Biphasic Luteinizing Hormone Secretion in Response to Gonadotropin-Releasing Hormone during Continuous Perifusion of Dispersed Rat Anterior Pituitary Cells: Changes in Total Release and the Phasic Components during the Estrous Cycle*
In vivo studies in the human and both in vivo and in vitro studies in animals have shown that GnRH-stimulated LH release is a function of the gonadal steroid environment. Furthermore, when GnRH is given in a sustained manner, the pattern of LH release is biphasic. Using an in vitro technique in which rat anterior pituitary cells were dispersed and perifusion was begun within 2 h after removal of the gland from the donor animal, we tested the hypothesis that both the overall secretion and the relative sizes of the two phases of GnRH-stimulated LH release would vary in a reproducible manner during the estrous cycle. Sprague-Dawley rats (aged 60–70 days) were killed at 0800 h on diestrus day 1, diestrus day 2, proestrus, and estrus and at 1400 h on proestrus. After 4 h of perifusion, during which a stable baseline of LH release was established, the cells were challenged for 5 h with 10 nM GnRH, and secretory rates of LH release were calculated. Basal LH release did not vary among the experimental groups. LH secretory rates (nanograms per min/107 cells; mean ± SEM) during the 5 h of GnRH infusion were similar on diestrus day 1 (3.47 ± 0.71) and diestrus day 2 (7.0 ± 1.03), rose significantly by 0800 h on proestrus (16.9 ± 2.54), became maximal on proestrus at 1400 h (20.1 ± 1.72), and fell on the day of estrus (8.1 ± 1.91). In each group, a biphasic response of LH to GnRH was noted, with the first phase ending and the second phase beginning at approximately 50 min into the infusion. Phase 1 (first 50 min of GnRH infusion) LH secretory rates (nanograms per min/107 cells; mean ± SEM) on diestrus day 1 (4.13 ± 0.63) and diestrus day 2 (4.45 ± 0.44) were no different, but had increased markedly on proestrus (21.0 ± 3.84 and 21.6 ± 1.91 at 0800 and 1400 h, respectively). On estrus, the phase 1 secretory rate (13.1 ± 2.08) was intermediate between those on proestrus and diestrus day 1. The phase 2 LH secretory rate (final 250 min of GnRH infusion) on diestrus day 2 (7.52 ± 1.16) was higher than that on diestrus day 1 (3.28 ± 0.84) and increased further on proestrus (16.17 ± 2.41 and 19.8 ± 1.83 at 0800 and 1400 h, respectively). Phase 2 release had fallen on the day of estrus (7.27 ± 2.03). When expressed as a ratio of secretory rates (phase 1 to phase 2), there was a rise between diestrus day 2 (0.64 ± 0.08) and proestrus (1.30 ± 0.14 and 1.12 ± 0.1 at 0800 and 1400 h, respectively), with the maximal value achieved on estrus (2.15 ± 0.29) and a subsequent fall on diestrus day 1 (1.86 ± 0.69). In summary, we found that, using dispersed and continuously perifused rat anterior pituitary cells, both GnRH-stimulated LH release and the relative sizes of the first and second phases of LH release vary in a reproducible manner during the estrous cycle. We suggest that the two phases of LH release may represent the functional pools of LH within the gonadotrope, described previously in in vivo studies.