Effect of Electroacupuncture on Reproductive Disorders and Insulin Resistance in a Murine Polycystic Ovary Syndrome Model

Polycystic ovarian syndrome (PCOS) is a common, complex, and heterogeneous endocrine and metabolic disorder. There is no standardized treatment, and it therefore requires individualized therapies according to the symptoms and pathogenesis of each patient. The present study aimed to determine the effect of electroacupuncture at the acupoints Zusanli (ST36), Sanyinjiao (SP6), and Neiguan (PC6) on reproductive disorders and insulin resistance in a murine model of PCOS induced by dehydroepiandrosterone (DHEA). Vaginal smear analysis was used to determine mice estrous cycle; intraperitoneal glucose and insulin tolerance tests were adopted to analyze metabolic characteristics; enzyme-linked immunosorbent assay was used to measure hormone levels; gene expression was quantified with real-time PCR; hematoxylin and eosin staining was used to observe ovarian morphology. We observed disordered estrous cycle, polycystic ovarian morphology, and higher levels of homeostasis model assessment-insulin resistance (HOMA-IR) and testosterone (T), indicating successful modeling of PCOS. DHEA increased levels of estrogen (E2), progesterone (P), testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH), and EA treatment restored them to levels seen in the control group. EA reduced the days in estrus caused by DHEA, improved the abnormal sex hormone receptor genes, and attenuated the DHEA-induced histomorphological changes in mouse ovaries. The average expressions of the androgen receptor (AR), estrogen receptor (ER), luteinizing hormone receptor (LHR), and follicle-stimulating hormone receptor (FSHR) genes in the ovary greatly increased after DHEA treatment and significantly decreased in the DHEA + EA group. After EA treatment, the cystic follicle (CF) number was reduced and corpora lutea (CL) increased in the DHEA + EA group compared to the DHEA group. EA improved glucose intolerance and insulin intolerance. Statistical analysis of intraperitoneal glucose tolerance test-area under curve (IPGTT-AUC) glucose levels revealed a significant decrease in DHEA group mice compared to the control and DHEA + EA groups. EA was found to restore fasting blood glucose, fasting serum insulin, and HOMA-IR. In summary, our study suggests that EA has a remarkable effect in the DHEA-induced murine PCOS model. Management of EA could improve estrous cycle, hormonal disorders, abnormal sex hormone receptors in ovaries, ovary morphology, and insulin resistance in PCOS mice.

The Effects of Separate and Combined Treatment of Male Rats with Type 2 Diabetes with Metformin and Orthosteric and Allosteric Agonists of Luteinizing Hormone Receptor on Steroidogenesis and Spermatogenesis

In men with type 2 diabetes mellitus (T2DM), steroidogenesis and spermatogenesis are impaired. Metformin and the agonists of luteinizing hormone/human chorionic gonadotropin(hCG)-receptor (LH/hCG-R) (hCG, low-molecular-weight allosteric LH/hCG-R-agonists) can be used to restore them. The aim was to study effectiveness of separate and combined administration of metformin, hCG and 5-amino-N-tert-butyl-2-(methylsulfanyl)-4-(3-(nicotinamido)phenyl)thieno[2,3-d]pyrimidine-6-carboxamide (TP3) on steroidogenesis and spermatogenesis in male rats with T2DM. hCG (15 IU/rat/day) and TP3 (15 mg/kg/day) were injected in the last five days of five-week metformin treatment (120 mg/kg/day). Metformin improved testicular steroidogenesis and spermatogenesis and restored LH/hCG-R-expression. Compared to control, in T2DM, hCG stimulated steroidogenesis and StAR-gene expression less effectively and, after five-day administration, reduced LH/hCG-R-expression, while TP3 effects changed weaker. In co-administration of metformin and LH/hCG-R-agonists, on the first day, stimulating effects of LH/hCG-R-agonists on testosterone levels and hCG-stimulated expression of StAR- and CYP17A1-genes were increased, but on the 3–5th day, they disappeared. This was due to reduced LH/hCG-R-gene expression and increased aromatase-catalyzed estradiol production. With co-administration, LH/hCG-R-agonists did not contribute to improving spermatogenesis, induced by metformin. Thus, in T2DM, metformin and LH/hCG-R-agonists restore steroidogenesis and spermatogenesis, with metformin being more effective in restoring spermatogenesis, and their co-administration improves LH/hCG-R-agonist-stimulating testicular steroidogenesis in acute but not chronic administration.

Autoimmunity to the Follicle-Stimulating Hormone Receptor (FSHR) and Luteinizing Hormone Receptor (LHR) in Polycystic Ovarian Syndrome

Hyperandrogenemia and ovulatory dysfunction are hallmarks of polycystic ovary syndrome (PCOS), pointing to a deranged hypothalamus-pituitary-ovarian (HPO) axis. An autoimmune etiology of PCOS is suspected in a subset of patients due to the relatively high concordance of PCOS with common autoimmune diseases. For this reason, we tested the hypothesis that natural autoantibodies (aAb) to the follicle-stimulating hormone receptor (FSHR) or luteinizing hormone receptor (LHR) are prevalent in PCOS. To this end, new luminometric assays for quantifying aAb to the FSHR (FSHR-aAb) or LHR (LHR-aAb) were developed using full-length recombinant human receptors as fusion proteins with luciferase as reporter. Prevalence of FSHR-aAb and LHR-aAb was determined in serum samples from healthy controls and PCOS patients. Steroid hormone profiles were compared between patients with and without FSHR-aAb or LHR-aAb. Signal linearity and detection ranges were characterized and both methods passed basic performance quality checks. The analysis revealed a relatively low prevalence, with 4 out of 430 samples positive for FSHR-aAb in the control versus 11 out of 550 samples in the PCOS group, i.e., 0.9% versus 2.0%, respectively. Similarly, there were only 5 samples positive for LHR-aAb in the control versus 2 samples in the PCOS group, i.e., 1.2% versus 0.4%, respectively. Samples positive for FSHR-aAb displayed steroid hormones in the typical range of PCOS patients, whereas the two samples positive for LHR-aAb showed relatively elevated free testosterone in relation to total testosterone concentrations with unclear significance. We conclude that the FSHR and LHR constitute potential autoantigens in human subjects. However, the prevalence of specific autoantibodies to these receptors is relatively low, both in control subjects and in women with PCOS. It is therefore unlikely that autoimmunity to the LHR or FSHR constitutes a frequent cause of hyperandrogenemia or ovulatory dysfunction in PCOS.

MeCP2 duplication causes hyperandrogenism by upregulating LHCGR and downregulating RORα

Duplication of MECP2 (methyl-CpG-binding protein 2) gene causes a serious neurological and developmental disorder called MECP2 duplication syndrome (MDS), which is usually found in males. A previous clinical study reported that MDS patient has precocious puberty with hyperandrogenism, suggesting increased MeCP2 may cause male hyperandrogenism. Here we use an MDS mouse model and confirm that MECP2 duplication significantly upregulates androgen levels. We show for the first time that MeCP2 is highly expressed in the Leydig cells of testis, where androgen is synthesized. Mechanistically, MECP2 duplication increases androgen synthesis and decreases androgen to estrogen conversion through either the upregulation of luteinizing hormone receptor (LHCGR) in testis, as a result of MeCP2 binds to G-quadruplex structure of Lhcgr promoter and recruits the transcription activator CREB1 or the downregulation of the expression of aromatase in testis by binding the CpG island of Rorα, an upstream regulator of aromatase. Taken together, we demonstrate that MeCP2 plays an important role in androgen synthesis, supporting a novel non-CNS function of MeCP2 in the process of sex hormone synthesis.

The Effects of Zearalenone on the Localization and Expression of Reproductive Hormones in the Ovaries of Weaned Gilts

This study aims to investigate the effects of zearalenone (ZEA) on the localizations and expressions of follicle stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), gonadotropin releasing hormone (GnRH) and gonadotropin releasing hormone receptor (GnRHR) in the ovaries of weaned gilts. Twenty 42-day-old weaned gilts were randomly allocated into two groups, and treated with a control diet and a ZEA-contaminated diet (ZEA 1.04 mg/kg), respectively. After 7-day adjustment, gilts were fed individually for 35 days and euthanized for blood and ovarian samples collection before morning feeding on the 36th day. Serum hormones of E2, PRG, FSH, LH and GnRH were determined using radioimmunoassay kits. The ovaries were collected for relative mRNA and protein expression, and immunohistochemical analysis of FSHR, LHR, GnRH and GnRHR. The results revealed that ZEA exposure significantly increased the final vulva area (p < 0.05), significantly elevated the serum concentrations of estradiol, follicle stimulating hormone and GnRH (p < 0.05), and markedly up-regulated the mRNA and protein expressions of FSHR, LHR, GnRH and GnRHR (p < 0.05). Besides, the results of immunohistochemistry showed that the immunoreactive substances of ovarian FSHR, LHR, GnRH and GnRHR in the gilts fed the ZEA-contaminated diet were stronger than the gilts fed the control diet. Our findings indicated that dietary ZEA (1.04 mg/kg) could cause follicular proliferation by interfering with the localization and expression of FSHR, LHR, GnRH and GnRHR, and then affect the follicular development of weaned gilts.

Bee bread mitigates down-regulation of steroidogenic genes, decreased spermatogenesis, and epididymal oxidative stress in male rats fed with high-fat diet

Background: The pituitary-gonadal axis plays an important role in steroidogenesis and spermatogenesis and by extension, fertility. Objectives: The aim of this study was to investigate the protective role of bee bread, which is a natural bee product, against obesity-induced decreases in steroidogenesis and spermatogenesis. Methods: Thirty-two adult male Sprague-Dawley rats weighing between 200-300 g were divided into four groups (n=8/group), namely: normal control (NC), high-fat diet (HFD), HFD plus bee bread administered concurrently for 12 weeks (HFD+B), HFD plus orlistat administered concurrently for 12 weeks (HFD+O) groups. Bee bread (0.5 g/kg) or Orlistat (10 mg/kg/day) was suspended in distilled water and given by oral gavage daily for 12 weeks. Results: Levels of follicle stimulating hormone, luteinizing hormone testosterone, and adiponectin, as well as sperm count, motility, viability, normal morphology and epididymal antioxidants decreased whereas levels of leptin and malondialdehyde, and sperm nDNA fragmentation increased significantly in HFD group relative to NC group. There were significant decreases in the testicular mRNA transcript and protein levels of androgen receptor, luteinizing hormone receptor, steroidogenic acute regulatory protein, cytochrome P450 enzyme, 3β-hydroxysteroid dehydrogenase (HSD) and 17β-HSD in the testes of the HFD group. Further, mount latency, mount frequency, intromission, ejaculation, penile iNOS and cGMP levels deceased significantly in the HFD group. Supplementation with bee bread significantly reduced leptin level and increased adiponectin level, enhanced sperm parameters and reduced sperm nDNA fragmentation, upregulated the levels of steroidogenic genes and proteins in HFD-induced obese male rats. Discussion and Conclusion: Bee bread improved spermatogenesis and steroidogenesis by upregulating steroidogenic genes. Therefore, bee bread may be considered as a potential supplementation to protect against infertility in overweight or obese men.

Expression and Purification of the Extracellular Domain of Luteinizing Hormone Receptor From Ovis Aries Testicle

The luteinizing hormone receptor (LHR) is a glycoprotein member of the G protein-coupled receptor superfamily. Physiologically, this receptor participates in corpus luteum formation and ovulation in females. In males, it acts in testosterone synthesis and spermatogenesis and is involved in some fertility disorders. RNA was extracted from Ovis aries testicles, and the corresponding cDNA was synthesized to amplify the lhr gene, termed lhr-bed here, consisting of 762 bp that encodes 273 amino acids of the extracellular domain of LHR. Thus, the lhr-bed was cloned into pJET1.2/blunt, subcloned into the pCOLD II expression vector and finally transformed into E. coli BL21 cells. Since the induced rLHR-Bed protein was found in the insoluble fraction, the purification protocol was modified as follows: induction at 25°C, denaturing conditions (8 M UREA and 0.1% CHAPS) and refolding in the column to increase solubility. The rLHR-Bed expression was corroborated by western blotting and mass spectrometry (MS) analysis. This successful method to obtain the recombinant LHR extracellular domain yields 0.2 mg/L of the protein with approximately 90% purity from a single chromatographic purification step. The present approach demonstrates the feasibility of obtaining large quantities of rLHR-Bed. This might be useful to accomplish future studies regarding the structure and functional analysis of the binding interplay with its ligand luteinizing hormone and its isoforms. Additionally, this biotechnological strategy might be used to improve and to develop new drugs for the treatment of reproductive disorders and might also be applied to species reproduction in the livestock industry.