Luteinizing Hormone Receptor Gene Expression Research Articles

Dynamics of gonadotropin and thienopyrimidine derivative TP03 effects on ovulation and ovarian steroidogenesis in Follimag-stimulated immature female rats

BACKGROUND: Gonadotropin preparations, particularly human chorionic gonadotropin (hCG), are commonly used to induce ovulation and treat reproductive disorders in women, albeit with associated side effects. Low-molecular-weight allosteric agonists of the luteinizing hormone receptor (LHR), such as thieno[2,3-d]pyrimidine derivatives, offer a potential alternative. AIM: This study aims to compare the effects of thieno[2,3-d]pyrimidine TP03 and hCG on ovarian weight, corpus luteum formation, and plasma levels of estradiol, progesterone, and luteinizing hormone in immature female rats pre-treated with Follimag®. It also examines their impact on ovarian gene expression related to LHR and steroidogenesis. MATERIALS AND METHODS: TP03 and hCG were administered 48 h after the Follimag® injection at a dose of 20 mg/kg (i.p.) and 15 IU/rat (s.c.), respectively. Parameters were assessed at 1, 2, 4, 8, 16, and 24 h after TP03 and hCG administration. Plasma hormone levels were measured via ELISA, and ovarian gene expression was analyzed using real-time PCR. RESULTS: TP03 increased ovarian weight, progesterone levels in the blood, and expression of steroidogenic genes encoding the cholesterol-transporting protein StAR and the cytochromes CYP11A1 and CYP17A1. TP03 also stimulated corpus luteum formation (16–24 h after treatment). The temporal dynamics of its stimulating effects were similar to those of hCG, although their magnitude was slightly inferior to those of gonadotropin. TP03-induced decrease in blood estradiol levels and aromatase gene expression in the ovaries was also more moderate. Unlike hCG, which suppressed LHR gene expression 8 h after treatment, TP03 maintained a high LHR gene expression, preserving ovarian sensitivity to endogenous luteinizing hormone. CONCLUSIONS: TP03 exhibits potential as an ovulation inducer with milder stimulating effects on ovarian steroidogenesis than hCG, which reduces the risks of developing ovarian hyperstimulation syndrome and resistance to gonadotropins.

Camel milk or silymarin could improve the negative effects that experimentally produced by aflatoxin B1 on rat’s male reproductive system

BackgroundCamel milk and silymarin have many different beneficial effects on several animal species. Meanwhile, Aflatoxins are mycotoxins with extraordinary potency that pose major health risks to several animal species. Additionally, it has been documented that aflatoxins harm the reproductive systems of a variety of domestic animals. The present design aimed to investigate the impact of aflatoxin B1 (AFB1) on rat body weight and reproductive organs and the ameliorative effects of camel milk and silymarin through measured serum testosterone, testes pathology, and gene expression of tumor necrosis factor (TNF-α), luteinizing hormone receptor (LHR), and steroidogenic acute regulatory protein (StAR) in the testes. A total of sixty mature male Wister white rats, each weighing an average of 83.67 ± 0.21 g, were used. There were six groups created from the rats. Each division had ten rats. The groups were the control (without any treatment), CM (1 ml of camel milk/kg body weight orally), S (20 mg silymarin/kg b. wt. suspension, orally), A (1.4 mg aflatoxin/kg diet), ACM (aflatoxin plus camel milk), and AS (aflatoxin plus silymarin).ResultsThe results indicated the positive effects of camel milk and silymarin on growth, reproductive organs, and gene expression of TNF-α, LHR, and StAR with normal testicular architecture. Also, the negative effect of AFB1 on the rat’s body weight and reproductive organs, as indicated by low body weight and testosterone concentration, was confirmed by the results of histopathology and gene expression. However, these negative effects were ameliorated by the ingestion of camel milk and silymarin.ConclusionIn conclusion, camel milk and silymarin could mitigate the negative effect of AFB1 on rat body weight and reproductive organs.

GnRHr, LHr, and Vg gene expression levels and ovarian development of G5 transgenic mutiara female catfish (Clarias gariepinus) after exposure photoperiod induction

This study aimed to determine the relative expression ratios of the genes gonadotrophin-releasing hormone receptor (GnRHr), luteinizing hormone receptor (LHr), vitellogenin (Vg) and β-actin genes as expression control internal of the G5 fish using real-time PCR in a photoperiod experiment with designed treatments (A: 8L-16D; B: 12L-12D; C: 16L-8D for transgenic fish; and A*: 8L-16D; B*: 12L-12D; C*: 16L-8D for nontransgenic fish) for 60 days of rearing period. Ovary maturation was evaluated in G5 transgenic mutiara catfish during different photoperiod induction. A short photoperiod (8L-16D) induced an high expression of GnRHr, LHr, and Vg genes (mean, 4.42 ± 0.53, 5.63 ± 0.42, and 6.67 ± 0.31, respectively), indicating the role of dark cycle in increasing the gene expressions involved in ovarian maturation of G5 transgenic mutiara catfish. The lowest GnRHr, LHr, and Vg gene expression levels were found in nontransgenic fish (C*) (mean, 1.27 ± 0.13, 1.38 ± 0.24, and 2.42 ± 0.33, respectively). The exposure of transgenic fish (CgGH insert content) to a long photoperiod (16L-8D) resulted in lower expression levels of GnRHr, LHr, and Vg (mean, 2.31 ± 0.27, 2.34 ± 0.25, and 4.49 ± 0.30, respectively) and lower levels of hormones Vg and E2 (mean, 295.16 ± 21.71 μg/mL and 0.25 ± 0.03 ng/mL, respectively) and in non-transgenic fish (mean, 163.54 µg/mL and 0.14 ng/mL, respectively). Short photoperiods (8L-16D and 12l-12D) led to oocyte maturation and higher GSI values (mean, 12.24 ± 0.53 and 10.24 ± 0.38, respectively) compared to long photoperiods (16L-8D). Conversely, a long photoperiod led to decreased GnRHr, LHr, and Vg expression levels, and Vg and E2 hormone levels, leading to the growth of immature oocytes and decreased GSI (mean, 3.93 ± 0.29) in nontransgenic fish. The presence of CgGH in G5 transgenic mutiara female catfish can maintain the growth of primary oocytes to secondary oocytes during the 16L-8D photoperiod induction.

Molecular insights into the effects of tetrachlorobisphenol A on puberty initiation in Wistar rats

Tetrachlorobisphenol A (TCBPA) is the chlorinated derivative of bisphenol A (BPA). Several studies have found that BPA adversely affects the reproductive activity largely through binding to estrogen receptors and the critical period of BPA exposure advances the vaginal opening time in the female offspring via the kisspeptin/G protein-coupled receptor 54 (KGG) system. However, whether TCBPA can affect puberty initiation via KGG and the roles of estrogen receptors in this process remain unknown. Therefore, this study investigated the influence of TCBPA on the onset time of puberty in Wistar rats and the related molecular mechanisms by combing in vitro GT1–7 cells and molecular docking. In female Wistar rats, TCBPA at ≥100 mg/kg bw/day (49.2 μmol/L in rat body) markedly advanced vaginal opening time and increased serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and gonadotropin-releasing hormone (GnRH). It also increased the relative gene expression of LH receptor (LHR), GnRH1, and FSH receptor (FSHR) in hypothalamic-pituitary-gonadal (HPG) axis tissues. In GT1–7 cells, TCBPA increased genes and proteins associated with KGG pathway and activated the extracellular-regulated protein kinase 1/2 (Erk1/2) and phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) pathways via G protein-coupled estrogen membrane receptor 1 (GPER1) and estrogen receptor alpha (ERα). Docking analyses supported its interactions with GPER1 and ERα, and treatment with specific inhibitors of ERα- and GPER1-modulated PI3K/Akt and Erk1/2 signaling suppressed its effects. Taken together, TCBPA-induced advancement of puberty initiation in Wistar rats thus results primarily from increased LH, GnRH, and FSH secretion together with GnRH1, FSHR, and LHR upregulation driven by ERα- and GPER1-modulated Erk1/2 and PI3K/Akt signaling. Our results provide new molecular insights into the reproductive toxicity of EDCs.

Differential expression of FSHR and LHR genes and proteins during development of rabbit ovarian follicles.

The development of an ovarian follicle is a complex process at the cellular and molecular level that is mainly regulated by follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR). To elucidate the contribution of these receptors to ovarian follicle development, it is necessary to determine their expression profiles during this biological process. Therefore, this study aimed to investigate the relationship between ovarian development pattern and the differential ovarian expression pattern of FSHR and LHR genes as well as proteins at different developmental stages. Ovaries were collected from 30 New Zealand rabbits at day 0 (birth), week 2 (neonate), week 4 (cub), week 16 (maturity), and day 18 pregnancy. Ovarian histology, and gene as well as protein expression were determined using light microscopy, real-time PCR and western blotting, respectively. The results showed that the expression levels of FSHR mRNA and protein increased coincidently with age and the growth of ovarian follicles. The levels of LHR mRNA and protein remained low from the day of birth until week 4 and became significantly higher by week 16 coinciding with appearance of growing and antral follicles, which have a defined thecal layer. FSHR gene and protein expression decreased with pregnancy, whereas LHR increased, reaching a peak level during pregnancy. It can be concluded that changes in FSHR and LHR gene and protein expression could be related to the growth and development of follicles, indicating the regulatory role for these receptors in rabbit folliculogenesis.

Anti-Müllerian hormone, testosterone, and insulin-like peptide 3 as biomarkers of Sertoli and Leydig cell function during deslorelin-induced testicular downregulation in the dog

The role of anti-Müllerian hormone (AMH) and insulin-like peptide 3 (INSL3) in male infertility is not fully understood. We used the downregulated testis as a model of gonadotropin-dependent infertility. Serum testosterone and AMH concentrations were studied in five adult male Beagles implanted (day 0) with 4.7 mg deslorelin (Suprelorin®, Virbac) (DES group). Testicular expression of LH receptor (LHR) and androgen receptor (AR), AMH, type 2 AMH receptor (AMHR2), INSL3 and its receptor (RXFP2) was evaluated 112 days (16 weeks) after deslorelin treatment by qPCR and immunohistochemistry, and compared to untreated adult (CON, n = 6) and prepubertal (PRE, n = 8) dogs. Serum testosterone concentration decreased significantly by the onset of aspermia on study day 14 (four dogs) or day 21 (one dog), and was baseline on day 105 (week 15). In contrast, serum AMH started to increase only after the onset of aspermia and reached the maximum detectable concentration of the assay by day 49-105 in individual dogs. Testicular LHR gene expression in DES was lower than in CON and PRE (P < 0.0001), while AR gene expression in DES was similar to CON and significantly higher than PRE (P < 0.0001). Testicular AMH expression in DES was intermediate compared to the lowest mRNA levels found in CON and the highest in PRE (P ≤ 0.006). AMHR2 gene expression was similar between groups. AMH protein was detected in Sertoli cells only, while AMHR2 immunoreactivity was principally detected in Leydig cells which appeared to be increased in DES. INSL3 and RXFP2 gene expression was significantly downregulated in the DES testis along with noticeably weak Leydig cell immunosignals compared to CON. In conclusion, deslorelin treatment caused testicular LH insensitivity without affecting androgen sensitivity, and de-differentiation of Sertoli and Leydig cells. In DES, upregulation of the AMH-AMHR2 feed-back loop and downregulation of the INSL3-RXFP2 feed-forward loop are paracrine-autocrine mechanisms that may additionally regulate testosterone production independent of gonadotropins. Our results support AMH and INSL3 as unique biomarkers and paracrine-autocrine regulators of testis function involved in the intimate interplay between Sertoli and Leydig cells.

Correlation between luteinizing hormone receptor gene expression in human granulosa cells with oocyte quality in poor responder patients undergoing in vitro fertilization: A cross-sectional study

Background: This study was performed to evaluate the role of luteinizing hormone (LH) and granulosa cell LH receptor (LH-R) in poor responder patients who underwent controlled ovarian stimulation. Expression levels of LH-R mRNA in granulosa cells was investigated and compared with oocyte morphology, oocyte maturity and fertilization rate. Methods: Granulosa cells were obtained from 30 patients who underwent in vitro fertilization (IVF) at Dr. Cipto Mangunkusumo Hospital, Jakarta. The patients were divided into two groups: group I (n=10) poor responders; and group II (n=20) non-poor responders. After the extraction of total RNA from granulosa cells, semi-quantitative RT-PCR was performed and the amount of LH-R mRNA was quantified. The relative values were calculated as the ratio of LH-R mRNA and actin beta mRNA. Statistical analysis was performed using Mann-Whitney test and Spearman correlation. Results: The relative value of LH-R mRNA was higher in group I compared with group II (27.37[0.00-28939.37] vs 0.00[0.00-7196.12]). Oocyte maturity (r=0.267) and morphology (r=0.267) in group I consistently showed a positive correlation with LH-R mRNA; in group II a negative correlation with LH-R mRNA was shown for oocyte maturity (r= -0.552) and morphology (r= -0.164). Group I had a positive correlation between LH-R expression with fertilization rate (r=0.430), and group II showed a negative correlation (r=-0.340). Conclusions: The expression of LH-R mRNA has a positive correlation with oocyte quality in poor responder patients and a negative correlation in non-poor responders. Our study suggests an optimal expression of LH- R mRNA in granulosa cells during controlled ovarian stimulation to obtain good quality oocytes.

Correlation between luteinizing hormone receptor gene expression in human granulosa cells with oocyte quality in poor responder patients undergoing in vitro fertilization: A cross-sectional study.

Background: This study was performed to evaluate the role of luteinizing hormone (LH) and granulosa cell LH receptor (LH-R) in poor responder patients who underwent controlled ovarian stimulation. Expression levels of LH-R mRNA in granulosa cells was investigated and compared with oocyte morphology, oocyte maturity and fertilization rate. Methods: Granulosa cells were obtained from 30 patients who underwent in vitro fertilization (IVF) at Dr. Cipto Mangunkusumo Hospital, Jakarta. The patients were divided into two groups: group I (n=10) poor responders; and group II (n=20) non-poor responders. After the extraction of total RNA from granulosa cells, semi-quantitative RT-PCR was performed and the amount of LH-R mRNA was quantified. The relative values were calculated as the ratio of LH-R mRNA and actin beta mRNA. Statistical analysis was performed using Mann-Whitney test and Spearman correlation. Results: The relative value of LH-R mRNA was higher in group I compared with group II (27.37[0.00-28939.37] vs 0.00[0.00-7196.12]). Oocyte maturity (r=0.267) and morphology (r=0.267) in group I consistently showed a positive correlation with LH-R mRNA; in group II a negative correlation with LH-R mRNA was shown for oocyte maturity (r= -0.552) and morphology (r= -0.164). Group I had a positive correlation between LH-R expression with fertilization rate (r=0.430), and group II showed a negative correlation (r=-0.340). Conclusions: The expression of LH-R mRNA has a positive correlation with oocyte quality in poor responder patients and a negative correlation in non-poor responders. Our study suggests an optimal expression of LH- R mRNA in granulosa cells during controlled ovarian stimulation to obtain good quality oocytes.

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.

Effects of Combined Estradiol-Sulpiride Treatment and Follicle Ablation on Vernal Transition in Mares: Evaluation of Plasma and Follicular Fluid Hormones and Luteinizing Hormone Receptor Gene Expression

This experiment assessed the hormonal production, secretory aspects, and changes in luteinizing hormone (LH) receptor gene expression of early induced ovulatory-sized follicles relative to the first ovulatory-sized follicles occurring naturally in the spring. Anovulatory mares were treated on January 21 with (1) 50 mg of estradiol cypionate (ECP, n = 8) alone or (2) with ECP followed by two 3-g sulpiride injections (n = 8), 5 and 12 days later. Half of each group also received complete follicle ablation via transvaginal aspiration before ECP treatment. Ovaries were scanned regularly until detection of a 32–35 mm follicle; follicular fluid was recovered via aspiration for analysis of hormonal concentrations. Blood was collected regularly to characterize plasma prolactin, LH, follicle stimulating hormone, progesterone, and estradiol concentrations. Mean date to first 35-mm follicle was earlier (P < .05) in sulpiride-treated mares: five of eight (63%) responded within 28 days of the first sulpiride injection. Ablation did not affect ovarian response. Plasma prolactin was stimulated (P < .0001) in ECP-sulpiride–treated mares for 16 days but did not dictate ovarian response. Estradiol stimulated plasma LH (P < .05), which was higher (P < .05) in treated mares that responded. There was no effect of treatment or ablation on follicular fluid concentrations of estradiol, progesterone, leptin, or insulin-like growth factor 1 or on LH receptor gene expression. These latter similarities indicate that ECP-sulpiride early induced follicles have apparently reached a degree of maturity equivalent to naturally occurring ovulatory-sized follicles later in the spring.

Luteinizing Hormone Receptor Gene and Regulator of G-protein Signaling 2 Gene Expression Level and Association with Oocyte Maturity in In vitro Fertilization/Intracytoplasmic Sperm Injection Cycle.

Aims:The aim is to study the relation and distribution in gene expression level of the luteinizing hormone receptor (LHR) gene and regulator of G-protein signaling 2 (RGS2) gene expression with oocyte maturation.Setting and Design:This cross-sectional study was undertaken in an instruction-based tertiary care infertility unit, department of obstetrics and gynecology.Materials and Methods:After controlled ovarian hyperstimulation, cumulus granulosa cells (CCs) from 59 oocytes among 18 women being treated by in vitro fertilization/intracytoplasmic sperm injection cycle technique from November 2015 to January 2016 were collected on the day of oocyte retrieval. Total RNA was extracted and converted to cDNA in individual oocytes. LHR and RGS2 gene levels were measured and analyzed using digital droplet polymerase chain reaction.Statistical Analysis:Gene expression level was analyzed using software STATA, version 14.0 (College Station, TX: StataCorp LP, USA).Results:CCs were obtained from 59 cumulus-oocyte complexes (COC), 46 COC from metaphase II (CCMII), 13 COC from metaphase I, and GV oocyte (CCMI + GV). The RGS2 gene expression level, when compared with the housekeeping gene in CCMII and CCMI + GV, was 0.15 (0.05–0.52) and 0.08 (0.02–0.27), respectively. The LHR gene expression when compared with the housekeeping gene in CCMII and CCMI + GV did not differ and was quite in the same value that was 0.02 (0.00–0.11) and 0.02 (0.00–0.06), respectively.Conclusion:This study showed that LHR gene expression did not differ in between oocyte groups. Even though the median of RGS2 gene expression was more in the mature oocyte group, the result was inconclusive due to scattering and overlapping of gene expression data between oocyte groups.

The expression of several reproductive hormone receptors can be modified by perfluorooctane sulfonate (PFOS) in adult male rats

This study was undertaken to evaluate the possible role of several reproductive hormone receptors on the disruption of the hypothalamic-pituitary-testis (HPT) axis activity induced by perfluorooctane sulfonate (PFOS). The studied receptors are the gonadotropin-releasing hormone receptor (GnRHr), luteinizing hormone receptor (LHr), follicle-stimulating hormone receptor (FSHr), and the androgen receptor (Ar). Adult male rats were orally treated with 1.0; 3.0 and 6.0 mg of PFOS kg−1 d−1 for 28 days. In general terms, PFOS can modify the relative gene and protein expressions of these receptors in several tissues of the reproductive axis. At the testicular level, apart from the expected inhibition of both gene and protein expressions of FSHr and Ar, PFOS also stimulates the GnRHr protein and the LHr gene expression. The receptors of the main hormones involved in the HPT axis may have an important role in the disruption exerted by PFOS on this axis.

Removal of spermatogonial stem cells (SSCs) from in vitro culture modulates testosterone synthesis in bovine

Niche cells, encompassing spermatogonial stem cells (SSCs) and controlling their development are believed to have a reciprocal communication with SSCs. The present study was conducted to evaluate the impact of the elimination of SSCs on testosterone production and genes contributing to testosterone synthesis. Following isolation, bovine testicular cells were cultured for 12 days on extracellular matrix-coated plates. In the germ cell-removed group, the SSCs were removed from the in vitro culture using differential plating; however, in the control group, no intervention in the culture was performed. Testosterone concentration and the gene expression of luteinizing hormone (LH) receptor (LHR), CYP17A1, and steroidogenic acute regulatory protein (StAR), were assessed on days 6 and 12. The concentration of testosterone was lower in the germ cell-removed than control group (P < 0.05). Moreover, quantitative real-time PCR revealed lower gene expression of LHR, CYP17A1, and StAR in the germ cell-removed group as compared with the control group (P < 0.05). In conclusion, the present study showed that elimination of SSCs from in vitro culture could diminish testosterone production via downregulation of factors contributing to testosterone synthesis. Additionally, this study provided evidence for the reciprocal relationship between SSCs and their niche cells.

Effect of the FSH receptor single nucleotide polymorphisms (FSHR 307/680) on the follicular fluid hormone profile and the granulosa cell gene expression in human small antral follicles.

The most pronounced effects of FSH signalling are potentially displayed in the follicle fluid, which acts as a reservoir for FSH-induced granulosa cell (GC) secreted hormones. This study investigates the effects of two common polymorphisms of FSHR, FSHR 307 (rs6165) and FSHR 680 (rs6166), by evaluating the hormone and gene expression profiles of human small antral follicles collected under physiological conditions in connection with fertility preservation. In total 69 women at various time during the menstrual cycle were included in this study. The intrafollicular hormone content of 179 follicular fluid samples and the gene expression levels of 85 GC samples were correlated to the genotype of both FSHR polymorphisms. The following parameters were evaluated: follicle diameter, levels of Anti-Müllerian hormone (AMH), progesterone, estradiol, testosterone and androstenedione and gene expression levels of FSHR, luteinizing hormone receptor (LHR), androgen receptor, aromatase cytochrome p450 (CYP19A1), AMH and AMH receptor II (AMHR2). There was 100% concordance between the FSHR 307 and the FSHR 680 genotypes: A/A (p.307Thr/Thr and p.680Asn/Asn), A/G (p.307Thr/Ala and p.680Asn/Ser) and G/G (p.307Ala/Ala and p.680Ser/Ser). Considering all follicles, compared with the other genotypes the G/G genotype was associated with significantly elevated gene expression levels for LHR, while AMHR2 gene expression levels were significantly reduced. In follicles 3-6 mm in diameter LHR gene expression was significantly increased, whereas AMH gene expression was significantly reduced for the G/G genotype. In follicles >6 mm, estradiol and CYP19A1 gene expression levels were significantly higher for the G/G genotype. In conclusion, significant changes were observed between the FSHR 307/680 polymorphisms in human small antral follicles collected under physiological FSH conditions.

Effects of luteinizing hormone receptor signaling in prostate cancer cells.

The importance of androgen signaling in prostate cancer (PC) is well described and prostate cancer cells retain the ability to directly synthesize androgens. Luteinizing hormone (LH) can induce expression of steroidogenic enzymes and trigger androgen production, but the regulation of this process is not well-described. Here, we explored the impact of silencing LH receptor (LHR) silencing on androgen synthesis and on several relevant signaling pathways in PC. LHR mRNA and protein expression was evaluated in LNCaP PC cells treated with LHR-siRNA. MTS assay was used to measure the effect of LHR-siRNA on proliferation in LNCaP and 22RV1 PC cells. Treated LNCaP and LAPC-3 cells were also assayed for differences in androgen synthesis and expression of steroidogenic enzymes, PSA, AR, and critical signaling molecules including PKA, ERK1/2, PI3K, AKT2, and HER2. We confirmed that functional LHR is expressed in both androgen-sensitive and castrate-resistant PC specimens. Treatment with LHR-siRNA effectively silenced LHR gene and protein expression and prevented LH-mediated proliferation and androgen synthesis in prostate cancer cells. LHR silencing also downregulated expression of AR, PSA, PKA, ERK1/2, PI3K, AKT2, and HER2. Collectively, these data demonstrate that silencing LHR expression suppresses androgen synthesis and signaling and the LH-LHR pathway may represent a viable therapeutic strategy in PC.

Gene expression of matrix metalloproteinases and LH receptors in mare follicular development

The period from the emergence of a dominant follicle until its formation requires tissue remodeling. Enzymes promoting collagen lysis, such as matrix metalloproteinases (MMPs), are fundamental for the process of extracellular matrix remodeling, which allows changes in ovarian tissue architecture during follicular growth. It has been suggested that the production of these enzymes may be affected by the rise in circulating concentrations of LH, which acts on the ovarian surface epithelium (OSE). The aim of this study was to determine the expression of MMP-1, MMP-2, and LH receptor (LHR) in the ovulation fossa and in the central portion of the equine ovary during follicular deviation and dominance. Ovaries of 12 cyclic mares were selected and subsequently divided into two groups: development (DEV) group and dominant (DOM) group. The DEV group consisted of ovaries from six animals whose follicles were less than 28 mm in diameter (follicular deviation), and the DOM group consisted of ovaries from six animals whose follicles measured 28 mm or more in diameter (dominant follicles). The latter group was divided into two subgroups: the group of ovaries with a dominant follicle (DOM-D) and the group of contralateral ovaries (DOM-C). Our results showed that mRNA for MMP-1, MMP-2, and LHR was present in the equine ovary during follicle development, in the ovulation fossa, and in the central portion of the ovary. MMP-1 and LHR gene expression was greater (P < 0.05) for the DOM-D group compared with the DOM-C group. In the DOM-D group, MMP-1, MMP-2, and LHR gene expression was greater (P < 0.05) in the ovarian stroma compared with the ovulation fossa. Using immunohistochemistry, OSE from the DOM group showed increased expression compared with the DEV group (P < 0.05). In conclusion, we demonstrated that MMP-1 and MMP-2 might be fundamental for events related to tissue remodeling, which occurs during follicular development until the formation of the dominant follicle. We also demonstrated the relationship between the gene expression of MMPs and the gene and protein expression of LHR, suggesting that LHR in the OSE might be an important factor to initiate the signaling cascade that culminates with the production of MMPs.

LH-Receptor Gene Expression in Human Granulosa and Cumulus Cells from Antral and Preovulatory Follicles

Human granulosa cells (GC) acquire LH receptor (LHR) expression during the follicular phase of the menstrual cycle. Currently, the precise follicular stage is unknown, and specific roles of LH in the follicular development are not fully understood. Our objective was to measure LHR gene expression on GC and cumulus cells (CC) from normal human follicles with diameters form 3-20 mm. At a university hospital, GC, CC, and the corresponding follicular fluid (FF) were collected from patients undergoing fertility preservation by having one ovary frozen and patients undergoing infertility treatment. Cells and fluids were isolated from surgically excised ovaries or from aspirated preovulatory follicles. We evaluated gene expression of LHR, FSHR, androgen receptor (AR), aromatase (CYP19a1), and AMHR2 normalized to the GAPDH expression and associated with FF levels of anti-Mullerian hormone, inhibin-B, and steroids. LHR expression was maximal in GC from preovulatory follicles before ovulation induction. A majority of 150 antral follicles (3-10 mm in diameter) showed LHR expression at approximately 10% of the maximum, and LHR expression showed significant associations with FSHR, AR, CYP19a1, and AMHR2 and with FF estradiol and progesterone. Levels of FSHR continued to decline in GC as the follicular diameter increased. The LHR gene is expressed in GC of human antral follicles throughout the follicular phase and is significantly associated with expression of the CYP19a1 gene and with the corresponding FF concentrations of estradiol and progesterone. LH appears to affect human follicular development during most the follicular phase in normal women.

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.

Bone Morphogenetic Protein-2 (BMP-2) Increases Gene Expression of FSH Receptor and Aromatase and Decreases Gene Expression of LH Receptor and StAR in Human Granulosa Cells

A growing body of evidence indicates that bone morphogenetic protein (BMP) cytokines play a key role in female fertility in mammals. BMP-2 is known to be expressed in the ovary of many species. In the present study, we examined the expression and function of BMP-2 in the human ovary. BMP-2 mRNA expression in the human ovary was evaluated by in situ hybridization. Human granulosa cells were obtained from in vitro fertilization patients. Human granulosa cells were cultured with recombinant BMP-2 or human chorionic gonadotrophin (HCG), followed by RNA extraction. BMP-2 expression was detected in granulosa cells of antral follicles but not of corpus luteum. The in vitro study showed that BMP-2 induced follicular stimulating hormone (FSH) receptor and aromatase expression, while decreasing luteinizing hormone (LH) receptor and steroidogenic acute regulatory protein expression in human granulosa cells. HCG decreased gene expression of BMP-2 and increased BMP and activin membrane-bound inhibitor (BAMBI), an antagonist of BMP-2. Expression and disappearance of BMP-2 might contribute to folliculogenesis and luteinization by regulating gonadotropin receptor expression in human granulosa cells. HCG can modulate BMP-2 function by controlling BMP-2 and BAMBI expression.

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.