Ovine placental lactogen (oPL) is produced by the conceptus trophectoderm and is secreted into both the maternal and fetal circulations. The present study was designed to examine in vivo the luteotropic effect of recombinant oPL (roPL), as determined by monitoring progesterone concentration and cycle length (experiment 1), and the antioxidative and antiapoptotic effects of roPL, as determined respectively by monitoring antioxidant enzymatic activity and apoptosis in the corpus luteum (CL) of cyclic ewes (experiment 2). We also studied whether roPL is capable of stimulating progesterone secretion in vitro by cultured luteal tissue of functionally active CL obtained from day-10 cyclic ewes (experiment 3) and day-60 pregnant ewes (experiment 4). Circulating concentrations of progesterone and cycle length were not affected by treatment of ewes with 80 microg/kg body weight per day of roPL (n = 4 ewes) for 10 days beginning on day 11 post-estrus, as compared with saline-treated ewes (n = 4 ewes). Luteolysis occurred between days 15 and 16 post-estrus in the four saline-treated ewes and in 3/4 roPL-treated ewes. The activities of the key antioxidant enzymes copper-zinc superoxide dismutase (Cu,Zn-SOD), manganese SOD (Mn-SOD), glutathione peroxidase (GPX), glutathione reductase (GSR) and glutathione S-transferase (GST) were unaffected by treatment of ewes with 80 microg/kg per day of roPL (n = 4 ewes) for 3 days, between days 11 and 14 post-estrus, as compared with saline-treated ewes (n = 4 ewes). In situ TUNEL method revealed that the number of apoptotic cells was not different between the two groups of ewes. There was no significant change in progesterone secretion by explants from day-10 estrous cycle (n = 3 ewes) or day-60 pregnancy (n = 3 ewes) CL cultured with different concentrations (10, 100 and 1000 ng/ml) of roPL, whereas treatment with oLH at the concentration of 100 or 1000 ng/ml caused a significant increase in progesterone secretion by explants from day-10 estrous cycle CL (P < 0.05) and by explants from day-60 pregnancy CL (P < 0.01). In conclusion, our results demonstrate that oPL has no luteotropic and/or luteoprotective actions in sheep, either in vivo or in vitro.

Anti-oxidative enzymes play a role in protecting cells from oxidative stress-induced cell death. The present study was conducted to evaluate whether the anti-oxidant and pro-oxidant enzymatic capacities of the sheep corpus luteum (CL) are correlated with steroidogenic and structural status of the gland during the estrous cycle. Steroidogenic activity, apoptosis and superoxide dismutase (SOD1 and SOD2), nitric oxide synthase (NOS), glutathione peroxidase (GPX), glutathione reductase (GSR) and glutathione S-transferase (GST) activities were determined in the CL at specific developmental stages of the luteal phase. The intensity of apoptotic DNA fragmentation, characteristic of physiological cell death, was much greater in CL at late luteal phase than at early and mid-luteal phase, concomitantly with the diminution in the plasma progesterone concentrations from mid-to late luteal phase. SOD1 and GPX activities increased from early to mid-luteal phase, and increased further at late luteal phase. SOD2 and GST activities were not different between early and mid-luteal phase, but increased at late luteal phase. GSR activity was not different between any luteal phase examined. NOS activity decreased from early to mid- and late luteal phase. These results show that the activities of SOD1, SOD2, NOS, GPX, GSR and GST in the sheep CL are subject to major changes during the estrous cycle, and that the anti-oxidant and pro-oxidant enzymatic capacities of luteal cells are not correlated with cell steroidogenic status and integrity during the late luteal phase.

The activities of copper, zinc-superoxide dismutase (SOD1), manganese SOD (SOD2), glutathione peroxidase (GPX), glutathione reductase (GSSG-R) and glutathione S-transferase (GST) were studied in sheep corpora lutea (CL) obtained on days 15, 40, 60, 80 and 128 of pregnancy. Maintained enzymatic activity of SOD1, SOD2, GPX, GSSG-R and GST were found in the sheep CL throughout pregnancy. Enzymatic activity of SOD1, GPX and GST increased significantly from day 15 to day 40 of pregnancy, and thereafter remained constant until day 128. SOD2 and GSSG-R activities were not different between any days of pregnancy examined. Apoptotic luteal cells identified by the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick-end labelling were very rarely observed, and their incidence (less than 0.5%) was not different between days of pregnancy. These results showed that the activities of antioxidant enzymes in the sheep CL are subject to major changes during early pregnancy, suggesting that the CL of early pregnancy may be rescued from luteolysis through increasing activities of key antioxidant enzymes and inhibition of apoptosis. Maintained levels of antioxidant enzymes in the CL throughout pregnancy may be linked to reactive oxygen species continuously generated in the steroidogenically active luteal cells, and may be involved in the maintenance of luteal steroidogenic activity and cellular integrity.