Berisha Bajram, Rodler Daniela, Schams Dieter, Sinowatz Fred, Pfaffl Michael W
Department of Animal Biotechnology, Faculty of Agriculture and Veterinary, University of Prishtina, Pristina, Kosovo.
Animal Physiology and Immunology Weihenstephan, Technical University of Munich, Munich, Germany.
Front Endocrinol (Lausanne). 2019 Jul 10;10:467. doi: 10.3389/fendo.2019.00467. eCollection 2019.
The aim of this study was to characterize the regulation pattern of prostaglandin family members namely prostaglandin F2alpha (PTGF), prostaglandin E2 (PTGE), their receptors (PTGFR, PTGER2, PTGER4), cyclooxygenase 2 (COX-2), PTGF synthase (PTGFS), and PTGE synthase (PTGES) in the bovine follicles during preovulatory period and early corpus luteum (CL). Ovaries containing preovulatory follicles or CL were collected by transvaginal ovariectomy ( = 5 cows/group), and the follicles were classified: (I) before GnRH treatment; (II) 4 h after GnRH; (III) 10 h after GnRH; (IV) 20 h after GnRH; (V) 25 h after GnRH, and (VI) 60 h after GnRH (early CL). In these samples, the concentrations of progesterone (P4), estradiol (E2), PTGF and PTGE were investigated in the follicular fluid (FF) by validated EIA. Relative mRNA abundance of genes encoding for prostaglandin receptors (PTGFR, PTGER2, PTGER4), COX-2, PTGFS and PTGES were quantified by RT-qPCR. The localization of COX-2 and PTGES were investigated by established immunohistochemistry in fixed follicular and CL tissue samples. The high E2 concentration in the FF of the follicle group before GnRH treatment (495.8 ng/ml) and during luteinizing hormone (LH) surge (4 h after GnRH, 574.36 ng/ml), is followed by a significant (P<0.05) downregulation afterwards with the lowest level during ovulation (25 h after GnRH, 53.11 ng/ml). In contrast the concentration of P4 was very low before LH surge (50.64 mg/ml) followed by a significant upregulation ( < 0.05) during ovulation (537.18 ng/ml). The mRNA expression of COX-2 increased significantely ( < 0.05) 4 h after GnRH and again 20 h after GnRH, followed by a significant decrease ( < 0.05) after ovulation (early CL). The mRNA of PTGFS in follicles before GnRH was high followed by a continuous and significant downregulation ( < 0.05) afterwards. In contrast, PTGES mRNA abundance increased significantely ( < 0.05) in follicles 20 h after GnRH treatment and remained high afterwards. The mRNA abundance of PTGFR, PTGER2, and PTGER4 in follicles before GnRH was high, followed by a continuous and significant down regulation afterwards and significant increase ( < 0.05) only after ovulation (early CL). The low concentration of PTGF (0.04 ng/ml) and PTGE (0.15 ng/ml) in FF before GnRH, increased continuously in follicle groups before ovulation and displayed a further significant and dramatic increase ( < 0.05) around ovulation (101.01 ng/ml, respectively, 484.21 ng/ml). Immunohistochemically, the granulosa cells showed an intensive signal for COX-2 and PTGES in follicles during preovulation and in granulosa-luteal cells of the early CL. In conclusion, our results indicate that the examined bovine prostaglandin family members are involved in the local mechanisms regulating final follicle maturation and ovulation during the folliculo-luteal transition and CL formation.
本研究的目的是表征排卵前期和早期黄体(CL)形成过程中,牛卵泡内前列腺素家族成员的调控模式,这些成员包括前列腺素F2α(PTGF)、前列腺素E2(PTGE)、它们的受体(PTGFR、PTGER2、PTGER4)、环氧化酶2(COX-2)、PTGF合酶(PTGFS)和PTGE合酶(PTGES)。通过经阴道卵巢切除术收集含有排卵前卵泡或CL的卵巢(每组 = 5头母牛),并对卵泡进行分类:(I)GnRH治疗前;(II)GnRH治疗后4小时;(III)GnRH治疗后10小时;(IV)GnRH治疗后20小时;(V)GnRH治疗后25小时;(VI)GnRH治疗后60小时(早期CL)。在这些样本中,通过经过验证的酶免疫分析(EIA)测定卵泡液(FF)中孕酮(P4)、雌二醇(E2)、PTGF和PTGE的浓度。通过逆转录定量聚合酶链反应(RT-qPCR)对编码前列腺素受体(PTGFR、PTGER2、PTGER4)、COX-2、PTGFS和PTGES的基因的相对mRNA丰度进行定量。通过既定的免疫组织化学方法,在固定的卵泡和CL组织样本中研究COX-2和PTGES的定位。GnRH治疗前卵泡组的FF中E2浓度较高(495.8 ng/ml),在促黄体生成素(LH)高峰期间(GnRH治疗后4小时,574.36 ng/ml),随后显著下调(P<0.05),在排卵时(GnRH治疗后25小时,53.11 ng/ml)达到最低水平。相比之下,LH高峰前P4浓度非常低(50.64 mg/ml),在排卵时(537.18 ng/ml)显著上调(P<0.05)。COX-2的mRNA表达在GnRH治疗后4小时和20小时显著增加(P<0.05),排卵后(早期CL)显著下降(P<0.05)。GnRH治疗前卵泡中PTGFS的mRNA水平较高,随后持续显著下调(P<0.05)。相反,GnRH治疗后20小时卵泡中PTGES的mRNA丰度显著增加(P<0.05),之后保持较高水平。GnRH治疗前卵泡中PTGFR、PTGER2和PTGER4的mRNA丰度较高,随后持续显著下调,仅在排卵后(早期CL)显著增加(P<0.05)。GnRH治疗前FF中PTGF(0.04 ng/ml)和PTGE(0.15 ng/ml)浓度较低,在排卵前卵泡组中持续增加,在排卵前后进一步显著急剧增加(P<0.05)(分别为101.01 ng/ml和484.21 ng/ml)。免疫组织化学显示,在排卵前卵泡和早期CL的颗粒黄体细胞中,颗粒细胞显示出强烈的COX-2和PTGES信号。总之,我们的结果表明,所检测的牛前列腺素家族成员参与了卵泡-黄体转化和CL形成过程中调节最终卵泡成熟和排卵的局部机制。