睾丸注射慢病毒生产转基因鸡的研究

【目的】采用睾丸注射慢病毒的方法生产转基因鸡以提高转基因效率,使转基因鸡的批量生产变得简单可行。【方法】用磷酸钙沉淀法包装慢病毒,对慢病毒包装体系中的质粒总量和HN Buffer的pH值进行优化,以包装出滴度较高的慢病毒;然后将慢病毒注射到受精鸡蛋的胚胎中验证其活性;最后,将慢病毒注射到8日龄公鸡的睾丸内,待其性成熟以后,将精液DNA呈阳性的公鸡与非转基因母鸡进行交配生产出转基因鸡。【结果】当90mm培养皿中质粒总量为50μg、HN Buffer的pH值为7.05时慢病毒的包装效率最高,在该条件下包装出的慢病毒滴度高达7.5×107 TU/mL;采用慢病毒胚胎注射途径成功地生产出增强绿色荧光蛋白(enhanced Green Fluorescent Protein,eGFP)转基因鸡;采用慢病毒睾丸注射途径生产的转基因鸡后代中能检测到eGFP目的基因,但未检测到其相关的表达。【结论】慢病毒睾丸注射途径能够将外源基因整合到精原干细胞中,并遗传给后代。     

三种重金属对中华稻蝗金属硫蛋白基因表达的影响

以栖息于稻田且主要取食水稻茎叶的中华稻蝗(Oxya chinensis)成虫为供试材料,经氯化镉、氯化铜和硫酸锌(Cd Cl2、Cu Cl2和Zn SO4)3种重金属急性染毒,采用RT-q PCR技术检测中华稻蝗2个金属硫蛋白基因(Oxya chinensis metallothionein,Oc MT1,Oc MT2)在精巢、卵巢和肌肉中的表达变化。结果表明,3种重金属均可诱导中华稻蝗MT基因不同程度的上调表达。经Cd急性处理后,3个器官组织中Oc MT1和Oc MT2表达水平在一定浓度范围内随着Cd浓度的增高而诱导表达上调;经Cu急性处理后,精巢和卵巢中Oc MT1在最低浓度时的诱导表达量最高,Oc MT2表达水平随着Cu浓度的增大而升高,肌肉中2个MT基因随着Cu浓度的增大而升高;经Zn处理后,Oc MT1和Oc MT2表达水平随着Zn浓度的升高而升高。由实验结果可以看出,3种重金属对中华稻蝗MT在不同器官组织均有诱导表达作用,但各浓度处理后其诱导表达水平不同,Oc MT对不同重金属的敏感性具有显著差异。     

水貂取皮期与配种期睾丸形态参数测定及组织学观察

为探明水貂睾丸的季节性变化规律,试验测定了取皮期和配种期雄性水貂睾丸的基础形态参数,通过石蜡切片、苏木精-伊红染色方法对取皮期和配种期水貂的睾丸及附睾进行了组织学结构特征比较。结果表明,水貂取皮期睾丸呈长卵圆形,平均长径、短径和厚径分别为16.88mm±1.52mm、10.90mm±0.85mm和10.28mm±1.03mm,体积为1.05cm3±0.23cm3,平均重量达到1.032 3g±0.263 3g;水貂配种期睾丸呈短卵圆形,各项形态生理参数、体积及重量极显著高于取皮期(P0.01)。在光镜下,与取皮期相比,配种期水貂睾丸生精小管内细胞层数和种类较多,包括精原细胞、初级精母细胞、次级精母细胞和精子细胞,生精小管与附睾管管腔中存在大量精子。而取皮期生精小管内细胞疏松,仅见少量精原细胞,并且配种期生精小管直径极显著高于取皮期(P0.01)。     

嫁虫戚精巢的组织学研究

本文研究了嫁虫戚雄性生殖系统的组织学。嫁虫戚的精巢结构同贻贝、栉孔扇贝的相似,也是由外膜及其内的无数生殖小管构成。外膜较薄,由平滑肌和少量结缔组织组成,只在局部区域含有单层柱状上皮细胞。生殖小管间充填着结缔组织及来自外膜的薄层肌肉,结缔组织中含有血管。生殖小管由位于基膜上的生殖上皮及由生殖上皮增殖出的精原细胞、初级精母细胞、次级精母细胞、精细胞和精子组成。精原细胞较大,圆形或椭圆形,核较大,与细胞同形,染色质呈颗粒状靠近核膜分布,核仁1个,圆形,明显。初级精母细胞和次级精母细胞结构相似。精细胞圆形,核染色深。精子呈长棒状,悬浮于生殖小管腔内。     

Pubertal development of male African catfish,Clarias gariepinus. In vitro steroidogenesis by testis and interrenal tissue and plasma levels of sexual steroids

In fish, sex steroids initiate and/or accelerate the maturation of the brain-pituitary-gonad axis. In order to obtain information on the steroid milieu during the pubertal development of male African catfish, we have monitored the conversion of [³H]-pregnenolone and [³H]-androstenedione by testis and [³H]-pregnenolone by interrenal tissue fragmentsin vitro. Pubertal development occurs between two and six months of age. Testicular development proceeds through four main stages that are characterised histologically by the presence of spermatogonia (stage I), spermatogonia and spermatocytes (stage II), spermatogonia, spermatocytes and spermatids (stage III), and all germ cells including spermatozoa (stage IV). 11β-Hydroxyandrostenedione and cortisol were the main products of testes and interrenal tissue, respectively, in all stages of the pubertal development; a change in the steroidogenic pattern was not observed during this period. The interrenal tissue displayed no significant conversion of [³H]-pregnenolone to 11-oxygenated androgens. Blood plasma was analyzed for the presence of five androgens; testosterone, 11β-hydroxytestosterone, 11β-hydroxyandrostenedione, androstenetrione, and 11-ketotestosterone. 11-Ketotestosterone was the quantitatively dominating androgen in the circulation at all stages of development, which was more pronounced in stages III and IV. The obvious differences between thein vitro andin vivo results, namely 11β-hydroxyandrostenedione being the main testicular productvs. 11-ketotestosterone dominating in the blood, may indicate that 11β-hydroxyandrostenedione is converted to 11-ketotestosterone at extratesticular sites.     

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.     

In vitro steroidogenesis by gonads of the Russian sturgeon, Acipenser gueldenstaedti Brandt

Ovaries and testes from the Russian sturgeon at different stages of sexual maturity were incubated with ;sup3;H-androstenedione or ³H-pregnenolone. The major metabolites of androstenedione in both sexes were testosterone and 5- and 5ß-androstanediols, but no evidence was found for the gonadal production of 11-oxygenated androgens. Pregnenolone was converted to 17-hydroxyprogesterone, androstenedione and testosterone together with reduced metabolites. 11-Oxygenated androgens were found in female serum by gas chromatography-mass spectrometry (GC-MS), in serum of both sexes by radioimmunoassay (RIA), and was detectable by RIA in interrenal but not gonadal tissue. The results suggest that sturgeon may differ from teleosts in that 11-oxygenation may take place in extragonadal tissues.     

In vitro synthesis of 20α-reduced and of 11- and 21-oxygenated steroids and their sulfates by testes of the goldfish (Carassius auratus): Testicular synthesis of corticosteroids

Testes from spermiating goldfish were incubated with [³H]17-hydroxyprogesterone. The major products in the unconjugated fraction were identified as androstenedione, androstenetrione, 11-β-hydroxyandrostenedione, 11-ketotestosterone, 17,20α-dihydroxy-4-pregnen-3-one (17,20αP) and 11-deoxycortisol. Testosterone was present predominantly in the glucuronide fraction, but yields were low (1–3%). The major components of the sulfate fraction were 17,20αP and 11-deoxycortisol. The identification of cortisone in low yield (< 2.5010) in both the free and sulfate fractions is the first report of corticosteroid biosynthesis by a teleost testis. The high yields of 17,20αP and 11-deoxycortisol and their sulphates suggests that their possible role in spermiation of the goldfish should be further investigated.     

犏牛PPP1R11基因的克隆及在睾丸中的表达规律研究

旨在克隆犏牛蛋白磷酸酶1调节亚基11(protein phosphatase 1 regulatory subunit 11,PPP1R11)基因,分析其在不同发育阶段睾丸中的表达与定位,为解析其在雄性生殖中的功能机制提供理论依据.本研究采集成年犏牛睾丸、附睾、心、肝、脾、肺、肾、大肠、小肠、胃、肌肉和脂肪组织(n=3...     

Retracted: Study of testis histology and sperm morphology in the Bunnei,Barbus sharpeyi (Gunther, 1874) induced by luteinizing hormone‐releasing hormone analogue and carp pituitary extract

This study was conducted to determine the effects of hormone treatment on testis structure in Barbus sharpeyi, as well as the morphology of sperm examined by scanning electronic microscopy (SEM). Male B. sharpeyi were divided into three groups (three fish per group) and injected with luteinizing hormone – releasing hormone analogue (LHRH–A₂) or carp pituitary extract (CPE). The first and second groups were treated with 10 μg kg^?1 LHRH–A₂ and metoclopramide (MET), and their testis were sampled pre‐ and Poststripping respectively. The third group received 2 mg kg^?1 CPE and were killed pre‐stripping. Based on the histological results obtained, the testicular connective tissue of the lumen was thicker, and seminal vesicles were of a lower volume, in fish injected with CPE in comparison to the other groups. After treatment with LHRH–A₂ and MET, not all spermatozoa within the testis were ejaculated, and only a small amount of sperm was obtained by abdominal stripping. The highest and lowest diameters of connective tissue within lobules were observed in fish receiving CPE and LHRH–A₂ treatments respectively. There was a significant difference (P < 0.05) in lobule space between the fish injected with the CPE and the fish injected with the LHRH–A₂ and MET. The SEM results revealed that the spermatozoa of B. sharpeyi were composed of a spherical to elliptical head, a cylindrical midpiece, and a lengthy flagellum. In conclusion, it was found that injection with LHRH–A₂ and MET improved the spermatogenic process in comparison to injection with CPE.