Generation of transgenic rats expressing enhanced green fluorescent protein in gonadotropin-releasing hormone neurons

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons govern reproductive function by controlling the release of gonadotropins from the pituitary. To facilitate identification of living GnRH neurons, here we attempted to generate transgenic rats that express enhanced green fluorescent protein (EGFP) in GnRH neurons. About 3 kb of rat GnRH promoter region was inserted into the EGFP reporter cassette, and the expression of EGFP fluorescence was confirmed in several cell lines following transient transfection. Then we successfully generated a transgenic rat by injecting linearized GnRH-EGFP transgene into the pronuclei of fertilized oocytes. The GnRH-EGFP transgenic rats expressed EGFP in the brain, but not in the ovary, testis or thymus. Immunohistochemical examination revealed that detectable EGFP fluorescence was confined to the cell body of GnRH-immunoreactive neurons in the septum and preoptic area, while no EGFP signal was discernible in the median eminence where abundant GnRH-immunoreactive fibers were observed. The mean percentage of EGFP-positive cells in the GnRH-positive cells was 76.3%. The GnRH-EGFP transgenic rats generated in the present study will enable characterization of properties of individual GnRH neurons.     

Involvement of anteroventral periventricular metastin/kisspeptin neurons in estrogen positive feedback action on luteinizing hormone release in female rats

Metastin/kisspeptin, the KiSS-1 gene product, has been identified as an endogenous ligand of GPR54 that reportedly regulates GnRH/LH surges and estrous cyclicity in female rats. The aim of the present study was to determine if metastin/kisspeptin neurons are a target of estrogen positive feedback to induce GnRH/LH surges. We demonstrated that preoptic area (POA) infusion of the anti-rat metastin/kisspeptin monoclonal antibody blocked the estrogen-induced LH surge, indicating that endogenous metastin/kisspeptin released around the POA mediates the estrogen positive feedback effect on GnRH/LH release. Metastin/kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) may be responsible for mediating the feedback effect because the percentage of c-Fos-expressing KiSS-1 mRNA-positive cells to total KiSS-1 mRNA-positive cells was significantly higher in the afternoon than in the morning in the anteroventral periventricular nucleus (AVPV) of high estradiol (E(2))-treated females. The percentage of c-Fos-expressing metastin/kisspeptin neurons was not different between the afternoon and morning in the arcuate nucleus (ARC). Most of the KiSS-1 mRNA expressing cells contain ERalpha immunoreactivity in the AVPV and ARC. In addition, AVPV KiSS-1 mRNA expressions were highest in the proestrous afternoon and lowest in the diestrus 1 in females and were increased by estrogen treatment in ovariectomized animals. On the other hand, the ARC KiSS-1 mRNA expressions were highest at diestrus 2 and lowest at proestrous afternoon and were increased by ovariectomy and decreased by high estrogen treatment. Males lacking the surge mode of GnRH/LH release showed no obvious cluster of metastin/kisspeptin-immunoreactive neurons in the AVPV when compared with high E(2)-treated females, which showed a much greater density of these neurons. Taken together, the present study demonstrates that the AVPV metastin/kisspeptin neurons are a target of estrogen positive feedback to induce GnRH/LH surges in female rats.     

The role of noradrenaline in the generation of the preovulatory LH surge in the ewe

Increasing plasma estrogen (E) levels during the follicular phase of the estrous cycle trigger the pre-ovulatory surge of gonadotropin-releasing hormone (GnRH)/LH. Noradrenaline (NA)-producing cells of the brain stem are involved in regulating GnRH cells and project to the preoptic area (POA) and bed nucleus of stria terminalis (BnST). Input to GnRH cells may be direct or indirect, via relay neurons in the POA/BnST. To investigate this, we ascertained whether an ₁-adrenergic antagonist would block/delay the LH surge in ovariectomised (OVX), E-treated ewes. E benzoate (EB) (50 μg) was injected (i.m.) and Doxazosin (100 nmol/h) or vehicle was infused into the third ventricle 2–26 h after EB injection. Doxazosin reduced the magnitude of the LH surge, but did not affect timing. To determine if NA is released in the POA/BnST of cyclic ewes, we immunostained dopamine-β-hydroxylase (DBH) in terminal fields. Reduced numbers of varicosities staining for DBH indicates release of NA. The number of varicosities immunostained for DBH was reduced in the dorsal and lateral BnST during the follicular phase and during the preovulatory LH surge compared to the luteal phase. These data suggest that noradrenergic mechanisms are involved in generation of the GnRH/LH surge via projections to the BnST and relay to GnRH cells. Since Doxasozin reduced the magnitude of the LH surge in the E-treated OVX ewe, and release of NA in cyclic ewes occurred during the follicular phase of the estrous cycle, we speculate that NA is a permissive factor in surge generation. Thus, increased noradrenergic activity is not a trigger mechanism for initiation of the surge.     

Inducible expression of enhanced green fluorescent protein by interleukin-1α, interleukin-1β and Toll-like receptor 2 promoters in goat mammary epithelial cells in response to bacterial challenges

The development of a bacteria-inducible expression system has several advantages compared with persistent expression of anti-bacterial proteins in milk to prevent and treat mastitis. The present study determined whether mastitis responsive promoters could regulate enhanced green fluorescent protein (EGFP) expression in goat mammary epithelial cells (GMECs) in response to challenges with Escherichia coli, Staphylococcus aureus or Streptococcus agalactiae. The level of expression of interleukin (IL)-1α was significantly increased in GMECs challenged with E. coli, S. aureus or S. agalactiae compared with untreated GMECs. IL-1β was induced by E. coli and S. aureus, while Toll-like receptor 2 (TLR2) was induced by E. coli only.GMECs were transfected with IL-1α, IL-1β and TLR2 promoter-EGFP reporter gene lentiviral expression vectors and the levels of expression of EGFP were measured by flow cytometry and Western blot analysis after bacterial challenge. EGFP expression driven by the IL-1α and IL-1β promoters was higher in GMECs challenged with E. coli, S. aureus or S. agalactiae than in untreated GMECs. There were no differences in EGFP expression driven by the TLR2 promoter between GMECs challenged with S. aureus or S. agalactiae and untreated GMECs, but EGFP expression was significantly increased in GMECs challenged with E. coli. Overall, these results indicate that the promoters of some bacteria-inducible genes can regulate EGFP expression in GMECs in response to bacterial challenges. This bacteria-inducible expression strategy could be used for production of mastitis resistant animals by regulating the expression of anti-bacterial proteins in the mammary gland.     

Posttranslational processing of progonadotropin-releasing hormone (PROGnRH) in ewes

Gonadotropin-releasing hormone (GnRH) is released from hypothalamic neurons into the hypophyseal-portal blood system following enzymatic cleavage of the decapeptide from a large precursor (proGnRH) molecule. The purpose of this study was to determine whether the ability of GnRH-producing neurons to synthesize and/or process proGnRH differed during physiological states associated with a suppressed and enhanced release of GnRH in ewes. Tissues were collected from ovariectomized ewes (OVX, N=4), OVX-estradiol treated ewes (OVX-E, N=5), and ewes (n=7) slaughtered 5 d after parturition (PP). Following euthanasia and exsanguination, stalk-median eminence (SME), medial-basal hypothalamus (MBH) and preoptic areas (POA) were collected. Concentrations of GnRH and proGnRH were determined by radioimmunoassay using specific antisera. Concentrations of GnRH in the SME did not differ (P>.05) between OVX-E and OVX ewes, but both groups contained less (P<.05) GnRH than the SME from PP ewes (4.4 ± 0.7, 12.1 ± 3.8 vs 24.3 ± 5.1 fmol/mg tissue, respectively). Concentrations of proGnRH in SME mimicked those of GnRH and were less (P<.05) in OVX-E ewes than PP ewes, but were not different (P>.05) from those in OVX ewes (.34 ± .34 vs 3.76 ± 1.53 and 1.7 ± .78 fmol/mg, respectively). In the MBH, OVX-E ewes had greater (P<.05) concentrations of GnRH than PP ewes (0.76 ± 0.29 vs 0.24 ± 0.04 fmol/mg) and OVX ewes were intermediate (0.41 ± 0.13 fmol/mg). No differences (P>.05) in concentrations of GnRH in the POA were detected among groups. Concentrations of proGnRH in MBH and POA were not different (P>.05) among groups. In summary, proGnRH is present in the SME which contains nerve terminals of GnRH-producing cells. Although concentrations of proGnRH and GnRH in the SME and MBH were affected by physiological state, ratio's of the prohormone:mature decapeptide remained constant. Therefore, alterations in posttranslational processing of the prohormone leading to formation of the mature GnRH-decapeptide were not demonstrated.     

Central injection of exogenous IL-1β in the control activities of hypothalamic-pituitary-gonadal axis in anestrous ewes

This study was performed to determine the effect of intracerebroventricular (icv) injection of interleukin (IL)-1β on the gene expression, translation and release of gonadotropin-releasing hormone (GnRH) and the GnRH receptor (GnRHR) gene expression in the hypothalamus of anestrous ewes. In the anterior pituitary gland (AP), the expression of genes encoding: GnRHR, β subunits of luteinizing hormone (LH) and folliculotropic hormone (FSH) was determined as well as the effect of IL-1β on pituitary gonadotropins release. The relative mRNA level was determined by real-time PCR, GnRH concentration in the cerebrospinal fluid (CSF) was assayed by ELISA and the plasma concentration of LH and FSH were determined by radioimmunoassay. Our results showed that icv injection of IL-1β (10 or 50 μg/animal) decreased the GnRH mRNA level in the pre-optic area (POA) (35% and 40% respectively; p ≤ 0.01) and median eminence (ME) (75% and 70% respectively; p ≤ 0.01) and GnRHR gene expression in ME (55% and 50% respectively; p ≤ 0.01). A significant decrease in GnRHR mRNA level in the AP in the group treated with the 50 μg (60%; p ≤ 0.01) but not with the 10 μg dose was observed. The centrally administrated IL-1β lowered also GnRH concentration in the CSF (60%; p ≤ 0.01) and reduced the intensity of GnRH translation in the POA (p ≤ 0.01). It was not found any effect of icv IL-1β injection upon the release of LH and FSH. However, the central injection of IL-1β strongly decreased the LHβ mRNA level (41% and 50%; p ≤ 0.01; respectively) and FSHβ mRNA in the case of the 50 μg dose (49%; p ≤ 0.01) in the pituitary of anestrous ewes. These results demonstrate that the central IL-1β is an important modulator of the GnRH biosynthesis and release during immune/inflammatory challenge.     

Neurogenic Differentiation of EGFP Gene Transfected Amniotic Fluid‐Derived Stem Cells from Pigs at Intermediate and Late Gestational Ages

The aims of this study were (i) to determine whether amniotic fluid‐derived stem cells (amniotic fluid‐derived stem; AFS cells) could be isolated from pigs at intermediate and late gestational ages, and (ii) to determine if these AFS cells could be differentiated in vitro into neural lineages following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Amniotic fluid‐derived stem cells were isolated from embryonic day 60 and day 110 porcine amniotic fluid respectively, and transfected with EGFP gene using lipofection. The transfected AFS cells were induced to differentiate into cells of neuronal lineages. Markers associated with undifferentiated AFS cells and their neural derivatives were tested by polymerase chain reaction. The results demonstrated that porcine AFS cells could be isolated at intermediate and late gestational ages and that transfected AFS expressed EGFP and could be induced to differentiate in vitro. Undifferentiated AFS cells expressed POU5F1, THY1 and SOX2, while following differentiation cells expressed markers for astrocytes (GFAP), oligodendrocytes (GALC) and neurons (NF, ENOS and MAP2).     

Sequences of canine glutamate decarboxylase (GAD) 1 and GAD2 genes, and variation of their genetic polymorphisms among five dog breeds

Glutamate decarboxylase (GAD) is the primary enzyme in the brain that catalyzes the synthesis of gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter. There are two isoforms named according to their molecular weights, GAD67 and GAD65, which are encoded by GAD1 and GAD2, respectively. To investigate the association between GAD genes and temperament in domestic dogs, Canis familiaris, we sequenced the full lengths of the coding regions of these genes and identified three single nucleotide polymorphisms (SNPs) in GAD1 and one in GAD2. When comparing genotype and allele frequencies of SNPs among five breeds with different behavioral traits, statistically significant interbreed differences were observed for three SNPs in GAD1. These results suggest that GAD1 SNPs may be useful for behavioral genetic studies in dogs.     

Expression of Adiponectin and its Receptors in the Porcine Hypothalamus During the Oestrous Cycle

Adiponectin is a hormonal link between obesity and reproduction, and its actions are mediated by two types of receptors: adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2). This study compares the expression levels of adiponectin and adiponectin receptor mRNAs and proteins in selected areas of the porcine hypothalamus responsible for GnRH production and secretion: the mediobasal hypothalamus (MBH), pre‐optic area (POA) and stalk median eminence (SME). The tissue samples were harvested on days 2–3, 10–12, 14–16 and 17–19 of the oestrous cycle. Adiponectin mRNA expression in MBH was significantly lower on days 14–16, whereas in SME, the most pronounced gene expression was found on days 2–3 of the cycle (p < 0.05). Adiponectin protein in MBH was most abundant on days 17–19 and in POA on days 2–3 (p < 0.05). Adiponectin protein expression in SME was at similar level throughout the most of the cycle with a statistically significant drop (p < 0.05) on days 14–16. AdipoR1 gene expression in POA was potentiated on days 2–3 and 10–12 of the oestrous cycle (p < 0.05). In SME, the highest AdipoR1 mRNA expression was noted on days 2–3 (p < 0.05). The concentrations of the AdipoR1 protein in POA were similar throughout the luteal phase (days 2–14 of the cycle), and they decreased on days 17–19 (p < 0.05). In SME, AdipoR1 protein expression peak occurred on days 2–3 (p < 0.05). The expression patterns of the AdipoR2 gene in MBH, POA and SME revealed the highest mRNA levels on days 2–3 of the cycle (p < 0.05). The highest content of AdipoR2 protein in MBH was reported on days 2–3 (p < 0.05), while in POA on days 17–19 and in SME on days 10–12 and 14–16 (p < 0.05). This study demonstrated that adiponectin and adiponectin receptor mRNAs and proteins are present in the porcine hypothalamus and that their expression levels are determined by the pig's endocrine status related to the oestrous cycle.     

Local administration of Neurokinin B in the arcuate nucleus accelerates the neural activity of the GnRH pulse generator in goats

Kisspeptin neurons in the arcuate nucleus (ARC), which co-express neurokinin B (NKB) and dynorphin A, are termed KNDy neurons. These neurons are candidates for the intrinsic gonadotropin-releasing hormone (GnRH) pulse generator. The central and peripheral administration of NKB or its receptor (NK3R) agonist evokes GnRH pulse generator activity and the subsequent pulsatile GnRH/luteinizing hormone (LH) secretion. However, the mechanism responsible for neural activation of the GnRH pulse generator in goats is unclear. We conducted electrophysiological and histochemical experiments to test the hypothesis that KNDy neurons receive NKB and that the signal is transmitted bilaterally to a population of KNDy neurons. Bilateral electrodes aimed at a cluster of KNDy neurons were inserted into the ovariectomized goat ARC. We observed the GnRH pulse generator activity, represented by characteristic increases in multiple-unit activity (MUA volleys). The unilateral administration of NKB or vehicle in the close vicinity of KNDy neurons under simultaneous MUA recording from both sides revealed that only NKB evoked MUA volley(s) immediately after administration. The timing of the MUA volley(s) evoked on the ipsilateral side was synchronized to that on the contralateral side. The double-labeled ISH for KISS1 and TACR3, which encode kisspeptin and NK3R, respectively, revealed that most KNDy neurons co-expressed TACR3. Therefore, NKB could directly stimulate KNDy neurons, following which the stimulatory signal is immediately transmitted to the entire population of KNDy neurons via connection with their fibers. This mechanism helps synchronize burst activity among KNDy neurons, thereby generating neural signals that govern pulsatile GnRH secretion.     

Electrophysiological and Morphological Evidence for Synchronized GnRH Pulse Generator Activity Among Kisspeptin/Neurokinin B/Dynorphin A (KNDy) Neurons in Goats

Neurons in the arcuate nucleus (ARC) that concomitantly express kisspeptin, neurokinin B (NKB) and dynorphin A are termed KNDy neurons and are likely candidates for the intrinsic gonadotropin-releasing hormone (GnRH) pulse generator. Our hypothesis is that KNDy neurons are functionally and anatomically interconnected to generate discrete neural signals that govern pulsatile GnRH secretion. Our goal was to address this hypothesis using electrophysiological and anatomical experiments in goats. Bilateral electrodes targeting KNDy neurons were implanted into ovariectomized goats, and GnRH pulse generator activity, represented by characteristic increases in multiple-unit activity (MUA volleys), was measured. Spontaneous and pheromone- or senktide (an NKB receptor agonist)-induced MUA volleys were simultaneously recorded from both sides of the ARC. An anterograde tracer, biotinylated dextran amine (BDA), was also injected unilaterally into the ARC of castrated male goats, and the distribution of fibers containing both BDA and NKB was examined using dual-labeling histochemistry. The results showed that MUA volleys, regardless of origin (spontaneous or experimentally induced), occur simultaneously between the right and left sides of the ARC. Tract tracing indicated that axons projecting from NKB neurons in the ARC were directly apposed to other NKB neuronal cells located bilaterally in the ARC. These results demonstrate that GnRH pulse generator activity occurs synchronously between both sides of the ARC in goats and that KNDy neurons are bilaterally interconnected in the ARC via NKB-containing fibers. Taken together, the results suggest that KNDy neurons form a neuronal circuit to synchronize burst activity among KNDy neurons and thereby generate discrete neural signals that govern pulsatile GnRH secretion.     

Distribution of glutamic acid decarboxylase, neurotensin, enkephalin, neuropeptide Y, and cholecystokinin neurons in the septo-preoptic region of male rats

Neurons in the lateral septum (LS) and preoptic area (POA) are known to play an inhibitory role in feminine sexual behavior regulation in male rats. In this study, the distribution of neurons containing glutamic acid decarboxylase (GAD) and of the peptidergic neurotransmitters neurotensin (NT), enkephalin (ENK), neuropeptide Y (NPY), and cholecystokinin (CCK), was examined immunohistochemically in the LS and POA of castrated male rats subcutaneously implanted with estrogen-containing Silastic tubes. Colchicine was injected into the lateral ventricle of the animals. The forebrain sections were immunostained for each substance. A large number of GAD-immunoreactive (ir) cells were found in the LS. Many NT-ir cells were seen in the intermediate and ventral parts of the LS at the rostral and middle levels. A considerable number of ENK-ir cells were scattered over the LS at the rostral and middle levels and were observed in the ventral part of the caudal LS. There were only a few NPY-ir cells in the LS. No CCK-ir cells were observed in the LS. In the POA, GAD-ir cells were observed in abundance. Many NT-ir cells were seen, especially in the medial preoptic nucleus. Some ENK-ir cells and a few NPY-ir cells were found in the medial POA. CCK-ir cells of the POA were restricted to the periventricular and paraventricular hypothalamic nuclei.     

Concentration and Distribution of Neuropeptide Y, Galanin, β-endorphin, Vasoactive Intestinal Peptide and Gonadotrophin-Releasing Hormone in the Hypothalamus of Gilts during Oestrogen-Induced Surge Secretion of Luteinizing Hormone

Contents The relationship of neuropeptide Y (NPY), galanin (GAL), β-endorphin (β-END) and vasoactive intestinal peptide (VIP) to GnRH neurons were determined during the estradiol-induced LH surge. In experiment 1, 16 ovariectomized (OVX) gilts received 15 μg estradiol benzoate (EB)/kg BW at 0800 h and were slaughtered at either 24 h (n = 5), 48 h (n = 6) or 72 h (n = 5) later and five were injected with corn oil vehicle (0 h controls). Concentrations of neuropeptides were determined in tissue extracts by RIA. In experiment 2, nine OVX gilts were injected with EB as in experiment 1 and killed at either 24, 48 or 72 h (n = 3) later and three were not injected with EB (0 h controls). Frozen sections were processed to localize neuropeptides. In experiment 1, all measured neuropeptides were highest in pituitary stalk median eminence (SME). The GnRH concentration was not different at any time point in medial basal hypothalamus (MBH), preoptic area (POA) or SME. The NPY content in MBH was lower at 24, 48 and 72 h after EB than at 0 h (p < 0.001), and lower in SME at 48 and 72 h than at 24 h (p < 0.05) and 0 h (p < 0.01), respectively. Concentration of GAL in SME was four times higher at 72 h than at 0, 24 or 48 h (p < 0.001). The VIP concentration increased in POA (p < 0.05) and MBH (p < 0.001) at 24 h and 72 h (p < 0.05). Concentration of VIP in SME was lower at 24 and 48 h than at 0 h (p < 0.05) and increased to more than twice (p < 0.05) by 72 h. Concentrations of β-END were not different at any time point in POA and MBH but the highest content of β-END in SME occurred at 24 h (p < 0.001). In experiment 2 a moderate number of GnRH-immunoreactive (IR) fibres were found in the periventricular area of the POA and in organum vasculosum of the laminae terminalis (OVLT). The GnRH-IR fibres formed networks in the external and internal layer of the median eminence (ME). At 24 h, GnRH-IR neurons and fibres in the POA and ME were more numerous and noticeable differences were found in the arcuate nucleus (ARC) and ventromedial nucleus (NVM). At 48 and 72 h, numbers of IR neurons and fibres were higher in the ARC and NVM, but no changes occurred in the POA and ME. The ARC contained a moderate number of NPY-IR fibres, but less numerous small cell bodies. Only a few NPY-IR perikarya and fibres were in the NVM and fibre density was similar at all times after EB injection. VIP-IR fibres were scarcely distributed mostly in the posterior POA and the internal layer of ME. The number of VIP-IR fibres was similar at all time points and regions. A moderate number of varicose β-END fibres supplied the POA, and they were especially dense near the OVLT, but the cell bodies were moderate in number and did not show strong immunoreactivity. In ME, ARC and NVM, the number of β-END immunoreactive structures was greater at 24 and 48 h than at 0 h. The number of β-END-IR nerve fibres in POA was higher at 72 h than at 0 h. Levels of all neuropeptides studied were similar in the POA and MBH and content of NPY, GAL and β-END was very high in the SME of the pig forebrain. The dynamic changes of NPY, GAL, VIP and β-END content in pig hypothalamus during the oestrogen-induced negative and positive feedback phases of LH secretion indicate their potential role in modulating GnRH release from the median eminence.     

初情期启动过程中小尾寒羊下丘脑GnRH基因甲基化状态及表达量关系研究

初情期启动的早晚关系到雌性动物的繁殖性能,GnRH是动物初情期启动过程中的关键基因,其启动子区甲基化状态与GnRH mRNA表达量之间的关系尚不清楚。本研究选择初情期前、临近初情期和初情期雌性小尾寒羊的下丘脑作为样本,利用亚硫酸氢盐测序(BSP)技术和实时荧光定量PCR(qRT-PCR)技术检测了初情期不同阶段小尾寒羊GnRH启动子区的甲基化状态和GnRH mRNA的表达量,并分析二者之间的关系。结果表明:小尾寒羊到达初情期时,GnRH基因启动子区甲基化水平显著降低(P0.05),尤其是在启动子区-570位点降低最明显,而随着初情期的启动GnRH mRNA表达量呈上升趋势。结果提示,初情期启动过程中,GnRH mRNA表达量升高可能与下丘脑GnRH基因启动子区特定位点甲基化水平降低存在一定的关系。     

京海黄鸡促性腺激素释放激素1基因克隆与原核表达研究

本试验根据GenBank公布的原鸡（Gallus gallus）促性腺激素释放激素1(gonadotropin releasing hormone 1,GnRH1)基因mRNA序列设计1对引物,采用RT-PCR方法,从京海黄鸡下丘脑组织克隆GnRH1基因编码区序列,对其进行生物信息学分析,并构建原核表达载体pET32a-GnRH1,在大肠杆菌BL21感受态细胞中表达。最终获得了包括编码区、大部分启动子区和部分3′区域在内的长度为352 bp京海黄鸡GnRH1基因序列,该核酸序列与火鸡、鸽子、人、牛、野猪、山羊、绵羊、藏羚羊、马和大鼠的GnRH1基因序列同源性分别为93%、81%、54%、58%、61%、76%、76%、59%、76%和66%。酶切测序鉴定结果显示,成功构建了原核表达载体pET32a-GnRH1;SDS-PAGE检测结果显示,构建的重组质粒在大肠杆菌BL21感受态细胞中成功表达,分子质量为25～28 ku,且上清表达量高于沉淀;Western blotting检测结果显示,在大肠杆菌BL21感受态细胞中表达的蛋白为目的蛋白,且主要为可溶性表达。结果表明,成功克隆了京海黄鸡GnRH1基因,构建了GnRH1原核表达体系,并成功表达目的蛋白,为后续相关研究打下了基础。     

GnRH antagonist inhibition of gonadotropin and steroid secretion in boars in vivo and steroid production in vitro

The hormone GnRH has a stimulatory effect on gonadotropin synthesis and secretion. The objective of the first study was to evaluate concentrations of FSH and LH in plasma of boars after successive treatment with SB75, a GnRH antagonist. Thirteen boars greater than 1 yr of age (eight White Composite [WC] and five Meishan [MS]) were injected once daily with SB75 (10 microg/kg of body weight) for 4 d. Plasma concentrations of LH and testosterone (T) decreased after 1 h from the first dose of SB75. After 12 h of treatment, LH gradually returned to pretreatment concentrations, but T remained suppressed (< 2 ng/mL) until after the last injection of SB75. There was a modest, but significant, reduction in FSH during treatment with SB75. The prolonged inhibitory effect of SB75 on suppression of plasma T concentrations, in the presence of pretreatment concentrations of LH, implied direct effects of SB75 at the testis. In the second experiment, testicular tissue from adult boars was incubated in the presence of three doses of human chorionic gonadotropin (hCG; 0, .5, and 5 IU) with SB75 (250 ng/mL) or with Deslorelin, a GnRH agonist (500 ng/mL). Samples of media were collected every hour for 3 h, and concentrations of T and estrone (E1) were determined by RIA. Concentrations of T and E1 increased with time in response to treatment with hCG. Co-treatment with SB75 decreased media concentrations of T (P < .01) and E1 (P < .03) compared to controls (77.9 vs 85.7 +/- 2.0 and 4.7 vs 5.3 +/- .2 ng/g). In contrast, treatment with Deslorelin had no effect on the amount of T (P > .50) or E1 (P > .26) released with all dosages of hCG. These results indicate that a GnRH antagonist has a direct effect on the testis, decreasing amounts of T and E1 released from the Leydig cells; however, treatment with a GnRH agonist had no direct effect on release of these gonadal steroids. Thus, it remains unresolved whether the site of action of GnRH antagonist on testicular steroidogenesis is through a testicular GnRH receptor or through some other mechanism.     

Stimulation of annexin A5 expression by gonadotropin releasing hormone (GnRH) in the Leydig cells of rats

The distribution and regulation of annexin A5 expression, a gonadotropin releasing hormone (GnRH) receptor regulated protein in gonadotropes and luteal cells, in the testes of rats were examined. Immunocytochemical staining revealed high levels of annexin A5 in the Leydig and endothelial cells and lower levels in the primary spermatocytes and sperm. Hemicastration significantly increased the annexin A5 content of the remaining testis within 24 h. Annexin A5 immunoreactivity was increased mainly in interstitial tissues including the peritubular cells, while some spermatocytes also showed higher intensity of annexin A5 in the remaining testis. Administration of hCG (50 IU) enhanced the testicular content of annexin A5 after 24 h. This treatment expanded the area of interstitial tissue in the testis and increased annexin A5 immunoreactivity, but the area of the of endothelial cells was unchanged. Similarly, human chorionic gonadotropin (hCG) enhanced annexin A5 expression in a primary culture of testis cells that consisted of mainly interstitial cells. Because GnRH stimulates the expression of annexin A5 in the gonadotropes and luteal cells, we examined the effect of GnRH on annexin A5 expression in the testes. We found that des-Gly10 [Pro9]-GnRH ethylamide (100 nM), a GnRH agonist, increased annexin A5 expression in cultured testis cells and that Cetrorelix (100 nM), a GnRH antagonist, inhibited the effect of hCG on annexin A5 expression. These results suggest that pituitary luteinizing hormone promotes annexin A5 synthesis in Leydig cells and that this effect could be mediated by local GnRH in the testis.     

GnRH受体在山羊结状神经节的分布

取山羊结状神经节5对,采用免疫组织化学SP法观察促性腺激素释放激素（Gonadotropin releasing hormon,GnRH）受体在结状神经节的分布特点。结果显示,GnRH受体在结状神经节上广泛分布,神经元、卫星细胞、神经纤维、血管肉皮细胞均有不同程度的免疫阳性染色。在神经元胞体中,细胞膜和细胞质有GnRH受体强阳性产物分布,核不着色;卫星细胞、血管内皮细胞也有强阳性产物分布;神经纤维存在弱阳性产物分布。图像分析结果表明,神经元中GnRH受体的相对表达量与其他非神经结构相比差异性显著（P〈0．05）。结果表明,山羊结状神经节中的GnRH受体主要存在于神经元中,山羊结状神经节中的内脏感觉传入神经元对GnRH具有反应性,提示GnRH结状神经节内脏感觉传入神经元的GnRH受体可能作为GnRH对内脏器官的内分泌调节和自主神经对内脏器官的神经调节这2种途径协调作用的节点。