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Luteal steroids are necessary to maintain the first 70-90 days of pregnancy in the mare. At 35 days postovulation, the resurgence of the primary corpus luteum (CL) coincides with the secretion of the fetal hormone eCG. In order to study the responsiveness of the primary CL to eCG, we have examined levels of luteal equine LH/CG receptors (eLH/CG-R) mRNAs by Northern blot analysis and measured concentrations of eLH/CG binding sites on luteal membranes using 125I-eLH saturation binding assays at three stages of gestation: before the onset of eCG secretion (Days 14-31), from onset to maximum eCG secretion (Days 38-62), and during decline of eCG secretion (Days 83-101). Multiple transcripts of eLH/CG-R (7, 5.7, 4.9, 3.9, 2.8, 1.8, 0.6 kilobase [kb]) were identified in the primary CL at all stages examined. Three of them (5.7, 2.8, 0.6 kb) coded for truncated eLH/CG-R lacking the transmembrane domain. The relative intensities of the four major transcripts tended to decrease (5.7 and 3.9 kb) or were steadily expressed (7 and 1.8 kb) during pregnancy. The affinity of eLH/CG binding sites did not change during pregnancy whereas the number of eLH/CG binding sites decreased significantly after the onset of eCG secretion. Nevertheless, levels of binding sites were still at 44.6% (Days 38-62) to 24.7% (Days 83-101) of those measured before the onset of eCG secretion. Taken together, the presence of eLH/CG-R mRNAs and of a substantial part of eLH/CG binding sites with high affinity suggest that the primary CL still expresses a high number of eLH/CG-R and remains responsive to eCG during early pregnancy.

The onset of eCG secretion in pregnant mares coincides with an increase in luteal steroid production and a relative shift toward androgen and estrogen synthesis. However, a cause-effect relationship between eCG and the shift in luteal steroidogenesis has not been demonstrated. In this study, we have investigated the effect of eCG on steroid production by the corpus luteum (CL) during equine pregnancy. All mares were supplemented with 44 mg altrenogest (a progestogen) per day on Days 18-50. Increasing doses of eCG were administered on Days 26-28, before the onset of endogenous eCG secretion, to four mares with and four mares without a functional CL (prostaglandin F2alpha administered on Day 18). Four mares with a functional CL received no exogenous eCG. In eCG-treated mares without a functional CL, progestin, androstenedione, and estrogen concentrations did not significantly increase after exogenous eCG administration or endogenous eCG secretion. In eCG-treated mares with a functional CL, progestin and estrogen production increased significantly after exogenous eCG administration and endogenous eCG secretion, whereas androstenedione concentrations tended to increase following exogenous eCG and increased significantly following endogenous eCG secretion. In mares with a functional CL that did not receive exogenous eCG, progestin and estrogen concentrations increased and androstenedione concentrations tended to increase only after the onset of endogenous eCG secretion. These data demonstrate that the increase in luteal steroidogenesis that coincides with the onset of eCG secretion is induced by eCG and results in an increase in luteal androgen and estrogen synthesis. Our findings support the hypothesis that eCG has a luteotropic action in pregnant mares.

In pregnant mares, eCG stimulates luteal androgen and estrogen production, increasing plasma concentrations 2- to 3-fold. To study how these changes are regulated, we examined the expression of mRNA for the steroidogenic enzymes 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), cytochrome P450 17 alpha-hydroxylase/17,20-lyase (P450 17 alpha), and cytochrome P450 aromatase (P450arom) in equine primary corpora lutea using Northern blot analyses. Three equine specific cDNAs were generated by reverse transcriptase polymerase chain reaction. When compared to human, bovine, and rat sequences, the nucleotide identities were 82%, 84%, and 76%, respectively, for 3 beta-HSD cDNA (843 base pairs [bp]); 79%, 80% and 66% for P450(17) alpha cDNA (541 bp); and 80%, 83% and 75% for P450arom cDNA (289 bp). The P450(17) alpha cDNA sequence demonstrated 99.6% nucleotide identity with the previously published sequence for equine testicular P450(17) alpha. Luteal tissue samples were collected at three times: diestrus (Days 8-10), early pregnancy before the onset of eCG secretion (Days 29-35), and early pregnancy after the onset of eCG secretion (Days 42-45). Although no significant changes were observed in 3 beta-HSD expression, P450(17) alpha and P450arom demonstrated stage-specific transcriptional regulation. Steady-state levels of P450(17) alpha mRNA were similar during diestrus and early pregnancy before the onset of eCG secretion but increased significantly after the onset of eCG secretion. Cytochrome P450arom mRNA levels decreased significantly after the onset of eCG secretion. Steady-state levels of P450arom mRNA were highest in luteal tissue collected during pregnancy before the onset of eCG secretion and intermediate during diestrus. Secretion of eCG appears to increase luteal estrogen synthesis by a transcriptional up-regulation of P450(17) alpha expression. These data suggest that availability of aromatizable androgens may be rate-limiting in luteal estrogen synthesis before the onset of eCG secretion.