Regulation of <i>α</i>-Melanocyte-Stimulating Hormone by Testosterone Is Associated with the Onset of Atopic Dermatitis Symptoms during Exercise

Objective: Atopic dermatitis (AD) symptoms are altered with exercise. However, no study has investigated the association between testosterone and AD. The aim of this study was to investigate the role of testosterone by which the strength and weakness of exercise affects the skin symptoms of AD. Methods: Specific pathogen-free (SPF) and conventional NC/Nga mice were used. NC/Nga mice spontaneously developed dermal symptoms similar to AD patients. Two exercises, mild (20 m/min, 60 min) and rigorous (25 m/min, 90 min), were carried out using a treadmill four times every alternate day. Furthermore, we administered testosterone (0, 5, 50, 500, and 5000 pg/mice) to non-exercised conventional NC/Nga mice. On the final day of this experiment, we analyzed the plasma levels of immunoglobulin E (IgE), interleukin (IL)-6, IL-4, IL-13, testosterone and α-melanocyte-stimulating hormone (α-MSH) by ELISA kit. Results: Symptoms manifested by NC/Nga mice were strongly exacerbated upon severe exercise but were ameliorated during mild exercise. Between mild and severe exercised conventional mice, the plasma level of IgE was not changed. On administering an equivalent amount of testosterone, depending on the exercise, AD-like symptoms in non-exercising NC/Nga mice were ameliorated with mild exercise and exacerbated through rigorous exercise. Plasma IL-6, IL-4, and IL-13 levels remained unchanged between +50 pg (mild) and +500 pg (severe) testosterone administration. Plasma α-MSH levels were elevated with +500 pg testosterone but decreased with +50 pg testosterone administration. Conclusion: The present results suggest that exercise largely mimics AD symptoms depending on the α-MSH and testosterone levels.

Influence of leptin on luteinizing hormone and follicle stimulating hormone secreted from cultured rat anterior pituitary cells

BACKGROUND： Leptin may regulate reproductive function via release of hypothalamic neuropeptide Y. However, it is unknown whether this regulatory effect is limited to the hypothalamus. OBJECTIVE： To detect the effect of different dosages of leptin on luteinizing hormone （LH） and follicle stimulating hormone （FSH） secretion from in vitro cultured rat anterior pituitary cells. DESIGN： Contrast study based on cells. SETTING： This study was performed in the Basic Institute of Chengde Medical College, Chengde City, Hebei Province, China from March to June 2007. MATERIALS： Eighteen female Wistar rats of three months of age, weighing 200-220 g, and of clean grade were used. Leptin was provided by Peprotech Company, DMEM culture medium by Invitrogen Company, and the radioimmunological kit by Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. METHODS： Three glandular organs were regarded as one group for culture of anterior pituitary cells. In the control group, saline was added to the culture medium instead of leptin. In the leptin group, leptin was prepared into different concentrations of 1×10^-12, 1×10^-11, 1×10^-9, 1×10^-7, and 1×10^-6 mol/L for stimulation of cultured cells. The culture supernatant was obtained at three hours after additional of saline/leptin. MAIN OUTCOME MEASURES： Contents of LH and FSH were detected by radioimmunology. RESULTS： Following leptin stimulation, LH release increased with increasing concentrations of leptin up to 1×10^-9 mol/L, where LH release peaked. LH release then progressively decreased with increasing leptin concentrations （P 〈 0.01）. LH release in the leptin （1×10^-12, 1×10^-11, 1×10^-7, and 1×10^-6 mol/L） groups was significantly higher than that in the control group （P 〈 0.01）. FSH content in the leptin （1×10^-11, 1×10^-9, and 1×10^-7 mol/L） groups was significantly higher than that in the control group （P 〈 0.01）. CONCLUSION： Leptin can directly affect pituitary tissue to promote the secretion of LH and FSH in a dose-dependent manner.

Maturation, proliferation and apoptosis of seminal tubule cells at puberty after administration of estradiol, follicle stimulating hormone or both

Aim： To assess proliferative and apoptotic potential of the seminiferous epithelium cells in relation to Sertoli cell maturation in newborn rats under the influence of estradiol, follicle stimulating hormone （FSH） or both agents given together. Methods： From postnatal day （PND） 5 to 15 male rats were daily injected with 12.5 μg of 1713-estradiol benzoate （EB） or 7.5 IU of human purified FSH （hFSH） or EB ＋ hFSH or solvents （control）. On postnatal day 16, autopsy was performed. Sertoli cell maturation/function was assessed by morphometry. Proliferation of the seminiferous epithelium cells was quantitatively evaluated using immunohistochemical labeling against proliferating cell nuclear antigen and apoptosis using the TUN-EL method. Results： Although EB inhibited Sertoli cell maturation and hFSH was not effective, a pronounced acceleration of Sertoli cell maturation occurred after EB ＋ hFSH. Whereas hFSH stimulated Sertoli cell proliferation, EB or EB ＋ hFSH inhibited Sertoli cell proliferation. All treatments significantly stimulated germ cell proliferation. Apoptosis of Sertoli cells increased 9-fold and germ cells 2-fold after EB, and was not affected by hFSH but was inhibited after EB ＋ hFSH. Conclusion： At puberty, estradiol inhibits Sertoli cell maturation, increases Sertoli and germ cell apoptosis but stimulates germ cell proliferation. Estradiol in synergism with FSH, but neither of the hormones alone, accelerates Sertoli cell maturation associated with an increase in germ cell survival. Estradiol and FSH cooperate to induce seminal tubule maturation and trigger first spermatogenesis.

Association between Two Polymorphisms of Follicle Stimulating Hormone Receptor Gene and Susceptibility to Polycystic Ovary Syndrome: a Meta-analysis

Objective To investigate the association between two polymorphisms of follicle stimulating hormone receptor （FSHR） gene and polycystic ovary syndrome （PCOS） susceptibility. Methods Case-control studies on relationship of Thr307Ala and Asn680Ser polymorphisms in FSHR gene and PCOS susceptibility were searched from PubMed, ISI web of knowledge, EBSCO, and China National Knowledge Infrastructure （CNKI） databases up to March 21, 2013. The pooled odds ratio （OR） and 95% confidence interval （CO were calculated using fixed- or random-effect model based on heterogeneity test in 5 genotype models analyses. Results A total of 11 studies were included in the Meta-analysis. The random-effect analysis showed Asn680Ser was significantly associated with the reduced susceptibility to PCOS with dominant model （Asn/Asn＋Asn/Ser vs. Ser/Ser, OR=0.83, 95% CI： 0.69-1.00）, recessive model （Asn/Asn vs. Asn/Ser＋ Ser/Ser, OR=0.84, 95% CI： 0.72-0.98）, homozygote comparison （Ash/Ash vs. Ser/Ser, 0R=0.79, 95% CI： 0.63-0.98）, and the allele contrast （Asn vs. Ser, OR=0.87, 95% CI： 0.79-0.97） respectively（P=0.02, I2=56.0%）, being protective factors for PCOS. However, no significant associations were found between Thr307Ala and PCOS. Conclusion There might be a significant association between Asn680Ser polymorphism and PCOS.

Urinary follicle stimulating hormone can be used as a biomarker to assess male reproductive function

Aim: To develop an algorithm for use in population-based studies to assess testicular function by measurements of totalurinary follicle stimulating hormone (FSH). Methods: Total concentrations of urinary FSH were measured in a groupof 44 men at the University of California, Davis (UCD) and were compared to FSH measurements in serum. On thebasis of these and other published data, a urinary FSH value of >2 ng/mg creatinine (Cr) was selected as the cutoffpoint to identify men with elevated serum FSH ( > 12 IU/L) or low sperm counts ( < 20 million/mL). Results: Thesensitivity and specificity of this algorithm for detecting elevated serum FSH in a group of 58 agricultural workers in thePeople's Republic of China were 100 % and 50 %, respectively. The sensitivity and specificity of this algorithm fordetecting low sperm counts in a population of 105 infertility patients at UCD were 58 % and 76 %, respectively.Conclusion: This test may have particular value in identifying populations with no evidence of testicular toxicity, andin which labor-intensive semen studies may not be feasible.

Elevated thyroid stimulating hormone levels are associated with metabolic syndrome in a Chinese community-based population of euthyroid people aged 40 years and older

This study investigated whether high-normal thyrotropin（TSH） levels are associated with metabolic syndrome in euthyroid Chinese people≥40 years old.Clinical and metabolic factors were assessed in 2,356 subjects（40-77 years old） with TSH levels in the normal range（0.35-5.00 mU/L）.Using 2.50 mU/L as the cut-off point of TSH level within the normal range,we divided subjects into the high-TSH（2.50-5.00 mU/L;n= 1,064） and low-TSH（0.35-2.50mU/L;n= 1,292） group.The results showed that the mean levels of body mass index（BMI）,total cholesterol（TC）,low density lipoprotein cholesterol（LDL-C）,and fasting plasma glucose（FPG） were higher in the high-TSH group and TSH levels were significantly positively con-elated with BMI,LDL-C,TC,and FPG.The prevalence of central obesity,hypertriglyceridemia,low high density lipoprotein cholesterol（HDL-C）,and high FPG（〉5.60 mmol/L） was significantly higher in females and subjects with high-TSH levels.Metabolic syndrome was also more prevalent in the high-TSH group.People over the age of 40 years with high-normal TSH levels had a 1.2-fold increased risk of metabolic syndrome,compared with those with low-normal TSII levels,after adjusting for age and gender.In conclusion,high normal TSH is a risk factor for metabolic syndrome in people ≥40 years old.

Fluoride Exposure,Follicle Stimulating Hormone Receptor Gene Polymorphism and Hypothalamus-pituitary-ovarian Axis Hormones in Chinese Women

The effects of fluoride exposure on thefunctions of reproductive and endocrine systemshave attracted widespread attention in academiccircle nowadays. However, it is unclear whether thegene-environment interaction may modify thesecretion and activity of hypothalamus-pituitary-ovarian （HPO） axis hormones. Thus, the aim of thisstudy was to explore the influence of fluorideexposure and follicle stimulating hormone receptor（FSHR） gene polymorphism on reproductivehormones in Chinese women. A cross sectionalstudy was conducted in seven villages of HenanProvince, China during 2010-2011. A total of 679women aged 18-48 years were recruited throughcluster sampling and divided into three groups, i.e.endemic fluorosis group （EFG）, defluoridationproject group （DFPG）, and control group （CG） basedon the local fluoride concentration in drinkingwater. The serum levels of gonadotropin releasinghormone （GnRH）, follicle stimulating hormone（FSH）, luteinizing hormone （LH）, and estradiol （E2）were determined respectively and the FSHRpolymorphism was detected by real time PCR assay.The results provided the preliminary evidenceindicating the gene-environment interaction onHPO axishormones in women.

Follicle-stimulating hormone autoantibody is involved in idiopathic spermatogenic dysfunction

Aim： To detect the anti-follicle-stimulating hormone （FSH） antibody in idiopathic infertile patients and fertile subjects in order to determine the role of this antibody in patients with spermatogenic dysfunction. Methods： The anti-FSH antibody in serum was detected by an enzyme-linked immunosorbent assay （ELISA）. The functional and structural integrity of the sperm membrane was evaluated with hypo-osmotic swelling （HOS） test and the ultrastructure of the spermatozoa was investigated by transmission electron microscopy （TEM）. Results： The extent of positive FSH antibody in the patients with oligozoospermia and/or asthenozoospermia was significantly higher than that in the fertile subjects and infertile patients with normal sperm concentration and motility, but it was significantly lower than that in the patients with azoospermia. The extent of anti-FSH antibody in the patients with azoospermia was significantly greater than that in patients with oligospermia and/or asthenospermia, infertile people with normal sperm density and motility and fertile people. The hypo-osmotic swelling test showed that the percentage of HOS-positive spermatozoa （swollen） was 45.1% ±3.5% in the FSH antibody-positive group and 59.1% ± 6.2% in the FSH antibody-negative control group. The percentage of functional membrane damage to spermatozoa was significantly higher in the anti- FSH antibody-positive group than in the control group. TEM showed that the outer acrosomal membrane was located far from the nucleus, and detachment of the acrosome was found in the FSH autoantibody-positive group. Conclusion： These data suggest that the presence of anti-FSH antibody is strongly correlated with the sperm quantity and quality in idiopathic male infertility. Anti-FSH antibody may be an important factor causing spermatogenic dysfunction and infertility.

The preparation and application of N-terminal 57 amino acid protein of the follicle-stimulating hormone receptor as a candidate male contraceptive vaccine

Follicle-stimulating hormone receptor （FSHR）, which is expressed only on Sertoli cells and plays a key role in spermatogenesis, has been paid attention for its potential in male contraception vaccine research and development. This study introduces a method for the preparation and purification of human FSHR 57-amino acid protein （FSHR-57aa） as well as determination of its immunogenicity and antifertility effect. A recombinant pET-28a（＋）-FSHR-57aa plasmid was constructed and expressed in Escherichia coil strain BL21 StarTM （DE3） and the FSHR-57aa protein was separated and collected by cutting the gel and recovering activity by efficient refolding dialysis. The protein was identified by Western blot and high-performance liquid chromatography analysis with a band of nearly 7 kDa and a purity of 97.4%. Male monkeys were immunized with rhFSHR-57aa protein and a gradual rising of specific serum IgG antibody was found which reached a plateau on day 112 （16 weeks） after the first immunization. After mating of one male with three female monkeys, the pregnancy rate of those mated with males immunized against FSHR-57aa was significantly decreased while the serum hormone levels of testosterone and estradiol were not disturbed in the control or the FSHR-57aa groups. By evaluating pathological changes in testicular histology, we found that the blood-testis barrier remained intact, in spite of some small damage to Sertoli cells. In conclusion, our study demonstrates that the rhFSHR-57aa protein might be a feasible male contraceptive which could affect sperm production without disturbing hormone levels.

Evaluation of steroidogenic capacity after follicle stimulating hormone stimulation in bovine granulosa cells of Revalor 200~ implanted heifers

Background： Heifers not used as breeding stock are often implanted with steroids to increase growth efficiency thereby altering hormone profiles and potentially changing the environment in which ovarian follicles develop. Because bovine granulosa cell culture is a commonly used technique and often bovine ovaries are collected from abattoirs with no record of implant status, the objective of this study was to determine if the presence of an implant during bovine granulosa cell development impacts follicle stimulating hormone-regulated steroidogenic enzyme expression. Paired ovaries were collected from 16 feedlot heifers subjected to 1 of 3 treatments： non-implanted （n = 5）, Revalor 200 for 28 d （n = 5）, or Revalor 200 for 84 d （n = 6）. Small follicle （1 to 5 mm） granulosa cells were isolated from each pair and incubated with phosphate buffered saline （n = 16） or 100 ng/mL follicle stimulating hormone （n = 16） for 24 h. Results： Granulosa cells of implanted heifers treated with follicle stimulating hormone produced medium concentrations of progesterone similar （P = 0.22） to non-implanted heifers, while medium estradiol concentrations were increased （P 〈 0.10） at 28 and 84 d compared to non-implanted heifers indicating efficacy of treatment. Additionally, real-time PCR analysis in response to follicle stimulating hormone treatment demonstrated a decrease in steroidogenic acute regulatory protein （P = 0.05） mRNA expression in heifers implanted for 84 d and an increase in P450 side chain cleavage mRNA in granulosa cells of heifers implanted for 28 （P 〈 0.10） or 84 d （P 〈 0.05） compared to non-implanted females. However, no difference in expression of 3-beta-hydroxysteroid dehydrogenase （P= 0.57） and aromatase （P = 0.23） were demonstrated in implanted or non-implanted heifers. Conclusions： These results indicate follicles which develop in the presence of high concentrations of androgenic and estrogenic steroids via an implant tend to demonstrate an altered capacity to respond to follicle stimulating hormone stimulation. Thus, efforts should be made to avoid the use of implanted heifers to study steroidogenesis in small follicle granulosa cell culture systems.

Stability of Human Follicle-Stimulating Hormone Receptor mRNA in Stably Transfected Cells

In order to assess the impact of mRNA degradation on steady state levels of follicle stimulating hormone receptor (FSHR) mRNA and on regulation of FSHR gene expression, the stability and half life of FSHR mRNA were determined in transfected cells expressing recombinant FSHR. Time dependent changes in FSHR mRNA content were determined by nuclease protection solution hybridization assay (NPA) or by qualitative reverse transcription competitive polymerase chain reaction (RT PCR) in cultured hFSHR YI cells, cell lines stably transfected with a human FSHR cDNA. FSHR mRNA content remained constant during 8 h control incubations of hFSHR Y1 cells (NPA, 2.9±0.3 μg/mg RNA; RT PCR, 2.7±0.3 μg/mg RNA). Actinomycin D (ActD, 5 μg/ml) inhibited mRNA synthesis, as assessed by incorporation of uridine into total RNA, by 90 % within 1 h in hFSHR Y1 cells. No effect of ActD on cellular morphology or viability was observed. ActD caused a time dependent decrease in FSHR mRNA content in hFSHR Y1 cell lines with a lag time of 1 h. There were no significant differences in the rate of FSHR mRNA degradation between the two methods of mRNA quantification. The half life of hFSHR mRNA was 3.6±0.2 h by NPA and 3.1±0.1 h by RT PCR. The results indicated that degradation of mRNA was an important process in maintenance of steady state expression of the FSHR gene in cells stably expressing recombinant receptor.

Quantification of Porcine Follicle-stimulating Hormone Receptor Messenger Ribonucleic Acid by Reverse Transcription competitive Polymerase Chain Reaction

An easy and reliable method was developed for construction and quantification of competitive templates, which shared the same sequence as the amplified target DNA except for a 20 bp insertion in the middle by recombinant polymerase chain reaction (PCR). Among the advantages of competitive PCR is that any predictable or unpredictable variable that affects amplification has the same effect on both target and competitor species and that the final ratio of amplified products reflects exactly the initial targets. The utilization of a thermostable reverse transcriptase in the RT step was proposed to overcome the problem of the efficiency of target cDNA synthesis. In addition, to obtain reliable measurements, it was recommended to perform four PCR with amounts of competitive template flanking the concentration of the target mRNA.

Cloning and Transcriptional Activity of Follicle-Stimulating Hormone Receptor Promoter in the Jintang Black Goat

[ Objective] To clone follicle-stimulating hormone receptor （FSHR） promoter in the Jintang black goat, study its transcriptional activity, and provide a basis for alternative splicing of FSHR gene. [Method] The total DNA were extracted from the womb of Jintang black goat, and one pair of primers were designed for amplification of FSHR promoter fragments, then the sequences and homology were analyzed. The FSHR promoter fragment was inserted into the pcFSHRB1 expression vector to substitute the CMV promoter and construct the pcFSHRB2 expression vector. The pcFSHRB1 and pcFSHRB2 expression vectors were transformed into HEK293 cells, respectively. Then these cells were collected after 24 and 48 h treatment with 2 mlU/ml follicle-stimulating hormone （FSH）, and the cAMP levels were detected. [Result] The FSHR promoter sequence of Jin- tang black goat had 34.2% homology to that of chicken and 41.6% to that of rat, respectively. The transcription initial site of FSHR was at -576 bp and its upstream sequences contained two TATA-boxes, four CAAT-boxes, one E-box and one Wl-box. After treating for 24 and 48 h, the cAMP levels of pcFSHRB2 were respectively 299.581 3 and 125.528 1 pmol/L; and that of pcFSHRB1 were respectively 120.057 1 and 109.940 7 pmoVL. [Conclusion] The FSHR promoter of Jintang black goat is a typical type 2 eukaryotic promoter, and it is also a strong promoter.

Follicle-stimulating Hormone(FSH) Induced Internalization of Porcine FSH Receptor in Cultured Porcine Granulosa Cells and Chinese Hamster Ovary Cells Transfected with Recombinant Porcine FSH Receptor cDNA

In order to study the fate of human follicle-stimulating hormone (FSH) when hormone binds to its receptor, a quick biochemical method that can differentiate between the surface-bound and internalized hormone was used to determine the internalization induced by FSH in cultured both porcine granulosa cells and Chinese hamster ovary (CHO) cells expressing recombinant porcine FSH receptor. The results showed that FSH was slowly internalized, and the internalized radioactivity (acid resistant) reached a peak 10-12 h after addition of 125 I-hFSH. It was suggested that FSHR do not get internalized rapidly under physiological circumstances precisely because the appropriate sequences are absent.

Effect of Follicle-Stimulating Hormone Dosage, Seasons and Treatment Frequency on Super-Ovulation in Goats

[ Objective] To explore the effects of FSH （ Follicle-stimulating hormone）, seasons and treatment frequency on super-ovulation in donor goats and to provide necessary data for research about embryo transfer and embryo biotechnology in Yangzhou area. [ Method] Two FSH dosages （240 and 300 I U）, two seasons （April- June and October- December）, and two treatment frequencies （one or two times） were used to induce super-ovulation in goats. [ Result] Both average ovulation point and number of transferable embryos were significantly different between the goats given 240 IU FSH and those given 300 lU FSH （average ovulation point, 10.12 vs 15.55; number of transferable embryos, 8.82 vs 13.15） at the 0.05 level. Both average ovulation point and number of transferable embryos were also significantly different between April -June and October- December （ average ovulation point, 9.05 vs 15.55; number of transferable embryos, 7.05 vs 13.15） at the 0.05 level. Super-ovulation effect was not significantly different between the two treatment frequencies. [ Conduslonl The FSH dosages and seasons have significant impact on super-ovu- lation, but repeat super-ovulation does not have the same impact.

The Determination and Evaluation of the Biological Activities for the Commercialization of Recombinant Follicle-Stimulating Hormone in Vitro

Follicle-stimulating hormone (FSH) plays a central role in mammals reproduction, with the actions of FSH mediated by follicle-stimulating hormone receptors (FSHRs) on the surface of target cells. The purposes of this study were to determine and evaluate the biological activities for the commercialization of recombinant follicle-stimulating hormone (rFSH) in vitro through the cellular internalization using cloned 293T-FSHR cell lines as target. Using imaging approaches we have found here that a little fluorescent signal from the surface of the cell transferred to the cytoplasm and accumulated around the nucleus by endocytosis. Compared with the control groups, the commercialization of rFSH have not the significant differences of internalization, but the rFSH have promoted the internalization of the fluorescent, suggested that this detection system might as a protocol for the bioactivity of recombinant therapeutic proteins in vitro.

Effects of Antisense Oligodeoxynucleotide to Follicle-stimulating Hormone Receptor on the Cell Proliferation and Apoptosis in Cells Derived from Human Ovarian Mucinous Cystadenocarcinoma in Vitro

The human ovarian mucinous cystadenocarcinoma （hOMC） cells were co-cultured with antisense oligodeoxynucleotide （antisense ODN）, nonsense ODN, and follicle-stimulating hormone （FSH） at different concentrations for the purpose of observing the effects of antisense ODN to FSH receptor （FSHR） on the proliferation and apoptosis of cultured hOMC cells in vitro. The inhibitory rates of growth were measured by using MTT method on the 2nd, 4th, 6th, 8th and 10th days after the interference of antisense ODN, nonsense ODN, and FSH, respectively. The apoptotic rates and the cell cycles were determined by means of flow cytometry, the apoptosis indexes were detected by using TUNEL, and the expression of caspase-3 was measured by using SP immunohistochemistry. Compared with that in the control group, the proliferative activity of hOMC cells was increased obviously in FSH groups （P〈0.05 or P〈0.01）, decreased distinctly in antisense ODN groups （P〈0.05 or P〈0.01）, and unchanged in nonsense ODN groups, respectively. Meanwhile, antisense ODN could significantly antagonize the FSH-promoted cell proliferative activity （P〈0.01）. Compared with those in the control group, the apoptotic rates and the expression of caspase-3 were dramatically increased in the mid- and high-dose antisense ODN groups （P〈0.05 or P〈0.01）, while the number of cells in G1/G0 phase was significantly decreased and that in S phase distinctly increased （P〈0.01）, There was no change in nonsense ODN groups （P〉0.05）, It was suggested that FSH may improve the development of hOMC cells, However, antisense ODN could inhibit proliferative activity and the FSH-promoted proliferative activity in hOMC cells, at the same time, antisense ODN could inhibit hOMC cell growth by inducing apoptosis.

Cloning and Expression Level Analysis of Melanocyte-stimulating Hormone Receptor 1 Gene(MC1R) in Alpacas with Different Coat Color

Specific primers for the MC1R gene of alpacas(GenBank EU1358800) were designed to amplify the cDNA sequence using RT-PCR to seek variation in the sequence and explore the relationship between the expression level of MC1R gene and alpaca coat color.The MC1R gene from white alpaca was cloned successfully and sequence analysis verified that the MC1R gene,encoding 317 amino acids,was 1081 bp in length.Compared with the existing sequence in GenBank,sequence identity was 99.9%and 7 mutations were found.Primers,designed from the sequence obtained,were used to assess the relative expression of MC1R in alpacas of different coat color using QRT-PCR and SPSS 13.0 software.Relative expression of MC1R in the skin of brown alpacas was 4.32 times higher than that in white alpacas after normalization with GAPDH(P【0.01),indicating that MC1R expression may be related to coat color of alpacas.

Polymorphism of Follicle-Stimulating Hormone Receptor Gene in Ningxia Tan Sheep

[ Objective] To study the polymorphism of follicle-stimulating hormone receptor （FSHR） gene in Ningxia Tan sheep and thus to provide a theoretical basis for breeding. I Methodl Genotypes of 111 healthy Ningxia Tan sheep were examined by polymerase chain reaction and restriction fragment length polymorphism （PCR-RFLP）. [ResultS] A 306-bp fragment was amplified. The PCR products digested with restriction enzyme Alu I showed polymorphism with three genotypes, L e., GG, CG and CC. The genotypic frequencies of GG, CG and CC were 0.135 1 （ 15 individuals）, 0.666 7 （74 individuals） and 0.198 2 （22 individuals）, respectively. The allele frequencies of G and C were 0.468 5 and 0.531 5, respectively.[ Conclusion] FSHR aene is Dolvmomhic in Ninaxia Tan Sheeo.

Gonadotrophin-releasing hormone-Ⅰ and -Ⅱ stimulate steroidogenesis in prepubertal murine Leydig cells in vitro

Aim： To study the effect and mechanism of gonadotrophin-releasing hormone （GnRH） on murine Leydig cell steroidogenesis. Methods： Purified murine Leydig cells were treated with GnRH-Ⅰ and -Ⅱ agonists, and testosterone production and steroidogenic enzyme expressions were determined. Results： GnRH-Ⅰ and -Ⅱ agonists significantly stimulated murine Leydig cell steroidogenesis 60%-80% in a dose- and time-dependent manner （P 〈 0.05）. The mRNA expressions of steroidogenic acute regulatory （STAR） protein, P450scc, 3β-hydroxysteroid dehydrogenase （HSD）, but not 17β-hydroxylase or 17β-HSD, were significantly stimulated by both GnRH agonists with a 1.5- to 3-fold increase （P 〈 0.05）. However, only 3β-HSD protein expression was induced by both GnRH agonists, with a 1.6- to 2-fold increase （P 〈 0.05）. Conclusion： GnRH directly stimulated murine Leydig cell steroidogenesis by activating 3β-HSD enzyme expression.