Active immunization with GnRH-tandem-dimer peptide in young male rats reduces serum reproductive hormone concentrations, testicular development and spermatogenesis.

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-ß and FSH-ß in the pituitary, LH receptor, FSH receptor, inhibin α, ßA and ßB 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.

Prenatal Exposures of Male Rats to the Environmental Chemicals Bisphenol A and Di(2-Ethylhexyl) Phthalate Impact the Sexual Differentiation Process.

The increasing incidence of reproductive anomalies, described as testicular dysgenesis syndrome, is thought to be related to the exposure of the population to chemicals in the environment. Bisphenol A (BPA) and di(2-ethylhexyl)phthalate (DEHP), which have hormonal and antihormonal activity, have attracted public attention due to their presence in consumer products. The present study investigated the effects of BPA and DEHP on reproductive development. Timed-pregnant female rats were exposed to BPA and DEHP by gavage from gestational days 12 to 21. Results showed that prenatal exposures to test chemicals exerted variable effects on steroidogenic factor 1 and GATA binding protein 4 protein expression and increased (P < .05) sex-determining region Y-box 9 and antimüllerian hormone protein in the infantile rat testis compared with levels in the control unexposed animals. Pituitary LHß and FSHß subunit protein expression was increased (P < .05) in BPA- and DEHP-exposed prepubertal male rats but were decreased (P < .05) in adult animals relative to control. Exposure to both BPA and DEHP in utero inhibited (P < .05) global DNA hydroxymethylation in the adult testis in association with altered DNA methyltransferase protein expression. Together the present data suggest that altered developmental programming in the testes associated with chemical exposures are related to the disruption of sexual differentiation events and DNA methylation patterns. The chemical-induced effects impact the development of steroidogenic capacity in the adult testis.

Indian Hedgehog and its targets in human endometrium: menstrual cycle expression and response to CDB-2914.

CONTEXT: Progesterone is critical for secretory endometrial differentiation in women, but its downstream mediators are poorly understood. OBJECTIVE: Our objective was to investigate endometrial expression of Indian Hedgehog (IHH) and genes involved in its signaling [smoothened (SMO), patched-1 (PTCH1), glioma-associated oncogene homolog 1 (GLI1), and GLI2] during the menstrual cycle and the effects of the selective progesterone receptor modulator CDB-2914 on its expression. DESIGN AND SETTING: Comparisons between normally cycling volunteers and women with symptomatic fibroids who received CDB-2914 or placebo were made at a clinical research center. PATIENTS AND INTERVENTIONS: Endometrial biopsy was performed on 34 volunteers, 17 additional women with fibroids. MAIN OUTCOME MEASURES: Endometrial expression of IHH, SMO, PTCH1, GLI1, and GLI2 by in situ hybridization and/or RT-PCR and IHH, GLI1, and PTCH1 immunohistochemistry were evaluated. RESULTS: RT-PCR showed expression of IHH, SMO, PTCH1, GLI1, and GLI2, with significant increases in IHH (5.2-fold) and GLI1 (3.6-fold) in endometrium exposed to CDB-2914 compared with placebo. In situ hybridization showed IHH mRNA expression in glands and stroma that was stronger in secretory samples. Among volunteers, IHH and GLI1 immunohistochemistry scores were higher in the secretory than proliferative phase in the nuclei and cytoplasm of glands and stroma (P=0.0002-0.04). Compared with follicular-phase controls, women exposed to CDB-2914 showed increased IHH expression in all compartments except stromal cytoplasm (P=0.0199-0.0423); GLI1 was up-regulated in glandular nuclei and cytoplasm compared with both volunteers and women receiving placebo (P≤0.0416). CONCLUSIONS: The temporal increase in endometrial IHH and GLI1 during the secretory phase, and their modulation by CDB-2914, suggests progestin regulation and a potential role in endometrial differentiation and implantation.

Development of real-time PCR assays in the study of gonadotropin subunits, follistatin and prolactin genes expression in the porcine anterior pituitary during the preovulatory period.

OBJECTIVES: Neural control of the anterior pituitary function consists of the interplay of neuropeptides action, gonadal steroid hormones and many other factors. The physiological effect of this regulatory action is the release and synthesis of protein hormones in the precise time and quantity. The main factor responsible for the gonadotropins release and synthesis is the gonadotropin-releasing hormone (GnRH). We must still study the modulation of the synthesis of the gonadotropins subunits - LHbeta, FSHbeta and alpha subunit by different forms of GnRH and by its analogs, in order to better understand the regulation of gonadotropin release and synthesis. THE AIM of this study was to develop real-time PCR assays of five candidate reference genes for normalization purposes in order to quantify target transcripts in anterior pituitary cells during the preovulatory period. Moreover, we focused on the influence of GnRH receptor antagonist (antide) treatment on mRNA expression levels of GPalpha, LHbeta, FSHbeta, FST(follistatin) and PRL(prolactin) genes in these cells. MATERIAL AND METHODS: Anterior pituitary cells were obtained from pituitary glands of four mature pigs at the preovulatory phase. Cells were incubated with or without antide and relative mRNA level of target genes was measured using the Applied Biosystems 7500 Real Time System. For an exact comparison of mRNA quantity, the stability of five reference genes, ACTB, B2M, GAPDH, RPL1, and TOP2B was evaluated to choose the most appropriate reference gene for qRT-PCR normalization in the pituitary cells. Expression stability of reference genes was calculated using the geNorm application. The developed method of PCR assay was applied to study gene expression in pig pituitary cells in short culture. RESULTS: The most stably expressed genes in the pituitary cells were GAPDH and TOP2B. The expression of ACTB, B2M and RPL1 appeared to be highly unstable. After normalization to the GAPDH/TOP2B, results showed that the mRNA expression of the FSHbeta gene was highest in comparison with LHbeta, GPalpha, FST and PRL genes (p<0.005). Pre-treatment of cells by the antide resulted in lower mRNA expression of these genes, while FSHbeta mRNA had a significantly lower expression (p<0.05) in comparison with control. CONCLUSIONS: Real-time PCR analysis of the expression of LHbeta, FSHbeta, alpha subunit, follistatin and prolactin genes in porcine anterior pituitary cells during the preovulatory period is suitable for the study of modulatory action of metal complexes with GnRH on the expression of these genes.

Fetal pituitary gonadotropin as an initial target of dioxin in its impairment of cholesterol transportation and steroidogenesis in rats.

Reproductive and developmental disorders are the most sensitive toxic effects caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD is thought to produce many, if not all, of these toxic effects by impairing steroidogenesis and/or steroid action during the prenatal or early postnatal stages. However, the mechanism of the antisex steroid effect of TCDD is not well understood. This study revealed that steroidogenic acute-regulatory protein (StAR), a key transporter of cholesterol for steroidogenesis, in the testes of fetal rats are down-regulated by maternal exposure to TCDD. It was also shown that many mRNAs of steroidogenetic enzymes, including cytochromes P450 11A1, 17, and 11B1 and 3beta-hydroxysteroid dehydrogenase, are reduced in fetuses of TCDD-treated dams in a testis-specific manner. The same was also observed for the expression of estrogen-alpha receptors and androgen receptors. Whereas StAR expression was not affected by TCDD in cultured fetal testis, the fetal serum content of LH, a pituitary regulator of StAR, was significantly reduced by TCDD. In agreement with this, pituitary expression of LHbeta subunit mRNA in fetuses was reduced by maternal exposure to TCDD, whereas the alpha-subunit remained unchanged. The reduction in LHbeta is suggested to occur by a mechanism different from the reduction in the GnRH level. Direct supply of exogenous gonadotropin to TCDD-exposed fetuses completely abolished the reduction of StAR expression. Taken together, these results demonstrate that TCDD impairs steroidogenesis in the fetus by targeting pituitary gonadotropins.

The effect of Nestorone on gonadotropic cells in pituitary of rats.

The implant containing Nestorone is a promising long-acting contraceptive especially suitable for lactating women. In this study, two experiments were designed to observe the effect of Nestorone on the gonadotropic cells in pituitary of rats for analyzing its antiovulation mechanism. In the first experiment, the ED50 of Nestorone on inhibiting ovulation was found to be 1.32 mg/kg. The serum luteinizing hormone (LH) levels were significantly lower 60 h after being treated with Nestorone at 8:30-9:00 a.m. on Day 2 (D2) of estrus. Image analysis showed that the average size of the LH cells in groups treated with Nestorone at 2 or 4 mg/kg was larger than that of the control. In the group treated with 4 mg/kg, most of gonadotropic cells were regular round in shape. And, abundant granules in cytoplasm were found in those cells, which indicated that the LH stored in cells was not released. In the second experiment, the rats were treated with Nestorone at 5 mg/kg at 11:30-12:00 a.m. on D2 of estrus. The normal or higher expression of LHbeta mRNA in pituitary suggested that the synthesis of LH was not inhibited by the treatment with Nestorone. The expression of PR mRNA in pituitary was significantly lower than that of the control at 33 h after treatment. This might be a direct effect of Nestorone, since there were no differences in the serum E2 and P4 levels between the treated and the control group. It is concluded that Nestorone prevents ovulation through inhibition of LH secretion and it has no effect on synthesis of LH. Progesterone receptors in pituitary might be involved in this process, but further study is needed to gain more evidence.