Jian-Pi-Yi-Shen decoction inhibits mitochondria-dependent granulosa cell apoptosis in a rat model of POF.

As a widely applied traditional Chinese medicine (TCM), Jian-Pi-Yi-Shen (JPYS) decoction maybe applied in curing premature ovarian failure (POF) besides chronic kidney disease (CKD). In vivo experiments, 40 female SD (8-week-old) rats were randomized into four groups, namely, control group (negative control), POF model group, JPYS treatment group, and triptorelin treatment group (positive control). JPYS group was treated with JPYS decoction (oral, 11 g/kg) for 60 days, and the triptorelin group was treated with triptorelin (injection, 1.5 mg/kg) for 10 days before the administration of cyclophosphamide (CTX) (50 mg/kg body weight) to establish POF model. We examined apoptosis, mitochondrial function, and target gene (ASK1/JNK pathway and mitochondrial fusion/fission) expression. In vitro experiments, the KGN human granulosa cell line was used. Cells were pretreated with CTX (20, 40, and 60 µg/mL) for 24 h, followed by JPYS-containing serum (2, 4, and 8 %) for 24 h. Thereafter, these cells were employed to assess apoptosis, mitochondrial function, and target gene levels of protein and mRNA. In vivo, JPYS alleviated injury and suppressed apoptosis in POF rats. In addition, JPYS improved ovarian function. JPYS inhibit apoptosis of granulosa cells through improving mitochondrial function by activating ASK1/JNK pathway. In vitro, JPYS inhibited KGN cell apoptosis through inhibited ASK1/JNK pathway and improved mitochondrial function. The effects of GS-49977 were similar to those of JPYS. During POF, mitochondrial dysfunction occurs in the ovary and leads to granulosa cell apoptosis. JPYS decoction improves mitochondrial function and alleviates apoptosis through ASK1/JNK pathway.

A critical point of male gonad development: neuroendocrine correlates of accelerated testicular growth in rats during early life.

Testis growth during early life is important for future male fertility and shows acceleration during the first months of life in humans. This acceleration coincides with the peak in gonadotropic hormones in the blood, while the role of hypothalamic factors remains vague. Using neonatal rats to assess this issue, we found that day 9 of life is likely critical for testis development in rats. Before this day, testicular growth was proportional to body weight gain, but after that the testes showed accelerated growth. Hypothalamic kisspeptin and its receptor mRNA levels begin to elevate 2 days later, at day 11. A significant increase in the mRNA levels for gonadotropin-releasing hormone (GnRH) receptors in the hypothalamus between days 5 and 7 was followed by a 3-fold decrease in GnRH mRNA levels in this brain region during the next 2 days. Starting from day 9, hypothalamic GnRH mRNA levels increased significantly and positively correlated with accelerated testicular growth. Triptorelin, an agonist of GnRH, at a dose that had no effect on testicular growth during "proportional" period, increased testis weights during the period of accelerated growth. The insensitivity of testicular growth to GnRH during "proportional" period was supported by inability of a 2.5-fold siRNA knockdown of GnRH expression in the hypothalamus of the 7-day-old animals to produce any effect on their testis weights. GnRH receptor blockade with cetrorelix was also without effect on testis weights during "proportional" period but the same doses of this GnRH antagonist significantly inhibited "accelerated" testicular growth. GnRH receptor mRNA levels in the pituitary as well as plasma LH concentrations were higher during "accelerated" period of testicular growth than during "proportional" period. In general, our data defined two distinct periods in rat testicular development that are primarily characterized by different responses to GnRH signaling.

Formulation and evaluation of an in situ gel forming system for controlled delivery of triptorelin acetate.

The novel physical hydrogels composed of chitosan or its water soluble derivatives such as carboxymethyl chitosan (CMCh) and sodium carboxymethyl chitosan (NaCMCh) and opened ring polyvinyl pyrrolidone (OP-PVP) were used as a controlled delivery system for triptorelin acetate, a luteinizing-releasing hormone agonist. The in situ gel forming system designed according to physical interactions such as chains entanglements and hydrophilic attractions especially h-bonds of chitosan and/or NaCMCh and OR-PVP. In order to increase in situ gel forming rate the chitosan microspheres prepared through spray drying technique. The chitosan or NaCMCh/OR-PVP blends prepared at different ratios (0.05, 0.10, 0.12, 0.16, 0.20 and 0.24) and suspended in sesame oil as non-aqueous vehicle at different solid content (10-30%). The suitable ratio of polymers with faster in situ gel forming rate was selected for in vivo studies. The gel formation and drug release from the system was evaluated both in vitro and in vivo. In vitro and in vivo results were compared with Diphereline SR 3.75mg, a commercially available controlled delivery system of triptorelin. In vitro release studies showed a sustained release profile for about 192h with first order kinetics. In vivo studies on male rats by determination of serum testosterone were confirmed the acceptable performance of in situ gel forming system compared with Diphereline SR in decreasing the serum testosterone level for 35days, demonstrating the potential of the novel in situ gel forming system for controlled delivery of peptides.

[Clinical values of triptorelin stimulating test in assessing hypothalamus-pituitary-gonad axis function in male patients with hypothalamus-pituitary-gonad axis disorders].

OBJECTIVE: To investigate the clinical values of luteinizing hormone-releasing hormone (LHRH) α (triptorelin) stimulating test in the differential diagnoses of hypothalamus-pituitary-gonad axis (HPGA) disorders. METHODS: A total of 229 male patients with various HPGA disorders were recruited for triptorelin stimulating test. And all patients were followed up for 12 - 48 months until a definite diagnosis was made. The values of triptorelin stimulating test in the differential diagnoses of HPGA disorders were assessed by examining the close relationship between LHmax and the final clinical diagnosis. RESULTS: (1) LH levels rose steady after an intramuscular injection of triptorelin 100 µg and the time of LHmax appeared at 45 - 60 min. (2) LHmax < 4 U/L indicated the function of HPGA was not activated. LHmax in the range of 4 - 12 U/L indicated the patients might have constitutional delayed puberty development. LHmax > 12 U/L indicated the fulfilled puberty development. CONCLUSION: Triptorelin stimulating test can precisely evaluate the functions of HPGA in various HPGA disorders and provide valuable information for the differential diagnoses in constitutional delayed puberty development, hypogonadotropic hypogonadism, central and peripheral precocious puberty disorders.

Dual targeting of tumor and endothelial cells by gonadotropin-releasing hormone agonists to reduce melanoma angiogenesis.

We showed previously that GnRH receptors are expressed in melanoma cells; their activation reduces cell growth and metastatic behavior. Here, we investigated whether GnRH agonists might affect the expression of genes involved in melanoma progression. By genome-wide transcriptomic and real-time PCR analysis, we first observed that GnRH agonists decrease the expression of the pro-angiogenic factor vascular endothelial growth factor (VEGF) (all isoforms) in BLM melanoma cells. Then, we demonstrated that GnRH agonists specifically decrease the expression of the VEGF165 isoform as well as its secretion from BLM cells. These data suggested that activation of GnRH receptors might reduce the pro-angiogenic behavior of melanoma cells. To verify this hypothesis, we treated BLM cells with a GnRH agonist; the conditioned medium from these cells was tested to assess its capability to stimulate human umbilical vein endothelial cell (HUVEC) motility. The migration of HUVECs towards the conditioned medium of GnRH agonist-treated BLM cells was significantly lower than the migration of HUVECs toward the conditioned medium of untreated cells. Thus, GnRH agonists reduce the pro-angiogenic behavior of melanoma cells through a decreased production of bioactive VEGF. We then found that GnRH receptors are also expressed on HUVECs and that GnRH agonists reduce their ability to proliferate and to form capillary-like tubes when stimulated by VEGF. These findings suggest that GnRH agonists exert an anti-angiogenic activity indirectly by decreasing VEGF secretion from tumor cells and directly by counteracting the pro-angiogenic activity of the growth factor. These data might lead to the development of novel targeted approaches for melanoma.

Pituitary and gonadal effects of GnRH (gonadotropin releasing hormone) analogues in two peripubertal female rat models.

Central precocious puberty is commonly treated by gonadotropin releasing hormone (GnRH) agonists. To compare modes of action and effectiveness of GnRH analogues and assess treatment combinations of agonistic (triptorelin) and antagonistic (cetrorelix acetate) GnRH analogues with established treatment, we used prepubertal 31-d-old ovariectomized female rats. Strongest inhibition of LH and FSH occurred after 2-d treatment with antagonist alone (LH 0.08 +/- 0.02 versus 3.2 +/- 0.56 ng/mL in controls; FSH 10.8 +/- 2.8 versus 44.2 +/- 5.0 ng/mL in controls, p < 0.001). Combined agonist/antagonist was second most effective of the treatments (after 5 d treatment, LH 0.52 +/- 0.15 versus 4.9 +/- 1.1 ng/mL in controls; p < 0.01). Pituitary gonadotropin subunit LHbeta mRNA levels were inhibited in all groups except controls, but pituitary GnRH receptor mRNA was stimulated by agonist yet unaffected by combined analogues. Explanted ovaries were incubated with either analogue, both 10-6 M. After 4 h, GnRH receptor mRNA levels were significantly reduced by antagonist but not agonist. To verify puberty-inhibiting effects of GnRH analogues, we used 26-d-old female rats with androgen-induced precocious puberty after injecting subcutaneously single 300 microg danazol on postnatal d 5. Single application of cetrorelix depot (cetrorelix embonate) reduced serum estradiol levels and pituitary LHbeta expression; GnRH receptor mRNA levels were down-regulated in the pituitary and ovary (p < 0.05). In androgen-induced precocious puberty model, single injection of antagonist effectively arrests premature hormonal activation and down-regulates pituitary and ovarian GnRH receptors. We conclude that GnRH analogue combination and especially antagonist alone treatment most directly suppress gonadotropin levels. This implies that early treatment gonadotropin flare-up associated with agonist treatment is avoidable.

Preclinical evaluation of targeted cytotoxic luteinizing hormone-releasing hormone analogue AN-152 in androgen-sensitive and insensitive prostate cancers.

PURPOSE AND EXPERIMENTAL DESIGN: To improve conventional chemotherapy, we developed cytotoxic analogues of luteinizing hormone-releasing hormone (LH-RH), which can be targeted to prostate cancers expressing LH-RH receptors. In view of pending clinical trials on cytotoxic LH-RH analogue AN-152, containing doxorubicin (DOX) linked to [D-Lys(6])-LH-RH, we investigated the effects of AN-152 on tumor growth of s.c. implanted androgen-sensitive LNCaP and MDA-PCa-2b prostate cancers, as well as androgen-independent C4-2 prostate cancers xenografted into the tibiae of nude mice. In the C4-2 study, serum prostate-specific antigen (PSA) levels were also measured. LH-RH receptors were analyzed by reverse transcription-PCR and ligand competition assay. We also evaluated whether AN-152 can affect mRNA expression of human epidermal growth factor receptor and HER-2 and -3 oncogenes RESULTS: After 32 days of treatment with AN-152, the growth of LNCaP cancers in castrated nude mice was strongly inhibited by 83% versus intact controls (P < 0.01) and 62% versus castrated controls (P < 0.05). In animals bearing MDA-PCa-2b prostate cancers, therapy with AN-152 for 25 days resulted in a 69% inhibition of tumor growth (P < 0.01 versus controls) and was more effective (P < 0.05) than equimolar doses of DOX or microcapsules of LH-RH agonist Decapeptyl. In nude mice bearing intraosseous C4-2 prostate cancers, treatment with AN-152 decreased serum PSA levels (P < 0.01) to 10.3 +/- 3.4 ng/ml from 24.8 +/- 4 ng/ml in controls, whereas DOX had no effect on PSA. The inhibitory effects of AN-152 on C4-2 tumors was accompanied by an increase in apoptosis and a decrease in tumor proliferation. Binding sites for LH-RH and the expression of mRNA for LH-RH receptors were found on s.c. C4-2 and MDA-PCa-2b tumors. The inhibition of MDA-PCa-2b tumors by AN-152 was associated with a significant decrease in mRNA expression for epidermal growth factor receptor, HER-2, and 3. CONCLUSIONS: Our findings suggest that cytotoxic analogue AN-152 could be considered for therapeutic trials in patients with advanced prostate carcinoma.

Endothelium-dependency of yohimbine-induced corpus cavernosum relaxation.

Development and maintenance of penile erection requires the relaxation of the smooth muscle cells in the cavernous bodies and is essentially mediated by nitric oxide (NO). The penile flaccid state is conversely maintained by the alpha adrenergic neuroeffector system and by other vasoconstrictors, such as endothelin-1 (ET-1). In this study we examined the mechanisms involved in yohimbine-induced relaxation in human and rabbit corpora cavernosa (CC). We essentially found that yohimbine not only blocks contractions induced by adrenergic agonists, but also by non-adrenergic substances, such as ET-1. This effect was unrelated to antagonism at the level of ET receptors, because yohimbine did not affect ET-1-induced increase in intracellular calcium in isolated CC cells. Conversely, our data suggest that yohimbine counteracts ET-1-induced contractions by interfering with NO release from the endothelium. In fact, yohimbine-induced CC relaxation was inhibited by the mechanical removing of the endothelium and by blocking NO formation or signalling via guanylate cyclase and cGMP formation. Conversely, yohimbine activity was strongly increased by inhibiting cGMP degradation. In an experimental model of hypogonadism, performed on rabbits by chronic treatment with a long-lasting GnRH agonist, the relaxant yohimbine activity was also decreased, but completely restored by androgen supplementation. This effect was evident only in preparations in which the main source of NO was present (endothelium) or in which NO formation was not impaired by L-NAME. Our data indicate that the relaxant effect of yohimbine is both endothelium and androgen-dependent. This might justify the lack of efficacy of this drug in treatment of some form of organic erectile dysfunction.

Autoregulation of the gonadotropin-releasing hormone (GnRH) system during puberty: effects of antagonistic versus agonistic GnRH analogs in a female rat model.

To address whether gonadotropin-releasing hormone (GnRH) regulates its own expression and the expression of its receptor in the hypothalamus and ovary, we treated five groups of prepubertal/peripubertal female rats from postnatal days 25-36 with either the GnRH agonist triptorelin (TRIP) or the GnRH antagonist cetrorelix (CET), each 10 or 100 microgram/day, or a placebo. We compared their effects regarding pubertal development, serum gonadotropins and the expression of GnRH and GnRH-receptor in the hypothalamus, pituitary, ovary and uterus. Onset of puberty was determined by vaginal opening, and expression levels of GnRH and GnRH-receptor were determined using either quantitative real-time PCR or competitive RT-PCR. Onset of puberty was retarded by both analogs but CET (100 microgram/day) inhibited while TRIP (10 and 100 microgram/day) stimulated serum gonadotropins (P<0.05). The expression of GnRH in the preoptic area did not show significant differences among the treatment groups but ovarian GnRH mRNA levels were significantly stimulated by CET (100 microgram/day). GnRH mRNA could not be detected in the uterus by either real-time PCR or competetive RT-PCR. The GnRH-receptor expression in the hypothalamus (preoptic area and mediobasal hypothalamus) did not vary among any of the groups, whereas in the pituitary GnRH-receptor mRNA levels were stimulated by TRIP (10 microgram/day) but inhibited by CET (100 microgram/day). In contrast, in the ovary GnRH-receptor mRNA levels were inhibited by both TRIP (100 microgram/day) and CET (100 microgram/day). Interestingly, the GnRH-receptor was even expressed in the uterus where it was strongly stimulated by both CET and TRIP in a dose-related manner. This shows that in addition to their different pituitary effects, the GnRH analogs cetrorelix and triptorelin exert different actions at the hypothalamic, ovarian and uterine level. This study also demonstrates an organ-specific regulation of GnRH and GnRH-receptor gene expression which is likely part of a local autoregulatory system. We conclude that the ovarian and uterine effects of GnRH analogs must be considered in addition to their known pituitary effects when deciding which GnRH analog is most suitable for treating precocious puberty.

Decreased bone density in elderly men treated with the gonadotropin-releasing hormone agonist decapeptyl (D-Trp6-GnRH).

Administration of GnRH agonist analogs to women may result in a hypoestrogenic state and bone mass reduction. In the present study we examined bone mineral density (BMD) and parameters of mineral metabolism in elderly men with benign prostatic hyperplasia before and during a hypoandrogenic state induced by the long-acting GnRH agonist D-Trp6-LHRH (decapeptyl). Our results showed that decapeptyl treatment caused a significant decrease in serum testosterone concentrations in all patients and resulted in a significant decrease in individual vertebral BMD in 10 of 17 patients. A significant decrease in BMD was observed in 5 patients after 6 months of treatment. Another 5 patients showed a decreased BMD only after 12 months. The mean serum concentrations of osteocalcin, phosphorus, and alkaline phosphatase activity increased after 6-12 months of treatment with decapeptyl. Serum calcium, vitamin D metabolites, and PTH concentrations remained unchanged during treatment. Urinary calcium excretion was slightly, but not significantly, increased after 6 months of treatment. These results demonstrate that long-acting GnRH agonist treatment may cause high turnover accelerated bone loss in some men during the first year of GnRH agonist treatment, as has been previously shown in women.