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.

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.

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.

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.

Concentraciones de gonadotropina coriónica humana en pacientes preeclámpticas y embarazadas normotensas sanas / Chorionic gonadotrophin concentrations in preeclamptic and healthy normotensive pregnant women

Objetivo. Comparar las concentraciones de gonadotropina coriónica humana en pacientes con preeclampsia y embarazadas normotensas sanas. Sujetos y métodos. Se seleccionó a un total de 100 pacientes. Se incluyó a 50 pacientes preeclámpticas como casos (grupo A) y un grupo control seleccionado por tener una edad y un índice de masa corporal similares al grupo de estudio que consistió en 50 embarazadas normotensas sanas (grupo B). Las muestras de sangre se recolectaron en todas las pacientes antes del parto e inmediatamente después del diagnóstico en el grupo B para determinar las concentraciones de gonadotropina coriónica humana. Resultados. No se encontraron diferencias significativas con relación a la edad materna, edad gestacional e índice de masa corporal al momento de la toma de la muestra (p=NS). Se encontraron diferencias estadísticamente significativa en las concentraciones de gonadotropina coriónica humana entre las pacientes en el grupo de estudio (grupo A: 47.661±18.124 mU/ml) y las pacientes del grupo control (grupo B: 27.459±13.329 mU/ml; p < 0,05). Se observó una correlación moderada, positiva y significativa con los valores de presión arterial sistólica (r=0,493; p<0,05) y con los valores de presión arterial diastólica (r=0,504; p<0,05). Conclusiones: Las pacientes preeclámpticas presentaron concentraciones significativamente más altas de gonadotropina coriónica humana al compararlo con embarazadas normotensas sanas (AU)

Objective. To compare chorionic gonadotrophin concentrations in preeclamptic and healthy normotensive pregnant women. Subjects and methods. One hundred patients were selected. Fifty preeclamptic patients were selected as cases (group A) and 50 healthy pregnant women with a similar age and body mass index to patients in group A were selected as controls (group B). Blood samples for chorionic gonadotrophin determination were collected in all patients before labor and immediately after diagnosis in the study group. Results. There were no significant differences in maternal age, gestational age or body mass index at sample collection (P=NS). Statistically significant differences in chorionic gonadotrophin concentrations were found between patients in group A (47.661+/- 18.124 mUI/mL) and patients in group B (27.459 +/- 13.329 mUI/mL; P<.05). There was a moderate, positive and significant correlation with values of systolic blood pressure (r = 0.493; P<.05) and values of diastolic blood pressure (r = 0.504; P<05). Conclusions. Chorionic gonadotrophin concentrations were significantly higher in preeclamptic patients than in healthy normotensive pregnant women (AU)

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.

Control of early ovarian follicular development.

Early follicular growth refers to the development of an ovarian follicle from the primordial to early antral phase. In sheep and cows these phases of growth can be classified by the configuration of granulosal cells in the largest cross-section of the follicle as types 1 (primordial), 1a (transitory) 2 (primary), 3 and 4 (preantral) and 5 (early antral). Follicles classified as type 1 may be highly variable within each species with respect to number of granulosal cells and diameter of oocyte. Much of the variation in granulosal cell composition of type 1 follicles may occur at formation and this may account for the variability in granulosal cell composition throughout subsequent stages of growth. There appear to be important differences among species (for example sheep and cattle) in the number and function of granulosal cells relative to the diameter of the oocyte during the initiation of follicular growth. There is evidence that most, if not all, of the growth phases from types 1 to 5 are gonadotrophin-independent and that follicles develop in a hierarchical manner. In sheep, cows and pigs, numerous growth factor, growth factor receptor and gonadotrophin receptor mRNAs and peptides (for example c-kit, stem cell factor, GDF-9, beta B and beta A activin/inhibin subunit, alpha inhibin subunit, follistatin, FGF-2, EGF, EGF-R, TGF beta 1,2 and 3 FSH-R and LH-R) are expressed in a phase of growth (for example types 1-5)-specific and cell-specific manner. However, the roles of many of these factors remain to be determined.

[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.

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.

Gonadotropin-releasing hormone receptor in gynecologic tumors. Frequent expression in adenocarcinoma histologic types.

BACKGROUND: Gonadotropin-releasing hormone (Gn-RH) analogs have been used in the therapy of the endocrine-dependent cancers. The authors attempted to determine the frequency with which Gn-RH receptor (Gn-RHR) is present in gynecological cancers. METHODS: Experiments were performed on gynecologic tumors that had been surgically removed and their cloned cell lines. Gn-RHR was characterized by [3H]Gn-RH binding to plasma membrane preparations. Gn-RHR messenger ribonucleic acid was determined by reverse transcription-polymerase chain reaction using oligonucleotide primers synthesized according to the published human Gn-RHR sequence. RESULTS: High affinity binding sites with nanomolar range of Kd and Gn-RHR mRNA were detected in a high proportion (over 90%) of the specimens from endometrium (6 of 6) and endometrial carcinomas (16 of 17), myometrium (6 of 6) and myomas (4 of 5), epithelial carcinoma (21 of 23), and stromal tumors (3 of 3) of the ovary. There was no substantial Gn-RHR in cervical carcinomas or germ cell-derived tumors of the ovary. Cloned cell lines gave identical results to those obtained in their respective mother tumors. CONCLUSIONS: We detected Gn-RHR in a wide range of the carcinomas and tissues originating from the endometrium and ovary, but not in the uterine cervix or germ cell-derived tumors. The expression of Gn-RH receptor raises the possibility that Gn-RH may play a direct regulatory role in the growth of these carcinomas, and provides a possible point of attack for therapeutic approaches using Gn-RH analogs in these malignancies.

Localization of two gonadotropin receptors in the salmon gonad by in vitro ligand autoradiography.

Receptors for two salmon gonadotropins, GTH I and GTH II, were localized by use of in vitro ligand autoradiography of coho salmon gonads at various stages of sexual maturation. The results in both sexes revealed the presence of two types of GTH receptors: type I (GTH-RI), which interacts with both GTHs, and type II (GTH-RII), which interacts specifically with GTH II. GTH-RI was found at all stages of spermatogenesis examined and was localized on cells that were most likely Sertoli cells; however, it could not be determined whether GTH-RI was also localized on Leydig cells. In contrast, GTH-RII was found only in Leydig cells in the testis from a spermiating fish. In the vitellogenic ovary, GTH-RI was localized in the thecal layer and intensely on granulosa cells; in the preovulatory follicle, in contrast, GTH-RI was found in the thecal layer and in interstitial connective tissue, but not in the granulosa layer. Among all the stages of oogenesis examined, only granulosa cells of the preovulatory follicle exhibited GTH-RII. The appearance of GTH-RII coincides well with the increase in plasma levels of GTH II that occurs during final oocyte maturation and spermiation in coho salmon. The nature, distribution, and timing of appearance of these two receptors can explain, at least in part, the results of previous studies on steroidogenic activities of the two GTHs. The present study also suggests the functional homology of salmon GTH I and GTH II to mammalian FSH and LH, respectively, during gonadal development.

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).

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)

Effect of estradiol and progesterone on myometrial LH/hCG receptors in pigs.

High affinity luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptors have been identified in porcine, rabbit and rat uteri and immunocytochemically demonstrated in the human uterus. We have now assessed the effect of estradiol and progesterone on the capacity and affinity of LH/hCG binding sites in crude membrane fractions of porcine myometria. Nineteen cross-bred gilts were ovariectomized at 6-7 months of age. Five weeks later, the experiment was conducted and gilts were given estradiol benzoate 2 mg (N = 5), progesterone 50 mg (N = 4) and 2 mg of estradiol benzoate plus 50 mg of progesterone in 2 ml of corn oil (N = 6), im for five consecutive days. Controls (N = 4) received 2 ml of the vehicle. Gilts were hysterectomized 24 h after the last injection. Blood samples for assays of LH, estradiol and progesterone were collected 1 h before hysterectomy. The numbers and affinities of unoccupied LH/hCG binding sites were characterized in all samples of myometrium. The results indicate that treatment with estradiol benzoate increases (p less than 0.01) the number of LH/hCG binding sites compared with gilts receiving corn oil. Progesterone treatment caused elevation in the number of LH/hCG receptors (p less than 0.05), when compared with estradiol alone (2.9 +/- 0.3 vs 1.2 +/- 0.1 fmol/mg protein, respectively). Combined administration of estradiol and progesterone increased receptor capacity to 2.7 +/- 0.4. Steroid treatment did not alter the affinity (Ka) of [125I]hCG binding to receptors and it varied from 1.8 +/- 0.8 to 2.9 +/- 0.2 x 10(11) l/mol.(ABSTRACT TRUNCATED AT 250 WORDS)

Increase in the expression of thyroid hormone receptors in porcine granulosa cells early in follicular maturation.

Thyroid hormone has been demonstrated to synergize with FSH to exert stimulatory effects on the differentiation of porcine granulosa cells. In order to further characterize the nature of thyroid hormone action on granulosa cells, the presence of triiodothyronine (T3) receptors in the nuclei of porcine granulosa cells was examined, and qualitatively and quantitatively compared during follicular maturation. Then, comparative abilities of granulosa cells from varying follicle stages to respond to T3 were assessed in terms of FSH-induced LH/hCG receptor formation and progesterone secretion. Furthermore, the expression of erb-A was analyzed using Northern blot hybridization of porcine granulosa cell RNA with a v-erb-A probe. Binding experiments with [125I]T3 showed that granulosa cell nuclei obtained from small follicles had a greater ability to bind [125I]T3 compared to those from large follicles. Scatchard analysis revealed the presence of nuclear T3 receptors with a single class of binding sites. There was little difference in the affinity of the T3 receptors during follicular maturation. By contrast, the number of the T3 receptors was higher in small follicle granulosa cells compared to that in large follicle granulosa cells. Thus, the increased T3 binding to small follicle granulosa cells relative to large follicle granulosa cells appears to be attributable to the increased number of the nuclear T3 receptors rather than to a change in the affinity. The magnitude of the stimulatory effects of T3 on granulosa cell functions was maximal in small follicle granulosa cells, but negligible in large follicle granulosa cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in the characteristics and distribution of rat luteal receptors for native and deglycosylated human choriogonadotropin.

Previous work has suggested that rat luteal cells have two populations of LH/hCG receptors that are located in different parts of the cell membrane. The possibility that these two receptor pools may have functional differences has been investigated through examination of the binding and action of native and deglycosylated hCG to different membrane fractions. Ovaries from eCG/hCG-primed immature female rats were separated into 1,000 x g (heavy) and 20,000 x g (light) particulate fractions. Increasing concentrations of NaCl had a biphasic effect on the binding of native and deglycosylated hCG to both membrane fractions, causing an increase in binding at low concentrations and a decrease in binding at higher concentrations. The binding of deglycosylated hCG to both membrane preparations and the binding of native hCG to light-membrane preparations was maximal at approximately the same NaCl concentration (50-65 mM). This was higher than the concentration of NaCl necessary for maximal binding of native hCG to the heavy-membrane preparation. In addition, maximal native hCG binding to this preparation occurred over a broader NaCl concentration range (15-65 mM). Equilibrium binding experiments showed differences in hCG binding to both fractions. In light membranes there were significantly more receptor sites for deglycosylated hCG (11.2 +/- 4.8 fmol/mg ovary) than for native hCG (4.8 +/- 0.7 fmol/mg ovary), with no significant different in affinity. In contrast, in heavy membranes the affinity for deglycosylated hCG (6.30 +/- 0.19.10(9) M-1), was significantly higher than that for native hCG (2.60 +/- 0.13.10(9) M-1), with no significant differences in receptor number.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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 human chorionic gonadotropin (hCG) on Neisseria gonorrhoeae invasion of and IgA secretion by human fallopian tube mucosa.

The possible effect of human chorionic gonadotropin (hCG) on the mucosal immune response and susceptibility of the fallopian tube mucosa to invasion by Neisseria gonorrhoeae (gonococci) was investigated in the fallopian tube organ culture (FTOC) model. Immunohistochemical and radioreceptor assay techniques showed specific high affinity binding of hCG in vitro to the apices of non-ciliated fallopian tube cells (Kd approximately 10(-9) M). Continuous exposure of the FTOC mucosa to hCG during infection with gonococci resulted in a marked increase (6- to 15-fold) in IgA secretion and significantly reduced gonococcal invasion (invasion score range 0.7 to 1.75) compared to infected control tissue which was not exposed to hCG (invasion score range 2.9 to 4.95, P less than or equal to 0.01). By contrast, exposure of the mucosa to hCG during the 24 h preceding gonococcal infection followed by the removal of hCG from the system at the time of infection resulted in enhanced gonococcal invasion (invasion score range 7.95 to 9.7, P less than 0.001). We conclude that hCG can modulate the mucosal immune response and susceptibility of fallopian tube epithelium to gonococcal invasion.

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.