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The heavy metal cadmium is proposed to be one of the environmental endocrine disruptors of spermatogenesis. Cadmium-induced inhibition of spermatogenesis is associated with a hormone secretion disorder. Letrozole is an aromatase inhibitor that increases peripheral androgen levels and stimulates spermatogenesis. However, the potential protective effects of letrozole on cadmium-induced reproductive toxicity remain to be elucidated. In this study, male mice were administered CdCl 2 (4 mg/kg BW) orally by gavage alone or in combination with letrozole (0.25 mg/kg BW) for 30 days. Cd exposure caused a significant decreases in body weight, sperm count, motility, vitality, and plasma testosterone levels. Histopathological changes revealed extensive vacuolization and decreased spermatozoa in the lumen. However, in the Cd + letrozole group, letrozole treatment compensated for deficits in sperm parameters (count, motility, and vitality) induced by Cd. Letrozole treatment significantly increased serum testosterone levels, which were reduced by Cd. Histopathological studies revealed a systematic array of all germ cells, a preserved basement membrane and relatively less vacuolization. For a mechanistic examination, RNA-seq was used to profile alterations in gene expression in response to letrozole. Compared with that in the Cd-treated group, RNA-Seq analysis showed that 214 genes were differentially expressed in the presence of letrozole. Gene ontology (GO) enrichment analysis and KEGG signaling pathway analysis showed that steroid biosynthetic processes were the processes most affected by letrozole treatment. Furthermore, we found that the expression of the testosterone synthesis-related genes LHCGR (luteinizing hormone/choriogonadotropin receptor) and Hsd3b6 (3 beta- and steroid delta-isomerase 6) was significantly downregulated in Cd‐treated testes, but these genes maintained similar expression levels in letrozole-treated testes as those in the control group. However, the transcription levels of inflammatory cytokines, such as IL-1β and IL-6, and oxidative stress-related genes (Nrf2, Nqo1, and Ho-1) showed no changes. The present study suggests that the potential protective effect of letrozole on Cd-induced reproductive toxicity might be mediated by the upregulation of LHCGR and Hsd3b6, which would beneficially increase testosterone synthesis to achieve optimum protection of sperm quality and spermatogenesis.

The pharmaceutical 17α-ethynylestradiol (EE2) is considered as an endocrine-disrupting chemical that interferes with male reproduction and hormonal activation. In this study, we investigated the molecular mechanism underlying EE2-regulatory testosterone release in vitro and in vivo . The results show that EE2 treatment decreased testosterone release from rat Leydig cells. Treatment of rats with EE2 reduced plasma testosterone levels and decreased the sensitivity of human chorionic gonadotropin (hCG). EE2 reduced luteinizing hormone receptor (LHR) expression associated with decreased cAMP generation by downregulation of adenylyl cyclase activity and decreased intracellular calcium-mediated pathways. The expression levels of StAR and P450scc were decreased in Leydig cells by treatment of rats with EE2 for 7 days. The sperm motility in the vas deferens and epididymis was reduced, but the histopathological features of the testis and the total sperm number of the vas deferens were not affected. Moreover, the serum dihydrotestosterone (DHT) level was decreased by treatment with EE2. The prostate gland and seminal vesicle atrophied significantly, and their expression level of 5α-reductase type II was reduced after EE2 exposure. Taken together, these results demonstrate an underlying mechanism of EE2 to downregulate testosterone production in Leydig cells, explaining the damaging effects of EE2 on male reproduction.

GnRH sterilization vaccines have been developed for various practical and clinical reasons. However, conjugation of GnRH peptide to carrier protein has many drawbacks, hampering the further commercialization of GnRH vaccines. In this study, a new nonconjugated GnRH vaccine, D-Lys6-GnRH-tandem-dimer peptide （TDK）, emulsified in Specol adjuvant was investigated for its immunocastration efficacy in young male rats. Prepubertal male rats were randomly allocated into three groups （n = 12）： control （no treatment）, surgically castrated or immunized against 100 μg TDK in Specol adjuvant at 6 weeks of age （with a booster 8 weeks later）. Blood samples （for antibody titers and hormone concentrations） were collected at 2-week intervals until rats were killed （18 weeks of age）. Compared to intact controls, active immunization against TDK reduced （P〈 0.05） serum concentrations of testosterone, inhibin B, LH and FSH, prevented the onset of spermatogenesis at puberty. Furthermore, mRNA expressions of GnRH receptor, LH-β and FSH-β in the pituitary, LH receptor, FSH receptor, inhibin α, βA and βB subunit in the testes were decreased in immunocastrated rats compared to intact controls （P 〈 0.05）. These results demonstrate for the first time that GnRH-tandem-dimer peptide emulsified in Specol is a promising veterinary sterilization medicine.

We recently showed that prenatal exposure to diesel exhaust (DE) disrupts spermatogenesis in mouse offspring. This study was undertaken to determine whether filtered DE in which 99.97% of diesel exhaust particles >0.3 μm in diameter were removed affects spermatogenesis in growing mice. After prenatal exposure to filtered DE for 2-16 days postcoitum, we examined daily sperm production (DSP), testicular histology, serum testosterone levels and mRNA expression of hormone synthesis process-related factors. In the filtered DE exposed group, DSP was markedly reduced at 12 weeks compared with the control group; clean air exposed group. Histological examination showed multinucleated giant cells and partial vacuolation in the seminiferous tubules of the exposed group. Testosterone was elevated significantly at 5 weeks. Moreover, luteinizing hormone receptor mRNA at 5 and 12 weeks, 17α-hydroxylase/C17-20-lyase and 17β-hydroxysteroid dehydrogenase mRNAs at 12 weeks were significantly elevated. These results suggest that filtered DE retains its toxic effects on the male reproductive system following prenatal exposure.

It has long been known that endometrial regeneration and proliferation is regulated by sex steroids, cytokines and various growth factors. The mechanisms responsible for such organized growth are still under investigation. Human chorionic gonadotropin/luteinizing hormone (hCG/LH) receptors have been found to be localized in human endometrium by immunocytochemistry. Gonadotropins have been widely used for hyperstimulation during in vitro fertilization (IVF) procedures; however, the direct effect of gonadotropin on the endometrium has not been adequately investigated yet. This study attempted to define the effect of gonadotropins on the proliferation of human endometrial stromal cells in vitro. Human endometrial stromal cells were obtained from hysterectomy specimens and cultured in serum-containing media for up to 72 h. The effects of adding 7.5, 15, 30, 150 mIU/ml of human menopausal gonadotropin (hMG) and follicle stimulating hormone (FSH), and 10, 100, 1,000, 10,000 mIU/ml of hCG on cumulative [(3)H]-thymidine incorporation in endometrial stromal cells were assessed. This study demonstrated that FSH and hMG induced significant inhibition in [(3)H]-thymidine uptake at all concentrations, respectively (P<0.05). In contrast to the above two hormones, hCG exerted inhibitory effect at concentrations of 1,000 and 10,000 mIU/ml (P<0.05). There was no evidence of dose-response correlations in all three gonadotropin experiments. These data imply that gonadotropins at the concentrations studied inhibit the proliferation of human endometrial stromal cells, at least, in short-term culture in vitro. Accordingly, we cannot negate the possibility that administered gonadotropin during ovarian hyperstimulation may directly influence the proliferation of human endometrial cells.

Human reproduction is dependent upon the actions of follicle-stimulating hormone (hFSH), luteinizing hormone (hLH), and chorionic gonadotropin (hCG). While the α subunits of these heterodimeric proteins can be interchanged without effect on receptor-binding specificity, their β subunits differ and direct hormone binding to either LH/CG or FSH receptors. Previous studies employing chemical modifications of the hormones, monoclonal antibodies, or synthetic peptides have implicated hCG β-subunit residues between Cys-38 and Cys-57 and corresponding regions of hLHβ and hFSHβ in receptor recognition and activation. Since the β subunits of hCG or hLH and hFSH exhibit very little sequence similarity in this region, we postulated that these residues might contribute to hormone specificity. To test this hypothesis we constructed chimeric hCG/hFSH β subunits, coexpressed them with the human α subunit, and examined their ability to interact with LH and FSH receptors and hormone-specific monoclonal antibodies. Surprisingly, substitution of hFSHβ residues 33-52 for hCGβ residues 39-58 had no effect on receptor binding or stimulation. However, substitution of hFSHβ residues 88-108 in place of the carboxyl terminus of hCGβ (residues 94-145) resulted in a hormone analog identical to hFSH in its ability to bind and stimulate FSH receptors. The altered binding specificity displayed by this analog is not attributable solely to the replacement of hCGβ residues 108-145 or substitution of residues in the \"determinant loop\" located between hCGβ residues 93 and 100.

Recent studies have shown that human and animal mammary gland carcinoma cell line express luteinizing hormone receptors (LHRs). We have examined the cytotoxic effect of Hecate-CGbeta conjugate, that is, fusion of a lytic peptide (Hecate) and a 15-amino acid fragment of the CGbeta-chain in vitro. To test the hypothesis that the Hecate-CGbeta conjugate selectively abolishes cells possessing LHR, estrogen dependent and independent human breast cancer cell lines (MCF-7; MDA-MB-231) and a mouse Leydig tumor cell line (BLT-1) were treated in vitro with Hecate-CGbeta conjugate and Hecate alone. Cytotoxic effects of the Hecate-CGbeta conjugate and the Hecate alone was measured by lactate dehydrogenase (LDH) release immediately after treatment. We observed that the Hecate-CGbeta conjugate selectively, in dose-dependent manner destroys cells possessing LHR in lower concentrations of preparate comparing to the Hecate alone and that the cytotoxic effect is strongly correlated with the number of LHR. Using Western blot analysis we characterized the LHR on membranes of MDA-MB-231, MCF-7 and BLT-1 tumor cell lines. In addition, we showed the evaluation of inhibition potential of the Hecate-CGbeta conjugate to LHR. At a concentration of 33 microM the conjugate inhibited (50%; IC50) the binding of CG to LHR. We suggest further development of this novel approach for the treatment of breast cancer by the Hecate-CGbeta for in vivo trials.

Purified testicular and ovarian luteinizing hormone/human chorionic gonadotropin (hCG) receptors are phosphorylated at serine and threonine residues by the catalytic subunit of the cAMP-dependent protein kinase (protein kinase A). Occupancy of the receptors by hCG significantly increased the rate but not the extent of phosphorylation. However, prolonged preincubation of receptors with hCG reduced the subsequent rate of receptor phosphorylation. Identical phosphopeptide maps were obtained for the phosphorylated ovarian and testicular receptors. The phosphorylated receptor, like the native receptor, bound to wheat germ lectin and hCG-Sepharose and migrated as a single band of Mr 90,000 (testis) and Mr 85,000 (ovary) on NaDodSO4/PAGE. Neuraminidase treatment of receptors caused reductions of molecular weight to 82,000 (testis) and 77,000 (ovary), and further treatment with O-Glycanase had minimal effect on molecular size. However, deglycosylation with N-Glycosidase and endoglycosidase F produced a single labeled polypeptide of Mr 59,000 for both gonadal receptors. Treatment of native receptors with neuraminidase caused no apparent change in binding of gonadotropin to blotted receptors, whereas deglycosylated receptors showed a major reduction in hormone binding. These results indicate that luteinizing hormone/hCG receptors are sialoglycoproteins with predominantly N-linked glycosyl residues that account for the size difference between testicular and ovarian receptors and that may participate in the interaction with gonadotropin. Receptor occupancy by agonist leads to a conformational change that facilitates its phosphorylation during initial binding and reduces the rate of phosphorylation after more prolonged exposure to hCG.

Naturally occurring mutations of G protein-coupled receptors (GPCRs) causing misfolding and failure to traffic to the cell surface can result in disease states. Some small-molecule orthosteric ligands can rescue such misfolded receptors, presumably by facilitating their correct folding and shuttling to the plasma membrane. Here we show that a cell-permeant, allosterically binding small-molecule agonist (Org 42599) rescues the folding and cell surface expression, and therefore target cell signaling, of mutant human luteinizing hormone (LH) receptors (A593P and S616Y) that cause Leydig cell hypoplasia in man. Both mutant receptors were retained in the cytoplasm whereas WT receptor localized at the cell membrane, and binding of LH to cells expressing the mutant receptors was markedly lower than to those expressing the WT receptor. Incubation with Org 42599 increased mutant receptor expression, cell surface localization, and the proportion of mutant receptor in the mature glycosylated form. Importantly, although LH stimulated little (S616Y) or no (A593P) activation of cells expressing mutant receptors, incubation of cells with Org 42599 facilitated rescue of expression and stimulation by the native ligand, LH. Although Org 42599 could activate these receptors, it could not displace ¹²⁵I-labeled human LH binding to the WT receptor, indicating that it acts in an allosteric manner. Here we demonstrate a small-molecule GPCR allosteric agonist that functionally rescues intracellularly retained mutant LH receptors by facilitating their cell surface expression. This approach may have application for treatment of infertile patients bearing such mutations and, more broadly, for other misfolded GPCR mutants resulting in human pathologic processes.

The increasing concern for the reproductive toxicity of abundantly used phthalates requires reliable tools for exposure risk assessment to mixtures of chemicals, based on real life human exposure and disorder-associated epidemiological evidence. We herein used a mixture of four phthalate monoesters (33% mono-butyl phthalate, 16% mono-benzyl phthalate, 21% mono-ethyl hexyl phthalate, and 30% mono-isononyl phthalate), detected in 1 st trimester urine of 194 pregnant women and identified as bad actors for a shorter anogenital distance (AGD) in their baby boys. Mice were treated with 0, 0.26, 2.6 and 13 mg/kg/d of the mixture, corresponding to 0x, 10x, 100x, 500x levels detected in the pregnant women. Adverse outcomes detected in the reproductive system of the offspring in pre-puberty and adulthood included reduced AGD index and gonadal weight, changes in gonadal histology and altered expression of key regulators of gonadal growth and steroidogenesis. Most aberrations were apparent in both sexes, though more pronounced in males, and exhibited a non-monotonic pattern. The phthalate mixture directly affected expression of steroidogenesis as demonstrated in a relevant in vitro model. The detected adversities at exposures close to the levels detected in pregnant women, raise concern on the existing safety limits for early-life human exposures and emphasizes the need for re-evaluation of the exposure risk.