Localization and expression of low-density lipoprotein receptor, steroidogenic acute regulatory protein, cytochrome P450 side-chain cleavage and P450 17-α-hydroxylase/C17-20 lyase in developing swine follicles:: in situ molecular hybridization and immunocytochemical studies

The present study utilizes in situ molecular hybridization and immunocytochemistry to investigate the follicular localization and expression of four thematically related sterol-metabolizing genes; low-density lipoprotein (LDL) receptor, steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (CYP11A) enzyme, and cytochrome P450 17 alpha -hydroxylase/C17-20 lyase (CYP17). To this end, semiquantitative analyses were applied to individual nonatretic follicles (N = 54) harvested from cycling gilts slaughtered on days 1, 3, 5, and 7 (N = 3 per day) following withdrawal of the progesterone agonist, altrenogest. In situ and immunocytochemical signal intensities were assigned numerical values of 0-3 corresponding to the degree of expression of each mRNA and its corresponding protein. LDL receptor mRNA and protein content was undectable in theca and granulosa cells on days 1, 3, and 5, acid then increased to low levels in theca cells on day 7. StAR message rose progressively in theca cells with follicular maturation, reaching a maximum on day 5, and then declining slightly on day 7 after the LH surge. In granulosa cells, small amounts of StAR mRNA and protein were detected on days 5 and 7. The amounts of CYP11A mRNA and protein were high in theca cells, and increased at each time point studied. Granulosa cells exhibited minimal CYP11A message on days 3, 5, and 7, while protein became detectable at low levels on day 7 only. Expression of CYP17 was localized exclusively in theca cells with protein and message content increasing unidirectionally to maxima on days 5 (RNA) and 7 (protein), respectively. Follicular fluid concentrations of androstenedione, and progesterone in contralateral ovaries correlated strongly and positively with accumulation of CYP17, and CYP11A proteins. In summary, these analyses demonstrate that preovulatory follicular development proceeds with the coordinate induction of pivotal genes and proteins that mediate the selective uptake, delivery and utilization of sterol substrate in granulosa and theca-cell steroidogenesis. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.