LHR Gene Expression Research Articles

Antioxidant, anti-inflammatory, and anti-reprotoxic effects of kaempferol and vitamin E on lead acetate-induced testicular toxicity in male rats.

The heavy metals cause repro-toxicity via oxidative stress and suppress the antioxidant enzymes. Kaempferol and vitamin E possess antioxidant properties that can counteract the deleterious heavy metals effects. The present study was directed to investigate the protective role of kaempferol, alone or with vitamin E, on testicular toxicity mediated by lead acetate in male rats. Fifty adult male rats were randomly grouped into five groups (n = 10): the control group received 5 ml distilled water, and the Pb group was intraperitoneally injected with 20 mg/kg of lead acetate. The Pb + Vitamin E group received Pb with 100 mg/kg of vitamin E, the Pb + KAF group received Pb with 50 mg/kg of kaempferol, the Pb + KAF + Vitamin E group received Pb with kaempferol and vitamin E for 6 weeks. The testicular levels of superoxide dismutase, catalase, steroidogenic enzyme, serum testosterone, follicle-stimulating hormone, interleukin (IL)-10, and sperm function were significantly decreased in the Pb group compared with all experimental groups. These parameters were significantly elevated in the Pb + KAF + Vitamin E group compared to other experimental groups. Lead acetate caused elevation in testicular malondialdehyde, nitric oxide, IL-6, IL-1β, tumor necrosis factor-α, nuclear factor kappa, and sperm abnormality compared to all treatment groups. All these parameters were significantly declined in the Pb + KAF + Vitamin E group and Pb + KAF group compared with the Pb group. The fold changes of pituitary follicle-stimulating hormone beta, gonadotropin-releasing hormone receptor, and luteinizing hormone beta, and testicular CYP11A1, LH receptor, and FHr gene expression were significantly upregulated in Pb + KAF + Vitamin E group compared with all experimental groups. In addition, KAF + Vitamin E has the potential to improve testicular regeneration in seminiferous tubules, Leydig, and Sertoli cells. Administration of kaempferol alone or with vitamin E can mitigate lead acetate-induced testicular toxicity in rats via its antioxidant and anti-inflammatory properties. The current research is the first to demonstrate that kaempferol can exert a preventive role in testicular dysfunction.

ВЛИЯНИЕ НИЗКОМОЛЕКУЛЯРНЫХ АЛЛОСТЕРИЧЕСКИХ АГОНИСТОВ РЕЦЕПТОРА ЛЮТЕИНИЗИРУЮЩЕГО ГОРМОНА НА ЕГО ЭКСПРЕССИЮ В СЕМЕННИКАХ САМЦОВ КРЫС

Human chorionic gonadotropin (hCG) and luteinizing hormone (LH) regulate testicular steroidogenesis by interacting with the orthosteric site located in the extracellular domain of the LH receptor (LHR). The use of hCG and LH in medicine is fraught with side effects caused by hyperactivation of LH-dependent cascades and the development of resistance of target cells to endogenous gonadotropins due to a decrease in the activity and expression of LHR. An alternative to gonadotropins is low-molecular-weight compounds which interact with the LHR transmembrane allosteric site. The aim of this work was to study the relationship between the steroidogenic effects of hCG and thieno[2,3-d]pyrimidine derivatives (TPDs) with an activity of LHR agonists and their ability to influence the expression of the LRH-encoding Lhr gene both in vitro, when acting on the primary culture of rat Leydig cells, and in vivo, when administered to male rats. hCG stimulated testosterone production with a high efficiency at an early stage of its effect on Leydig cells (after 30 min) and upon a single administration to male rats (after 3 h), exceeding TPDs in activity by this parameter. After 1–3 h of acting on Leydig cells and long-term administration to male rats, the steroidogenic effect of hCG decreased and became comparable to that of TPDs. In Leydig cells and rat testes, hCG suppressed Lhr gene expression, which was partially restored in vivo on days 7–10 being accompanied by a slight increase in the steroidogenic effect of hCG, though to a significantly smaller extent than on the first day. The treatment with TP03, which was the most active of the TPDs studied, had a little effect on Lhr gene expression in Leydig cells but significantly increased it in rat testes after long-term administration of the drug. The steroidogenic effect of TP03 positively correlated with Lhr gene expression. When TP03 was applied to aging rats, its steroidogenic effect decreased, most likely, due to the absence of its stimulating effect on LHR expression. Thus, after long-term administration, TPDs exert a moderately expressed, stable over time, stimulating effect on testosterone production but do not decrease LHR expression, which prevents testicular resistance to endogenous gonadotropins under conditions of steroidogenesis stimulation.

Lactational exposure of polychlorinated biphenyls impair Leydig cellular steroidogenesis in F1 progeny rats

The present study was aimed to determine the effects of lactational exposure of PCBs (Aroclor 1254) on Leydig cellular steroidogenesis in F1 progeny rats. Lactating dams were orally treated by gavage with different doses of PCBs (1, 2 and 5mg/kg b.wt./day). Male progenies were sacrificed on PND60. Our results demonstrated that exposure to PCBs decreased the body weight, testis weight and anogenital distance (AGD) index in the F1 progeny rats. Importantly, PCBs exposure reduced the serum levels of LH, testosterone and estradiol. Interestingly, PCBs caused a decrease in the Leydig cell population along with decreased activities of steroidogenic enzymes 3β- and 17β-HSD. Additionally, we observed a significant decrease in LHR, SR-B1, StAR protein, Cyp11a1, 3β-HSD, Cyp17a1, 17β-HSD, 5α-reductase, Cyp19a1 and AR gene expression in the Leydig cells of progeny rats. In conclusion, our study demonstrates that lactational exposure of PCBs alters Leydig cellular steroidogenesis in the F1 progeny rats.

The polymorphic insertion of the luteinizing hormone receptor “insLQ” show a negative association to LHR gene expression and to the follicular fluid hormonal profile in human small antral follicles

The luteinizing hormone receptor (LHCGR) has a little studied polymorphic 6 bp insertion (rs4539842/insLQ). This study has evaluated the insLQ polymorphism in relation to potential associations with hormonal characteristics of human small antral follicles (hSAFs).In total, 310 hSAFs were collected from 86 women undergoing fertility preservation. Analysis included hormonal profile of 297 follicular fluid (FF) samples and 148 corresponding granulosa cells samples were evaluated by qPCR for selected genes.Significantly reduced and non-detectable mRNA levels of anti-Müllerian hormone receptor II (AMHR2) and LHCGR, respectively, were observed for insLQ/insLQ compared to –/insLQ and the −/− genotypes. Moreover, LHCGR and CYP19a1 together with oestradiol and inhibin-B were significantly increased in –/insLQ compared to the −/− genotype. The homozygous insLQ genotype showed strong significant associations to GC specific genes LHCGR and CYP19a1, which may translate into significant changes in FF hormone profiles and an altered LH signaling.

Analysis of LH receptor in canine ovarian follicles throughout the estrous cycle

The aim of this study was to determine the mRNA LHR and LHR protein expression pattern in the canine ovarian follicles at different stage of development throughout the estrous cycle. Dog ovaries were obtained from 1-6y bitches at proestrus/estrus, anestrus and diestrus stages following ovariohysterectomy. Follicular cells were mechanically recovered from follicles distributed into four types (preantral, small antral, medium antral and large antral). Total RNA extraction was performed and the evaluation of gene expression levels was achieved by relative quantification q-PCR analysis. Intrafollicular amounts of LHR were assessed by western blot method. All results were evaluated by ANOVA. The expression levels of mRNA LHR in follicular cells were observed in every stage of development, however this gene expression varied over the estrous cycle. LHR transcripts increased (P < 0.05) from preantral to antral stage. There were not differences in LHR gene expression among follicles at preantral stages; however, at antral stages the lowest (P < 0.05) LHR mRNA expression was found at anestrus and the highest (P < 0.05) at proestrus/estrus. The LHR protein was also detected in dog follicles in all reproductive phases with patterns varying with stage of follicular development over the reproductive cycle. The antibody against human LHR revealed two bands at ∼90 and ∼67 kDa, probably representing the matured protein and its precursor respectively. Both bands LHR appeared already at preantral follicles increasing (P < 0.05) with growth. A high proportion of LHR was presented as immature forms in all follicles stages during different phases of the estrous cycle. In conclusion, the gene and protein of LHR are differentially expressed in dog follicles over the estrous cycle, increasing with growth and the precursor protein is the most predominant LHR form present in canine follicles.

Pre-translational regulation of luteinizing hormone receptor in follicular somatic cells of cattle

Differential regulation of LHR in theca cells (TC) and granulosa cells (GC) is important for normal follicular development. Unlike TC, GC only acquire LH-responsiveness during the later stages of antral follicle development. This study tested the hypothesis that differential LH-responsiveness in these two cell types may be due, in part, to shifts in cellular patterns of alternatively spliced LHR mRNA transcripts which may not be obvious from analysis of total LHR gene expression. It also further explored the role of translation inhibition by an LHR binding protein (LHBP), normally associated with the production of endogenous cholesterol. LHR mRNA variation arises as a result of the alternative splicing of two variable deletion sites (VDS) designated 5′ VDS and 3′ VDS, and it was proposed that differences in cell sensitivity to LH may be due in part to variations in the pattern of the mRNA expression of the receptor variants. The outcomes of the present study support a dynamic multi-facetted regulation of LHR during pre-translation. Not only did the ratio between variants change during antral follicle growth and in vitro cell differentiation but also between TC and GC. Regulation could also be linked to LH concentration feedback mechanisms as the absence of LH caused cultured TC to markedly up-regulate amounts of LHR mRNA. In both TC and GC, LHR mRNA was greatly reduced after treatment to block mevalonate production in the de novo cholesterol pathway, adding further support for a regulatory mechanism linked to enriched cellular amounts of mevalonate kinase.

Size matters: Associations between the androgen receptor CAG repeat length and the intrafollicular hormone milieu

Granulosa cell (GC) expressed androgen receptors (AR) and intrafollicular androgens are central to fertility. The transactivating domain of the AR contains a polymorphic CAG repeat sequence, which is linked to the transcriptional activity of AR and may influence the GC function. This study aims to evaluate the effects of the AR CAG repeat length on the intrafollicular hormone profiles, and the gene expression profiles of GC from human small antral follicles.In total, 190 small antral follicles (3–11 mm in diameter) were collected from 58 women undergoing ovarian cryopreservation for fertility preservation. The biallelic mean of the CAG repeat lengths were calculated for each woman, and grouped in three groups: Long CAG repeats (23–26 mean CAG); medium CAG repeats (20.5–22.5 mean CAG) and short CAG repeats (17.5–20.0 mean CAG). The following parameters were measured: follicle diameter, intrafollicular levels of Anti-Müllerian Hormone (AMH), progesterone, oestradiol, testosterone and androstenedione, and GC gene expression levels of FSHR, LHR, AR, CYP19A1, and AMH.The long CAG repeat lengths were associated with significantly decreased testosterone levels, as compared to medium CAG repeats (P = 0.05) and short CAG repeats (P = 0.003). Furthermore, in follicles 3–6 mm in diameter, the long CAG repeats were associated with significantly increased LHR and CYP19A1 gene expression levels compared to short CAG repeat lengths (P = 0.004 and P = 0.04 respectively), and significantly increased LHR expression compared to medium CAG repeat lengths (P = 0.03).In conclusion, long CAG repeat lengths in the AR were associated to significant attenuated levels of androgens and an increased conversion of testosterone into oestradiol, in human small antral follicles.

Effects of melatonin on the proliferation and apoptosis of sheep granulosa cells under thermal stress.

The cross-talk between oocyte and somatic cells plays a crucial role in the regulation of follicular development and oocyte maturation. As a result, granulosa cell apoptosis causes follicular atresia. In this study, sheep granulosa cells were cultured under thermal stress to induce apoptosis, and melatonin (MT) was examined to evaluate its potential effects on heat-induced granulosa cell injury. The results demonstrated that the Colony Forming Efficiency (CFE) of granulosa cells was significantly decreased (heat 19.70% ± 1.29% vs. control 26.96% ± 1.81%, p < 0.05) and the apoptosis rate was significantly increased (heat 56.16% ± 13.95%vs. control 22.80% ± 12.16%, p < 0.05) in granulosa cells with thermal stress compared with the control group. Melatonin (10−7 M) remarkably reduced the negative effects caused by thermal stress in the granulosa cells. This reduction was indicated by the improved CFE and decreased apoptotic rate of these cells. The beneficial effects of melatonin on thermal stressed granulosa cells were not inhibited by its membrane receptor antagonist luzindole. A mechanistic exploration indicated that melatonin (10−7 M) down-regulated p53 and up-regulated Bcl-2 and LHR gene expression of granulosa cells under thermal stress. This study provides evidence for the molecular mechanisms of the protective effects of melatonin on granulosa cells during thermal stress.

Allelic asymmetry of the Lethal hybrid rescue (Lhr) gene expression in the hybrid between Drosophila melanogaster and D. simulans: confirmation by using genetic variations of D. melanogaster

In the cross between Drosophila melanogaster females and D. simulans males, hybrid males die at the late larval stage, and the sibling females also die at later stages at high temperatures. Removing the D. simulans allele of the Lethal hybrid rescue gene (Lhr (sim) ) improves the hybrid incompatibility phenotypes. However, the loss-of-function mutation of Lhr (sim) (Lhr (sim0) ) does not rescue the hybrid males in crosses with several D. melanogaster strains. We first describe the genetic factor possessed by the D. melanogaster strains. It has been suggested that removing the D. melanogaster allele of Lhr (Lhr (mel) ), that is Lhr (mel0) , does not have the hybrid male rescue effect, contrasting to Lhr (sim0) . Because the expression level of the Lhr gene is known to be Lhr (sim) > Lhr (mel) in the hybrid, Lhr (mel0) may not lead to enough of a reduction in total Lhr expression. Then, there is a possibility that the D. melanogaster factor changes the expression level to Lhr (sim) < Lhr (mel) . But in fact, the expression level was Lhr (sim) > Lhr (mel) in the hybrid irrespectively of the presence of the factor. At last, we showed that Lhr (mel0) slightly improves the viability of hybrid females, which was not realized previously. All of the present results are consistent with the allelic asymmetry model of the Lhr gene expression in the hybrid.

LH receptor gene expression in cumulus cells in women entering an ART program

Luteinizing hormone (LH) exerts its actions through its receptor (LHR), which is mainly expressed in theca cells and to a lesser extent in oocytes, granulosa and cumulus cells. The aim of the present study was the investigation of a possible correlation between LHR gene and LHR splice variants expression in cumulus cells and ovarian response as well as ART outcome. Forty patients undergoing ICSI treatment for male factor infertility underwent a long luteal GnRH-agonist downregulation protocol with a fixed 5-day rLH pre-treatment prior to rFSH stimulation and samples of cumulus cells were collected on the day of egg collection. RNA extraction and cDNA preparation was followed by LHR gene expression investigation through real-time PCR. Furthermore, cumulus cells were investigated for the detection of LHR splice variants using reverse transcription PCR. Concerning LHR expression in cumulus cells, a statistically significant negative association was observed with the duration of ovarian stimulation (odds ratio = 0.23, p = 0.012). Interestingly, 6 over 7 women who fell pregnant expressed at least two specific types of LHR splice variants (735 bp, 621 bp), while only 1 out of 19 women that did not express any splice variant achieved a pregnancy. Consequently, the present study provide a step towards a new role of LHR gene expression profiling as a biomarker in the prediction of ovarian response at least in terms of duration of stimulation and also a tentative role of LHR splice variants expression in the prediction of pregnancy success.

Molecular Characterization and Gene Expression of Japanese Eel (Anguilla japonica) Gonadotropin Receptors

A luteinizing hormone receptor (lhr) cDNA with high identity to other fish lhrs was fully cloned from the ovary of the Japanese eel (Anguilla japonica). The genes for two gonadotropin receptors (Gthr), follicle-stimulating hormone receptor (fshr) and lhr, were differentially expressed during oogenesis, which was artificially induced by salmon pituitary extract, a gonadotropin-rich source. Transcript abundance of fshr was significantly elevated at the early vitellogenic stage and peaked at the late vitellogenic stage, while lhr gene expression rapidly induced at the late vitellogenic stage and thereafter remained at a high level. The abundance of fshr and lhr transcripts was highest in the ovary in female eels. In addition to the ovary, forebrain was a major site for the fshr transcript, although the level did not change with reproductive status. Furthermore, it was examined how eel Gthrs were activated by two mammalian chrionic gonadtropin (CG), equine CG (eCG) and human CG (hCG), that have been used for study of fish reproduction as substitutes for homologous Gths. Both CGs specifically activated eel Lhr, but not Fshr, although the degree of effectiveness was different; thus the concentration of hCG (0.1 ng/ml) required for significant activation of Lhr was much lower than that of eCG (100 ng/ml). These data on gene expression and ligand-activation of Gthrs suggest that Fsh and Lh act differentially in the regulation of reproductive function in Japanese eel.

Characterization of serum-free buffalo granulosa cell culture and analysis of genes involved in terminal differentiation from FSH- to LH-responsive phenotype

In the present study, buffalo granulosa cells were cultured under serum-free conditions and characterized to study the changes in gene expression associated with the transition of granulosa cells from estrogen- to progesterone-secreting phenotype. The cells were cultured in vitro under completely serum-free conditions for 8 d. Gene expression and hormone analysis showed that on day 4 granulosa cells exhibit FSH responsiveness with preovulatory phenotype having highest CYP19 gene expression and 17β-estradiol production, whereas a significant increase in transcript abundance of STAR, CYP11, and HSD3B genes accompanied with an increase in progesterone production was observed on day 8. Cells treated with LH on day 4 followed by gene expression analysis at 1, 2, 4, 6, 18, and 24 h showed significant increase in transcripts of LH-responsive genes. In conclusion, culture condition used in the present study showed that granulosa cells were FSH responsive and attained attributes of granulosa cells of dominant follicles at day 4 with highest CYP19 and LHR gene expression beyond which they acquired the ability to luteinize and thus were more LH responsive. In addition, after LH treatment, analysis of early LH-responsive genes ( EGR2, RUNX1, and NR4A1) on day 4 showed that granulosa cells at this stage in culture exhibits phenotype similar to that of preovulatory follicles before LH surge in vivo and corresponds to the in vivo transition of well-orchestrated gene expression profile after LH surge. The characterized culture conditions represent a suitable in vitro model for analysis of genes involved in terminal differentiation of granulosa cells from FSH- to LH-responsive phenotype during folliculogenesis in buffalo.

The expression of pituitary FSHβ and LHβ mRNA and gonadal FSH and LH receptor mRNA in the chicken embryo

In avian species, synthesis of sex steroids by embryonic gonads is regulated by luteinizing hormone (LH) and follicle-stimulating hormone (FSH). In order to elucidate the role of the two gonadotropins in gonadal axis development during the second half of chicken embryogenesis, pituitary expression of LH beta subunit (LHbeta) and FSH beta subunit (FSHbeta) mRNAs as well as gonadal expression of LH and FSH receptor (LHR and FSHR) mRNAs were determined on days 11 (E11) and 17 (E17) of embryonic development and after hatching (D1). In the pituitary of the female embryo, the gene expression of FSHbeta was the lowest on E11 and increased on E17. In the male pituitary, the expression of FSHbeta did not differ among the studied days. The FSHbeta mRNA expression on E11 was higher in the male than in the female pituitary gland. The expression of LHbeta mRNA in the female pituitary increased on D1 in comparison to E11. In the male pituitary gland, the expression of LHbeta gene was relatively constant. The expression of mRNA encoding FSHR in the ovary increased on E17, while in testes it did not differ among the studied days. There were no significant alterations in LHR gene expression in the ovary or in the testes in the examined period however, the gene expression on E17 was higher in the ovary than in the testes. We observed positive correlations between the pituitary FSHbeta mRNA expression and ovarian expression of FSHR mRNA (r = 0.63; p<0.01) as well as between LHbeta mRNA and LHR mRNA in the testes (r=0.65; p<0.01). The reported alterations in gene expression of FSHbeta, LHbeta and their receptors between sexes and among the stages of embryonic development indicate time- and sex-dependent action of gonadotropins in gonads of chicken embryos.

TRICHOSTATIN A INDUCED DEREPRESSION OF THE HUMAN LUTEINIZING HORMONE RECEPTOR GENE BY CELL‐SPECIFIC PHOSPHATASE RELEASE FROM THE PROMOTER

The histone deacetylase inhibitor, Trichostatin A (TSA), induces de‐repression of the human Luteinizing Hormone receptor (hLHR) 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/PKCzeta cascade phosphorylates Sp1 at Ser641, which is essential for the release of the repression of LHR transcription. These studies have revealed a critical role of serine/threonine protein phosphatases PP2A and PP1 in TSA‐induced activation of LHR gene transcription in a cell type‐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 HDAC, respectively at the promoter. This favors the phophorylation of Sp1 mediated by the PI3K/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 PI3K/PKCzeta and phosphatases could be critical for up‐ and down‐regulation of LHR gene expression in physiological and pathological settings.

LHR splicing variants and gene expression in the marmoset monkey

In the marmoset monkey, the LHR type II, lacking exon 10, is the native receptor type. We characterised the LHR splicing pattern in marmoset testes and the adrenals during puberty and in pre- and postpubertal ovaries and quantified mRNA LHR expression in the testis. We detected 11 LHR splicing variants expressed at similar levels and generated by exon skipping and/or usage of cryptic splice sites. No preferred splicing variant during pubertal maturation was observed in both sexes. Testicular and adrenal LHR expression levels did not significantly change with age. However, a significant increase during pubertal maturation for the serum testosterone/ LHR ratio indicated that testosterone secretion increases in the presence of constant LHR mRNA expression levels. We conclude that LHR splicing in the marmoset displays a homogenous pattern and that the main function of the LHR is established in the testis, reaching its highest efficiency during pubertal maturation.

Biphasic Action of Prolactin in the Regulation of Murine Leydig Tumor Cell Functions

We investigated in this study the effects of ovine PRL on endocrine functions of cultured murine Leydig tumor cells (mLTC-1). The parameters studied were the activation of signal transduction systems involving cAMP and intracellular free Ca2+, the expression of Janus kinase 2 (JAK2), expression and function of LH and PRL receptors (R), expression of the steroidogenic acute regulatory (StAR) protein, and stimulation of steroidogenesis. Very similar biphasic dose- and time-dependent responses of all the parameters studied were found upon PRL stimulation, comprising a fast inhibition within 24 h in response to high PRL doses (≥30 μg/liter), and a slow stimulation, between 48–72 h, in response to lower PRL doses (1–10μ g/liter). In addition, extracellular Ca2+ (1.5 mmol/liter) increased the effect of PRL on human CG (hCG)-stimulated StAR messenger RNA expression and progesterone (P) production. Importantly, the biphasic effects of PRL on LHR gene expression and hCG-mediated P production were abolished in the presence of anti-PRL antiserum, demonstrating specificity of PRL action. The PRL effects on StAR expression, and steroid and cAMP production, apparently reflect its effects on LHR function. The relevance of the PRL effects observed in mLTC-1 cells was supported by demonstration of similar PRL responses in hCG-stimulated testosterone production of isolated mouse Leydig cells. Collectively, these findings clearly demonstrate the biphasic regulatory actions of PRL, and clarify some facets of the controversial role of this hormone in Leydig cell function.

Biphasic action of prolactin in the regulation of murine Leydig tumor cell functions.

We investigated in this study the effects of ovine PRL on endocrine functions of cultured murine Leydig tumor cells (mLTC-1). The parameters studied were the activation of signal transduction systems involving cAMP and intracellular free Ca(2+), the expression of Janus kinase 2 (JAK2), expression and function of LH and PRL receptors (R), expression of the steroidogenic acute regulatory (StAR) protein, and stimulation of steroidogenesis. Very similar biphasic dose- and time-dependent responses of all the parameters studied were found upon PRL stimulation, comprising a fast inhibition within 24 h in response to high PRL doses (>/=30 microgram/liter), and a slow stimulation, between 48-72 h, in response to lower PRL doses (1-10 microgram/liter). In addition, extracellular Ca(2+) (1.5 mmol/liter) increased the effect of PRL on human CG (hCG)-stimulated StAR messenger RNA expression and progesterone (P) production. Importantly, the biphasic effects of PRL on LHR gene expression and hCG-mediated P production were abolished in the presence of anti-PRL antiserum, demonstrating specificity of PRL action. The PRL effects on StAR expression, and steroid and cAMP production, apparently reflect its effects on LHR function. The relevance of the PRL effects observed in mLTC-1 cells was supported by demonstration of similar PRL responses in hCG-stimulated testosterone production of isolated mouse Leydig cells. Collectively, these findings clearly demonstrate the biphasic regulatory actions of PRL, and clarify some facets of the controversial role of this hormone in Leydig cell function.

Regulation of function of the murine luteinizing hormone receptor promoter by cis- and trans-acting elements in mouse Leydig tumor cells

The transcriptional activity of various lengths of the 5′-untranslated region (UTR) of the murine LH receptor (R) gene were studied using the luciferase reporter gene in transiently transfected mouse Leydig tumor cells (mLTC-1). Chinese hamster ovarian (CHO) and HeLa cells were used as controls. The basal transcriptional promoter activity in mLTC-1 cells resided within the first 173 base pairs (bp) of the 5′-UTR. Placing and LHR promoter fragment (bases −715 −56 ) in front of the Herpes simplex virus thymidine kinase (TK) minimal promoter resulted in a 7-fold increase in luciferase activity. Deletion of bases −56 to −173 of the above construct totally abolished the increased luciferase activity, brought about by the LHR promoter sequences. Basically similar results on LHR promoter function were observed using CHO cells. In contrast, no LHR promoter activity was detected in HeLa cells, indicating a cell specific nature of its function. The first 173 bp promoter domain is GC-rich, with several SP-1 binding domains, and it bound specifically nuclear proteins isolated from mLTC-1 and HeLa cells. RNAse protection assays reconfirmed the presence of several transcription initiation sites within the first 310 bp of the 5′-UTR, also in the absence of the cognate LHR coding sequences. The most distal site at bp −310 did not function in the absence of the first 173 bp of the 5′-UTR. Other transcription initiation sites were identified closer to the translation initiation site. hCG (50 μg/l), 8-bromo (Br)-cAMP (100 μmol/l) and cholera toxin (100 μg/l) displayed qualitatively similar negative effects on the LHR promoter activity in the transfected mLTC-1 cells when the constructs containing at least the first 565 bp of the LHR 5′-UTR were used, but the inhibitory effects were greatly decreased in constructs containing ≤304 bp of the promoter region. Hence, the hCG cAMP associated inhibitory effects interact with region(s) located mainly between bp −565 and −305 of the LHR promoter. The inhibitory role of cAMP on LHR gene expression was also confirmed at the level of hCO-binding and hCG stimulated cAMP production of mLTC-1 cells. In conclusion, the current results elucidate the cis- and trans-acting elements in the regulation of expression of the murine LHR gene in cultured mouse Leydig cells. The minimal basal promoter activity is within the first 173 nucleotides of the 5′-UTR and the structural elements of the negative LHR regulation by the cognate hormone and elevated cAMP levels are mainly located within nucleotides −305 to −565 of the 5′-UTR. The function of the murine LHR promoter is similar to, though not identical with that of the rat, but at variance with that of the human LHR gene.

Ontogeny of follicle-stimulating hormone receptor gene expression in the rat testis and ovary

The ontogeny of the follicle-stimulating hormone (FSH) receptor (R) gene expression was studied in the rat testis and ovary between day 12.5 or 14.5 of fetal life (f), respectively, and adulthood. In Northern blots hybridized with a cRNA probe corresponding to a part of the extracellular domain of the FSHR, specific hybridization to testicular RNA was detected from day f18.5, and to ovarian RNA from postnatal day 7 onwards. The main transcripts in the testis were at all ages 7.0 kb and 2.5 kb in size. In the ovary, the main transcript was always 2.5 kb in size. In order to increase the sensitivity of mRNA detection, the FSHR gene expression was also analyzed using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique with primer pairs corresponding to the near full-length FSHR mRNA or to its extracellular domain. The specificity of the PCR products was verified by Southern hybridization using a nested 32P-labeled cDNA probe. The results indicated that the expression of the extracellular domain of the FSHR was first detected on day f14.5 in the testis and on day f20.5 in the ovary. The full-length mRNA appeared in both sexes 2 days later, which is in agreement with earlier measurements of appearance of FSHR binding in the rat testis (day f17.5) and ovary (day 3 post partum). In situ hybridization using an antisense cRNA probe for FSHR demonstrated that, as early in development as specific hybridization was detected, it was confined to the Sertoli cells in the testis and to granulosa cells in the ovary. When compared with the developmental onset of the LHR gene expression (our earlier data), a major difference was observed in the ovary; the message encoding the extracellular LHR domain appeared > 10 days earlier than that corresponding to the full-length LHR message. In the case of mRNAs for the testicular LHR, and for FSHR of both sexes, the difference between the developmental appearance of the truncated and full-length RNA forms was only 2 days.