A nerve growth factor from the venom of Chinese cobra (Naja naja atra) and its effects on male reproductive system in rats

A nerve growth factor (NGF) was isolated from the venom of Chinese cobra (Naja naja ntr a) by ion exchange chromatography, gel filtration and fast protein liquid chromatography (FPLC). The N-terminal sequence of 22 amino acid residues was identical with other NGFs previously purified from the venom of the same genus. The NGF monomer molecular weight was estimated to be 13 500 by reducing SDS-PAGE and the isoelectric point was determined to be 7.2 by isoelectric focusing electrophoresis. NGF improved the epididymal sperm motility of male rats and increased the pregnancy rate and fetus number of mated female rats. The serum levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) of male rats administrated NGF + gossypol was lower than that of male rats administrated gossypol. Histological sections of testes and epididymides showed that NGF reduced the destructive effects of gossypol on rat testes. (C) 1999 Elsevier Science Inc. All rights reserved.

Dynamic changes in ovarian follicles measured by ultrasonography during gonadotropin stimulation in rhesus monkeys

The objective was to study dynamic changes of ovaries in rhesus macaques stimulated by gonadotropins to identify an indicator for predicting ovarian response to stimulation. Twenty-one cycling monkeys were given 36 IU/d recombinant human follicle-stimulat

THE POSSIBLE INVOLVEMENT OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR IN LUTEOLYSIS OF RHESUS-MONKEY

Changes of plasminogen activators (PA) during different stages of development of the corpus luteum, and their possible physiological role in luteolysis were studied in rhesus monkeys. It was demonstrated for the first time that monkey corpus luteal cells not only produce PA, but that the function of the corpus luteum is also closely related to the activity of this enzyme system. Generally, the life span for a corpus luteum in monkey is approximately 14-16 days, its demise beginning thereafter. In the present study, we found that urokinase in the corpus luteum is higher on day 5 and day 10 after human chorionic gonadotrophin injection, while the tissue type (t) PA is mainly produced on day 13 when luteolysis may take place. Progesterone production remained high on day 5 and day 10 and decreased dramatically from day 13, indicating the important role of tPA but not urokinase (u) PA in suppressing luteal function. When purified tPA (but not uPA) monoclonal antibody was added to luteal cell culture to neutralize endogenously produced tPA activity, progesterone production in the cells was increased significantly. Interestingly, prolactin alone was capable of increasing PA production by luteal cells; prolactin together with luteinizing hormone, however, had a synergistic luteotrophic effect.

Molecular characterization of Chinese sturgeon gonadotropins and cellular distribution in pituitaries of mature and immature individuals

Chinese sturgeon (Acipenser sinensis) is a rare and endangered species, and also an important resource for the sturgeon aquaculture industry. To understand molecular characterization of Chinese sturgeon gonadotropins (GTHs), we cloned the full-length cDNAs of gonadotropin subunits common alpha (GTH-alpha), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from a pituitary cDNA library of mature female. Two subtypes of GTH-alpha were identified. The nucleotide sequences of A. sinensis common alpha I (AsGTH-alpha I), common alpha II (AsGTH-alpha II), FSH beta (AsFSH beta) and LH beta (AsLH beta) subunit cDNAs are 345, 363, 387 and 414 bp in length, and encode mature peptides of 115, 121, 129 and 138 aa, respectively. Then, three polyclonal antibodies were prepared from the in vitro expressed AsGTH-alpha I, AsFSH beta and AsLH beta mature proteins, respectively. Significant expression differences were revealed between immature and mature sturgeon pituitaries. Western blot detection and immunofluoresence localization revealed the existence of three-gonadotropin subunits (AsGTH-alpha, AsFSH beta and AsLH beta) in mature sturgeon pituitaries, but only AsFSH beta was detected in immature individual pituitaries during early stages in the sturgeon life, and obvious difference was observed between males and females. In males, AsFSH beta was expressed in 4-year-old individuals, whereas in females, AsFSH beta was just expressed in 5-year-old individuals. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Alleviation of pre-exposure of mouse brain with low-dose C-12(6+) ion or Co-60 gamma-ray on male reproductive endocrine damages induced by subsequent high-dose irradiation

Irradiation has been widely reported to damage organisms by attacking on proteins, nucleic acid and lipids in cells. However, radiation hormesis after low-dose irradiation has become the focus of research in radiobiology in recent years. To investigate the effects of pre-exposure of mouse brain with low-dose C-12(6+) ion or Co-60 gamma (gamma)-ray on male reproductive endocrine capacity induced by subsequent high-dose irradiation, the brains of the B6C3F(1) hybrid strain male mice were irradiated with 0.05 Gy of C-12(6+) ion or Co-60 gamma-ray as the pre-exposure dose, and were then irradiated with 2 Gy as challenging irradiation dose at 4 h after pre-exposure. Serum pituitary gonadotropin hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), testosterone, testis weight, sperm count and shape were measured on the 35th day after irradiation. The results showed that there was a significant reduction in the levels of serum FSH, LH, testosterone, testis weight and sperm count, and a significant increase in sperm abnormalities by irradiation of the mouse brain with 2 Gy of C-12(6+) ion or Co-60 gamma-ray. Moreover, the effects were more obvious in the group irradiated by C-12(6+) ion than in that irradiated by Co-60 gamma-ray. Pre-exposure with low-dose C-12(6+) ion or Co-60 gamma-ray significantly alleviated the harmful effects induced by a subsequent high-dose irradiation.

Inhibitory effects of the androgenic gland on ovarian development in the mud crab Scylla paramamosain

Isolation and characterization of androgenic hormone in decapod crustaceans depend on an effective bioassay of its action. In the present study, the effect of androgenic gland on ovarian development in the mud crab Scylla paramamosain was investigated with a view to develop a bioassay for androgenic hormone. Ovarian regression with degeneration of oocytes occurred in some female crabs implanted with androgenic gland in vivo. In vitro incubation of ovarian tissues at secondary vitellogenesis in extract of androgenic gland resulted in a significant decrease in amino acid uptake by the tissues. We propose that this inhibitory effect could be established as an effective bioassay for the isolation of androgenic hormone in the mud crab. (c) 2005 Published by Elsevier Inc.

Estradiol stimulates growth hormone production in female goldfish

The effects of estradiol (E(2)) on growth hormone (GH) production was investigated in gonad-intact female goldfish. It was first necessary to generate a specific antibody for use in immunocytochemistry, Western, and dot-blot analyses of GH production. To accomplish this, grass carp GH (gcGH) cDNA was cloned by the reverse transcription polymerase chain reaction (RT-PCR) and expressed in Echerichia coli and a specific polyclonal antibody to recombinant gcGH was generated in the rabbit. In Western blot, the anti-gcGH antibody specifically immunoreacted with recombinant gcGH, purified natural common carp GH, and with a single 21.5-kDa GH form from pituitary extracts of grass carp, common carp, goldfish, and zebrafish but not salmon, trout, or tilapia. Intraperitoneal injection of the recombinant gcGH enhanced the growth rates of juvenile common carp demonstrating biological activity of this GH preparation. Electron microscopic studies showed that the anti-gcGH-I antibody specifically reacted with GH localized in the secretory granules of the goldfish somatotroph. Using anti-gcGH-I in a dot-blot assay, it was found that in vivo implantation of solid silastic pellets containing E(2) (100 mu g/g body weight for 5 days) increased pituitary GH content by 150% in female goldfish. In a second, independent study employing a previously characterized anticommon carp GH antibody for radioimmunoassay, it was found that E(2) increased pituitary GH content by 170% and serum GH levels by approximately 350%. The E(2)-induced hypersecretion of GH and increase in pituitary GH levels was not associated with changes in steady-state pituitary GH mRNA levels, suggesting that this sex steroid may enhance GH synthesis at the posttranscriptional or translational level. Previous observations indicate that GH can stimulate ovarian E(2) production. The present results show that E(2) can in turn stimulate GH production, indicating the existence of a novel pituitary GH-ovarian feedback system in goldfish. (C) 1997 Academic Press.

Effect of age and breeding season on the developmental capacity of oocytes from unstimulated and follicle-stimulating hormone-stimulated rhesus monkeys

Effects of age and season on the developmental capacity of oocytes from unstimulated and FSH-stimulated rhesus monkeys were examined, Immature cumulus-oocyte complexes were matured in vitro in modified CMRL-1066 medium containing 20% bovine calf serum and

17 beta-Estradiol and progesterone improve in-vitro cytoplasmic maturation of oocytes from unstimulated prepubertal and adult rhesus monkeys

BACKGROUND: Effects of 17beta-estradiol and progesterone on rhesus monkey oocyte maturation in vitro were evaluated by embryo development subsequent to IVF. METHODS AND RESULTS: In experiment 1, immature cumulus-oocyte complexes collected from unstimulated adult females during the non-breeding season were matured in modified medium CMRL-1066 containing various combinations of gonadotrophins (FSH + LH), estradiol and/or progesterone. Formation of morulae and blastocysts was greatest in oocytes matured in medium containing estradiol and/or progesterone, with or without gonadotrophins (morula 38-46%, blastocyst 14-20%) than in control oocytes matured without estradiol or progesterone (morula 14%, blastocyst 0%). In experiment 2, cumulus-oocyte complexes from unstimulated prepubertal female monkeys were matured in medium with gonadotrophins, estradiol or progesterone. The best development to the morula stage was obtained with oocytes matured with gonadotrophins and estradiol or gonadotrophins and progesterone (43 and 25 morulae, respectively), while control oocytes matured with gonadotrophins but without steroid hormones gave the poorest morula developmental response (12%). However, there was no difference in blastocyst development across all groups (0-3%). CONCLUSIONS: These results demonstrate that during rhesus monkey oocyte maturation in vitro: (i) estradiol or progesterone can improve oocyte developmental competence; (ii) immature oocytes from prepubertal versus adult females have differential responses to challenge with estradiol or progesterone.

Waterborne exposure to fluorotelomer alcohol 6:2 FTOH alters plasma sex hormone and gene transcription in the hypothalamic-pituitary-gonadal (HPG) axis of zebrafish

Fluorotelomer alcohols (FTOHs) have shown estrogenic activity in vitro and in vivo, but the mechanism of this activity is not known. In this study, 18-week-old zebrafish (Danio rerio) were exposed to 0, 0.03, 0.3 and 3.0 mg/l 1H, 1H, 2H, 2H-perfluorooctan-1-ol (6:2 ETCH) for 7 days, and the effects on plasma sex hormone levels were measured followed by use of real-time PCR to examine selected gene expression in hypothalamic-pituitary-gonadal (HPG) axis and liver. Exposure to 6:2 FTOH significantly increased plasma estradiol (E2) and testosterone (T) levels in both males and females. Furthermore, the ratio of T/E2 was reduced in females while increased in males. In females, the increase of E2 was accompanied by up-regulated hepatic estrogenic receptor alpha (ER alpha) and vitellogenin (VTG1 and VTG3) expression. In males, the elevation of the T level is consistent with the up-regulation of cytochrome P450 c17 alpha-hydroxylase, 17, 20-lase (CYP17) and the down-regulation of cytochrome P450 aromatase A (CYP19A). The present study demonstrated that waterborne exposure to 6:2 FTOH alter plasma sex hormone levels and the ratio of T/E2, as well as the transcriptional profiles of some genes in the HPG axis and liver. The results suggested that FTOHs may disturb fish reproduction through endocrine disrupted activity. (C) 2009 Elsevier B.V. All rights reserved.

Seasonal variations of estrogenic compounds and their estrogenicities in influent and effluent from a municipal sewage treatment plant in China

The seasonal variations of estrogenic compounds and the estrogenicities of influent and effluent were investigated by OF chemical analysis and in vitro assay in a municipal sewage treatment plant in Wuhan (China). The levels of eight estrogenic compounds, including 17 beta-estradiol (E-2) estrone (E-1), estriol (E-3) diethylstilbestrol (DES), 17 alpha-ethinylestradiol, nonylphenol (NP), 4-tert-octylphenol (OP), and bisphenol A (BPA), were measured by gas chromatography-mass spectrometry. Total estrogenic activity of sewage was quantitatively assessed using primary cultured hepatocytes of male Megalobrama amblycephala Yih using vitellogenin as a biomarker. The E-2 equivalents (EEQs) obtained from the chemical analysis were consistent with those measured by bioassay. The natural (E-1, E-2, and E-3) and synthetic (DES) estrogens, as well as NP, were the main contributors of the total EEQs of influent and effluent in the present study. The levels of natural estrogens E-1 and E-3 in the influent and effluent were higher in winter than in summer, whereas the situation for NP and OP was the reverse. The levels of E-2, DES, and BPA varied little among different seasons. 17 alpha-Ethinylestradiol was not detected in the influent and effluent. The estrogenicities of the influent and of the primary and secondary effluents were all higher in summer than in winter. Estrogenic activities in winter mainly originated from natural (E-1, E-2, and E-3) and synthetic (DES) estrogens, whereas the increase of EEQs in summer was contributed by NP The results from chemical analysis and bioassay demonstrate that estrogenic compounds cannot be entirely removed by the existing sewage treatment process, which should be further improved to protect aquatic ecosystems and human health.

Serum concentrations of gonadotropins and steroid hormones of Neophocaena phocaenoides asiaeorientalis in middle and lower regions of the Yangtze River

As a relatively isolated and unique freshwater population, the Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis) is the most endangered subpopulation of this species. The objective of this study was to improve our understanding of their reproductive endocrinology by measuring (radioimmunoassay) serum concentrations of FSH, LH, estradiol (E-2), progesterone (P-4) and testosterone (T-2) in free-ranging animals. Blood samples were collected from 66 Yangtze finless porpoises (41 males and 25 females) captured in the middle and lower reaches of the Yangtze River. Based on significant variation of serum T-2 concentrations in males with body length (BL) > 138 cm, we inferred that they were mature; in these animals, there were significant seasonal variations in serum T-2 concentrations, with the highest concentrations in March and April (502.0 +/- 319.8 ng/dL, mean +/- S.D.) and the lowest in December (79.4 +/- 83.2 ng/dL). Serum T-2 concentrations positively correlated with serum concentrations of LH and weakly correlated with serum concentrations of E-2, whereas there was a significant negative correlation between serum LH and FSH concentrations in males > 138 cm. The smallest apparently pregnant female porpoise had a BL of 130 cm. Serum P-4 concentrations ranged from 13.2 to 112.4 ng/mL (43.9 +/- 28.3 ng/mL) in pregnant females, and fluctuated under 1.0 ng/mL in non-pregnant females with BL > 130 cm. Serum LH concentrations were significantly higher in non-pregnant females > 130 cm versus those females < 130 cm. To our knowledge, this is the first study of endocrine-related maturity and seasonal breeding characteristics of the Yangtze finless porpoise. (c) 2006 Elsevier Inc. All rights reserved.

Reproductive seasonality and maturity of male Neophocaena phocaenoides asiaeorientalis in captivity: A case study based on the hormone evidence

To provide hormone evidence on reproductive seasonality and maturity of the Yangtze finless porpoise, the authors monitored the monthly variations of serum reproductive hormones of a male Yangtze finless porpoise in captivity via radioimmunoassay from 1997 to 2003. It was demonstrated that the immature animal had a serum T level of 28-101ngdL -1 (6552ngdL -1 ), reached puberty with the serum T concentration ranging between 250 and 590ngdL -1 (390130ngdL -1 ), and attained reproductive maturity when the serum T level went beyond 1120180ngdL -1 in the breeding season. The body length growth of the animal showed a pattern similar to the serum T variations during its adolescent period from the age of 4 to 6 years. Moreover, the serum T concentration of the male porpoise exhibited significant seasonal variations, indicating that its breeding season may start as early as March and end as late as September.