249 THE DIRECT AND CUMULUS-MEDIATED EFFECTS OF PROLACTIN ON BOVINE OOCYTES

Abstract

Prolactin (PRL) is an important regulator of female reproduction, as indicated using PRL receptor knockout mice (Bole-Feysot C et al. 1998 Endocrinol. Rev. 19, 225–268). The presence of PRL receptors or their mRNA has been shown in different follicular compartments of various mammalian species, including oocytes of sheep and mice (Picazo RA et al. 2004 Reproduction 128, 545–553; Kiapekou E et al. 2005 Reprod. Biomed. Online 10, 339–346). The aim of the present study was to characterize the direct and cumulus-mediated pathways of PRL signaling into bovine oocytes. The expression of PRL receptor mRNA in follicular cells was detected by reverse transcription polymerase chain reaction (RT-PCR) analysis. For oocytes, nested PCR was used. In addition, a total of 707 COC and 689 denuded oocytes (DO) from follicles 2 to 8 mm in diameter were cultured for 6 h (COC), 8 h (DO), or 24 h (COC and DO) in the presence or absence of 50 ng mL^–1 bovine PRL (20 IU mg^–1; Research Center for Endocrinology, Moscow, Russia). The following systems for COC and DO culture were used: (1) TCM-199 containing 10% fetal calf serum (system 1) and (2) DMEM containing 10 U mL^–1 pregnant mare’s serum gonadotropin (PMSG), 5 U mL^–1 hCG (Intervet Scandinavia, Copenhagen, Denmark), and 5% estrous cow serum (system 2). The nuclear status of oocytes was evaluated by Tarkowski’s cytogenetic method. After IVM in system 2, a portion of the oocytes (348 COC and 311 DO) underwent IVF and IVC, and the embryo development was tracked until the blastocyst stage. All treatments were repeated 4 to 7 times. Results were expressed as mean ± SEM. Arcsine-transformed data were analyzed by two-way ANOVA. Messenger RNA expression of long and short isoforms of PRL receptor was revealed in both bovine oocytes and cumulus cells. In system 1, PRL raised the rate of DO remaining at the diplotene stage by 8 h of culture (from 32.6 ± 3.0 to 47.5 ± 4.7%, P < 0.05), whereas there was no effect of PRL on the meiotic resumption in COC. By contrast, the hormone exerted a stimulatory action on the meiotic progression in the presence of cumulus cells by increasing the proportion of oocytes reaching the telophase I or metaphase II stages during 24 h of maturation (from 61.9 ± 1.2 to 73.5 ± 0.9%, P < 0.05). In system 2, PRL did not affect nuclear maturation of either cumulus-enclosed or cumulus-free oocytes, with the maturation rate varying between 93.9 ± 2.7 and 95.2 ± 2.4% (COC) and between 85.6 ± 3.0 and 83.0 ± 4.0% (DO). When added to IVM system 2, PRL raised the cleavage and blastocyst rates only in the case of COC (from 72.2 ± 2.4 to 80.2 ± 2.1%, P < 0.05, and from 20.5 ± 3.6 to 40.9 ± 4.2%, P < 0.01, respectively). These findings suggest functioning of the direct and cumulus-mediated pathways of PRL signaling into bovine oocytes, with the hormone affecting meiosis via both pathways.

We thank A. D. Roed for expert technical assistance with IVF and IVC. This research was supported in part by RFBR (project no. 07-04-01485). I.L. was the recipient of an Erasmus Mundus fellowship.