Role of estradiol as a biological amplifier of gonadotropin action in the ovary: In vitro studies using swine granulosa cells and homologous lipoproteins

Swine granulosa cells cultured under serum-free conditions in vitro exhibit significant responsivity to the stimulatory actions of estradiol (E₂) and FSH or LH. Under these conditions, granulosa cells harvested from either immature or mature Graafian follicles synthesized significantly increased quantities of progesterone in response to homologous low density lipoprotein (LDL) and, to a lesser degree, homologous high density lipoprotein (HDL) particles. The effects of LDL and HDL were dose dependent and saturable. The stimulatory influence of E₂, FSH, or LH alone was significantly enhanced in the presence of pig LDL or HDL. Moreover, the synergism between E₂ and FSH or between E2 and LH was significantly augmented by porcine LDL and, to a lesser degree, porcine HDL. To assess the physiological relevance of these observations, the lipoprotein contents of swine blood and follicular fluid were determined by heparin-manganese precipitation and after differential ultracentrifugation. The majority (>70%) of cholesterol in pig blood resided in the LDL fraction, but follicular fluid was essentially devoid of LDL. On the other hand, follicular fluid contained large quantities of a presumptive HDL species with a density between 1.063-1.210. The HDL particle in follicular fluid was further characterized by agarose gel and sodium dodecyl sulfate-polyacrylamide gel electrophoreses, which demonstrated an α-migrating species whose major apoprotein exhibited an apparent mol wt of 28,000 and comigrated with human apoprotein A-l. Analytical ultracentrifugation of the pig follicular fluid HDL revealed a sedimentation coefficient (S_{20, w}) of 4.93, similar to that of serum HDL (S_{20, w}= 5.0). The physiological relevance of the HDL particle purified from follicular fluid was further demonstrated by its ability to significantly increase progesterone production by granulosa cells cultured under serum-free conditions in vitro. In summary, we have demonstrated striking responsivity of cultured pig granulosa cells to exogenously supplied LDL and, to a lesser degree, HDL, with further stimulation when cells are treated with estrogen and/or LH and FSH. Although LDL is the predominant lipoprotein in swine blood, it is essentially undetectable in the antral fluid of the intact Graafian follicle. Thus, the unambiguous in vitro responsiveness of granulosa cells to LDL that we observe suggests that the marked increase in availability of blood-borne LDL to granulosa-luteal cells that presumptively occurs at ovulation would contribute significantly to augmented rates of progesterone biosynthesis by luteal tissues in the pig. In contrast, the presence of a high concentration of specific HDL in follicular fluid which equals or exceeds that shown to be maximally effective in vitro suggests that the availability of HDL to granulosa cells is not limiting to progresterone production before ovulation. Most importantly, our in vitro demonstration that estrogen and FSH or LH can significantly enhance responsiveness to LDL suggests that these hormones may act to prepare granulosa cells in developing follicles for effective postovulatory utilization of the large quantities of LDL that become available via circulating blood.