Early preantral follicles of the domestic cat express gonadotropin and sex steroid signaling potential.

Key biomolecular processes, which regulate primordial ovarian follicle dormancy and early folliculogenesis in mammalian ovaries, are not fully understood. The domestic cat is a useful model to study ovarian folliculogenesis and is the most relevant for developing in vitro growth methods to be implemented in wild felid conservation breeding programs. Previously, RNA-sequencing of primordial (PrF), primary (PF), and secondary follicle (SF) samples from domestic cat implicated ovarian steroidogenesis and steroid reception during follicle development. Here, we aimed to identify which sex steroid biosynthesis and metabolism enzymes, gonadotropin receptors, and sex steroid receptors are present and may be potential regulators. Differential gene expression, functional annotation, and enrichment analyses were employed and protein localization was studied too. Gene transcripts for PGR, PGRMC1, AR (steroid receptors), CYP11A1, CYP17A1, HSD17B1 and HSD17B17 (steroidogenic enzymes), and STS (steroid metabolizing enzyme) were significantly differentially expressed (Q values of ≤0.05). Differential gene expression increased in all transcripts during follicle transitions apart from AR which decreased by the secondary stage. Immunohistochemistry localized FSHR and LHCGR to oocytes at each stage. PGRMC1 immunostaining was strongest in granulosa cells, whereas AR was strongest in oocytes throughout each stage. Protein signals for steroidogenic enzymes were only detectable in SFs. Products of these significantly differentially expressed genes may regulate domestic cat preantral folliculogenesis. In vitro growth could be optimized as all early follicles express gonadotropin and steroid receptors meaning hormone interaction and response may be possible. Protein expression analyses of early SFs supported its potential for producing sex steroids.

Comparative messenger RNA expression of FSHß, LHß, FSHR, LHR, and ERß in high and low prolific goat breeds.

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) have a central role in follicle growth and maturation, but no clear differences between breeds with different ovulation rates have been found. Therefore, this study investigated mRNA expression of FSHß, LHß, FSH receptor (FSHR), LH receptor (LHR), and estrogen receptor-ß (ERß) genes in prolific Lezhi black (LB) goats and nonprolific Tibetan (TB) goats by real-time PCR. Follicles and pituitaries were recovered from goats at 12-24 h after onset of estrus. Real-time PCR analysis revealed that the expression levels of FSHß and LHß mRNA were significantly higher (p < 0.01) in pituitary of LB than in TB does, but the expression levels of FSHR and LHR mRNA in follicle of TB were greater (p < 0.05). Expression level of follicular ER ß was not different between the two breeds. Data provide evidence that the greater ovulation rate in the LB goat as compared to the TB breed is associated with a greater gonadotropin expression during follicular phase.

Distribution of LPXRFa, a gonadotropin-inhibitory hormone ortholog peptide, and LPXRFa receptor in the brain and pituitary of the tilapia.

In vertebrates, gonadotropin-releasing hormone (GnRH) and gonadotropin-inhibitory hormone (GnIH), respectively, regulate reproduction in positive and negative manners. GnIH belongs to the LPXRFa family of peptides previously identified in mammalian and nonmammalian vertebrates. Studying the detailed distribution of LPXRFa as well as its receptor (LPXRFa-R) in the brain and pituitary is important for understanding their multiple action sites and potential functions. However, the distribution of LPXRFa and LPXRFa-R has not been studied in teleost species, partially because of the lack of fish-specific antibodies. Therefore, in the present study, we generated specific antibodies against LPXRFa and its receptor from Nile tilapia (Oreochromis niloticus), and examined their distributions in the brain and pituitary by immunohistochemistry. Tilapia LPXRFa-immunoreactive neurons lie in the posterior ventricular nucleus of the caudal preoptic area, whereas LPXRFa-R-immunoreactive cells are distributed widely. Double immunofluorescence showed that neither LPXRFa-immunoreactive fibers nor LPXRFa-R is closely associated or coexpressed with GnRH1, GnRH3, or kisspeptin (Kiss2) neurons. In the pituitary, LPXRFa fibers are closely associated with gonadotropic endocrine cells [expressing luteinizing hormone (LH) and follicle-stimulating hormone (FSH)], with adrenocorticomelanotropic cells [corticotropin (ACTH) and α-melanotropin (α-MSH)], and with somatolactin endocrine cells. In contrast, LPXRFa-R are expressed only in LH, ACTH, and α-MSH cells. These results suggest that LPXRFa and LPXRFa-R signaling acts directly on the pituitary cells independent from GnRH or kisspeptin and could play multiple roles in reproductive and nonreproductive functions in teleosts. J. Comp. Neurol. 524:2753-2775, 2016. © 2016 Wiley Periodicals, Inc.

Effect of inhibin from primate Sertoli cells and GnRH on gonadotropin subunit mRNA in rat pituitary cell cultures.

Partially purified inhibin from primate Sertoli cell culture medium (pSCl) suppresses both LH and FSH secretion from cultured rat pituitary cells stimulated with GnRH. To examine the mechanism of action of pSCl, we have measured steady state levels of mRNAs for the gonadotropin subunits in pituitary cell cultures exposed to 10 nM GnRH for 6 h in control or pSCl-containing medium (short term) and after 72-h pretreatment with pSCl or control medium (long term). Messenger RNA levels were determined by Northern analysis using specific cDNA probes for rat FSH beta, LH beta, and the common alpha-subunit. In the long term experiments, pSCl inhibited GnRH-stimulated release of FSH (47.4 +/- 3.3% of control), LH (69.2 +/- 2.3%), and free glycoprotein alpha-subunit (74.2 +/- 4.5%), and intracellular FSH declined to 88.4 +/- 3.5% of control. Concentrations of the subunit mRNAs were all decreased: FSH beta to 54.4 +/- 5.0%, LH beta to 79.6 +/- 9.4%, and alpha to 70.8 +/- 8.7% of control. In the short-term experiments, pSCl also suppressed FSH, LH, and alpha-subunit secretion to 75.9 +/- 3.6%, 79.5 +/- 2.1%, and 90.9 +/- 1.8% of control, respectively. Intracellular LH and alpha-subunit levels were significantly increased in cells treated for 6 h with GnRH and pSCl (155 +/- 18%, 145 +/- 14% of control), while FSH was comparable to control. After 6 h, pSCl selectively reduced the level of mRNA for FSH beta (56.5 +/- 5.8% of control).(ABSTRACT TRUNCATED AT 250 WORDS)

Agonist-induced phosphorylation of the luteinizing hormone/chorionic gonadotropin receptor expressed in a stably transfected cell line.

Much of the definitive work on G-protein-coupled receptor phosphorylation and its impact on receptor function has been performed with the catecholamine receptors. Evidence for receptor phosphorylation is lacking, however, for G-protein-coupled receptors that bind larger ligands, such as LH/CG. Using immunoprecipitation techniques and a clonal cell line stably transfected with the LH/CG receptor, we show here for the first time that exposure of cells to hCG induces phosphorylation of its cognate receptor. The hCG-induced increase in receptor phosphorylation requires receptor activation because it cannot be elicited with a hCG antagonist and is mediated at least in part by the cAMP second messenger system. This hypothesis is supported by the finding that the hCG-induced receptor phosphorylation is greatly reduced (but not abolished) in a cell line that overexpresses cAMP phosphodiesterase and that receptor phosphorylation can be induced by activation of endogenous cAMP synthesis with prostaglandin E2 or by addition of 8-bromo-cAMP. Last, we show that LH/CG receptor phosphorylation can be induced with a phorbol ester, but not with a calcium ionophore. We also examined a potential correlation between LH/CG receptor phosphorylation and uncoupling of the receptor from its effector. Although the phorbol ester-induced phosphorylation of the LH/CG receptor can be correlated with uncoupling, other experiments indicate that hCG-induced uncoupling of the LH/CG receptor can occur under conditions where the cAMP-mediated receptor phosphorylation is greatly reduced (or abolished).

The presence of gonadotropin receptors in nonpregnant human uterus, human placenta, fetal membranes, and decidua.

The possible presence of gonadotropin receptors in nonpregnant human uterus and human fetoplacental unit was investigated by light microscope immunocytochemistry using a monoclonal antibody to rat luteal hCG/LH receptors. The receptor antibody cross-reacted with human and bovine hCG/LH receptors and appears to be directed against the receptor rather than other proteins, including HLA class I antigens. Uterus and fetoplacental unit contained receptor antibody-binding sites, which indicates the presence of hCG/LH receptors. In the endometrium these receptors were present in glandular and luminal epithelial cells as well as in stromal cells. In the myometrium the receptors were detected in circular and elongated myometrial smooth muscle and vascular smooth muscle. Comparison of immunostaining intensities, which indicates the presence of different amounts of receptors, revealed that luminal and glandular epithelial cells contained more receptors than stromal cells. These cells, in turn, contained more receptors than myometrial and vascular smooth muscle. All cells in secretory phase uterine specimens contained more receptors than corresponding cells from the proliferative phase of the cycle. Midpregnancy placenta, amniotic epithelium, chorionic cytotrophoblasts, and decidual cells contained hCG/LH receptors. At term pregnancy, while receptors in fetal membranes and decidua continue to be detected, placental tissues did not show any detectable receptors unless the tissues were pretreated with neuraminidase. This indicated that term pregnancy placenta contain hCG/LH receptors masked by sialic acid residues. Comparison of immunostaining intensities suggested that syncytiotrophoblasts contained more receptors than cytotrophoblasts at midpregnancy; mesenchymal cells or blood vessels contained no detectable receptors. There were more receptors in decidua than in fetal membranes at mid- and term pregnancy. While the amniotic epithelial receptors decreased, the receptors in chorionic cytotrophoblasts and decidual cells increased from mid- to term pregnancy. In summary, hCG/LH receptors were demonstrated in the nonpregnant human uterus, human placenta, fetal membranes, and decidua. This indicates that hCG/LH may directly regulate functions of these tissues by endocrine, autocrine, or paracrine mechanisms.

The expression of human chorionic gonadotropin/human luteinizing hormone receptors in human gestational trophoblastic neoplasms.

Normal human placental trophoblasts have recently been shown to contain receptors for hCG/hLH. The present studies investigated the expression of these receptors in hyperplastic and anaplastic trophoblasts in gestational trophoblastic neoplasms. The results demonstrated that both hydatidiform moles and choriocarcinomas contained receptor messenger RNA (mRNA) and receptor protein. A variety of nontrophoblast tumors, on the other hand, contained neither receptor mRNA nor receptor protein. Choriocarcinomas contained more receptor mRNA and receptor protein than hydatidiform moles which in turn contained more than normal human placenta. Midluteal phase human corpus luteum contained more receptor mRNA than normal human placenta and about the same as choriocarcinomas. The hyperplastic and anaplastic trophoblasts in hydatidiform moles and choriocarcinomas contained more receptor immunostaining than the normal trophoblasts in the same tissue or those from normal placentas from about the same gestational age. The receptor immunostaining increased as the degree of trophoblast hyperplasia increased in hydatidiform moles. Anaplastic trophoblasts of choriocarcinomas contained a similar amount of receptor immunostaining as severely hyperplastic trophoblasts of hydatidiform moles. Invading anaplastic trophoblasts of choriocarcinoma contained greater amount of receptor immunostaining than the surrounding endometrial stromal and myometrial smooth muscle cells. In summary, this is the first study to our knowledge demonstrating the expression of hCG/hLH receptor gene in gestational trophoblastic neoplasms. The increased receptor expression in these neoplasms suggests that hCG, via its receptors, could play a fundamental and previously unsuspected autocrine role in the regulation of trophoblast transformation, growth, invasion, and high hCG secretion.

Effect of short-term starvation on reproductive hormone gene expression, secretion and receptor levels in male rats.

The effects of 4-6 days of food deprivation on the pituitary-testicular function of adult male rats were studied. Fasting decreased body weights on average by 23% (P less than 0.01) and those of seminal vesicles by 55% (P less than 0.01) in 4 days. No consistent changes were found in testicular and ventral prostate weights. The pituitary levels of gonadotrophin-releasing hormone (GnRH) receptors decreased by 50% (P less than 0.01). Serum and pituitary levels of LH, FSH and prolactin decreased by 25-50% (P less than 0.01 for all). Testicular and serum levels of testosterone decreased by 70-80%, testicular LH receptors by 26%, those of prolactin by 50% (P less than 0.01 for all), but those of FSH remained unaffected. Acute (2 h) stimulation by a GnRH agonist (buserelin, 10 micrograms/kg i.m.) resulted in similar LH, FSH and testosterone responses in the fasted and control animals, and human chorionic gonadotrophin (hCG) stimulation (30 IU/kg i.m.) in similar increases in testosterone. A 42% decrease was found in pituitary content of mRNA of the common alpha subunit (P less than 0.05), but the mRNAs of the LH- and FSH-beta chains and prolactin were unaffected by fasting for 4 days. When the same mRNAs were measured after 6 days of fasting, the decrease of the mRNA of FSH-beta also became significant (50%, P less than 0.01). In contrast, the mRNA of LH-beta was increased twofold (P less than 0.01) at this time and serum LH levels were similar in control and starved animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Agonist activity of mammalian gonadotropin-releasing antagonists in chicken gonadotropes reflects marked differences in vertebrate gonadotropin-releasing receptors.

The pharmacology of mammalian and avian gonadotropin-releasing (GnRH) receptors differs for agonist analogues. We have therefore compared the activities of mammalian-based GnRH antagonists in sheep and chicken gonadotropes to further elucidate the different structural requirements of the receptors. The antagonist activities of ten GnRH analogues were compared in cultured sheep and chicken pituitary cells by determining the dose required to cause a 50% inhibition of luteinizing hormone secretion (IC50) induced by GnRH at its half-maximal concentration (EC50). Nine analogues showed high antagonist activity in the sheep bioassay. Analogue IC50s varied between half and twice ((1.22-6.06) x 10(-10) M) the GnRH EC50 (3 x 10(-10) M). One of these peptides exhibited partial agonist activity. In contrast, eight of the analogues showed low antagonist activity in chicken pituitary cells, with IC50s varying from 46 to 1490 times ((1.4-44.7) x 10(-7) M) the GnRH EC50 (3 x 10(-9) M) and had a different order of potencies compared with that in the sheep. Furthermore, two analogues did not display antagonist activity at all in the chicken bioassay, but acted as pure agonists, stimulating LH secretion. These findings demonstrate marked differences in pharmacology between the avian and mammalian pituitary GnRH receptors and emphasize that GnRH antagonists, selected for their efficacy in mammals, cannot necessarily be used for physiological studies in non-mammalian vertebrates. The distinctly different pharmacology of the receptors and structural requirements of analogues for agonist/antagonist activity establish a basis for identifying receptor features involved in ligand-induced signal propagation using chimaeras of cloned sheep and chicken receptors.

Influence of temperature on the function of Sertoli and Leydig cells of human testes.

The influence of temperature on Leydig and Sertoli cell functions was investigated in two experiments. One experiment was performed with testes of men with or without varicocele, because the temperature differs between right and left testes of men with left varicocele and right testes of men without varicocele. There were no significant differences in follicle-stimulating hormone (FSH), human chorionic gonadotropin (hCG) receptors, and testosterone (T) concentrations among the testes. The other experiment was performed with the use of testicular organ culture. Specific binding sites for FSH, hCG, and T production were similar in cultured testes maintained at 33 degrees and 37 degrees C for 7 days. It is concluded that high temperature may not disturb Leydig and Sertoli cell functions in the short term.

Comparisons of endocrinological and testis parameters in 18-month-old Ile de France and Romanov rams.

Endocrinological and testis parameters of adult 18-month-old Ile de France (IF) and Romanov (Ro) rams were compared during sexual season. Testis weights, total volumes of intertubular tissue, and of blood and lymph vessels, total seminiferous tubule length, rete testis flow rate and daily production of germ cells were significantly higher in IF than in Ro rams. These variations originated from differences in Sertoli cell numbers, which were established before puberty. When daily productions of germ cells, of ABP or of RTF were expressed per Sertoli cell, they were higher in Ro than in IF rams. Quality of spermatids, as measured by their cellular size prior to elongation, was lower in Ro than in IF. The number of FSH-binding sites per Sertoli did not differ between the two breeds but FSH plasma levels were higher in Ro than in IF rams. Total numbers of Leydig cells per testis, their individual size or their LH-binding capacity did not differ significantly between the two breeds. However, the ratio of mean testosterone upon mean LH plasma levels were greater in Ro than in IF rams while both breeds had identical LH mean plasma levels.

Endocrine profiles, testicular gonadotropin receptors and sperm production in hemi-castrated ram lambs.

The effects of hemi-castration upon compensatory hypertrophy, serum gonadotropin and testosterone concentrations, testicular gonadotropin receptors and daily sperm production (DSP) were studied in 10 crossbred ram lambs. At 4 mo of age lambs were either hemi-castrated (HC; n = 5) or left intact (INT; n = 5). Blood samples were collected every 2 h for the first 24 h post-surgery, every 6 h for the next 24 h and then three times weekly for the following 14 wk. Serial blood samples (15-min intervals for 8 h) were collected during the 4th, 8th and 12th week following hemi-castration. Individual mean testicular and epididymal weights increased (P less than .05) 48 and 33% in HC compared with INT rams, respectively. Serum follicle stimulating hormone (FSH) increased (P less than .05) within 8 h after HC, reached peak concentrations within 1 wk and remained elevated for 4 wk before returning to concentrations of INT rams. Neither mean serum luteinizing hormone (LH) nor pulse patterns of LH or FSH were different (P greater than .05) between these two groups at any period examined. Serum testosterone (T) concentrations were lower (P less than .05) during the first 48 h post-surgery in HC rams, but by 1 wk concentrations were similar (P greater than .05) to those in INT rams. Remaining testes from HC and INT rams were removed at 7 mo of age, 3 mo after initial gonadal manipulation. On a per-testis basis there were more (P less than .05) LH and FSH receptors in HC than INT rams, respectively; however, concentrations of receptors were not different (P greater than .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of exogenous insulin and body condition on metabolic hormones and gonadotropin-induced follicular development in prepuberal gilts.

To determine influences of insulin and body condition on follicular growth, prepuberal gilts (n = 16) treated with pregnant mare's serum gonadotropin (PMSG) were used in a 2 X 2 factorial experiment with main effects of insulin (0 or .4 IU/kg every 12 h beginning at 1800 on the day before PMSG) and backfat depth (moderate, 25 +/- .8; high, 32 +/- .7 mm; P less than .0001). Body weights were similar. Blood sampling was at 6-h intervals for analyses of LH, FSH, growth hormone (GH), glucagon, cortisol, insulin, insulin-like growth factor-I (IGF-I), plasma urea nitrogen (PUN), nonesterified fatty acids (NEFA), testosterone, estradiol-17 beta, and progesterone. Ovaries were removed 75 h after PMSG treatment, and visible small (less than or equal to 3 mm), medium (4 to 6 mm), large (greater than or equal to 7 mm), and macroscopically atretic follicles were counted. Administration of insulin increased IGF-I in fluid of medium follicles (108.8 vs 60.7 ng/ml; SEM = 13.3; P less than .05). Neither insulin nor fatness affected hCG binding by granulosa cells (12.5 +/- 1.6 ng/10(6) cells) or numbers of large (16.7 +/- 2.6) and medium (10.4 +/- 2.3) follicles. However, insulin increased the number of small follicles (58.9 vs 29.9; SEM = 9.7; P less than .05) and reduced the number of atretic follicles (3.8 vs 11.3; SEM = 1.1; P less than .05). The predominant effect of insulin on reducing number of atretic follicles was in the small size class (.6 vs 6.9; SEM = .6, P less than .01). Follicular fluid estradiol and progesterone were not affected by treatments; however, testosterone concentrations in large follicles were lower in gilts with higher backfat (32.5 vs 59.9 ng/ml; SEM = 4.0; P less than .05). Systemic LH, FSH, glucagon, cortisol, PUN, NEFA, estradiol, and testosterone were not affected by insulin or level of feeding. However, GH was lower in gilts that had higher backfat (overall average of 3.2 vs 2.8 ng/ml; SEM = .1; P less than .05). Insulin reduced atresia and altered intrafollicular IGF-I independently of body condition and without sustained effects on other hormones.

Effect of porcine somatotropin on number of granulosa cell luteinizing hormone/human chorionic gonadotropin receptors, oocyte viability, and concentrations of steroids and insulin-like growth factors I and II in follicular fluid of lean and obese gilts.

Prepubertal gilts of obese (n = 24) or lean (n = 24) genetic lines were injected (s.c.) daily with 0, 2, or 4 mg of porcine somatotropin (pST) for 6 wk starting at 160 d of age to determine whether pST affects follicular function. Blood and ovaries were collected at slaughter 24 h after the last injection. Surface follicles greater than or equal to 1.0 mm in diameter were counted, and pools of follicular fluid (FFL) and granulosa cells were collected from 1.0- to 3.9-mm (small) and 4.0- to 6.9-mm (medium) follicles. Oocytes were collected from small and medium follicles and evaluated for maturational stage and viability. Porcine somatotropin increased (P less than .08) the numbers of small but not the numbers of medium follicles per gilt (P greater than .10). Oocyte maturation and viability were not affected by pST or genetic line. Porcine somatotropin increased (P less than .05) concentrations of insulin-like growth factor I (IGF-I) in serum and FFL of both obese and lean gilts; IGF-I was lower (P less than .01) in lean gilts. Treatment with pST decreased (P less than .05) IGF-II in FFL of lean but not in that of obese gilts. Dose of pST and line had no effect on concentrations of progesterone in FFL of small or medium follicles or on concentrations of estradiol in FFL of small follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunobiology of human chorionic gonadotropin.

PIP: A comprehensive review of the immunobiology of human chorionic gonadotropin (hCG), including the structure of both alpha and beta chains, immunogenicity of various segments and epitopes of each, secretion and function of the hormone, determinants of receptor recognition, and finally, clinical studies of possible contraceptive beta-hCG-based vaccines, is presented. hCG is composed of 2 glycosylated peptides. The alpha subunit is identical to that found in hLH, hFSH and hTSH. The beta subunit, which is limiting in the sense that it is secreted in smaller amounts, defines the biological activity of hCG. hCG is secreted throughout pregnancy from 170 hours after fertilization to a peak at 8-10 weeks of and is essential for maintenance of early pregnancy by progesterone secreted by the corpus luteum. Although native hCG evokes antibodies, they cross react with LH, so such a vaccine would not be useful for contraception. Beta-hCG has been purified and also produced by monoclonal antibodies, and shown to produce antibodies and infertility in baboons. Phase I clinical trials of immunologically purified beta-hCG complexed to tetanus toxoid were conducted on 63 women in an international study in the mid-1970s, but results were mixed in terms of antibody titer and duration. New vaccines have been designed based on more sophisticated adjuvants, beta- hCG-terminal peptides, and polyvalent vaccines and are being tested in 4 Phase I trials currently, sponsored by the Population Council, the Indian government-sponsored program, and the WHO.^ieng

[Gonadotropin releasing hormone and its receptor in the tissue of human hepatocellular carcinoma].

OBJECTIVE: To provide the basis for studying the relationship between gonadotropin releasing hormone(GnRH) and the regulation of hepatocarcinoma cell proliferation and differentiation, we observed the localization and distribution of GnRH and its receptor(GnRH-R) in various differentiated hepatocarcinoma tissues. METHODS: GnRH and its receptor were detected in 33 hepatocellular carcinoma(HCC) by immunohistochemical SABC technique. RESULTS: GnRH and its receptor had the same distributive pattern in hepatocarcinoma cells. The positive signal was found mainly on the surface and in the cytoplasm of hepatoma cells, with enhanced staining in the cytoplasm surrounding the nucleus; no positive signal was revealed in the nucleus. The GnRH positive rate (94%; 16/17) of edmondson grade I-II HCC was higher than that of grade III HCC (50%; 5/10) and grade IV HCC(17%; 1/6), (P < 0.05 and P < 0.001 respectively). In grades I-II HCC GnRH-R positive rate was 88% (15/17), which was higher than 17% (1/6) in grade IV HCC (P < 0.01). Although GnRH and GnRH-R positive rates in every grade HCC were lower than those in their related peri-cancerous tissues, the differences were not significant (P > 0.05). CONCLUSION: The expression of GnRH and GnRH-R in the tissues of hepatocarcinoma is related to their degree of differentiation.

Immunolocalization of GnRHRI, gonadotropin receptors, PGR, and PGRMCI during follicular development in the rabbit ovary.

The aim of this study was to investigate the presence and localization of gonadotropin-releasing hormone receptor-I (GnRHRI), gonadotropin receptors (FSHR, LHR), progesterone receptor (PGR), and progesterone receptor membrane-binding component-I (PGRMCI) in the different developmental stages of the rabbit follicle. The ovaries were collected from four healthy New Zealand white rabbits, and the mRNA expression and protein levels of GnRHRI, FSHR, LHR, PGR, and PGRMCI were examined with real-time PCR and immunohistochemistry. The results showed that GnRHRI, FSHR, LHR, PGR, and PGRMCI mRNA was expressed in the ovary; furthermore, we show cell-type specific and follicular development stage-specific expression of these receptors at the protein level. Specifically, all of the receptors were detected in the oocytes from the primordial to the tertiary follicles and in the granulosa and theca cells from the secondary and tertiary follicles. In the mature follicles, all receptors were primarily localized in the granulosa and theca cells. In addition, LHR was also localized in the granulosa cells from the primordial and primary follicles. With follicular development, the expression level of all of the receptors, except GnRHRI, in the follicles showed a tendency to decrease because the area of the follicle increased sharply. The expression level of GnRHRI, FSHR, and PGR in the granulosa and theca cells showed an increasing trend with ongoing follicular development. Interestingly, the expression level of FSHR in the oocytes obviously decreased from the primary to the tertiary follicles, whereas LHR in the oocytes increased from the secondary to tertiary follicles. In conclusion, the expression of GnRHRI, the gonadotropin receptors, PGR, and PGRMCI decreased from the preantral follicles (primordial, primary, and secondary follicles) to the tertiary follicles. The expression of GnRHRI and LHR in the oocytes increased from the secondary to the tertiary follicles, whereas FSHR decreased from the primary to the tertiary follicles. The expression of GnRHRI and PGR in the granulosa and theca cells increased from the secondary to the mature follicles. These observations suggest that these receptors play roles in follicular development and participate in the regulation of follicular development.

Gonadotropin-releasing hormone stimulates prolactin release from lactotrophs in photoperiodic species through a gonadotropin-independent mechanism.

Previous studies have provided evidence for a paracrine interaction between pituitary gonadotrophs and lactotrophs. Here, we show that GnRH is able to stimulate prolactin (PRL) release in ovine primary pituitary cultures. This effect was observed during the breeding season (BS), but not during the nonbreeding season (NBS), and was abolished by the application of bromocriptine, a specific dopamine agonist. Interestingly, GnRH gained the ability to stimulate PRL release in NBS cultures following treatment with bromocriptine. In contrast, thyrotropin-releasing hormone, a potent secretagogue of PRL, stimulated PRL release during both the BS and NBS and significantly enhanced the PRL response to GnRH during the BS. These results provide evidence for a photoperiodically modulated functional interaction between the GnRH/gonadotropic and prolactin axes in the pituitary gland of a short day breeder. Moreover, the stimulation of PRL release by GnRH was shown not to be mediated by the gonadotropins, since immunocytochemical, Western blotting, and PCR studies failed to detect pituitary LH or FSH receptor protein and mRNA expressions. Similarly, no gonadotropin receptor expression was observed in the pituitary gland of the horse, a long day breeder. In contrast, S100 protein, a marker of folliculostellate cells, which are known to participate in paracrine mechanisms within this tissue, was detected throughout the pituitaries of both these seasonal breeders. Therefore, an alternative gonadotroph secretory product, a direct effect of GnRH on the lactotroph, or another cell type, such as the folliculostellate cell, may be involved in the PRL response to GnRH in these species.

[Comparative study on human chorionic gonadotropin receptor in normal human ovary with ovarian tumors].

Human Chorionic Gonadotropin (HCG) is major physiological luteotropic factors for the human corpus luteum. The observations strongly suggest that the human ovary possesses a gonadotropin receptor in the cell membrane. We studied the HCG receptor in normal human ovary and ovarian tumors. Twenty-three human ovarian specimens and 16 ovarian tumor specimens were obtained from women patients having gynecological surgery. Ovaries were homogenized and sonicated. The homogenates were centrifuged at 2000 g for 15 min. After sucrose density gradient ultracentrifugation (78,000 g, 4 h), two fractions were collected from layer of 33% and interface between 33% and 37%. Thirty micrograms of ovarian protein, 8 ng 125I-HCG and unlabeled HCG in a final volume of 0.5 ml of 0.05 mol/L Tris buffer were incubated at 30 degrees C for 2 h. The results were shown in the table.

Developmental changes in the properties of gonadotropin receptors in the ovarian follicles of amago salmon (Oncorhynchus rhodurus) during oogenesis.

The presence of specific, saturable, high-affinity gonadotropin receptors was demonstrated in membrane preparations from preovulatory ovarian follicles of amago salmon (Oncorhynchus rhodurus), including intact follicles, isolated thecal layers, and isolated granulosa cells. Optimum conditions for the binding study using the amago salmon receptor system were similar to those previously reported for postovulatory ovaries of the same species (A. Kanamori, H. Kagawa, and Y. Nagahama, 1987, Gen. Comp. Endocrinol. 66, 210-217). Scatchard analysis of chum salmon gonadotropin (CSG-SII) binding to the membrane fraction suggested the presence of high-affinity binding sites in the intact follicles, isolated thecal layers, and isolated granulosa cells at all stages of development. The dissociation constant is consistent with those reported for gonadotropin receptors in several teleost gonads and in other vertebrate classes (about 0.1-1 nM). During oogenesis, the number of binding sites per follicle increased from about 20 to about 60 pg. Similarly an increase in binding sites was observed with granulosa cells and thecal layers during oogenesis. These findings show an increase in the number of gonadotropin receptors in the follicles and are in good temporal agreement with the developmental changes in follicular steroidogenesis in response to gonadotropin. The increase in gonadotropin receptors in the thecal layer was associated with the increased capacity for production of testosterone, whereas the increase in gonadotropin receptors in the granulosa cells was associated with an increase in gonadotropin sensitivity in terms of 20 beta-hydroxysteroid dehydrogenase activation.