Expression and localization of relaxin in the ovary of the mare

Abstract

Immunoreactive, chromatographic and molecular techniques were used to study the expression of relaxin in mare ovaries at different stages of the oestrous cycle. Relaxin in follicular fluid ranged from 1.6 to 2.5, from 1.4 to 5.2, from 1.2 to 6.7 and from 1.0 to 3.5 ng ml-1 in small (≤ 2 cm), medium (> 2 ≤ 3 cm), medium-large (> 3 ≤ 4 cm) and large (> 4 cm) follicles, respectively, and total content of fluid relaxin per follicle increased (P < 0.05) with follicular size. When subjected to reverse phase HPLC analysis, follicular fluid yielded absorbance profiles corresponding closely to those of purified relaxin, and immunoreactive peaks in follicular fluid fractions measured by radioimmunoassay matched peaks of the relaxin standard. While relaxin was localized immunocytochemically to granulosa and theca cells of preovulatory follicles, northern blot and reverse transcriptase-PCR followed by Southern blot analysis failed to detect a relaxin transcript in these tissues. A single relaxin transcript (428 bp) corresponding to mRNA encoding relaxin was identified in early, mid- and late stage corpora lutea but not in corpora haemorrhagica or albicantia. Northern blot analysis revealed a weakly expressed 1 kb transcript in total cellular RNA from mature corpora lutea. In situ hybridization studies localized the mRNA to the large luteal cells of mature corpora lutea and relaxin protein was detected by immunocytochemistry in the same tissue. This is the first report demonstrating relaxin in the equine ovary and its expression by luteal cells, thereby suggesting a role for relaxin in follicular or corpus luteum function in cyclic mares.