Theca Research Articles

Obesity modulates cell-cell interactions during ovarian folliculogenesis

SummaryObesity is known to affect female reproduction, as evidenced by obese patients suffering from subfertility and abnormal oogenesis. However, the underlying mechanisms by which obesity impairs folliculogenesis are poorly documented. Here, we performed comprehensive single-cell transcriptome analysis in both regular diet (RD) and obese mouse models to systematically uncover how obesity affects ovarian follicle cells and their interactions. We found an increased proportion of Inhbb highly expressed granulosa cells (GCs) among all the GC subpopulations in obese mice. Under obese conditions, excessive androgen secreted from endocrine theca cells (ETCs) may contribute to the imbalanced change of GC subtypes through ETCs-GCs interactions. This is alleviated by enzalutamide, an androgen receptor antagonist. We also identified and confirmed typical GC markers, such as Marcks and Prkar2b, for sensitive evaluation of female fertility in obesity. These data represent a resource for studying transcriptional networks and cell-cell interactions during folliculogenesis under physiological and pathological conditions.

Simulated Microgravity Induces the Proliferative Inhibition and Morphological Changes in Porcine Granulosa Cells.

Astronauts are always faced with serious health problems during prolonged spaceflights. Previous studies have shown that weightlessness significantly affects the physiological function of female astronauts, including a change in reproductive hormones and ovarian cells, such as granulosa and theca cells. However, the effects of microgravity on these cells have not been well characterized, especially in granulosa cells. This study aimed to investigate the effects of simulated microgravity (SMG) on the proliferation and morphology of porcine granulosa cells (pGCs). pGC proliferation from the SMG group was inhibited, demonstrated by the reduced O.D. value and cell density in the WST-1 assay and cell number counting. SMG-induced pGCs exhibited an increased ratio of cells in the G0/G1 phase and a decreased ratio of cells in the S and G2/M phase. Western blot analysis indicated a down-regulation of cyclin D1, cyclin-dependent kinase 4 (cdk4), and cyclin-dependent kinase 6 (cdk6), leading to the prevention of the G1-S transition and inducing the arrest phase. pGCs under the SMG condition showed an increase in nuclear area. This caused a reduction in nuclear shape value in pGCs under the SMG condition. SMG-induced pGCs exhibited different morphologies, including fibroblast-like shape, rhomboid shape, and pebble-like shape. These results revealed that SMG inhibited proliferation and induced morphological changes in pGCs.

Fever and Abdominal Pain in a 10-year-old with Autism.

Fever and Abdominal Pain in a 10-year-old with Autism.

Polycystic ovary rat model exposure to 150 kHz intermediate frequency: hypothalamic-pituitary-ovarian axis at the receptor, cellular, tissue, and hormone levels

IntroductionThe hypothalamic-pituitary-ovarian (HPO) axis is the principal regulator of the reproductive system. The neurons in the arcuate nucleus of the hypothalamus signal the basophilic cells of the anterior pituitary to release luteinizing hormone (LH) and follicle stimulating hormone (FSH), which bind to the granulosa and theca cells of a follicle in the ovary to promote healthy follicular development. Disruption of this process at any time can lead to polycystic ovaries and, if left untreated, can lead to Polycystic Ovarian Syndrome (PCOS), one of the leading causes of infertility. A novel treatment option using 150 kHz Intermediate Frequency (IF) Electromagnetic Radiation (EMR) has been proposed to monitor the effect of this frequency during cystic development.MethodsTo prove this, an experiment was conducted to study the effect of whole-body exposure to 150 kHz EMR for 8 weeks at receptor, cellular, tissue and hormonal levels on the HPO axis of 25 young cyclic female rats.ResultsThe results showed that 150 kHz EMR did not affect the histoarchitecture of neurons of arcuate nucleus of the hypothalamus of PCO-induced rats. It was also found that the number of basophilic cells of the pituitary gland was increased and the immunoreactivity of LH and FSH secretion increased. This EMR field also decreased the development of follicular cysts in the ovary and possibly increased the immunoreactivity of the LH and FSH receptors as well on the theca and granulosa cells of follicles in the ovary.ConclusionThere are still many limitations to this study. If properly evaluated, the results of this experiment could help develop a new non-invasive treatment option for women with PCOS in the near future.

Beneficial Effects of Neurotrophin-4 Supplementation During in vitro Maturation of Porcine Cumulus-Oocyte Complexes and Subsequent Embryonic Development After Parthenogenetic Activation.

Neurotrophin-4 (NT-4) is a neurotrophic factor that plays an important role in follicular development and oocyte maturation. However, it is not yet known whether NT-4 is related to oocyte maturation and follicular development in pigs. This study aims to investigate the effects of NT-4 supplementation during in vitro maturation (IVM) of porcine oocytes and subsequent embryonic development after parthenogenetic activation (PA). First, NT-4 and its receptors (TrkB and p75NTR) were identified through fluorescent immunohistochemistry in porcine ovaries. NT-4 was mainly expressed in theca and granulosa cells; phospho-TrkB and total TrkB were expressed in theca cells, granulosa cells, and oocytes; p75NTR was expressed in all follicular cells. During IVM, the defined maturation medium was supplemented with various concentrations of NT-4 (0, 1, 10, and 100 ng/mL). After IVM, the nuclear maturation rate was significantly higher in the 10 and 100 ng/mL NT-4 treated groups than in the control. There was no significant difference in the intracellular reactive oxygen species levels in any group after IVM, but the 1 and 10 ng/mL NT-4 treatment groups showed a significant increase in the intracellular glutathione levels compared to the control. In matured cumulus cells, the 10 ng/mL NT-4 treatment group showed significantly increased cumulus expansion-related genes and epidermal growth factor (EGF) signaling pathway-related genes. In matured oocytes, the 10 ng/mL treatment group showed significantly increased expression of cell proliferation-related genes, antioxidant-related genes, and EGF signaling pathway-related genes. We also investigated the subsequent embryonic developmental competence of PA embryos. After PA, the cleavage rates significantly increased in the 10 and 100 ng/mL NT-4 treatment groups. Although there was no significant difference in the total cell number of blastocysts, only the 10 ng/mL NT-4 treatment group showed a higher blastocyst formation rate than the control group. Our findings suggest that supplementation with the 10 ng/mL NT-4 can enhance porcine oocyte maturation by interacting with the EGF receptor signaling pathway. In addition, we demonstrated for the first time that NT-4 is not only required for porcine follicular development, but also has beneficial effects on oocyte maturation and developmental competence of PA embryos.

Histological and Ultrastructural Characterization of the Gonads of the Grunting Toadfish Allenbatrachus grunniens (Linnaeus, 1758) from the Pranburi River Estuary, Thailand

Information on the reproductive biology of toadfish remains limited. In this study, we examined the structure and development of gonads in the grunting toadfish Allenbatrachus grunniens (Linnaeus, 1758) using morphological, histological and ultrastructural methods. The fish were collected from the Pranburi River Estuary, Thailand, during the annual reproductive period for this species (January to December 2018). The ovary of this species was paired and had elongated sac-like structures parallel to the kidneys and the digestive tract. In females, we identified 4 oocyte differentiation phases in the ovary including oogonia proliferation phase, a primary growth phase that was further classified into 2 steps (perinucleolar and oil droplets-cortical alveolar steps), and a secondary growth phase that also contained 2 steps (secondary growth and full-grown oocyte steps) and post-ovulatory phases, indicating an asynchronous pattern in ovarian development for this species. Transmission electron microscopy showed the 4 layers including the zona pellucida, basement membrane, granulosa cells and theca cells, all of which initially appeared in the oil droplets-cortical alveolar stage. The zona pellucida and the granulosa cells were highly developed during the secondary growth stage. The granulosa cells contained abundant smooth endoplasmic reticulum near the mitochondria. In males, the spermatogenesis was classified into spermatogonium to spermatozoa. Finally, we associated the morphological gonadal developments (stage I - IV) and the gonadosomatic index (GSI value) with the cellular developmental processes in both sexes. These results help integrate various levels of reproductive observations, which will be applied to understanding the reproductive cycle and development for aquaculture.

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

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

Generation of offspring-producing 3D ovarian organoids derived from female germline stem cells and their application in toxicological detection

In vitro production of oocytes capable of producing offspring has exciting potential applications in reproductive medicine. Here, we generated and characterized an ovarian organoid model derived from female germline stem cells using a three-dimensional culture system. We show that this model generated normal offspring and detected drug toxicity. The ovarian organoids could produce oocytes and exhibited endocrine functions. Single-cell analysis of ovarian organoids identified six ovarian cell lineages, such as germ, granulosa and theca cells, and produced gene-expression signatures for each cell type. Investigation of the expression patterns of genes related to meiosis and gene ontogeny analysis for germ cell clusters showed that a germ cell population was maintained in the ovarian organoids. Moreover, flow cytometric analysis confirmed that the population of germ cells could be maintained on the organoids and showed that ascorbic acid treatment had a beneficial effect of germ cell population maintenance on the organoids. Furthermore, we demonstrated the successful production of offspring from oocytes derived from ovarian organoids. Finally, we showed the ovarian organoids had the potential to drug toxicological detection. For example, we found that salinomycin impaired the formation of ovarian organoids and germ cell population maintenance by inducing apoptosis. These results indicate that the female germline stem cell-derived ovarian organoids represent a valuable model system for generating oocytes that can yield offspring, and provide a novel model for drug screening and toxicological detection.

Effect of the spatial\u2013temporal specific theca cell Cyp17 overexpression on the reproductive phenotype of the novel TC17 mouse

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

ANTI-FERTILITY ACTIVITY OF LEAVES AND STEM PARTS OF ARTABOTRYS ODORATISSIMUS. (ROXB.) R.BR. ANNONACEAE

The present study was done to evaluate the effects of Artabotrys odoratissimus in regulation of fertility in female albino wistar rats. Two successive solvent extracts (Ethanolic and Aqueous) of the leaves and stem of Artabotrys odoratissimus used at two dose levels 250, 500 mg/kg and 200, 400 mg/kg respectively to evaluate antifertility in female albino wistar rats by interfering the growth and release of eggs from the ovaries (ovulation). To observe the effect of various treatments on rats the rats were divided into six groups. After 21 days treatment, it was observed that the growth rate, weight gain and obesity of the rats, violent behavior of the rats like lateral attacks, biting and the muricidal behavior significant increase in Test- Iand Test- IV groups in compared with others group. The serum cholesterol and estrogen levels were increased tremendously in the Test- I, whereas estrogen levels decreased when compared to other groups. In histopathology of ovary the area of the largest follicle and the thickness of its follicular wall that is the theca cell layer and granulosa cell layer were calculated. The ovarian follicles and corpora lutea (CL) at different stages of development and regression, and the theca and granulosa cell layer were analyzed profoundly

Metabolic exchanges between the oocyte and its environment: focus on lipids.

Finely regulated fatty acid (FA) metabolism within ovarian follicles is crucial to follicular development and influences the quality of the enclosed oocyte, which relies on the surrounding intra-follicular environment for its growth and maturation. A growing number of studies have examined the association between the lipid composition of follicular compartments and oocyte quality. In this review, we focus on lipids, their possible exchanges between compartments within the ovarian follicle and their involvement in different pathways during oocyte final growth and maturation. Lipidomics provides a detailed snapshot of the global lipid profiles and identified lipids, clearly discriminating the cells or fluid from follicles at distinct physiological stages. Follicular fluid appears as a main mediator of lipid exchanges between follicular somatic cells and the oocyte, through vesicle-mediated and non-vesicular transport of esterified and free FA. A variety of expression data allowed the identification of common and cell-type-specific actors of lipid metabolism in theca cells, granulosa cells, cumulus cells and oocytes, including key regulators of FA uptake, FA transport, lipid transformation, lipoprotein synthesis and protein palmitoylation. They act in harmony to accompany follicular development, and maintain intra-follicular homeostasis to allow the oocyte to accumulate energy and membrane lipids for subsequent meiotic divisions and first embryo cleavages.

257 Awardee Talk: Pursuit of Novel Factors that Regulate Ovarian Follicular Function

Abstract As follicles grow, theca cells (TC) and granulosa cells (GC) must proliferate with minimal differentiation while thecal vascularization increases so that follicles do not prematurely ovulate or luteinize before the oocyte is fully mature. In the early 2000s we used Affymetrix microarrays to discover several unique genes involved in ovarian follicular development. Thrombospondin and fibroblast growth factor (FGF) 2 receptor genes were stimulated by IGF1 in porcine GC. We compared GC gene expression in bovine cystic versus normal follicles and discovered several novel genes including Indian hedgehog protein (IHH), FGF9, brain ribonuclease (BRB), and G protein-coupled receptor 34 (GPR34), leading to identification of roles for these proteins in ovarian follicular development. During the past 10 years, follow-up TC microarray and mechanistic studies have identified FGF9 control of cell cycle proteins, tight junction proteins, and microRNA 221 (MIR221), and that the mitogenic and steroidogenic responses to the major trophic hormones of the ovary (including IGF1, LH and FSH) are altered by overexpression of MIR221 in GC. In addition, we discovered that: 1) FGF9 stimulates GC and TC mitosis while inhibiting steroidogenesis; 2) FGF9 induces E2 transcription factor (E2F)-1, E2F-8 and cyclin D1 (CCND1), and that both IGF1 and vascular endothelial growth factor-A (VEGFA) synergize with FGF9 to further induce E2F8 and CCND1 mRNA; 3) FGF9 induces the nuclear protein UHRF1; and 4) an E2F inhibitor blocks the stimulatory and inhibitory effects of FGF9 on GC proliferation and steroidogenesis, respectively, and down-regulates UHRF1 mRNA and up-regulates VEGFA mRNA. Thus, aberrant production of FGF9 and the factors it induces/inhibits may lead to vascular dysfunction and ovarian disorders such as ovarian cysts. With additional research, knowledge about these newly identified factors may be used to help the livestock industry improve reproductive efficiency via new treatments for estrous synchronization, superovulation and cystic ovaries.

PSX-A-24 Late-Breaking: Testosterone production by theca cells of large preovulatory follicles is affected by growth hormone depending on the hen age and the presence of granulosa cells

Abstract Testosterone produced by theca cells may be involved in regulating of the growth and ovulation of hen preovulatory follicles (Rangel, Gutierrez, Gen Comp Endocrinol, 203:250, 2014). In the current research, we studied effects of growth hormone (GH), a known regulator of the hen ovarian function, on in vitro testosterone production by the theca layer (TL) from the two largest yellow follicles in relation to the hen age and the presence of the granulosa layer (GL). Young hens with long clutch (YLC, 32–33 week-old, >10 eggs per clutch) and old hens with short clutch (OSC, 74–76 week-old, 3–6 eggs per clutch) were used. After isolation, TL from F1 and F2 follicles (n = 8–9) was cultured for 18 h in two systems, separately or together with the corresponding GL, in the presence or absence of chicken GH (25 ng/ml). Concentrations of testosterone in the spent media were measured by ELISA. The data were analyzed by RM-ANOVA. In the case of separate TL culture, GH did not change significantly testosterone production in both follicles of YLC hens and reduced it from 338±105 to 152±52 fmol/mg tissue (P < 0.05) in F1 follicles of OSC hens. When TL was cultured in the presence of GL, GH enhanced 1.8-2.6-fold (P < 0.05) the secretion of testosterone in the case of F1 follicles and decreased it 1.8-2.5-fold (P < 0.05) in the case of F2 follicles in both young and old hens. Regardless of the treatment, follicular size or culture system, the production of testosterone in OSC hens was 2–5 times higher than in YLC hens. The results indicate that the interaction between TL and GL changes the steroidogenic response of theca cells from preovulatory follicles to GH in young and old hens. Furthermore, testosterone production is obviously increased with reproductive aging of laying hens. The study was supported by RFBR (19-016-00216).

Immuno-localization of estrogen receptor (ER) and progesterone receptor (PR) in the buffalo ovary in relation to their plasma hormonal levels

In this study, we examined the tissue distribution of estrogen receptor alpha (ERα) and progesterone receptor (PR) in different compartments of the buffalo ovary during follicular and luteal phases of the estrous cycle. The receptors were localized by immunohistochemistry. Image analysis was done to quantify the immune reactivity. ERα was localized in various cell types of buffalo ovaries differentially during follicular and luteal phases of the estrous cycle. Immunoreactivity of ERα was detected in the primordial, primary, secondary and tertiary follicles, atretic follicles, in cells of the deep and superficial stroma, and the tunica albuginea. Specific immunostaining was observed with anti-ERα antibodies in the nuclei of follicular cells/granulosa cells and theca cells. No reaction was observed in the ovarian surface epithelium. In the growing follicle and secondary follicle, the immunoreaction for these receptors was strong. While in the tertiary follicles weak immunoreactions were recorded in the granulosa cells and theca cells. The progesterone receptors (PR) as revealed by immunohistochemistry were localized in the nuclei of different groups of ovarian cells. It was detected in the primordial, primary, secondary and tertiary follicles, atretic follicles, in cells of the deep and superficial stroma, and the tunica albuginea and surface epithelium. PR was localized in follicular cells of preantral and antral follicles, the stroma of the ovary, endothelial cells of blood vessels. PR positivity was found in one or two granulosa cells of primordial and primary follicles, with moderate immunoreaction, but no staining in oocytes. In the antral follicles, both granulosa cells, as well as theca cells, were immunostained for PR. In the obliterative atretic follicles, the invading stromal cells were highly positive for PR. Follicular cells of the primordial follicle and granulosa cells and theca cells of tertiary follicles had statistically higher percentage positive cells in the follicular phase as compared to the luteal phase. No staining was observed in the negative controls.

Imunolocalização de angiotensina-(1-7), angiotensina II e enzima-conversora de angiotensina 2 em ovário de cabras

There is increasing evidence as to the participation of the ovarian renin-angiotensin system in important reproductive processes. The inhibition of the angiotensin-converting enzyme (ACE) caused an increase in the rate of ovulation and pregnancy in the artificial insemination protocol has fixed time (TFIA). This study aimed to investigate the presence and location of Ang II, Ang- (1-7) and ACE2 in goat ovaries and the possibility of the involvement of these peptides in previous results. Ten ovaries from goats were collected in a slaughterhouse, washed in buffered PBS, perfused with protease inhibitor solution and processed for immunohistochemistry protocol. The search for peptides was performed using the avidin–biotin–peroxidase method. A strong immunoreactivity for Ang II in theca cells of antral follicles and corpus luteum was observed. Antral follicles (theca cells), corpus luteum and oocyte cytoplasm in early antral follicles exhibited strong immunoreactivity for Ang-(1-7). There was strong immunoreactivity for ACE2 in the cytoplasm of luteal cells and theca cells of antral follicles. In this study, for the first time, the presence and location of Ang II, Ang-(1-7) and ACE2 are reported in goat ovary, suggesting that there is participation in follicular development, oocyte maturation and corpus luteum development.

Inositols: From Established Knowledge to Novel Approaches.

Myo-inositol (myo-Ins) and D-chiro-inositol (D-chiro-Ins) are natural compounds involved in many biological pathways. Since the discovery of their involvement in endocrine signal transduction, myo-Ins and D-chiro-Ins supplementation has contributed to clinical approaches in ameliorating many gynecological and endocrinological diseases. Currently both myo-Ins and D-chiro-Ins are well-tolerated, effective alternative candidates to the classical insulin sensitizers, and are useful treatments in preventing and treating metabolic and reproductive disorders such as polycystic ovary syndrome (PCOS), gestational diabetes mellitus (GDM), and male fertility disturbances, like sperm abnormalities. Moreover, besides metabolic activity, myo-Ins and D-chiro-Ins deeply influence steroidogenesis, regulating the pools of androgens and estrogens, likely in opposite ways. Given the complexity of inositol-related mechanisms of action, many of their beneficial effects are still under scrutiny. Therefore, continuing research aims to discover new emerging roles and mechanisms that can allow clinicians to tailor inositol therapy and to use it in other medical areas, hitherto unexplored. The present paper outlines the established evidence on inositols and updates on recent research, namely concerning D-chiro-Ins involvement into steroidogenesis. In particular, D-chiro-Ins mediates insulin-induced testosterone biosynthesis from ovarian thecal cells and directly affects synthesis of estrogens by modulating the expression of the aromatase enzyme. Ovaries, as well as other organs and tissues, are characterized by a specific ratio of myo-Ins to D-chiro-Ins, which ensures their healthy state and proper functionality. Altered inositol ratios may account for pathological conditions, causing an imbalance in sex hormones. Such situations usually occur in association with medical conditions, such as PCOS, or as a consequence of some pharmacological treatments. Based on the physiological role of inositols and the pathological implications of altered myo-Ins to D-chiro-Ins ratios, inositol therapy may be designed with two different aims: (1) restoring the inositol physiological ratio; (2) altering the ratio in a controlled way to achieve specific effects.

Knockdown of Ptprn-2 delays the onset of puberty in female rats

In the present study, we evaluated how Ptprn-2 (encoding tyrosine phosphatase, receptor type, N2 polypeptide protein) affects the onset of puberty in female rats. We evaluated the expression of Ptprn-2 mRNA and protein in the hypothalamus-pituitary-ovary axis of female rats using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunofluorescence at infancy, prepuberty, puberty, peripuberty, and adulthood. We evaluated the effects of Ptprn-2 gene knockdown on different aspects of reproduction-related biology in female rats, including the expression levels of puberty-related genes in vivo and in vitro, the time to onset of puberty, the concentration of serum reproductive hormones, the morphology of ovaries, and the ultrastructure of pituitary gonadotropin cells. Our results demonstrated that PTPRN-2 was primarily distributed in the arcuate nucleus (ARC), periventricular nucleus (PeN), adenohypophysis, and the ovarian follicular theca, stroma, and granulosa cells of female rats at various stages. Ptprn-2 mRNA levels significantly varied between peripuberty and puberty (P < 0.05) in the hypothalamus and pituitary gland. In hypothalamic cells, Ptprn-2 knockdown decreased the expression of Ptprn-2 and Rfrp-3 mRNA (P < 0.05) and increased the levels of Gnrh and Kiss-1 mRNA (P < 0.05). Ptprn-2 knockdown in the hypothalamus resulted in delayed vaginal opening compared to the control group (n = 12, P < 0.01), and Ptprn-2, Gnrh, and Kiss-1 mRNA levels (P < 0.05) all decreased, while the expression of Igf-1 (P < 0.05) and Rfrp-3 mRNA (P < 0.01) increased. The concentrations of FSH and P4 in the serum of Ptprn-2 knockdown rats were lower than in control animals (P < 0.05). Large transverse perimeters and longitudinal perimeters (P < 0.05) were found in the ovaries of Ptprn-2 knockdown rats. There were fewer large secretory particles from gonadotropin cells in adenohypophysis tissue of the Ptprn-2 knockdown group compared to the control group. This indicates that Ptprn-2 knockdown can regulate levels of Gnrh, Kiss-1, and Rfrp-3 mRNA in the hypothalamus, regulate the concentration of serum FSH and P4, and alter the morphology of ovarian and gonadotropin cells, delaying the onset of puberty in female rats.

Prenatal exposure to an environmentally relevant phthalate mixture alters ovarian steroidogenesis and folliculogenesis in the F1 generation of adult female mice

Phthalates are a family of chemicals that can be found in plastic and personal care products used by consumers every day and they are known endocrine disrupting chemicals that can disrupt female reproduction. In previous studies, an environmentally relevant phthalate mixture was shown to affect female reproduction in a transgenerational manner. However, limited information was available on the effect of phthalate mixtures on ovarian steroidogenesis and folliculogenesis. Ovarian steroidogenesis is important for producing hormones needed for reproduction and ovarian regulation, and folliculogenesis is essential for the development of ovarian follicles and successful fertility. Thus, this study tested the hypothesis that prenatal exposure to an environmentally relevant phthalate mixture adversely affects ovarian steroidogenesis and folliculogenesis in the F1 generation of adult female mice. Pregnant dams (F0 generation) were orally dosed with vehicle control or a phthalate mixture (20 μg/kg/day-500 mg/kg/day) daily from gestational day 10 to birth, and the adult F1 females were the offspring of the dosed dams. The ovaries of the F1 generation were collected at postnatal day 60. One ovary was used for histological examination of the numbers and percent of different follicle types. The other ovary was used to measure expression of theca and granulosa cell enzymes. Additionally, sera were collected for measuring hormone levels. The results indicate that prenatal exposure to the phthalate mixture decreases hormone levels and gene expression, alters the transitioning of follicle types, and leads to a higher incidence of atresia in the F1 generation offspring.

ERβ Regulation of Gonadotropin Responses during Folliculogenesis.

Gonadotropins are essential for regulating ovarian development, steroidogenesis, and gametogenesis. While follicle stimulating hormone (FSH) promotes the development of ovarian follicles, luteinizing hormone (LH) regulates preovulatory maturation of oocytes, ovulation, and formation of corpus luteum. Cognate receptors of FSH and LH are G-protein coupled receptors that predominantly signal through cAMP-dependent and cAMP-independent mechanisms that activate protein kinases. Subsequent vital steps in response to gonadotropins are mediated through activation or inhibition of transcription factors required for follicular gene expression. Estrogen receptors, classical ligand-activated transcriptional regulators, play crucial roles in regulating gonadotropin secretion from the hypothalamic–pituitary axis as well as gonadotropin function in the target organs. In this review, we discuss the role of estrogen receptor β (ERβ) regulating gonadotropin response during folliculogenesis. Ovarian follicles in Erβ knockout (ErβKO) mutant female mice and rats cannot develop beyond the antral state, lack oocyte maturation, and fail to ovulate. Theca cells (TCs) in ovarian follicles express LH receptor, whereas granulosa cells (GCs) express both FSH receptor (FSHR) and LH receptor (LHCGR). As oocytes do not express the gonadotropin receptors, the somatic cells play a crucial role during gonadotropin induced oocyte maturation. Somatic cells also express high levels of estrogen receptors; while TCs express ERα and are involved in steroidogenesis, GCs express ERβ and are involved in both steroidogenesis and folliculogenesis. GCs are the primary site of ERβ-regulated gene expression. We observed that a subset of gonadotropin-induced genes in GCs, which are essential for ovarian follicle development, oocyte maturation and ovulation, are dependent on ERβ. Thus, ERβ plays a vital role in regulating the gonadotropin responses in ovary.

Germ cells are not essential for sexual dimorphism of gonads in common carp, C. carpio L.

Common carp is the fourth most cultivated fish species in the world. Sexually dimorphic growth of common carp makes its sex differentiation an important research topic. The main objective of this study was to investigate the role of germ cells in sex differentiation of common carp. We generated the germ cell-deficient common carp through knocking down dead end gene with a morpholino antisense oligonucleotide during the embryogenesis. In the common carp without germ cells, we found about half of the gonads (type I gonads) contain granulosa cells and theca cells, which were the same with somatic cells we observed in the wild-type ovaries. The remaining half of the gonads (type II gonads) contain Sertoli cells and Leydig cells, which were the same with somatic cells in the wild-type testes. Furthermore, the expression of sex differentiation related genes was detected in the gonads without germ cells. It was found cyp19a1a was mainly expressed in the type I gonads, and the cyp19a1a mRNA level is much higher in each type I gonad than in the type II gonad. While dmrt1 was mainly expressed in the type II gonads, and the dmrt1 mRNA level is much higher in each type II gonad than in the type I gonad. Our comprehensive analysis showed that the gonadal structure and expression of genes related with sex differentiation were sexually dimorphic in the germ cell-deficient carp. These findings are valuable for discovering the underlying mechanism of common carp sex differentiation which is crucial for cultivating monosexual carp for aquaculture.