Modulating effect of growth hormone on the functional state of cultured cells from hen preovulatory follicles

Somatotropic hormone (STH) is an important positive modulator of ovarian function in mammals. Local production of STH and the expression of the corresponding specific receptors were also detected in hen ovarian follicles, which indicates the participation of this hormone in the endocrine/paracrine control of folliculogenesis in birds. Nevertheless, the role of STH in the regulation of growth of avian follicles at the final stage of maturation is still not clear.Objective: To study in vitro the effect of STH on the proliferative activity and apoptotic changes of granulosa and theca cells from preovulatory follicles of domestic hens.Materials and methods. Young laying hens aged 34-35 weeks with a long clutch were used in the experiments. Granulosa and theca cells were isolated from the largest yellow follicle in the hierarchy (F1). The cells were cultured in a medium containing 10% fetal bovine serum until a monolayer was formed, and then for 24 h in the medium without serum in the absence (control) or in the presence of STH at various concentrations (1-100 ng/ml). The proliferative activity and apoptotic changes in the cells were assessed by immunocytochemical assay, based on the expression level of proliferating cell nuclear antigen PCNA and pro-apoptotic protein Bax, respectively.Results. The proportion of PCNA-positive granulosa cells increased 1.3-1.8 times (P<0.01-0.05) as compared to control with increasing the content of STH in the medium to 10-100 ng/ml. Furthermore, within this concentration range, the studied hormone reduced 1.2-1.6 times (P<0.05) the relative number of granulosa cells with the positive reaction to Bax. The sensitivity of theca cells to the growth-stimulating effect of STH was lower than that of granulosa cells. Such the effect of STH led to an increase in the proportion of PCNA-positive thecal cells by 1.2-1.3 times (P<0.05) and was detected only at concentrations of 25 and 100 ng/ml. Meanwhile, STH (25-100 ng/ml) increased 1.3 times (P<0.05) the level of Bax expression in theca cells.Conclusions. The results of the present study indicate the stimulating effect of STH in vitro on the proliferative activity of granulosa and theca cells from the most mature hen preovulatory follicle. In addition, STH is able to reduce the expression of the pro-apoptotic protein Bax in granulosa cells and increase this expression in thecal cells. Thus, the data obtained indicate the possible participation of STH in the regulation of growth and development of follicles at the final stage of maturation during the period of maximum egg-laying intensity in laying hens.

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

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, &gt;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 &lt; 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 &lt; 0.05) the secretion of testosterone in the case of F1 follicles and decreased it 1.8-2.5-fold (P &lt; 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).

Immunolocalization of angiotensin-(1-7), angiotensin II and angiotensin-converting enzyme 2 in goat ovary

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.

Immunohistochemistry and Expression Profile of Estrogen Receptor 2 Gene in Different Grade Size Ovarian Follicles of Leizhou Black Ducks

BackgroundEstrogen receptor 2 (ESR2) plays significant biological roles in the reproductive system and ovarian follicle development. This study, therefore, aimed to reveal the expression pattern and cell-specific localization of ESR2 in the ovarian follicles of Leizhou black ducks. MethodFour laying Leizhou black ducks at 43 weeks old were annihilated and different grade-sized follicles were collected for immunohistochemistry and expression profile study. The follicles were grouped into seven (7) as small white follicles (SWF), large white follicles (LWF), small yellow follicles (SYF), large yellow follicles (LYF), follicle 5 (F5), follicle 2 (F2), and follicle 1 (F1). ResultsThe qRT/PCR results displayed that ESR2 mRNA was expressed in all follicles with the highest (P < 0.05) level of expression found in F1 compared to other follicles. Immunohistochemistry analysis of the cell-specific localization of ESR2 protein revealed that ESR2 was distributed in both granulosa and theca cells region in all the follicles examined. There was a significantly higher localization of ESR2 protein in the granulosa cells than the theca cells of SWF, SYF, LYF, F2, and F1. Comparatively, ESR2 was highly expressed in the granulosa cells of LYF than in all the other follicles. ConclusionThese results provide theoretical knowledge for the in-depth study of the related biological functions of the ESR2 gene and its application at the cellular level.

Wingless-type mouse mammary tumor virus integration site (WNT) regulation of bovine theca cells

Ovarian paracrine mediation by components of the wingless-type mouse mammary tumor virus integration site ligands (WNT1 to 11) and their receptors, Frizzled family members (FZD1 to 10), has been proposed. Secreted truncated forms of FZD proteins (e.g., SFRP4) block the action of WNT ligands. Dickkopf-1 (DKK1) is another WNT antagonist, and R-spondin-1 (RSPO1) is one of a group of four secreted proteins that enhance WNT/β-catenin signaling. Our hypothesis was that granulosa cells signal theca cells (TC) via SFRP4, DKK1, RSPO1 and WNT secretion to regulate TC differentiation and proliferation. Therefore, in vitro experiments were conducted to study the effects of WNT family member 3A (WNT3A), WNT5A, RSPO1, DKK1, insulin-like growth factor 1 (IGF1), bone morphogenetic protein 7 (BMP7), Indian hedgehog (IHH), and fibroblast growth factor-9 (FGF9) on bovine TC proliferation and steroidogenesis. Theca cells of large (8 to 20 mm) and small (3 to 6 mm) follicles were collected from bovine ovaries, TC monolayers were established in vitro, and treated with various doses of recombinant human WNT3A, WNT5A, RSPO1, DKK1, IGF1, FGF9, BMP7, IHH and/or ovine LH in serum-free medium for 48 h. In experiment 1 using LH-treated TC, IGF1, IHH and WNT3A increased (P &lt; 0.05) cell numbers and androstenedione production, whereas WNT3A and BMP7 inhibited (P &lt; 0.05) progesterone production. In experiment 2, FGF9 blocked (P &lt; 0.05) the WNT3A-induced increase in androstenedione production in LH plus IGF1-treated TC. In experiment 3, RSPO1 further increased (P &lt; 0.05) LH plus IGF1-induced progesterone and androstenedione production. In experiment 4, SFRP4 and DKK1 alone had no significant effect on TC proliferation or progesterone production of large-follicle TC, but both blocked the inhibitory effect of WNT5A on androstenedione production. In contrast, DKK1 alone inhibited (P &lt; 0.05) small-follicle TC androstenedione production whereas SFRP4 was without effect. We conclude that the ovarian TC WNT system is functional in cattle, with WNT3A increasing proliferation and androstenedione production of TC.

New Insights on Folliculogenesis and Follicular Placentation in Marine Viviparous Fish Black Rockfish (Sebastes Schlegelii)

Background: In viviparous fish, a considerable degree of variation in placental structures have been described. However, no distinct structures are reported in Scorpaenidae.Results: In this study, we demonstrate a new type of folliculogenesis and follicular placentation in Sebastes schlegelii. Before copulation, the germinal epithelium gradually surrounds the oocytes and develops to individually follicles with a stalk-like structure hanging on the ovigerous lamella, which ensures each follicle have access to spermatozoa after copulation. From stage V to early gestation, the cyp17-I highly expressed accompanied by cyp19a1a signals disappearance, and 11-ketotestosterone level keeps rising and peaks at blastula stage, while 17β-estradiol declines to the bottom. Meanwhile, the theca cells rapidly proliferate and invade outwards forming a highly hypertrophied and folded microvillous placenta. This unbalance of hormone might be an important factor driving the theca cells proliferation and invasion. Additionally, some conserved genes related to mammalian placentation are significantly high expression in follicular placenta suggesting the high convergence in vertebrate placenta evolution.Conclusions: This finding provided a new type of placentation pattern for viviparous teleost between the intrafollicular gestation and intraluminal gestation.

Prostaglandin F2 Alpha Triggers the Disruption of Cell Adhesion with Cytokeratin and Vimentin in Bovine Luteal Theca Cells

Intermediate filaments (IFs) maintain cell–cell adhesions and are involved in diverse cellular processes such as cytokinesis, cell migration and the maintenance of cell structure. In this study, we investigated the influence of prostaglandin F2 alpha (PGF2α) on cytokeratin and vimentin IFs, Rho-associated protein kinase (ROCK), and cell-cell adhesion in bovine luteal theca cells (LTCs). The luteal cells were isolated from bovine corpus luteum (CL), and the LTCs were treated with 0, 0.01, 0.1 and 1.0 mM PGF2α. Cytokeratin, vimentin and desmoplakin proteins were disrupted and the ROCK protein was significantly increased in PGF2α-treated LTCs. In addition, cell–cell adhesion was significantly (p < 0.05) decreased in the PGF2α-induced LTCs compared to control group (0 mM PGF2α). In conclusion, PGF2α affected the adhesion of cell to cell via disruption of desmoplakin, cytokeratin and vimentin, additionally increasing ROCK in bovine LTCs. These results may provide a better understanding of the mechanism of bovine CL regression.

Creating an Artificial 3-Dimensional Ovarian Follicle Culture System Using a Microfluidic System

We hypothesized that the creation of a 3-dimensional ovarian follicle, with embedded granulosa and theca cells, would better mimic the environment necessary to support early oocytes, both structurally and hormonally. Using a microfluidic system with controlled flow rates, 3-dimensional two-layer (core and shell) capsules were created. The core consists of murine granulosa cells in 0.8 mg/mL collagen + 0.05% alginate, while the shell is composed of murine theca cells suspended in 2% alginate. Somatic cell viability tests and hormonal assessments (estradiol, progesterone, and androstenedione) were performed on days 1, 6, 13, 20, and 27. Confocal microscopy confirmed appropriate compartmentalization of fluorescently-labeled murine granulosa cells to the inner capsule and theca cells to the outer shell. Greater than 78% of cells present in capsules were alive up to 27 days after collection. Artificially constructed ovarian follicles exhibited intact endocrine function as evidenced by the production of estradiol, progesterone, and androstenedione. Oocytes from primary and early secondary follicles were successfully encapsulated, which maintained size and cellular compartmentalization. This novel microfluidic system successfully encapsulated oocytes from primary and secondary follicles, recapitulating the two-compartment system necessary for the development of the mammalian oocyte. Importantly, this microfluidic system can be easily adapted for sterile, high throughput applications.