Novel Plasma Membrane Androgen Receptor SLC39A9 Mediates Ovulatory Changes in Cells of the Monkey Ovarian Follicle.

Follicular androgens are important for successful ovulation and fertilization. The classical nuclear androgen receptor (AR) is a transcription factor expressed in the cells of the ovarian follicle. Androgen actions can also occur via membrane androgen receptor SLC39A9. Studies in fish ovary demonstrated that androgens bind to SLC39A9 and increase intracellular zinc to regulate ovarian cell function. To determine if SLC39A9 is expressed and functional in the key cell types of the mammalian ovulatory follicle, adult female cynomolgus macaques underwent ovarian stimulation. Ovaries or ovarian follicular aspirates were harvested at 0, 12, 24, and 36 hours after human chorionic gonadotropin (hCG). SLC39A9 and AR mRNA and protein were present in granulosa, theca, and vascular endothelial cells across the entire 40-hour ovulatory window. Testosterone, bovine serum albumin-conjugated testosterone (BSA-T), and androstenedione stimulated zinc influx in granulosa, theca, and vascular endothelial cells. The SLC39A9-selective agonist (-)-epicatechin also stimulated zinc influx in vascular endothelial cells. Taken together, these data support the conclusion that SLC39A9 activation via androgen induces zinc influx in key ovarian cells. Testosterone, BSA-T, and androstenedione each increased proliferation in vascular endothelial cells, indicating the potential involvement of SLC39A9 in ovulatory angiogenesis. Vascular endothelial cell migration also increased after treatment with testosterone, but not after treatment with BSA-T or androstenedione, suggesting that androgens stimulate vascular endothelial cell migration through nuclear AR but not SLC39A9. The presence of SLC39A9 receptors and SLC39A9 activation by follicular androstenedione concentrations suggests that androgen activation of ovarian SLC39A9 may regulate ovulatory changes in the mammalian follicle.

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.

Evaluation of Lipids and Lipid-Related Transcripts in Human and Ovine Theca Cells and an in Vitro Mouse Model Exposed to the Obesogen Chemical Tributyltin.

BACKGROUND: Exposure to obesogenic chemicals has been reported to result in enhanced adipogenesis, higher adipose tissue accumulation, and reduced ovarian hormonal synthesis and follicular function. We have reported that organotins [tributyltin (TBT) and triphenyltin (TPT)] dysregulate cholesterol trafficking in ovarian theca cells, but, whether organotins also exert lipogenic effects on ovarian cells remains unexplored. OBJECTIVE: We investigated if environmentally relevant exposures to organotins [TBT, TPT, or dibutyltin (DBT)] induce lipid dysregulation in ovarian theca cells and the role of the liver X receptor (LXR) in this effect. We also tested the effect of TBT on oocyte maturation and neutral lipid accumulation, and lipid-related transcript expression in cumulus cells and preimplantation embryos. METHODS: Primary theca cell cultures derived from human and ovine ovaries were exposed to TBT, TPT, or DBT (1, 10, or 50 ng/ml). The effect of these chemical exposures on neutral lipid accumulation, lipid abundance and composition, lipid homeostasis-related gene expression, and cytokine secretion was evaluated using liquid chromatography-mass spectrometry (LC-MS), inhibitor-based methods, cytokine secretion, and lipid ontology analyses. We also exposed murine cumulus-oocyte complexes to TBT and evaluated oocyte maturation, embryo development, and lipid homeostasis-related mRNA expression in cumulus cells and blastocysts. RESULTS: Exposure to TBT resulted in higher intracellular neutral lipids in human and ovine primary theca cells. In ovine theca cells, this effect was dose-dependent, independent of cell stage, and partially mediated by LXR. DBT and TPT resulted in higher intracellular neutral lipids but to a lesser extent in comparison with TBT. More than 140 lipids and 9 cytokines were dysregulated in TBT-exposed human theca cells. Expression of genes involved in lipogenesis and fatty acid synthesis were higher in theca cells, as well as in cumulus cells and blastocysts exposed to TBT. However, TBT did not impact the rates of oocyte maturation or blastocyst development. DISCUSSION: TBT induced dyslipidemia in primary human and ovine theca cells, which may be responsible for some of the TBT-induced fertility dysregulations reported in rodent models of TBT exposure. https://doi.org/10.1289/EHP13955.

In vitro effects of two environmental toxicants, beauvericin and glyphosate in Roundup, on cell numbers and steroidogenesis of bovine ovarian cells.

Beauvericin is an emerging Fusariotoxin naturally occurring in cereal grains throughout the world whereas glyphosate (N-phosphonomethyl-glycine) is a non-selective systemic herbicide used worldwide. The purpose of this study is to evaluate a newly developed ovarian cell culture system (that includes both granulosa and theca cells) as an in vitro model for toxicological studies. Specifically, the effects of beauvericin and glyphosate in formulation with Roundup on ovarian cell numbers and steroid production were evaluated. Ovaries collected from cattle without luteal structures were sliced into 30-70 pieces each, and granulosa and theca cells were collected. Harvested cells were cultured for 48 h in 10% fetal bovine serum-containing medium followed by 48 h in serum-free medium containing testosterone (500 ng/mL; as an estrogen precursor) with the following eight treatments: (1) controls, (2) FSH (30 ng/mL) alone, (3) FSH plus insulin-like growth factor-1 (IGF1; 30 ng/mL), (4) FSH plus IGF1 plus beauvericin (3 µM), (5) FSH plus IGF1 plus glyphosate in Roundup (10 µg/mL), (6) FSH plus IGF1 plus fibroblast growth factor 9 (FGF9, 30 ng/mL), (7) a negative control without added testosterone, and (8) IGF1 plus LH (30 ng/mL) with basal medium without added testosterone. In the presence of FSH, IGF1 significantly increased cell numbers, estradiol and progesterone production by severalfold. Glyphosate in Roundup formulation significantly inhibited IGF1-induced cell numbers and estradiol and progesterone production by 89-94%. Beauvericin inhibited IGF1-induced cell numbers and estradiol and progesterone by 50-97% production. LH plus IGF1 significantly increased androstenedione secretion compared with controls without added testosterone indicating the presence of theca cells. In conclusion, the present study demonstrates that toxicological effects of beauvericin and glyphosate in Roundup formulation are observed in a newly developed ovarian cell model system and further confirms that both glyphosate and beauvericin may have the potential to impair reproductive function in cattle.

Increased oxidative stress is associated with hyperandrogenemia in polycystic ovary syndrome evidenced by oxidized lipoproteins stimulating rat ovarian androgen synthesis in vitro.

PURPOSE: To determine the relationship between serum total testosterone (TT) levels and oxidative stress indices in patients with polycystic ovary syndrome (PCOS), and to investigate the effect of oxidative stress on androgen synthesis and its mechanism in rat ovarian theca-interstitial (T-I) cells. METHODS: Clinical, hormonal, metabolic, and oxidative stress parameters were analyzed in a cross-sectional case-control study including 626 patients with PCOS and 296 controls. The effects of oxidized low-density lipoprotein (ox-LDL) and oxidized high-density lipoprotein (ox-HDL) on cell proliferation, TT secretion, and expression of key enzymes involved in testosterone synthesis were evaluated in T-I cells. RESULTS: Serum TT levels were elevated with an increase in ox-LDL levels, whereas glutathione concentrations were lower in the high-TT subgroup than in the low-TT subgroup. The average ovarian volume and ox-LDL and malondialdehyde levels were significant predictors of TT levels in the multivariate regression models. In a rat ovarian T-I cell model, lipoprotein and oxidized lipoprotein treatments stimulated proliferation and promoted testosterone secretion. The mRNA and protein levels of 17α-hydroxylase were significantly higher in oxidized lipoprotein-treated cells than those in lipoprotein-treated cells. The mRNA levels of cholesterol side chain cleavage enzyme and steroidogenic acute regulatory protein were also significantly higher in ox-HDL-treated cells than in HDL-treated cells. CONCLUSIONS: Oxidative stress can promote androgen production by up-regulating the expression of testosterone synthesis-related enzymes in vitro and may be an essential factor in elevating serum TT levels in patients with PCOS.

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.

Effects of exogenous energy on synthesis of steroid hormones and expression characteristics of the CREB/StAR signaling pathway in theca cells of laying hen.

Energy and the cAMP-response element binding protein (CREB)/steroidogenic acute regulatory protein (StAR) signaling pathway play important roles in steroid hormone production and follicular development in hens. This present study aimed to investigate the effects of exogenous energy on the synthesis of steroid hormones and the expression characteristics of the CREB/StAR signaling pathway in theca cells of laying hen. The primary theca cells of small yellow follicles were randomly divided into 6 treatments and cultured in medium with glucose concentrations of 1, 1.5, 3, 4.5, 6, and 7.5 mg/mL for 48 h. It was found that growth was robust and cell outlines were clear when cells were cultured with 1, 1.5, 3, and 4.5 mg/mL glucose, but cell viability was diminished and cell density decreased after exposure to glucose at 6 and 7.5 mg/mL for 48 h. Cell viability showed an increasing and then decreasing quadratic response to increasing glucose concentration in culture (r2 = 0.688, P < 0.001). The cell viability of theca cells cultured with 4.5 mg/mL glucose was greater than those cultured with 1, 1.5, 6, and 7.5 mg/mL glucose (P < 0.05). The concentration of estradiol in the medium containing 3 mg/mL glucose was higher than in medium containing 1, 1.5, and 6 mg/mL glucose (P < 0.05). There was an increasing and then decreasing quadratic correlation between progesterone concentrations and glucose concentrations (r2 = 0.522, P = 0.002). The concentration of progesterone in medium with 4.5 mg/mL glucose was higher than in medium with 1 and 7.5 mg/mL glucose (P < 0.05). There was an increasing and then decreasing quadratic correlation between the relative expression of CREB1 (r2 = 0.752, P < 0.001), StAR (r2 = 0.456, P = 0.002), CYP1B1 (r2 = 0.568, P < 0.001), and 3ß-HSD (r2 = 0.319, P = 0.018) in theca cells of laying hens and glucose concentrations after treatment with different glucose concentrations for 48 h. After treatment with 4.5 mg/mL glucose, the expression of StAR, CYP1B1, and 3ß-HSD genes were increased compared to treatment with 1, 1.5, 3, 6, and 7.5 mg/mL glucose (P < 0.001). There was an increasing and then decreasing quadratic correlation between glucose concentrations and protein expression of CREB1 (r2 = 0.819, P < 0.001), StAR (r2 = 0.844, P < 0.001), 3ß-HSD (r2 = 0.801, P < 0.001), and CYP11A1 (r2 = 0.800, P < 0.001) in theca cells of laying hens. The protein expression of CREB1, StAR, and 3ß-HSD in theca cells cultured with 4.5 mg/mL glucose was higher than in other groups (P < 0.001). The results indicate that the appropriate glucose concentration (4.5 mg/mL) can improve the synthesis of steroid hormones in theca cells of laying hens through the upregulation of key genes and proteins in the CREB/StAR signaling pathway.

Expression of genes and enzymes involved in ovarian steroidogenesis in relation to human follicular development.

Introduction: Granulosa cells (GCs) and theca cells (TCs) play a pivotal role in human ovarian steroidogenesis, facilitating the conversion of cholesterol into sex steroids that regulate normal reproductive function. This study aims to explore the expression patterns of key enzymes that govern human ovarian steroidogenesis throughout follicle development, employing both genomic and immunological methodologies. Methods: Follicles and GCs obtained from women undergoing ovarian tissue cryopreservation (OTC) and in vitro fertilisation treatment were utilized. Gene expression data were obtained from a Chinese study using RNA sequencing and from microarray data generated in our laboratory to comprehensively analyse gene expression profiles across distinct stages of follicular development. To corroborate the localisation of key enzymes within GCs and TCs, immunohistochemistry analyses utilizing colourimetric and fluorescent techniques were conducted. Results: Steroidogenesis-related enzymes displayed low gene expression levels during early follicle development. However, a notable upregulation of HSD3B2 was observed in GCs as follicles progressed to the antral/preovulatory stage, confirmed consistently using both microarray and RNA sequencing methodologies. Furthermore, immunohistochemical analyses effectively demonstrated that HSD3B2 were not only expressed in GCs, but co-localised with CYP17A1 within a specific subset of TCs surrounding human small antral follicles. Contributing to an enhanced progesterone production during the second half of the follicular phase was a significant upregulation of CYB5A in both microarray and RNA-seq datasets as follicles transition from the antral stage to the pre-ovulatory stage. Moreover, an augmented expression of DHCR24 and LDLR in both types of data, along with HMGCR expression expression in the microarray data, indicates increased substrate availability for ovarian steroidogenesis. Discussion: This study confirms and extends that GCs gradually augment expression of HSD3B2 thereby enhancing their capacity for progesterone synthesis as follicles reach the size of selection at around 10 mm in diameter. This is supported by the expression CYB5A and possibly augmented availability of steroid precursors. A subset of TCs exhibit concurrent expression of CYP17A1 and HSD3B2, collectively contributing to the synthesis of 17-hydroxyprogesterone. These data significantly enhance our understanding of the dynamic regulation of progesterone throughout the process of follicular development.

Comparing ovarian expression of sperm acrosome associated 3 protein in young and adult queens.

The purpose of this research was to quantify sperm acrosome associated 3 protein expression in the ovaries of young (3.0 ± 0.9 months, n = 11) and adult (10.4 ± 2.8 months, n = 11) queens. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded feline ovarian sections. Ovaries were obtained following routine ovariohysterectomy of queens. Cellular expression of sperm acrosome associated 3 protein was measured in primordial, primary, secondary, and tertiary follicles using an image-analysis software's red, green, and blue stack and manual thresholding functions. The oocyte nucleus, ooplasm, granulosa cells, and theca cells were outlined using the freehand selection tool and mean grey value was recorded. Results from each cellular location were compared between age groups using a Student's t-test and between follicle stages using an analysis of variance. Compared to adult queens, younger queens had significantly greater sperm acrosome associated 3 protein expression in granulosa cells of primary, secondary, and tertiary follicles. Also, theca cells of secondary and tertiary follicles had significantly greater sperm acrosome associated 3 protein expression in younger queens compared to adult queens. The oocyte nucleus of primordial, primary, and secondary follicles had significantly greater sperm acrosome associated 3 protein expression in younger queens compared to adult queens. However, sperm acrosome associated 3 protein expression within the ooplasm did not differ significantly between age groups of any follicle type. More research is needed to determine what role sperm acrosome associated 3 protein may play in female fertility in animals as well as what mechanisms regulate ovarian sperm acrosome associated 3 protein expression over time.

Real-life exposure to Fusarium toxins deoxynivalenol and zearalenone triggers apoptosis and activates NLRP3 inflammasome in bovine primary theca cells.

Cattle are deemed less susceptible to mycotoxins due to the limited internal exposure resulting from rumen microbiota activity. However, the significant amounts of Fusarium mycotoxins deoxynivalenol (DON) and zearalenone (ZEN) frequently detected in bovine follicular fluid samples suggest that they could affect ovarian function. Both mycotoxins trigger several patterns of cell death and activate the NLRP3 inflammasome in the intestine. In vitro studies have reported a number of adverse effects on bovine oocytes. However, the biological relevance of such findings with regard to realistic concentrations of DON and ZEN in bovine follicular fluid is still not clear. Hence, it is important to better characterize the effects of dietary exposure to DON and ZEN on the bovine ovary. Using bovine primary theca cells, this study investigated the effects of real-life patterns for bovine ovary exposure to DON and ZEN, but also DON metabolite DOM-1, on cell death and NLRP3 inflammasome activation. Exposure to DON starting from 0.1 µM significantly decreased theca cell viability. The kinetics of phosphatidylserine translocation and loss of membrane integrity showed that ZEN and DON, but not DOM-1, induce an apoptotic phenotype. qPCR analysis of the expression of NLRP3, PYCARD, IL-1ß, IL-18, and GSDMD in primary theca cells at concentrations of mycotoxin previously reported in cow follicular fluid clearly indicated that DON and DOM-1 individually and in mixture, but not ZEN, activate NLRP3 inflammasome. Altogether, these results suggest that real-life dietary exposure of cattle to DON may induce inflammatory disorders in the ovary.

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.

Developmental and hormonal regulation of FBN1 and OR4M1 mRNA in bovine granulosa cells.

Recent studies have reported hormonal regulation of expression of fibrillin 1 (FBN1), the gene that encodes asprosin, in bovine theca cells, however, hormonal regulation of gene expression of FBN1 and the asprosin receptor, olfactory receptor 4M1 (OR4M1), has not been evaluated in granulosa cells (GC). This study was designed to characterize FBN1 and OR4M1 gene expression in GC during development of bovine dominant ovarian follicles, and to determine the hormonal regulation of FBN1 and OR4M1 mRNA expression in GC. GC FBN1 mRNA abundance was greater (P < 0.05) in medium (5.1-8 mm) estrogen inactive (EI) follicles than in large (>8.1 mm) or small (1-5 mm) EI follicles. In comparison, GC OR4M1 mRNA abundance was greater (P < 0.05) in small EI follicles than in large or medium EI follicles. Abundance of OR4M1 mRNA in GC of follicles collected on days 3 to 4 (early growth phase) and on days 5 to 6 (late growth phase) was similar, whereas FBN1 mRNA abundance was greater (P < 0.05) on days 5 to 6 vs days 3 to 4. Hormonal regulators for FBN1 mRNA abundance in cultured small-follicle GC were identified: TGFß1 causing a 2.45-fold increase, WNT3A causing a 1.45-fold increase, and IGF1 causing a 65% decrease. Steroids, leptin, insulin, growth hormone, follicle stimulating hormone, fibroblast growth factor 9 and epidermal growth factor had no effect on FBN1 mRNA abundance. Abundance of OR4M1 mRNA in GC was regulated by progesterone with 3.55-fold increase, but other hormones did not affect GC OR4M1 mRNA abundance. Findings indicate that both FBN1 and OR4M1 gene expression are hormonally and developmentally regulated in bovine follicles, and thus may affect asprosin production and its subsequent role in ovarian follicular function in cattle.

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.

The Mycotoxin De-Epoxy-Deoxynivalenol (DOM-1) Increases Endoplasmic Reticulum Stress in Ovarian Theca Cells.

Deoxynivalenol (DON) is a major mycotoxin present in animal feed and negatively affects growth and reproduction in farm species, including pigs and cattle. The mechanism of DON action involves the ribotoxic stress response (RSR), and it acts directly on ovarian granulosa cells to increase cell death. In ruminants, DON is metabolized to de-epoxy-DON (DOM-1), which cannot activate the RSR but has been shown to increase cell death in ovarian theca cells. In the present study, we determined if DOM-1 acts on bovine theca cells through endoplasmic stress using an established serum-free cell culture model and to assess whether also DON activates endoplasmic stress in granulosa cells. The results show that DOM-1 increased the cleavage of ATF6 protein, increased the phosphorylation of EIF2AK3, and increased the abundance of cleaved XBP1 mRNA. Activation of these pathways led to an increased abundance of mRNA of the ER stress target genes GRP78, GRP94, and CHOP. Although CHOP is widely associated with autophagy, inhibition of autophagy did not alter the response of theca cells to DOM-1. The addition of DON to granulosa cells partially increased ER stress pathways but failed to increase the abundance of mRNA of ER stress target genes. We conclude that the mechanism of action of DOM-1, at least in bovine theca cells, is through the activation of ER stress.

Human theca arises from ovarian stroma and is comprised of three discrete subtypes.

Theca cells serve multiple essential functions during the growth and maturation of ovarian follicles, providing structural, metabolic, and steroidogenic support. While the function of theca during folliculogenesis is well established, their cellular origins and the differentiation hierarchy that generates distinct theca sub-types, remain unknown. Here, we performed single cell multi-omics analysis of primary cell populations purified from human antral stage follicles (1-3 mm) to define the differentiation trajectory of theca/stroma cells. We then corroborated the temporal emergence and growth kinetics of defined theca/stroma subpopulations using human ovarian tissue samples and xenografts of cryopreserved/thawed ovarian cortex, respectively. We identified three lineage specific derivatives termed structural, androgenic, and perifollicular theca cells, as well as their putative lineage-negative progenitor. These findings provide a framework for understanding the differentiation process that occurs in each primordial follicle and identifies specific cellular/molecular phenotypes that may be relevant to either diagnosis or treatment of ovarian pathologies.

Ligusticum chuanxiong promotes the angiogenesis of preovulatory follicles (F1-F3) in late-phase laying hens.

Ligusticum chuanxiong (CX) is a traditional Chinese medicine that is widely planted throughout the world. CX is one of the most important and commonly used drugs to enhance blood circulation. The preovulatory follicles in laying hens have a large number of blood arteries and meridians that feed the follicles' growth and maturation with nutrients, hormones, and cytokines. With the extension of laying time, preovulatory follicles angiogenesis decreased gradually. In this study, we studied the mechanism of CX on preovulatory follicles angiogenesis in late-phase laying hens. The results show that CX extract can increase the angiogenesis of preovulatory follicles (F1-F3) of late-phase laying hens. CX extract can promote vascular endothelial growth factor receptor 2 (VEGFR2) phosphorylation in preovulatory follicles theca layers, promote the proliferation, invasion and migration through PI3K/AKT and RAS/ERK signaling pathways in primary follicle microvascular endothelial-like cells (FMECs). In addition, CX extract can up-regulate the expression of hypoxia inducible factor α (HIF1α) in granulosa cells (GCs) and granulosa layers through PI3K/AKT and RAS/ERK signaling pathways, thereby promoting the secretion of vascular endothelial growth factor A (VEGFA). In conclusion, the current study confirmed the promoting effect of CX extract on the preovulatory follicles angiogenesis, which sets the stage for the design of functional animal feed for late-phase laying hens.

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.

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.

AMH inhibits androgen production in human theca cells.

Both excessive ovarian production of AMH and androgen are important features of polycystic ovary syndrome (PCOS). Present study aimed to explore the direct effect of AMH on androgen production in human theca cells. Primary cultured human theca cells were treated with AMH, an ALK2 (the BMP type 1 receptor) inhibitor and an ALK5 (the TGFß type 1 receptor) inhibitor. AMH significantly suppresses the expression of the androgen synthesis-related enzyme CYP17A1 and reduces the production of androstenedione and testosterone in normal human theca cells and PCOS theca cells. Inhibitors of ALK2/3 and ALK5 antagonize the effect of AMH on the expression of CYP17A1. Although both ALK5 and ALK2 interact with AMHR2 in the presence of AMH, AMH activated neither TGFßR-Smads (Smad 2/3) nor BMPR-Smads (Smad 1/5/8). Our data suggested that AMH suppresses androgen synthesis-related enzyme CYP17A1 expression and inhibits androgen production in human theca cells, which process may be mediated by ALK2 and ALK5.

Effect of Angelica Sinensis extract on the angiogenesis of preovulatory follicles (F1-F3) in late-phase laying hens.

In order to form follicles and ovulate normally, there must be abundant blood vessels. Angelica sinensis (Oliv.) Diels (AS), as a traditional Chinese medicinal herb, has the effects of tonifying the blood and activating the blood circulation. However, the effect of AS on angiogenesis in hen-follicles remains to be discovered. In this study, we identified vascular richness, granulosa layer thickness, expression of platelet endothelial cell adhesion molecule-1 (CD31) and the content of vascular endothelial growth factor A (VEGFA) in granulosa layers to elucidate the effect of AS extract on angiogenesis in preovulatory follicles (F1-F3) of late-phase laying hens (75 wk). Based on network pharmacology, we predicted beta-sitosterol, ferulic acid, and caffeic acid as the main active components of AS, and hypoxia-inducible factor-1α (HIF1α), vascular endothelial growth factor receptor 2 (VEGFR2) as hub targets of AS in angiogenesis. The intersection targets were enriched by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and the hub targets were verified by immunofluorescence and western blot. Molecular docking of active components with hub targets was performed and verified in vitro. The results showed that AS extract promoted angiogenesis in preovulatory follicles and increased granulosa cell layer thickness, CD31 expression and content of VEGFA. Experiments in vitro and in vivo demonstrated that AS extract promoted the expression of HIF1α and VEGFA, up-regulated the phosphorylation levels of VEGFR2. These results further demonstrated the reliability of molecular docking and network pharmacology findings. In summary, AS extract can promote angiogenesis in the preovulatory follicles in late-phase laying hens.