171 Effect of progesterone and prolactin on the developmental competence of bovine oocytes during the terminal phase of in vitro maturation

Abstract

To date, considerable progress has been achieved in in vitro production (IVP) technologies in cattle; however, developmental potentials of oocytes matured in vitro remain low compared with in vivo-matured oocytes. Thus, a better understanding of different aspects of oocyte maturation may allow us to increase the embryo development rate. Our study was aimed to assess the effects of progesterone (P4) and prolactin (PRL) on the bovine oocyte developmental competence. Bovine cumulus-enclosed oocytes (CEO) were matured using either one-step or two-step maturation conditions. For the one-step protocol, CEO were cultured for 24 h in TCM-199 supplemented with 10% fetal calf serum (FCS), 10 μg mL⁻¹ porcine FSH, and 10 μg mL⁻¹ ovine LH (standard medium). For the two-step procedure, CEO were first cultured for 16 h in the standard medium (n = 1263) and then transferred to 1 of 3 experimental media and cultured for additional 8 h in either absence or presence of either P4 (50 ng mL⁻¹) or bovine PRL (50 ng mL⁻¹). The 3 media tested in the two-step maturation were (1) TCM-199 containing 10% FCS (group 1), (2) TCM-199 containing 3 mg mL⁻¹ BSA (group 2), or (3) Fert-TALP medium supplemented with 6 mg mL⁻¹ BSA (group 3). Fert-TALP was selected because it can potentially be used throughout maturation and fertilization. Following in vitro maturation, all oocytes underwent an IVF/in vitro culture procedure as described previously (Singina et al. 2014 Reprod. Fertil. Devel. 26, 154). The embryo development was evaluated at Days 2 and 7 for cleavage and blastocyst rates. In addition, obtained blastocysts were fixed, and the total cell number and the level of apoptosis were determined using 4′,6-diamidino-2-phenylindole (DAPI) and TUNEL staining. The data from 4 to 5 replicates (113-159 oocytes per treatment) were analysed by ANOVA. For oocytes matured for 24 h in the one-step culture, the cleavage rate, blastocyst rate, total cell number, and apoptotic nuclei per blastocyst were 66.1 ± 1.1, 23.7 ± 2.0, 71.4 ± 9.1, and 4.8 ± 1.2%, respectively. For the two-step culture, the cleavage rate did not differ from that of the one-step culture system, ranging from 64.8 to 76.5%. Also, no effects of the two-step systems were observed on total cell number (63.0-78.8) or the proportion of apoptotic nuclei (3.3-5.3%) at the blastocyst stage. The culture of CEO in group 1 (without the supplements) had a reduced blastocyst rate (17.4 ± 0.4%; P < 0.05) compared with the standard one-step maturation group, and the addition of P4 (but not PRL) improved the blastocyst yield (P < 0.05). Furthermore, when P4 (but not PRL) was added to group 2 and group 3 media, blastocyst rates increased significantly (32.9 ± 3.1 and 32.8 ± 2.7%, respectively) compared with those of the one-step group (P < 0.05), but did not differ from those of untreated groups 2 and 3 (26.2 ± 2.7 and 30.0 ± 3.0%, respectively). Our data indicate that P4 supplementation during the terminal phase of two-step IVM can enhance the developmental competence of bovine oocytes and that the nature of this effect depends on the composition of IVM medium, whereas no effect of PRL supplementation was observed.

The study was supported by RFBR (No. 17-29-08035).