Role of Frequency and Amplitude of Repetitive HCG Stimulations for Sustained Progesterone Secretion from the Bovine Corpus Luteum In Vitro

Summary

This investigtion aimed at evaluating a role for frequencies and amplitudes of repeated HCG stimulations for the optimal maintenance of progesterone (P₄) secretion from the bovine corpus luteum in vitro. Slices (100-120 mg) of midluteal bovine corpora lutea were perifused with medium M199 (0.05% BSA, pH 7.2, 38.5 °C) and the perifusion effluent collected at 15 minute intervals for 20-29 hours. Unstimulated P₄ release (n = 5) was distinctly pulsatile (by Pulsar pulse algorithm), with pulses occurring every 90±6 minutes (mean±SEM) and pulse amplitudes of 14.4±1.1 ng. Conversely, no pulses were detected in two control perifusions. Unstimulated P₄ release increased during the first 5 perifusion hours (from 39.3±4.6 to 50.3±5.6 ng/15 min, p < 0.01), but then appeared to decline (to 29.3±1.3 ng/15 min, p < 0.05) towards the end of the perifusion periods. Hourly pulses of HCG (6.7 mM) did not change the P₄ pulse amplitudes (16.6±2.0 ng), the pulse periodicities (105±15 min) and overall release rates (34.7±5.7 ng/15 min), nor did they prevent the decline in P₄ secretion towards the end of perifusions (n = 5). In contrast, 2-hourly HCG stimulations maintained stable P₄ release rates throughout the perifusion periods (34.7±6.8 ng/15 min), with P₄ pulses of similar amplitudes (14.7±1.7 ng), but of lower periodicities (135±2 min, p < 0.05) than during Unstimulated conditions (n = 5). HCG pulses at less frequent intervals (4-hourly, 6-hourly, n = 8) increased the P₄ release (from 33.7±5.5 to 92.6±12.8 ng/15 min, p < 0.01). They also enhanced the P₄ pulse amplitudes (to 32±4.7 ng, p < 0.05), but slowed the periodicities (to 311±85 min, p < 0.05). Continuous HCG stimulations for 11-20 hours during a total 20 hour perifusion period (n = 7) enhanced the P₄ release (from 49±5.6 to 94.1±5.8 ng/15 min after 5.8±0.9 hours, p < 0.05) and the pulse amplitudes (to 38.6±5.2 ng, p < 0.01), but decreased the frequencies (to 242±45 min, p < 0.05). A terminal decline in P₄ secretion at the end of continuous stimulations could be reversed by intermittent hourly HCG stimulations. P₄ release during 2-hourly HCG administrations at doubled concentrations (13.45 mM) increased the overall P₄ release rates (to 88.7±7.6 ng/15 min, p < 0.05) and pulse amplitudes (to 38.3±4.6 ng, p < 0.01), but slowed the pulse frequencies (to 170±39 min, p < 0.05) (n = 4). These observations demonstrate an autonomous episodic P₄ release from the bovine corpus luteum in vitro. The intrinsic P₄ pulse periodicity may be maintained and the P₄ secretion be optimized by HCG stimulations administered at intervals comparable to those observed during the midluteal phase in vivo. The terminal decline in P₄ secretion during prolonged perifusions or continuous HCG stimulations appears to relate rather to a P₄ depletion in the corpus luteum than to a loss of receptor sensitivity.

Key words

HCG - Progesterone - Perifusion - Corpus Luteum