The maturation of the salmon GnRH system and its regulation by gonadal steroids in masu salmon

Pituitary gonadotropin (GTH) secreting cells and brain gonadotropin-releasing hormone (GnRH) secreting neurons are known to be subjected to feedback control by gonadal steroid in teleosts. In masu salmon, Oncorhynchus masou, salmon GnRH (sGnRH) neurons in the ventral telencephalon (VT) and the preoptic area (POA) are involved in the control of GTH cells because sGnRH synthesis in these areas is activated with gonadal maturation. In this study, we attempted to clarify mechanisms of feedback control of sGnRH neurons by gonadal steroids. We examined the effects of 17-methyltestosterone (MT) on sGnRH synthesis in yearling and 2-year-old female fish (which were immature during experimentation in May), and the effects of castration on sGnRH synthesis in underyearling precocious male fish in August. sGnRH synthesis in the POA, but not in the VT, was increased by MT administration in 2-year-old females only, indicating higher sensitivity to MT in the preoptic sGnRH neurons. Castration increased sGnRH synthesis in the VT but not in the POA. These results suggest that sGnRH neurons in the VT and those in the POA are differentially regulated by gonadal steroids.     

Gonadotropin-releasing hormone and gonadotropin in goldfish and masu salmon

Reproductive activities in vertebrates are regulated by an endocrine system, consisting of the brain-pituitary-gonad axis. In teleosts, gonadotropin-releasing hormone (GnRH) in the brain stimulates gonadotropin (GTH) release in the pituitary gland, but because of lack of the portal vessel, it is not known when and how much GnRH is released for the regulation of GTH release. There are multiple molecular types of GnRH in teleosts and several distinct populations of GnRH neurons in the brain. However, we do not know which types and populations of GnRH neurons regulate reproductive activities. Here we summarize our recent studies on GnRH and GTH in masu salmon Oncorhynchus masou and goldfish Carassius auratus. Immunocytochemistry showed the location and molecular types of GnRH neurons. Salmon (sGnRH) and chicken-II GnRH (cGnRH-II) neuronal fibers were widely distributed in the brain of both masu salmon and goldfish. Only sGnRH fibers were observed in the pituitary of masu salmon, whereas both sGnRH and cGnRH-II fibers were observed in the goldfish pituitary, indicating that species specific GnRH profiles are involved in the regulation of pituitary function in teleosts. A series of experiments in masu salmon and goldfish suggest that among GnRH neuron populations GnRH neurons in the ventral telencephalon and the hypothalamus regulate GTH release, and that GnRH of the terminal nerve origin is not essential to gonadal maturation and ovulation. The biological function of other GnRH neurons remains unkown. Two GTHs appear to be characteristic of teleost; however, regulation of reproduction by these GTHs is a question that remains to be elucidated. In salmonid species, it is proposed that GTH I stimulates early gonadal development, whereas GTH II acts in later stages. When GTH expression was examined in goldfish, both GTH I and II mRNA levels in the pituitary gland showed increases in accordance with gonadal development, unlike the sequential expression of GTH subunits in salmonids. The expression of these GTH subunit mRNAs were affected by water temperature, starvation, and steroid hormones in goldfish, but in what manner these two GTHs regulate gonadal development remains to be clarified.     

Gonadotropin-releasing hormone (GnRH) neuronal systems in the pejerrey, Odontesthes bonariensis (Atheriniformes)

The brain of the pejerrey (Odontesthes bonariensis) has recently been shown to contain three forms of gonadotropin-releasing hormone (GnRH): salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II) and pejerrey GnRH (pjGnRH), nevertheless neuroanatomical studies on the distribution of these peptides are lacking. In this study we investigated the distribution of immunoreactive GnRH in the brain of adult pejerrey. Four different policlonal antisera and a monoclonal antibody against different GnRH variants were applied on cryosections and visualized using the ABC method. Three antisera (PBL#49, sGnRH#2 and cII741) revealed three different immunoreactive areas: the terminal nerve ganglion (at the junction between the olfactory bulbs and the anterior telencephalon), the preoptic area just anterior to the hypothalamus and the midbrain tegmentum. Fibers immunoreactive to GnRH were detected in different brain areas: the olfactory bulbs, the ventral thelencephalon, the hypothalamus, the mesencephalic area and an important innervation entering into the pituitary gland. Two other antibodies (LRH13 and s1668) labeled the two nuclei corresponding to the forebrain but not the midbrain tegmentum. As both antibodies have low crossreactivity to cGnRH-II, the data suggest that this group of cells express cGnRH-II. In summary, three different areas with immunoreactivity to GnRH were detected in the pejerrey brain. The distribution of sGnRH, pjGnRH and cGnRH-II expressing neurons, is discussed.     

GnRH systems in masu salmon and barfin flounder

Multiple forms of the gonadotropin-releasing hormone (GnRH) exist in teleost fish. A salmonid fish, masu salmon Oncorhynchus masou has salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II). sGnRH neurons were scattered from the olfactory nerve through the ventral telencephalon (VT) and the preoptic area (POA). sGnRH but not cGnRH-II was detected in the pituitary. sGnRH mRNA levels in the VT and the POA increased during gonadal maturation, suggesting that sGnRH neurons in these areas are involved in gonadal maturation. sGnRH neurons were first detected in a cluster near the olfactory epithelium 40 days after fertilization. sGnRH neurons were not detected in the brain by the olfactory epithelia lesion, suggesting that sGnRH neurons are derived from the olfactory epithelium. A pleuronectiform fish, barfin flounder Verasper moseri has sGnRH, cGnRH-II and seabream GnRH (sbGnRH). sGnRH and cGnRH-II-immunoreactive fibers were observed throughout the brain, but not in the pituitary. sbGnRH neurons were located in the POA and sent fibers to the pituitary, indicating that sbGnRH is involved in GTH secretion. Judging from the location of neuronal somata and their projections, it is indicated that three GnRH systems exist in the barfin flounder; the TN-, the MT- and the POA-GnRH system. However, in masu salmon, clear anatomical identification of the TN- and the POA-GnRH system is difficult, because the GnRH neurons located in the ventral forebrain are consecutive and the GnRH form produced in these neurons is the same (sGnRH). Thus, it is suggested in masu salmon that sGnRH neurons are derived from the olfactory epithelium, migrate into the brain and play different roles according to the location in the brain.     

Central regulation of reproduction in teleosts

As in other vertebrates, reproduction in teleosts depends upon interactions taking place along the brain-pituitary-gonads axis. At the central level, these interactions involve at least three types of factors:A gonadotrophin-releasing factor which has recently been isolated from chum salmon brain extracts. This decapeptide, whose structure is (Trp⁷-Leu⁸)-LHRH, appears to have a widespread distribution among teleosts, and is less active that LHRH or LHRH analogues in releasing gonadotrophin from the teleost pituitary. Immunohistochemical and quantitative studies have demonstrated that Gn-RH neurons are mainly located in the ventral telencephalon and the preoptic area, while projections are found in the entire brain and the pituitary gland.A gonadotrophin release-inhibiting factor has been demonstrated in the anterior preoptic region of the goldfish and a large set of data suggests that dopamine has GRIF activity in goldfish, and in other teleost species, by direct action on the gonadotrophs. Accordingly, a dopaminergic preoptico-hypophyseal pathway could be demonstrated in the goldfish brain.Sex steroids exert, depending on the dosages, either a negative feedback in sexually mature fish or a positive feedback in immature fish. Such a positive feedback is caused by estrogens and aromatizable androgens. Accordingly, the brain of teleosts contains high levels of aromatase activity in particular in the telencephalon and anterior hypothalamus. The distribution of estrogens concentrating cells within the brain is consistent with possible interactions with Gn-RH or catecholaminergic neurons at the level of certain brain territories.These data are discussed in relation with the functional significance of different brain areas where interactions between these different factors possibly take place, in particular the terminal nerve, the ventral telencephalon, the preoptic area and nucleus lateralis tuberis.     

Distinct expression of GnRH genes in the red seabream brain

This paper reports the molecular cloning of a cDNA encoding the precursor of seabream gonadotropin-releasing hormone (prepro-sbGnRH) and the localization of salmon GnRH (sGnRH) and seabream GnRH (sbGnRH) expressing neurons in the brain of the red seabream (Pagrus major). The cloned prepro-sbGnRH cDNA has a 285 bps open reading frame encoding a 23 amino acid signal peptide, a 10 amino acid sbGnRH, the cleavage site (Gly-Lys-Arg), and a 59 amino acid GnRH-associated peptide. The expression of sGnRH and sbGnRH peptides, and prepro-sGnRH and prepro-sbGnRH mRNA were studied using immunocytochemistry and non-radioactive in situ hybridization, respectively. We found cell bodies that reacted positively with both the sGnRH cRNA probe and anti-sGnRH serum, but not with the sbGnRH cRNA probe or anti-sbGnRH serum in the ganglion of the terminal nerve. Cell bodies that reacted positively with the sbGnRH cRNA probe, anti-sbGnRH serum, and anti-sGnRH serum, but negatively with the sGnRH cRNA probe were found in the preoptic area (POA). Immunocytochemistry showed that a distinct bundle of axons arises in the POA which projected to the pituitary gland. These results suggest that sbGnRH is the most relevant hypophysiotropic form of GnRH.     

Role of neuropeptide Y (NPY) in the regulation of reproduction: study based on catfish model

Significance of NPY in the regulation of GnRH–LH axis was evaluated. Considerable NPY immunoreactivity was seen in the components like olfactory system, basal telencephalon, preoptic and tuberal areas, and the pituitary gland that serve as neuroanatomical substrates for processing reproductive information. Close anatomical association as well as colocalizations of NPY and GnRH were seen in the olfactory receptor neurons, olfactory nerve fibers and their terminals in the glomeruli, ganglion cells of nervus terminalis, medial olfactory tracts, fibers in the ventral telencephalon and pituitary. In the pituitary, NPY fibers seem to innervate the GnRH as well as LH cells. Intracranial administration of NPY resulted in significant increase in the GnRH immunoreactivity in all the components of the olfactory system. In the pituitary, NPY augmented the population of GnRH fibers and LH cells. HPLC analysis showed that salmon GnRH content in the olfactory organ, bulb, preoptic area+telencephalon and pituitary was also significantly increased following NPY treatment. NPY may play a role in positive regulation of GnRH throughout the neuraxis and also up-regulate the LH cells in the pituitary.     

Immunohistochemical localization of three GnRH systems in brain and pituitary of Japanese flounder

ABSTRACT:   To clarify the possible roles of gonadotropin-releasing hormone (GnRH) in the reproduction of Japanese flounder Paralichthys olivaceus , localization of salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II), and sea bream GnRH (sbGnRH) immunoreactive (ir) cell bodies and fibers in the brain and pituitary were examined together with follicle stimulating hormone (FSH) and luteinizing hormone (LH)-ir cells in the pituitary by immunohistochemistry. sGnRH-ir cell bodies were localized in the ventromedial part of the rostral olfactory bulb and cGnRH-II-ir cell bodies were restricted to the midbrain tegmentum, while sbGnRH-ir cell bodies were evident in the preoptic area. sGnRH-ir fibers were distributed throughout the brain, especially abundant in the forebrain. cGnRH-II-ir fibers were also scattered in many areas of the brain with abundance in the midbrain, but sbGnRH-ir fibers were observed in the preoptic–hypothalamic area and innervated the pituitary. In the pituitary, neither sGnRH-ir fibers nor cGnRH-II-ir fibers were found, but sbGnRH-ir fibers were profuse in the neurohypophysis and invaded the proximal pars distalis, targeting FSH and LH cells. These results suggest that three GnRH systems can play different physiological roles in the brain of Japanese flounder. Among them, sbGnRH is considered to be involved in reproduction by stimulating gonadotropin secretion, while sGnRH and cGnRH-II can function as a neurotransmitter and/or neuromodulator within the brain in this species.     

The GnRH systems in the brain and pituitary of normal and hCG treated European silver eels

The distribution of immunoreactive GnRH was studied in the brain and pituitary gland of normal and human chorionic gonadotrophin (hCG) injected silver eels. It was found that the general organization of GnRH systems in this species is similar to that reported in other teleosts. Cell bodies were present in the olfactory bulbs, ventral telencephalon, periventricular hypothalamus and dorsal tegmentum. No positive perikarya could be detected in the preoptic region. Only scarce fibers were observed in the proximal neurohypophysis. Treatment with hCG does not modify the distribution of GnRH but it increases the density of positive structures, in particular at the level of the pituitary. The results are discussed in relation with the present status of knowledge of the mechanisms underlying the blockage of sexual maturation in the European eel at the silver stage.     

GnRH and gpcr: laser-captured single cell gene profiling

We have developed a novel single cell real-time quantitative PCR technique, which incorporates harvesting marker-identified single cells using laser-capture. Here, for the first time in a vertebrate species, using this innovative single cell gene profiling technique, we report the presence of G-protein coupled receptors in individual gonadotropin-releasing hormone (GnRH) neurons and endocrine cells of the pituitary of the tilapia Oreochromis niloticus. The differential expression of multiple combinations of three GnRH receptor types (R1, R2 and R3) in individual gonadotropic and nongonadotropic cells demonstrates cellular and functional heterogeneity. The differential use of GnRH receptors in corticotropes, melanotropes and thyrotropes during gonadal maturation and reproductive behaviors suggests new roles for these hormones. Further, we provide evidence of the structure of a novel nonmammalian G-protein coupled receptor (GPR54) for kisspeptins, encoded by Kiss-1 gene, which is highly conserved during evolution and expressed in GnRH1, GnRH2 and GnRH3 neurons. We hypothesize GPR54 stimulates GnRH secretion and is crucial for pubertal maturation. We speculate, the use of this method will allow the identification and quantification of known and unknown genes in single cells, which would greatly facilitate our understanding of the complex interactions that govern the physiology of individual cells in vertebrates species.     

Pituitary gonadotropin-releasing hormone (GnRH) receptor activity in goldfish and catfish: seasonal and gonadal effects

The goldfish pituitary contains two classes of gonadotropin-releasing hormone (GnRH) binding sites, a high affinity/low capacity site and a low affinity/high capacity site (Habibiet al. 1987a), whereas the catfish pituitary contains a single class of high affinity GnRH binding sites (De Leeuwet al. 1988a). Seasonal variations in pituitary GnRH receptor binding parameters, and the effect of castration on pituitary GnRH receptor binding were investigated in goldfish and catfish, respectively. In goldfish, GnRH receptors undergo seasonal variation with the highest pituitary content of both high and low affinity sites occurring during the late stages of gonadal recrudescence. The observed changes in pituitary GnRH receptor content correlate closely with responsiveness to a GnRH agonistin vivo in terms of serum gonadotropin (GTH) levels. In catfish, castration results in a two-fold increase in pituitary GnRH receptor content, which can be reversed by concomitant treatment with androstenedione, but not by the non-aromatizable androgen 11β-hydroxyandrostenedione; changes observed in GnRH receptor content correlate with variations in serum GTH levels and responsiveness to a GnRH agonist. In summary, the present study provides a clear evidence for seasonal variation in pituitary GnRH receptor activity in goldfish, and demonstrates a gonadal feedback mechanism regulating GnRH receptor activity in the catfish pituitary.     

GnRH_A和紫萁抑制大黄鱼性腺早熟的机制

应用脑垂体组织生理学和免疫组织化学方法，对GnRH-A和紫萁抑制大黄鱼性早熟的作用机制进行了研究。结果表明，性早熟大黄鱼脑垂体许多促性腺激素分泌细胞的胞质出现空泡，提示性早熟的原因可能是由于GtH细胞提早进入分泌活动所致。长期服用促性腺激素释放激素类似物的养殖大黄鱼（没有性早熟），它的脑垂体GtH细胞对GnRH抗独特型发生弱的免疫阳性反应，而对照组性早熟鱼仍出现强的反应，表明实验组大黄鱼脑垂体GtH细胞对GnRH-A的应答能力下降，出现脱敏效应，这可能与GnRH-A抑制大黄鱼性早熟有关。养殖大黄鱼长期服用紫萁，它的脑垂体GtH细胞膜上GnRH受体则没有出现脱敏现象，提示紫萁抑制大黄鱼性早熟的机制不同于GnRH类似物，确切机制有待进一步研究。     

Extrapituitary gonadotropin-releasing hormone (GnRH) binding sites in goldfish

In teleosts, as in other vertebrates, the secretion of pituitary gonadotropin (GTH) is mediated by the hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH). Recent findings in teleosts indicate that GnRH receptors are not restricted to the pituitary gonadotropes and are also associated with somatotropes as well as being present in a number of other tissues. In the present study, we provide novel information on GnRH binding in a number of extrapituitary tissues in goldfish. However, we do not intend to provide full characterization of GnRH binding sites in various extrapituitary tissues in goldfish as this would clearly be outside the scope of this paper. In this study we examined GnRH binding in a number of extrapituitary tissues in goldfish and observed specific binding in ovary, testis, brain, liver and kidney. No specific GnRH binding was observed in muscle, skin, gut, gill and heart. In general, the present findings together with the results of other studies carried out in our laboratory demonstrate that mature goldfish ovary and testis contain two classes of GnRH binding sites, high affinity/low capacity and low affinity/high capacity sites with binding characteristics similar to those of the pituitary GnRH receptors. The brain of goldfish was also found to contain two classes of GnRH binding sites, a super-high affinity/low capacity and a low affinity/high capacity sites. Furthermore, study of goldfish liver and kidney demonstrated the presence of a single class of GnRH binding sites with characteristics different from those of pituitary, ovary, testis and brain. Overall, it is evident that goldfish contains a family of GnRH binding sites which can be classified into four groups based on binding affinities: 1) A class of high affinity binding sites present in the pituitary, ovary and testis, 2) a class of super high affinity sites so far only detected in the brain, 3) a class of intermediate-affinity GnRH binding sites in the liver and kidney, and 4) a class of low affinity binding sites present in all the tissues containing specific GnRH binding sites except for liver and kidney.     

The origin of the mammalian form of GnRH in primitive fishes

The presence of neuroendocrine hormones in extant agnathan fishes suggests that a method of control involving these hormones was operating 500–600 million years ago in emerging vertebrates. Data on a limited number of species show that several members of the GnRH family of peptides may have arisen in non-teleost fishes. Lamprey (Petromyzon marinus) GnRH has a unique composition and has not been detected in other vertebrates. It is not yet clear whether the chicken II GnRH-like molecule arose in cartilaginous fishes, but a chromatographically and immunologically similar molecule is found in dogfish (Squalus acanthias) and ratfish (Hydrolagus colliei). Finally, a mammalian GnRH-like molecule is detected in three primitive bony fish: sturgeon (Acipenser transmontanus), reed fish (Calamoichthys calabaricus), and alligator gar (Lepidosteus spatula). Minor forms are also present, but are not yet characterized. Clearly, the basic structure of GnRH peptides was established in primitive fish. In contrast, at least three other identified forms of GnRH have been detected in teleosts or tetrapods: Salmon I, catfish I, and chicken I GnRH. Evidence for the presence of members of the GnRH family and the neurohypophysial hormone family in primitive fishes argues for the importance of neuroendocrine control throughout the history of vertebrates.     

斑马鱼端脑的微细形态与结构

为了深入了解斑马鱼端脑的微细形态和超微结构,采用光镜和电镜技术对斑马鱼端脑进行观察研究。斑马鱼端脑由左、右嗅球和左、右大脑半球构成。嗅球前方有一对嗅神经,后端伸出嗅茎与大脑半球联系。光镜下,嗅球组织结构从外向内依次为上皮层、神经纤维层、小细胞层和内部细胞层。大脑半球外部覆盖很薄的大脑皮,基部为纹状体,两者之间的腔隙为公共脑室。纹状体由神经核团和神经纤维构成。神经核团分布于纹状体周缘,主要有连前核、背嗅核、侧嗅核、视前核和脚内核等。神经纤维包括横行的前连合,纵行和斜行的中央嗅束和侧嗅束。电镜下,嗅球分层明显,可观察到僧帽细胞、神经胶质细胞和众多突触。大脑半球中可见神经胶质细胞、突触以及血脑屏障系统等。斑马鱼端脑形态结构与大多数硬骨鱼相似,但有个别核团存在差异。实验结果可为斑马鱼神经生物学模型的建立与应用提供有效的理论基础。     

Positive feedback control by the gonads on gonadotropin (GTH) and gonadoliberin (GnRH) levels in experimentally matured female silver eels,Anguilla anguilla

Treatment of sham-operated female silver eels with carp pituitary extract stimulated ovarian development and induced increases in pituitary gonadotropin (GTH) and gonadoliberin (GnRH) contents. Both effects of carp pituitary extract were abolished in ovariectomized eels, indicating the involvement of the gonads. Endogenous sexual steroids, the secretion of which was increased during sexual maturation, should be responsible for the stimulation of GTH and GnRH levels. Ovariectomy itself had no significant effect on pituitary GTH and GnRH contents, reflecting the fact that, at the silver stage, sexual steroid levels are too low to exert any significant effect on pituitary GTH and GnRH. The positive feedback control exerted by the gonads on GTH and GnRH levels during sexual maturation, in the eel as well as in some other teleosts, would produce an amplification of the pubertal stimulation of the hypothalamo-pituitary-gonadal axis.     

Distribution of three GnRHs in the brain and pituitary of the wild Japanese flounder Paralichthys olivaceus

ABSTRACT:   Wild adult maturing and immature female Japanese flounder Paralichthys olivaceus were collected in June 2004 and January 2005, respectively, to clarify a possible role of gonadotropin-releasing hormones (GnRHs) in reproduction. Levels of salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II) and sea bream GnRH (sbGnRH) in the brain and pituitary were examined by time-resolved fluoroimmunoassay. Three forms of GnRHs were detected in the discrete brain at various levels. In the pituitary of both maturing and immature fish, sbGnRH was abundant together with a pronounced amount of sGnRH, whereas cGnRH-II was almost below the detectable limit. In maturing fish, levels of sbGnRH were high in the telencephalon, hypothalamus and pituitary, while levels of sbGnRH of immature fish were very low in these regions. These results indicate that sbGnRH is mainly responsible for gonadotropin secretion, and that sbGnRH in the anterior part of the brain is associated with gonadal maturation in the Japanese flounder.     

中华鳖GnRH 1基因cDNA克隆及表达特征

为了解GnRH基因在中华鳖(Pelodiscus sinensis)性腺和胚胎发育过程中的表达特征,采用cDNA末端快速扩增(RACE)技术从中华鳖全脑中获得与生长生殖调控密切相关的GnRH1基因全长cDNA,并运用实时荧光定量PCR(qRT-PCR)技术检测GnRH1在成鳖不同组织和胚胎发育时期的表达水平。结果显示:中华鳖GnRH1基因cDNA全长546 bp,其中5′非编码区(5′UTR)99 bp,3′非编码区(3′UTR)168 bp,开放阅读框(ORF)279 bp,编码92个氨基酸,分子质量为10.23 ku,理论等电点pI为5.65,具有N端信号肽(1~23 aa)、核心十肽区域(24~33 aa)、断裂位点GKR(34~36 aa)及相关肽区域(37~92 aa),符合GnRH蛋白典型结构特征。系统进化树结果显示,中华鳖GnRH1基因和绿海龟(Chelonia mydas)、墨西哥箱龟(Terrapene carolina mexicana)及西部锦龟(Chrysemys picta bellii)GnRH1基因聚为一支。qRT-PCR结果表明,GnRH1基因在中华鳖雌雄个体的8个组织中均有表达,在脑和性腺组织中高表达,且具有性别差异,雄性中华鳖中的表达显著高于雌性(P<0.05);在10个胚胎发育时期均表达,且随发育时间的后移,表达量显著增加,在第16期达到峰值。GnRH1基因可能在中华鳖生长及性腺分化中具有重要作用。     

促黄体素释放激素和多巴胺拮抗物对鲇促性腺激素释放的作用

以珠江流域鲇为研究对象，分别在性腺发育早期、性腺发育晚期、性腺发育成熟与产卵前期、性腺退化期注射促黄体素释放激素类似物和多巴胺D2受体拮抗物，处理后6h、12h、24h测定血液中的促性腺激素水平变化。结果表明，在性腺发育的各个时期，促黄体素释放激素类似物和多巴胺D2受体拮抗物联合注射能显著剂激鲇促性腺激素分泌；鲇脑垂体促性腺激素的分泌受下丘脑释放的促性腺素释放激素和多巴胺的双重调节，多巴胺只能抑制促性腺素释放激素诱导的促性腺激素分泌。在建立鲇人工繁殖技术时，可采用促黄体素释放激素类似物和多巴胺D2受体拮抗物联合注射方法。