Search

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. • Queens ≤4 months old had significantly greater SPACA3 expression in granulosa cells. • Queens ≤4 months old had significantly greater SPACA3 expression in theca cells. • Queens ≤4 months old had significantly greater SPACA3 expression in oocyte nuclei. • Age did not affect SPACA3 expression within the feline ooplasm. [ABSTRACT FROM AUTHOR]

In avian, follicular selection is a key molecular event that can determine avian egg production. Theca cells (TC) are the main components of follicles, the molecular mechanisms about TCs physiological differences during follicle selection stage are still unclear. This study revealed significant differences in proliferation, apoptosis, lipid synthesis, and steroid secretion levels between prehierarchical theca cells (phTC) and hierarchical theca cells (hTC) of Tianfu meat-type geese. A total of 1,559 differentially expressed genes (DEG) and 71 differentially expressed miRNAs (DEM) were identified between phTCs and hTCs, respectively. Functional enrichment analysis results showed that 143 DEGs were enriched in the pathways related to cell proliferation/apoptosis and lipid/steroid metabolism. Protein-protein interaction (PPI) network results indicated the 143 DEGs have functional interactions. Additionally, the predicted target genes of 71 DEMs were jointly analyzed with the above 143 DEGs, and the results showed that 15 DEMs and 17 DEGs with targeted relationships were found. Among them, miR-202-5p was significantly down-regulated both in hTCs and hierarchical theca layers, and target prediction results showed that miR-202-5p may affect TCs proliferation/apoptosis by targeting CHPT1 to regulate the expression levels of CCN1 / FOXO3 ; meanwhile, may affect TCs lipid/steroid metabolism and proliferation/apoptosis by targeting CHPT1 to regulate the expression levels of p53 / ABCA1 / SREBP -2. This study provides new insights into the regulatory mechanisms of TCs physiological differences during goose follicle selection. [ABSTRACT FROM AUTHOR]

Objective: To characterize circulating insulin-like factor 3 (INSL3) in different stages of ovarian insufficiency and its role in the evaluation of premature ovarian insufficiency (POI).Design: Retrospective cohort study.Setting: University-based center for reproductive medicine.Patient(s): A total of 145 women, including 48 patients with POI (25 IU/L < follicle-stimulating hormone [FSH] ≤40 IU/L), 49 with biochemical POI (bPOI) (10 IU/L < FSH ≤25 IU/L) and 48 age-matched control women with normal ovarian reserve (FSH <10 IU/L), retrospectively included from the reproductive hospital affiliated with Shandong University between 2017 and 2019.Intervention(s): Levels of INSL3 in the serum and follicular fluid assayed with a commercial radioimmunoassay.Main Outcome Measure(s): Level of INSL3 in serum and follicular fluid among control women and patients with bPOI and POI, its association with different ovarian reserve markers, and its predictive value for bPOI and POI.Result(s): The serum INSL3 level continuously declined with the progress of ovarian insufficiency. It showed strong negative association with FSH (-0.655) and luteinizing hormone (-0.433), but positively correlated with antimüllerian hormone (0.617), inhibin B (0.400), antral follicle count (0.630), and testosterone (0.180). Additionally, the circulating INSL3 served as a good predictor for bPOI and POI. No statistically significant difference of INSL3 levels in follicular fluid was observed between bPOI patients and control women.Conclusion(s): For the first time our study has revealed an INSL3 deficiency in women with POI, indicating that circulating INSL3 could serve as a promising theca-cell specific marker for POI. Future research on the role of INSL3 in modulating follicular development, steroidogenesis, and POI pathogenesis is warranted. [ABSTRACT FROM AUTHOR]

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 (r 2 = 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 (r 2 = 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 (r 2 = 0.752, P < 0.001), StAR (r 2 = 0.456, P = 0.002), CYP1B1 (r 2 = 0.568, P < 0.001), and 3β-HSD (r 2 = 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 3β-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 (r 2 = 0.819, P < 0.001), StAR (r 2 = 0.844, P < 0.001), 3β-HSD (r 2 = 0.801, P < 0.001), and CYP11A1 (r 2 = 0.800, P < 0.001) in theca cells of laying hens. The protein expression of CREB1, StAR, and 3β-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. [ABSTRACT FROM AUTHOR]

Theca cells (TCs) are essential to folliculogenesis by contributing to steroidogenesis. However, the in vitro growth (IVG) of oocytes co-cultured with TCs has not yet been examined. In the present study, we investigated the feasibility of the IVG of bovine oocyte-cumulus-granulosa cell complexes (OCGCs) co-cultured with TCs and the developmental competence of co-cultured oocytes. OCGCs and TCs were co-cultured without steroid hormone addition for 12 days. Steroidogenesis, the viability of OCGCs, and TC numbers during co-culture were assessed every 4 days. After IVG, oocytes were matured and the nuclear status was evaluated. Some oocytes were inseminated and cultured to examine blastocyst development. During the co-culture, androstenedione production by TCs was only observed during the first 4 days (1.1 ng/well) while estradiol-17β was continuously produced, peaking during the second 4 days (0.5 ng/well). The number of TCs decreased to ∼60% of the seeding number (4.0 × 104 cells/well) during the first 4 days, and was maintained thereafter. The majority of co-cultured OCGCs (82.7%) survived after 12-day IVG. Only a few OCGCs (6.2%) survived in the OCGC culture without TCs (p < 0.01); however, the addition of androstenedione to the culture medium markedly improved survivability to 80.1%, which was similar to that in the co-culture with TCs. In the subsequent development of oocytes derived from the co-culture, 58.3% reached metaphase II stage, 58.7% cleaved, and 17.3% developed to blastocysts, which were similar values to those of oocytes cultured with the addition of androstenedione. In conclusion, TC-produced androgen contributes to OCGC growth and the acquisition of subsequent embryonic developmental competence. • Theca cells can produce androstenedione and support oocyte development in vitro. • However, theca cells showed quick spontaneous luteinization. [ABSTRACT FROM AUTHOR]

Abstract Ethnopharmacological relevance Polycystic Ovarian Syndrome (PCOS) is a complex endocrine and reproductive disorder. A main hallmark includes increased androgen production. The root of Paeonia lactiflora Pall. (Bai Shao) is used in Chinese herbal medicine for reproductive disorders, however its effects and mechanisms on ovarian theca cells has not yet been fully elucidated. Aim of the study The aim of this study was to evaluate effect of paeoniflorin extract (PFE), the main constituents of Bai Shao, on androgen production in ovarian theca cells. Materials and methods Primary murine theca cells were treated with concentrations of PFE (1–100 µg/mL) in the presence of dexamethasone (10 µM) with media-only treated cells used as the control. After 24 h, culture media was collected for biochemistry assays of testosterone and progesterone. Expression of key steroidogenic enzymes, cholesterol side-chain cleavage (CYP11A1) and 17α-hydroxylase (CYP17A1) was characterized using immunofluorescence staining, immunoblotting and qRT-PCR. Results Dexamethasone significantly enhanced testosterone secretion (P < 0.05 vs. the control cells). PFE reversed over-production of testosterone induced by dexamethasone in a dose-dependent manner. The treatment with PFE also normalized production of progesterone in dexamethasone-treated cells. Expression of CYP11A1 and CYP17A1 in the theca cells were visualised by immunofluorescence staining. All doses of PFE significantly inhibited CYP17A1 expression detected by immunoblotting, but only 100 µg/mL of PFE downregulated CYP11A1 expression and reduced CYP11A1 significantly in dexamethasone-treated theca cells. Conclusions PFE may reduce over-secretion of testosterone in theca cells through downregulation of CYP17A1 and CYP11A1. These findings provide scientific evidence to treat ovarian hyperandrogenism with the root of Paeonia lactiflora Pall. Graphical abstract fx1 [ABSTRACT FROM AUTHOR]

Abstract The theca cell layer of the ovarian follicle secretes growth factors that impact the function of granulosa cells. One such factor is fibroblast growth factor 18 (FGF18) that causes apoptosis of granulosa cells, however it is not known if FGF18 induces apoptosis also in theca cells. Addition of recombinant FGF18 to bovine theca cells in vitro inhibited steroidogenesis but, in contrast to previous data in granulosa cells, decreased the incidence of apoptosis. FGF18 activated typical FGF signaling pathways in theca cells, which was not previously observed in granulosa cells. The transcription factor Early Growth Response-1 (EGR1) was a target of FGF18 action; overexpression and knock-down experiments demonstrated that EGR1 is a major upstream component of FGF signaling in theca cells and that it directs cell fate toward proliferation. These data suggest that FGF18 is mitogenic for theca cells while being pro-apoptotic in granulosa cells. Graphical abstract Image 1 Highlights • FGF18 stimulates theca cell proliferation. • FGF18 stimulates EGR1 mRNA levels. • EGR1 overexpression stimulates FGF signaling pathways. • EGR1 is anti-apoptotic in theca cells. [ABSTRACT FROM AUTHOR]

We reported previously that fibroblast growth factor 9 (FGF9) acts as an antidifferentiation factor, stimulating proliferation of granulosa cells (GCs) and theca cells (TCs) while suppressing hormone-induced steroidogenesis of these cells. How FGF9 acts to simultaneously suppress steroidogenesis and stimulate proliferation remains to be fully elucidated. Thus, this study was undertaken to clarify the effects of FGF9 on the TC transcriptome. Ovaries were obtained from beef heifers at a local abattoir, TCs were isolated from large antral follicles, and cultured with or without 30 ng/mL of FGF9 for 24 h in the presence of LH and IGF-1. After treatment, total RNA was extracted from TC and processed for microarray using Affymetrix GeneChip Bovine Genome Arrays (n = 4/group). Transcriptome analysis comparing FGF9-treated TC with control TC using 1.3-fold cutoff, and a P < 0.05 significance level identified 355 differentially expressed transcripts, with 164 elements upregulated and 191 elements downregulated by FGF9. The ingenuity pathway analysis (IPA) was used to investigate how FGF9 treatment affects molecular pathways, biological functions, and the connection between molecules in bovine TC. The IPA software identified 346 pathways in response to FGF9 in TC involved in several biological functions and unveiled interesting relationships among genes related to cell proliferation (eg, CCND1 , FZD5, and MYB ), antioxidation/cytoprotection (eg, HMOX1 and NQO1 ), and steroidogenesis (eg, CYP11A1 and STAR ). Overall, genes, pathways, and networks identified in this study painted a picture of how FGF9 may regulate folliculogenesis, providing novel candidate genes for further investigation of FGF9 functions in ovarian follicular development. [ABSTRACT FROM AUTHOR]

Paired pigeons only lay 2 eggs in a laying period, which is closely related to ovarian follicle development, but this process is not well understood. In this study, 60 pairs of 12-mo-old White King pigeons were selected and serum and follicles were collected at 4 stages of laying interval (LI), including the first (LI1), the third (LI3), the fifth (LI5), and the seventh day (LI7). Morphological results showed that paired pigeons normally had 2 preovulatory follicles and the second-largest follicle (F2) developed from LI3 and had been selected in LI5. Prehierarchical follicles were coupled and hierarchical, which was in accordance with its clutch size. The P 4 concentration increased gradually from LI1 to LI5, reaching a maximum of 30.67 ng/mL in LI5 and decreasing to 27.83 ng/mL in LI7 (P < 0.05). The levels of T in LI1 and LI5 were higher than LI3 and LI7 (P < 0.05), although there was no significant difference in E 2 in LI (P > 0.05), but it stayed at high levels. In the TCs of the largest follicle (F1), HSD3B1 mRNA and HSD17B1 mRNA levels peaked in LI7. The expression pattern of CYP17A1 and CYP19A1 was similar, increasing from LI3 to LI5 and then decreasing. In the TCs of F2, the expressions of HSD3B1 and CYP17A1 had no significant difference between LI5 and LI7 (P > 0.05), while the expression pattern of HSD17B1 and CYP19A1 was the opposite. In TCs of SF1, HSD3B1 mRNA level peaked in LI3 while CYP19A1 mRNA levels peaked in LI7. The expression of CYP17A1 had a minor change (P > 0.05) and the expression pattern of HSD17B1 was similar to F1. It was concluded that the morphological characteristics of follicles during the LI for the first time, including the number and diameter of small follicles (SFs) and hierarchical follicles in pigeon and the concentrations of steroid hormones and expressions of steroidogenic genes in TCs of different follicles could explain the growth and selection of 2 preovulatory follicles. This study facilitates further research into the regulation of ovulation and egg production in pigeons. [ABSTRACT FROM AUTHOR]

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 TGFβ 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 TGFβR-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. • AMH suppresses CYP17A1 expression in normal human theca cells and PCOS theca cells. • AMH reduces the androgen production in normal human theca cells and PCOS theca cells. • ALK2 and ALK5 interact with AMHR2 in the presence of AMH in human theca cells. • ALK2 and ALK5 may mediate the regulation of androgen production by AMH in theca cells. [ABSTRACT FROM AUTHOR]

Fibroblast growth factor 9 (FGF9) has been suggested to act as an antidifferentiation factor in cattle by reducing steroidogenesis and increasing cell proliferation in granulosa (GC) and theca (TC) cells. The objective of this study was to characterize FGF9 mRNA abundance in GC and TC during development of dominant follicles in dairy cattle. Estrous cycles of nonlactating dairy cattle were synchronized, and ovaries were collected on either d 3 to 4 (n = 8) or 5 to 6 (n = 8) postovulation for GC and TC RNA extraction from small (1-5 mm), medium (5.1-8 mm), and large (8.1-18 mm) follicles for PCR analysis. The FGF9 mRNA abundance was greater in GC than in TC. In GC, FGF9 mRNA abundance was greater in small, medium, and large estrogen-inactive [i.e., concentrations of estradiol (E2) < progesterone (P4)] follicles than in large E2-active (i.e., concentrations of E2 > P4) follicles at both early (d 3-4) and late (d 5-6) growing phases of first dominant follicle. Abundance of FGF9 mRNA increased in medium-sized follicles from early to late growing phase of the dominant follicle. In TC, FGF9 mRNA abundance was greater in large E2-inactive follicles than in large E2-active follicles on d 3 to 4 postovulation; no significant differences in TC FGF9 mRNA existed among follicle types on d 5 to 6 postovulation. Correlations among levels of follicular fluid hormones and FGF9 mRNA levels revealed significant negative correlations between GC FGF9 mRNA abundance and follicular fluid E2 (r = -0.68), free IGF-1 (r = -0.63), and E2-to-P4 ratio (r = -0.58). In summary, abundance of FGF9 mRNA in GC and TC increases in medium-sized follicles during development of dominant follicles and is less in dominant E2-active than subordinate E2-inactive follicles, suggesting that FGF9 signaling could contribute to normal follicle development and steroidogenesis in dairy cattle. [ABSTRACT FROM AUTHOR]

Objective To explore the temporal expression in granulosa and theca cells of key members of the MMP and ADAMTS families across the periovulatory period in women to gain insight into their possible roles during ovulation and early luteinization. Design Experimental prospective clinical study and laboratory-based investigation. Setting University medical center and private IVF center. Animal and Patient(s) Thirty-eight premenopausal women undergoing surgery for tubal ligation and six premenopausal women undergoing assisted reproductive techniques. Intervention(s) Administration of hCG and harvesting of follicles by laparoscopy and collection of granulosa-lutein cells at oocyte retrieval. Main Outcome Measure(s) Expression of mRNA for matrix metalloproteinase (MMPs) and the A disintegrin and metalloproteinase with thrombospondin-like motifs (ADAMTS) in human granulosa cells and theca cells collected across the periovulatory period of the menstrual cycle and in cultured granulosa-lutein cells after hCG. Localization of MMPs and ADAMTSs by immunohistochemistry. Result(s) Expression of MMP1 and MMP19 mRNA increased in both granulosa and theca cells after hCG administration. ADAMTS1 and ADAMTS9 mRNA increased in granulosa cells after hCG treatment, however, thecal cell expression for ADAMTS1 was unchanged, while ADAMTS9 expression was decreased. Expression of MMP8 and MMP13 mRNA was unchanged. Immunohistochemistry confirmed the localization of MMP1, MMP19, ADAMTS1, and ADAMTS9 to the granulosa and thecal cell layers. Conclusion(s) The collection of the dominant follicle throughout the periovulatory period has allowed the identification of proteolytic remodeling enzymes in the granulosa and theca compartments that may be critically involved in human ovulation. These proteinases may work in concert to regulate breakdown of the follicular wall and release of the oocyte. [ABSTRACT FROM AUTHOR]

Myricetin (a flavonol) and piceatannol (a stilbenoid) are naturally occurring phenolic compounds in red wine with cardio-protective and anti-carcinogenic effects, but their potential reproductive effects have not been investigated. Thus, the present study was designed to determine if myricetin and piceatannol can directly affect ovarian function using bovine granulosa cells (GC) and theca cells (TC) as in vitro model systems to evaluate effects on cell proliferation and steroid production. In Experiment 1 and 2, myricetin and piceatannol at 30 μM blocked insulin-like growth factor 1 (IGF1)-induced progesterone production by GC without affecting GC numbers. In contrast, myricetin stimulated IGF1-induced estradiol production, whereas piceatannol at 30 μM inhibited IGF1-induced estradiol production by 90% in GC. In Experiment 3 and 4, TC androstenedione and progesterone production and TC proliferation was inhibited by myricetin and piceatannol at 30 μM. In Experiment 5, piceatannol (30 μM) reduced the Fusarium mycotoxin, beauvericin (6 μM)-induced inhibition on progesterone production and cell proliferation. Myricetin (30 μM) reduced the inhibitory effect of beauvericin on estradiol but not progesterone production or cell proliferation. In conclusion, the red wine phenols, myricetin and piceatannol, directly affected GC and TC steroidogenesis, and were able to reduce some of the inhibitory effects of beauvericin on GC function. • The grape phenol, myricetin stimulated granulosa cell estradiol production. • Myricetin inhibited granulosa and theca cell progesterone production. • The grape phenol, piceatannol inhibited IGF1-induced theca cell proliferation. • Piceatannol inhibited granulosa and theca cell steroid production. • Inhibitory effects of beauvericin were partially attenuated by grape phenols. [ABSTRACT FROM AUTHOR]

• FGFR1c and FGFR2c mRNA abundance is least in granulosa cells of dominant follicles. • Estradiol decreased FGFR2c mRNA abundance in granulosa cells. • Androgen increased FGFR1c, FGFR2c and FGFR4 mRNA abundance in granulosa cells. • FGFR3c and FGFR4 mRNA abundance in granulosa cells is similar among follicle types. • FGFR2c and FGFR3c mRNA abundance in theca cells is similar among follicle types. The various fibroblast growth factors (FGF) regulate their function via binding to 4 main FGF receptor (FGFR) subtypes and their splice variants, FGFR1b, FGF1c, FGFR2b, FGFR2c and FGFR3c and FGFR4 , but which of these FGFR are expressed in the granulosa (GC) and theca cells (TC), the 2 main cell layers of ovarian follicles, or change during follicular development is unknown. We hypothesized that FGFR1c, FGFR2c and FGFR3c (but not FGFR4) gene expression in GC (but not TC) would change with follicular development. Hence, the objective of this study was to determine if abundance of FGFR1c, FGFR2c, FGFR3c , and FGFR4 mRNA change according to follicular size, steroidogenic status, and days post-ovulation during growth of first-wave dominant follicles in Holstein cattle exhibiting regular estrous cycles. Estrous cycles of non-lactating dairy cattle were synchronized, and ovaries were collected on either d 3 to 4 (n = 8) or d 5 to 6 (n = 8) post-ovulation for GC and TC RNA extraction from small (1–5 mm), medium (5.1 to 8 mm) or large (8.1–18 mm) follicles for real-time PCR analysis. In GC, FGFR1c and FGFR2c mRNA relative abundance was greater in estrogen (E2)-inactive (ie, concentrations of E2 < progesterone, P4) follicles of all sizes than in GC from large E2-active follicles (ie, E2 > P4), whereas FGFR3c and FGFR4 mRNA abundance did not significantly differ among follicle types or days post-estrus. In TC, medium E2-inactive follicles had greater FGFR1c and FGFR4 mRNA abundance than large E2-active and E2–inactive follicles on d 5 to 6 post-ovulation whereas FGFR2c and FGFR3c mRNA abundance did not significantly differ among follicle types or day post-estrus. In vitro experiments revealed that androstenedione increased abundance of FGFR1c, FGFR2c and FGFR4 mRNA in GC whereas estradiol decreased FGFR2c mRNA abundance. Neither androstenedione nor estradiol affected abundance of the various FGFR mRNAs in cultured TC. Taken together, the findings that FGFR1c and FGFR2c mRNA abundance was less in GC of E2-active follicles and FGFR1c and FGFR4 mRNA was greater in TC of medium inactive follicles at late than at early growing phase of the first dominant follicle support an anti-differentiation role for FGF and their FGFR as well as support the idea that steroid-induced changes in FGF and their receptors may regulate selection of dominant follicles in cattle. [ABSTRACT FROM AUTHOR]

Abstract: Abundance of IGF-2 receptor (IGF2R), FSH receptor (FSHR), and LH receptor (LHCGR) mRNA in granulosa cells (GCs) or theca cells (TCs) or both cells as well as estradiol (E2), progesterone (P4), and androstenedione concentrations in follicular fluid were compared in cows genetically selected (Twinner) or not selected (control) for multiple ovulations and twin births. Cows were slaughtered at day 3 to 4 (day 3) and day 5 to 6 (day 5) of an estrous cycle, and ovaries, follicular fluid, GCs, and TCs were collected. The two largest (F1 and F2) E2-active (EA) and E2-inactive (EI) follicles were selected according to their E2-to-P4 ratio and diameter. Androstenedione levels in EA F1 and F2 follicles were 5-fold greater (P < 0.05) in Twinner cows than in control cows on day 3 but did not differ on day 5. Twinner cows also had greater (P < 0.05) E2 and P4 concentrations, whereas steroid levels in EI follicles did not differ (P > 0.10) between genotypes. In EA F2 follicles, IGF2R levels in GCs were greater (P < 0.05) in control cows than in Twinner cows on day 3 and day 5, whereas IGF2R mRNA in TCs did not differ (P > 0.10). On day 3, FSHR mRNA levels were greater (P < 0.05) in GCs of EA F1 and EI F2 follicles of control cows than of Twinner cows. LH receptor mRNA expression was less in GCs and greater in TCs of EA F2 follicles in control cows than in Twinner cows (P < 0.05). We hypothesize that reduced GC IGF2R expression in F2 follicles of Twinner cows may play a role in the development of 2 or more dominant follicles. [Copyright &y& Elsevier]

Abstract: The aim of this study was to create a microcapsule system simulating native follicle structure by introducing microcarrier culture to microencapsulation for providing endogenous female hormones. Granulosa and theca cells of rat follicles were isolated. Granulosa cells were grown on microcarriers and enclosed together with theca cells in alginate–chitosan–alginate microcapsules. The cell viability and female hormone secretion were investigated in vitro. The microcapsules were transplanted to ovariectomized rats and the serum levels of estradiol and progesterone were measured for 60 days. The microencapsulated granulosa cells growing on microcarriers exhibited enhanced viability and promoted secreting ability of estradiol and progesterone compared with those without the microcarriers. Co-microencapsulation of granulosa cells and theca cells markedly elevated estradiol secretion in vitro. Transplantation of co-microencapsulated granulosa cells on microcarriers and theca cells maintained serum estradiol and progesterone at normal levels for 60 days. Microcarrier cell culture has been proved to be an effective method to enhance the viability of granulosa cells in microcapsules. Moreover, the transplantation of microcapsules enclosing granulosa cells on microcarriers and theca cells may be promising to provide endogenous female hormones for menopausal syndrome treatment. [Copyright &y& Elsevier]

Objective: To test whether and to what extent inhibin mediates Cyp17 messenger RNA (mRNA) expression in theca cells (TCs) in response to FSH stimulation of granulosa cells (GCs). Design: Ex vivo and in vitro experimental study. Setting: University. Animal(s): Immature female Sprague Dawley rats. Intervention(s): Ovarian tissue explants and isolated theca cell preparations with or without GCs were treated with FSH, inhibin, inhibin antibody, or β-glycan antibody. Main Outcome Measure(s): As a key enzyme in androgen production, Cyp17 mRNA levels were measured by real-time reverse transcription–polymerase chain reaction. Result(s): After 24 hours, Cyp17 mRNA expression was dose-dependently increased by FSH in ovarian tissue explants and theca cells, suggesting that paracrine factor(s) secreted from GCs in response to FSH mediates Cyp17 mRNA expression in TCs. Antibodies against inhibin and inhibin coreceptor, β-glycan, blocked the stimulatory effect of FSH on Cyp17 mRNA expression. However, inhibin alone did not increase Cyp17 mRNA level to the same extent. Conclusion(s): These findings suggest a role for inhibin in the paracrine regulation of TC Cyp17 mRNA expression by GCs influenced by FSH; however, other paracrine factors produced by GCs by virtue of FSH seem to be required. [ABSTRACT FROM AUTHOR]

Abstract: Alterations of ovarian follicle morphology and function have been well documented in women with PCOS. These include increased numbers of growing preantral follicles, failure of follicle growth beyond the mid-antral stage, evidence of granulosa call degeneration, and theca cell hyperplasia. Functional abnormalities include paradoxical granulosa cell hyperresponsiveness to FSH which is clinically linked to ovarian hyperstimulation during ovulation induction. In addition, there is likely a primary theca cell defect that accounts for the majority of excess androgen production in this disorder. The precise mechanisms responsible for altered follicle function are not completely clear. However, several factors appear to influence normal advancement of follicle development as well as impair ovarian steroidogenesis. These include intra- as well as extraovarian influences that distort normal ovarian growth and disrupt steroid production by follicle cells. [Copyright &y& Elsevier]

Abstract: Although luteinizing hormone (LH) affects androstenedione (A4) and progesterone (P4) production in theca cells, it is still unknown how LH influences molecular mechanism of A4 and P4 production. To examine the relationship between LH and transcription factors involved in A4 and P4 production, ovarian theca cells were cultured in the presence or absence of high concentrations of LH for 24h (pre-treatment with high concentration of LH) and then cultured in the presence or absence of low concentration of LH for 48h. Low LH enhanced production of A4 and P4, and expressions of CYP17 and StAR mRNA in theca cells without pre-treatment with high LH. In addition, low LH stimulated the expression of SF-1 protein in nuclear fractions from theca cells with or without pre-treatment with high LH. The binding of SF-1 to the CYP17 and StAR promoter regions increased in theca cells treated with low LH. Although GATA-4 and GATA-6 are both found in the nuclear fraction but not in the cytosol of theca cells, low LH enhanced the binding of GATA-6, but not of GATA-4, to the CYP17 promoter region without pre-treatment with high LH. Acetylation histone H3 in StAR and CYP17 promoter regions were changed by different LH-dosage. Overall, we showed that LH regulates the production of A4 and P4 by affecting the nuclear localization and switching of transcription factors in theca cells and that target transcription factors involved in steroid production in theca cells are changed by different LH concentration. [Copyright &y& Elsevier]

Abstract: Resistin is an adipokine that has not been extensively studied in cattle but is produced by adipocytes in greater amounts in lactating versus non-lactating cattle. Seven experiments were conducted to determine the effect of resistin on proliferation, steroidogenesis, and gene expression of theca and granulosa cells from small (1–5mm) and/or large (8–22mm) cattle follicles. Resistin had no effect on IGF-I-induced proliferation of large-follicle theca cells or small-follicle granulosa cells, but decreased IGF-I-induced proliferation of large-follicle granulosa cells. Resistin weakly stimulated FSH plus IGF-I-induced estradiol production by large-follicle granulosa cells, but had no effect on IGF-I- or insulin-induced progesterone and androstenedione production by theca cells or progesterone production by granulosa cells of large follicles. In small-follicle granulosa cells, resistin attenuated the stimulatory effect of IGF-I on progesterone and estradiol production of small-follicle granulosa cells. RT-PCR measuring abundance of side-chain cleavage enzyme (CYP11A1), aromatase (CYP19A1), FSH receptor (FSHR) and LH receptor (LHCGR) mRNA in large- and small-follicle granulosa cells indicated that resistin reduced the stimulatory effect of IGF-I on CPY11A1 mRNA abundance in large-follicle granulosa cells but had no effect on CYP19A1, FSHR or LHCGR mRNA abundance in large- or small-follicle granulosa cells. Resistin had no effect on CYP11A1, CYP17A1 or LHCGR mRNA abundance in theca cells. These results indicate that resistin preferentially inhibits steroidogenesis of undifferentiated (small follicle) granulosa cells and inhibits proliferation of differentiated (large follicle) granulosa cells, indicating that the ovarian response to resistin is altered during follicular development. [Copyright &y& Elsevier]

Abstract: Chronic, subclinical intramammary infection depresses fertility. We previously found that 30% of subclinical mastitic cows exhibit delayed ovulation, low circulating estradiol levels, and delayed luteinizing hormone surge. We examined the function of preovulatory follicles of cows experiencing subclinical mastitis or a past event of acute clinical mastitis. Cows were diagnosed for mastitis by somatic cell count and bacteriological examination. All clinical infections were caused by Escherichia coli, and most subclinical infections were caused by Streptococcus dysgalactiae and coagulase-negative staphylococci. On day 6 of the cycle, cows received PGF2α; 42 h later, follicular fluids and granulosa cells or theca cells were aspirated from preovulatory follicles in vivo or following slaughter, respectively. Overall, follicular estradiol and androstenedione concentrations in the subclinical group (n = 28) were 40% lower (P < 0.05) than those in uninfected cows (n = 24) and lower than in past clinical mastitic cows (n = 9). Distribution analysis revealed a clear divergence among subclinical cows: one-third (9/28) exhibited low follicular estradiol; the other two-thirds had normal levels similar to all uninfected (P < 0.01) and most clinical cows (P < 0.08) that had normal follicular estradiol levels. Subclinical normal-estradiol cows had twofold higher (P < 0.05) circulating estradiol concentrations and sevenfold and fourfold higher (P < 0.05) follicular androstenedione levels and estradiol-to-progesterone ratio, respectively, than subclinical low-estradiol cows. Follicular progesterone level was not affected. Reduced expression (P < 0.05) of LHCGR in theca and granulosa cells, CYP11A1 (mRNA and protein) and CYP17A1 in theca cells, and CYP19A1 in granulosa cells may have contributed to the lower follicular steroid production in the subclinical low-estradiol subgroup. StAR and HSD3B1 in theca cells and FSHR in granulosa cells were not affected. Mastitis did not alter follicular growth dynamics, and no carryover effect of past clinical mastitis on follicular function was detected. These data indicate that a considerable proportion (one-third) of subclinical mastitic cows have abnormal follicular steroidogenesis, which can explain the reproductive failure associated with this disease. [Copyright &y& Elsevier]

Abstract: Orphan nuclear receptor NR4A1, a member of the nuclear receptor superfamily, is widely expressed in different cell types and mediates diverse biological processes. Recent emerging evidence suggests that NR4A1 is involved in the transcriptional regulation of several steroidogenic enzyme genes in gonads and adrenals. However, its function in ovarian theca cells remains to be defined. In the present study, immunohistochemical staining of NR4A1 in healthy human ovaries indicate that it is expressed in theca cells and granulosa cells. In an effort to explore the function of NR4A1 in the transcript regulation of steroidogenic enzyme genes responsible for ovarian theca cell steroidogenesis, we constructed recombinant adenovirus AdCMV-NR4A1 and AdH1-NR4A1 to enhance or knockdown the expression of NR4A1 in theca cells, respectively. The expression patterns of StAR, CYP11A1, CYP17 and HSD3B2 were subsequently analyzed by real-time RT-PCR. Moreover, concentrations of testosterone in the spent medium were measured by radioimmunoassay. Our results show that overexpression of NR4A1 in theca cells stimulates the expression of StAR, CYP11A1, CYP17 and HSD3B2, leading to increased testosterone production. Conversely, knockdown of the endogenous NR4A1 exhibits a significant decrease in StAR, CYP11A1, CYP17 and HSD3B2 expression and testosterone production. Since expression of NR4A1 in the endocrine organs is known to be regulated by both cAMP/PKA mediated hormones, ACTH and LH, forskolin (FSK), an activator of cAMP/PKA pathway, was applied to the cultured follicles. FSK rapidly increases the NR4A1 mRNA levels followed by an increase in StAR, CYP11A1, CYP17 and HSD3B2. Collectively, our results outline a previously unrecognized role for NR4A1 in the transcriptional regulation of StAR, CYP11A1, CYP17 and HSD3B2 in ovarian theca cells. Modulation of these steroidogenic enzymes by NR4A1 could influence the capacity of the ovarian theca cells to produce androgen. [Copyright &y& Elsevier]

Abstract: In the mammalian ovary, the microvasculature in the thecal layer of follicles is associated with follicular development. Apelin and its receptor, APJ, are expressed in the tissues and organs which include the vasculature. The aims of the present study were to examine the mRNA expression of apelin and the APJ receptor in granulosa cells and theca tissue of bovine follicles and the effects of steroid hormone and gonadotrophins on the expression of these genes in cultured granulosa cells and theca cells. The expression of apelin mRNA was not found in the granulosa cells of bovine follicles. The expression of the APJ gene was increased in granulosa cells of estrogen-inactive dominant follicles. The expression of apelin mRNA increased in theca tissues of estrogen-inactive dominant follicles. APJ expression in theca tissues increased with follicle growth. Progesterone stimulated the expression of APJ mRNA in the cultured granulosa cells. FSH stimulated the expression of APJ mRNA in the cultured granulosa cells. LH induced the expression of apelin and APJ receptor mRNAs in cultured theca cells. Taken together, our data indicate that the APJ receptor in granulosa cells and both apelin and the APJ receptor in theca tissues are expressed in bovine ovary, that APJ in granulosa cells may be involved in the appearance of the cell apoptosis, and that LH stimulates the expression of apelin and APJ genes in theca cells. [Copyright &y& Elsevier]

Abstract: Adiponectin is an adipokine that has been implicated in insulin resistance, a condition associated with polycystic ovarian syndrome in humans, but whether adiponectin can directly affect ovarian theca or granulosa cell function is unknown. Therefore, to determine the effects of adiponectin on proliferation, steroidogenesis and gene expression of large-follicle theca and granulosa cells, experiments were conducted using bovine ovarian cell cultures. RT-PCR was used to elucidate the effects of adiponectin on gene expression of CYP11A1 and LH receptor (LHR) in large-follicle theca and granulosa cells, as well as expression of CYP17A1 in theca cells and CYP19A1 in granulosa cells. Adiponectin decreased (P <0.05) insulin-induced progesterone and androstenedione production as well as attenuated IGF-I-induced LHR, CYP11A1, and CYP17A1 gene expression in theca cells. In contrast, adiponectin decreased (P <0.05) LHR mRNA abundance in granulosa cells but did not affect steroidogenic enzyme gene expression in granulosa cells. Adiponectin had no effect (P >0.10) on proliferation of large-follicle theca cells. RT-PCR also revealed that abundance of mRNA for the adiponectin receptor (ADIPOR2) was greater (P <0.05) in large-follicle than in small-follicle theca cells and did not significantly differ between small- and large-follicle granulosa cells. In cultured theca cells, LH increased (P <0.05) and IGF-I decreased (P <0.05) ADIPOR2 mRNA abundance. These results indicate that the inhibitory effects of adiponectin on steroidogenesis are primarily localized to theca cells and that the response of theca cells to adiponectin (i.e., ADIPOR2) may be regulated by LH and IGF-I. [Copyright &y& Elsevier]

Abstract: Ovarian follicular growth and dominance are controlled by a series of hormonal and intraovarian events including a decrease in intrafollicular IGF-binding proteins −2, −4 and −5 levels. Proteolytic enzymes such as pregnancy-associated plasma protein-A (PAPP-A) degrade IGFBPs and increase bioavailability of IGF-I and -II during follicular development. The objective of this study was to determine the effect of IGF-I, IGF-II, insulin (INS), LH, FSH, estradiol (E2), leptin or cortisol on ovarian PAPP-A mRNA levels. Granulosa (GC) from small (SM) (1–5mm) and large (LG) (8–22mm) follicles as well as theca cells (TC) from LG follicles were collected from bovine ovaries and cultured for 48h in medium containing 10% FCS and then treated with various hormones in serum-free medium for an additional 24h. Cells were treated with various concentrations (3–500ng/ml) and combinations of IGF-I, IGF-II, FSH, LH, E2, INS, leptin and (or) cortisol for 24h (Experiments 1–10). PAPP-A mRNA levels were measured using quantitative real-time RT-PCR. In SM-GC and LG-GC, none of the treatments significantly affected (P >0.10) PAPP-A mRNA abundance. In LG-TC, IGF-I, LH or cortisol did not affect (P >0.10) PAPP-A mRNA levels, whereas INS with or without LH decreased (P <0.05) PAPP-A mRNA. E2 alone decreased PAPP-A mRNA levels in LG-TC, and E2 amplified the insulin-induced inhibition of PAPP-A mRNA abundance in LG-TC. We conclude that control of PAPP-A mRNA abundance in granulosa and theca cells differs, and that E2 may be part of an intraovarian negative feedback system which may reduce the bioavailable IGFs in the theca layer during growth and selection of follicles. [Copyright &y& Elsevier]

Abstract: Theca cells are the endocrine cells associated with ovarian follicles that play an essential role in fertility by producing the androgen substrate required for ovarian estrogen biosynthesis. Theca cells differentiate from the interfollicular stroma in response to proteins secreted from growing follicles. The most common endocrine cause of infertility is associated with excessive proliferation of theca cells and ovarian hyperandrogenism. Cell facts: [-] ovarian androgen-producing cells; [-] are associated only with developing follicles; [-] over-activity of theca cells causes infertility due to hyperandrogenism; [-] under-activity of theca cells causes infertility due to lack of estrogen. Theca cells: androgen-producing cells in the ovary. [Copyright &y& Elsevier]

Objective: To correlate serum and intrafollicular E2 levels in women receiving GnRH agonist and recombinant human (h)FSH for IVF. Design: Prospective study. Setting: Academic IVF center. Patient(s): Twenty-three nonhirsute ovulatory women. Intervention(s): In vitro fertilization cycles were classified as high- or low-normal response, according to whether the amount of recombinant hFSH administered was below or above, respectively, the mean recombinant hFSH given to all women combined. Serum bioactive LH and immunoreactive FSH were measured during pituitary desensitization preceding recombinant hFSH and repeated at oocyte retrieval. The first mature follicle aspirated from either ovary had a mean diameter >15 mm. Main Outcome Measure(s): Serum and follicular fluid (FF) insulin, bioactive LH, immunoreactive FSH, and steroid levels. Result(s): Basal serum gonadotropin levels were comparable between female groups. Serum immunoreactive FSH levels, but not bioactive LH levels, at oocyte retrieval were greater in cycles of low-normal vs. high-normal response, whereas maximum serum E2 levels and total oocyte counts were similar between both groups. Despite elevated FF immunoreactive FSH levels, FF E2 was decreased in low-normal response cycles. Intrafollicular T and P also were decreased and increased, respectively, in cycles of low-normal response, although FF bioactive LH levels were comparable in both groups. Conclusion(s): Intrafollicular E2 is decreased in cycles of reduced ovarian responsiveness to GnRH agonist and recombinant hFSH and is not predicted by maximal serum E2 or number of oocytes retrieved. [Copyright &y& Elsevier]

Abstract: To determine if insulin-like growth factor (IGF)-1 and -2, FSH, or leptin alter IGF-binding protein (IGFBP)-2, -3, -4, and -5 mRNA levels in bovine granulosa and (or) theca cells, granulosa and theca cells were collected from bovine ovarian follicles, plated for 48h in 10% FCS and then treated for 24h in serum-free medium containing various hormone treatments arranged in three different experiments. Amounts of IGFBP-2, -3, -4, and -5 mRNA were quantitated using fluorescent quantitative real-time RT-PCR. Neither 100ng/ml of IGF-1 nor IGF-2 had an effect (P > 0.10) on IGFBP-2, -3, -4, or -5 mRNA levels in small-follicle (1–5mm; Experiment 1) granulosa cells. In large-follicle (>7.9mm; Experiment 2) granulosa cells, 100ng/ml of IGF-1 increased (P < 0.05) IGFBP-2 mRNA levels above controls and 3ng/ml of IGF-1; 100ng/ml of IGF-1 also decreased (P < 0.10) IGFBP-5 mRNA levels compared to 3ng/ml of IGF-1 or FSH or 100ng/ml leptin, while 100ng/ml of IGF-2 had no effect (P > 0.10) on IGFBP-2, -3, -4, and -5 mRNA levels (Experiment 2). At the doses tested, leptin and FSH had no effect (P > 0.10) on IGFBP-2, -3, -4, and -5 mRNA levels in large-follicle granulosa cells. In theca cells, IGF-2 decreased (P < 0.05) IGFBP-2 mRNA levels, but had no effect on IGFBP-3 or -4 mRNA expression (Exp. 3); IGF-1 did not affect (P > 0.10) thecal IGFBP-2, -3 or -4 mRNA levels. In contrast, IGF-1 but not IGF-2 increased (P < 0.01) thecal IGFBP-5 mRNA levels. Ligand blotting revealed that both IGF-1 and -2 increased IGFBP-2 and -5 (protein) and had no effect on IGFBP-3 (protein), whereas IGF-1 (but not IGF-2) increased IGFBP-4 (protein), suggesting IGFBP-2, -4, and -5 are post-transcriptionally regulated. These results suggest that expression of IGFBP-2, -3, -4, and -5 mRNA by granulosa and theca cells are differentially regulated by IGF-1 and -2, therefore discretely modulating the amount of bio-available IGFs to these cells depending upon the specific hormonal milieu. [Copyright &y& Elsevier]

The effects of estradiol, insulin, and gonadotropins on levels of insulin-like growth factor binding protein (IGFBP)-2, -3, -4, and -5 mRNA levels in bovine granulosa and theca cells were evaluated in vitro using serum-free medium containing various hormone treatments arranged in four different experiments. Amounts of IGFBP-2, -3, -4 and -5 mRNA were quantitated using fluorescent quantitative real-time RT-PCR. In small-follicle (1–5 mm) granulosa cells, follicle-stimulating hormone (FSH) in the presence or absence of insulin increased (P<0.05) IGFBP-3 mRNA but did not change IGFBP-2, -4, or -5 mRNA levels; estradiol was without effect on IGFBP-2, -3, -4, or -5 mRNA levels in the absence of insulin but increased (P<0.05) IGFBP-2 mRNA levels in the presence of insulin. Luteinizing hormone (LH) in the absence (but not presence) of insulin increased (P<0.05) small-follicle granulosa cell IGFBP-3 mRNA levels. In large-follicle (>7.9 mm) granulosa cells, insulin alone increased (P<0.05) IGFBP-2 gene expression while LH, FSH, and estradiol were without effect (P>0.10). Estradiol (3 and 300 ng/ml) decreased (P<0.05) IGFBP-5 mRNA levels in large-follicle granulosa cells. In theca cells, insulin decreased (P<0.05) IGFBP-4 expression, but had no effect (P>0.10) on IGFBP-2, -3, or -5 mRNA levels. Estradiol decreased (P<0.05) IGFBP-2, -3, and -4 mRNA levels but had no effect on IGFBP-5 mRNA levels in theca cells. LH had no effect on levels of IGFBP-2, -3, -4, or -5 mRNA in theca cells. These results indicate that expression of IGFBP-2, -3, -4, and -5 mRNA by granulosa and theca cells are differentially regulated by estradiol, insulin and gonadotropins, therefore discretely modulating the amount of bioavailable IGFs to these cells depending upon the specific hormonal stimuli. In particular, these studies are the first in cattle to show that estradiol selectively inhibits IGFBP-2, -3, and -4 gene expression in theca cells, inhibits IGFBP-5 gene expression in large-follicle granulosa cells, and stimulates IGFBP-2 gene expression in small-follicle granulosa cells. [Copyright &y& Elsevier]

The aim of the presented study was to evaluate the effects of PCB 126 and PCB 153 on granulosa and theca cell apoptosis. Granulosa and theca cells were collected from small, medium, and large preovulatory porcine follicles and cultured as monolayers. Cells were initially cultured for 24 h to allow attachment to the plates. Media were changed and 100 pg/ml PCB 126 or 100 ng/ml PCB 153 were added. After 48 h, granulosa and theca cells were fixed for assessment of the number of apoptotic cells utilizing a Hoechst staining technique or frozen for measurement of caspase-3 activity. Media were collected for testosterone concentration analysis from theca cell cultures or estradiol from granulosa cell cultures. Neither PCB 153 nor PCB 126 had an effect on testosterone secretion by theca cells collected from small and medium size follicles, while both PCBs decreased testosterone secretion by large follicles. The decrease in testosterone secretion by large follicles under the influence of both PCBs was paralleled by a suppression of caspase-3 activity and a decreased incidence of apoptotic bodies. Neither of the PCBs had an effect on estradiol secretion by granulosa cells collected from small and medium size follicles, while both PCBs increased estradiol in granulosa cells collected from large follicles. PCB-associated increased estradiol secretion by granulosa cells collected from large follicles was accompanied by suppression of caspase-3 activity and a decreased incidence of apoptotic bodies. In conclusion, we have presented evidence that in preovulatory follicles PCBs inhibit both theca and granulosa cells apoptosis. Therefore, an exposure to PCBs may cause alterations in the pattern of terminal differentiation of follicles and attenuate spontaneous elimination of atretic follicles. [Copyright &y& Elsevier]

Wilms' tumor gene WT1 encodes a nuclear transcriptional factor, which has been shown to regulate granulosa cell steroidogenesis in bovine; however, it is not known whether the functions of theca cells are regulated by WT1. Here, we determined the effects of this gene on theca cell proliferation, apoptosis, and steroidogenesis in vitro. In cultured bovine theca cells, the downregulation of WT1 increased the secretion of progesterone but had no effect on proliferation and apoptosis. WT1 includes the variants WT1(+KTS) and WT1(−KTS), which differ by 3 amino acids KTS (lysine, threonine, and serine). WT1(±KTS) upregulation increased the messenger RNA (mRNA) expression of STAR and CYP17A1 and decreased the progesterone secretion and CYP11A1 mRNA expression. In contrast to WT1(+KTS), WT1(−KTS) upregulation also decreased the mRNA expression of 3β-HSD. In both variants, WT1(−KTS) has more obvious effects. In conclusion, WT1 can decrease progesterone secretion, likely due in part to the inhibition of CYP11A1 and 3β-HSD. • WT1 was found to decrease progesterone production. • The decrease of progesterone production likely due in part to the inhibition of CYP11A1 and 3β-HSD. • In both variants, WT1(−KTS) has more obvious effects. [ABSTRACT FROM AUTHOR]

Polycystic ovary syndrome (PCOS) is a clinical syndrome characterized by hyperandrogenism, oligo/anovulation, and polycystic ovary. Autophagy is an intracellular system that degrades cytosolic proteins and organelles. The relationship between autophagy and PCOS has not been clarified. We found that p62 and ubiquitin were significantly increased in theca cells of women with PCOS using immunohistochemistry. Autophagy inhibition by palmitic acid and chloroquine in bovine theca cells increased p62 and ubiquitin and induced the expression of cytochrome P450 17A1 (CYP17A1) and plasminogen activator inhibitor-1 (PAI-1) mRNA. Furthermore, palmitic acid and chloroquine exposure significantly increased reactive oxygen species (ROS) and activated p38 and c-Jun N-terminal kinase (JNK). Inhibition of p38 and JNK significantly reduced CYP17A1 and PAI-1 mRNA expression. We showed that inhibition of autophagy in theca cells may have contributed to the pathogenesis of PCOS, based on CYP17A1 and PAI-1 mRNA expression via the ROS/p38 and JNK signalling pathways. • P62 and ubiquitin were increased in theca cells with polycystic ovary syndrome. • Autophagy inhibition by palmitic acid and chloroquine induce CYP17A1 and PAI-1 expression. • Palmitic acid and chloroquine stimulate ROS/p38 and JNK pathway. • Inhibition of p38 and JNK ameliorate CYP17A1 and PAI-1 expression upregulated by palmitic acid and chloroquine. [ABSTRACT FROM AUTHOR]

Overexpression of the transcription factor, E2F8 , has been associated with ovarian cancer. Objectives of this study were to determine: 1) if E2F8 gene expression in granulosa cells (GC) and theca cells (TC) change with follicular development, and 2) if E2F8 mRNA abundance in TC and GC is hormonally regulated. Using real-time PCR, E2F8 mRNA abundance in GC and TC was greater (P < 0.05) in small than large follicles. FGF9 induced an increase (P < 0.05) in E2F8 mRNA abundance by 1.6- to 7-fold in large-follicle (8–20 mm) TC and GC as well as in small-follicle (1–5 mm) GC. Abundance of E2F8 mRNA in TC was increased (P < 0.05) with FGF2, FGF9 or VEGFA treatments alone in vitro , and concomitant treatment of VEGFA with FGF9 increased (P < 0.05) abundance of E2F8 mRNA above any of the singular treatments; BMP4, WNT3A and LH were without effect. IGF1 amplified the stimulatory effect of FGF9 on E2F8 mRNA abundance by 2.7-fold. Collectively, our studies show for the first time that follicular E2F8 is developmentally and hormonally regulated indicating that E2F8 may be involved in follicular development. • E2F8 mRNA abundance is greater in small versus large follicles. • E2F8 mRNA in theca cells and granulosa cells was induced by FGF9. • E2F8 mRNA induced by FGF9 in theca cells is enhanced by IGF1 and VEGFA. • E2F8 may be involved in follicular growth of cattle. [ABSTRACT FROM AUTHOR]