Targeted deletion of NR2F2 and VCAM1 in theca cells impacts ovarian follicular development: insights into polycystic ovary syndrome?†.

Defining features of polycystic ovary syndrome (PCOS) include elevated expression of steroidogenic genes, theca cell androgen biosynthesis, and peripheral levels of androgens. In previous studies, we identified vascular cell adhesion molecule 1 (VCAM1) as a selective androgen target gene in specific NR2F2/SF1 (+/+) theca cells. By deleting NR2F2 and VCAM1 selectively in CYP17A1 theca cells in mice, we documented that NR2F2 and VCAM1 impact distinct and sometimes opposing theca cell functions that alter ovarian follicular development in vivo: including major changes in ovarian morphology, steroidogenesis, gene expression profiles, immunolocalization images (NR5A1, CYP11A1, NOTCH1, CYP17A1, INSL3, VCAM1, NR2F2) as well as granulosa cell functions. We propose that theca cells impact follicle integrity by regulating androgen production and action, as well as granulosa cell differentiation/luteinization in response to androgens and gonadotropins that may underlie PCOS.

METTL14 mediates nerve growth factor-stimulated testosterone synthesis in porcine theca cells†.

Ovarian theca cells produce testosterone, which acts as a vital precursor substance for synthesizing estrogens during follicular development. Nerve growth factor (NGF) has been shown to participate in reproductive physiology, specifically to follicular development and ovulation. There is currently no available data on the impact of NGF on testosterone synthesis in porcine theca cells. Furthermore, m6A modification is the most common internal modification in eukaryotic mRNAs that are closely associated with female gametogenesis, follicle development, ovulation, and other related processes. It is also uncertain whether the three main enzymes associated with m6A, such as Writers, Erasers, and Readers, play a role in this process. The present study, with an in vitro culture model, investigated the effect of NGF on testosterone synthesis in porcine theca cells and the role of Writers-METTL14 in this process. It was found that NGF activates the PI3K/AKT signaling pathway through METTL14, which regulates testosterone synthesis in porcine theca cells. This study will help to further elucidate the mechanisms by which NGF regulates follicular development and provide new therapeutic targets for ovary-related diseases in female animals. Summary Sentence  The present study investigated the effect of NGF on testosterone synthesis in porcine theca cells. It was found that NGF activates the PI3K/AKT signaling pathway through METTL14, which regulates testosterone synthesis in porcine theca cells.

Characterization of ovarian progenitor cells for their potential to generate steroidogenic theca cells in vitro.

In brief: Progenitor cells with ovulation-related tissue repair activity were identified with defined markers (LGR5, EPCR, LY6A, and PDGFRA), but their potentials to form steroidogenic cells were not known. This study shows that the cells can generate progenies with different steroidogenic activities. Abstract: Adult mammalian ovaries contain stem/progenitor cells necessary for folliculogenesis and ovulation-related tissue rupture repair. Theca cells are recruited and developed from progenitors during the folliculogenesis. Theca cell progenitors were not well defined. The aim of current study is to compare the potentials of four ovarian progenitors with defined markers (LY6A, EPCR, LGR5, and PDGFRA) to form steroidogenic theca cells in vitro. The location of the progenitors with defined makers was determined by immunohistochemistry and immunofluorescence staining of ovarian sections of adult mice. Different progenitor populations were purified by magnetic-activated cell sorting (MACS) and/or fluorescence-activated cell sorting (FACS) techniques from ovarian cell preparation and were tested for their abilities to generate steroidogenic theca cells in vitro. The cells were differentiated with a medium containing LH, ITS, and DHH agonist for 12 days. The results showed that EPCR+ and LGR5+ cells primarily distributed along the ovarian surface epithelium (OSE), while LY6A+ cells distributed in both the OSE and parenchyma. However, PDGFRA+ cells were exclusively located in interstitial compartment. When the progenitors were purified by these markers and differentiated in vitro, LY6A+ and PDGFRA+ cells formed steroidogenic cells expressing both CYP11A1 and CYP17A1 and primarily producing androgens, showing characteristics of theca-like cells, while LGR5+ cells generated steroidogenic cells devoid of CYP17A1 expression and androgen production, showing a characteristic of progesterone-producing cells (granulosa- or lutea-like cells). In conclusion, progenitors from both OSE and parenchyma of adult mice are capable of generating steroidogenic cells with different steroidogenic capacities, showing a possible lineage preference.

Progesterone and 17-hydroxy-progesterone concentrations in follicular fluid and serum reflect their production in granulosa and theca cells.

RESEARCH QUESTION: How is the production of progesterone (P4) and 17-hydroxy-P4 (17-OH-P4) regulated between theca cells and granulosa cells during the follicular phase, during ovulation and after transformation into a corpus luteum? DESIGN: Three cohorts were examined: (i) 31 women undergoing natural and stimulated cycles, with serum hormone measurements taken every 3 days; (ii) 50 women undergoing ovarian stimulation, with hormone concentrations in serum and follicular fluid assessed at five time points during final follicle maturation; and (iii) 12 women undergoing fertility preservation, with hormone concentrations evaluated via the follicular fluid of small antral follicles. RESULTS: In the early follicular phase, theca cells primarily synthesized 17-OH-P4 while granulosa cells produced limited P4, maintaining the P4:17-OH-P4 ratio <1. As follicles reached follicle selection at a diameter of approximately 10 mm, P4 synthesis in granulosa cells was up-regulated, but P4 was mainly accumulated in follicular fluid. During final maturation, enhanced activity of the enzyme HSD3B2 in granulosa cells enhanced P4 production, with the P4:17-OH-P4 ratio increasing to >1. The concentration of 17-OH-P4 in the luteal phase was similar to that in the follicular phase, but P4 production increased in the luteal phase, yielding a P4:17-OH-P4 ratio significantly >1. CONCLUSIONS: The P4:17-OH-P4 ratio reflects the activity of granulosa cells and theca cells during the follicular phase and following luteinization in the corpus luteum. Managing the function of granulosa cells is key for reducing the concentration of P4 during ovarian stimulation, but the concerted action of FSH and LH on granulosa cells during the second half of the follicular phase makes this complex.

Novel Insight into the mechanism of di (2-ethylhexyl) phthalate (DEHP) impairing early follicle development.

Di (2-ethylhexyl) phthalate (DEHP), an artificially synthetic plasticizer, is a widespread environmental endocrine disruptor, which has raised substantial concern among the public about its potential reproductive toxicity effects. Taking large amounts of DEHP disrupts the normal functioning of the ovaries, however, the toxicological effects and the mechanisms by which DEHP impairs fetal folliculogenesis remain poorly understood. Our research aims to elucidate the associations between utero exposure to DEHP and fetal folliculogenesis in offspring. In this research, we monitored the spatiotemporal and expression levels of GDF9-Hedgehog (Hh) pathway-related genes during postnatal days 3-14, confirming initially the potential associations between defects in theca cell development and the downregulation of GDF9-Hh signaling. Moreover, utilizing an ovarian organ in vitro culture model, rescue validation experiments demonstrated that the addition of recombinant GDF9 protein effectively alleviate the theca cell damage caused by DEHP, thus supporting the aforementioned associations. In conclusion, our findings validate the significant role of the GDF9-Hh pathway in the enduring reproductive toxicity resulting from prenatal exposure to DEHP.

The Interstitial Gland as a Source of Pro- or Anti-Senescent Cells during Chinchilla Rabbit Ovarian Aging.

The aging ovary in mammals leads to the reduced production of sex hormones and a deterioration in follicle quality. The interstitial gland originates from the hypertrophy of the theca cells of atretic follicles and represents an accumulative structure of the ovary that may contribute to its aging. Here, reproductive and mature rabbit ovaries are used to determine whether the interstitial gland plays a crucial role in ovarian aging. We demonstrate that, in the mature ovary, interstitial gland cells accumulate lipid droplets and show ultrastructural characteristics of lipophagy. Furthermore, they undergo modifications and present a foamy appearance, do not express the pan-leukocyte CD-45 marker, and express CYP11A1. These cells are the first to present an increase in lipofuscin accumulation. In foamy cells, the expression of p21 remains low, PCNA expression is maintained at mature ages, and their nuclei do not show positivity for H2AX. The interstitial gland shows a significant increase in lipofuscin accumulation compared with the ovaries of younger rabbits, but lipofuscin accumulation remains constant at mature ages. Surprisingly, no accumulation of cells with DNA damage is evident, and an increase in proliferative cells is observed at the age of 36 months. We suggest that the interstitial gland initially uses lipophagy to maintain steroidogenic homeostasis and prevent cellular senescence.

Follicle development in pigs: State of the art.

Understanding the factors and pathways involved with recruitment, atresia, and selection of follicles in the pig, may provide insight into approaches to limit fertility failures. Antral follicles depend upon FSH to the 2-3 mm stage, become codependent upon LH at 4-5 mm, and rely on LH when >5 mm. Within the follicle, gonadotropin binding, steroids, growth factors, and inhibin interact to determine the fate of the follicle. Continuous recruitment appears likely for follicles, and once >1 mm, they may have a limited period for survival, before selection or atresia. If true, then the number of healthy follicles that can respond to a hormone signal for selection, could vary by size and development stage. Which follicles are selected may depend upon their age, numbers of capillaries, granulosa and thecal cells, and FSH and LH receptors. This might also suggest that factors such as management, nutrition, and stress in prior weeks, could affect different cohorts of follicles to determine which of those from the ovarian population will be selected.

Low temperature plasma protects against inflammatory agents-mediated dysfunction of theca cells via enhancing MANF expression.

BACKGROUND: Low temperature plasma (LTP) exerts a protective effect in inflammation via enhancing MANF expression. Hyperactivation and dysfunction of theca cells induced by inflammatory agents is accompanied by polycystic ovary syndrome (PCOS), which is a common reproductive and endocrine disorder. However, the effect of LTP on theca cells is still unknown. METHODS AND RESULTS: Theca cells were stimulated with IL-1ß or TNF-α for 12 h, then treated with LTP for 100 s. After 8 h, medium supernatant and theca cells were collected. Production of androgen from theca cells were detected by ELISA. The PCNA and Annexin V levels in theca cells were detected by using immunofluorescent staining. The levels of PCNA, BCL-2 and BAX were evaluated by western blot and qPCR. MTT assay was used to detect the viability of theca cells. The proportions of apoptosis of theca cells were detected by Flow cytometry. The mRNA levels of androgenic genes were detected by qPCR. The MANF levels in medium supernatant and cell lysate were detected by using ELISA, western and qPCR. BIP and CHOP expressions were detected by using western blot and qPCR. We found that LTP irradiation decreased inflammatory agents-induced upregulation of androgen and androgenic genes in theca cells. And LTP irradiation relieves IL-1ß or TNF-α-induced pathological proliferation and apoptosis in theca cells. In terms of mechanism, LTP irradiation increased MANF level in theca cells to inhibit BIP and CHOP expression. CONCLUSION: These evidences suggest the protective effect of LTP on theca cells in inflammatory microenvironment, and LTP has the potential clinical application of PCOS.

Sphingosine-1-phosphate regulation of luteinising hormone-induced steroidogenesis and proliferation of bovine theca cells in vitro.

CONTEXT: Sphingosine-1-phosphate (S1P) is synthesised by follicle granulosa cells under the influence of follicle-stimulating hormone and seems to be necessary for the biological effects of this gonadotrophin. AIMS: To determine if luteinising hormone (LH) increases S1P production and if this sphingolipid, either induced by LH or added to culture media, regulates steroidogenesis and cell viability in bovine theca cells. METHODS: We used bovine theca cell cultures treated with: S1P (0, 0.1, 1 and 10µM; Experiment 1), LH (0, 0.02, 0.2 and 2ngmL-1 ; Experiment 2) and LH (0.02ngmL-1 ) plus a sphingosine kinase inhibitor (SKI-178; 0, 5 and 10µM; Experiment 3). KEY RESULTS: Treatment with S1P did not affect (P >0.05) theca cell viability or their ability to produce progesterone and testosterone. LH (0.02ngmL-1 ) increased (P <0.05) S1P production, and stimulated the expression of phosphorylated sphingosine kinase-1 (pSPHK1). However, the inhibition of SPHK1, by a specific SPHK1 inhibitor (SKI-178), reduced (P <0.05) cell viability and progesterone secretion. Additionally, the use of SKI-178 increased theca cell testosterone production (P<0.05). CONCLUSIONS: S1P added to culture media did not affect cell viability or steroid synthesis. However, LH stimulated the production of S1P, by increasing phosphorylation of SPHK1 in theca cells. This intracellular S1P was inhibitory on testosterone production but augmented progesterone and viable cell number. IMPLICATIONS: These results suggest a novel signalling pathway for LH in theca cells and underline the importance of S1P in the regulation of steroid synthesis.

Co-culture of human cryopreserved fragmented ovarian tissue with theca progenitor cells derived from theca stem cells.

PURPOSE: Despite the significant advances in the in vitro development of human primordial follicles, it is still a challenging approach with great potential for improvements. Therefore, the present study aimed to investigate the effect of a feeder layer of human theca progenitor cells (hTPCs) on the development of primordial follicles embedded in human ovarian tissue. METHODS: Fragments of frozen-thawed ovarian tissue were activated using the vanadate-derivative dipotassium bisperoxo (5-hydroxy-pyridine-2-carboxylic) oxovanadate (V) and kit ligand for 24 h. Then, the specimens were divided into the co-culture and mono-culture groups and were cultured with and without a hTPC feeder layer for 6 days, respectively. Afterward, the follicles were counted and classified, and the hormone levels and expression levels of apoptosis- and folliculogenesis-related genes were assessed. RESULTS: Both culture groups showed significant follicle growth (P < 0.05). However, the co-culture group had a significantly higher number of growing follicles compared to the other group (P < 0.05). Moreover, the expression levels of ZP1, ZP2, ZP3, BMP-7, AMH, and GDF9 were significantly higher in the co-culture group compared to the other group (P < 0.05), while the expression levels of P53 and CASP3 were significantly lower (P < 0.05). Also, the concentrations of estradiol, progesterone, testosterone, and androstenedione were significantly higher in the co-culture group compared to the other group (P < 0.05). CONCLUSION: The present study results provided novel evidence on the direct role of hTPCs in the growth and development of human primordial follicles. However, there is a need for future studies to illustrate the underlying mechanisms. Schematic summary of the results. According to our results, the expression of ZP1, ZP2, ZP3, and GDF9 in the oocytes, AMH in the granulosa cells, and BMP4 in the theca cells of the co-culture group were significantly higher than those of the mono-culture and non-culture groups, while the expression of apoptotic genes (BAX, CASP3, and P53) was significantly lower. Moreover, the co-culture group showed significantly increased levels of estradiol, progesterone, testosterone, and androstenedione in its culture media compared to the mono-culture groups.

Classification of Atretic Small Antral Follicles in the Human Ovary.

The reproductive lifespan in humans is regulated by a delicate cyclical balance between follicular recruitment and atresia in the ovary. The majority of the small antral follicles present in the ovary are progressively lost through atresia without reaching dominance, but this process remains largely underexplored. In our study, we investigated the characteristics of atretic small antral follicles and proposed a classification system based on molecular changes observed in granulosa cells, theca cells, and extracellular matrix deposition. Our findings revealed that atresia spreads in the follicle with wave-like dynamics, initiating away from the cumulus granulosa cells. We also observed an enrichment of CD68+ macrophages in the antrum during the progression of follicular atresia. This work not only provides criteria for classifying three stages of follicular atresia in small antral follicles in the human ovary but also serves as a foundation for understanding follicular degeneration and ultimately preventing or treating premature ovarian failure. Understanding follicular remodeling in the ovary could provide a means to increase the number of usable follicles and delay the depletion of the follicular reserve, increasing the reproductive lifespan.

Endothelial cell-derived fibroblast growth factor-18 regulates ovarian function in sheep.

Increasing the efficiency of farm animal reproduction is necessary to reduce the environmental impact of food production systems. One approach is to increase the number of healthy eggs (oocytes) produced per female for fertilization, thus it is important to understand factors that decrease oocyte health. One paracrine factor that decreases ovarian follicle growth is fibroblast growth factor 18 (FGF18) secreted by cells in the theca layer of the ovarian follicle, however the factors that regulate FGF18 secretion are unknown. In this study we hypothesized that FGF18 secretion is controled by intrafollicular factors and is linked to fertility, which we tested by using cell culture and sheep genetic models in vivo. Separation of theca cell populations revealed that FGF18 messenger RNA (mRNA) is located mainly in thecal endothelial rather than endocrine cells, and immunohistochemistry localized FGF18 protein to microvessels in the theca layer in situ. Culture of ovine theca-derived endothelial cells was used to demonstrate stimulation of FGF18 mRNA and protein abundance by bone morphogenetic protein 4 (BMP4), a growth factor derived from theca endocrine cells. Taking advantage of a sheep genetic model, we demonstrate reduced ovarian and peripheral FGF18 concentrations in the hyperprolific Booroola ewe harboring the FecBB mutation in BMPR1B. These data suggest a novel control of fertility by follicular endothelial cells, in which theca endocrine cells secrete BMP4 that stimulates the secretion of FGF18 from thecal endothelial cells, which in turn diffuses into the granulosa cell layer and promotes apoptosis.

Follicle isolation methods reveal plasticity of granulosa cell steroidogenic capacity during mouse in vitro follicle growth.

Follicles are the functional unit of the ovary and several methods have been developed to grow follicles ex vivo, which recapitulate key events of oogenesis and folliculogenesis. Enzymatic digestion protocols are often used to increase the yield of follicles from the ovary. However, the impact of these protocols on the outermost theca and granulosa cells, and thereby follicle function, is not well defined. To investigate the impact of enzymatic digestion on follicle function, we collected preantral follicles from CD1 mice either by enzymatic digestion (Enzy-FL) or mechanical isolation (Mech-FL) and compared follicle growth, steroidogenesis and cell differentiation within an encapsulated in vitro follicle growth system which maintains the 3D architecture of the oocyte and its surrounding somatic cells. Follicles were encapsulated in 0.5% alginate and cultured for 8 days. Compared with Enzy-FL, Mech-FL grew more rapidly and produced significantly higher levels of androstenedione, estradiol and progesterone. The expression of theca-interstitial cell marker genes, Cyp17a1, which encodes 17-hydroxylase/17, 20-lyase and catalyzes the hydroxylation of pregnenolone and progesterone to 17-hydroxypregnenolone and 17-hydroxyprogesterone, and the conversion of these products into dehydroepiandrosterone and androstenedione, and Star, which encodes a transport protein essential for cholesterol entry into mitochondria, were also higher in Mech-FL than in Enzy-FL. Mech-FL maintained an intact theca-interstitial layer on the outer edge of the follicle that phenocopied in vivo patterns as confirmed by alkaline phosphatase staining, whereas theca-interstitial cells were absent from Enzy-FL from the onset of culture. Therefore, preservation of the theca cell layer at the onset of culture better supports follicle growth and function. Interestingly, granulosa cells in the outermost layers of Enzy-FL expressed CYP17A1 by Day 4 of culture while maintaining inhibin α-subunit expression and a cuboidal nucleus. Thus, in the absence of theca-interstitial cells, granulosa cells have the potential to differentiate into androgen-producing cells. This work may have implications for human follicle culture, where enzymatic isolation is required owing to the density of the ovarian cortex.

Heat shock proteins exhibit distinct spatiotemporal expression patterns in the domestic cat (Felis catus) ovary during the oestrous cycle.

Heat shock proteins (HSP) are significant regulators of cell proliferation, differentiation and apoptosis. HSP participate in ovarian physiology through proliferative and apoptotic mechanisms and the modulation of sex steroid receptor functions. We investigated whether the expression and localisation patterns of HSP in the domestic cat ovary vary with the oestrous cycle stage. Immunohistochemical analysis revealed cell type-specific localisation patterns of HSPD1/HSP60, HSPA/HSP70, HSPC/HSP90 and HSPH/HSP105 in several ovarian cells of the domestic cat, including oocytes, follicular (granulosa and theca cells) and luteal cells, stromal and thecal interstitial cells, stromal cells, and vascular endothelial and smooth muscle cells during the anoestrous, follicular and luteal phases of the oestrous cycle. Western blot results showed that the expression of three HSP (HSPD1/HSP60, HSPA/HSP70 and HSPH/HSP105) varied with the oestrous cycle stage. While the maximal expression of HSPD1/HSP60 and HSPH/HSP105 occurred during the luteal phase, the expression of HSPA/HSP70 was minimal. The expressions of HSPA/HSP70 and HSPH/HSP105 were low during the follicular phase compared to the anoestrous phase. In conclusion, the alterations that occur in the expression of HSP in the domestic cat ovary during the different stages of the oestrous cycle imply that these proteins participate in the regulation of ovarian function under different physiological conditions.

Transcriptional regulation of CYP19A1 expression in chickens: ESR1, ESR2 and NR5A2 form a functional network.

Aromatase, encoded by CYP19A1, is responsible for the conversion of androgen to estrogen, which plays a vital role in the development and function of the ovary and functions in many other physiological processes in both sexes. Instead of being expressed in ovarian granulosa cells, as in mammals, CYP19A1 is expressed in chickens in the theca cells of ovarian follicles, and the mechanism of CYP19A1 expression regulation remains unknown. Here, using immunofluorescence and western blotting assay, we first confirmed that CYP19A1 and FOXL2 (Forkheadbox L2) were coexpressed in pre-granulosa cells of female chicken embryonic gonads, while FOXL2 did not affect aromatase expression at embryonic stages. Second, our research showed that CYP19A1, ESR1 (estrogen receptor alpha), ESR2 (estrogen receptor beta) and NR5A2 (liver receptor homologue-1) were coexpressed in the theca cell layers of chicken small yellow follicles. There was cross-talk between CYP19A1 and candidate transcription factors (ESR1, ESR2 and NR5A2), which was identified by generating a reliable theca cell culture model. Using luciferase assays in theca cells and chicken embryonic fibroblast (DF-1) cells, the results suggested that ESR1 and NR5A2 had potential effects on CYP19A1 promoter activity in chickens. Overexpression of ESR1, ESR2 and NR5A2 in chicken embryonic fibroblast (DF-1) cells upregulated the protein expression of CYP19A1, mutually restricted each other and formed a potential regulatory network to coordinate the expression of CYP19A1. To conclude, our results indicated that FOXL2 cannot regulate the expression of CYP19A1 at chicken embryonic stages and after sexual maturity, ESR1, ESR2 and NR5A2 form a functional network to affect the expression of CYP19A1. These results laid a foundation for further research on the transcriptional regulation of chicken aromatase.

Effects of IL-18 on the proliferation and steroidogenesis of bovine theca cells: Possible roles in the pathogenesis of polycystic ovary syndrome.

Interleukin 18 (IL-18) is a pleiotropic pro-inflammatory cytokine and is associated with arrested follicle development and anovulation which are the typical pathological changes of PCOS. Theca cells (TCs) have a key role in follicular growth and atresia. But whether IL-18 can directly affect ovarian TCs function is unknown. Therefore, the objective of this study was to determine the effect of IL-18 on proliferation and steroidogenesis of bovine TCs and to explore the biological effect of IL-18 on folliculogenesis. This work revealed that at 300-1000 pg/mL, IL-18 led to a time- and dose-dependently increase in cell proliferation (P < .05). IL-18 increased 17-hydroxyprogesterone (17OHP4) and androstenedione (A2) secretion with up-regulation of key steroidogenesis-related genes CYP11A1 and CYP17A1 (P < .05). Furthermore, our data demonstrated that the IL-18R protein is predominantly expressed in small-follicle (3-6 mm) TCs than large follicles (8-22 mm) by immunohistochemistry. We also found that the stimulation effects of IL-18 on TCs can be reversed with the addition of IL-18BP as early as at 4 hours of culture and reached the peak at 16 hours. We conclude that IL-18 appears to target TCs in bovine, and suggest an important role for this cytokine in ovarian function. Present findings further validate potential effects of IL-18 in the conditions associated with follicular dysplasia and excessive growth of ovarian TCs (such as PCOS). But additional research is needed to further understand the mechanism of action of IL-18 in theca cells as well as its precise role in folliculogenesis.

Effect of the spatial-temporal specific theca cell Cyp17 overexpression on the reproductive phenotype of the novel TC17 mouse.

BACKGROUND: In the ovarian follicle, the Theca Cells (TCs) have two main functions: preserving morphological integrity and, importantly, secreting steroid androgen hormones. TCs express the essential enzyme 17α-hydroxylase/17,20-desmolase (CYP17), which permits the conversion of pregnenolone and progesterone into androgens. Dysregulation of CYP17 enzyme activity due to an intrinsic ovarian defect is hypothesized to be a cause of hyperandrogenism in women. Androgen excess is observed in women with polycystic ovary syndrome (PCOS) resulting from excess endogenous androgen production, and in transgender males undergoing exogenous testosterone therapy after female sex assignment at birth. However, the molecular and morphological effects of Cyp17 overexpression and androgen excess on folliculogenesis is unknown. METHODS: In this work, seeking a comprehensive profiling of the local outcomes of the androgen excess in the ovary, we generated a transgenic mouse model (TC17) with doxycycline (Dox)-induced Cyp17 overexpression in a local and temporal manner. TC17 mice were obtained by a combination of the Tet-dependent expression system and the Cre/LoxP gene control system. RESULTS: Ovaries of Dox-treated TC17 mice overexpressed Cyp17 specifically in TCs, inducing high testosterone levels. Surprisingly, TC17 ovarian morphology resembled the human ovarian features of testosterone-treated transgender men (partially impaired folliculogenesis, hypertrophic or luteinized stromal cells, atretic follicles, and collapsed clusters). We additionally assessed TC17 fertility denoting a perturbation of the normal reproductive functions (e.g., low pregnancy rate and numbers of pups per litter). Finally, RNAseq analysis permitted us to identify dysregulated genes (Lhcgr, Fshr, Runx1) and pathways (Extra Cellular Matrix and Steroid Synthesis). CONCLUSIONS: Our novel mouse model is a versatile tool to provide innovative insights into study the effects of Cyp17 overexpression and hyperandrogenism in the ovary.

Cells responding to hedgehog signaling contribute to the theca of ovarian follicles.

Cell-fate mapping was used to identify cells that respond to the hedgehog (HH) signaling pathway and that are incorporated into the theca cell layer during ovarian follicle development. Expression of Gli1 is increased by HH signaling and can be used as a marker of cells responsive to HH in reporter mice. In transgenic Gli1ERcre/tdT mice, injection of tamoxifen (TAM) induces cre-mediated recombination and expression of td tomato (tdT) which leads to permanent fluorescent marking of cells expressing Gli1 and their progeny. The identity of tdT-positive cells was determined by co-staining ovaries for endothelial cells (CD31), pericytes (CSPG4), vascular smooth muscle cells (VSMC; smooth muscle actin) and steroidogenic cells (cytochrome P450 17A1). Gli1ERcre/tdT mice were injected with TAM on the day of birth. Cells positive for tdT in 2-day-old mice were identified as pericytes, located primarily in the medulla of the ovary in close proximity to endothelial cells. In both prepubertal mice and adult mice treated with equine chorionic gonadotropin to induce the formation of preovulatory follicles, tdT-positive cells were located within the theca cell layer and were identified as pericytes, VSMC and steroidogenic theca cells. Granulosa cells are known to express two HH ligands, Indian HH and desert HH (DHH). In DHHcre/tdT reporter mice, endothelial cells were marked as tdT-positive indicating that endothelial cells, in addition to granulosa cells, express Dhh in the ovary. These findings suggest that HH signaling may stimulate the development of the vasculature along with steroidogenic capacity of the theca layer during follicle development.

The immunoexpression patterns of fibroblast growth factors in the pregnant and postpartum rat ovary.

Fibroblast growth factors (FGFs) are polypeptides involved in the regulation of oogenesis and folliculogenesis by inducing ovarian mitogenic, homeostatic and angiogenic activity. This study was aimed at determining the localisation of FGF ligands (FGF1 and FGF2) and FGF receptor 2 (FGFR2) in the rat ovary by immunohistochemical analyses, at pregnancy and the postpartum period. During pregnancy and the postpartum period, positive FGF1 immunoreactions were observed in the nucleus and cytoplasm of germinative epithelial cells, granulosa cells of follicles in different developmental stages, theca interna cells, interstitial cells, luteal cells and atretic follicles. FGF2 immunoreactivity was strong in the cytoplasm of the endothelial cells and smooth muscle cells of the ovarian blood vessels and in the smooth muscle cells of the ovarian cortex and medulla. Strong FGFR2 immunoreactivity was observed in the stromal cells surrounding the blood vessels and rete ovarii. Immunoreaction intensity of the FGF1, FGF2 and FGFR2 had relatively similar abundances between the periods examined. Considering that FGFs act as local regulators in oogenesis, folliculogenesis, follicular atresia, ovulation, corpus luteum formation and regression and angiogenesis, this study supports the idea that FGFs may also be involved in these physiological functions in rat ovaries during pregnancy and postpartum period.

Prenatal programming by testosterone of follicular theca cell functions in ovary.

In mammalian ovaries, the theca layers of growing follicles are critical for maintaining their structural integrity and supporting androgen synthesis. Through combining the postnatal monitoring of ovaries by abdominal magnetic resonance imaging, endocrine profiling, hormonal analysis of the follicular fluid of growing follicles, and transcriptomic analysis of follicular theca cells, we provide evidence that the exposure of ovine fetuses to testosterone excess activates postnatal follicular growth and strongly affects the functions of follicular theca in adulthood. Prenatal exposure to testosterone impaired androgen synthesis in the small antral follicles of adults and affected the expression in their theca cells of a wide array of genes encoding extracellular matrix components, their membrane receptors, and signaling pathways. Most expression changes were uncorrelated with the concentrations of gonadotropins, steroids, and anti-Müllerian hormone in the recent hormonal environment of theca cells, suggesting that these changes rather result from the long-term developmental effects of testosterone on theca cell precursors in fetal ovaries. Disruptions of the extracellular matrix structure and signaling in the follicular theca and ovarian cortex can explain the acceleration of follicle growth through altering the stiffness of ovarian tissue. We propose that these mechanisms participate in the etiology of the polycystic ovarian syndrome, a major reproductive pathology in woman.