Relationship between Serum Luteinizing Hormone Levels and the Ability of Porcine Granulosa Cells to Luteinize and Respond to Exogenous Luteinizing Hormone in Culture*

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Abstract
A group of 16 normally cycling pigs was subjected to bilateral ovariectomy at various times on the day before (day−1) and the day of estrus (day 0). Blood samples were assayed for LH by RIA. The granulosa cells were harvested from the large (6–12 mm), medium (3–5 mm), and small (1–2 mm) follicles of each pair of ovaries and cultured for 8 days in medium 199 plus 15% pig serum. Some of the granulosa cells were cultured in the presence of 1.0 μg/ml ovine LH. Progesterone was measured in the spent culture medium after 2, 4, 6, and 8 days. At the end of culture, the cultures were examined morphologically for luteinization after staining with oil Red 0 and hematoxalin. When LH levels were low (0.6–1.5 ng/ml) on day −1 of the estrous cycle, granulosa cells harvested from 6- to 7-mm follicles failed to luteinize morphologically in culture, exhibited a fibroblastic type morphology, and contained few lipid droplets. Their ability to secrete progesterone into the culture medium was poor. After 2, 4, and 6 days, these cultures secreted 2.1, 0.86, and 0.85 pg/cellday, respectively. The addition of 1.0 μg ovine LH led to a 700–2300% stimulation of progesterone secretion. Granulosa cells harvested from 7- to 12-mm follicles on day −1 of the estrous cycle when LH levels were high (2.6–9.2 ng/ml) luteinized 60–70% in the absence of exogenous LH and secreted elevated amounts of progesterone corresponding to 31, 23, 19.4, and 12.8 pg/cell·day after 2,4, 6, and 8 days of culture, respectively. They responded poorly to exogenous LH. Granulosa cells harvested from 9- to 11-mm follicles during day −1 of the estrous cycle when LH levels were intermediate (1.6–2.5 ng/ml) luteinized partially morphologically and secreted progesterone in amounts intermediate between the group having low and high LH levels. On the first day of estrus (day 0), granulosa cells harvested from 10- to 11-mm follicles luteinized morphologically and secreted elevated amounts of progesterone which were 42.9 and 18.2 pg/cell·day after 2 and 4 days of culture, respectively. Granulosa cells harvested from small 1- to 2-mm follicles of ovaries obtained anytime during days −1 and 0 of the estrous cycle failed to luteinize. It can be concluded that porcine granulosa cells must be exposed to elevated amounts of LH before they can luteinize spontaneously in culture. Exposure to elevated amounts of LH in vivo renders the granulosa cells refractory to further addition of LH in vitro. (Endocrinology106: 317, 1980)