Luteinizing Hormone Receptor Transcription Research Articles

AKAP13 Enhances CREB1 Activation by FSH in Granulosa Cells.

Granulosa cells (GCs) must respond appropriately to follicle-stimulating hormone (FSH) for proper follicle maturation. FSH activates protein kinase A (PKA) leading to phosphorylation of the cyclic AMP response element binding protein-1 (CREB1). We identified a unique A-kinase anchoring protein (AKAP13) containing a Rho guanine nucleotide exchange factor (RhoGEF) region that was induced in GCs during folliculogenesis. AKAPs are known to coordinate signaling cascades, and we sought to evaluate the role of AKAP13 in GCs in response to FSH. Aromatase reporter activity was increased in COV434 human GCs overexpressing AKAP13. Addition of FSH, or the PKA activator forskolin, significantly enhanced this activity by 1.5- to 2.5-fold, respectively (p < 0.001). Treatment with the PKA inhibitor H89 significantly reduced AKAP13-dependent activation of an aromatase reporter (p = 0.0067). AKAP13 physically interacted with CREB1 in co-immunoprecipitation experiments and increased the phosphorylation of CREB1. CREB1 phosphorylation increased after FSH treatment in a time-specific manner, and this effect was reduced by siRNA directed against AKAP13 (p = 0.05). CREB1 activation increased by 18.5-fold with co-expression of AKAP13 in the presence of FSH (p < 0.001). Aromatase reporter activity was reduced by inhibitors of the RhoGEF region, C3 transferase and A13, and greatly enhanced by the RhoGEF activator, A02. In primary murine and COV43 GCs, siRNA knockdown of Akap13/AKAP13 decreased aromatase and luteinizing hormone receptor transcripts in cells treated with FSH, compared with controls. Collectively, these findings suggest that AKAP13 may function as a scaffolding protein in FSH signal transduction via an interaction with CREB, resulting in phosphorylation of CREB.

Interaction of positive coactivator 4 with histone 3.3 protein is essential for transcriptional activation of the luteinizing hormone receptor gene

The luteinizing hormone receptor (LHR) is essential for sexual development and reproduction in mammals. We have established that Sp1 has a central role in derepression of LHR gene transcription induced by Trichostatin A (TSA) in MCF7 cells. Moreover, the co-activator PC4 which associates directly with Sp1 at the LHR promoter is essential for TSA-mediated LHR transcription. This study explores interactions of PC4 with histone proteins, which presumably triggers chromatin modifications during LHR transcriptional activation. TSA treatment of MCF7 cells expressing PC4-Flag protein induces acetylation of histone 3 (H3) and immunoprecipitation (IP) studies revealed its interaction with PC4-Flag protein. MS/MS analysis of the protein complex obtained after IP from TSA treated samples detected H3.3 acetylated at K9, K14, K18, K23 and K27 as a PC4 interacting protein. The association of PC4 with H3.3 was corroborated by IP and re-ChIP using H3.3 antibody. Similarly, IP and re-ChIP showed association of PC4 with H3 acetylated protein. Knockdown of PC4 in MCF7 cells reduced H3.3 enrichment, H3 acetylation at the Lys sites and LHR promoter activity in TSA treated cells despite an increase in H3 and H3.3 protein induced by TSA, linking PC4 to H3 acetylation and LHR transcription. Depletion of H3.3 A/B in MCF7 cells impair chromatin accessibility and enrichment of Pol II and TFIIB at the LHR promoter and its activation, resulting in marked reduction of LHR gene expression. Together, these findings point to the critical role of PC4 and its association with acetylated H3.3 in TSA-induced LHR gene transcription.

Interaction of Positive Coactivator 4 with Histone 3.3 Protein is Essential for Transcriptional Activation of the Luteinizing Hormone Receptor Gene

The luteinizing hormone receptor (LHR) is a member of the G protein‐coupled receptor family which is essential for sexual development and reproduction in mammals. Previous studies from our laboratory established that Sp1 is important for basal LHR promoter activity and has central role in derepression of LHR gene transcription induced by the histone deacetylase inhibitor Trichostatin A (TSA) in MCF‐7 cancer cells. Moreover, our studies revealed that the co‐activator PC4 which associates directly with Sp1 at the LHR promoter is essential for TSA‐mediated LHR transcription. The present study explores interactions of PC4 with histone proteins which presumably triggers chromatin modifications during LHR transcriptional activation induced by TSA. TSA treatment of MCF‐7 cells transiently expressing PC4‐Flag protein induces acetylation of H3 and immunoprecipitation (IP) studies revealed its interaction with PC4‐Flag protein. Mass spectrometry analysis from the protein complex obtained after IP with PC4 flag in TSA treated MCF‐7 samples detected H3.3 acetylated at lysine residues (K9, K14, K18, K23, K27 and K36) as a PC4 interacting protein. This association of PC4 with H3.3 was corroborated by re‐ChIP. In subsequent studies by IP and reChip analysis using specific antibodies against acetylated H3 protein for the lysine residues, also confirmed PC4 interaction with acetylated H3 protein. Knockdown of PC4 in MCF‐7 cells significantly reduced H3 protein acetylation at these sites and LHR promoter activity in TSA treated cells despite an increase in H3 and H3.3, linking PC4 expression to H3.3 recruitment and its acetylation. Depletion of H3.3 A/B in MCF7 cells significantly impair recruitment of Pol II and TFIIB to the LHR promoter and its activation induced by TSA. In this study, we have shown interaction between PC4 and acetylated H3.3 in LHR induced derepression by TSA. Together these findings point to the critical role of PC4 and it is associated acetylated H3.3 protein in TSA‐induced LHR gene transcription.Support or Funding InformationIntramural Research Program, National Institute of Child Health and Human Development, NIHThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Examining sources of variation in HPG axis function among individuals and populations of the dark-eyed junco

Gonadal steroids are important mediators of traits relevant to fitness, and thus may be targets of selection. However, more knowledge is needed about sources of variation along the endocrine axes that may contribute to functional variation in steroid levels. In a controlled captive environment, we studied males of two closely related subspecies of the dark-eyed junco (Junco hyemalis) that differ in testosterone-related phenotype, asking whether they also differ in testosterone (T), and assessing the contribution of the sequential links of the hypothalamic–pituitary–gonadal axis. When males of both subspecies were challenged with gonadotropin-releasing hormone (GnRH), they were similar in circulating luteinizing hormone (LH) and T responses. When challenged with exogenous LH, they again produced levels of T similar to one another, and to the levels produced in response to GnRH. However, the smaller, less ornamented, and less aggressive subspecies had greater abundance of mRNA for LH receptor in the testes and for androgen receptor in the rostral hypothalamus, suggesting potential differences in regulatory feedback. We suggest that circulating hormone levels may be less prone to evolutionary change than the responsiveness of individual hormone targets. Among individuals, T titers were highly repeatable whether males were challenged with GnRH or with LH, but LH produced in response to GnRH did not covary with T produced in response to LH. Testis mass, but not LH receptor transcript abundance, predicted individual variation in T responses. These data implicate the gonad, but not the pituitary, as an important source of individual variation in T production.

English

Bone morphogenetic proteins (BMPs), belonging to the transforming growth factor-beta family, are crucial factors in follicular growth and development in the mammalian ovary. In this study, we examined the effects of BMP2, 4, 6 and 7 on goat granulosa cells by in vitro culture. The results showed that BMP2, 4 and 7 increased follicle-stimulating hormone receptor (FSHR) level in goat granulosa cells whereas they decreased luteinizing hormone receptor (LHR) level in both transcript and protein levels; however, BMP6 upregulated LHR transcript and protein level in goat granulosa cells, whereas it had no effect on FSHR level. FSHR knockdown increased granulosa cell apoptosis. Our data provided the i¬rst evidence that BMP2, 4, 6 and 7 may inhibit granulosa cell apoptosis through the regulation of FSHR and/or LHR level. These i¬ndings provided new insight into the biological functions of BMP2, 4, 6 and 7 for follicular development in goat ovary. Key words: Granulosa cell, bone morphogenetic proteins (BMPs), follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR)

Circadian Clock genes Per2 and clock regulate steroid production, cell proliferation, and luteinizing hormone receptor transcription in ovarian granulosa cells

Circadian Clock genes are associated with the estrous cycle in female animals. Treatment with Per2 and Clock siRNAs decreased the number of granulosa cells and LHr expression in follicle-stimulating hormone FSH-treated granulosa cells. Per2 siRNA treatment did not stimulate the production of estradiol and expression of P450arom, whereas Clock siRNA treatment inhibited the production of estradiol and expression of P450arom mRNA. Per2 and Clock siRNA treatment increased and unchanged, respectively, progesterone production in FSH-treated granulosa cells. Similarly, expression of StAR mRNA was increased by Per2 siRNA and unchanged by Clock siRNA. Our data provide a new insight that Per2 and Clock have different action on ovarian granulosa cell functions.

Coactivator Function of Positive Cofactor 4 (PC4) in Sp1-directed Luteinizing Hormone Receptor (LHR) Gene Transcription

The LHR has an essential role in sexual development and reproductive function, and its transcription is subjected to several modes of regulation. In this study, we investigated PC4 coactivator function in the control of LHR transcription. Knockdown of PC4 by siRNA inhibited the LHR basal promoter activity and trichostatin A (TSA)-induced gene transcriptional activation and expression in MCF-7 cells. While overexpression of PC4 alone had no effect on the LHR gene, it significantly enhanced Sp1- but not Sp3-mediated LHR transcriptional activity. PC4 directly interacts with Sp1 at the LHR promoter, and this interaction is negatively regulated by PC4 phosphorylation. The coactivator domain (22-91 aa) of PC4 and DNA binding domain of Sp1 are essential for PC4/Sp1 interaction. ChIP assay revealed significant occupancy of PC4 at the LHR promoter that increased upon TSA treatment. Disruption of PC4 expression significantly reduced TSA-induced recruitment of TFIIB and RNAP II, at the promoter. PC4 functions are beyond TSA-induced phosphatase release, PI3K-mediated Sp1 phosphorylation, and HDAC1/2/mSin3A co-repressor release indicating its role as linker coactivator of Sp1 and the transcriptional machinery. These findings demonstrated a critical aspect of LHR modulation whereby PC4 acts as a coactivator of Sp1 to contribute to the human of LHR transcription.

PC4 is a coactivator for basal and TSA‐induced Luteinizing Hormone Receptor (LHR) transcription

The LHR has an essential role in sexual development and reproductive function, and its transcription is subject to several modes of regulation and multiple effectors. In this study we investigated PC4 coactivator function in the control of LHR transcription. In MCF‐7 and JAR cells, PC4 knockdown with siRNA significantly reduced the basal LHR promoter activity. While over‐expression of PC4 alone had no effect on the LHR promoter activity, it synergistically enhanced Sp1‐ but not Sp3‐mediated LHR transcriptional activity and GST‐pull down showed that PC4 interacted with Sp1. Moreover, DNA affinity precipitation assay revealed the association of PC4 with Sp1/Sp3 binding sites of the LHR gene promoter. This association was markedly increased by dephosphorylation of PC4 with treatment of calf intestinal phosphatase, suggesting a role of phosphorylation in the modulation of PC4/Sp1 interaction. Furthermore, PC4 was found to be involved in the activation of LHR gene expression by TSA in MCF‐7 cells, since the induced LHR transcription/expression was substantially reduced by &gt;90% after knockdown of PC4 protein. Chromatin immunoprecipitation assay further showed significant occupancy of PC4 in the LHR promoter. Taken together, these studies have demonstrated another layer of LHR modulation whereby PC4 acts as a coactivator of Sp1 to contribute to the basal and TSA‐induced derepression of LHR transcription.

PC4 Coactivator Mediates Sp1‐driven Luteinizing Hormone Receptor Transcription

The luteinizing hormone receptor (LHR) has an essential role in sexual development and reproductive function by participating in gonadal maturation and regulation of steroidogenesis and gametogenesis. Transcription of the LHR gene is subject to several modes of regulation by multiple effectors through its core promoter, which contains two activating Sp1/Sp3 binding domains and an inhibitory motif recognized by EAR2 and EAR3/COUP‐TF1. The present studies demonstrate that PC4 acts as a coactivator of Sp1 to mediate Sp1‐dependent LHR expression. In MCF‐7 breast cancer cells and choriocarcinoma JAR cells, knockdown of PC4 expression with its specific siRNA significantly reduced LHR promoter activity. Co‐transfection of PC4 with an Sp1 or Sp3 expression plasmid showed that PC4 synergistically enhanced Sp1 but not Sp3‐activated LHR expression in reporter gene analyses, indicative of a functional interaction between PC4 and Sp1. In support of this, DNA affinity precipitation assay revealed the sequence‐specific association of PC4 with Sp1/Sp3 binding sites of the LHR gene promoter. Moreover, dephosphorylation of PC4 by calf intestinal phosphatase substantially increased its interaction with the Sp1 binding sites, suggesting a role of phosphorylation in modulation of the PC4/Sp1 interaction. Taken together, these studies have demonstrated that PC4 functions as a coactivator of Sp1 in LHR regulation.

Unlocking Repression of the Human Luteinizing Hormone Receptor Gene by Trichostatin A-induced Cell-specific Phosphatase Release

Our previous studies demonstrated that the histone deacetylase inhibitor, trichostatin A (TSA), induces derepression of the human luteinizing hormone receptor (LHR) gene by de-recruitment of the pRB homologue p107 repressor from the promoter in JAR and MCF-7 cancer cells. TSA initiates a mechanism whereby the phosphatidylinositol 3-kinase/protein kinase zeta (PKCzeta) cascade phosphorylates Sp1 at Ser-641, which is essential for the release of the repression of LHR transcription. The present studies have revealed that dissociation of serine/threonine protein phosphatases PP2A and PP1 from the LHR promoter mediates TSA-induced activation of LHR gene transcription in a cell-specific manner. Changes in chromatin structure induced by TSA cause the release of PP2A in JAR cells or of PP1 in MCF-7 cells, which is associated with Sp1 directly or through histone deacetylase 1/2, respectively, at the promoter. This favors the phosphorylation of Sp1 mediated by the phosphatidylinositol 3-kinase/PKCzeta pathway, which in turn causes the release of the p107 inhibitor from Sp1 and marked transcriptional activation of the LHR. These findings reveal the importance of phosphatases in the control of LHR transcription, where the balance between phosphatidylinositol 3-kinase/PKCzeta and phosphatases could be critical for up- and down-regulation of LHR gene expression in physiological and pathological settings.

PKC alpha‐Induced Derepression of the Human Luteinizing Hormone Receptor (LHR) through ERK‐Mediated Sp1 Phosphorylation

Transcription of the LHR is subject to repression/derepression through various modes and multiple effectors. The present studies revealed that PKCα/ERK signaling is essential for activation of the LHR gene induced by phorbol‐12‐myristate‐13‐acetate (PMA) in Hela cells. While this effect was attributable to PKCα activity, the ERK pathway was the downstream effector in LHR activation. PMA caused significant enhancement of Sp1 phosphorylation at serine residue(s), which was blocked by inhibition of PKCα or ERK. The interaction of activated phospho‐ERK with Sp1 and ERK's association with the LHR promoter points to Sp1 as a direct target of ERK. Following Sp1 phosphorylation the HDAC/mSin3A repressor complex dissociated from Sp1 sites, histone 3 was acetylated, and TFIIB and Pol II were recruited. In addition, overexpression of a constitutively active PKCα (PKCαCA) which strongly activated LHR transcription in MCF‐7 cells (devoid of PKCα), induced Sp1 phosphorylation at serine residue(s) and caused de‐recruitment of the HDAC1/mSin3A complex from the promoter. These effects were negated by co‐transfection of dominant negative PKCα. These studies have revealed a novel regulatory signaling mechanism of transcriptional control in which the LHR is derepressed through PKCα/ERK‐mediated Sp1 phosphorylation, causing the release of HDAC1/mSin3A complex from the promoter.

Transcription of Key Genes Regulating Gonadal Steroidogenesis in Control and Ketoconazole- or Vinclozolin-Exposed Fathead Minnows

This study evaluated changes in the expression of steroidogenesis-related genes in male fathead minnows exposed to ketoconazole (KTC) or vinclozolin (VZ) for 21 days. The aim was to evaluate links between molecular changes and higher level outcomes after exposure to endocrine-active chemicals (EACs) with different modes of action. To aid our analysis and interpretation of EAC-related effects, we first examined variation in the relative abundance of steroidogenesis-related gene transcripts in the gonads of male and female fathead minnows as a function of age, gonad development, and spawning status, independent of EAC exposure. Gonadal expression of several genes varied with age and/or gonadal somatic index in either males or females. However, with the exception of aromatase, steroidogenesis-related gene expression did not vary with spawning status. Following the baseline experiments, expression of the selected genes in male fathead minnows exposed to KTC or VZ was evaluated in the context of effects observed at higher levels of organization. Exposure to KTC elicited changes in gene transcription that were consistent with an apparent compensatory response to the chemical's anticipated direct inhibition of steroidogenic enzyme activity. Exposure to VZ, an antiandrogen expected to indirectly impact steroidogenesis, increased pituitary expression of follicle-stimulating hormone beta-subunit as well as testis expression of 20beta-hydroxysteroid dehydrogenase and luteinizing hormone receptor transcripts. Results of this study contribute to ongoing research aimed at understanding responses of the teleost hypothalamic-pituitary-gonadal axis to different types of EACs and how changes in molecular endpoints translate into apical outcomes reflective of either adverse effect or compensation.

Dual mechanisms of regulation of transcription of luteinizing hormone receptor gene by nuclear orphan receptors and histone deacetylase complexes

The luteinizing hormone receptor (LHR), a member of the G protein-coupled, seven transmembrane receptor family, is essential for normal sexual development and reproductive function. LHR are expressed primarily in the gonads, but also are found in non-gonadal and cancer tissues. LH acts through LH receptors in Leydig cells to maintain general metabolic processes and steroidogenic enzymes, and in the ovary enhances follicular development and steroidogenesis in granulosa and luteal cells. The major transcriptional start sites of the LHR gene are located within the 176 bp promoter domain. In the rat, the LHR gene is constitutively inhibited by upstream sequences (−176/−2056 bp) in several cell systems, while in the human only a minor inhibitory effect was observed in JAR and HeLa cells (>20%). The TATA-less human promoter is driven by Sp1 and Sp3 transactivators that bind to two Sp1 domains at −79 bp [Sp1(I)] and −119 bp [Sp1(II)] (from ATG) with additive effects. An imperfect estrogen receptor half-site response element direct-repeat within the LHR promoter is an inhibitory locus. Endogenous orphan receptors, EAR2 and EAR3/COUP-TFI, bind this motif and repress promoter activity by 70%. TR4 also binds this motif and stimulates promoter activity (up to 2.5-fold). This is reversed by coexpression of EAR2 or EAR3/COUP-TFI through competitive binding to this site. Comparative studies of hDR and rDR orphan receptors binding and function revealed sequence-specific requirements. The A/C mismatch between hDR and rDR is responsible for the lack of TR4 binding and function in the rat. The G 3′-adjacent to the hDR core is important for EAR2/EAR3-COUP-TFI high-affinity binding. The Sp1-1 site is critical for EAR3/COUP-TFI repression, with minor participation for EAR2, and is not involved in the TR4 effect. Interaction of EAR3/COUP-TFI with Sp1 perturbs association of TFIIB with Sp1, independently of HDACs, and caused impairment of LHR transcription. Other aspect of control is through HDAC/mSin3A mechanism. Inhibition of HDACs by TSA increases LHR promoter activity in JAR cells (40-fold), association of acetylated H3/H4 with the LHR promoter, recruitment of Pol II to the promoter, and LHR mRNA levels. A multiprotein complex is recruited to the hLHR promoter via interaction with Sp1/Sp3: HDACs dock directly to Sp1-1 bound DNA and indirectly to Sp3-1 bound DNA through RbAp48, while mSin3A interacts HDACs and potentiates HDAC1-mediated repression. Our studies have demonstrated that orphan receptor–ERE complexes, and the HDAC1–HDAC2–mSin3A complex have important roles in the regulation of LHR gene transcription by interaction with Sp1/Sp3, and by region-specific changes in histone acetylation and Pol II recruitment within the LHR promoter.

Expression and actions of both the follicle stimulating hormone receptor and the luteinizing hormone receptor in normal ovarian surface epithelium and ovarian cancer

The ability of gonadotropins to act on and regulate normal ovarian surface epithelial (OSE) cells and ovarian cancer cells was investigated. Bovine OSE was used as a model to study normal OSE. Results demonstrate that follicle stimulating hormone (FSH) and the luteinizing hormone (LH) like molecule, human chorionic gonadotropin (hCG), can both stimulate ( 3H)-thymidine incorporation into DNA in normal OSE cells. Similar results were obtained using either purified hormones or recombinant human hormones. A human ovarian cancer cell-line OCC1 was also stimulated to grow in response to FSH and hCG, but the growth of a different human ovarian cancer cell-line SKOV3 was not affected. In addition to effects on cell growth, gonadotropins also stimulated growth factor expression. Both FSH and hCG stimulated steady state levels of keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and kit ligand (KL) mRNA in OSE cells. Previously, KGF, HGF, and KL have been shown to stimulate OSE growth. Both follicle stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) were observed in OSE cells by Northern blot analysis. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed on fresh and cultured OSE cells. Normal OSE was found to express FSHR and LHR both in vivo and in vitro. The PCR reaction products were sequenced and found to provide a 100% homology with the bovine gonadotropin receptor sequences previously reported. FSHR and LHR transcripts were also detected in gonadotropin responsive OCC1 cells, but not in the gonadotropin insensitive SKOV3 cells. Observations support the hypothesis that gonadotropins may influence some ovarian cancers. In summary, the current study demonstrates the novel observation that both the FSHR and LHR are expressed by bovine OSE and selected ovarian cancers. Interestingly, the actions of FSH and LH to promote OSE growth may in part be mediated indirectly through an elevation in the expression of autocrine growth factors (KGF, HGF, and KL). Ovarian cancer is more common in conditions with elevated gonadotropins such as post-menopausal women. Therefore, gonadotropin actions on the OSE are postulated to be a potential factor in the onset and progression of some ovarian cancers.

Decreased Luteinizing Hormone Receptor mRNA Expression in Human Ovarian Epithelial Cancer

Objectives. The objective of this study was to examine the distribution and cellular localization of luteinizing hormone receptor (LHR) in ovarian epithelial tumors (OETs) and their presumed precursor lesions—ovarian epithelial inclusions (OEIs). The clinicopathologic correlation of the receptor expression in OET was also examined.Methods. Fifteen microdissected samples of ovarian surface epithelium (OSE), 20 OEIs from benign ovaries, and 141 OETs, including 48 cystadenomas, 33 borderline tumors, 60 carcinomas, and 5 metastatic cancers, were examined for LHR expression by using reverse transcription polymerase chain reaction and in situ hybridization. LHR expression in tumor epithelium and tumor stroma was analyzed separately. The clinicopathologic correlation data were analyzed by standard analysis of variance and contingency table methods.Results. LHR expression was identified in the majority of OSE and OEI samples. In OETs, LHR positivity was found in the epithelial cells in 27% of cases and in the stromal compartment in 37% of cases. LHR-positive stromal cells were mainly luteinized cells. Within the tumor epithelium, LHR expression was detected in 42% of benign, 24% of borderline, and 17% of malignant OETs. LHR expression in tumor stroma showed a similar trend of reduction from benign to malignant OETs. Within the 17 carcinomas, LHR was expressed in the epithelium in 47% of grade 1, 12% of grade 2, and only 5% of grade 3 cancers. The mean age of the LHR-positive group was younger than that of the receptor-negative patients. Compared with mucinous and other types of OETs, serous OETs showed higher LHR expression in the epithelium. Compared with the OETs removed in the different menstrual phases, OETs in the secretory phase showed higher LHR in the tumor stroma than in the proliferative phase. No receptor mRNA was detected in the epithelium of five carcinomas metastatic to the ovary. LHR transcription splicing variants from a single previous report were confirmed in this study.Conclusions. Malignant OETs have significant reduction of LHR expression compared with precursor lesions and benign and borderline OETs. LHR expression shows a steady decline from low-grade to high-grade ovarian cancer. The presence of LHR receptor in tumor epithelium suggests that luteinizing hormone in serum may have direct influence on tumor growth, whereas the receptor in tumor stroma may be indicative of a paracrine function of LH in the development of OETs.

Gene expression of luteinizing hormone receptor and steroidogenic enzymes during Leydig cell development.

Testicular Leydig cells (LC) are rapidly and selectively destroyed by an injection of ethane dimethane sulfonate (EDS). LC regeneration occurs in the testis of the EDS-treated rats from the differentiation of the precursor Leydig cells (PLC). This study was designed to investigate the patterns of change in the mRNAs for the luteinizing hormone receptor (LHR) and the steroidogenic enzymes, cholesterol side chain cleavage (P-450scc) and 17 alpha-hydroxylase (P-450(17 alpha)) during LC regeneration from PLCs. Mature (60 days of age) Sprague-Dawley male rats received a single intraperitoneal injection of EDS and were killed at different times between days 2 and 60 post-treatment. PLC- and LC-enriched fractions were isolated from the testes of the EDS-treated rats and age-matched control rats using a collagenase digestion-Percoll gradient method. Total RNA was extracted from these cell populations and subjected to Northern blot analysis. The LC fraction isolated from testes of control rats expressed four major transcripts of the LHR, sized 1.8, 2.5, 4.2 and 7.0 kb. The undifferentiated PLC fraction from controls expressed only a truncated form, the 1.8 kb transcript. This truncated LHR transcript was also the only LHR mRNA species detected in PLCs at day 2 post-EDS treatment. In contrast, all four transcripts of the LHR were detected in the PLC fraction at day 10 post-EDS treatment. The levels of the full length 7.0 kb transcript increased thereafter and reached a peak between days 24 and 36 post-EDS treatment in the PLC fraction. Concomitant with the increase in the 7.0 kb transcript, the truncated 1.8 kb transcript decreased in amount and reached a nadir between days 16 and 36 post-treatment. The changes observed in this cell fraction reflect the process of differentiation of PLCs into LCs. At day 45 post-EDS treatment, the level of the 7.0 kb transcript decreased while the 1.8 kb form increased in the PLC fraction, reflecting the completion of LC regeneration from this cell fraction. By day 60 post-EDS treatment, the levels of the 1.8 kb transcript rose to the value observed in undifferentiated control PLCs and the other transcripts were no longer detected in the PLC fraction, indicating that cells in the PLC fraction were again in an undifferentiated stage. Messenger RNAs for both the steroidogenic enzymes, P-450scc and P-450(17 alpha) were expressed in the control LC fraction. Neither of these two mRNAs were detected in the PLC fraction of the control rats. P-450scc and P-450(17 alpha) mRNAs were first expressed in the PLC fraction at day 10 post-EDS treatment. Thereafter, the levels of P-450scc and P-450(17 alpha) mRNAs increased in the PLC fraction and reached a peak between days 24 and 36 and days 24 and 45 post-EDS treatment respectively. P-450scc and P-450(17 alpha) mRNAs were no longer expressed in the PLC fraction at day 60 post-EDS treatment. These patterns also reflect the process of differentiation of PLCs into functional LCs. These results demonstrate for the first time that PLCs in the control testis are undifferentiated and do not express functional LHR and steroidogenic enzymes or their mRNAs. The PLCs are characterized, however, by the expression of a truncated 1.8 kb transcript of the LHR mRNA. Functional LHR and steroidogenic enzymes are expressed in PLCs only during their differentiation into LCs after EDS treatment. Subsequent to LC regeneration, the PLCs return to an undifferentiated stage.

Expression of Messenger Ribonucleic Acids of Luteinizing Hormone and Follicle-Stimulating Hormone Receptors in Granulosa and Theca Layers of Chicken Preovulatory Follicles

Expression of luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR) mRNAs was demonstrated in the granulosa and theca layers of the large preovulatory follicles of the chicken ovary by Northern hybridization and reverse transcription-polymerase chain reaction (RT-PCR). Northern hybridization results showed multiple LHR and FSHR mRNA transcripts and the predominant species were 2.3 and 2.5 kb, respectively. The highest abundance of LHR mRNA was found in the granulosa layer of the largest follicle (F1follicle), while the abundance remained low in the granulosa layers of the third (F3) and fifth largest (F5) follicles. FSHR mRNA abundance was the highest in the granulosa layer of F5follicle, but decreased in the granulosa layers of F3and F1follicles. In the theca layers of all three follicles examined LHR and FSHR mRNAs were extremely low. These results were confirmed by RT-PCR experiment which involved coamplification of LHR or FSHR mRNA and β-actin mRNA as the internal control. The LHR PCR product was sequenced and indicated 92.2% homology with the corresponding region of the quail LHR cDNA. This study indicated that the marked increase in expression of LHR mRNA in granulosa layer of the F1follicle might be important for LHR protein synthesis and succeeding bonus progesterone production in F1follicle destined to ovulation. However, higher expression of FSHR mRNA in the granulosa layer of the less mature follicles may be involved in the differentiation and maturation of granulosa cells in these follicles.

The luteinizing hormone receptor

Both major and incremental advances in the past decade have provided important insights into the structure, function, and regulation of the luteinizing hormone receptor (LHR) and its gene. The availability of the crystal structure of the extracellular domain of the follicular stimulating hormone receptor-hormone complex, and its applicability to other members of the gonadotropin receptor family, has facilitated prediction of the localized areas of specific interaction between the LHR and its cognate hormone. This has been supported by the incorporation of information derived from mutational and chimeric studies. The nature of the structural and molecular changes involved in the initiation and propagation of signaling is beginning to be revealed by the various models. It is likely that components of each domain of the LHR, the hinge region of the extracellular domain, and the LH/human chorionic gonadotrophin α-1,3 loops participate in the receptor activation. Naturally occurring and created mutations have provided fertile ground for modeling, leading to plausible proposals for the regions of the receptor that participate in receptor coupling functions. The knockout mouse model has demonstrated the lack of participation of the hormone/receptor in fetal gonadal development, in contrast with the situation in the human, where the essentiality of the LHR is exemplified by Leydig cell hypoplasia. The association of an LHR polymorphism with breast cancer age of onset and severity has been documented. Significant advances have been achieved in studies on LHR gene structure and regulation of transcription. Two independent mechanism of repression/derepression have been identified. Studies on the epigenetic control of LHR transcription have revealed the combined importance of promoter methylation status; and changes in histone acetylation/methylation in the association/release of inhibitory complexes from the promoter, and recruitment of the preinitiation complex and polymerase II. Furthermore, major advances await elucidation of the participation and molecular mechanisms of signaling pathways involved in the transcription of the LHR.

Ligand-lnduced Down-Regulation of Testicular and Ovarian Luteinizing Hormone (LH) Receptors Is Preceded by Tissue-Specific Inhibition of Alternatively Processed LH Receptor Transcripts

Down-regulation of plasma membrane receptors by homologous hormones has been found in diverse cell types. In testicular Leydig and ovarian luteal cells, treatment with LH/hCG decreases LH receptor content. Although suppression of LH-binding sites may result from ligand-induced receptor internalization, sequestration, and/or phosphorylation, the gonadotropins may also regulate receptor mRNA levels. We examined the regulation of testis LH receptor mRNAs in adult rats that received 10 or 200 IU hCG, using cRNA probes derived from the 5' extracellular domain (EC) or the 3' transmembrane domain (TM) of the rat receptor cDNA. Probe EC hybridized to predominant signals of 7 and 1.8 kilobases (kb) and weaker signals of 4.2 and 2.5 kb. However, probe TM hybridized to the three larger forms of the LH receptor mRNA, but not to the 1.8-kb species, suggesting that the latter form lacks the transmembrane domain. After 6 and 12 h of treatment with 200 or 10 IU hCG, respectively, hybridization to the larger mRNA species decreased by more than 60%, preceding decreases in testicular [125I]hCG binding. These transcripts were further inhibited (greater than 93%) between 24-72 h after hCG treatment and returned to 40% and 100% of control levels by days 6 and 9, respectively. In contrast, the truncated 1.8-kb LH receptor transcript was not affected by hCG treatment, indicating a differential suppressive effect of the ligand on its receptor mRNA levels. In the ovary, hybridization to probe EC revealed four transcripts with similar sizes as those found in the testes, with a predominant 7-kb species.(ABSTRACT TRUNCATED AT 250 WORDS)