To evaluate the effect of nitric oxide (NO.) in human corpus luteum (CL) function, we investigated the expression and the presence of NO. synthase (NOS) in the human CL and the action of NO. on the in vitro luteal steroid production. The expression of endothelial NOS (eNOS) in early, mid-, and late CL was assessed by reverse transcriptase polymerase chain reaction (RT-PCR) and the immunohistochemical study was performed in human CL histological sections by using monoclonal antibodies (MAbs) against the distinct NOS isoforms. In addition, seven human mid-CLs were enzymatically dispersed, and the cells were cultured with NO. donor compounds. Steroid production was measured in the culture media by specific radioimmunoassay. The results show that the expression of eNOS was highly detected in mid- and early CL, and to a lesser extent, in late CL. Meanwhile, the immunohistochemical study indicated that both isoenzymes of NOS were expressed in mid-human CL, eNOS being the more abundant isoform present. On the other hand, functional studies showed that NO. donors (L-arginine [L-Arg] and sodium nitroprusside) elicited an inhibitory action on steroid synthesis, preferentially on estradiol production by the luteal cell cultures (p < 0.05). In conclusion, the NO-NOS system is present in the human CL, and it may serve as a modulator of the in vitro human luteal steroidogenesis.
Flow cytometry analysis of luteal cells revealed that an important proportion of these cells are leukocytes. The percentage of leukocytes was higher in the early (42 +/- 4) and late (35 +/- 3) luteal phases than in the mid-luteal (24 +/- 2) phase. However, the proportion of macrophages did not differ between the luteal stages. The flow cytometric properties correlated with cellular size and granularity were not reliable as discriminators of luteal cell subpopulations. Therefore, to assess the contribution of luteal leukocytes, these cells were completely removed from luteal cell suspensions (total cells), by a negative selection procedure (immunomagnetic separation). The functional role of leukocytes in mid-luteal steroidogenesis was assessed, in total as well as leukocyte-depleted cells. Progesterone production was found to have increased 2.2-fold in leukocyte-depleted cell cultures, in comparison with total cells under basal conditions. However, the response to human chorionic gonadotrophin (HCG) was 36% lower under the latter conditions. Oestradiol production was not significantly modified under basal or HCG-treated conditions. In leukocyte-depleted cells, the concentration of interleukin (IL)-1beta decreased 5-fold in comparison with total cell cultures, suggesting that leukocytes are the principal source of IL-1beta. In summary, the results of the present investigation suggest functional interactions between the immune system and steroidogenic cells of the human corpus luteum.
To evaluate the effect of reactive oxygen species in human corpus luteum function, we investigated whether hydrogen peroxide (H2O2) affects the in vitro luteal cell production of steroids. H2O2 treatment (1.0-100 microM) of mid and late luteal cell cultures elicited a dose-dependent decrease in basal progesterone production. However, treatment of mid luteal cells with a low concentration of H2O2 (0.01 microM) significantly stimulated progesterone secretion (P < 0.05). In addition, H2O2 (100 microM) markedly inhibited human chorionic gonadotropin (hCG)-stimulated progesterone and estradiol secretion. cAMP production was enhanced (2.4-fold, P < 0.05) by hCG treatment of luteal cells. The addition of H2O2 (0.1-100 microM) to hCG-stimulated luteal cell cultures elicited a decrease in cAMP concentration (P < 0.05) and in the specific binding of radiolabeled hCG by luteal cells. Progesterone and estradiol production stimulated by dibutyryl cAMP were significantly inhibited by H2O2 (P < 0.05). These findings suggest that H2O2 interferes with basal steroid production and, in hCG-stimulated conditions, it may inactivate the gonadotropin-receptor complex. The anti-steroidogenic action of H2O2 therefore raises the possibility of a modulatory role of H2O2 in human luteal steroidogenesis.
It has been reported that the mammalian corpus luteum is composed mainly of two subpopulations of luteal cells (large and small) of different morphology and function. The aims of this study were first to characterize cytologically the human corpus luteum throughout the luteal phase, and second to establish the in-vitro steroidogenic capacity of a well-defined human mid-luteal cell system. The results show that the most predominant (> 70%) cell shape, is polyhedric, and the number of cells per unit area is significantly different in the early, mid- and late corpus luteum (P < 0.005). Moreover, small cells (< 22 microns) were most common (56.8%) in all tissues analysed. On the other hand, both subpopulations synthesized progesterone, oestradiol and testosterone, although a significantly greater production of basal steroids was observed in large luteal cells (P < 0.05). Nevertheless, the response of small cells to human chorionic gonadotrophin (HCG) was significantly greater (P < 0.05) than that of large cells, in agreement with the preferential specific binding obtained for [125I]HCG to the small cell subpopulation. In summary, these results indicate that the human corpus luteum possesses distinct cell types, which may be related to endocrine function and its control.
The objectives of this investigation were to examine in vivo insulin like-growth factor-I (IGF-I) secretion by the human midcorpora lutea (mid-CL) and the effects of IGF-I, hCG, FSH, and human GH on progesterone (P) production by CL cells obtained from patients at laparotomy. We first examined whether the CL produces IGF-I by measuring IGF-I levels in the ovarian vein from the ovary bearing the CL. The IGF-I concentration in the ovarian vein bearing the CL (206 +/- 31 ng/mL) was significantly increased compared to the concentration in the contralateral ovarian vein (179.2 +/- 32 ng/mL; P < 0.05). Luteal cells isolated from mid-CL were cultured in serum-free medium 199 in the presence and absence of hCG, FSH, GH, and graded concentrations of IGF-I. At the end of the incubation period (24 h), P levels in the medium were measured by RIA. The treatment with IGF-I (0.1-10 ng/mL) showed a dose-dependent stimulatory action of IGF-I on P synthesis in the luteal cell system, being maximal between 5-10 ng/mL. The treatment with hCG (10 IU/mL), IGF-I (5 ng/mL), and GH (1000 ng/mL) increased basal P synthesis by 300%, 80%, and 30%, respectively (P < 0.001 and P < 0.05). FSH (100 ng/mL), either alone or in combination with IGF-I, failed to stimulate P synthesis. Treatment with IGF-I monoclonal antibody (1:5000) completely reduced P synthesis induced by 5 ng/mL IGF-I and slightly reduced basal P synthesis as well as GH-stimulated P synthesis by human midluteal cells. To further evaluate the specific role of IGF-I on luteal steroidogenesis, IGF-I receptor was identified by chemical cross-linking of [125I]IGF-I to mid-CL membranes. Experiments conducted in the absence and presence of unlabeled IGF-I (500 ng) revealed proteins with characteristics of the type I IGF receptor. These results are consistent with multihormonal regulation of P synthesis by the human mid-CL. hCG and IGF-I play a major role in the stimulation of P synthesis and, to a lesser extent, human GH. These in vivo and in vitro data suggest that the CL is a site of secretion, action, and reception of IGF-I during the midluteal phase.
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