrition observed in E11.5 Fancg−/− embryos are features consistent with delayed migration of PGCs along the migratory pathway to the genital ridges. Moreover, we show that an inhibitor of RAC1 mitigates the abnormal migratory pattern observed in Fancg−/− PGCs.

Characterization of the sex differentiation and gonadal development in small yellow croaker (Larimichthys polyactis) and its hybrid (L. polyactis ♀ × L. crocea ♂).

Interspecific hybridization has been considered as a possible approach to improve biological traits and has been applied in aquaculture practices. In the present study, artificial hybridization was carried out in the small yellow croaker (SYC; Larimichthys polyactis) ♀ × large yellow croaker (LYC; L. crocea) ♂ by artificial insemination, and the processes of sex differentiation and gonadal development in SYC and its hybrid were investigated under controlled conditions. Histological analysis of SYC larvae showed that migrating primordial germ cells (PGCs) were observed at 4days post-hatching (dph), a genital ridge was formed on the dorsal side of the peritoneum at 6 dph, and a pair of primary gonads was first observed at 10 dph. Signs of the differentiated ovary and ovarian cavity were observed at 45 dph. However, some presumptive testes showed alterations in morphology, including an increase in the number of oocytes and an enhanced basophilia at 50 dph. These presumptive testes seemed to alter again, and numerous gonial cells were arranged in cyst-like groups with several degenerating oocytes that developed into residual body-like structures during 60-90 dph. Compared with SYC, the hybrid had a lower number of PGCs and showed retarded gonadal development at the early stage. Ovarian differentiation in the hybrid was observed at 50 dph, while testicular differentiation occurred at 60 dph. The presence of vitellogenic oocytes and spermatozoa at 360 dph in the hybrids suggested that hybrid individuals can undergo successful gametogenesis in females and males, respectively. Overall, the present results suggest that morphological sex differentiation occurred at 40 and 50 dph in SYC and its hybrid, respectively, both of which have normal gametogenesis. Moreover, some level of heterosis (hybrid vigor) occurred in the growth of the hybrid (total length and body weight) compared with that in the growth of SCY over time. Gonadal development of the hybrid was also found to be advanced at 360 dph. The present information will contribute to the potential use and management of these fish for aquaculture.

Concerted morphogenesis of genital ridges and nephric ducts in the mouse captured through whole-embryo imaging.

During development of the mouse urogenital complex, the gonads undergo changes in three-dimensional structure, body position and spatial relationship with the mesonephric ducts, kidneys and adrenals. The complexity of genital ridge development obscures potential connections between morphogenesis and gonadal sex determination. To characterize the morphogenic processes implicated in regulating gonad shape and fate, we used whole-embryo tissue clearing and light sheet microscopy to assemble a time course of gonad development in native form and context. Analysis revealed that gonad morphology is determined through anterior-to-posterior patterns as well as increased rates of growth, rotation and separation in the central domain that may contribute to regionalization of the gonad. We report a close alignment of gonad and mesonephric duct movements as well as delayed duct development in a gonad dysgenesis mutant, which together support a mechanical dependency linking gonad and mesonephric duct morphogenesis.

Sacrococcygeal Teratoma : A Tumor at the Center of Embryogenesis.

Sacrococcygeal teratoma (SCT) is an extragonadal germ cell tumor (GCT) that develops in the fetal and neonatal periods. SCT is a type I GCT in which only teratoma and yolk sac tumors arise from extragonadal sites. SCT is the most common type I GCT and is believed to originate through epigenetic reprogramming of early primordial germ cells migrating from the yolk sac to the gonadal ridges. Fetal SCT diagnosed in utero presents many obstetrical problems. For high-risk fetuses, fetal interventions (devascularization and debulking) are under development. Most patients with SCT are operated on after birth. Complete surgical resection is the key for tumor control, and the anatomical location of the tumor determines the surgical approaches. Incomplete resection and malignant histology are risk factors for recurrence. Approximately 10–15% of patients have a tumor recurrence, which is frequently of malignant histology. Long-term surveillance with monitoring of serum alpha fetoprotein and magnetic resonance imaging is required. Survivors of SCT may suffer anorectal, urological, and sexual sequelae later in their life, and comprehensive evaluation and care are required.

MicroRNAs in amniotic fluid and maternal blood plasma associated with sex determination and early gonad differentiation in cattle†.

We hypothesized that sexually dimorphic differences exist in the expression of miRNAs in amniotic fluid (AF) and maternal blood plasma (MP) in association with the process of sex determination and gonad differentiation in cattle. Amniotic fluid and MP were collected from six pregnant heifers (three carrying a single male and three a single female embryo) following slaughter on Day 39 postinsemination, coinciding with the peak of SRY expression. Samples (six AF and six MP) were profiled using an miRNA Serum/Plasma Focus PCR Panel. Differentially expressed (DE) miRNAs were identified in AF (n = 5) and associated MP (n = 56) of male vs. female embryos (P < 0.05). Functional analysis showed that inflammatory and immune response were among the 13 biological processes enriched by miRNAs DE in MP in the male group (FDR < 0.05), suggesting that these sex-dependent DE miRNAs may be implicated in modulating the receptivity of the dam to a male embryo. Further, we compared the downstream targets of the sex-dependent DE miRNAs detected in MP with genes previously identified as DE in male vs. female genital ridges. The analyses revealed potential targets that might be important during this developmental stage such as SHROOM2, DDX3Y, SOX9, SRY, PPP1CB, JARID2, USP9X, KDM6A, and EIF2S3. Results from this study highlight novel aspects of sex determination and embryo–maternal communication in cattle such as the potential role of miRNAs in gonad development as well as in the modulation of the receptivity of the dam to a male embryo.

Visualization of X chromosome reactivation in mouse primordial germ cells in vivo.

ABSTRACTX chromosome inactivation (XCI), determined during development, remains stable after embryonic cell divisions. However, primordial germ cells (PGCs) are exceptions in that XCI is reprogrammed and inactivated X chromosomes are reactivated. Although interactions between PGCs and somatic cells are thought to be important for PGC development, little is known about them. Here, we performed imaging of X chromosome reactivation (XCR) using the ‘Momiji’ mouse system, which can monitor the X chromosome's inactive and active states using two color fluorescence reporter genes, and investigated whether interactions would affect XCR in PGCs. Based on their expression levels, we found that XCR of the Pgk1 locus began at embryonic day (E)10.5 and was almost complete by E13.5. During this period, PGCs became distributed uniformly in the genital ridge, proliferated, and formed clusters; XCR progressed accordingly. In addition, XCR of the Pgk1 locus preceded that of the Hprt locus, indicating that the timing of epigenetic memory erasure varied according to the locus of each of these X-linked genes. Our results indicate that XCR proceeds along with the proliferation of PGCs clustered within the genital ridge.This article has an associated First Person interview with the first author of the paper.

Female Germ Cell Development, Functioning and Associated Adversities under Unfavorable Circumstances.

In the present era, infertility is one of the major issues which restricts many couples to have their own children. Infertility is the inability to achieve a clinical pregnancy after regular unprotected sexual intercourse for the period of one year or more. Various factors including defective male or female germ cell development, unhealthy and improper lifestyles, diseases like cancer and associated chemo-or-radiation therapies, congenital disorders, etc., may be responsible for infertility. Therefore, it is highly important to understand the basic concepts of germ cell development including primordial germ cell (PGC) formation, specification, migration, entry to genital ridges and their molecular mechanisms, activated pathways, paracrine and autocrine signaling, along with possible alteration which can hamper germ cell development and can cause adversities like cancer progression and infertility. Knowing all these aspects in a proper way can be very much helpful in improving our understanding about gametogenesis and finding possible ways to cure related disorders. Here in this review, various aspects of gametogenesis especially female gametes and relevant factors causing functional impairment have been thoroughly discussed.

Marker counter-selection via CRISPR/Cas9 co-targeting for efficient generation of genome edited avian cell lines and germ cells

Efficient isolation of genetically modified cells that are phenotypically indistinguishable from the unmodified cells remains a major technical barrier for the broader utilization of CRISPR/Cas9. Here, we report a novel enrichment approach to select the genome engineered cells by co-targeting a genomically integrated GFP gene along with the endogenous gene of interest (GOI). Using this co-targeting approach, multiple genomic loci were successfully targeted in chicken (DF1) and quail (CEC-32) fibroblast cell lines by transient transfection of Cas9 and guide RNAs (gRNAs). Clonal isolation of co-targeted DF1 cells showed 75% of cell clones had deletion of GFP and biallelic deletion of the GOI. To assess the utility of this approach to generate genome modified animals, we tested it on chicken primordial germ cells (PGCs) expressing GFP by co-targeting with gRNAs against GFP and endogenous ovomucoid (OVM) gene. PGCs enriched for loss of GFP and confirmed for OVM deletion, derived by co-targeting, were injected into Hamburger and Hamilton stage 14–15 chicken embryos, and their ability to migrate to the genital ridge was confirmed. This simple, efficient enrichment approach could easily be applied to the creation of knock-out or edited cell lines or animals.

Genome-wide DNA methylation dynamics during epigenetic reprogramming in the porcine germline

BackgroundPrior work in mice has shown that some retrotransposed elements remain substantially methylated during DNA methylation reprogramming of germ cells. In the pig, however, information about this process is scarce. The present study was designed to examine the methylation profiles of porcine germ cells during the time course of epigenetic reprogramming.ResultsSows were artificially inseminated, and their fetuses were collected 28, 32, 36, 39, and 42 days later. At each time point, genital ridges were dissected from the mesonephros and germ cells were isolated through magnetic-activated cell sorting using an anti-SSEA-1 antibody, and recovered germ cells were subjected to whole-genome bisulphite sequencing. Methylation levels were quantified using SeqMonk software by performing an unbiased analysis, and persistently methylated regions (PMRs) in each sex were determined to extract those regions showing 50% or more methylation. Most genomic elements underwent a dramatic loss of methylation from day 28 to day 36, when the lowest levels were shown. By day 42, there was evidence for the initiation of genomic re-methylation. We identified a total of 1456 and 1122 PMRs in male and female germ cells, respectively, and large numbers of transposable elements (SINEs, LINEs, and LTRs) were found to be located within these PMRs. Twenty-one percent of the introns located in these PMRs were found to be the first introns of a gene, suggesting their regulatory role in the expression of these genes. Interestingly, most of the identified PMRs were demethylated at the blastocyst stage.ConclusionsOur findings indicate that methylation reprogramming in pig germ cells follows the general dynamics shown in mice and human, unveiling genomic elements that behave differently between male and female germ cells.

Extensive nuclear gyration and pervasive non-genic transcription during primordial germ cell development in zebrafish.

ABSTRACTPrimordial germ cells (PGCs) are the precursors of germ cells, which migrate to the genital ridge during early development. Relatively little is known about PGCs after their migration. We studied this post-migratory stage using microscopy and sequencing techniques, and found that many PGC-specific genes, including genes known to induce PGC fate in the mouse, are only activated several days after migration. At this same time point, PGC nuclei become extremely gyrated, displaying general broad opening of chromatin and high levels of intergenic transcription. This is accompanied by changes in nuage morphology, expression of large loci (PGC-expressed non-coding RNA loci, PERLs) that are enriched for retro-transposons and piRNAs, and a rise in piRNA biogenesis signatures. Interestingly, no nuclear Piwi protein could be detected at any time point, indicating that the zebrafish piRNA pathway is fully cytoplasmic. Our data show that the post-migratory stage of zebrafish PGCs holds many cues to both germ cell fate establishment and piRNA pathway activation.

Study on the Function and Mechanism of Lin28B in the Formation of Chicken Primordial Germ Cells

Simple SummaryIn this study, we explored the function and molecular mechanism of Lin28B in the formation of chicken primordial germ cells (PGCs) in detail. Our results indicate that Lin28B participates in the formation of PGCs through let-7a-3p, which set a theoretical foundation for improving the function and mechanism of the Lin28 family in the formation of PGCs.Lin28A and Lin28B are two homologues of the same family of RNA binding proteins (RBPs). The function and molecular mechanism of Lin28A in the formation of primordial germ cells (PGCs) are very clear, but the related research on Lin28B is rarely reported. Here, we found that the overexpression of Lin28B can promote the formation of PGC in vivo. Furthermore, the overexpression of Lin28B also resulted in the inhibition of totipotency gene expression and upregulated the PGCs marker genes, and a significant increase in the number of PGCs in genital ridge, as detected by Periodic Acid-Schiff(PAS) staining. However, the inhibited Lin28B expression showed completely opposite results, which were confirmed on the PGC induction model in vitro. Mechanistically, we found that the overexpression of Lin28B can inhibit the maturation of let-7a-3p, and the results of high-throughput sequencing indicated that let-7a-3p was a negative regulator of the formation process of PGCs. Therefore, we conclude that our results determine that Lin28B participates in the formation of PGCs through let-7a-3p, which set a theoretical foundation for improving the function and mechanism of Lin28 family in the formation of PGCs.

Pattern of Gonadal Sex Differentiation in the Rice Field Frog Hoplobatrachus rugulosus (Anura: Dicroglossidae).

Sex differentiation during gonadal development is diversified among anuran amphibian species. In this study, the anuran experimental species Hoplobatrachus rugulosus was examined. The pattern of gonadal sex differentiation was observed by morphological and histological approaches. The gonad was observed morphologically at Gosner stage 33, while distinct testis and ovary were evident from 3-4 weeks after metamorphosis ended. Histological analysis showed that genital ridge formation began at stage 25 and ovarian differentiation began at stage 36. The developing ovary appeared with numerous primary oogonia, which developed into oocytes, while the medulla regressed to form an ovarian cavity. During metamorphosis, only an ovary was observed. Testicular differentiation seemed to begin later, during the first week after metamorphosis, and occurred via an intersex condition. The intersex gonads contained developing testicular tissue with both normal and atretic oocytes. The fully developed testis was first identified at 6 weeks after metamorphosis. Comparing the times of gonadal differentiation and somatic development revealed that the ovary exhibited a basic rate of differentiation while the testis exhibited a retarded one. These results establish that males of this species develop later than do females, and the testis develops through an intersex gonad, as is evident from its seminiferous cord formation, the presence of testis-ova, and atretic oocytes in the tissue. Thus, the pattern of gonadal sex differentiation in H. rugulosus is an undifferentiated type, in which only female gonads are observed during metamorphosis and intersex and male gonads are observed later. These results are crucial for further research on the sexual development of anurans.

Germline Specific Expression of a vasa Homologue Gene in the Viviparous Fish Black Rockfish (Sebastes schlegelii) and Functional Analysis of the vasa 3' Untranslated Region.

Germ cells play a key role in gonad development. As precursors, primordial germ cells (PGCs) are particularly important for germline formation. However, the origination and migration patterns of PGCs are poorly studied in marine fish, especially for viviparous economic species. The vasa gene has been widely used as a germ cell marker to identify a germline because vasa RNA is a component of germ plasm. In this study, we described the expression pattern of black rockfish (Sebastes schlegelii) vasa (Ssvas) in gonadal formation and development by in situ hybridization. The results showed that Ssvas failed in localization at the cleavage furrows until the late gastrula stage, when PGCs appeared and migrated to the genital ridge and formed elongated gonadal primordia at 10 days after birth. This study firstly revealed the PGCs origination and migration characteristics in viviparous marine fish. Furthermore, we microinjected chimeric mRNA containing EGFP and the 3′untranslated region (3′UTR) of Ssvas into zebrafish (Danio rerio) and marine medaka (Oryzias melastigma) fertilized eggs for tracing PGCs. We found that, although Sebastes schlegelii lacked early localization, similar to red seabream (Pagrus major) and marine medaka, only the 3′UTR of Ssvas vasa 3′UTR of black rockfish was able to label both zebrafish and marine medaka PGCs. In comparison with other three Euteleostei species, besides some basal motifs, black rockfish had three specific motifs of M10, M12, and M19 just presented in zebrafish, which might play an important role in labeling zebrafish PGCs. These results will promote germ cell manipulation technology development and facilitate artificial reproduction regulation in aquaculture.

Molecular characterization of XX maleness

Androgens and anti-Mullerian hormone (AMH), secreted by the foetal testis, are responsible for the development of male reproductive organs and the regression of female anlagen. Virilization of the reproductive tract in association with the absence of Mullerian derivatives in the XX foetus implies the existence of testicular tissue, which can occur in the presence or absence of SRY. Recent advancement in the knowledge of the opposing gene cascades driving to the differentiation of the gonadal ridge into testes or ovaries during early foetal development has provided insight into the molecular explanation of XX maleness.