Cloning and Expression of Gonadotrophin Releasing Hormone Receptor in Apis cerana cerana

[Objective] This study was to clone the GnRH （gonadotropin-releasing hormone）, and to investigate its expression in Apis cerana cerana. [Method] The cDNA sequence of GnRHR gene was amplified from Apis cerana cerana by using RT-PCR techniques. It was conducted with bioinformatics analysis and the in situ hybridization histochemistry of its expression products was studied. [Result] The sequence analy- sis showed that the full cDNA sequence was 1 050 bp with the open reading frame of 1 050 bp, and it encoded 349 amino acid residues. The deduced amino sequence included 7 transmembrane regions, and the predicted molecular mass and isoelectric point were 40.6 kD and 9.54, respectively. The cluster analysis showed that the GnRHR from ＇,4. cerana cerana had close relationship to the GnRHR II from other insects. In situ hybridization showed that Bee-GnRHR staining was specifically localized to the brain, intestine, fat body and testis. [Conclusion] The results indicated that the GnRHR provided molecular bond for the reproduction and metabolism for insects, and suggested a functional role for bee-GnRHR signaling in the coupling of reproduction activities and environment conditions.

水貂缺乳的防治

水貂缺乳的原因大多是因为妊娠期和哺乳期饲料营养不足;妊娠期母貂过肥或过瘦;母貂患乳房炎、及其它重性疾病,致使乳汁分泌减少或停止;内分泌机能失调也可导致母貂缺乳或无乳。

New understandings of the genetic basis of isolated diopathic central hypogonadism

Idiopathic hypogonadotropic hypogonadism is a rare disease that is characterized by delayed/absent puberty and/or infertility due to an insufficient stimulation of an otherwise normal pituitary-gonadal axis by gonadotrophin-releasing hormone （GnRH） action. Because reduced or normal luteinizing hormone （LH）/follicle-stimulating hormone （FSH） levels may be observed in the affected patients, the term idiopathic central hypogonadism （ICH） appears to be more appropriate. This disease should be distinguished from central hypogonadism that is combined with other pituitary deficiencies. Isolated ICH has a complex pathogenesis and ~s fivefold more prevalent in males. ICH frequently appears in a sporadic form, but several familial cases have also been reported. This finding, in conjunction with the description of numerous pathogenetic gene variants and the generation of several knockout models, supports the existence of a strong genetic component. ICH may be associated with several morphogenetic abnormalities, which include osmic defects that, with ICH, constitute the cardinal manifestations of Kallmann syndrome （KS）. KS accounts for approximately 40% of the total ICH cases and has been generally considered to be a distinct subgroup. However, the description of several pedigrees, which include relatives who are affected either with isolated osmic defects, KS, or normo-osmic ICH （nlCH）, justifies the emerging idea that ICH is a complex genetic disease that is characterized by variable expressivity and penetrance. In this context, either multiple gene variants or environmental factors and epigenetic modifications may contribute to the variable disease manifestations. We review the genetic mechanisms that are presently known to be involved in ICH pathogenesis and provide a clinical overview of the 227 cases that have been collected by the collaborating centres of the Italian ICH Network.

Identification and distribution of gonadotropin-releasing hormone-like peptides in the brain of horseshoe crab Tachypleus tridentatus

Gonadotropin-releasing hormone （GnRH） is a crucial peptide for the regulation of reproduction. Using immunological techniques, we investigated the presence of GnRH in horseshoe crab Tachypleus tridentatus. Octopus GnRH-like immunoreactivity, tunicate GnRH-like immunoreactivity, and lamprey GnRH-I-like immunoreactivity were detected in the neurons and fibers of the protocerebrum. However, no mammal GnRH-like immunoreactivity or lamprey GnRH-LII-like immunoreactivity was observed. Our results suggest that a GnRH-like factor, an ancient peptide, existed in the brain of T. tridentatus and may be involved in the reproductive endocrine system.

Recent Possibilities for the Diagnosis and Pharmacological Control of Pregnancy Loss in Dairy Cow

One of the most recent techniques for the diagnosis of EP （early pregnancy） in cattle on the farm is B-mode ultrasonography. Under field conditions, acceptable results may be achieved with ultrasonography from days 25 to 30 post-AI. The reliability of the test greatly depends on the frequency of the transducer used, the skill of the examiner, the criterion used for a positive PD （pregnancy diagnosis） and the position of the uterus in the pelvic inlet. Pregnancy protein assays （PAG, PSPB） may provide an alternative method to ultrasonography for determining EP or LEM/EFM （late embryonic/early foetal mortality） in the cow. Although early pregnancy factor is the earliest specific indicator for fertilization, its detection is entirely dependend on the use of the RIT （rosette inhibition test）, therefore its use in the field needs further inventions. Preventive pharmaceutical treatments with hCG （human chorionic gonadotropin）, GnRH （gonadotropin realising hormone）, PRID （progesterone-releasing intravaginal device） or CIDR （controlled internal drug release） inserts at different time periods at or post-AI may have some benefit in decreasing PL （pregnancy loss）, however further examinations are warranted to determine how and when these treatments may influence PL in the field.

The Characteristics of vasa Gene from Japanese Sea Bass(Lateolabrax japonicas) and Its Response to the External Hormones

The RNA helicase Vasa is an important regulator of primordial germ cell development. Its function in mature fish, espe- cially the hormone-related differences in maturing male fish has seldom been documented. In this study, a full length cDNA sequence of the vasa gene was cloned from Japanese sea bass, Lateolabraxjaponicas, and it was namedjsb-vasa. Homology analysis showed thatjsb-vasa was closely related to its teleost homologs. The spatial distribution ofjsb-vasa indicated that it was only highly ex- pressed in testis, showing its germ cell-specific expression pattern. During the testicular development cycle, jsb-vasa was highly expressed during early period of spermatogenesis, and reduced when spermatogenesis advanced. In addition, the jsb-vasa gene ex- pression was significantly inhibited at 6 h, 12 h and 24 h after injecting hCG （human ehorionic gonadotropin） and GnRHa （Gonad- otropin-releasing hormone analogue）, indicating thatjsb-vasa gene may play an important role in spermatogenesis of Japanese sea bass, and be under the regulation of external sex hormones.

垂体下调药物剂量与卵巢血流阻力关系的研究

目的 探讨卵巢刺激周期垂体下调药物剂量与卵巢血流阻力指数的关系。方法 将 30例接受体外受精 -胚胎宫腔内移植的不育女性随机分为大剂量垂体下调药物组 (A组 ,达必佳 10 0 μg/d) ,及小剂量垂体下调药物组 (B组 ,达必佳 5 0 μg/d) ,两组均于月经周期第 8d及注射人绒毛膜促性腺激素 (HCG)日进行经阴道彩色多普勒超声检查 ,测量双侧卵巢动脉阻力指数 (RI)及卵泡发育情况。结果 B组月经周期第 8d卵巢动脉RI、应用人绝经期促性腺激素 (HMG)总量及天数显著小于A组。所有病例月经周期第 8d及注射HCG日含直径 >10mm卵泡较多一侧卵巢动脉的RI显著小于对侧。但两组妊娠率差异无显著性意义。结论 卵巢刺激周期应用低剂量垂体下调药物可降低卵泡期卵巢动脉RI ,减少促性腺激素使用天数及总量 ,但不影响妊娠成功率。

Central administration of Orphanin FQ inhibits GnRH secretion by ORL1 receptor in the median eminence of freely moving ovariectomized rats

Objective This study aimed to investigate the possible role of Orphanin FQ(OFQ) in the regulation of hypothalamic gonadotropin-releasing hormone(GnRH) secretion. Methods The method of push-pull perfusion and radioimmunoassay(RIA) were adopted to examine the secretory profile of GnRH in the median eminence(ME) in freely moving ovariectomized(OVX) rats after intracerebroventricular(icv) injection of OFQ and/or [Nphe1]NC(1-13)NH2(NC13),a competitive antagonists of the opioid receptor-like 1 receptor(ORL1 recep...

Correlation of expression of preprothyrotropin-releasing hormone and receptor with rat testis development

Objective To investigate the expression regulation of thyrotrophin-releasing hormone (TRH) and TRH receptor (TRH-R), and their role in the development of rat testis.Methods Oligonucleotide primers were designed from the sequences of rat hypothalamus prepro TRH (ppTRH) and pituitary TRH-R cDNA for reverse transcription polymerase chain reaction (RT-PCR). Specific fragments of ppTRH and TRH-R cDNA were cloned and sequenced. Expression plasmids containing ppTRH and TRH-R genes were then constructed, and expression was found in E.coli DH5-α. ppTRH and TRH-R mRNA in the testis was quantitated in RNA samples prepared from rats at different developmental stages by real time quantitative RT-PCR.Results The quantitative analyses demonstrated that no ppTRH and TRH mRNA could be detected at the earliest stage (day 8). ppTRH and TRH mRNA signals were detected on day 15 and increased progressively on days 20, 35, 60 and 90. Conclusion Our results suggest that rat testis could specifically express TRH and TRH-R, and the transcriptions of ppTRH and TRH-R genes in the rat testis were development-dependent. The acquirement of expressed products for ppTRH and TRH-R can be used for further research on the physiological significance of TRH and TRH-R expression in rat testis.

The cAMP-mediated protein kinase signal transduction pathway is involved in the pyrogenic effect of CRH in rats

To determine whether the cyclic adenosine monophosphate (cAMP) mediated protein kinase signal transduction pathway is involved in the pyrogenic action of corticotropin releasing hormone (CRH) in rats.Methods Corticotropin releasing hormone, 2', 3 '-dideoxyadenosine (DDA) and adenosine-3', 5'(cyclic) monophosphorothionate, Rp-lsomer (Rp-cAMPS), were administered intracerebroventricularly (i.c.v.). The colonic temperature was measured using a thermistor, and the content of cAMP in the hypothalamus was determined by radioimmunoassay. Hypethalemic incubation was used to assess the effects of CRH on the content of cAMP in the hypothalamus in vitro.Results Microinjection (i.c.v.) of CRH (2.5 μg, 5.0 μg and 10 μg) caused increases in colonic temperature and the hypothalemus cAMP level in conscious rats. CRH increased hypothalemus cAMP level in vitro. The pyrogenic effects of CRH were abolished or markedly inhibited by prior injection (i. c. v. ) of an adenylate cyclase inhibitor, DDA (30 μg), or an inhibitor of cAMP-dependent protein kinase, Rp-cAMPS (15 μg).Conclusion cAMP mediates the pyrogenic action of centrally administered of CRH in rats, and protein kinase A may play an important role in the central CRH-induced fever. The cAMP-dependent protein kinase signal transduction pathway may be involved in the central mechanisms of the pyrogenic action of CRH in rats.

Characterization of urocortin in human pregnancy

Objective To examine whether urocortin is produced locally to regulate utero-placental vascular tone during pregnancy.Methods We examined the distribution of urocortin in human placenta, fetal membranes and uterine tissue at term in the presence and absence of labor using a urocortin antibody produced in our laboratory and the immunoperoxidase staining method. Subsequently, we tested urocortin secretion from chorio-decidual cells in vitro using an immunoblot technique. Then, we tested whether urocortin is present in maternal plasma throughout gestation using a radioimmunoassay. A Sephadex G-50 column was used to examine whether immunoreactive urocortin (IR-urocortin) in maternal plasma is the same as synthetic urocortin.Results IR-urocortin was observed in vascular smooth muscle of myometrium decidual stromal cells, syncytiotrophoblast and amnion epithelium. No differences in staining intensity for urocortin were detected between tissues obtained in the absence or presence of labor. Staining intensity for IR-urocortin was greatest in the decidua, suggesting this may be the main site of urocortin production. Positive staining for urocortin was observed in 40% of chorio-decidual cells with 34% of these cells secreting urocortin under basal conditions. Urocortin was detectable in maternal plasma from 16 weeks gestation and concentrations did not change as gestation progressed. IR-urocortin in the maternal plasma eluted from a Sephadex G-50 column at the same site as synthetic urocortin and had a calculated retention co-efficient of 0.44.Conclusion This study indicates that urocortin is produced by the decidua during human pregnancy and is detectable in maternal plasma. These data are consistent with the hypothesis that urocortin is produced locally by the decidua and may act to regulate utero-placental blood flow.

Bilateral regulatory action of corticotropin-releasing hormone on immune-mediated inflammation

In trauma, infection and hemorrhagic shock derived stress, primary and secondary injury may result in severe derangement in the internal environment. The abnormal changes of immune-mediated inflammation interfere its pathogenesis and development directly. In recent years, various aspects of neuroendocrine responses, especially the regulatory effects of hypothalamic-pituitary-adrenal and sympathetico-adrenomedullary axes in inflammatory diseases have been the focus of research. Most importantly,corticotropin-releasing hormone （CRH） acts as a key player in the regulation of interactions between neuroendocrine and immunity both directly and indirectly. The paper summarized the recent development of CRH in the immune-mediated inflammation.

Effect of soothing liver therapy on oocyte quality and growth differentiation factor-9 in patients undergoing in vitro fertilization and embryo transfer

OBJECTIVE:To investigate the effect of Soothing liver therapy on infertile women undergoing in vitro fertilization and embryo transfer(IVF-ET)and to explore its mechanism.METHODS:Fifty-eight women with tubal infertility were randomized into two groups:30 in an experimental group treated with Xiaoyao powder(Shugan)plus gonadotropin-releasing hormone analog(GnRHa)/follicle-stimulating hormone(FSH)/human chorionic gonadotropin(hCG)and 28 in the control group who were treated with GnRHa/FSH/hCG only.The total gonadotropin(Gn)doses required,endometrial thickness,oocyte numbers,high quality embryo production rate and pregnancy rate of the two groups were compared.The concentration of growth differentiation factor-9(GDF-9)in follicular fluid was detected by western blotting and the expression of GDF-9 mRNA in granulosa cells was measured using reverse tran-scription-polymerase chain reaction amplification.RESULTS:In the experimental group,the Gn dose was significantly lower than that in the control group;the endometrial thickness,high quality embryo production and pregnancy rates were significantly higher and the expression of GDF-9 mRNA was also significantly higher than in the control group(all P<0.05).CONCLUSION:Shugan treatment can improve the pregnancy rate of women with tubal infertility;its mechanism is possibly related to the increased expression of GDF-9 in granulosa cells.

Anxiety-related cell-type-specific neural circuits in the anterior-dorsal bed nucleus of the stria terminalis

The bed nucleus of the stria terminalis(BNST) plays a critical role in regulating anxiety, yet the involved specific cell types and their connections functioning in anxiety-related behaviors remains elusive. Here we identified two cell subpopulations—corticotropin-releasing hormone-positive(CRH+) and protein kinase C-δ-positive(PKC-δ+) neurons—each displayed discrete emotionally valenced behaviors in the anterior-dorsal BNST(ad BNST). Using whole-cell patch-clamp recordings and virus-assisted circuit tracing techniques, we delineated the local and long-range connectivity networks in a cell-type-specific manner. The results show that the CRH+ and PKC-δ+ neurons received inputs from similar brain regions and exhibited significant differences in the downstream projection density. In addition, in vivo calcium imaging as well as gain-and loss-of-function studies characterized the physiological response properties and the functional heterogeneities in modulating anxiety, further suggesting the similarity and individuality between the two ad BNST cell types. These results provide novel insights into the circuit architecture of ad BNST neurons underlying the functionally specific neural pathways that relate to anxiety disorders.

Effect of Chaihushugan San on expression of the Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase pathway in the hippocampi of perimenopausal rats induced by immobilization stress

OBJECTIVE: We wished to study the impact of Chaihushugan San(CSS) on the behavior of perimenopausal rats with liver-Qi stagnation(LQS) and to investigate the effect of CSS on signal transduction of the Raf/mitogen-activated protein kinase(MEK)/extracellular signal-regulated kinase(ERK) cascade in the hippocampi of rats induced by immobilization.METHODS: Twenty 52-week-old female rats were divided into two groups by the random number table method: model control group(MCG) and CSS group(CSSG), with 10 rats in each group.Ten-week-old female rats were used as the normal control group(NCG). CSS effects were assessed using rats exposed to immobilization stress by measuring body weight and sucrose consumption, serum hormone levels, and observing performance in the open field test(OFT). Molecular mechanisms were examined by measuring the effect of CSS on expression of Raf1, MEK1/2 and ERK1/2 m RNA in hippocampi using quantitative real-time polymerase chain reaction and by measuring levels of these proteins and related phospho-proteins using Western blotting.RESULTS: Perimenopausal rats with LQS had decreased locomotor activity; reduced sucrose consumption; and increased serum levels of corticotropin releasing hormone(CRH) and corticosterone(CORT). Activation of hippocampal Raf/MEK/ERK cascade was suppressed significantly in the MCG,and activation was increased after 21 days of CSS treatment.CONCLUSION: CSS has significant effects upon relief of the symptoms of LQS in immobilization-induced rats. The mechanism underlying this action might(at least in part) be mediated by reversal of disruption of the Raf/MEK/ERK pathway.

Endogenous glucocorticoid increases the basal level of Treg-Th17 balance under early phase of stress

Objective： To explore the changes of Treg-Thl 7 balance influenced by corticosterone, major ef- fect hormone ofhypothalamic-pituitary-adrenal （HPA） axis under running stress. Methods： Atotal of 25 corticotropin-releasing hor- mone （CRH） wildtype （CRH＋/＋） and knockout （CRH-/-） mice were adopt and divided into 4 groups as follows： CRH＋/＋ ctrl, CRH＋/＋ stress, CRH-/- ctrl and CRH-/- stress. All mice in stress groups were under 2 h running. After 1 h, blood plasma in all groups was collected and the ex- pression of corticosterone and IL- 17A was detected by ELISA. Meanwhile, unicell suspensions of peripheral lymph node and spleen in each group were prepared too and stained by PE-CD4 and FITC-CD25, then the changes of Treg （CD4＋CD25＋） in different groups were checked by flow cytometry; all data were statistically analyzed by the software of WinMDI 2.9, SPSS 11.5, Origin 7.5 and Matlab 2-D and 3-D plot function. Results： The levels of corticosterone were signifi- cantly higher in stress groups than that in correspond- ing control groups （P〈0.05）, especially in CRH＋/＋ stress group （P〈0.01）. However, the changes of Tregs were not obvious between stress groups and control groups with respective genotypes （P〈0.05）. Compared with that in CRH＋/＋ control group, the ratio of Treg and the expres- sion of IL-17A in CRH-/- stress group were significantly higher than those in control group （P〈0.05）. Combined with the expression levels of corticosterone, Treg and Thl7, our study suggests that endogenous glucocorti- cold with basal level may cause the changes in Treg- Thl7 balance. Moreover, as the corticosterone level increases, the expression of Treg and Thl7 appears to manifest antagonistic fluctuant status with a rising ten- dency in general. Conclusion： Endogenous glucocorticoid under early stage of stress may increase the function of T lymphocyte immunity to some extent.