The simultaneous subcutaneous injections of dihydrotestosterone (0.5 mg/kg) and/or ovine prolactin (5 or 50 IU/kg) to mature castrate rats for periods of 5–10 days led to an augmentation of prostate gland levels of RNA, DNA and protein. Of the various lobes of the prostate gland, the dorsal portion seemed to be particularly sensitive to the effects of the combined hormone treatment. In general, the highest dose of prolactin (50 IU/kg) and the longer duration of administration (10 days) produced the most consistent augmentation responses between dihydrotestosterone and prolactin as compared to those rats receiving only androgen. These findings reveal that certain lobes of the prostate are more responsive to hormonal interactions, but do not clarify the mechanisms involved in causing such enhancements of prostate cell growth and proliferation.
Incubation of swine granulosa cells in chemically defined medium selectively deficient in calcium ions markedly impaired progesterone production in response to submaximal and maximally stimulating concentrations of LH. Accumulation of progesterone in response to LH was reduced significantly in both cells and medium, without a discernible shift in the time-course of progestin production. The reduction in progesterone accumulation could not be accounted by increased formation of the catabolite 20 alpha-hydroxypregn-4-en-3-one. In addition, progesterone secretion basally or in response to exogenously supplied pregnenolone was not altered in calcium-deficient incubations. Administration of verapamil or diltiazem, organic inhibitors of net transmembrane calcium uptake, also suppressed LH-stimulated progesterone production. Conversely, micromolar concentrations of the divalent cation ionophore A23187 significantly enhanced the stimulatory effects of LH. The mechanisms of calcium action were examined further in relation to the cAMP effector system. Calcium deprivation significantly suppressed the dose-dependent accumulation of cAMP in granulosa cells treated with LH but had no effect on basal levels. Omission of calcium ions from the extracellular medium also markedly impaired production of progesterone in response to 8-bromo-cAMP, cholera toxin, or 3-isobutyl-l-methylxanthine. The present studies suggest that calcium ions significantly modulate LH-stimulated progesterone biosynthesis in isolated ovarian cells in vitro. Specific regulatory actions of calcium ions in granulosa cells may be exerted at several levels, including LH-stimulated cAMP accumulation and at intracellular loci distal to the actual generation of cAMP.
The role of PRL in the development of male accessory sex organs remains unresolved. In the present studies, the influence of PRL and testosterone upon DNA synthesis and cell proliferation was examined in the anterior prostate gland (AP) and the seminal vesicles (SV) of mice. Hormonal effects on DNA synthesis were evaluated by examining the in vitro incorporation of [3H]thymidine into DNA in relation to temporal alterations in tissue DNA content. In intact mice, PRL (300 IU/kg daily) produced peak stimulation of [3H]thymidine incorporation into DNA by SV after 3 days of treatment. In contrast, PRL failed to alter [3H]thymidine incorporation by the AP. Only in SV did PRL injections lead to elevated levels of tissue DNA. Injections of testosterone (0.75 or 7.5 mg/kg daily for 3 days) to castrated mice also produced significant stimulation of labeled thymidine incorporation into DNA by AP and SV. Concomitant injections of PRL (150 IU/kg) and testosterone (0.75 or 7.5 mg/kg) enhanced the stimulatory effects of this androgen on DNA synthesis and DNA content in the SV, but not in the AP. When PRL alone was administered to castrated mice, it failed to affect either [3H]thymidine incorporation into DNA by AP and SV or accessory sex organ weights and DNA contents. The results of these studies suggest that PRL enhances the effects of androgens upon DNA synthesis and growth of the seminal vesicles, but not of the anterior prostate glands of mice.
We studied the mechanism(s) by which calcium ions modulate progesterone biosynthesis by isolated swine granulosa cells incubated in chemically defined medium in vitro. In selectively calcium-deficient incubations, the capacity of 8-bromo-cAMP to stimulate pregnenolone synthesis from endogenous sterol substrate was significantly impeded. This effect of calcium ions was specific, because calcium ions did not influence basal pregnenolone production or alter progesterone production in response to exogenously supplied cholesterol substrate. Moreover, calcium ions did not modify other biosynthetic processes in granulosa cells, such as de novo synthesis of cholesterol from [14C]acetate or the aromatization of testosterone to 17β-estradiol. The possible role of calmodulin in mediating calcium's actions in pig granulosa cells was tested by measuring the calmodulin content of these cells and assessing the functional responses to classical calmodulin antagonists. By immunoassay, swine granulosa cells contained high concentrations of calmodulin, viz. 4.21–4.88 μg calmodulin/mg protein. Moreover, calmodulin antagonists inhibited LH-stimulated progesterone production with the following rank order of potencies [estimated by half-maximally inhibitory concentrations (ID50)]: penfluridol (1 μm), trifluoroperazine (9 μm), chlorpromazine (95 μm), and trifluoperazine sulfoxide (>300 μm). In addition, the nonphenothiazine calmodulin antagonist W7 inhibited stimulated progesterone production with an ID50 of 16.7 μm. W5 was less active. None of these antagonists significantly suppressed LH-stimulated cAMP generation at the low concentrations capable of inhibiting progesterone production. The effects of calcium ions seemed to depend upon the availability of intracellular pools of calcium, because TMB-8, an inhibitor of intracellular calcium mobilization, effectively suppressed LH-stimulated progesterone production (ID50, 18 μm). However, even 100 μm TMB-8 failed to alter basal progesterone production or suppress LH-stimulated cAMP generation in these cells. In summary, the present studies indicate that calcium ions significantly modulate LH's stimulation of pregnenolone biosynthesis from endogenous cholesterol substrate in swine ovarian cells. Calcium does not influence basal pregnenolone production, estrogen synthesis from androgen substrate, de novo biosynthesis of cholesterol from [14C]acetate, or progesterone production from exogenously supplied sterol substrate. Further studies indicate that these actions of calcium may be mediated in part via calmodulin, since: 1) pig granulosa cells contain high concentrations of calmodulin, and 2) both phenothiazine and naphthalenesulfonamide antagonists of camodulin significantly impair the maximal stimulatory effects of LH on progesterone production. These antagonists also impede the stimulatory actions of exogenously supplied cAMP, but do not suppress LH's ability to augment intracellular cAMP generation. Thus, we suggest that calcium ions modulate LH action in ovarian cells in part by calcium-calmodulin-dependent mechanisms. Such mechanisms seem to regulate steroidogenesis at a site(s) distal to LHstimulated cAMP production, probably at the level of delivery or utilization of endogenous sterol substrate in the side-chain cleavage reaction. (Endocrinology114: 441, 1983)
We have used a novel competitive inhibitor of acyl coenzyme A:cholesterol acyltransferase (ACAT), Sandoz compound 58-035 [3-(decyldimethyl-silyl)N-[2-(4-methylghenylph- phenylethyl propanamide], to assess the importance of the cholesterol esterification reaction in ovarian steroidogenesis. Compound 58-035 markedly (≫96%) inhibited ACAT activity of swine ovarian microsomes in a dose-dependent (0.1–3.5 μg/ml) fashion. In addition, treatment of cultured granulosa pells with this fatty acylamide effectively (≫98%) suppressed hormonally stimulated cholesterol esterification, as assessed by the incorporation of [3H]oleic acid into cholesteryl ester. Accordingly, we used this inhibitor to test the role of cholesterol esterification in ovarian cells. In cultures with limited or no serum supplementation, long term (2- to 6-day) treatment of granulosa cells with compound 58-035 significantly increased basal progesterone production and amplified by 2- to 10-fold the stimulatory actions of trophic hormones, such as estradiol, FSH, estradiol combined with FSH, or insulin. The amplifying effect of ACAT inhibition on hormone- stimulated progesterone production could be mimicked by providing exogenous cholesterol substrate in the form of low density lipoprotein (LDL). Cotreatment with compound 58-035 and LDL resulted in no further augmentation of steroidogenesis. In contrast to the facilitative effects of compound 58-035 in longer term cultures, this ACAT inhibitor did not alter progesterone biosynthesis acutely (2–20 h) in swine or hamster ovarian cells. These observations suggest that there is an obligatory partitioning of some sterol into the ester pool in granulosa cells. In times of diminished availability of cholesterol, inhibition of the esterification pathway can make additional cholesterol available for use in steroid hormone biosynthesis. Thus, in the intact Graafian follicle, where LDL cholesterol delivery to granulosa cells and intracellular cholesteryl ester stores are limited, regulation of the ACAT reaction may significantly modulate rates of progesterone biosynthesis. The present results indicate that the use of a selective inhibitor of cholesterol esterification can permit one to probe the functional significance of the esterification reaction in steroidogenic cells. (Endocrinology116: 25–30, 1985)
