Wohlres-Viana S, Arashiro E K N, Minare T P, Fernandes C A C, Grazia J G V, Siqueira L G B, Machado M A, Viana J H M
Universidade Federal de Juiz de Fora, Juiz de Fora, MG, 36036-900, Brazil.
Universidade Federal Fluminense, Niteroi, RJ, 24230-340, Brazil.
Theriogenology. 2019 Mar 1;126:68-74. doi: 10.1016/j.theriogenology.2018.12.004. Epub 2018 Dec 3.
The aim of this study was to evaluate the pattern of expression of LHCGR isoforms in Gir heifers characterized as good (10.3 ± 1.2 ova/embryos per flush, n = 5) or poor responders (1.1 ± 0.3 ova/embryos per flush, n = 5) to superovulation protocols. In both groups, an adapted ultrasound-guided follicular aspiration system was used to collect granulosa cells from 8 mm follicles formed either during a synchronized, non-stimulated follicular wave (no stimulation control, NS) or on the fourth day of a superovulation protocol (SOV) induced with 200 IU of pFSH. The recovered follicular fluid was centrifuged and granulosa cells were washed with NaCl 0.9% and kept in RNAlater. RNA extraction was performed using a commercial RNeasy Micro Kit and eluted samples were quantified and reverse transcribed using the commercial Superscript III kit. cDNA samples were amplified by real-time PCR using a primer to target LH/hCG receptor gene - not selective for LHCGR isoforms (total LHCGR) - and four sets of isoforms selective primers (S1, S10, S10 + 11, and S11). Analyses were performed using the REST software and expression levels are shown as mean ± SEM. Under physiological conditions (NS), poor responders had a higher expression of total LHCGR (4.9 ± 1.7 fold-change, P < 0.01) as well as isoforms S10, S11 and S10 + 11, compared to good responders. In both phenotypes, superovulation down-regulated total LHCGR expression (-0.5 ± 0.2 and -0.9 ± 0.0 for good and poor responders, respectively; P < 0.05). However, in poor responders the exogenous FSH treatment up-regulated the S10 (2.4 ± 2.0; P < 0.05), S10 + 11 (3.8 ± 3.2; P < 0.01), and S1 isoforms (1.8 ± 1.3; P < 0.05), compared to good responders We conclude that down-regulation of total LHCGR, associated to up-regulation of their inactive isoforms, may have compromised follicle development and thus contributed to the low efficiency of superovulation in heifers with a poor responder phenotype.
本研究的目的是评估促黄体生成素/人绒毛膜促性腺激素受体（LHCGR）亚型在对超排方案反应良好（每次冲卵平均获得10.3±1.2个卵子/胚胎，n = 5）或反应较差（每次冲卵平均获得1.1±0.3个卵子/胚胎，n = 5）的吉尔小母牛中的表达模式。在两组中，均使用改良的超声引导卵泡抽吸系统，从同步化、未刺激的卵泡波（无刺激对照，NS）期间形成的8毫米卵泡或用200国际单位垂体促卵泡素（pFSH）诱导的超排方案（SOV）第4天形成的8毫米卵泡中收集颗粒细胞。将回收的卵泡液离心，颗粒细胞用0.9%氯化钠洗涤，并保存在RNA Later中。使用商业RNeasy Micro试剂盒进行RNA提取，对洗脱的样品进行定量，并使用商业Superscript III试剂盒进行逆转录。使用靶向促黄体生成素/人绒毛膜促性腺激素受体基因的引物（对LHCGR亚型无选择性，即总LHCGR）和四组亚型选择性引物（S1、S10、S10 + 11和S11）通过实时PCR扩增cDNA样品。使用REST软件进行分析，表达水平以平均值±标准误表示。在生理条件下（NS），与反应良好的小母牛相比，反应较差的小母牛总LHCGR以及亚型S10、S11和S10 + 11的表达更高（变化倍数为4.9±1.7，P < 0.01）。在两种表型中，超排均下调了总LHCGR的表达（反应良好和反应较差的小母牛分别为-0.5±0.2和-0.9±0.0；P < 0.05）。然而，与反应良好的小母牛相比，在反应较差的小母牛中，外源性促卵泡素处理上调了S10（2.4±2.0；P < 0.05）、S10 + 11（3.8±3.2；P < 0.01）和S1亚型（1.8±1.3；P < 0.05）。我们得出结论，总LHCGR的下调及其无活性亚型的上调可能损害了卵泡发育，从而导致反应较差表型的小母牛超排效率低下。
