Fetal Ovaries Research Articles

Characterization of the effects of the steroidogenesis inhibitor Ketoconazole on the human fetal ovary

Characterization of the effects of the steroidogenesis inhibitor Ketoconazole on the human fetal ovary

Stem Cells in Adult Mice Ovaries Form Germ Cell Nests, Undergo Meiosis, Neo-oogenesis and Follicle Assembly on Regular Basis During Estrus Cycle.

Very small embryonic-like (VSELs) and ovarian (OSCs) stem cellsare located in adult mammalian ovary surface epithelium (OSE). OSCs can expand long-term and differentiate into oocyte-like structures in vitro and have resulted in birth of fertile pups. Lineage tracing studies have provided evidence to suggest OSCs differentiation into oocytes in vivo. But how these stem cells function under normal physiological conditions has not yet been well worked out. Besides studying STRA-8 and SCP-3 expression in enzymatically isolated OSE cells smears, mice were injected BrdU to track mitosis, meiosis and follicle assembly. H&E stained OSE cells during late diestrus and proestrus showed VSELs undergoing asymmetrical cell divisions to give rise to slightly bigger OSCs which in turn underwent symmetrical cell divisions followed by clonal expansion (rapid expansion with incomplete cytokinesis) during early estrus to form germ cell nests (GCN). OCT-4, SSEA-1, MVH and DAZL positive cells in GCN expressed Erα, Erβ and FSHR, were interconnected by ring canals (TEX-14), showed mitochondrial aggregation (Cytochrome C) and Balbiani Body (TRAL). Apoptosis in 'nurse' cells was marked by PARP and putative oocytes were clearly visualized. BrdU was detected in cells undergoing mitosis/meiosis and also in an oocyte of secondary follicle. FACS sorted, green fluorescent protein (GFP) positive VSELs upon transplantation resulted in GFP positive GCN suggesting crucial role for VSELs in adult ovaries. Results suggest that various events described during oogenesis and follicle assembly in fetal ovaries are recapitulated on regular basis in adult ovary and result in the formation of follicles.

ZSWIM7 Is Associated With Human Female Meiosis and Familial Primary Ovarian Insufficiency.

BackgroundPrimary ovarian insufficiency (POI) affects 1% of women and is associated with significant medical consequences. A genetic cause for POI can be found in up to 30% of women, elucidating key roles for these genes in human ovary development.ObjectiveWe aimed to identify the genetic mechanism underlying early-onset POI in 2 sisters from a consanguineous pedigree.MethodsGenome sequencing and variant filtering using an autosomal recessive model was performed in the 2 affected sisters and their unaffected family members. Quantitative reverse transcriptase PCR (qRT-PCR) and RNA sequencing were used to study the expression of key genes at critical stages of human fetal gonad development (Carnegie Stage 22/23, 9 weeks post conception (wpc), 11 wpc, 15/16 wpc, 19/20 wpc) and in adult tissue.ResultsOnly 1 homozygous variant cosegregating with the POI phenotype was found: a single nucleotide substitution in zinc finger SWIM-type containing 7 (ZSWIM7), NM_001042697.2: c.173C > G; resulting in predicted loss-of-function p.(Ser58*). qRT-PCR demonstrated higher expression of ZSWIM7 in the 15/16 wpc ovary compared with testis, corresponding to peak meiosis in the fetal ovary. RNA sequencing of fetal gonad samples showed that ZSWIM7 has a similar temporal expression profile in the developing ovary to other homologous recombination genes.Main conclusionsDisruption of ZSWIM7 is associated with POI in humans. ZSWIM7 is likely to be important for human homologous recombination; these findings expand the range of genes associated with POI in women.

Sexual Differentiation and Primordial Germ Cell Distribution in the Early Horse Fetus.

Simple SummaryIn horses, gonadal development and sexual differentiation occur during early fetal life. This is accompanied by primordial germ cell differentiation and migration to the gonad site. However, little is known about the time when these processes take place and the mechanisms behind them. Additionally, no information is available regarding number and distribution of primordial germ cells in the equine gonad. During development and differentiation, gonads can be affected by stressors causing infertility, but this condition can only be diagnosed in post-pubertal animals. Herein we show that equine gonads develop asynchronously between male and female fetuses, and that the number and distribution of primordial germ cells is influenced by fetal sex. By day 45 of pregnancy, equine gonads were clearly differentiated, while migration of primordial germ cells still occurred at this stage. No sign of genital duct regression was seen until day 60 of pregnancy.It was the aim of this study to characterize the development of the gonads and genital ducts in the equine fetus around the time of sexual differentiation. This included the identification and localization of the primordial germ cell population. Equine fetuses between 45 and 60 days of gestation were evaluated using a combination of micro-computed tomography scanning, immunohistochemistry, and multiplex immunofluorescence. Fetal gonads increased in size 23-fold from 45 to 60 days of gestation, and an even greater increase was observed in the metanephros volume. Signs of mesonephros atrophy were detected during this time. Tubular structures of the fetal testes were present from day 50 onwards, whereas cell clusters dominated in the fetal ovary. The genital ducts were well-differentiated and presented a lumen in all samples. No sign of mesonephric or paramesonephric duct degeneration was detected. Expression of AMH was strong in the fetal testes but absent in ovaries. Irrespective of sex, primordial germ cells selectively expressed LIN28. Migration of primordial germ cells from the mesonephros to the gonad was detected at 45 days, but not at 60 days of development. Their number and distribution within the gonad were influenced (p < 0.05) by fetal sex. Most primordial germ cells (86.8 ± 3.2% in females and 84.6 ± 4.7% in males) were characterized as pluripotent according to co-localization with CD117. However, only a very small percentage of primordial germ cells were proliferating (7.5 ± 1.7% in females and 3.2 ± 1.2% in males) based on co-localization with Ki67. It can be concluded that gonadal sexual differentiation in the horse occurs asynchronously with regard to sex but already before 45 days of gestation.

P–624 Doxorubicin exposure detrimentally effects establishment of early ovarian reserve, ex-vivo mouse model

Abstract Study question Does exposure to chemotherapy agents used to treat cancer during pregnancy cause significant decrease in early establishment of the ovarian reserve? Summary answer Significant oocyte loss is noted following exposure of early mouse ovaries to doxorubicin. This apoptotic decline is not seen with cisplatin, docetaxel or paclitaxel exposure. What is known already Chemotherapy has been shown to adversely affect the ovarian reserve of pre-pubertal and reproductive age females. However, little is known regarding the effects in the next generation following treatment in pregnancy beyond observational population studies that examined rates of birth defects and developmental delays. Reproductive function in offspring develops over time and would be difficult to quantify in these short-term studies. An ex-vivo mouse ovary culture offers the ability to closely mimic maternal treatment level serum concentrations and directly evaluate the impact on oocyte number and cell death markers. Study design, size, duration In mice, the ovarian reserve is matured postnatally, mimicking the biologic second trimester activity of the human ovary. Wild-type C57bl/6 pups were collected on postnatal day 0. Ovaries were cultured in hanging well organ culture media with addition of DMSO or a chemotherapy agent. Immunofluorescence was used to quantify oocyte number and density. Power calculation showed a N of 11 per drug would be needed to demonstrate a decrease of ⅔ or more. Participants/materials, setting, methods: 83 ovaries were cultured, sectioned and analyzed in duplicate. Planned analysis at serum max and mid concentrations was performed at 48 hours and 5 days. Given clinically variation in concentrations of cisplatin, additional concentration samples were added. After noting the degradation following exposure to doxorubicin, additional samples at earlier time points of 12 and 24 hours of exposure were added for dynamic evaluation. Means were calculated and then compared using a 2-way ANOVA. Main results and the role of chance Doxorubicin exposure during establishment of the ovarian reserve resulted in a significant loss of oocyte number and density. At 12 hours a 22% decrease was noted; this increased to a loss of 91% of oocytes by 48 hours of exposure. The oocyte density fell from 693 oocytes/ mm² in the control to only 63 oocytes/ mm² in the serum max concentration (p = 0.003). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) revealed early impact within the stroma by 12 hours with widespread apoptotic changes by 24 hours. Treatment with cisplatin resulted in a phenotypic change in the oocyte population with preservation of smaller, more peripheral cells. The average oocyte density remained similar to control even at the highest clinical concentrations, 536 oocytes/ mm² compared to 570 oocytes/ mm² (p = 0.772). Docetaxel and paclitaxel demonstrated an increase in oocyte number and density, though not enough to reach significance. The oocyte density 5 days following docetaxel exposure was 802 oocytes/ mm², a 41% increase (p = 0.12). The oocyte density 5 days following paclitaxel exposure was 780 oocytes/ mm², a 37% increase (p = 0.817). The drug exposure did impact stromal cells, as noted in TUNEL images. Limitations, reasons for caution This ex-vivo mouse model offers tight control of chemotherapy concentration, it does not account for filtration and modification by the placenta. Longer term cultures may also demonstrate that temporary arrest in small oocytes, such as those exposed to cisplatin, do not thrive and later progress to apoptosis. Wider implications of the findings: Doxorubicin is employed as the most common chemotherapy during pregnancy. Utilization may be to the serious detriment of the younger generation. If other alternatives are clinically effective they should be considered. No other models have explored the effect of fetal ovary exposure to chemotherapy on the establishment of ovarian reserve. Trial registration number Not applicable

TCF21+ mesenchymal cells contribute to testis somatic cell development, homeostasis, and regeneration in mice

Testicular development and function rely on interactions between somatic cells and the germline, but similar to other organs, regenerative capacity declines in aging and disease. Whether the adult testis maintains a reserve progenitor population remains uncertain. Here, we characterize a recently identified mouse testis interstitial population expressing the transcription factor Tcf21. We found that TCF21lin cells are bipotential somatic progenitors present in fetal testis and ovary, maintain adult testis homeostasis during aging, and act as potential reserve somatic progenitors following injury. In vitro, TCF21lin cells are multipotent mesenchymal progenitors which form multiple somatic lineages including Leydig and myoid cells. Additionally, TCF21+ cells resemble resident fibroblast populations reported in other organs having roles in tissue homeostasis, fibrosis, and regeneration. Our findings reveal that the testis, like other organs, maintains multipotent mesenchymal progenitors that can be potentially leveraged in development of future therapies for hypoandrogenism and/or infertility.

Maternal protein restriction affects fetal ovary development in sheep.

SummaryMaternal malnutrition has important developmental consequences for the foetus. Indeed, adverse fetal ovarian development could have lifelong impact, with potentially reduced ovarian reserve and fertility of the offspring. This study investigated the effect of maternal protein restriction on germ cell and blood vessel development in the fetal sheep ovary. Ewes were fed control (n = 7) or low protein (n = 8) diets (17.0 g vs 8.7 g crude protein/MJ metabolizable energy) from conception to day 65 of gestation (gd65). On gd65, fetal ovaries were subjected to histological and immunohistochemical analysis to quantify germ cells (OCT4, VASA, DAZL), proliferation (Ki67), apoptosis (caspase 3) and vascularisation (CD31). Protein restriction reduced the fetal ovary weight (P < 0.05) but had no effect on fetal weight (P > 0.05). The density of germ cells was unaffected by maternal diet (P > 0.05). In the ovarian cortex, OCT4+ve cells were more abundant than DAZL+ve (P < 0.001) and VASA+ve cells (P < 0.001). The numbers, density and estimated total weight of OCT4, DAZL, and VASA+ve cells within the ovigerous cords were similar in both dietary groups (P > 0.05). Similarly, maternal protein restriction had no effect on germ cell proliferation or apoptotic indices (P > 0.05) and the number, area and perimeter of medullary blood vessels and degree of microvascularisation in the cortex (P > 0.05). In conclusion, maternal protein restriction decreased ovarian weight despite not affecting germ cell developmental progress, proliferation, apoptosis, or ovarian vascularity. This suggests that reduced maternal protein has the potential to regulate ovarian development in the offspring.Lay summaryVariations in a mother’s diet during pregnancy can influence her offspring’s growth and might cause fertility problems in the offspring in later life. We investigated whether reducing the protein fed to sheep during early pregnancy affects their daughters’ ovaries. We then compared our findings to the offspring of sheep on a complete diet. We measured ovary size and estimated the number of germ cells (cells that become eggs) they contained. We used cell markers to assess potential changes in the pattern of germ cell growth, division, and death, and how the ovarian blood supply had developed. We found that protein restriction reduced ovary size. However, the pattern of germ cell development, growth, or death was not altered by poor diet and blood vessels were also unaffected. This suggests that maternal diet can change ovarian development by an unknown mechanism and might reduce future fertility in their offspring.

Altered germline cyst formation and oogenesis in Tex14 mutant mice.

ABSTRACTDuring oocyte differentiation in mouse fetal ovaries, sister germ cells are connected by intercellular bridges, forming germline cysts. Within the cyst, primary oocytes form via gaining cytoplasm and organelles from sister germ cells through germ cell connectivity. To uncover the role of intercellular bridges in oocyte differentiation, we analyzed mutant female mice lacking testis-expressed 14 (TEX14), a protein involved in intercellular bridge formation and stabilization. In Tex14 homozygous mutant fetal ovaries, germ cells divide to form a reduced number of cysts in which germ cells remained connected via syncytia or fragmented cell membranes, rather than normal intercellular bridges. Compared with wild-type cysts, homozygous mutant cysts fragmented at a higher frequency and produced a greatly reduced number of primary oocytes with precocious cytoplasmic enrichment and enlarged volume. By contrast, Tex14 heterozygous mutant germline cysts were less fragmented and generate primary oocytes at a reduced size. Moreover, enlarged primary oocytes in homozygous mutants were used more efficiently to sustain folliculogenesis than undersized heterozygous mutant primary oocytes. Our observations directly link the nature of fetal germline cysts to oocyte differentiation and development.

In utero Exposure to Excessive Estrogen Impairs Homologous Recombination and Oogenesis via Estrogen Receptor 2 in Mice.

The association between the accumulation of synthetic chemicals with estrogenic activity and risks to oogenesis has become a growing concern. This study indicates that in utero estrogen exposure can affect homologous recombination in early oogenesis and influence the reproductive potential and lifespan of female offspring. We conducted this study in developing mouse ovaries using two different models: oral doses administered to the mother, and fetal ovary cultures. Our analyses of meiotic fetal oocytes suggest that 17-β-estradiol induces gross aberrations in prophase I events, including delayed meiotic progression, increased unrepaired DNA damage, and altered homologous recombination levels. These effects were mainly mediated by estrogen receptor 2 (ESR2) activation. Mid-gestation exposure to estrogen also led to delayed primordial folliculogenesis after birth, impaired follicle development after prepuberty, and ultimately reduced the total litter size of the offspring. This raises the concern that maternal exposures to substances activating ESR2 may compromise the fertility of the exposed female fetus.

Immunuhistochemical features of the ovaries’ structure in case of fetuses with a gestational term of 21-28 weeks from mother, whose pregnancy was complicated by chronic infections of lower genital tracts

The following article presents results of the complex research on postulation of immunohistochemical features of the ovaries’ structure in case of fetuses with a gestational term of 21-28 weeks from mothers with HILGT comparing to ones in case of fetuses from mothers with a physiological pregnancy. The materials of the research are: 25 fetuses from mothers with HILGT as well as 25 fetuses from mothers with physiological pregnancy. All fetuses had died antenatally and inranatally, as a result of violation of uteroplacental and umbilical-uterine circulation. Research methods: macroscopic, organometric, histological, immunohistochemical, morphometric, statistical. The organometric method revealed a clear decrease of main indexes of the fetuses’ ovaries from mothers with a complicated pregnancy comparing to ones in case of organs of fetuses from healthy mothers. Morphometric method revealed a clear decrease of a relative volume of cortical substance as well as a clear increase of a relative volume of brain substance in the gonads of fetuses from the main group; as well as it showed a clear decrease of an index of follicular tissue’ relative volume, likewise a clear increase of an index of interstitial component relative volume.The staining by picrofuxin by van Gieson method revealed a massive growth of the connective tissue in all strucutral components of organs of fetuses from mothers with HILGT relatively to gonads of fetuses from healthy mothers. Using of MCAT to main types of collagens allowed to reveal a violation of maturation of main types of collagens in the structure of connective tissue of fetal gonads in the main group. In the follicular component of the fetuses’ ovaries in the main group are appearing mature, cystic-artretic and growing forms of follicles. Immunohistochemical method revealed an increased number of an apoptotically altered eggs in the ovaries of fetuses from mothers with HILGT comparing to one in organs of fetuses from mothers with physiological pregnancy. The peroxidase method revealed an increased endotheline-producing activity of the vascular component as arterial, like a venous type; likewise a clear decrease of the hormone-producing activity in the ovaries of fetuses from mothers with HILGT comparing to such indexes in fetal organs of fetuses from mothers with physiological pregnancy. The complex of changes in the ovaries of fetuses with a 21-28 weeks of gestational term, which was described, was formed under the influence of the chronic hypoxia, prolonged antigenic stimulation as well as hormonal violations in the mother-placenta-fetus system, which is a leading link of the infectious pathology’ pathogenesis. The aforementioned complex of the structural and functional changes in the ovaries of fetuses from mothers with HILGT in a gestational term of 21-28 weeks indicates violations in implementation and formation of the fetal gonads, as well as it could further lead to disorders of the germinal function in the female organism.

Long non-coding RNA Xist regulates oocyte loss via suppressing miR-23b-3p/miR-29a-3p maturation and upregulating STX17 in perinatal mouse ovaries

The fecundity of female mammals is resolved by the limited size of the primordial follicle (PF) pool formed perinatally. The establishment of PF pool is accompanied by a significant programmed oocyte death. Long non-coding RNAs (lncRNA) are central modulators in regulating cell apoptosis or autophagy in multiple diseases, however, the significance of lncRNAs governing perinatal oocyte loss remains unknown. Here we find that Yin-Yang 1 (YY1) directly binds to the lncRNA X-inactive-specific transcript (Xist) promoter and facilitates Xist expression in the perinatal mouse ovaries. Xist is highly expressed in fetal ovaries and sharply downregulated along with the establishment of PF pool after birth. Gain or loss of function analysis reveals that Xist accelerates oocyte autophagy, mainly through binding to pre-miR-23b or pre-miR-29a in the nucleus and preventing the export of pre-miR-23b/pre-miR-29a to the cytoplasm, thus resulting in decreased mature of miR-23b-3p/miR-29a-3p expression and upregulation miR-23b-3p/miR-29a-3p co-target, STX17, which is essential for timely control of the degree of oocyte death in prenatal mouse ovaries. Overall, these findings identify Xist as a key non-protein factor that can control the biogenesis of miR-23b-3p/miR-29a-3p, and this YY1-Xist-miR-23b-3p/miR-29a-3p-STX17 regulatory axis is responsible for perinatal oocyte loss through autophagy.

Expression Patterns of Analgesic Metabolising Machinery in 1st and 2nd Trimester Human Fetal Liver and Gonads

Use of over-the-counter analgesics during pregnancy is widespread globally. Most analgesic compounds can freely diffuse through the placental feto-maternal interface and reach the developing fetus. Current literature suggests an endocrine disruptor (ED) potential of in utero exposure to these compounds. The liver is the primary site of contact with EDs in the fetus. Exposure of the fetal gonads can also alter reproductive function with potential intergenerational effects. We aimed to characterise the metabolic capability of these fetal organs. RNA sequencing was performed in 80 second trimester human fetal livers and 48 fetal gonads (balanced for fetal age and fetal sex). Samples were collected from elective terminations of normal pregnancies (liver 11-19 weeks, FeGo study: REC 04/S0802/21, and gonads 6-17 weeks, as previously described1. RNA was extracted and Illumina NextSeq was used to produce 76 bp single end (liver) or paired end 2x50 bp (gonads) sequencing reads. Reads were quality controlled, aligned to the human reference genome and quantified at gene regions. Statistical analyses involved an ANOVA model of two-way interactions between fetal sex and fetal age. All organs expressed phase I and II metabolising enzymes and drug transporters involved in the pharmacokinetic and pharmacodynamic pathways of over-the-counter analgesics. The human fetal liver expressed ABCC2, ABCC3, ABCC4 and ABCG2 receptors at similar levels between males and females. Expression of cytochrome p450 enzymes CYP2A6, CYP2C8, CYP2C9, CYP2E1, CYP3A4 involved in metabolism of the analgesics paracetamol and ibuprofen, all increased with gestational age in the liver. Expression of GSTM1, GSTP1, GSTT1, SULT1A1, SULT1A3, SULT1A4, SULT1E1, SULT2A1, UGT2B4, UGT2B7 and UGT2B15 metabolising enzymes also increased during gestation, while fetal hepatic GSTP1 expression showed a significant 2-way interaction between both sex and age. Fetal gonads expressed ABCC4 and ABCG2 transporters, with transcript levels demonstrating significant sex-specific and gestational age differences. Fewer analgesic metabolising enzymes were expressed in the gonads than the fetal liver, including CYP2E1, GSTP1 and SULT1A1, all significantly altered by gestation and fetal sex. Our results reveal expression of major analgesics metabolic and transport components within the human fetal liver, ovaries and testes between gestation weeks 7-19. Significant sex alterations in transcript levels also suggest sexually dimorphic metabolic activity of these organs during fetal life. In conclusion, analgesics can be transported into fetal liver and gonad cells and metabolised into bioactive forms, posing toxicity risks for the developing fetus.1. Lecluze E, Rolland AD, Filis P, et al. Dynamics of the transcriptional landscape during human fetal testis and ovary development. Hum Reprod. 2020;35(5):1099-1119.

Immunohistochemical Detection of Cancer-Testis Antigen PRAME

Cancer-testis (CT) antigens were identified by their ability to elicit T- or B-cell immune responses in the autologous host. They are typically expressed in a wide variety of neoplasms and in normal adult tissues are restricted to testicular germ cells. PReferentially expressed Antigen of Melanoma (PRAME) is a member of the family of nonclassical CT antigens being expressed in a few other normal tissues besides testis. Interestingly, knowledge about the protein expression of many CT antigens is still incomplete due to the limited availability of reagents for their immunohistochemical detection. Here, we tested several commercially available serological reagents and identified a monoclonal antibody suitable for the immunohistochemical detection of PRAME in formalin-fixed paraffin-embedded specimens. We also tested a wide array of normal and neoplastic tissues. PRAME protein expression in normal tissues is congruent with original molecular data being present in the testis, and at low levels in the endometrium, adrenal cortex, and adult as well as fetal ovary. In tumors, there is diffuse PRAME immunoreactivity in most metastatic melanomas, myxoid liposarcomas, and synovial sarcomas. Other neoplasms such as seminomas and carcinomas of various origins including endometrial, serous ovarian, mammary ductal, lung, and renal showed an intermediate proportion of cases and variable extent of tumor cells positive for PRAME protein expression. As seen with other CT antigens, hepatocellular and colorectal carcinoma, Leydig cell tumors, mesothelioma, and leiomyosarcoma are poor expressers of PRAME.

Intercellular bridges coordinate the transition from pluripotency to meiosis in mouse fetal oocytes.

Meiosis is critical to generating oocytes and ensuring female fertility; however, the mechanisms regulating the switch from mitotic primordial germ cells to meiotic germ cells are poorly understood. Here, we implicate intercellular bridges (ICBs) in this state transition. We used three-dimensional in toto imaging to map meiotic initiation in the mouse fetal ovary and revealed a radial geometry of this transition that precedes the established anterior-posterior wave. Our studies reveal that appropriate timing of meiotic entry across the ovary and coordination of mitotic-meiotic transition within a cyst depend on the ICB component Tex14, which we show is required for functional cytoplasmic sharing. We find that Tex14 mutants more rapidly attenuate the pluripotency transcript Dppa3 upon meiotic initiation, and Dppa3 mutants undergo premature meiosis similar to Tex14 Together, these results lead to a model that ICBs coordinate and buffer the transition from pluripotency to meiosis through dilution of regulatory factors.

Abnormal early folliculogenesis due to impeded pyruvate metabolism in mouse oocytes†.

Fetal ovarian germ cells show characteristic energy metabolism status, such as enhanced mitochondrial metabolism as well as glycolysis, but their roles in early folliculogenesis are unclear. We show here that inhibition of pyruvate uptake to mitochondria by UK5099 in organ cultures of fetal mouse ovaries resulted in repressed early folliculogenesis without affecting energy production, survival of oocytes, or meiosis. In addition, the abnormal folliculogenesis by UK5099 was partially rescued by α-ketoglutarate and succinate, intermediate metabolites in the TCA cycle, suggesting the importance of those metabolites. The expression of TGFβ-related genes Gdf9 and Bmp15 in ovarian germ cells, which are crucial for folliculogenesis, was downregulated by UK5099, and the addition of recombinant GDF9 partially rescued the abnormal folliculogenesis induced by UK5099. We also found that early folliculogenesis was similarly repressed, as in the culture, in the ovaries of a germ cell-specific knockout of Mpc2, which encodes a mitochondria pyruvate carrier that is targeted by UK5099. These results suggest that insufficient Gdf9 expression induced by abnormal pyruvate metabolism in oocytes results in early follicular dysgenesis, which is a possible cause of defective folliculogenesis in humans.

Histochemical features of the structure of fetal ovaries in different stages of gestation from mothers with physiological pregnancy

In the following article specific hystochemical features of the fetal ovaries’ structure from mothers with the physiological pregnancy are considered. All fetals were divided into subgroups by taking into consideration the term of gestation, as well as main stages of fetal gonads’ formation and laying. Namely: 21-28 weeks, 29-36 weeks, 37-40 weeks. All fetuses had died intranatally, as a result of acute uterine-placental circulation. The course of pregnancy in all cases was psychological, according to appropriate medical documentation. In the aim of reaching the scientific goal the following research methods were used: macroscopic, organometric, histological, histochemichal, morphometric, statistical. The comprehensive study has revealed the following features of the ovaries’ structure, depending on gestational term: weight, length, thickness, width and volume of the ovaries were reaching their minimum levels in case of fetuses with a gestational age of 21-28 weeks, while the maximum one was reached in case of fetuses on 37-42 weeks of gestation. All gonads are represented by cortical and cerebral matter, with the constant ratio despite of different gestational term. According to the growth of pregnancy term the number of germ cells decreases, while the number of apoptotically altered forms oppositely increases. Primarily it is because of the psychological cells’ death during ovarian formation.The follicular component at all stages of gestation is represented by primordial and primary follicles; at the 37-40 weeks though the atretic forms and cystically altered specimens are appearing. Moreover, in accordance with the growth of the pregnancy term, the number of primordial follicles decreases, while oppositely, the number of primary ones increases. The aforementioned changes lead to the decrease in the relative volume of follicular tissue and to the increase of the relative volume of the interstitial component. By the following histochemical methods (Felgen-Rossenbeck reaction, Brache reaction, Schiff-reaction) an increase in the functional activity of the fetal ovaries was revealed, by reaching its maximum point during gestation of the 37-40 weeks. The aforementioned features correspond with gestational terms as well as with stages of the ovarian development of the fetus. That is why they could be used as a control group during leading a research on characteristical features of fetal gonads structure of fetuses from mothers with complicated pregnancies.

New insights into the GDF9-Hedgehog-GLI signaling pathway in human ovaries: from fetus to postmenopause.

Are glioma-associated oncogene homolog 1, 2, and 3 (GLI1, 2, and 3) and protein patched homolog 1 (PTCH1) specific markers for precursor theca cells in human ovaries as in mouse ovaries? To study the GDF9-HH-GLI pathway and assess whether GLI1 and 3 and PTCH1 are specific markers for precursor theca cells in the human ovary, growth differentiation factor 9 (GDF9), Indian Hedgehog (IHH), Desert Hedgehog (DHH), Sonic Hedgehog (SHH), PTCH1 and GLI1, 2 and 3 were investigated in fetal (n=9), prepubertal (n=9), reproductive-age (n=15), and postmenopausal (n=8) human ovarian tissue. Immunohistochemistry against GDF9, IHH, DHH, SHH, PTCH1, GLI1, GLI2, and GLI3 was performed on human ovarian tissue sections fixed in 4% formaldehyde and embedded in paraffin. Western blotting was carried out on extracted proteins from the same samples used in the previous step to prove the antibodies' specificity. The quantitative real-time polymerase chain reaction was performed to identify mRNA levels for Gdf9, Ihh, Gli1, Gli2, and Gli3 in menopausal ovaries. Our results showed that, in contrast to mice, all studied proteins were expressed in primordial follicles of fetal, prepubertal, and reproductive-age human ovaries and stromal cells of reproductive-age and postmenopausal ovaries. Intriguingly, Gdf9, Ihh, and Gli3 mRNA, but not Gli1 and 2, was detected in postmenopausal ovaries. Moreover, GLI1, GLI3, and PTCH1 are not limited to a specific population of cells. They were spread throughout the organ, which means they are not specific markers for precursor theca cells in human ovaries. These results could provide a basis for understanding how this pathway modulates follicle development and ovarian cell steroidogenesis in human ovaries.

Blocking estrogen-induced AMH expression is crucial for normal follicle formation.

ABSTRACTIn mammals, primordial follicles assembled in fetuses or during infancy constitute the oocyte resources for life. Exposure to 17beta-estradiol and phytogenic or endocrine-disrupting chemicals during pregnancy and/or the perinatal period leads to the failure of normal follicle formation. However, the mechanisms underlying estrogen-mediated abnormal follicle formation and physiological follicle formation in the presence of endogenous natural estrogen are not well understood. Here, we reveal that estrogen receptor 1, activated by estrogen, binds to the 5′ region of the anti-Mullerian hormone (Amh) gene and upregulates its transcription before follicle formation in cultured mouse fetal ovaries. Ectopic expression of AMH protein was observed in pregranulosa cells of these explants. Furthermore, the addition of AMH to the culture medium inhibited normal follicle formation. Conversely, alpha-fetoprotein (AFP) produced in the fetal liver reportedly blocks estrogen action, although its role in follicle formation is unclear. We further demonstrated that the addition of AFP to the medium inhibited ectopic AMH expression via estrogen, leading to successful follicle formation in vitro. Collectively, our in vitro experiments suggest that upon estrogen exposure, the integrity of follicle assembly in vivo is ensured by AFP.

In-utero exposure to HT-2 toxin affects meiotic progression and early oogenesis in foetal oocytes by increasing oxidative stress.

HT-2 toxin (HT-2), a mycotoxin produced by Fusarium species, is detected in a variety of cereal grain-based human food and animal feed. Apart from its well-established immunotoxicity and haematotoxicity, it also causes reproductive disorders. In the present study, we revealed the adverse effects of HT-2 on early oogenesis at the foetal stage. Pregnant mice were orally administered with HT-2 for 3 days at mid-gestation. Oocytes from female foetuses exposed to HT-2 displayed defects in meiotic prophase, including unrepaired DNA damage, elevated recombination levels, and reduced expression of meiotic-related genes. Subsequently, increased oxidative stress was observed in the foetal ovaries exposed to HT-2, along with the elevated levels of reactive oxygen species, malondialdehyde, catalase, and superoxide dismutase 1/2, thereby resulting in impaired mitochondrial membrane potential and cell apoptosis. Furthermore, pre-treatment with urolithin A, a natural compound with antioxidant activities, partially reversed the delayed meiotic process by alleviating oxidative stress. Since early oogenesis is essential to determine female fertility in adult life, this study indicated that brief maternal exposure to HT-2 toxin may compromise the fertility of a developing female foetus.

Identification of regulatory elements required for Stra8 expression in fetal ovarian germ cells of the mouse

In mice, the entry of germ cells into meiosis critically depends on the expression of stimulated by retinoic acid gene 8 (Stra8). Stra8 is expressed specifically in pre-meiotic germ cells of females and males, at fetal and postnatal stages respectively, but the mechanistic details of its spatiotemporal regulation are yet to be defined. In particular, there has been considerable debate regarding whether retinoic acid is required, in vivo, to initiate Stra8 expression in the mouse fetal ovary. We show that the distinctive anterior-to-posterior pattern of Stra8 initiation, characteristic of germ cells in the fetal ovary, is faithfully recapitulated when 2.9 kb of the Stra8 promoter is used to drive eGFP expression. Using in vitro transfection assays of cut-down and mutant constructs we identified two functional retinoic acid responsive elements (RAREs) within this 2.9 kb regulatory element. We also show that the transcription factor DMRT1 enhances Stra8 expression, but only in the presence of RA and the most proximal RARE. Finally, we used CRISPR/Cas9-mediated targeted mutation studies to demonstrate that both RAREs are required for optimal Stra8 expression levels, in vivo.