AQP8 participates in oestrogen-mediated buffalo follicular development by regulating apoptosis of granulosa cells

Aquaporins (AQPs), a family of small membrane-spanning proteins, are involved in fluid transport, cell signalling and reproduction. Regulating AQP8 expression influences apoptosis of granulosa cells (GCs), ovarian folliculogenesis, oogenesis and early embryonic development in mice, but its role has never been investigated in other species. The aim of the present study was to characterize the AQP8 function in buffalo follicular development. The expression pattern of AQP8 in buffalo follicle was analysed by immunohistochemistry method. 17β-Estradiol (E2) or oestrogen receptor antagonist ICI182780 was used to treat GCs cultured in vitro, and the expression of AQP8 was detected using qRT-PCR. Its roles in apoptosis of buffalo GCs were investigated by shRNA technology. AQP8 was found to be expressed higher in secondary follicles (p < .05), and its mRNA level in GCs was upregulated by E2 via receptor-mediated mechanism in a dose-dependent manner. A 732-bp buffalo AQP8 coding region was obtained, which was highly conserved at the amino acid level among different species. AQP8-shRNA2 had more effective inhibition on target gene than AQP8-shRNA1 (66.49% vs. 58.31%) (p < .05). Knockdown of AQP8 induced GCs arrested at G2/M stage and occurred apoptosis. Compared with the control group, higher Caspase9 expression were observed in AQP8-shRNA2 lentivirus infected GCs (p < .05), while Bcl-2 and Bax expression levels had no obvious change (p > .05). Altogether, the above results indicate that AQP8 is involved in oestrogen-mediated regulation of buffalo follicular development by regulating cell cycle progression and apoptosis of GCs.     

水牛水通道蛋白9基因克隆及其表达分析

本研究旨在对水牛水通道蛋白9 (aquaporins 9,AQP9) 基因进行克隆,并对其在水牛不同组织中的表达规律及其在水牛卵巢和睾丸组织中的表达差异进行探索。根据GenBank上黄牛AQP9基因序列(登录号:NM_001205833.1)设计特异性引物,以水牛睾丸组织cDNA为模板,应用RT-PCR方法扩增AQP9基因编码区片段;运用生物信息学方法分析其核苷酸序列的保守性和氨基酸的理化性质;应用实时荧光定量PCR技术分析AQP9基因在水牛组织中的表达情况;免疫组织化学方法分析AQP9蛋白在不同发育阶段水牛卵泡及睾丸组织中的表达差异。结果表明,克隆获得了888 bp的水牛AQP9基因编码区序列,其编码295个氨基酸。多重序列比较显示,水牛AQP9核苷酸序列与牛、猪、绵羊和人相应序列相似性分别为99%、90%、97%、88%;氨基酸序列的同源性分别为99%、86%、97%、83%,系统进化树分析结果推测,AQP9基因在物种进化过程中具有高度保守性。实时荧光定量PCR结果显示,AQP9基因在水牛肝脏、肺脏、大脑、皮肤、睾丸和卵巢组织中有不同程度的表达,在肝脏组织中表达最高,皮肤和睾丸次之,肺脏和卵巢表达较低。免疫组化结果显示,在卵巢组织中,AQP9蛋白表达随卵泡发育时期的不同而变化,并随着卵泡发育其表达逐渐增强;在睾丸组织中,AQP9蛋白在各级精母细胞和间质细胞中均有表达。结果提示,成功克隆得到水牛AQP9基因序列;AQP9在水牛卵巢和睾丸中的表达及其功能可能与水牛卵泡发育和精子发生有重要的关联。     

Cloning of Buffalo AQP9 Gene and Analysing its Expression in the Buffalo Tissues

Cloning buffalo AQP9 gene and analyzing its expression in buffalo tissues.A pair of primers was designed according to the released bovine AQP9 sequences in GenBank,which was used to clone buffalo AQP9 gene.The AQP9 gene was amplified by RT-PCR,whose nucleotide sequence and protein structure were analyzed by bioinformatics methods.The expression of AQP9 in buffalo tissues was assayed by Real-time quantitative PCR.The expression of AQP9 gene in buffalo ovary and testis tissue was detected by immunohistochemical staining method.The results showed that the cloned ORF length of buffalo AQP9 gene was 888 bp,which coded 295 amino acids.The results of multiple sequence comparison showed that the nucleotide sequence of buffalo AQP9 shared 99%,90%,97% and 88% homologeous compared with that of Bos taurus,Sus scrofa,Ovis ariessis and Homo sapiens,respectively,while shared 99%,86%,97%,83% homologeous for amino acids,respectively.Phylogenetic tree analysis indicated that AQP9 gene was highly conservative in the evolutionary process.Real-time quantitative PCR results showed that AQP9 gene expressed in buffalo liver,lung,brain,skin,testis and ovary tissues with different levels,had the most abundant expression in liver,followed by in skin and testis,less observed in lung and ovary.The results of immunohistochemical staining showed that the expression of AQP9 protein varied with the development of buffalo ovarian tissue,and gradually enhanced with follicle development.In testicular tissue,AQP9 protein expressed in spermatocyte and leydig cells of developmental stage testis.These results indicated that we had successfully cloned buffalo AQP9 gene sequences.The expression and its function of AQP9 in buffalo ovaries and testes might play an important role in follicle development and spermatogenesis.     

海兰褐蛋鸡ZP3基因结构及其在卵泡中表达规律分析

旨在探讨ZP3基因在海兰褐蛋鸡中的表达特性，为后期研究ZP3基因在蛋鸡卵泡发育中的作用提供理论支撑。本研究以产蛋期50周龄（50 W）海兰褐蛋鸡为试验对象，利用RACE技术克隆ZP3基因全长，并对编码区进行生物信息学分析。选择健康、体重相近的海兰褐蛋鸡6只，分别取心、肝、肺、肾、卵巢、胸肌、腿肌和不同等级卵泡构建表达谱。卵巢颗粒细胞经不同浓度（PBS、5 ng·mL^-1、10 ng·mL^-1、20 ng·mL^-1）促卵泡素（follicle-stimulating hormone，FSH）处理后，提取总RNA，采用实时荧光定量检测ZP3基因在各个样品的表达水平。结果表明，鸡ZP3基因全长1 415 bp，其中编码区1 341 bp，共编码446个氨基酸，位于10号染色体上，包含9个外显子；其亲缘关系与猪最远；跨膜结构预测表示，其含有一个跨膜结构域和一个信号肽区域；对其亲疏水性进行分析，预测该蛋白为弱的疏水性蛋白，蛋白质结构主要由无规则卷曲构成。组织表达谱显示，ZP3基因在鸡的卵巢中相对表达量最高。各等级卵泡ZP3表达分析显示，随着卵泡发育，ZP3基因在成熟卵泡（F1）颗粒细胞层表达量最高；在使用FSH处理等级颗粒细胞后，发现ZP3表达量显著上调（P<0.05），暗示ZP3基因在颗粒细胞中受到FSH激素调节。本试验对ZP3基因的结构进行了分析并预测该基因存在跨膜结构和信号肽区域。基因表达谱分析结果显示，ZP3基因在卵泡发育过程中表达量逐渐升高且主要在颗粒细胞中表达，可能受到FSH的调节作用，因此预测，ZP3基因可能与海兰褐蛋鸡繁殖功能相关。     

Expression Analysis of C-type Natriuretic Peptide and Its Receptor in Buffalo Follicles

In order to investigate the expression pattern of C-type natriuretic peptide（CNP）and its receptors（NPR）in buffalo follicles,firstly,the mRNA expression level of ANP,BNP,CNP,NPR1 and NPR2 in ovary were assayed by Real-time quantitative PCR;Secondly,the protein expression of CNP and NPR2 in buffalo follicles were detected by immunohistochemical stainning method;Lastly,the mRNA expression level of CNP and NPR2 in granule cells and cumulus cells were assayed by Real-time quantitative PCR.The results showed that CNP gene and its receptor NPR2 mainly expressed in buffalo ovary,the protein of CNP and NPR2 expressed in all stages of follicles,CNP mainly expressed in mural granulosa cells and NPR2 primarily presented in cumulus cells,the mRNA expression of CNP gene in granule cells was significantly higher than cumulus cells（P<0.05),whereas the mRNA expression of NPR2 gene in cumulus cells was significantly higher than granule cells（P<0.05).In conclusion,among the main members of natriuretic peptides and its receptors,CNP and NPR2 presented strong expression in buffalo ovary.CNP mainly expressed in mural granulosa cells,but NPR2 primarily presented in cumulus cells which arounding oocytes.     

C型利钠肽及其受体在水牛卵泡中的表达分析

为了探明C型利钠肽（C-type natriuretic peptide,CNP）及其受体（natriuretic peptide receptor,NPR）在水牛卵泡中的表达模式,本研究首先采用实时荧光定量PCR技术检测水牛卵巢中利钠肽家族主要成员A型、B型、C型利钠肽（ANP、BNP、CNP）及其Ⅰ型、Ⅱ型受体（NPR1、NPR2）的mRNA表达情况,然后利用免疫组化技术对水牛卵泡中CNP及NPR2蛋白进行定位,最后利用实时荧光定量PCR技术检测颗粒细胞和卵丘细胞中CNP及NPR2的mRNA表达规律。结果显示,水牛卵巢主要表达CNP及NPR2,且在各级卵泡中均有表达,其中,CNP主要在壁层颗粒细胞中表达,NPR2主要在卵丘细胞中表达;颗粒细胞上CNP mRNA表达水平显著高于卵母细胞周围的卵丘细胞（P<0.05）,而卵丘细胞上NPR2 mRNA表达水平显著高于颗粒细胞（P<0.05）。综上所述,在利钠肽主要家族成员和受体中,CNP和NPR2在水牛卵巢中呈现强表达,CNP主要在壁层颗粒细胞中表达,而NPR2主要在卵母细胞周围的卵丘细胞中表达。     

Effect of growth differentiation factor‐9 (GDF‐9) on the progression of buffalo follicles in vitrified–warmed ovarian tissues

To improve the reproductive performance of water buffalo to level can satisfy our needs, the mechanisms controlling ovarian follicular growth and development should be thoroughly investigated. Therefore, in this study, the expressions of growth differentiation factor‐9 (GDF‐9) in buffalo ovaries were examined by immunohistochemistry, and the effects of GDF‐9 treatment on follicle progression were investigated using a buffalo ovary organ culture system. Frozen–thawed buffalo ovarian follicles within slices of ovarian cortical tissue were cultured for 14 days in the presence or absence of GDF‐9. After culture, ovarian slices were fixed, sectioned and stained. The follicles were morphologically analysed and counted. Expression pattern of GDF‐9 was detected in oocytes from primordial follicles onwards, besides, also presented in granulosa cells. Moreover, GDF‐9 was detected in mural granulosa cells and theca cells of pre‐antral follicles. In antral follicles, cumulus cells and theca cells displayed positive expression of GDF‐9. In corpora lutea, GDF‐9 was expressed in both granulosa and theca lutein cells. After in vitro culture, there was no difference in the number of primordial follicles between cultured plus GDF‐9 and cultured control that indicated the GDF‐9 treatment has no effect on the primordial to primary follicle transition. GDF‐9 treatment caused a significant decrease in the number of primary and secondary follicles compared with controls accompanied with a significant increase in pre‐antral and antral follicles. These results suggest that a larger number of primary and secondary follicles were stimulated to progress to later developmental stages when treated with GDF‐9. Vitrification/warming of buffalo ovarian tissue had a little remarkable effect, in contrast to culturing for 14 days, on the expression of GDF‐9. In conclusion, treatment with GDF‐9 was found to promote progression of primary follicle that could provide an alternative approach to stimulate early follicle development and to improve therapies for the most common infertility problem in buffaloes (ovarian inactivity).     

Regulation of secondary follicle growth by theca cells and insulin-like growth factor 1

Ovaries contain follicles at various stages of development, including primordial, primary, secondary, antral and Graafian follicles. Although the growth of these follicles is controlled to maintain regular ovulation, the mechanism through which this occurs remains unclear. In our study, we found that the growth rate of cultured secondary follicles separated from mice ovaries differed between follicles. After 4 days of culture, the size of some secondary follicles was markedly increased, while that of others had either slightly increased, remained unchanged or shrunk. We compared the expression levels of growth factors between these secondary follicles and found that the growth rate of cultured secondary follicles correlated with the expression level of insulin-like growth factor 1 (Igf1) mRNA. Igf1 mRNA expression level in secondary follicles containing theca cells was higher than that in secondary follicles without theca cells, and the granulosa cell proliferation around follicles containing theca cells was increased. Furthermore, an IGF1 inhibitor also inhibited the granulosa cell proliferation, and administration of IGF1 to secondary follicles without growth promoted granulosa cell proliferation. These results indicated that the theca cells of secondary follicles induced the expression of IGF1 and promoted the follicle growth.     

DDR1基因过表达对水牛乳腺上皮细胞的影响

为揭示盘状结构域受体1（discoidin domain receptor1,DDR1）基因对水牛泌乳性能的影响,本研究构建了水牛DDR1基因真核表达载体,并对其最佳转染时间进行摸索,同时分析DDR1基因过表达对水牛乳腺上皮细胞的影响。琼脂糖凝胶电泳检测结果显示,载体片段大小与目标载体片段大小一致,均为8.8 kb,测序结果显示其与目的片段序列匹配率为100%。细胞转染试验结果显示,DDR1基因过表达最佳转染时间为48 h。细胞增殖检测结果显示,DDR1基因过表达组与对照组细胞相对荧光值差异不显著（P>0.05）。细胞凋亡检测结果显示,DDR1基因过表达对水牛乳腺上皮细胞的晚期凋亡率（19.87% VS 17.49%）无影响（P>0.05）,但极显著增加了水牛乳腺上皮细胞早期凋亡率（6.48% VS 1.35%,P<0.01）。同时,DDR1基因过表达上调了水牛乳腺上皮细胞中抑凋亡基因BCL-2和XIAP的表达,而下调了促凋亡基因P53的表达。此外,DDR1基因过表达显著提高了水牛乳腺上皮细胞的迁移率（66.26% VS 58.76%,P<0.05）。综上,试验成功构建了水牛DDR1基因真核表达载体并证明了DDR1基因过表达促进了水牛乳腺上皮细胞的早期凋亡和迁移,为今后进一步研究水牛乳腺发育和泌乳性能提供了一定理论依据。