Several recent studies have elucidated the structure of the mammalian LH/hCG receptor; as reported in the present work, we have developed a series of monoclonal antibodies (mAbs) against the rat ovarian LH/hCG receptor using highly purified receptor as immunogen and by screening hybridomas with purified LH/hCG receptors. The mAbs were able to specifically immunoprecipitate LH/hCG receptors from solubilized preparations of rat ovarian membranes as well as from partially purified preparations. Western blotting with mAb P1B4 detected a probable receptor dimer and a receptor fragment in rat and porcine ovarian tissue but not in other tissues. This mAb also partially inhibited hCG binding to rat and porcine ovarian tissues. The receptor mAbs were able to inhibit hCG-induced progesterone synthesis in cultured human and porcine granulosa cells without affecting cAMP- and FSH-induced progesterone synthesis. The mAb P1B4 was used to demonstrate that the majority of ovarian receptors are internalized after hCG treatment and that in pseudopregnant rats receptors are present in the rough endoplasmic reticulum and in microvesicles. Bovine corpus luteal cells also contained P1B4 binding sites, as detected by immunohistochemical technique. Taken together, these results suggest that the mAbs are specific for the LH/hCG receptor, mAb P1B4 recognizes an epitope that is highly conserved among mammals, and this epitope is probably in the extracellular domain.

Although the binding, internalization, and regulation of LH, PRL, and their respective receptors have been extensively studied, it is not known whether the receptors are coordinately regulated. Using double labeling experiments, we have previously shown that receptor-bound LH and PRL can be colocalized in identical endosomes of granulosa cells. We hypothesize that high levels of PRL may induce a heterologous down-modulation of LH receptors, consequently reducing ovarian responsiveness to further gonadotropin stimulation. In this study we used a novel procedure to enrich endosomes containing internalized PRL and to determine whether unoccupied LH receptors were cointernalized in granulosa cells. Porcine granulosa cells were obtained from medium-sized (3-5 mm) follicles and cultured for 4 days in the presence of FSH. Fluorescein isothiocyanate-labeled PRL (FITC-PRL) was used as a ligand to induce internalization of PRL receptors and as a marker to label endosomes. Granulosa cells were incubated with FITC-PRL at either 4 or 37 C for various times. At the end of the incubation, cells were trypsinized to remove surface receptors and then homogenized. The postnuclear fraction containing endosomes and other subcellular organelles was sorted using a FACStar Plus cell sorter. Results from 14 separate sorting experiments showed that 1) FITC-PRL-treated cells exhibited a sorting pattern distinct from that of FITC-BSA-treated or untreated cells; 2) excess unlabeled PRL partially shifted the sorting profile to one similar to that in controls; 3) the differences in sorting profiles were not due to free FITC; and 4) using this method, it was possible to isolate FITC-PRL-containing endosomes that were virtually devoid of other contaminating subcellular particles. Fluorescently positive (FITC-PRL-containing) organelles were collected and assayed for LH receptors using [125I]hCG as a tracer. When the cells were incubated with FITC-PRL at 37 C for 3 h, the number of available LH receptors (as determined by [125I]hCG binding) was 37% higher in particles containing FITC-PRL than in those devoid of FITC-PRL. If the cells were allowed to preincubate with FITC-PRL at 4 C for 10-16 h before raising the temperature to 37 C, the number of available LH receptors in FITC-PRL-containing endosomes was about 7-fold higher than that in FITC-negative endosomes. Results from this study suggest that PRL not only induces internalization of its own receptor, but also causes down-modulation of unoccupied LH receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Macrophages and their secretory products, cytokines, play an integral role in many reproductive processes. In this study we examined the effect of conditioned media from cultured human peritoneal macrophages on progesterone production by granulosa cells and the role of calcium in this process. Macrophages were pretreated with various concentrations of a calcium channel blocker (verapamil) or a calcium ionophore (A23187). Macrophage-conditioned media (MCM) or cell-free media that contained calcium channel modifiers were added at three dose levels to cultured porcine granulosa cells. Progesterone production and LH receptor content were determined. Macrophage-conditioned media alone elevated basal progesterone production, but significantly attenuated granulosa cell LH receptor content. These effects were neither potentiated nor suppressed by pretreating macrophages with verapamil. However, production of the LH receptor lowering factor(s) appeared to be suppressed by calcium ionophore. We conclude that (1) one or more factors produced by macrophages have a net stimulatory effect on basal progesterone production and these factor(s) may not be calcium-dependent and (2) macrophage-derived secretory products reduce granulosa cell LH receptor content. The production of these factor(s) may be calcium-dependent.

Recent evidence indicates that factors produced by immune cells (cytokines) may play a role in ovarian function. To explore this possibility, we examined the effects of conditioned medium obtained from cultures of either unstimulated splenocytes (splenocyte-conditioned medium; SCM) or concanavalin A-stimulated splenocytes (CAS) on estrogen and progesterone production by porcine granulosa cells. Granulosa cells were obtained from small (less than 3 mm) or large (greater than 7 mm) follicles and treated with increasing doses of SCM or CAS in the presence or absence of pFSH (100 ng/ml) for 24 h at 37 degrees C. In granulosa cells obtained from small follicles it was found that both SCM and CAS evoked a dose-dependent increase in estrogen but not progesterone production. Estrogen production was no further enhanced by the presence of FSH. Additionally, SCM was able to augment FSH-stimulated progesterone production by these cells, whereas CAS had no effect. Identical treatment of granulosa cells obtained from large follicles demonstrated that both SCM and CAS caused dose-dependent increases in estrogen as well as progesterone production. In response to CAS, FSH augmented progesterone production but exerted a biphasic on estrogen production (inhibiting at lower doses while stimulating at higher doses). In contrast, SCM had no effect on FSH-stimulated estrogen production. Additional controls indicated that the above results could not be attributed to either concanavalin A or serum. Taken together, these findings suggest that cytokines can exert significant effects over granulosa cell steroidogenesis and further imply that these factors may play an important role in the differentiation and developmental regulation of granulosa cell function.