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ABSTRACT: 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.

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

Context: New animal welfare legislation and ethical guidelines encourage alternative approaches for canine contraception, instead of surgical gonadectomy which is considered invasive and unjustified in healthy dogs. Aims: Reversible contraception might be achieved by inhibition of aromatase (CYP19), an enzyme catalysing the conversion of androgens to oestrogens. This study provides insights into the spatio-temporal expression and distribution of aromatase in canine ovarian tissue. Methods: Ovarian tissue was collected from 39 healthy and sexually mature bitches during different stages of the oestrous cycle: pro-oestrus (n = 8), oestrus (n = 12), dioestrus (n = 9) (luteal phase) and anoestrus (n = 10). Localisation of cytochrome P450 aromatase was determined by immunohistochemistry. Key results: Aromatase activity in the dog is high during pro-oestrus, ovulation and early dioestrus. Comparing types of follicles and corpora lutea, the highest aromatase abundance was found in antral follicles and luteinising follicles, whereas corpora lutea and early antral follicles showed an intermediate presence of the enzyme. Interesting was the high abundance of aromatase in luteinising theca interna cells, prevailing over granulosa cells. Conclusions and implications: Understanding of cells involved in oestradiol production is important for targeted inhibition of oestradiol synthesis, possibly offering an approach for contraception and suppression of oestrus. In clinical practice, contraception in female dogs is traditionally achieved by surgical gonadectomy. As this method is discouraged by new animal welfare legislation in some parts of the world and is also related to several side effects, alternative possibilities for contraception are needed. Here, we report the distribution of aromatase in the canine ovary during different stages of the oestrous cycle. In-depth knowledge of oestrogen synthesis in the female dog is mandatory for targeted inhibition of oestradiol, possibly by the use of aromatase inhibitors. Photograph by M. P. Kowalewski. [ABSTRACT FROM AUTHOR]

ABSTRACT: 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 3β-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 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 (r2 = 0.819, P < 0.001), StAR (r2 = 0.844, P < 0.001), 3β-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 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.

Summary: Background: In the United States, the carrying capacity of the land has been overwhelmed with the wild horse and burro populations. Identification of a long‐term contraception method is needed to control growing populations as current methods cannot quell these growing numbers. Sperm acrosome‐associated 3 protein (SPACA3) is a protein expressed in both male and female reproductive systems. SPACA3 may prove to be a viable contraception target in horses in immunosterilant research. Objectives: The objective of the current study was to evaluate and compare ovarian immunoexpression of SPACA3 between domesticated and free‐roaming mares. This information may lead to the development of a SPACA3 immunosterilant to reduce free‐roaming horse and burro populations. Study design: Non‐randomised comparative study design. Methods: Routine immunohistochemistry was performed on ovarian sections from domesticated (n = 8) and free‐roaming mares (n = 8). At least three representative images of each follicle stage (primordial, primary, secondary, tertiary) from each ovary were digitally captured and analysed using Image J software. Expression of SPACA3 was quantified in cellular locations (granulosa or theca) specific to each follicle stage: primordial, primary, secondary, and tertiary. Results: Domesticated horses had higher SPACA3 expression compared to free‐roaming mares in granulosa cells of primordial (p = 0.0022), primary (p < 0.001), secondary (p = 0.025), and tertiary follicles (p = 0.0078). In theca cells, domesticated horses had higher SPACA3 expression in tertiary follicles (p = 0.022), but not secondary follicles (p = 0.089). The highest SPACA3 expression was observed in the granulosa cells of tertiary follicles (p = 0.022). Main limitations: Varying background colours of representative images taken of ovaries, leading to slight technical issues in Image J software. Conclusions: This is the first report quantifying SPACA3 immunoexpression during folliculogenesis and describing the spatial and temporal expression of SPACA3 in the equine ovary. This information may lead to the development of a SPACA3 immunosterilant to reduce free‐roaming horse and burro populations. [ABSTRACT FROM AUTHOR]

Polycystic ovary syndrome (PCOS) is a common and frequent disease and always leads endocrine and metabolic disorder among women in reproductive age. Ovary is the main organ involved in polycystic ovary syndrome, and its function impairment will lead to reproductive dysfunction. Some recent studies have demonstrated that autophagy plays an important role in the pathogenesis of PCOS, and there are many different mechanisms that affect autophagy and the occurrence of PCOS, and they provide a new direction for us to predict the mechanism of PCOS. In this review, we discuss the role of autophagy in different ovarian cells: granulosa cells, oocytes, and theca cells, and introduce the important role that they play in the progress of PCOS. The main purpose of this review is to provide the research background and some relevant suggestions for our future work in autophagy and help us better explore the pathogenesis and autophagy mechanisms of PCOS. Furthermore, it will help us gain a new insight of the pathophysiology and treatment of PCOS. [ABSTRACT FROM AUTHOR]

Kisspeptin, a hypothalamic neuropeptide encoded by the KISS1 gene, has a pivotal role in promoting gonadotrophin-releasing hormone secretion in mammals. Kisspeptin and its receptor (KISS1R) are also expressed in certain peripheral tissues including gonads, suggesting intra-gonadal roles. Such actions at the level of the bovine ovary have not been explored previously. The current aims were to determine whether KISS1 and KISS1R are expressed in the bovine ovary and whether kisspeptin or a kisspeptin antagonist can modulate ovarian steroid production by cultured ovarian cells. Granulosa cells (GC) and theca interna cells (TC) were collected from antral follicles (3–18 mm) categorized into five class sizes. Early, mid and regressing corpora lutea (CL) were also collected for RT-qPCR analysis of KISS1 and KISS1R expression. Bovine TC and GC cultured under both non-luteinizing (serum-free) and luteinizing (serum-supplemented) conditions were treated for 4 days with kisspeptin-10 (10–10–10–6M) or kisspeptin antagonist (kp234; 10–10–10–6M), alone and in combination with either follicle-stimulating hormone (GC), luteinizing hormone (TC) or forskolin (luteinized GC/TC). Steroid secretion (GC: oestradiol, progesterone; TC: androstenedione, progesterone; luteinized GC/TC: progesterone) was measured by ELISA and viable cell number determined by neutral red uptake assay. KISS1 and KISS1R transcripts were detected in TC, GC and CL with significant differences between follicle categories and CL stages. However, neither kisspeptin-10 nor kisspeptin antagonist affected steroid secretion or viable cell number in any of the four ovarian cell culture models. As such, the hypothesis that kisspeptin has a direct intraovarian role to modulate follicular or luteal steroidogenesis, or cell proliferation/survival, is not supported.

ABSTRACT: 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 P4 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 E2 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.

Yaks (Bos grunniens) are the only bovine species that adapt well to the harsh high-altitude environment in the Qinghai-Tibetan plateau. However, the reproductive adaptation to the climate of the high elevation remains to be elucidated. Cell composition and molecular characteristics are the foundation of normal ovary function which determines reproductive performance. So, delineating ovarian characteristics at a cellular molecular level is conducive to elucidating the mechanism underlying the reproductive adaption of yaks. Here, the single-cell RNA-sequencing (scRNA-seq) was employed to depict an atlas containing different cell types with specific molecular signatures in the yak ovary. The cell types were identified on the basis of their specifically expressed genes and biological functions. As a result, a cellular atlas of yak ovary was established successfully containing theca cells, stromal cells, endothelial cells, smooth muscle cells, natural killer cells, macrophages, and proliferating cells. A cell-to-cell communication network between the distinct cell types was constructed. The theca cells were clustered into five subtypes based on their biological functions. Further, CYP11A1 was confirmed as a marker gene for the theca cells by immunofluorescence staining. Our work reveals an ovarian atlas at the cellular molecular level and contributes to providing insights into reproductive adaption in yaks. [ABSTRACT FROM AUTHOR]

The formation of functional eggs (oocyte) in ovarian follicles is arguably one of the most important events in early mammalian development since the oocytes provide the bulk genetic and cytoplasmic materials for successful reproduction. While past studies have identified many genes that are critical to normal ovarian development and function, recent studies have highlighted the role of mechanical force in shaping folliculogenesis. In this review, we discuss the underlying mechanobiological principles and the force-generating cellular structures and extracellular matrix that control the various stages of follicle development. We also highlight emerging techniques that allow for the quantification of mechanical interactions and follicular dynamics during development, and propose new directions for future studies in the field. We hope this review will provide a timely and useful framework for future understanding of mechano-signalling pathways in reproductive biology and diseases.

KRAS plays critical roles in regulating a range of normal cellular events as well as pathological processes in many tissues mediated through a variety of signaling pathways, including ERK1/2 and AKT signaling, in a cell-, context- and development-dependent manner. The in vivo function of KRAS and its downstream targets in gonadal steroidogenic cells for the development and homeostasis of reproductive functions remain to be determined. To understand the functions of KRAS signaling in gonadal theca and interstitial cells, we generated a Kras mutant (tKrasMT) mouse line that selectively expressed a constitutively active KrasG12D in these cells. KrasG12D expression in ovarian theca cells did not block follicle development to the preovulatory stage. However, tKrasMT females failed to ovulate and thus were infertile. The phosphorylated ERK1/2 and forkhead box O1 (FOXO1) and total FOXO1 protein levels were markedly reduced in tKrasMT theca cells. KrasG12D expression in theca cells also curtailed the phosphorylation of ERK1/2 and altered the expression of several ovulation-related genes in gonadotropin-primed granulosa cells. To uncover downstream targets of KRAS/FOXO1 signaling in theca cells, we found that the expression of bone morphogenic protein 7 (Bmp7), a theca-specific factor involved in ovulation, was significantly elevated in tKrasMT theca cells. Chromosome immunoprecipitation assays demonstrated that FOXO1 interacted with the Bmp7 promoter containing forkhead response elements and that the binding activity was attenuated in tKrasMT theca cells. Moreover, Foxo1 knockdown caused an elevation, whereas Foxo1 overexpression resulted in an inhibition of Bmp7 expression, suggesting that KRAS signaling regulates FOXO1 protein levels to control Bmp7 expression in theca cells. Thus, the anovulation phenotype observed in tKrasMT mice may be attributed to aberrant KRAS/FOXO1/BMP7 signaling in theca cells. Our work provides the first in vivo evidence that maintaining normal KRAS activity in ovarian theca cells is crucial for ovulation and female fertility. [ABSTRACT FROM AUTHOR]

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

Objective: To investigate the direct effect of growth differentiation factor 9 (GDF9) on androgen production in human theca cells., Design: Experimental study., Setting: Tertiary hospital-based research laboratory., Patient(s): Women who underwent in vitro fertilization and intracytoplasmic sperm injections at our clinic were included in this study., Intervention(s): Primary cultured human theca cells from women undergoing in vitro fertilization and intracytoplasmic sperm injection treatment were treated with GDF9, an activin receptor-like kinase 5 (ALK5) inhibitor, and a SMAD4 agonist., Main Outcome Measure(s): The expression of androgen synthesis-related genes StAR, CYP17A1, and LHCGR, levels of androstenedione and testosterone, phosphorylation of SMAD2/3, and the interaction between bone morphogenic protein-activated type II receptor and ALK5 were evaluated using reverse transcription-quantitative polymerase chain reaction, Western blot, enzyme-linked immunosorbent assays, and coimmunoprecipitation assays, respectively., Result(s): Growth differentiation factor 9 decreased StAR, CYP17A1, and LHCGR expression levels in human theca cells, which was prevented by treatment with the ALK5 inhibitor, and suppressed production of androgen in human theca cells. Growth differentiation factor 9 increased SMAD2/3 phosphorylation, and the ALK5 inhibitor also suppressed this effect. Bone morphogenic protein-activated type II receptor and ALK5 bound to each other after GDF9 stimulation. The SMAD4 agonist kartogenin also decreased messenger RNA levels of StAR and CYP17A1 and protein levels of StAR in human theca cells., Conclusion(s): Growth differentiation factor 9 can activate the bone morphogenic protein-activated type II receptor-ALK5-SMAD2/3 signaling pathway, suppress CYP17A1 expression, and decrease androgen production in human theca cells., Competing Interests: Declarations of competing interest X.G. has nothing to disclose. Y.Z. has nothing to disclose. Y.L. has nothing to disclose. R.W. has nothing to disclose. L.H. has nothing to disclose. C.H. has nothing to disclose. M.C. has nothing to disclose., (Copyright © 2023 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

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]

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]

The formation of functional eggs (oocyte) in ovarian follicles is arguably one of the most important events in early mammalian development since the oocytes provide the bulk genetic and cytoplasmic materials for successful reproduction. While past studies have identified many genes that are critical to normal ovarian development and function, recent studies have highlighted the role of mechanical force in shaping folliculogenesis. In this review, we discuss the underlying mechanobiological principles and the force-generating cellular structures and extracellular matrix that control the various stages of follicle development. We also highlight emerging techniques that allow for the quantification of mechanical interactions and follicular dynamics during development, and propose new directions for future studies in the field. We hope this review will provide a timely and useful framework for future understanding of mechano-signalling pathways in reproductive biology and diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Biswas, Ng, Prabhakaran and Chan.)

• 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]

KRAS plays critical roles in regulating a range of normal cellular events as well as pathological processes in many tissues mediated through a variety of signaling pathways, including ERK1/2 and AKT signaling, in a cell-, context- and development-dependent manner. The in vivo function of KRAS and its downstream targets in gonadal steroidogenic cells for the development and homeostasis of reproductive functions remain to be determined. To understand the functions of KRAS signaling in gonadal theca and interstitial cells, we generated a Kras mutant ( tKrasMT ) mouse line that selectively expressed a constitutively active Kras G12D in these cells. Kras G12D expression in ovarian theca cells did not block follicle development to the preovulatory stage. However, tKrasMT females failed to ovulate and thus were infertile. The phosphorylated ERK1/2 and forkhead box O1 (FOXO1) and total FOXO1 protein levels were markedly reduced in tKrasMT theca cells. Kras G12D expression in theca cells also curtailed the phosphorylation of ERK1/2 and altered the expression of several ovulation-related genes in gonadotropin-primed granulosa cells. To uncover downstream targets of KRAS/FOXO1 signaling in theca cells, we found that the expression of bone morphogenic protein 7 ( Bmp7 ), a theca-specific factor involved in ovulation, was significantly elevated in tKrasMT theca cells. Chromosome immunoprecipitation assays demonstrated that FOXO1 interacted with the Bmp7 promoter containing forkhead response elements and that the binding activity was attenuated in tKrasMT theca cells. Moreover, Foxo1 knockdown caused an elevation, whereas Foxo1 overexpression resulted in an inhibition of Bmp7 expression, suggesting that KRAS signaling regulates FOXO1 protein levels to control Bmp7 expression in theca cells. Thus, the anovulation phenotype observed in tKrasMT mice may be attributed to aberrant KRAS/FOXO1/BMP7 signaling in theca cells. Our work provides the first in vivo evidence that maintaining normal KRAS activity in ovarian theca cells is crucial for ovulation and female fertility., Competing Interests: Z-JL is currently employed by the Company Alltech Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sun, Wang, Liu, Guo, Li, Cao, Ko, Lan and Lei.)