Influence of the Hinge Region and Its Adjacent Domains on Binding and Signaling Patterns of the Thyrotropin and Follitropin Receptor

Glycoprotein hormone receptors (GPHR) have a large extracellular domain (ECD) divided into the leucine rich repeat (LRR) domain for binding of the glycoprotein hormones and the hinge region (HinR), which connects the LRR domain with the transmembrane domain (TMD). Understanding of the activation mechanism of GPHRs is hindered by the unknown interaction of the ECD with the TMD and the structural changes upon ligand binding responsible for receptor activation. Recently, our group showed that the HinR of the thyrotropin receptor (TSHR) can be replaced by those of the follitropin (FSHR) and lutropin receptor (LHCGR) without effects on surface expression and hTSH signaling. However, differences in binding characteristics for bovine TSH at the various HinRs were obvious. To gain further insights into the interplay between LRR domain, HinR and TMD we generated chimeras between the TSHR and FSHR. Our results obtained by the determination of cell surface expression, ligand binding and G protein activation confirm the similar characteristics of GPHR HinRs but they also demonstrate an involvement of the HinR in ligand selectivity indicated by the observed promiscuity of some chimeras. While the TSHR HinR contributes to specific binding of TSH and its variants, no such contribution is observed for FSH and its analog TR4401 at the HinR of the FSHR. Furthermore, the charge distribution at the poorly characterized LRR domain/HinR transition affected ligand binding and signaling even though this area is not in direct contact with the ligand. In addition our results also demonstrate the importance of the TMD/HinR interface. Especially the combination of the TSHR HinR with the FSHR-TMD resulted in a loss of cell surface expression of the respective chimeras. In conclusion, the HinRs of GPHRs do not only share similar characteristics but also behave as ligand specific structural and functional entities.     

Evidence for Follicle-stimulating Hormone Receptor as a Functional Trimer

Follicle-stimulating hormone receptor (FSHR), a G-protein coupled receptor, is an important drug target in the development of novel therapeutics for reproductive indications. The FSHR extracellular domains were observed in the crystal structure as a trimer, which enabled us to propose a novel model for the receptor activation mechanism. The model predicts that FSHR binds Asnα⁵²-deglycosylated FSH at a 3-fold higher capacity than fully glycosylated FSH. It also predicts that, upon dissociation of the FSHR trimer into monomers, the binding of glycosylated FSH, but not deglycosylated FSH, would increase 3-fold, and that the dissociated monomers would in turn enhance FSHR binding and signaling activities by 3-fold. This study presents evidence confirming these predictions and provides crystallographic and mutagenesis data supporting the proposed model. The model also provides a mechanistic explanation to the agonist and antagonist activities of thyroid-stimulating hormone receptor autoantibodies. We conclude that FSHR exists as a functional trimer.     

Induction of G protein-coupled estrogen receptor (GPER) and nuclear steroid hormone receptors by gonadotropins in human granulosa cells

Estradiol and progesterone mediate their actions by binding to classical nuclear receptors, estrogen receptor α (ERα) and estrogen receptor β (ERβ) and progesterone receptor A and B (PR-A and PR-B) and the non-classical G protein-coupled estrogen receptor (GPER). Several animal knock-out models have shown the importance of the receptors for growth of the oocyte and ovulation. The aim of our study was to identify GPER in human granulosa cells (GC) for the first time. Moreover, the effect of different doses of gonadotropins on estrogen and progesterone receptors in the human ovary should be investigated as follicle stimulating hormone (FSH) and luteinizing hormone (LH) are also responsible for numerous mechanisms in the ovary like induction of the steroid biosynthesis. Human GC were cultured in vitro and stimulated with different doses of recombinant human FSH or LH. Receptor expression was analyzed by immunocytochemistry and quantitative real-time RT-PCR. GPER could be identified for the first time in human GC. It could be shown that high concentrations of LH increase GPER protein expression. Furthermore FSH and LH increased ERβ, PR-A and PR-B significantly on protein level. These findings were verified for high doses of FSH and LH on mRNA level. ERα was not affected with FSH or LH. We assume that gonadotropins induce GPER, ERβ and PR in luteinized granulosa cells.     

Follicle-stimulating hormone and luteinizing hormone mediate the androgenic pathway in Leydig cells of an evolutionary advanced teleost

The endocrine pathways controlling vertebrate spermatogenesis are well established in mammals where the pituitary gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) exclusively activate the FSH receptor (FSHR) in Sertoli cells and the LH/choriogonadotropin receptor (LHCGR) in Leydig cells, respectively. In some teleosts, however, it has been shown that Lh can cross-activate the Fshra ortholog, and that Leydig cells coexpress the Lhcgrba and Fshra paralogs, thus mediating the androgenic function of Fsh in the testis. Here, we investigated whether these proposed mechanisms are conserved in an evolutionary advanced pleuronectiform teleost, the Senegalese sole (Solea senegalensis). Transactivation assays using sole Fshra- and Lhcgrba-expressing cells and homologous single-chain recombinant gonadotropins (rFsh and rLh) showed that rFsh exclusively activated Fshra, whereas rLh stimulated both Lhcgrba and Fshra. The latter cross-activation of Fshra by rLh occurred with an EC(50) 4-fold higher than for rFsh. Both recombinant gonadotropins elicited a significant androgen release response in vitro and in vivo, which was blocked by protein kinase A (PKA) and 3beta-hydroxysteroid dehydrogenase inhibitors, suggesting that activation of steroidogenesis through the cAMP/PKA pathway is the major route for both Lh- and Fsh-stimulated androgen secretion. Combined in situ hybridization and immunocytochemistry using cell-specific molecular markers and antibodies specifically raised against sole Fshra and Lhcgrba demonstrated that both receptors are expressed in Leydig cells, whereas Sertoli cells only express Fshra. These data suggest that Fsh-mediated androgen production through the activation of cognate receptors in Leydig cells is a conserved pathway in Senegalese sole.     

The G Protein-Coupled Estrogen Receptor (GPER) Is Expressed in Two Different Subcellular Localizations Reflecting Distinct Tumor Properties in Breast Cancer

Introduction

The G protein-coupled estrogen receptor (GPER) is a novel estrogen receptor that mediates proliferative effects induced by estrogen but also by tamoxifen. The aim of our study was to analyze the frequency of GPER in a large collective of primary invasive breast carcinomas, with special emphasis on the subcellular expression and to evaluate the association with clinicopathological parameters and patient overall survival.

Methods

The tissue microarrays from formalin-fixed, paraffin embedded samples of primary invasive breast carcinomas (n = 981) were analyzed for GPER expression using immunohistochemistry. Expression data were compared to the clinicopathological parameters and overall survival. GPER localization was also analyzed in two immortalized breast cancer cell lines T47D and MCF7 by confocal immunofluorescence microscopy.

Results

A predominantly cytoplasmic GPER expression was found in 189 carcinomas (19.3%), whereas a predominantly nuclear expression was observed in 529 cases (53.9%). A simultaneous comparable positive expression of both patterns was found in 32 of 981 cases (3.2%), and negative staining was detected in 295 cases (30%). Confocal microscopy confirmed the occurrence of cytoplasmic and nuclear GPER expression in T47D and MCF7. Cytoplasmic GPER expression was significantly associated with non-ductal histologic subtypes, low tumor stage, better histologic differentiation, as well as Luminal A and B subtypes. In contrast, nuclear GPER expression was significantly associated with poorly differentiated carcinomas and the triple-negative subtype. In univariate analysis, cytoplasmic GPER expression was associated with better overall survival (p = 0.012).

Conclusion

Our data suggest that predominantly cytoplasmic and/or nuclear GPER expression are two distinct immunohistochemical patterns in breast carcinomas and may reflect different biological features, reason why these patterns should be clearly distinguished in histological evaluations. Prospective studies will be needed to assess whether the expression status of GPER in breast carcinomas should be routinely observed by clinicians, for instance, before implementing endocrine breast cancer treatment.     

Effect of resistin on granulosa and theca cell function in cattle

Resistin is an adipokine that has not been extensively studied in cattle but is produced by adipocytes in greater amounts in lactating versus non-lactating cattle. Seven experiments were conducted to determine the effect of resistin on proliferation, steroidogenesis, and gene expression of theca and granulosa cells from small (1-5mm) and/or large (8-22 mm) cattle follicles. Resistin had no effect on IGF-I-induced proliferation of large-follicle theca cells or small-follicle granulosa cells, but decreased IGF-I-induced proliferation of large-follicle granulosa cells. Resistin weakly stimulated FSH plus IGF-I-induced estradiol production by large-follicle granulosa cells, but had no effect on IGF-I- or insulin-induced progesterone and androstenedione production by theca cells or progesterone production by granulosa cells of large follicles. In small-follicle granulosa cells, resistin attenuated the stimulatory effect of IGF-I on progesterone and estradiol production of small-follicle granulosa cells. RT-PCR measuring abundance of side-chain cleavage enzyme (CYP11A1), aromatase (CYP19A1), FSH receptor (FSHR) and LH receptor (LHCGR) mRNA in large- and small-follicle granulosa cells indicated that resistin reduced the stimulatory effect of IGF-I on CPY11A1 mRNA abundance in large-follicle granulosa cells but had no effect on CYP19A1, FSHR or LHCGR mRNA abundance in large- or small-follicle granulosa cells. Resistin had no effect on CYP11A1, CYP17A1 or LHCGR mRNA abundance in theca cells. These results indicate that resistin preferentially inhibits steroidogenesis of undifferentiated (small follicle) granulosa cells and inhibits proliferation of differentiated (large follicle) granulosa cells, indicating that the ovarian response to resistin is altered during follicular development.     

蒙药乌力吉-18对大鼠下丘脑-垂体-卵巢轴相关激素及受体表达的影响

目的:探讨蒙药乌力吉-18对大鼠下丘脑-垂体-卵巢轴相关激素及受体的影响。方法:选取40只健康雌性未孕SD大鼠,随机分为空白组、对照组、乌力吉-18高、低2个剂量组,每组10只。空白组灌胃等体积蒸馏水,对照组灌胃逍遥丸,高、低剂量组分别灌胃2.0 g·kg^-1·d^-1、1.0 g·kg^-1·d^-1乌力吉-18,连续给药31学艺术d。采用酶联免疫吸附法测定血清促性腺激素释放激素(GnRH)、促卵泡生成素(FSH)、黄体生成素(LH)、雌二醇(E₂)及孕酮(PROG)的含量;免疫组化法检测下丘脑组织促性腺激素释放激素(GnRH)、垂体组织促性腺激素释放激素受体(GnRHR)的表达;以蛋白免疫印迹技术检测卵巢组织促卵泡生成素受体(FSHR)、黄体生成素受体(LHR)蛋白表达量。以实时荧光定量PCR检测卵巢组织中FSHR、LHR基因表达量。结果:与空白组比较,乌力吉-18低剂量组可明显升高血清LH含量(P<0.05),上调下丘脑组织GnRH、垂体组织GnRHR表达及卵巢组织FSHR、LHR蛋白表达(P<0.05);乌力吉-18高剂量组可显著升高血清FSH、LH、E₂含量(P<0.05),上调下丘脑组织GnRH表达及卵巢组织FSHR表达量(P<0.05),并可显著升高卵巢组织中FSHR、LHR基因表达量(P<0.05);对照组可明显升高血清E₂含量(P<0.05)。结论:蒙药乌力吉-18可明显升高血清FSH、LH及E₂的含量,促进下丘脑组织GnRH、垂体组织GnRHR及卵巢组织中FSHR、LHR的表达,表明乌力吉-18能够对下丘脑-垂体-卵巢轴相关激素及受体表达产生影响。     

An activated human follicle-stimulating hormone (FSH) receptor stimulates FSH-like activity in gonadotropin-deficient transgenic mice

FSH mediates its testicular actions via a specific Sertoli cell G protein-coupled receptor. We created a novel transgenic model to investigate a mutant human FSH receptor (FSHR(+)) containing a single amino acid substitution (Asp567Gly) equivalent to activating mutations in related glycoprotein hormone receptors. To examine the ligand-independent gonadal actions of FSHR(+), the rat androgen-binding protein gene promoter was used to direct FSHR(+) transgene expression to Sertoli cells of gonadotropin-deficient hypogonadal (hpg) mice. Both normal and hpg mouse testes expressed FSHR(+) mRNA. Testis weights of transgenic FSHR(+) hpg mice were increased approximately 2-fold relative to hpg controls (P < 0.02) and contained mature Sertoli cells and postmeiotic germ cells absent in controls, revealing FSHR(+)-initiated autonomous FSH-like testicular activity. Isolated transgenic Sertoli cells had significantly higher basal ( approximately 2-fold) and FSH-stimulated ( approximately 50%) cAMP levels compared with controls, demonstrating constitutive signaling and cell-surface expression of FSHR(+), respectively. Transgenic FSHR(+) also elevated testosterone production in hpg testes, in the absence of circulating LH (or FSH), and it was not expressed functionally on steroidogenic cells, suggesting a paracrine effect mediated by Sertoli cells. The FSHR(+) response was additive with a maximal testosterone dose on hpg testicular development, demonstrating FSHR(+) activity independent of androgen-specific actions. The FSHR(+) response was male specific as ovarian expression of FSHR(+) had no effect on hpg ovary size. These findings reveal transgenic FSHR(+) stimulated a constitutive FSH-like Sertoli cell response in gonadotropin-deficient testes, and pathways that induced LH-independent testicular steroidogenesis. This novel transgenic paradigm provides a unique approach to investigate the in vivo actions of mutated activating gonadotropin receptors.     

mRNA expression pattern of gonadotropin receptors in bovine follicular cysts

Follicular growth and steroidogenesis are dependent on gonadotropin binding to their receptors in granulosa and theca cells of ovarian follicles. The aim of the present study was to evaluate the expression patterns of follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHCGR) in ovarian follicular structures from cows with cystic ovarian disease (COD) as compared with those of regularly cycling cows. Relative real-time RT-PCR analysis showed that the expression of FSHR mRNA in granulosa cells was highest in small antral follicles, then decreased significantly as follicles increased in size, and was lowest in cysts. FSHR mRNA was not detected in the theca cells of any follicular category, including cysts. LHCGR mRNA expression in granulosa cells was significantly higher in large antral follicles than in cysts, and not detected in granulosa cells of small and medium antral follicles. In theca cells, the expression level of LHCGR mRNA in medium antral follicles was higher than in small and large antral follicles, whereas that in follicular cysts it was similar to those in small and medium antral follicles, but higher than that in large antral follicles. Our findings provide evidence that there is an altered gonadotropin receptor expression in bovine cystic follicles, and suggest that in conditions characterized by altered ovulation, such as COD, changes in the signaling system of gonadotropins may play a fundamental role in their pathogenesis.     

Functional Differences of Invariant and Highly Conserved Residues in the Extracellular Domain of the Glycoprotein Hormone Receptors

Multiple interactions exist between human follicle-stimulating hormone (FSH) and the N-terminal hormone-binding fragment of the human FSH receptor (FSHR) extracellular domain (ECD). Binding of the other human glycoprotein hormones to their cognate human receptors (luteinizing hormone receptor (LHR) and thyroid-stimulating hormone receptor (TSHR)) was expected to be similar. This study focuses on amino acid residues in β-strands 2 (Lys⁷⁴), 4 (Tyr¹²⁴, Asn¹²⁹, and Thr¹³⁰), and 5 (Asp¹⁵⁰ and Asp¹⁵³) of the FSHR ECD identified in the human FSH·FSHR ECD crystal structure as contact sites with the common glycoprotein hormone α-subunit, and on noncontact residues in β-strands 2 (Ser⁷⁸) and 8 (Asp²²⁴ and Ser²²⁶) as controls. These nine residues are either invariant or highly conserved in LHR and TSHR. Mutagenesis and functional characterization of these residues in all three human receptors allowed an assessment of their contribution to binding and receptor activation. Surprisingly, the six reported α-subunit contact residues of the FSHR ECD could be replaced without significant loss of FSH binding, while cAMP signaling potency was diminished significantly with several replacements. Comparative studies of the homologous residues in LHR and TSHR revealed both similarities and differences. The results for FSH/FSHR were analyzed on the basis of the crystal structure of the FSH·FSHR ECD complex, and comparative modeling was used to generate structures for domains, proteins, and complexes for which no structures were available. Although structural information of hormone-receptor interaction allowed the identification of hormone-receptor contact sites, functional analysis of each contact site was necessary to assess its contribution to hormone binding and receptor activation.     

Human follicle-stimulating hormone (FSH) receptor interacts with the adaptor protein APPL1 in HEK 293 cells: potential involvement of the PI3K pathway in FSH signaling

Selection of a dominant follicle that will ovulate likely occurs by activation of cell survival pathways and suppression of death-promoting pathways in a mechanism involving FSH and its cognate receptor (FSHR). A yeast two-hybrid screen of an ovarian cDNA library was employed to identify potential interacting partners with human FSHR intracellular loops 1 and 2. Among eight cDNA clones identified in the screen, APPL1 (adaptor protein containing PH domain, PTB domain, and leucine zipper motif; also known as APPL or DIP13alpha) was chosen for further analysis. APPL1 appears to coimmunoprecipitate with FSHR in HEK 293 cells stably expressing FSHR (293/FSHR cells), confirming APPL1 as a potential FSHR-interacting partner. The phosphorylation status of members of the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway was also examined because of the proposed role of APPL1 in the antiapoptotic PI3K/Akt pathway. FOXO1a, also referred to as forkhead homologue in rhabdomyosarcoma, is a downstream effector in the pathway and tightly linked to expression of proapoptotic genes. FOXO1a, but not the upstream kinase Akt, is rapidly phosphorylated, and FOXO1a is thereby inactivated when 293/FSHR cells are treated with FSH. In addition, FSHR coimmunoprecipitates with Akt. The identification of APPL1 as a potential interactor with FSHR and the finding that FOXO1a is phosphorylated in response to FSH provide a possible link between FSH and PI3K/Akt signaling, which may help to delineate a survival mechanism whereby FSH selects the dominant follicle to survive.     

Effects of quinestrol on reproductive hormone expression, secretion, and receptor levels in female Mongolian gerbils (Meriones unguiculatus)

Quinestrol, a synthetic estrogen with marked estrogenic effects and prolonged activity, has potential as a contraceptive for Mongolian gerbils. The objective of this study was to describe the effects of quinestrol on reproductive hormone expression, secretion, and receptor levels in female Mongolian gerbils. Serum and pituitary concentrations of follicle stimulating hormone (FSH) and luteinizing hormone (LH) were decreased, whereas serum concentrations of estradiol (E2) and progesterone (P4) were increased after quinestrol treatment; the effects were both time- and dose-dependent. Furthermore, quinestrol downregulated expression of FSHβ and LHβ mRNA in the pituitary gland, as well as FSH receptor (FSHR) and estrogen receptor (ER) β in the ovary. However, it up-regulated mRNA expression levels of ERα and progesterone receptor (PR) in the pituitary gland and uterus, as well as mRNA for LH receptor (LHR) and PR in the ovary (these effects were time- and dose-dependent). In contrast, quinestrol had no significant effects on the mRNA expression levels of ERα in the ovary, or the gonadotropin α (GtHα) subunit in the pituitary gland. We inferred that quinestrol impaired synthesis and secretion of FSH and LH and that the predominant ER subtype in the pituitary gland of Mongolian gerbils may be ERα. Overall, quinestrol disrupted reproductive hormone receptor expression at the mRNA level in the pituitary-gonadal axis of the Mongolian gerbil.     

Insulin-Like Growth Factor 1 Receptor Is a Prognostic Factor in Classical Hodgkin Lymphoma

The interaction between the tumor cells in classical Hodgkin lymphoma (cHL) and the microenvironment includes aberrant activity of receptor tyrosine kinases. In this study we evaluated the expression, functionality and prognostic significance of Insulin-like growth factor-1 receptor (IGF-1R) in cHL. IGF-1R was overexpressed in 55% (44/80) of cHL patients. Phosphorylated IGF-1R was detectable in a minority of the IGF-1R positive tumor cells. The overall survival (OS, 98%) and 5-year progression-free survival (PFS, 93%) was significantly higher in IGF-1R positive cHL patients compared to IGF-1R negative patients (OS 83%, p = .029 and PFS 77%, p = .047, respectively). Three cHL cell lines showed expression of IGF-1R, with strong staining especially in the mitotic cells and expression of IGF-1. IGF-1 treatment had a prominent effect on the cell growth of L428 and L1236 cells and resulted in an increased phosphorylation of IGF1R, Akt and ERK. Inhibition of IGF-1R with cyclolignan picropodophyllin (PPP) decreased cell growth and induced a G2/M cell cycle arrest in all three cell lines. Moreover, a decrease in pCcd2 and an increase in CyclinB1 levels were observed which is consistent with the G2/M cell cycle arrest. In conclusion, IGF-1R expression in HRS cells predicts a favorable outcome, despite the oncogenic effect of IGF-1R in cHL cell lines.     

Expression of luteinizing hormone and follicle-stimulating hormone receptor in the dog prostate

A possible role for gonadotrophins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the prostate physiology has been suggested in humans and rats. This study aimed at investigating the presence of receptors for LH and FSH (LHR and FSHR) in the canine prostate. Prostates were collected at post mortem from 6 clinically healthy, sexually intact beagles free from any prostatic disorder. Tissue was sampled from dorsal, middle and ventral regions of each prostate. Immunohistochemical localization was performed on wax-embedded sections using polyclonal antibodies for LHR or FSHR. The pattern and intensity of staining in the parenchyma (glandular epithelium) and stroma were determined using a semiquantitative histologic assessment. Receptors for LH and FSH were consistently present in both the glandular epithelium and the stroma in all tissue samples examined. Expression for both receptors was higher in the glandular epithelium than the stroma of all prostatic regions (P < 0.001). In the glandular epithelium, LHR (P < 0.01) and FSHR (P < 0.05) expression was lower in the lateral than the other regions, and there was no difference between dorsal and ventral regions. However, variations in the expression for LHR and FSHR among prostatic regions were not found in the stroma. These findings have demonstrated that LHR and FSHR are expressed in the dog prostate, and the variation observed in their levels of expression among its regions and tissue layers suggests a potential role of gonadotrophins LH and FSH in the regulation of the prostate physiology, particularly the glandular epithelium.     

Manganese-Induced Effects on Testicular Trace Element Levels and Crucial Hormonal Parameters of Hyline Cocks

Manganese (Mn) is an essential element required for normal development and reproduction. However, little is known about the reproductive toxicity of Mn in birds. To investigate the Mn-induced toxicity on testicular trace element levels and crucial hormonal parameters on male reproduction in birds, 50-day-old male Hyline cocks were fed either a commercial diet or a Mn-supplemented diet. The changes in contents of copper (Cu), iron (Fe), zinc (Zn), and calcium (Ca) in testis were detected. Hormonal parameters were evaluated including the levels of testosterone (T), luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), triiodothyronine (T3), and thyroxine (T4) in the serum. The mRNA levels of luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR) were determined in this study. The results showed that Mn was accumulated in testis, and the content of Cu, Fe, Zn, and Ca decreased. Exposure to Mn significantly lowered the content of T, LH, FSH, and the mRNA expression levels of LHR and FSHR. Levels of T3 and T4 appeared with a decreased tendency, and TSH presented no obvious regularity. It indicated that Mn exposure resulted in the disbalance of testicular trace elements and influenced hormone levels in the molecular level, which may be possible underlying reproductive toxicity mechanism induced by Mn.     

The role of luteinizing hormone in regulating gene expression during selection of a dominant follicle in cattle

At approximately 8.5 mm in diameter, the future dominant follicle is "selected" for continued growth in cattle. In the present study, cows were treated with a gonadotropin-releasing hormone receptor antagonist, acyline, just before follicle selection (near 7.8 mm) to investigate the role of LH in changing mRNA concentrations during selection of a dominant follicle. The ovaries containing the expected dominant follicle (EDF; first largest follicle) and expected largest subordinate follicle (ESF) were removed after 12 or 24 h of treatment. Real-time PCR was used to determine mRNA concentrations. ELISA was used to measure testosterone and 17beta-estradiol (E(2)) and radioimmunoassay to measure androstenedione (A(4)) in follicular fluid. Concentrations of E(2) were greater in EDF than in ESF of untreated cows near the time of follicle selection (12 h) or at 12 h after selection (24 h). Testosterone, E(2), and A(4) were all dramatically decreased by acyline treatment at both times. In theca cells, acyline treatment reduced CYP17A1 (P450 17alpha) in EDF and STAR (steroidogenic acute regulatory protein) in both EDF and ESF but did not alter CYP11A1 (P450scc). In granulosa cells (GCs), LHCGR (luteinizing hormone [LH] receptor) was much greater in EDF than in ESF at both time of selection (739% greater) and 12 h after selection (2837% greater) and was decreased by acyline in EDF (87% decrease). The mRNA for CYP19A1 (cytochrome P450 aromatase) and PAPPA (pregnancy-associated plasma protein-A) tended to be greater in EDF than in ESF at follicle selection, and both mRNAs were much greater at 12 h after selection, with acyline significantly decreasing PAPPA mRNA after 24 h of treatment. The mRNA for FSHR (follicle-stimulating hormone receptor) was not different in EDF versus ESF and was not altered by acyline. Thus, induction of LHCGR mRNA in GCs is an early event during the follicle selection process, and surprisingly, expression of LHCGR mRNA is dependent on circulating LH. Production of follicular A(4), testosterone, and E(2) are also acutely related to LH but due to changes in expression of STAR and CYP17A1 in TC.