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  • Author: F.F.G. Rommerts x
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F. F. G. Rommerts

Androgens are required for maintenance of spermatogenesis and for stimulation of the accessory glands. The peripheral androgen concentration is determined mainly by the steroidogenic activities of Leydig cells which are regulated by luteinizing hormone (LH), other hormones and also by locally produced factors. The androgen level in the testis is also, therefore, under the influence of LH. Since Leydig cells are the source of androgens, much higher levels are present in the testis than in peripheral androgen-dependent organs. There has been a long-running debate as to whether this high intratesticular concentration of androgen is essential for maintenance of spermatogenesis, or whether it is a prerequisite for the secretion of androgens. It has been suggested that locally produced factors operate to keep intratesticular testosterone levels high, especially in between LH pulses or at those specific stages of spermatogenesis which require high testosterone levels (Sharpe, 1984, 1986). Support for this hypothesis was

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F. F. G. ROMMERTS, F. H. DE JONG, J. A. GROOTEGOED and H. J. VAN DER MOLEN

Biochemical properties of isolated Leydig cells, Sertoli cells and spermatocytes from rat testes have been investigated after in-vivo or in-vitro exposure of these cells to abdominal temperature (37 °C). The rate of production of testosterone and pregnenolone by isolated Leydig cells from cryptorchid and normal testes from mature rats was not different. Production of pregnenolone by mitochondria prepared from cryptorchid testes was 6·7 times higher than production by mitochondria from normal testes.

Sertoli cells prepared from immature rats and incubated in vitro at 32 or 37 °C showed, on day 1 of the culture period, an initial twofold increase in the secretion of androgen-binding protein which was absent after 6 days in culture. In contrast, incorporation of [3H]leucine into secreted proteins was stimulated twofold on day 1 as well as by day 6 of culture. Secretion of oestradiol was increased 30-fold by day 6 when compared with the level found on day 1 when cells had been cultured at 37 °C and the increased secretion of oestradiol was maintained for approximately 2 days when the temperature of incubation was decreased to 32 °C Spermatocytes isolated from seminiferous tubules incubated for 20 h at 37 °C were active in the synthesis of RNA. No degeneration of these cells was observed in testes of 25-day-old rats 5 days after experimental cryptorchidism, whereas under similar conditions massive degeneration of spermatocytes was shown in the testes of mature rats.

These results suggest that the effects of temperature on the different testicular cells greatly depend on the experimental conditions used to study the effect of temperature.

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K. J. Teerds, D. G. de Rooij, F. F. G. Rommerts and C. J. G. Wensing

ABSTRACT

The formation of new Leydig cells in adult male rats was studied after the complete destruction of the original population by ethane dimethane sulphonate (EDS). Following administration of EDS, proliferating interstitial cells were labelled in a pulse-chase experiment by way of three [3H]thymidine injections on days 2, 3 and 4 after EDS administration. Some of the newly formed Leydig cells found 14 days after EDS administration were labelled with [3H]thymidine, indicating that these Leydig cells were derived from precursor cells, most likely mesenchymal cells, that had incorporated [3H]thymidine at days 2, 3, or 4 after EDS administration. At 21 days after EDS administration, the total number of Leydig cells (labelled plus unlabelled) had increased 7- to 16-fold compared with the number of cells that were present 14 days after EDS had been administered.

In a second series of experiments, [3H]thymidine was given 2 h before the rats were killed (short-term labelling experiment). In this experiment it was shown that the proliferative activity of the mesenchymal cells, which are presumed to be the precursors of the Leydig cells, after a considerable increase at day 2 after EDS administration, had returned to the control level at day 7. However, the total number of mesenchymal cells (labelled plus unlabelled) remained increased from 2 to 49 days after EDS administration. This indicated that the majority of the new Leydig cells which were formed from day 14 onwards probably did not derive from differentiating mesenchymal cells. The labelling index of the Leydig cells was approximately 100 times higher 21 days after EDS administration than that of the untreated controls, showing that many Leydig cells were formed by proliferation of the newly formed Leydig cells. Thereafter, the labelling index of the Leydig cells gradually decreased, whereas the total number of Leydig cells still increased threefold. At 49 days after EDS administration, the number of Leydig cells was approximately 80% of that in normal adult rats.

It is concluded that the regeneration of Leydig cells after EDS administration is the result of two successive waves of proliferation, namely of the precursor cells (mesenchymal cells) and of the newly formed Leydig cells.

Journal of Endocrinology (1990) 126, 229–236

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K. J. Teerds, D. G. de Rooij, C. J. G. Wensing and F. F. G. Rommerts

ABSTRACT

Several studies have shown that the cytotoxic agent ethane-1,2-dimethane sulphonate (EDS) specifically destroys Leydig cells in the adult rat testis. It has also been reported that when rats are pretreated with human chorionic gonadotrophin (hCG), administration of EDS does not result in the complete destruction of the Leydig cell population. It has been suggested that hCG pretreatment 'protects' Leydig cells against the cytotoxic action of EDS. In the present study the underlying principles for this resistance to the cytotoxic effects of EDS have been investigated. Within 48 h of the start of daily hCG treatment the number of nuclear profiles of Leydig cells (henceforth called relative number of Leydig cells) had increased from 1014 ± 40 to 1368 ± 30 cells per 1000 Sertoli cell nuclei. Previous experiments have indicated that these newly formed Leydig cells probably develop from differentiating Leydig cell precursors. When EDS is administered concomitantly with the third injection of hCG (2 days after the start of hCG treatment), the relative number of Leydig cells surviving EDS treatment was 388 ± 52 per 1000 Sertoli cells. Hence, there is a similarity between the increase in the relative number of Leydig cells after 2 days of hCG treatment and the relative number of EDS-resistant Leydig cells. The Leydig cells that survived EDS administration showed characteristics which also occur in developing Leydig cells in the immature testis. It is concluded that, in rats pretreated with hCG for 2 days before EDS administration, new Leydig cells with some immature characteristics are formed. One of these characteristics is that these cells are insensitive to EDS.

Journal of Endocrinology (1992) 134, 85–90

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R. Molenaar, F. F. G. Rommerts and H. J. van der Molen

ABSTRACT

The presence of non-specific esterase activity is correlated with different Leydig cell characteristics: 3β-hydroxysteroid dehydrogenase (3β-HSD), human chorionic gonadotrophin binding and LH-stimulated steroid production. This indicates that esterase can be used as a marker enzyme for Leydig cells. Esterase, however, has also been used as a marker enzyme for macrophages. We have compared, using biochemical and histochemical techniques, the esterase activity of Leydig cell preparations from mature and immature rats and of preparations enriched in testicular or peritoneal macrophages. Leydig cells were identified by staining for 3β-HSD, and macrophages by phagocytosis of fluorescent beads. Leydig cell preparations from mature rats showed an approximately 400-fold higher esterase activity than peritoneal macrophage preparations and an approximately 50-fold higher activity than testicular macrophage preparations. Leydig cell preparations from mature rats showed a 60-fold higher esterase activity than Leydig cell preparations from immature rats.

Differences in esterase activity were also demonstrated histochemically. Leydig cells from mature rats showed positive esterase staining after 30 s at room temperature. Testicular macrophages showed esterase activity after staining for 3 min. Only approximately 25% of the 3β-HSD-positive cells from immature rats showed esterase activity after staining for 6 min. Esterase is therefore a useful marker enzyme for Leydig cells from mature rats and can be of help in studies concerning the development of these cells.

J. Endocr. (1986) 108, 329–334

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F. F. G. ROMMERTS, B. Ch. KRÜGER-SEWNARAIN, J. A. GROOTEGOED and H. J. VAN DER MOLEN

SUMMARY

Binding ability of androgen-binding protein (ABP) in concentrated media from Sertoli cell cultures and in various preparations of cytosols from rat testicular tissue have been estimated using dialysis and polyacrylamide gel electrophoresis (PAGE). Equilibrium dialysis in the presence of 1 nm-17β-hydroxy-5α-androstan-3-one appears to be the most convenient and reliable method for estimation of ABP if no other binding proteins are present. PAGE can be applied for estimation of ABP in samples which also contain other steroid-binding proteins. Estimated binding activities in culture media measured by PAGE and dialysis were essentially similar. Binding of androgens to ABP estimated with PAGE in cytosols prepared after sonication of testicular tissue was much lower than in cytosols obtained after homogenization using a Potter–Elvehjem homogenizer. When ABP was added to cytosols prepared after sonication or homogenization, approximately 50 and 90% respectively of the original binding of androgens to ABP was recovered.

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F. F. G. Rommerts, J. W. Hoogerbrugge and H. J. van der Molen

ABSTRACT

After the addition of charcoal-treated testicular fluid to Leydig cells isolated from 22-day-old rats, pregnenolone production could be increased to a maximum of tenfold within 30 min in a dose-dependent manner. Testicular fluid, but not serum, further increased pregnenolone formation threefold when pregnenolone production by Leydig cells was stimulated by the addition of LH-releasing hormone (fourfold), LH (25-fold) and 22R-hydroxycholesterol (300-fold). The effect of testicular fluid on steroid production in the presence of 22R-hydroxycholesterol was not inhibited by cycloheximide whereas cycloheximide completely inhibited the effect of LH. It appears unlikely that steroids, lipoproteins or other plasma components constitute the stimulatory agents in testicular fluid. The biologically active principles may be locally produced factors with a molecular weight > 25 000. Similar biological activities could be shown in testicular lymph from boars but not in systemic lymph from boars nor in charcoal-treated bovine follicular fluid. The presumably locally produced factor(s) may amplify the effect of LH and can thus act as a local modulator(s).

J. Endocr. (1986) 109, 111–117

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D. M. Stocco, K. J. Teerds, M. van Noort and F. F. G. Rommerts

ABSTRACT

The biochemical activities involved in the maintenance of Leydig cell functions, and the effects of hypophysectomy and human chorionic gonadotrophin (hCG) on these functions are largely unknown. In the present study, adult hypophysectomized rats were used as a model to determine the effects of these treatments on a number of biochemical and morphological parameters. After 33 days of hypophysectomy, the morphology of the Leydig cells had been drastically altered. In addition, α-naphthol and β-naphthol esterase activity as well as the steroidogenic capacity of the Leydig cells were greatly reduced at this time. In contrast, the level of sterol carrier protein 2 (SCP2), a Leydig cell-specific protein, was affected by hypophysectomy much less than the other parameters measured. Two daily injections of hCG to rats hypophysectomized for 31 days resulted in no change in the morphology of the Leydig cells, or in their proliferative activity. Non-specific esterase activities were also unaffected by 2 days of treatment with hCG. However, two injections of hCG to rats hypophysectomized for 31 days resulted in nearly complete restoration of steroidogenic capacity, and a 3·5-fold increase in the level of SCP2. These findings indicate that hypophysectomy results in significant morphological and biochemical changes in Leydig cells, and that hCG is capable of restoring some of these capacities within a short time.

Journal of Endocrinology (1990) 126, 367–375

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A. P. N. Themmen, J. W. Hoogerbrugge, F. F. G. Rommerts and H. J. van der Molen

ABSTRACT

The stimulation of steroid production in Leydig cells by LH is accompanied by increased cyclic AMP levels, activation of protein kinase A, increased phosphorylation of at least six phosphoproteins and requires protein synthesis. However, an LH-releasing hormone agonist (LHRH-A) can stimulate steroid production without stimulation of cyclic AMP levels. In the present study we have shown that LH action involves calcium fluxes through the plasma membrane, in addition to activation of protein kinase A. The action of LHRH-A, in contrast, does not require calcium fluxes and is not potentiated by 1-methyl-3-isobutylxanthine, indicating that cyclic AMP is not involved. Extracellular calcium is required for the action of both LH and LHRH-A. An increase in intracellular calcium concentration due to the effect of ionophore A23187 did not stimulate steroidogenesis and had deleterious effects on intracellular adenosinetriphosphate levels. LH and 4β-phorbol-12-myristate-13-acetate (PMA), an activator of protein kinase C, both stimulated phosphorylation of proteins of 17 000 and 33 000 mol. wt, whereas LHRH-A had no effect. However, compared with the effect of LH, PMA had a much smaller effect on steroid production, indicating that even if protein kinase C may be activated by LH its role in the regulation of steroid production may be less important than the role of protein kinase A. Action of LHRH-A does not appear to be mediated by calcium fluxes, protein kinase C activation or active protein phosphorylation.

J. Endocr. (1986) 108, 431–440

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M. van Noort, F.F.G. Rommerts, A. van Amerongen and K.W.A. Wirtz

ABSTRACT

In testis tissue from mature rats the non-specific lipid transfer protein (nsLTP), also called sterol carrier protein2 (SCP2), is concentrated in the Leydig cells and cannot be detected in Sertoli cells or germinal cells. Conclusions were reached after cell fractionation studies with normal testis tissue and after selective destruction of Leydig cells or germinal cells in vivo.

The amount of nsLTP (SCP2) in testis tissue increased twofold 48 h after two daily injections of human chorionic gonadotrophin (100 i.u., s.c.) and decreased twofold after plasma luteinizing hormone levels were suppressed to almost undetectable levels with silicone elastomer implants containing testosterone.

The specific localization in the Leydig cells and the luteinizing hormone-dependent cellular concentration of nsLTP/SCP2 support the possibility that this protein could play a role in the regulation of steroidogenesis by regulating the availability of cholesterol for the P450 side-chain cleavage enzyme in the mitochondria of Leydig cells.