Female Fetal Gonads Research Articles

Gene expression profiles of bovine genital ridges during sex determination and early differentiation of the gonads†

Most current knowledge of sex determination in mammals has emerged from mouse and human studies. To investigate the molecular regulation of the sex determination process in cattle, we used an RNA sequencing strategy to analyze the transcriptome landscape of male and female bovine fetal gonads collected in vivo at key developmental stages: before, during, and after SRY gene activation on fetal days D35 (bipotential gonad formation), D39 (peak SRY expression), and D43 (early gonad differentiation). Differentially expressed genes (DEGs) were identified in male vs. female germinal ridges and among group genes showing similar expression profiles during the three periods. There were 143, 96, and 658 DEG between males and female fetuses at D35, D39, and D43, respectively. On D35, genes upregulated in females were enriched in translation, nuclear export, RNA localization, and mRNA splicing events, whereas those upregulated in males were enriched in cell proliferation regulation and male sex determination terms. In time-course experiments, 767 DEGs in males and 545 DEGs in females were identified between D35 vs. D39, and 3157 DEGs in males and 2008 in females were identified between D39 vs. D43. Results highlight unique aspects of sex determination in cattle, such as the expression of several Y chromosome genes (absent in mice and humans) before SRY expression and an abrupt increase in the nuclear expression of SOX10 (instead of SOX9 expression in the Sertoli cell cytoplasm as observed in mice) during male determination and early differentiation.

A Comparative Proteome Profile of Female Mouse Gonads Suggests a Tight Link between the Electron Transport Chain and Meiosis Initiation

Generation of haploid gametes by meiosis is a unique property of germ cells and is critical for sexual reproduction. Leaving mitosis and entering meiosis is a key step in germ cell development. Several inducers or intrinsic genes are known to be important for meiotic initiation, but the regulation of meiotic initiation, especially at the protein level, is still not well understood. We constructed a comparative proteome profile of female mouse fetal gonads at specific time points (11.5, 12.5, and 13.5 days post coitum), spanning a critical window for initiation of meiosis in female germ cells. We identified 3666 proteins, of which 473 were differentially expressed. Further bioinformatics analysis showed that these differentially expressed proteins were enriched in the mitochondria, especially in the electron transport chain and, notably, 9 proteins in electron transport chain Complex I were differentially expressed. We disrupted the mitochondrial electron transport chain function by adding the complex I inhibitor, rotenone to 11.5 days post coitum female gonads cultured in vitro. This treatment resulted in a decreased proportion of meiotic germ cells, as assessed by staining for histone γH2AX. Rotenone treatment also caused decreased ATP levels, increased reactive oxygen species levels and failure of the germ cells to undergo premeiotic DNA replication. These effects were partially rescued by adding Coenzyme Q10. Taken together, our results suggested that a functional electron transport chain is important for meiosis initiation. Our characterization of the quantitative proteome of female gonads provides an inventory of proteins, useful for understanding the mechanisms of meiosis initiation and female fertility.

Overactive type 2 cannabinoid receptor induces meiosis in fetal gonads and impairs ovarian reserve

Type 2 cannabinoid receptor (CB2R) has been proposed to promote in vitro meiotic entry of postnatal male germ cells and to maintain the temporal progression of spermatogenesis in vivo. However, no information is presently available on the role played by CB2R in male and female fetal gonads. Here we show that in vitro pharmacological stimulation with JWH133, a CB2R agonist, induced activation of the meiotic program in both male and female fetal gonads. Upon stimulation, gonocytes initiated the meiotic program but became arrested at early stages of prophase I, while oocytes showed an increased rate of meiotic entry and progression toward more advanced stage of meiosis. Acceleration of meiosis in oocytes was accompanied by a strong increase in the percentage of γ-H2AX-positive pachytene and diplotene cells, paralleled by an increase of TUNEL-positive cells, suggesting that DNA double-strand breaks were not correctly repaired during meiosis, leading to oocyte apoptosis. Interestingly, in vivo pharmacological stimulation of CB2R in fetal germ cells through JWH133 administration to pregnant females caused a significant reduction of primordial and primary follicles in the ovaries of newborns with a consequent depletion of ovarian reserve and reduced fertility in adult life, while no alterations of spermatogenesis in the testis of the offspring were detected. Altogether our findings highlight a pro-meiotic role of CB2R in male and female germ cells and suggest that the use of cannabis in pregnant female might represent a risk for fertility and reproductive lifespan in female offspring.

The Function of PRDM9 on Mammalian Recombination Hotspots

PRDM9 (PR domain containing 9) is one kind of histone trimethylase which catalyzes the H3K4 trimethylation, and possesses the activity of transcription factor. PRDM9 transcripts were only expressed in germ cells entering meiotic prophase in female fetal gonads and in postnatal testis, whose deficiency results in sterility. The zinc finger motif of mammalian PRDM9 rapidly evolves, which is respond to the sequences of recombination hotspots. Several researches indicated that PRDM9 was involved in the binding to recombination hotspots and initiation of recombination. These progresses are important for understanding of species evolution and the mechanism of genetic recombination.

Cytometry of DNA mutation and modification

MORE than 100 years after the discovery of chromosomes, new techniques for their investigation are still being developed and the knowledge obtained about their structure and function continues to grow. Comparative genomic hybridization (CGH) allows the analysis of genomic instability at the chromosomal level (by 10 Mbp resolution). This fluorescencebased staining method can easily detect larger genomic changes such as deletions or additions, aneuploidy, ring chromosomes, or micronuclei (1). These nuclear variations in size and shape and genomic instability are mostly hallmarks of dedifferentiated cancer cells (2). Collection of single cells in the presence or absence of micronuclei with subsequent whole genome amplification and single-cell CGH were applied to detect genomic aberrations in individual HT-1080 fibrosarcoma cells. Currently, not only high-throughput array-based CGH but also high-throughput methods such as single nucleotide polymorphism (SNP) arrays (with up to >10 kbp resolution), expression arrays, and ChIP-on-chip arrays are available to study gene alterations in human cancers. A SNP is a DNA sequence variation occurring when a single nucleotide (A, T, C, or G) differs between chromosomes in an individual or between individuals in the population. For example, the triplet ACG to ATG contains a difference in a single nucleotide. SNP allele variations are commonly found comparing geographical or ethnic groups. Their greatest importance in biomedical research is that there are variations in the DNA sequences of humans which are related to diseases or cancer development (3,4). SNPs are also thought to be key enablers in realizing the concept of preventive or personalized medicine. The fine mapping and deciphering of specific cancer phenotypes is taking advantage of molecular-profiling studies based on genomewide approaches. The third genome-scanning method is spectral karyotyping (SKY) which is capable of identifying aberrations in all chromosomes simultaneously (5). It is a fluorescence microscope-based approach that uses five fluorophores, single and in combinations, to uniquely label DNA from each chromosome. By means of computer separation (classification) of spectra, spectrally overlapping chromosome-specific DNA probes can be resolved, and all human chromosomes are simultaneously identified. SKY is much more efficient (and cost-effective) than using individual chromosome paint probe 1 (or 2 or 3) at a time to identify chromosome aberrations. In situ hybridization with this complex DNA probe is used in metaphase spreads. This method can identify those chromosome aberrations that are not apparent by G-banding, as well as aberrations in solid tumor metaphases where Gbanding is suboptimal. SKY is a recently developed cytogenetic technique for cancer diagnosis and research on genetic disorders. By simultaneously viewing the multiple labeled specimens in different color channels, it facilitates the detection of subtle chromosomal aberrations (6,7). From these technologies, CGH is probably the most frequently applied assay to scan copy number variations. It uses, like SKY, mitotic cells for analysis. The analysis of the meiotic (reducing) cell division is marginal in cancer research. Currently, the interest is increasing in the detailed analysis of the reproduction and germ cell producing meiosis. Traditionally, meiosis has been more extensively studied in males because of easier access to large amounts of meiotic tissue. Relatively little is known about meiosis in females. Recently, Wojtasz et al. (8) described criteria that allow discrimination and identification of germ cells and somatic cells in cell suspension of female fetal gonads. Side and forward scattering without staining is suitable for isolation of germ cell populations with higher than 90% purity. These sorted cells can then be used in downstream immunofluorescence and RT-PCR applications. The DNA content analysis of the male germ cells is prevalent in cytometry; also the analysis of human spermatozoa is a

Effects ofin vitroculture of mouse fetal gonads on subsequent ovarian developmentin vivoand oocyte maturationin vitro

Under organ culture, female fetal gonads in mice cannot develop beyond the preantral follicle stage unless the follicles are individually isolated and cultured again. In this study, we investigated the effect of in vitro culture of female fetal gonads before transplantation on subsequent in vivo development. The gonads derived from female fetuses 12.5 days postcoitum were organ-cultured for 0, 7 and 14 days, and then were grafted underneath the kidney capsules of severe combined immunodeficient mice and recovered at 21, 14 and 7 days post-transplantation, respectively. The histological analysis of the grafts showed that the in vitro culture of the fetal gonads restricted follicular development to the antral follicle stage post-transplantation. In the grafts cultured for 14 days, particularly, no antral follicle was observed. However, the oocytes in these follicles had grown to around 65 microm in diameter and had competence to resume meiosis in vitro. When the fetal gonads were grafted after culture for 7 and 14 days, 13.0% and 6.8% of the oocytes progressed to the metaphase II stage, respectively. These data showed significant differences (P < 0.05) in comparison with the control group (25.3%). Our results indicate that the in vitro culture of female fetal gonads before transplantation affects the subsequent in vivo development of both follicular cells and oocytes, and in vitro oocyte maturation. However, this effect seems to be more severe in terms of follicular development when compared with oocyte growth and maturation.

Normalizing Gene Expression Levels in Mouse Fetal Germ Cells1

Real-time PCR has become a popular method to analyze transcription of genes that are developmentally regulated during organogenesis of the testes and ovaries. However, the heterogenous cell populations and commitment to strikingly different developmental pathways of the germ and somatic cells in these organs complicate analysis of this process. The selection of suitable reference genes for quantifying gene expression in this system is essential, but to date it has not been sufficiently addressed. To rectify this problem, we have used fluorescence-activated cell sorting to purify germ cells from mouse fetal testes and ovaries and examined 16 common housekeeping genes for their suitability as reference genes. In pure populations of germ cells isolated from Embryonic Day 12.5 (E12.5) to E15.5 male and female gonads, Mapk1 and Sdha were identified as the most stable reference genes. Analysis of the heterogenous fraction of gonadal somatic cells revealed that Canx and Top1 were stable in both sexes, whereas a comparative analysis of germ and somatic cell populations identified Canx and Mapk1 as suitable reference genes through these developmental stages. Application of these reference genes to quantification of gene expression in developing gonads revealed that past assays, which employed nonverified reference genes, have in some cases provided misleading gene expression profiles. This study has identified suitable reference genes to directly compare expression profiles of genes expressed in germ and somatic cells of male and female fetal gonads. Application of these reference genes to expression analysis in fetal germ and somatic cells provides a more accurate system in which to profile gene expression in these tissues.

Fluorescence activated cell sorting of live female germ cells and somatic cells of the mouse fetal gonad based on forward and side scattering

Analysis of female mammalian germ cells has been hindered by difficulties in isolating high purity germ cell populations from embryonic and fetal gonads. Meiotic prophase stage oocytes are particularly difficult to isolate due to the lack of suitable surface markers. Oct4 promoter driven GFP expression has been used to distinguish germ cells/oocytes (GFP positive) from somatic cells (GFP negative), however, the requirement for transgenic animals has limited the use of this technique. We analyzed the side- and forward scattering properties of living cell populations obtained from fetal ovaries of Oct4-GFP transgenic and wild-type mice. On the basis of these measurements, we defined criteria that allow the discrimination and identification of germ cells and somatic cells within cell suspensions of nontransgenic female fetal gonads. The described method is suitable for the isolation of populations of germ cells and somatic cells of higher than 90% purity. We also demonstrated that the sorted cells can be used in downstream immunofluorescence and RT-PCR applications. Hence, we conclude that side and forward scattering based sorting of female germ cells is a valuable tool that will benefit the understanding of female gametogenesis.

ATRA and KL promote differentiation toward the meiotic program of male germ cells.

While it is known that Retinoic Acid (RA) induces meiosis in mouse female fetal gonads, the mechanisms which regulate this process during spermatogenesis are poorly understood. We show that the All trans RA derivative (ATRA) and Kit Ligand (KL) increase meiotic entry of postnatal mouse spermatogonia in vitro without synergism. Competence to enter meiosis is reached by spermatogonia only at the stage in which they undergo Kit-dependent divisions. Besides increasing Kit expression in spermatogonia, ATRA also upregulates KL expression in Sertoli cells. Both ATRA and KL increase the expression of Stimulated by Retinoic Acid Gene 8 and Dmc1, an early meiotic marker. A specific Kit tyrosine kinase inhibitor prevents the increase in the number of meiotic cells induced by both the two factors, suggesting that they converge on common Kit-dependent signalling pathways. Meiotic entry induced by ATRA and KL is independent from their ability to affect germ cell viability, and is mediated by the activation of PI3K and MAPK pathways through Kit autophosphorylation. ATRA-induced phosphorylation of the two downstream kinases is mediated by a non-genomic mechanism.These data suggest that RA may control the timing of meiosis by influencing both the somatic and the germ cell compartment of the postnatal testis through the activation of the KL/Kit system.

Conserved and divergent patterns of expression of DAZL, VASA and OCT4 in the germ cells of the human fetal ovary and testis.

BackgroundGerm cells arise from a small group of cells that express markers of pluripotency including OCT4. In humans formation of gonadal compartments (cords in testis, nests in ovary) takes place during the 1st trimester (6–8 weeks gestation). In the 2nd trimester germ cells can enter meiotic prophase in females whereas in males this does not occur until puberty. We have used qRTPCR, Westerns and immunohistochemical profiling to determine which of the germ cell subtypes in the human fetal gonads express OCT4, DAZL and VASA, as these have been shown to play an essential role in germ cell maturation in mice.ResultsOCT4 mRNA and protein were detected in extracts from both 1st and 2nd trimester ovaries and testes. In ovarian extracts a marked increase in expression of VASA and DAZL mRNA and protein occurred in the 2nd trimester. In testicular extracts VASA mRNA and protein were low/undetectable in 1st trimester and increased in the 2nd trimester whereas the total amount of DAZL did not seem to change. During the 1st trimester, germ cells were OCT4 positive but did not express VASA. These results are in contrast to the situation in mice where expression of Vasa is initiated in Oct4 positive primordial germ cells as they enter the gonadal ridge. In the 2nd trimester germ cells with intense cytoplasmic staining for VASA were present in both sexes; these cells were OCT4 negative. DAZL expression overlapped with both OCT4 and VASA and changed from the nuclear to the cytoplasmic compartment as cells became OCT4-negative. In males, OCT4-positive and VASA-positive subpopulations of germ cells coexisted within the same seminiferous cords but in the ovary there was a distinct spatial distribution of cells with OCT4 expressed by smaller, peripherally located, germ cells whereas DAZL and VASA were immunolocalised to larger (more mature) centrally located cells.ConclusionOCT4, DAZL and VASA are expressed by human fetal germ cells but their patterns of expression are temporally and spatially distinct. In the 1st trimester OCT4 was detected in most germ cells. In the 2nd trimester the onset of expression of VASA was associated with the formation of oocytes and spermatogonia both of which were OCT-4 negative. Relocation of DAZL from nucleus to cytoplasm paralleled the down regulation of OCT4 and the onset of expression of VASA. These data reveal similarities between the expression of key regulatory proteins within germ cells as they mature in male and female fetal human gonads suggesting that in the female these maturational changes are not determined by entry into meiosis.

Expression of AMH in Female Fetal Intersex Gonads in the Bovine

Anti Müllerian Hormone, AMH, is believed to be the main agent in the freemartin syndrome. Supposing an active role of freemartin gonads in AMH secretion, in the present study, we aimed at investigating the presence and the localization of AMH producing cells either in fetal or in adult freemartin gonads. Our finding of positive AMH cells in a 26-week-old freemartin fetus indicates an active role of masculinized freemartin gonads in AMH secretion. However, the positive reaction, limited to few cells grouped in 'nests' in proximity to testis cord-like structures, supports a chimeric origin of such cells, migrated from the male co-twin. No adult freemartin, irrespective from the degree of masculinization, showed any AMH positive cell.

Sex‐specific windows for high mRNA expression of DNA methyltransferases 1 and 3A and methyl‐CpG‐binding domain proteins 2 and 4 in human fetal gonads

DNA methyltransferases (DNMTs) and 5-methyl-CpG-binding domain proteins (MBDs) are involved in the acquisition of parent-specific epigenetic modifications in human male and female germ cells. Reverse Northern blot analyses demonstrated sex-specific differences in mRNA expression for the maintenance DNMT1 and the de novo DNMT3A in developing testis and ovary. In fetal testis DNMT1 and DNMT3A expression peaked in mitotically arrested spermatogonia around 21 weeks gestation. In fetal ovary transcriptional upregulation of DNMT1 and DNMT3A occurred during a very brief period at 16 weeks gestation, when the oocytes proceeded through meiotic prophase. Fetal gonads showed several fold higher DNMT3A expression levels than fetal brain and adult tissues. The most abundant DNMT3A isoform in fetal testis and ovary was DNMT3A2, whereas in all other analyzed tissues DNMT3A1 predominated. The catalytically inactive DNMT3A3 isoform was also present at relatively high levels in developing gonads and may perform a regulatory function(s). In both male and female fetal gonads expression of genes for MBD2 and MBD4, which may be implicated in chromatin remodeling of methylated genomic DNA sequences, was tightly linked to DNMT expression. We propose that the sex-specific time windows for concomitant upregulation of DNMT1, DNMT3A, MBD2, and MBD4 are associated with prenatal remethylation of the human male and female germ line.

Investigation of the fate of Sry‐expressing cells using an in vivo Cre/loxP system

Sry (sex-determining region on Y chromosome) is expressed in the undifferentiated, bipotential genital ridges of mammalian XY fetuses. The expression of Sry initiates testis development, but the lineage of Sry-expressing cells is unclear. In this study, double-transgenic mice were analyzed using the Cre/loxP system. Cre under the control of the Sry promoter was expressed in the fetal gonads of transgenic mice similarly to endogenous Sry. The Sry/Cre-transgenic mice were crossed with CAG(cytomegalovirus immediate-early enhancer, chicken beta-actin promoter and fusion intron of chicken beta-actin and rabbit beta-globin)/loxP/CAT/loxP/LacZ-transgenic mice, in which the transgene expressed beta-galactosidase after a Cre-mediated recombination event. Sertoli cells, germ cells of testes and granulosa cells of ovaries of double-transgenic mice stained positive with X-gal. Cre expression was detected in germ cells and peritubular/Sertoli cells in adult testes. It is not clear whether beta-galactosidase expression in the Sertoli cells of the testes occurred as a result of Cre expression in the adult or in the fetal gonads. These analyses indicate that cells expressing Sry-inducing factors in female fetal gonads become granulosa cells.

A histone H3 methyltransferase controls epigenetic events required for meiotic prophase

Epigenetic modifications of histones regulate gene expression and chromatin structure. Here we show that Meisetz (meiosis-induced factor containing a PR/SET domain and zinc-finger motif) is a histone methyltransferase that is important for the progression of early meiotic prophase. Meisetz transcripts are detected only in germ cells entering meiotic prophase in female fetal gonads and in postnatal testis. Notably, Meisetz has catalytic activity for trimethylation, but not mono- or dimethylation, of lysine 4 of histone H3, and a transactivation activity that depends on its methylation activity. Mice in which the Meisetz gene is disrupted show sterility in both sexes due to severe impairment of the double-stranded break repair pathway, deficient pairing of homologous chromosomes and impaired sex body formation. In Meisetz-deficient testis, trimethylation of lysine 4 of histone H3 is attenuated and meiotic gene transcription is altered. These findings indicate that meiosis-specific epigenetic events in mammals are crucial for proper meiotic progression.

Subcellular and molecular mechanisms regulating anti-Müllerian hormone gene expression in mammalian and nonmammalian species.

Anti-Müllerian hormone (AMH) is best known for its role as an inhibitor of the development of female internal genitalia primordia during fetal life. In the testis, AMH is highly expressed by Sertoli cells of the testis from early fetal life to puberty, when it is downregulated by the action of testosterone, acting through the androgen receptor, and meiotic spermatocytes, probably acting through TNFalpha. Basal expression of AMH is induced by SOX9; GATA4, SF1, and WT1 enhance SOX9-activated expression. When the hypothalamic-pituitary axis is active and the negative effect of androgens and germ cells is absent, for example, in the fetal and neonatal periods or in disorders like androgen insensitivity, FSH upregulates AMH expression through a nonclassical cAMP-PKA pathway involving transcription factors AP2 and NFkappaB. The maintenance and hormonal regulation of AMH expression in late fetal and postnatal life requires distal AMH promoter sequences. In the ovary, granulosa cells express AMH from late fetal life at low levels; DAX1 and FOG2 seem to be responsible for negatively modulating AMH expression. Particular features are observed in AMH expression in nonmammalian species. In birds, AMH is expressed both in the male and female fetal gonads, and, like in reptiles, its expression is not preceded by that of SOX9.

Wilms' tumor suppressor gene (WT1) as a target gene of SRY function in a mouse ES cell line transfected with SRY.

With the aim of identifying the gene(s) located downstream from SRY, we transfected an ES cell line with XX karyotype, TMA-18, with a Sry DNA construct and established cell lines, TS18-1 and TS18-2, where the transfected Sry was expressed in the functional linear mRNA form. Among the five potential SRY-target genes examined, i.e., MIS, SF1, P450arom, Sox9 and WT1, only the expression of WT1 was induced de novo by the unscheduled expression of Sry in the transfected cell lines. No clear indication of Sry-induced enhancement of Sox9 expression was obtained in the present series of experiments. Function of a yet unidentified gene(s) located on the Y chromosome might be needed for the up-regulation of Sox 9 expression which takes place during the development of male gonads. Quantitative RT-PCR analysis of the patterns of WT1 expression in developing fetal gonads revealed that although both male and female fetal gonads express WT1, male gonads invariably expressed WT1 mRNA at higher levels than female ones after the Sry expression. Immunohistochemical analysis of the male fetal gonads between 10.5 and 13.5 dpc demonstrated the presence of strong WT1 immunoreactivity in Sertoli cells of the primordial testes. Suggestions were made in the past indicating that both SF1 and WT1 proteins might be active in a common pathway upstream from Sry. Our results showed that WT1 is located downstream, rather than upstream from Sry and behaves independently from SF1. Analysis using an appropriate in vitro system will be essential to understand the molecular mechanisms of SRY action within cells.

Fetal androgens and sexual mimicry in spotted hyaenas (Crocuta crocuta).

Sexual differentiation of the Müllerian duct system and gonad in female fetuses conformed to the general mammalian pattern, whereas the external genital anlage in male and female fetuses developed into the male facies. Interstitial cells occurred in the primary germinal cords of both male and female fetal gonads and are suggested to be the source of androgen production in spotted hyaena fetuses. Maternal transfer of androgens to the fetus via the placenta was negligible. Male fetuses had higher gonadal and plasma concentrations of testosterone than female fetuses, and vice versa for androstenedione. Plasma testosterone was nearly as high in a female 31-day-old fetus as in the twin male, and masculinization of the genital tubercle probably results from an episode of androgen secretion by the fetal gonad although the Wolffian ducts in the female do not respond.