The effect of testicular macrophages, macrophage-conditioned medium and interleukin-1alpha on bank vole Leydig cell steroidogenesis.

It has already been accepted that the function and activity of the testis is regulated not only by gonadotrophins, but also by many locally produced factors and by cell-cell interactions. That is why the aim of our work was to determine whether macrophages and/or their products have an influence on Leydig cell steroidogenic activity. The source of Leydig cells and macrophages were male bank voles of spring and autumn generations, reared in 18 light:6 dark or 6 light:18 dark (18L:6D or 6L:18D) conditions for 7-8 weeks. The Leydig cells were growing in monocultures or in co-cultures with macrophages (testicular or peritoneal), either as control or hCG-stimulated ones. To some of the cultures 6 IU/ml of interleukin 1alpha (IL-1alpha) was added. After then the cells were analysed morphologically, histochemically, and radioimmunologically. In the present study we found many differences in morphology and steroidogenic activity of Leydig cells obtained from different photoperiods. Leydig cells from a long day formed monolayer contrary to the cells from a short one growing as single cells or in clusters. Moreover, Leydig cells from a long photoperiod produced more testosterone and were sensitive to the stimulatory effect of both testicular macrophages and testicular macrophage-conditioned medium. They were also more sensitive to the inhibitory influence of IL-1alpha.

Effect of prostaglandins on hormonal function of cultured rat Leydig cells.

The effect of PGF2 alpha and its analogues on androgen production and activity of delta 5,3 beta-hydroxysteroid dehydrogenase in rat Leydig cells in vitro was investigated. Prostaglandin of the F type inhibit the enzyme activity and hormone secretion by cultured Leydig cells. This effect was considerably stronger in Leydig cells isolated from mature rats, than by Leydig cells from immature animals.

Response to gonadotropic stimulation of cultured rat luteal cells isolated from corpora lutea of early pregnancy. Cytochemical and biochemical studies.

The hormonal activity of corpora lutea isolated from pregnant rat was examined on 1, 2, 3, 4, 5, 6, 15, and 20th day of pregnancy. The cells were grown as a monolayers up to 6 days at 37 degrees C in Medium 199 supplemented with 10% calf serum. The concentrations of progesterone and estrogens were measured using appropriate radioimmunoassays [1, 7] respectively. Luteal cells were cultured with the addition of the following amounts of hormones: 100 ng LH, 10 i.u. HCG, 100 ng PRL and 150 ng estradiol 17 beta. Cytochemical and histochemical observation of the activity of delta 5, 3 beta-hydroxysteroid dehydrogenase (delta 5, 3 beta-HSD) were also carried out. The addition of LH and HCG to culture medium of cells collected on day 1 and 2 of pregnancy caused increased histochemical reaction for delta 5, 3 beta-HSD and progesterone secretion. It was only on day 3 of pregnancy that the influence of PRL was observed. On day 4 corpus luteum cells began to respond to exogenous estradiol. On day 5 the sensitivity of corpus luteum to exogenous hormones disappeared but the intensive hormonal activity of the corpus luteum marked by the high level of progesterone, was maintained.

The effect of prostaglandin F2 alpha and its analogues on progesterone, estradiol and androgen production by the cultured bovine luteal cells.

The effect of PGF2 alpha and its analogues on progesterone, estradiol and androgen production in cow corpus luteum in vitro was investigated. The cells derived from cow corpora lutea (CL) and collected in the early and middle luteal phases of the oestrus cycle were cultured as monolayers. The inhibitory effect was not apparent during the first 48 hr of culture, but appeared after this time and persisted through the remainder of the culture period. The direct luteolytic influence of PGF2 alpha was observed in the cultured cells and showed that this compound can act independently of the blood supply.

Immunolocalization of androgen receptors in testicular cells of prepubertal and pubertal pigs.

In the testis, androgen receptors are known to mediate autocrine and paracrine effects of androgens on Leydig cell function and spermatogenesis. The pig presents some unusual features with regard to the synthesis of testosterone and estrogens in the male gonads. In testes from prepubertal males, testosterone level was lower than in testes from adult boars, while estrogen secretion was relatively high and comparable to that of mature porcine gonad. Immunolocalization of androgen receptors and intensity of immunohistochemical staining was age-dependent. In testis sections from adult boars, androgen receptors were found in nuclei of all somatic cells such as Leydig cells, Sertoli cells, and peritubular-myoid cells, whereas in sections from immature pigs only in the Leydig cell cytoplasm showed positive immunoreaction for androgen receptors. In control tissue sections incubated with omission of the primary antibody, no positive staining was observed. Detection of the androgen receptors in testicular cells of the pig is important for understanding of their central role in mediating androgen action.

Cytochrome P450 aromatase in the testis of immature and mature pigs.

Aromatization of androgens into estrogens is performed by a microsomal enzyme, the cytochrome P450 aromatase. A direct approach for identifying the cellular source of aromatase is the use of immunohistochemistry with a specific antibody that recognizes aromatase. The pig presents some unusual features with regard to the synthesis of testosterone and estrogens in the male gonads. In testes from prepubertal males, testosterone level measured radioimmunologically, was lower than in testes from adult pig, while estrogen secretion was relatively high and comparable to that of mature porcine gonads. Immunolocalization of aromatase in testes from both immature and mature pigs was confined to the Leydig cell cytoplasm. The intensity of immunohistochemical staining indicated the presence of unsynchronous Leydig cell population. Other somatic cells and germ cells were negative for aromatase. In control tissue sections, incubated in the absence of the primary antibody or in the presence of normal rabbit serum, no positive staining was observed. Western blot analysis revealed one major band of aromatase about 50-52 kDa in testes from both immature and mature pigs.

Histochemical evaluation of delta 5,3 beta-OHSD activity in two types of porcine corpora lutea and granulosa cells in tissue culture.

Activity of delta 5,3 beta-hydroxysteroid dehydrogenase (delta 5,3-OHSD) in cultured porcine corpus luteum cells of early and middle luteal phase and granulosa cells was histochemical investigated. Control cultures as well as cultured with addition of luteinizing hormone (LH), prolactin, human chorionic gonadotrophin (HCG), and estradiol 17 beta were carried on. The stimulatory influence of LH and HCG on 3 cell types has been stated. Prolactin and estradiol stimulated only the cells of corpus luteum from early luteal phase.

Organ culture as a model of studying follicular development and function of postnatal mouse ovaries.

Ovarian organ culture was used to study the influence of various gonadotropin hormones (100 ng FSH, 100 ng LH, 100 ng LH added together with 100 ng FSH; 1 i.u. hCG or 10 i.u. PMSG) on growth and development of follicles as well as on steroid secretion by ovaries of postnatal, 15-day-old mice. Ovaries were aseptically removed and single organs were placed on a piece of lens paper which was supported by a stainless steel grid in the small organ culture dish. The cultures were maintained in medium M199 supplemented with 5% of calf serum in a CO2 incubator at 37 degrees C. The morphological changes and steroid secretion measured by appropriate RIAs were studied. The stages of follicular development and the incidence of particular types of follicles were scored. Progesterone and estradiol were detected in the medium by radioimmunoassay. Differences between control and gonadotropin stimulated ovaries were found in the number of the ovarian follicles in more advanced maturation stages. There was increased number of multilaminar and antral follicles in FSH and FSH plus LH treated cells. The adding of gonadotropin hormones to the culture medium stimulated significantly progesterone secretion. The most significant effect was observed in media of cultures treated with LH and hCG. As to estradiol secretion the highest stimulatory effect was seen in cultures supplemented with FSH alone, together with LH and with PMSG. The organ culture technique applied in the current study could be a suitable model of studying the interaction of various factors as well as its effect on ovarian differentiation and on selection of dominant follicles. This system allows maintaining the structural integrity of the whole ovary, thus in the physiological functional status of the organ.

Evaluation of the physiological value of porcine luteal cells isolated in various stages of the luteal phase: tissue culture approach.

Porcine luteal cells were collected from corpora lutea in four different stages of the luteal phase and cultured as monolayers. Progesterone (P4) secretion was assayed using radioimmunoassays (Gregoraszczuk, 1991). Luteal cells cultured from porcine corpora lutea collected in the early luteal phase maintained steroidogenic capacity for 6 days in culture until the time comparable with midluteal corpora lutea. Luteal cells collected from mature and regressing corpora lutea did not dedifferentiate during 2 days of culture. After this time secretion of progesterone decreased to undetectable amounts characteristic of old corpora lutea. The regression in the culture progressed. The results demonstrate that the degree of the decline of progesterone depends on the type of corpus luteum, which is connected to particular time intervals of the luteal phase. Before starting experiments it is necessary to take into consideration the stage of the luteal phase from which the material is collected for culture. This study provides evidence that long term culture is useful for investigating a variety of aspects of luteal function only if cells are collected in the early luteal phase. Short term culture is suitable for investigation of cells collected from mid and late luteal phase. Regulation of luteal function is dependent on stage of the luteal phase.

Luteinizing hormone-induced modifications of the cytoskeleton in cultured mouse Leydig cells.

Modifications of cytoskeleton organization during the culture of mouse Leydig cells in the presence or absence of luteinizing hormone (LH) have been demonstrated. The main changes were observed in the distribution of microfilaments. Stress fibres dispersed or even disappeared after 6 or 12 h of LH treatment. Concurrently, no substantial changes in microtubule and intermediate filament organization were found. The presence of tubulin or microtubule-associated protein in the mid-bodies and nuclei was noticed with the use of A8B3 monoclonal antibody. The increased amount of protein detected by this antibody was correlated with enhancement of androgen secretion and proliferation of cultured Leydig cells.

Time- and dose-dependent antigonadotropic activity of oxidation products of gallic acid and pyrogallol on Leydig cells in vitro.

Auto-oxidation products of plant phenolics in alkaline medium, such as gallic acid and pyrogallol were used to show antigonadotropic activity. The complex mixture of oxidation products was extracted from aqueous medium successively by ethyl ether and ethyl acetate. The fractions obtained were tested on a model of mouse Leydig cells in vitro. All compounds used inhibited luteinizing hormone-stimulated testosterone secretion during 6 and 24 h culture whereas basal secretion was stimulated by pyrogallol oxidation products. Not only low molecular weight substances extracted by organic solvents but also the remaining water soluble, dark brown, high molecular weight products were found to be antigonadotropically active.

The influence of PRL, LH alone and in combination upon progesterone secretion by porcine luteal cells in aggregate culture.

Corpora lutea removed from ovarian of cycling pigs in early luteal phase (1-3 days) were used. After enzyme dispersion the luteal cells were suspended in medium M 199 supplemented with 10% of calf serum. Cultures were carried out in triplicate and incubated at 37 degrees C for 24 h. After that time the following hormones were added to the culture medium; 100 ng LH, or 100 ng PRL or 100 ng LH plus 100 ng PRL. The cells were incubated with hormone for 6, 12, 24 and 48 h. LH added to the medium resulted in the increase of progesterone secretion (P less than 0.05) only during 6 h incubation (140% of control progesterone). Stimulatory effect of PRL was observed only after 12 h incubation (300% of control progesterone; P less than 0.001). The addition of LH plus PRL decreased progesterone secretion after 6 h incubation (33.3% of control progesterone; P less than 0.05), a little stimulatory effect being observed after 24 h incubation (141% of control progesterone). This study confirmed the results obtained with monolayer cultures previously described. PRL appeared to be a luteotrophic hormone which is responsible for the increase of progesterone secretion by the early developing corpus luteum of pig. From these data it was concluded that luteal cells may require either LH or prolactin but not both at a time to sustain higher level of progesterone secretion.