Granulosa Layer Of Preovulatory Follicles Research Articles

Active immunization against AMH reveals its inhibitory role in the development of pre-ovulatory follicles in Zhedong White geese

The aim of this study was to investigate the effects of active immunization against recombinant Anti-Müllerian hormone (AMH) protein on the ovarian follicular development, egg production, and molecular regulatory mechanisms in broody-prone Zhedong White geese. For this, a recombinant goose AMH protein was expressed using a prokaryotic expression system. Fifty incubating geese from the same genetic background were selected and equally divided into two groups. The immunization group was actively immunized against the recombinant goose AMH protein, whereas the control group was immunized against bovine serum albumin (BSA). Immunization against AMH accelerated ovarian follicular development and increased clutch sizes by one to two eggs in two consecutive laying-incubation cycles. Furthermore, immunization against AMH upregulated the mRNA transcription levels of the FSH-beta gene in the pituitary gland, and FSHR, 3beta-HSD, and Smad4 genes in the granulosa layer of pre-ovulatory follicles; however, immunization downregulated the expression of the OCLN gene in the granulosa layer of pre-ovulatory follicles, and Smad5 and Smad9 genes in the granulosa layer of SYFs. These results suggest that AMH might hinder ovarian follicular development by decreasing both pituitary FSH secretion as well as ovarian follicular sensitivity to FSH. The latter molecular mechanism could be fulfilled by regulating Smad5 or Smad9 signals in SYFs, as well as the FSHR and Smad4 signals that affect progesterone synthesis and yolk deposition in the pre-ovulatory follicles.

Expression of aquaporin 4 in the chicken ovary in relation to follicle development.

In the mammalian ovary, aquaporins (AQPs) are thought to be involved in the regulation of fluid transport within the follicular wall and antrum formation. Data concerning the AQPs in the avian ovary is very limited. Therefore, the present study was designed to examine whether the AQP4 is present in the chicken ovary, and if so, what is its distribution in the ovarian compartment of the laying hen. Localization of AQP4 in the ovarian follicles at different stage of development was also investigated. After decapitation of hens the stroma with primordial follicles and white (1-4mm), yellowish (4-8mm), small yellow and the three largest yellow pre-ovulatory follicles F3-F1 (F3<F2<F1; 20-36mm) were isolated from the ovary. The granulosa and theca layers were separated from the pre-ovulatory follicles. The AQP4 mRNA and protein were detected in all examined ovarian compartments by the real-time PCR and Western blot analyses, respectively. The relative expression of AQP4 was depended on follicular size and the layer of follicular wall. It was the lowest in the granulosa layer of pre-ovulatory follicles and the highest in the ovarian stroma as well as white and yellowish follicles. Along with approaching of the largest follicle to ovulation the gradual decrease in AQP4 protein level in the granulosa layer was observed. Immunoreactivity for AQP4 was present in the granulosa and theca cells (theca interna≥theca externa>granulosa). The obtained results suggest that AQP4 may take part in the regulation of water transport required for follicle development in the chicken ovary.

Effect of Monochromatic Light on Expression of Estrogen Receptor (ER) and Progesterone Receptor (PR) in Ovarian Follicles of Chicken

Artificial illumination is widely used in modern poultry houses and different wavelengths of light affect poultry production and behaviour. In this study, we measure mRNA and protein abundance of estrogen receptors (ERs) and progesterone receptors (PRs) in order to investigate the effect of monochromatic light on egg production traits and gonadal hormone function in chicken ovarian follicles. Five hundred and fifty-two 19-wk-old laying hens were exposed to three monochromatic lights: red (RL; 660 nm), green (GL; 560 nm), blue (BL; 480 nm) and control cool white (400–760 nm) light with an LED (light-emitting diode). There were 4 identical light-controlled rooms (n = 138) each containing 3 replicate pens (46 birds per pen). Water was supplied ad libitum and daily rations were determined according to the nutrient suggestions for poultry. Results showed that under BL conditions there was an increase in the total number of eggs at 300 days of age and egg-laying rate during the peak laying period. The BL and GL extended the duration of the peak laying period. Plasma melatonin was lowest in birds reared under BL. Plasma estradiol was elevated in the GL-exposed laying hens, and GL and BL increased progesterone at 28 wk of age. In the granulosa layers of the fifth largest preovulatory follicle (F5), the third largest preovulatory follicle (F3) and the largest preovulatory follicle (F1), ERα mRNA was increased by BL and GL. Treatment with BL increased ERβ mRNA in granulosa layers of F5, F3 and F1, while GL increased ERβ mRNA in F5 and F3. There was a corresponding increase in abundance of the proteins in the granulosa layers of F5, with an increase in PR-B, generated via an alternative splice site, relative to PR-A. Treatment with BL also increased expression of PR mRNA in all of the granulosa layers of follicles, while treatment with GL increased expression of PR mRNA in granulosa layers of SYF(small yellow follicle), F5 and F1. These results indicate that blue and green monochromatic lights promote egg production traits via stimulating gonadal hormone secretion and up-regulating expression of ERs and PRs. Changes in PR-B protein suggest that this form of the progesterone receptor is predominant for progesterone action in the granulosa layers of preovulatory follicles in chickens during light stimulation.

Effect of 3,3′,5-triiodothyronine and 3,5-diiodothyronine on progesterone production, cAMP synthesis, and mRNA expression of STAR, CYP11A1, and HSD3B genes in granulosa layer of chicken preovulatory follicles

In vitro studies were performed to assess whether stimulatory effects of triiodothyronine (T 3) on progesterone (P 4) production in a granulosa layer (GL) of chicken preovulatory follicles are associated with 3′,5′-cyclic adenosine monophosphate (cAMP) synthesis and mRNA expression of STAR protein, CYP11A1, and HSD3B. Effects of 3,5-diiodothyronine (3,5-T 2) on steroidogenic function in these follicles were also investigated. The GL of F3 to F1 follicles was incubated in medium supplemented with T 3 or 3,5-T 2, LH, or forskolin (F), and a combination of each iodothyronine with LH or F. Levels of P 4 and cAMP in culture media were determined by RIA. Expression of genes involved in P 4 synthesis (ie, STAR protein, CYP11A1, and HSD3B) in the GL of F3 to F1 follicles incubated in medium with T 3 or 3,5-T 2 and their combination with LH was performed by real-time PCR. Triiodothyronine increased basal and LH- and F-stimulated P 4 secretion by preovulatory follicles. The 3,5-T 2 elevated P 4 synthesis by F3, had no effect on F2 follicles, and diminished P 4 production by the GL of F1 follicles. It had no effect on LH-stimulated P 4 production; however, it augmented F-stimulated P 4 production by F2 and F1 follicles. Although T 3 did not affect basal and F-stimulated cAMP synthesis by the GL of preovulatory follicles, it increased LH-stimulated synthesis of this nucleotide. However, 3,5-T 2 elevated F-stimulated cAMP synthesis in F3 and F2 follicles; it did not change basal and LH-stimulated cAMP production. Triiodothyronine decreased basal STAR and CYP11A1 mRNAs in F3 follicles, increased them in F1 follicles, and elevated HSD3B mRNA levels in F1 follicles. Triiodothyronine augmented LH-stimulated STAR, CYP11A1, and HSD3B mRNA levels in F2 and CYP11A1 in F1 follicles. However, T 3 decreased LH-stimulated STAR and HSD3B mRNA levels in F1 follicles. The 3,5-T 2 did not affect basal STAR and CYP11A1 mRNA expression in all investigated follicles; however, it decreased LH-stimulated STAR expression in F2 and F1 ones. The effects of 3,5-T 2 caused elevated basal but diminished LH-stimulated HSD3B mRNA levels. In conclusion, data indicate that both iodothyronines are involved in P 4 production in the GL of chicken preovulatory follicles acting alone and additively with LH. Effects of iodothyronines depend on follicle maturation and are associated with modulation of cAMP synthesis and STAR, CYP11A1, and HSD3B mRNA expression. We suggest that iodothyronines participate in maturation and ovulation of chicken follicles.

Immunolocalization of androgen receptor and steroidogenic enzymes in cumuli oophori of pre- and post-ovulatory rats

Immunolocalization of 3β-hydroxysteroid dehydrogense (3β-HSD), cytochrome P450c17 (P450c17) and androgen receptor (AR) were investigated in rat cumuli oophori (COCs) of late pre-ovulatory follicles and in post-ovulatory COCs bearing fertilized oocytes. A gradient of intensity of 3β-HSD immunolabelling was observed in the granulosa layer of pre-ovulatory follicles, with almost negative immunolabelling in COCs and with the strongest immunoreaction in the mural granulosa cells. Post-ovulatory COCs showed strong 3β-HSD immunolabelling in the peripheral regions and weak labelling near the oocyte, suggestive of responsiveness of cumulus cells to an anti-luteinizing effect exerted by the fertilized oocyte. In pre-ovulatory follicles, a weak P450c17 immunopositivity was limited to expanded cumulus granulosa cells and the positive labelling persisted in post-ovulatory COCs. P450c17 immunopositivity was also found in ampullary epithelial cells. A strong AR immunopositivity was confined mainly to the COCs in pre-ovulatory follicles and a similar immunoreaction was present in the granulosa cells of ovulated COCs. Simultaneous AR and cytochrome P450c17 immunolabelling in the pre- and post-ovulatory COCs is suggestive of an intra- and paracrine androgen regulation of the cumulus granulosa cell function.

Influence of triiodothyronine (T 3) on secretion of steroids and thyroid hormone receptor expression in chicken ovarian follicles

The present study was designed to (1) assess the role of triiodothyronine (T 3) with regard to in vitro steroid hormone secretion by chicken ovarian follicles; (2) determine whether T 3 influences the in vivo function of the pituitary-ovarian axis in the hen; and (3) detect expression of thyroid hormone receptor (TR) mRNA in chicken ovarian follicles. In the first experiment, laying hens were decapitated 22.5 h before ovulation. White prehierarchical follicles (1–8 mm) and fragments of theca and granulosa layers of the 3 largest yellow preovulatory follicles F3-F1 (22–35 mm) were incubated in a medium supplemented with T 3 (0, 0.1, 1, 10, 100, or 1000 ng/mL) or ovine luteinizing hormone (LH) (10 ng/mL) in combination with doses of T 3 (1, 10, and 100 ng/mL). Triiodothyronine decreased basal and LH-stimulated estradiol secretion by white follicles and the theca layer of all preovulatory follicles. On the other hand, it increased progesterone secretion by F2 and F1 follicles. In the second experiment, hens were injected 1 h after ovulation with saline (control) or T 3 (10 μg/100 g body weight, intraperitoneally). Results indicated that exogenous T 3 decreased plasma concentrations of LH and estradiol and increased plasma concentrations of progesterone. In the third experiment, using reverse transcription polymerase chain reaction (RT-PCR) analysis, expression of thyroid hormone receptor (TRα and TRβ0), mRNA was detected in all of the ovarian compartments. The expression of TRα mRNA was relatively greater in comparison with TRβ0. There were no differences between white ovarian follicles in the expression of TRα and TRβ0 mRNA. A considerably higher TRα and lower TRβ0 expression was detected in the granulosa layer of preovulatory follicles in comparison with the theca layer. In conclusion, the data indicate that thyroid hormones acting via nuclear receptors are involved in regulation of the pituitary-ovarian axis and processes associated with follicle growth and maturation.

Inhibition of ovulation by a lipoxygenase inhibitor involves reduced cyclooxygenase-2 expression and prostaglandin E2 production in gonadotropin-primed immature rats

Potential roles of cyclooxygenase (COX) pathway of arachidonic acid (AA) metabolism are established in a murine model of induced ovulation. Pharmacological inhibition of an alternative lipoxygenase (LOX) pathway has been shown to cause defective ovulation, but the mechanism is still undefined. This study investigated the effects of two LOX inhibitors and their time dependency on ovulation and COX activity in gonadotropins (eCG and human chorionic gonadotropin (hCG))-primed immature rats. Intra-ovarian bursal treatment with a general LOX inhibitor nordihydroguaiaretic acid (NDGA) at 0 h post-hCG (hCG0h) dose dependently inhibited ovulation rate. The drug was still but less effective when treated at hCG6h. A more specific inhibitor, 3,4-dihydroxyphenyl ethanol (DPE) was also inhibitory when treated at hCG0h but not at hCG6h. Interestingly, treatment with DPE at hCG0h resulted in attenuated expression of immunoreactive PTGS2 in granulosa layers and concomitant decrease in ovarian prostaglandin E(2) (PGE(2)) content at hCG8h. NDGA treatment reduced immunoreactive PTGS2. Ovulatory impairment by both inhibitors was prevented by systemic administration of PGE(2) at hCG6h. Immunohistochemistry revealed the expression of ALOX5 and ALOX12 in both thecal and granulosa layers of preovulatory follicles and, notably, the augmented immunoreactivities during 8 h after hCG treatment. Our results indicate the probable presence of multiple LOX isoforms and that specific inhibition of LOX at an early stage of hCG-signaling led to reduced PTGS2 activity and thus defective ovulation. They reveal a probable relationship between two pathways of AA metabolism and account at least partly for the mechanism by which the LOX inhibitor causes impaired ovulation.

Effects of Daidzein on Messenger Ribonucleic Acid Expression of Gonadotropin Receptors in Chicken Ovarian Follicles

Effects of daidzein on expression of mRNA of gonadotropin receptors [follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR)] were evaluated in ovarian follicles of ISA laying hens that were 13 mo old in the postpeak period of egg laying. The hens were randomly allocated as control and daidzein-treated groups, with daidzein supplemented to the basal diet at the level of 10 mg/kg for 7 wk. The granulosa layers of preovulatory follicles (F1, F2, F3, F4, F5) and follicular layers of the small yellow follicle (SYF), large white follicle (LWF), and atretic follicle were collected. The mRNA expression of related genes was measured by semiquantitative reverse transcription PCR. Results showed that daidzein significantly increased the egg-laying rate (P < 0.05) and the number of SYF and LWF (P < 0.05). The relative abundance of the FSHR mRNA decreased in the granulosa layers from F5 to F1, but LHR mRNA displayed the opposite trend in developmental changes. Treatment with daidzein resulted in increased expression of FSHR mRNA in LWF, SYF, and granulosa layers of F4 to F2 and LHR mRNA in granulosa layers of F4 and F1 (P < 0.05). These results indicated that dietary supplementation of daidzein upregulated mRNA expression of gonadotropin receptors to improve follicle development in chicken developing follicles and laying performance after the peak laying period.

Two Steroidogenic Pathways Present in the Chicken Ovary: Theca Layer Prefers Δ 5 Pathway and Granulosa Layer Prefers Δ 4 Pathway

It has been suggested that there is a shift in steroidogenic pathways of the theca layer from Δ 5 to Δ 4 during follicular maturation in the chicken ovary. However, the preferred steroid metabolic pathway in the theca and granulosa layers of small and preovulatory follicles has not been identified. Therefore, the goal of our study was to identify the preferred steroidogenic pathways used by these follicles. Previously, we found that small follicles that had not entered the follicular hierarchy did not use cholesterol but did use pregnenolone (P 5) to produce testosterone and estradiol. For this reason, P 5 was used as the substrate for steroid production in the present study. The theca layer of small follicles (2–4 mm in diameter), and the theca and granulosa layers of the fifth largest (F5), third largest (F3), and largest (F1) preovulatory follicles were isolated from the chicken ovary 22 hr before ovulation. The granulosa layer of small follicles was excluded from the study because it is steroidogenically incompetent. Tissues were incubated with [ 3H]-P 5 for 1 hr at 37° C. Production of steroid metabolites was determined on the basis of comparison with known steroid standards using two-dimensional thin layer chromatography (2D-TLC). Conversion of [ 3H]-P 5 to [ 3H]-DHEA was greater than that of [ 3H]-P 4 in the theca layer of small and preovulatory follicles. Even though the amount of [ 3H]-P 4 was less than that of [ 3H]-DHEA, theca cells from F3 and F1 follicles did produce [ 3H]-P 4. In contrast to the theca layer, conversion of [ 3H]-P 5 to [ 3H]-P 4 was greater than that of [ 3H]-DHEA in the granulosa layer of preovulatory follicles. Conversion of [ 3H]-P 5 to [ 3H]-DHEA by the granulosa layer gradually decreased as the follicle matured and approached ovulation. We conclude that both Δ 5 and Δ 4 pathways are functional in theca and granulosa cells. However, in contrast to earlier speculation, the theca layer preferentially metabolizes steroids via the Δ 5 pathway regardless of the maturational stage of the follicle, whereas the granulosa layer of preovulatory follicles preferentially metabolizes steroids via the Δ 4 pathway.

Temporal changes in inhibin subunit mRNAs during atresia of preovulatory follicles in the rat.

This study aimed to investigate the time course of disappearance of the mRNAs of the various subunits of inhibin in follicles which become atretic. An animal model was used in which atresia of preovulatory follicles could be studied in a chronological order. Injection of gonadotrophin-releasing hormone (GnRH) antagonist (20 microg) at the morning of pro-oestrus (P) blocked ovulation and the 10-12 preovulatory follicles became gradually atretic. A second injection was given the next day to prevent delayed ovulation. The rate of atresia could be delayed by simultaneous administration of a subovulatory dose of human chorionic gonadotrophin (hCG) (0.5 IU) and could be advanced by administration of a fivefold larger amount of GnRH antagonist. Functional activity of follicles becoming atretic was studied by measuring oestradiol production after incubation of individual follicles for 4 h. Follicles isolated 24 h after the first injection of GnRH antagonist (P+24) already secreted significantly less oestradiol in vitro than follicles isolated at pro-oestrus, although they were morphologically not different from pro-oestrous follicles. Follicles isolated at P+24 from hCG-treated rats secreted more oestradiol compared with follicles from rats not treated with hCG. In contrast, follicles isolated at P+24 from rats that were given a fivefold larger amount of GnRH antagonist secreted less oestradiol. Once this model was validated, temporal changes in inhibin subunit mRNAs in follicles undergoing atresia were measured by in situ hybridization and RNase protection assay. In situ hybridization showed abundant alpha- and betaA-subunit mRNA in the whole granulosa layer of preovulatory follicles at P and P+24, while betaB-subunit mRNA was restricted to the antral layer and cumulus. At P+48 the amount of alpha- and betaA-subunit mRNA had declined and was restricted to the cumulus, whereas betaB-subunit mRNA was absent. In the atretic follicles present at P+72 and P+96, mRNAs of all three inhibin subunits were absent. Administration of 0.5 IU hCG delayed the decline in the amount of alpha, betaA and betaB mRNA in preovulatory follicles at P+48. RNase protection assay of inhibin subunits in isolated follicles revealed no changes between P and P+24. However, at P+48, the mRNAs of alpha- and betaA-subunits were decreased. Expression of the mRNA of betaB-subunit declined gradually from P to P+48. The present study demonstrates that in follicles which are becoming atretic, mRNAs of alpha- and betaA-subunits decline simultaneously with the appearance of pycnotic cells in the granulosa layer, while betaB-subunit mRNA declines earlier, simultaneously with the decrease in the ability to secrete oestradiol in vitro.

Molecular cloning of inhibin/activin beta A-subunit complementary deoxyribonucleic acid and expression of inhibin/activin alpha- and beta A-subunits in the domestic hen.

A chicken inhibin/activin beta A-subunit cDNA clone has been isolated and used to detect the beta A-subunit mRNA in the hen. A cDNA library of the hen granulosa layer was screened with a rat inhibin beta A-subunit cDNA probe, and a cDNA of 1540 bp was cloned and sequenced. The clone contains the entire open reading frame of 1272 nucleotides encoding 424 amino acids. It shows 78% nucleotide identity (85% in deduced amino acids) in the full-length coding region and 85% nucleotide identity (98% in deduced amino acids) in the 116-amino acid C-terminal mature region to the rat beta A-subunit cDNA clone. The predicted positions of one N-linked glycosylation site and all cysteine residues are fully conserved as well. Northern blot analysis showed that the major 8.4-kb inhibin/activin beta A-mRNA band was primarily expressed in the granulosa layer of the preovulatory follicles. It was also detected in much less abundance in the theca layer, thyroid gland, heart, muscle, intestine, adrenal gland, liver, lung, spinal cord, and spleen. When a chicken inhibin alpha-subunit cDNA probe was used, the 1.7-kb major band of the inhibin alpha-subunit mRNA was detected most strongly in the granulosa layer and less abundantly in the postovulatory follicle, theca layer, testis, heart, and adrenal gland. Moreover, a distinct 1.3-kb alpha-mRNA was detected in the muscle and lung. The present cloning study extends our understanding of the inhibin/activin beta A-subunit cDNA from studied species to the chicken and shows that the beta A-subunit is highly conserved during evolution. Our data indicate that the mRNAs for inhibin/activin alpha- and beta A-subunits are widely present in a variety of chicken tissues but that the granulosa layer of large preovulatory follicles is the primary site of expression. The present study also suggests that both inhibin and activin may serve as local factors to regulate cell function in chicken tissues.

Steroid concentrations in isolated theca and granulosa layers of preovulatory follicles during the ovulatory cycle of the domestic hen.

The purpose of the present investigation was to measure the concentrations of progesterone (P4), testosterone (T) and estradiol-17 beta (E2) in isolated theca and granulosa layers of the five preovulatory follicles of the domestic hen. The largest follicle (F1), the second largest (F2), third largest (F3), fourth largest (F4) and fifth largest (F5) follicles were removed at 24, 18, 12, 6 and 2 h before the expected ovulation. Theca and granulosa layers were isolated and P4, T, E2 and protein concentrations determined. Protein concentrations of the granulosa and theca layers increased 5- and 15-fold, respectively, during the five ovulatory cycles prior to ovulation. As the follicle approached ovulation, there was a linear decrease in E2 concentration of the theca layer with the most significant decrease (P less than 0.001) occurring between 24 and 18 h of the ovulatory cycle. In the F3 and F4 theca layers, there was a significant increase (P less than 0.005) in E2 at 6 h of the ovulatory cycle. Fluctuations in T concentrations in theca and granulosa layers were similar. There was a significant increase (P less than 0.05) in T in both layers of the F2, F3 and F4 follicles at 6 h followed by a decrease (P less than 0.005) in the theca layers at 2 h of the ovulatory cycle. The P4 concentration of the granulosa layer increased gradually during follicular maturation, with the greatest increase occurring in the F2 and F1 granulosa layers between 18 and 12 h of the ovulatory cycle.(ABSTRACT TRUNCATED AT 250 WORDS)