NOD1和NOD2基因在牦牛雄性生殖器官中的表达检测

为了解NOD1和NOD2基因在牦牛生殖系统中的表达情况,从脾脏组织总RNA中RT-PCR扩增牦牛NOD1和NOD2基因,半定量RT-PCR分析NOD1和NOD2 mRNA在牦牛雄性生殖组织的表达水平。结果显示,NOD1在脾脏、睾丸、附睾头、附睾体、附睾尾、输精管和阴茎中均相对低表达,而NOD2在脾脏、睾丸、附睾头、附睾体、附睾尾、输精管和阴茎中均相对高表达。结果表明,NOD1和NOD2在牦牛雄性生殖组织中广泛表达,提示其在牦牛生殖过程中可能发挥一定的作用。     

猕猴两性畸形

１９８９年８月，我们见到１例８岁猕猴具有两性器官。雄性器官包括一只睾丸、附睾、输精管、精囊腺、前歹腺和阴茎。雌性器官为一个卵巢、输卵管、子宫和阴道。综上所述，此例猕猴是两性畸形，且雄性生殖器官发育略占优势。     

6种蝙蝠精子储存现象的组织学观察

精子储存是蝙蝠的生殖对策之一,有些种类的蝙蝠能将成熟精子在交配前(雄性)或交配后(雌性)储存在附睾或者输卵管及子宫内数月。采用冰冻切片和苏木精-伊红(H.E)染色法,观察了6种蝙蝠的睾丸、附睾、卵巢、输卵管和子宫,发现5种蝙蝠有精子储存现象。另外,本文还讨论了不同生殖对策蝙蝠的地理分布。     

小鼠子宫颈部位输卵管蛋白表达的初步研究

用RT-PCR及免疫印迹法检测证实小鼠子宫颈粘膜上皮细胞具表达输卵管蛋白的能力,宫颈部所获得的输卵管蛋白转录产物, 310到 714的405bp的cDNA片段经序列测定及blast比较表明与输卵管部位表达的输卵管蛋白完全一致(100%)。小鼠子宫颈输卵管蛋白在动情周期的表达波动情况与输卵管粘膜上皮的类似,其表达于小鼠动情期明显增加。Westernblot显示小鼠子宫颈提取物中存在两种不同分子量(60KD,30KD)的输卵管蛋白。原位杂交结果则提示子宫颈粘膜上皮细胞含丰富的输卵管蛋白mRNA信号。通过"原体中风"实验未发现输卵管蛋白在体外对精子活动有影响,其功能尚须进一步分析。     

蛋白激酶H11基因在小鼠子宫中的表达及其性激素调节

为研究蛋白激酶H11基因在生殖系统中的作用,我们采用半定量RT-PCR和原位杂交方法,研究了蛋白激酶H11基因在小鼠中的组织特异性表达,在妊娠初始期胚胎植入位点、妊娠期子宫和胎盘以及正常动情周期子宫中的表达及其受性激素的调节。结果发现:蛋白激酶H11基因在小鼠多种组织中都有表达,在卵巢及子宫等一些生殖相关的组织中表达水平较高;妊娠初始期,蛋白激酶H11基因在小鼠子宫内膜植入位点处有明显的高表达,其mRNA定位于腔上皮细胞和基质细胞中。在动情周期中,蛋白激酶H11基因在动情前期子宫中表达水平较低;卵巢切除模型显示雌激素和孕激素均可显著上调蛋白激酶H11基因的表达。以上结果提示蛋白激酶H11可能参与了胚胎植入过程中腔上皮细胞凋亡和基质细胞增殖与蜕膜化以及动情周期小鼠子宫内膜细胞的功能调节[动物学报51(3):462-468,2005]。     

不同发育阶段雄性小鼠睾丸组织mEppin基因mRNA的表达差异研究

目的实时荧光定量PCR检测雄性小鼠不同发育阶段睾丸组织中m Eppin基因的mRNA表达差异,为探究m Eppin基因表达与雄性生殖相关性提供生物学数据。方法分别选取胚胎期、50日龄、90日龄雄性ICR小鼠各6只,共18只,取各组小鼠睾丸组织提取总RNA,实时荧光定量PCR方法检测m Eppin基因mRNA表达水平。结果不同发育阶段小鼠睾丸m Eppin基因表达水平差异明显。胚胎组小鼠中没有检测到m Eppin基因mRNA表达,50日龄小鼠和90日龄小鼠中均检测到m Eppin基因mRNA有较高表达水平。其中,90日龄小鼠睾丸中m Eppin基因mRNA的相对表达水平是50日龄小鼠的2.65倍。结论本研究结果表明m Eppin基因mRNA的表达在小鼠睾丸组织中具阶段特异性。     

Fancm基因敲除小鼠构建及其表型分析

目的构建范可尼贫血通路Fancm基因敲除小鼠,研究Fancm基因缺失对小鼠生理功能,特别是雄性生殖器官的影响。方法采用CRISPR/Cas9技术,获得Fancm基因敲除小鼠。分析FANCM蛋白在野生型和Fancm^-/-小鼠睾丸组织中的表达。统计Fancm^-/-小鼠的出生率、体重、性别比例及子代生育情况,分析血液常规指标。组织形态学研究雄性Fancm^-/-小鼠睾丸生理病理表型。结果敲除Fancm基因ATG区域,获得稳定遗传的C57BL/6背景Fancm^-/-小鼠。Fancm^-/-小鼠睾丸中FANCM蛋白表达完全丢失。Fancm^-/-小鼠无明显的胚胎致死现象,但雌性Fancm-/-小鼠数目显著少于雄性Fancm^-/-小鼠。同窝Fancm^-/-小鼠比较野生型体重无明显区别,部分血常规指标有显著性差异。Fancm^-/-小鼠有明显的生殖能力缺陷。雄性Fancm^-/-小鼠睾丸有显著的发育缺陷,其生精细胞凋亡增加、细胞周期阻滞,影响睾丸发育与精子的生成。结论成功获得稳定遗传C57BL/6背景Fancm^-/-小鼠,Fancm基因参与小鼠的生长发育,特别是雄性生殖器官功能的维持及调控。     

VEGF、VEGFR2在青春期大鼠睾丸、附睾及附睾精子上的表达

目的通过对血管内皮生长因子(VEGF)及其受体VEGFR2在青春期大鼠睾丸及附睾表达的研究,探讨其在雄性生殖器官中的作用。方法采用免疫组化法检测VEGF、VEGFR2在SD大鼠睾丸和附睾的表达定位,用免疫荧光法检测它们在大鼠附睾精子上的表达定位。结果VEGF及VEGFR2在青春期大鼠睾丸和附睾组织中均有表达。在睾丸中,VEGF主要表达于精原细胞胞质、精子细胞发育中的顶体、Sertoli细胞胞质及精子残余体内,Leydig细胞胞质也有阳性表达;VEGFR2主要表达于精子细胞发育中的顶体和间质细胞胞质。在附睾中,VEGF表达于附睾管上皮所有主细胞胞质内;而VEGFR2表达于附睾管头段和尾段上皮主细胞胞质内,体段免疫染色阴性。免疫荧光显示,VEGF与VEGFR2都与精子头部顶体、尾部颈段、中段和主段相结合,末段未见阳性荧光。结论VEGF及VEGFR2在大鼠的睾丸和附睾中均有表达,其表达定位具有细胞特异性和区域特异性,提示其可能在大鼠睾丸精子发生和附睾精子成熟中发挥重要作用。     

长期低浓度七氟醚吸入对雄性小鼠生殖功能的影响

目的:探讨长期低浓度七氟醚吸入对雄性小鼠生殖功能的影响。方法:雄性C57小鼠20只随机分为吸醚组和对照组,吸醚组给予连续45 d,每天4 h吸入0.2%七氟醚。吸醚第36 d起每只雄性小鼠与4只雌性小鼠合笼交配。合笼结束后,雄性小鼠称重,计算睾丸指数,观察睾丸形态学变化,检测附睾精子活力和血清激素水平,统计子代小鼠数量和性别比例。结果:长期低浓度七氟醚吸入后雄性小鼠体重增长明显减少,精子活力显著降低,血清卵泡刺激素(FSH),促黄体生成素(LH),瘦素(LEP)显著升高,雄激素(T)水平无显著差异。雌性小鼠受孕率、子代数量及性别比例无显著差异。结论:长期低浓度七氟醚吸入对雄性小鼠具有一定的生殖毒性,影响其血清激素水平和精子活力,但受孕率和子代性别比无显著差异。     

合欢皮总皂苷对雄性小鼠的抗生育作用研究

目的探讨合欢皮总皂苷对雄性小鼠的抗生育作用。方法将60只健康的雄性性成熟昆明小鼠随机分成3个试验组和1个对照组,各试验组小鼠灌胃540 mg·(kg·d)-1,270 mg·(kg·d)-1和135 mg·(kg·d)-1的合欢皮总皂苷水溶液,对照组自由饮水,灌胃1周后按雌雄比2∶1合笼,继续对雄鼠灌胃至19 d,采集雄性小鼠的血液并测定血液生化指标,剖检灌胃雌性小鼠,统计雌性小鼠的怀孕率,测定雄性小鼠睾丸与附睾的脏器系数及附睾精子活力,并固定睾丸和附睾,研究其组织学变化。结果与对照组相比较,雄性小鼠灌胃合欢皮总皂苷后,导致雌性小鼠的怀孕率降低,睾丸脏器系数和附睾精子活率极显著降低(P<0.01),精子畸形率极显著升高(P<0.01)。光学显微镜下,试验组小鼠睾丸和附睾均有一定程度的损失,睾丸生精细胞排列疏松,间质减少,可见曲细精管内有多核巨噬细胞浸润,各级生精细胞脱落。附睾具正常的管腔结构,但能明显观察到管腔内精子几乎消失。结论合欢皮总皂苷对雄性小鼠具有抗生育作用,主要通过影响精子的生成及破坏生精组织而达到。     

牦牛Atg5基因克隆及其在输卵管、卵巢和子宫中的表达定位

自噬是一种保守的细胞内降解过程,在多种生物体内证实自噬有重要的生物学意义。但是自噬在牦牛这一高原特色物种体内的研究未见报道。因此为探索正常生理条件下自噬相关基因在牦牛生殖过程中的表达,本研究分别采集牦牛不同繁殖周期（卵泡期、黄体期及妊娠期）的主要生殖器官（输卵管、卵巢、子宫）,克隆牦牛自噬相关基因5（Autophagy related 5,Atg5）基因并进行生物信息学分析;利用qRT-PCR检测Atg5 基因在组织中的相对表达量;并采用蛋白质免疫印迹（Western-blot, WB）检测Atg5和Atg5-Atg12（Autophagy related 12,自噬相关基因12）复合体在不同组织中的表达水平;免疫组织化学方法分析Atg5在各生殖器官中的分布特征。结果显示,成功克隆牦牛Atg5基因在进化过程中高度保守,编码的蛋白质为可溶性的非跨膜蛋白;qRT-PCR和WB检测结果显示Atg5和Atg5-Atg12复合体在牦牛输卵管、卵巢和子宫中均有表达。其中卵泡期卵巢Atg5-Atg12表达显著高于黄体期和妊娠期,卵泡期Atg5的表达却显著低于黄体期和妊娠期;黄体期输卵管Atg5-Atg12表达量显著高于卵泡期和妊娠期,妊娠期输卵管中Atg5的表达量显著高于卵泡期和黄体期;卵泡期和妊娠期子宫中Atg5-Atg12的表达量显著高于黄体期,而黄体期子宫中的Atg5的表达量又显著高于妊娠期和卵泡期,在蛋白水平上Atg5和Atg5-Atg12的表达呈负相关。免疫组织化学结果显示Atg5在输卵管黏膜上皮,卵巢卵泡膜、颗粒层、生殖上皮、黄体细胞,子宫内膜和子宫腺体均有表达。研究结果表明自噬在牦牛体内与其他物种相似具有保守性,通过检测Atg5-Atg12复合体在牦牛生殖器官中的表达,推测自噬可能参与牦牛生殖生理过程的调控。该研究结果对自噬在其他大型哺乳动物以及高寒低氧环境中动物的研究具有借鉴意义,有助于自噬参与其他动物生殖生理作用机制的研究。       

Gene expression and localization of insulin-like growth factors and their receptors throughout amelogenesis in rat incisors.

Insulin-like growth factors (IGFs) are expressed in many tissues and control cell differentiation, proliferation, and apoptosis. In teeth, the temporo-spatial pattern of expression IGFs and their receptors has not been fully characterized. The purpose of this study was to obtain a comprehensive profile of their expression throughout the life cycle of ameloblasts, using the continuously erupting rat incisor model. Upper incisors of young male rats were fixed by perfusion, decalcified, and embedded in paraffin. Sections were processed for in situ hybridization and immunohistochemistry. mRNA and protein expression profiles IGF-I, IGF-II, IGF-IR, and IGF-IIR mRNA were essentially identical. At the apical loop of the incisor, very strong signals were seen in the outer enamel epithelium while the inner enamel epithelium showed a moderate reaction. In the region of ameloblasts facing pulp, inner enamel epithelium cells were still moderately reactive while signals over the outer enamel epithelium were slightly reduced. In the region of ameloblasts facing dentin and the initial portion of the secretory zone, signals in ameloblasts were weak while those over the outer enamel epithelium were strong. In the region of postsecretory transition, signals in both ameloblasts and papillary layer cells gradually increased. In maturation proper, signals in ameloblasts appeared as alternating bands of strong and weak reactivities, which corresponded to the regions of ruffle-ended and smooth-ended ameloblasts, respectively. Papillary layer cells also showed alternations in signal intensity that matched those in ameloblasts. These results suggest that the IGF family may act as an autocrine/paracrine system that influences not only cell differentiation but also the physiological activity of ameloblasts.     

Cellular expression of monocarboxylate transporters in the female reproductive organ of mice: implications for the genital lactate shuttle

The present study examined the cellular localization of monocarboxylate transporters (MCTs), glucose transporters (GLUTs), and some glycolysis-related molecules in the murine female genital tract to demonstrate existence of lactate/pyruvate-dependent energy systems. MCT1, a major MCT subtype, was localized selectively in the ovarian granulosa, oviductal-ciliated cells, and vaginal epithelium; all localizations were associated with intense expressions of glycolytic enzymes. MCT1 was localized in the cell membrane of granulosa cells, including fine processes extending from cumulus cells toward oocytes. The cumulus cells and oocytes showed intense signals for lactate dehydrogenase (LDH)-A and -B, respectively. The basolateral membrane of oviductal-ciliated cells expressed MCT4 as well as MCT1, while adjacent non-ciliated cells contained an intense immunoreactivity for aldolase-C, a glycolytic enzyme. The expression of GLUTs in the ovary was generally weak with an intense expression of GLUT1 only in some vascular endothelia. The oviductal epithelium expressed GLUT1 and GLUT3, respectively, in the basolateral and apical membrane of non-ciliated cells. In the vagina, the basal layers of epithelium were immunolabeled for MCT1 with the entire length of cell membrane, and expressed abundantly both GLUT1 and LDH-A. The findings correspond well with the rich existence of lactate in the genital fluids and strongly suggest the active transport of lactate/pyruvate in the female reproductive tract, which provides favorable conditions for oocytes, sperms, and zygotes.     

富含脯氨酸小蛋白-2在小鼠子宫中的表达及调节

富含脯氨酸小蛋白(Sprrs)参与构建复层扁平上皮的角化细胞壳（CE）,它们在子宫单层上皮中的作用还不清楚.采用RNA印迹和半定量RT-PCR方法,研究了Sprr2在小鼠动情周期和妊娠子宫中的表达及其激素调控.实验结果发现:Sprr2在动情前期和动情期表达上调,而动情后期和间情期表达下调.在妊娠初期表达迅速下调,直至临产期表达重新受到诱导并在产后达到高峰.鉴于其在不同生殖阶段子宫中独特的表达模式和在复层上皮中保护性的功能,推测Sprr2与子宫对交配和分娩所产生的应激反应有关.     

Patterns of heterogeneous expression of pannexin 1 and pannexin 2 transcripts in the olfactory epithelium and olfactory bulb

Pannexins form membrane channels that release biological signals to communicate with neighboring cells. Here, we report expression patterns of pannexin 1 (Panx1) and pannexin 2 (Panx2) in the olfactory epithelium and olfactory bulb of adult mice. In situ hybridization revealed that mRNAs for Panx1 and Panx2 were both expressed in the olfactory epithelium and olfactory bulb. Expression of Panx1 and Panx2 was mainly found in cell bodies below the sustentacular cell layer in the olfactory epithelium, indicating that Panx1 and Panx2 are expressed in mature and immature olfactory neurons, and basal cells. Expression of Panx2 was observed in sustentacular cells in a few locations of the olfactory epithelium. In the olfactory bulb, Panx1 and Panx2 were expressed in spatial patterns. Many mitral cells, tufted cells, periglomerular cells and granule cells were Panx1 and Panx2 positive. Mitral cells located at the dorsal and lateral portions of the olfactory bulb showed weak Panx1 expression compared with those in the medial side. However, the opposite was true for the distribution of Panx2 positive mitral cells. There were more Panx2 mRNA positive mitral cells and granule cells compared to those expressing Panx1. Our findings on pannexin expression in the olfactory system of adult mice raise the novel possibility that pannexins play a role in information processing in the olfactory system. Demonstration of expression patterns of pannexins in the olfactory system provides an anatomical basis for future functional studies.     

BMPs are necessary for stomach gland formation in the chicken embryo: a study using virally induced BMP-2 and Noggin expression

Epithelial-mesenchymal interactions are necessary for the normal development of various digestive organs. In chicken proventriculus (glandular stomach), morphogenesis and differentiation of the epithelium depend upon the inductive signals coming from underlying mesenchyme. However, the nature of such signals is still unclear despite extensive analyses carried out using experimental tissue recombinations. In this study we have examined the possible involvement of bone morphogenetic proteins (BMPs) in the formation of stomach glands in the chicken embryo. Analysis of the expression patterns of BMP-2, -4 and -7 showed that these BMPs were present in the proventricular mesenchyme prior to the initiation of the proventricular gland formation. BMP-2 expression, in particular, was restricted to the proventriculus among anterior digestive organs. Virus-mediated BMP-2 overexpression resulted in an increase in the number of glands formed. Moreover, ectopic expression of Noggin, which antagonizes the effect of BMPs, in the proventricular mesenchyme or epithelium, led to the complete inhibition of gland formation, indicating that BMP signals are necessary for the proventricular gland formation. These findings suggest that BMPs are of prime importance as mesenchymal signals for inducing proventricular glands.     

Oxidative damage increases and antioxidant gene expression decreases with aging in the mouse ovary

Oxidative stress has been implicated in various aspects of aging, but the role of oxidative stress in ovarian aging remains unclear. Our previous studies have shown that the initiation of apoptotic cell death in ovarian follicles and granulosa cells by various stimuli is initiated by increased reactive oxygen species. Herein, we tested the hypothesis that ovarian antioxidant defenses decrease and oxidative damage increases with age in mice. Healthy, wild-type C57BL/6 female mice aged 2, 6, 9, or 12 mo from the National Institute on Aging Aged Rodent Colony were killed on the morning of metestrus. Quantitative real-time RT-PCR was used to measure ovarian mRNA levels of antioxidant genes. Immunostaining using antibodies directed against 4-hydroxynonenal (4-HNE), nitrotyrosine (NTY), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) was used to localize oxidative lipid, protein, and DNA damage, respectively, within the ovaries. TUNEL was used to localize apoptosis. Ovarian expression of glutathione peroxidase 1 (Gpx1) increased and expression of glutaredoxin 1 (Glrx1), glutathione S-transferase mu 2 (Gstm2), peroxiredoxin 3 (Prdx3), and thioredoxin 2 (Txn2) decreased in a statistically significant manner with age. Statistically significant increases in 4-HNE, NTY, and 8-OHdG immunostaining in ovarian interstitial cells and follicles were observed with increasing age. Our data suggest that the decrease in mRNA expression of mitochondrial antioxidants Prdx3 and Txn2 as well as cytosolic antioxidants Glrx1 and Gstm2 may be involved in age-related ovarian oxidative damage to lipid, protein, DNA, and other cellular components vital for maintaining ovarian function and fertility.     

Insulin-like growth factor 1 is required for survival of transit-amplifying neuroblasts and differentiation of otic neurons

Neurons that connect mechanosensory hair cell receptors to the central nervous system derive from the otic vesicle from where otic neuroblasts delaminate and form the cochleovestibular ganglion (CVG). Local signals interact to promote this process, which is autonomous and intrinsic to the otic vesicle. We have studied the expression and activity of insulin-like growth factor-1 (IGF-1) during the formation of the chick CVG, focusing attention on its role in neurogenesis. IGF-1 and its receptor (IGFR) were detected at the mRNA and protein levels in the otic epithelium and the CVG. The function of IGF-1 was explored in explants of otic vesicle by assessing the formation of the CVG in the presence of anti-IGF-1 antibodies or the receptor competitive antagonist JB1. Interference with IGF-1 activity inhibited CVG formation in growth factor-free media, revealing that endogenous IGF-1 activity is essential for ganglion generation. Analysis of cell proliferation cell death, and expression of the early neuronal antigens Tuj-1, Islet-1/2, and G4 indicated that IGF-1 was required for survival, proliferation, and differentiation of an actively expanding population of otic neuroblasts. IGF-1 blockade, however, did not affect NeuroD within the otic epithelium. Experiments carried out on isolated CVG showed that exogenous IGF-1 induced cell proliferation, neurite outgrowth, and G4 expression. These effects of IGF-1 were blocked by JB1. These findings suggest that IGF-1 is essential for neurogenesis by allowing the expansion of a transit-amplifying neuroblast population and its differentiation into postmitotic neurons. IGF-1 is one of the signals underlying autonomous development of the otic vesicle.