Immunohistochemical and qPCR determination of the expression and serum level of anti‐Müllerian hormone in pre‐pubertal,intact and ovarian remnant syndrome detected bitches

The aim of this study was to determine the serum concentrations, ovarian presence and expression of anti‐Müllerian hormone (AMH) in pre‐pubertal, bitches with signs of ovarian remnant syndrome (ORS) and intact bitches. In addition, we aimed to verify the suitability of serum AMH concentrations for diagnostic purposes in sterilized bitches and/or in suspected cases of ORS in the field of veterinary medicine. For this purpose, 36 healthy female dogs divided into six groups: proestrus, oestrus, dioestrus, anoestrus, pre‐pubertal and ORS. Serum AMH concentrations were determined by electrochemiluminescence immunoassay, and ovarian presence and distribution of AMH was confirmed by immunohistochemical and qPCR techniques. According to the results of qPCR, while the expression values of AMH were at the highest concentrations in the proestrus and oestrus, there was a statistically significant decrease in these values at the later stages of the cycle (p < 0.05). According to hormone analysis, the serum AMH values of the ORS group had decreased significantly compared with the proestrus and oestrus (p < 0.05). Although serum AMH levels of ORS group were increased compared with anestrus and pre‐pubertal groups, this increase was statistically non‐significant (p > 0.05). Immunohistochemically, AMH expression was first observed in the granulosa cells of primordial follicles in folliculogenesis. Expression values were the highest in the proestrous and oestrus groups, but values from bitches in later stages of the cycle were statistically significant decrease in comparison with these groups (p < 0.05). As a result, AMH concentration and expression were found to be higher in proestrus and oestrus than in other periods (p < 0.05). In addition, the measurable level of AMH concentration in bitches with ORS is an indication that it can be used in the diagnosis of ORS.     

TGF-β家族成员调控家禽卵泡发育的研究进展及展望

与哺乳动物不同,家禽的卵泡发育无明显发情周期,其卵泡发育遵循严格的等级发育体系。禽类卵泡的等级发育是一个复杂的调节过程,受卵泡细胞内外的内分泌激素、自分泌/旁分泌因子的共同调节。促卵泡刺激素(Follicle stimulating hormone,FSH)和促黄体激素(Luteinizing hormone,LH)在调控卵泡发育、卵母细胞成熟和排卵中起着重要的作用。此外,局部产生的生长因子如转化生长因子β超家族(Transforming growth factor superfamily,TGF-β)成员,包括抗缪勒氏激素(Anti-Müllerian hormone,AMH)、骨形态蛋白家族(Bone morphogenetic proteins,BMPs)、抑制素(Inhibin)和激活素(Activin)等,与促性腺激素一起协同调控卵泡的生长发育、卵母细胞的成熟等过程。文章综述了TGF-β家族成员,包括AMH、BMPs、抑制素、激活素在家禽卵泡发育中的作用及其未来的研究方向。     

Relationship among ovarian follicular status,developmental competence of oocytes,and anti‐Müllerian hormone levels: A comparative study in Japanese wild boar crossbred gilts and Large White gilts

The aim of this study was to investigate the ovarian follicular development, developmental competence of oocytes, and plasma anti‐Müllerian hormone (AMH) levels of Japanese wild boar crossbred (wild hybrid) gilts, whose litter size is inferior to that of European breeds. Ovary and plasma samples were collected from two different breeds of gilts (wild hybrid and Large White breeds). The ovaries from the wild hybrid gilts had a lower average numbers of secondary follicles and vesicular follicles in ovarian cross‐sections and of good quality oocytes collected from ovarian follicles as compared with those from Large White gilts (p < 0.05). The development rate to the blastocyst stage of good quality oocytes after in vitro maturation, fertilization and culture was also lower (p < 0.05) in wild hybrid gilts than in Large White gilts. Plasma AMH levels with >0.16 ng/ml were detected in 8.3% of the examined wild hybrid gilts and 33% of the Large White gilts. These results indicate that the low reproductive performance of wild hybrid breed may result in part from low numbers of vesicular follicles and good quality oocytes, and low developmental competence of oocytes. Moreover, plasma AMH levels may support low number of vesicular follicles in ovaries of wild hybrid gilts.     

血浆抗苗勒氏管激素水平预测荷斯坦牛胚胎生产能力的研究初报

为了验证抗苗勒氏管激素（AMH）作为标记物预测荷斯坦牛胚胎的生产能力,为选择供体牛进行胚胎生产提供一种简便有效的方法。本研究测定了10头供体牛在促性腺激素注射前后血浆中AMH浓度,结果表明促性腺激素处理前后AMH浓度无明显差异（P〉0.05）。试验还测定分析了30头供体牛血浆中AMH浓度与胚胎生产数量之间的关系,结果表明,AMH水平与卵巢的储备功能及卵巢反应性有关,与获得的胚胎数量呈显著正相关（r=0.60,P〈0.01,n=30）,可以作为一种预测荷斯坦牛超数排卵后胚胎生产能力的标记物。     

Anti-Mullerian hormone and its relationship to ovulation response and fertility in timed AI Bos indicus heifers

This study evaluated the association between plasma anti-Mullerian hormone (AMH) concentration and fertility in Nelore (Bos indicus) heifers submitted to timed artificial insemination (TAI). At the onset of the synchronization protocol, heifers (n = 289) received a subcutaneous P4 ear implant (3 mg) and 2 mg of oestradiol benzoate. Eight days later, the P4 implant was removed and 0.5 mg of oestradiol cypionate, prostaglandin (0.265 mg, i.m.) and equine chorionic gonadotropin (300 UI, i.m.) was administered, and TAI was performed 48 hr after ear implant removal. Ovarian ultrasound evaluations were performed to measure number of ovarian follicles, dominant follicle size and ovulation response. Pregnancy diagnosis was performed by ultrasound 30 days after AI. Heifers with greater circulating AMH had more antral follicles, a smaller dominant follicle near timed ovulation and lower ovulation response to the timed AI protocol compared to heifers with lower circulating AMH. Although AMH and pregnancy outcome had a quadratic-shaped pattern, AMH was not significantly associated with fertility. In conclusion, heifers with lower AMH had larger follicles towards the end of the synchronization protocol and greater ovulation responses, whereas greater circulating AMH was unrelated to conception success.     

供体牛外周血AMH浓度对荷斯坦青年奶牛体内胚胎生产效率的影响

旨在研究供体牛超数排卵（简称超排）过程中不同时间外周血AMH浓度与荷斯坦青年奶牛体内胚胎生产效率的相关性,优化供体牛筛选标准,提高荷斯坦青年奶牛体内胚胎生产效率。本试验共选用96头荷斯坦青年奶牛进行超排处理,分别在孕酮阴道栓（CIDR）埋置、人工授精和胚胎回收当天通过尾根采集外周血检测抗缪勒管激素（anti-Müllerian hormone,AMH）浓度,将不同时期的外周血AMH浓度按照高（浓度分布最高的25%）、中等（浓度分布中间的25%~75%）、低（浓度分布最低的25%）分为3组,并检验了这3组之间头均回收胚胎数、头均可用胚胎数、头均退化胚胎数和头均未受精数等指标间的差异。结果表明：1）外周血AMH浓度检测结果显示,从CIDR埋置至胚胎回收的全过程AMH浓度整体呈下降趋势;2）CIDR埋置当天AMH高浓度组头均回收胚胎数（9.38±1.24）显著高于中等浓度组（6.47±0.56）（P<0.05）,说明CIDR埋置当天AMH浓度高的供体牛超排效果更好,回收胚胎数更多;但是,结果也表明,高浓度组头均未受精卵母细胞数显著高于其他两组（P<0.05）,说明部分卵母细胞未能成功受精,这可能是由于高浓度组牛只更多的卵泡优势化,排卵周期延长导致部分卵母细胞未能完成正常受精;3）人工授精和胚胎回收当天的AMH数据分析结果均表明,3组间各项胚胎生产指标均无显著性差异（P>0.05）;4）通过对供体牛体重记录数据分析发现,不同体重牛只间的体内胚胎生产效率差异也不显著（P>0.05）,但是体况适中组牛只的可用胚胎数、回收胚胎数优于其他两组,尤其是优于体重偏胖组。因此,CIDR埋置时,外周血AMH浓度可以作为青年荷斯坦供体牛筛选标准之一,且供体牛选择时宜选用体况适中的牛只。     

Anti-Muellerian hormone levels in plasma of Holstein-Friesian heifers as a predictive parameter for ovum pick-up and embryo production outcomes

The aim of this study was to investigate whether plasma anti-Muellerian hormone (AMH) levels of Holstein-Friesian heifers could be used to predict ovum pick-up (OPU) and embryo production outcomes. Plasma samples and data were collected from 64 heifers, which underwent repeated OPU with subsequent in vitro embryo production followed by embryo flushing after superovulation. AMH levels were significantly positively correlated with the number of follicles aspirated per OPU session (r = 0.45), recovered oocytes per OPU (r =0.43) and in vitro produced embryos per OPU (r = 0.28). No significant correlations between AMH and in vivo produced embryos were ascertained. Our results suggest that correlations between AMH and outcomes of an OPU-IVF program are too low to use AMH as a precise predictive parameter for the success of a particular OPU procedure in Holstein-Friesian heifers. However, AMH can help to identify groups of very good or very poor oocyte donors.     

奥利亚罗非鱼AMH基因结构及其表达

抗缪勒氏管激素(antiMullerian hormone, AMH),也称缪勒氏管抑制物质(mullerian inhibiting substance, MIS),为肽类生长因子,属于TGFβ生长和分化因子家族。应用RTPCR和RACE法,从奥利亚罗非鱼精巢组织中分离出AMH的cDNA。分离到3′UTR长为621 bp和168 bp的两条cDNA,除了长度不一,两者之间只存在两个碱基的差别。5′UTR为 27 bp,阅读框为1 545 bp,翻译成514个氨基酸,其中,AMH-N域237个氨基酸,TGFβ域93个氨基酸。同源性分析显示,AMH保守性偏低,奥利亚罗非鱼与其它鱼类的相似性为32%~60%,与哺乳类动物只有19%~21%,而TGF-β域的保守性相对较高,与其它鱼类的相似性为54%~72%。基因结构分析显示,奥利亚罗非鱼AMH存在6个内含子,和斑马鱼AMH结构相似,和人、鼠AMH有较大差别,它们缺少奥利亚罗非鱼和斑马鱼AMH基因共有的内含子Ⅰ和Ⅵ。然而,启动子转录因子分析结果表明,奥利亚罗非鱼AMH启动子中也存在SRY, SF-1, GATA-4, Sox5, Sox9等结合位点。使用Taqman探针分析奥利亚罗非鱼AMH在成鱼不同组织中的表达以及鱼苗、鱼种、成鱼性腺中的表达量,结果显示AMH在成鱼的卵巢、精巢中均有表达,而在脑、肝、肌肉以及心脏中不表达。AMH在鱼苗性腺中不表达,在鱼种、成鱼中雄鱼AMH表达量显著高于雌鱼,而鱼种雄鱼AMH表达量是成鱼雄鱼的10倍。