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

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

ABSTRACT

The effects of pure FSH and/or LH preparations on the number of Leydig cells and their function in immature hypophysectomized rats have been investigated. As a result of hypophysectomy at the age of 17–18 days, the number of recognizable Leydig cells per testis decreased, as did the steroidogenic capacity in vivo and in vitro. Treatment with 64 μg FSH on both 22 and 23 days of age, did not affect the number of recognizable Leydig cells. In contrast, two injections of LH (10 μg) caused a sixfold increase in the number of Leydig cells, but had a negative effect on spermatogenesis. These stimulatory and inhibitory effects of LH diminished when FSH was added. Treatment with FSH for 7 days caused a twofold increase in the number of Leydig cells when compared with hypophysectomized controls. 3β-Hydroxysteroid dehydrogenase (3β-HSD) and esterase activity in Leydig cells also increased under the influence of FSH. The pregnenolone production per Leydig cell in the presence of 5-cholesten-3β,22(R)-diol (22R-hydroxycholesterol) as substrate showed a sevenfold increase. Plasma testosterone levels 2 h after injection of human chorionic gonadotrophin in intact rats and hypophysectomized FSH-treated rats were the same. Following LH treatment for 7 days, the number of Leydig cells proved to be 11 times higher, and 3β-HSD and esterase activity were not different from intact controls. The testicular pregnenolone production was four- to fivefold higher when compared with untreated hypophysectomized rats. However, pregnenolone production per Leydig cell in LH-treated rats was only slightly different from the hypophysectomized controls.

In conclusion, FSH treatment caused an increase in the number and steroidogenic activity of Leydig cells, and LH had a major effect on the number of Leydig cells, but did not stimulate the steroidogenic capacity.

Journal of Endocrinology (1989) 120, 97–106

ABSTRACT

The effect of 100IU human chorionic gonadotrophin (hCG) on testicular capillary blood flow was studied in adult male rats using a ¹³³Xe clearance method and a radioactive microsphere technique. To investigate the role of Leydig cells in regulation of testicular blood flow after treatment with hCG, rats were pre-treated with ethane dimethylsulphonate (EDS) which selectively destroys mature Leydig cells. Six hours after treatment with hCG, testicular blood flow decreased in control and hypophysectomized rats to 25–50% of normal values, but not in EDS-pretreated animals. Prostaglandin E₂ levels were also determined 6 h after an injection of hCG. A 300-fold increase in the concentration of prostaglandin E₂ occurred in normal testis tissue. This rise was markedly inhibited if EDS was given 3 days before administration of hCG. Furthermore, 6 h after administration of hCG, the filling of the testicular capillary bed with methylacrylate was decreased, while in control rats and rats treated with EDS and hCG, complete filling of the capillaries was seen. Cell degeneration in some subcapsular seminiferous tubules was observed 6–10 days after treatment with hCG.

The results suggest that the hCG-induced precapillary vasoconstriction, probably mediated (in part) by prostaglandins, causes reduction in testicular blood flow 6 h after administration of hCG, and may result in cell damage.

J. Endocr. (1988) 117, 51–57

ABSTRACT

The influence of LH levels on the proliferation and differentiation of possible Leydig cell precursors was investigated in adult rats, after the destruction of the existing Leydig cells with the cytotoxic drug ethane dimethyl sulphonate (EDS). In rats bearing a testosterone implant which prevented the rise in plasma LH levels and kept them within the normal range after the destruction of the Leydig cells, the proliferative activity of possible Leydig cell precursors still increased seven- to eightfold 2 days after EDS administration. Apparently, in this situation, locally produced factors, and not LH, may play a role in the stimulation of proliferation. The proliferative activity of the possible precursor cells could be further stimulated by treating rats with daily injections of human chorionic gonadotrophin (hCG) following EDS administration. It was concluded that the proliferative activity of possible Leydig cell precursors is probably regulated by both paracrine and endocrine factors.

Almost no Leydig cells were formed in the rats bearing a testosterone implant during the first 4 weeks after EDS administration. When these rats were treated with hCG, starting 28 days after administration of EDS, a substantial number of Leydig cells was found after 2 days, and these cells also showed 3β-hydroxysteroid dehydrogenase (3β-HSD) and α-naphtyl esterase (α-NE) activity. When hCG treatment was started at 14 or 21 days after EDS administration, some cells with the nuclear characteristics of Leydig cells were present after 2 days, but no 3β-HSD or α-NE activity could be detected. Finally, when hCG treatment was started directly after EDS administration, a considerable number of Leydig cells was found 14 days after EDS, and some of these cells already showed 3β-HSD and α-NE activity. It is concluded that precursor cells are able to develop into advanced precursor cells at normal LH levels, and that the rate of development of new Leydig cells strongly depends upon LH/hCG levels.

Journal of Endocrinology (1989) 122, 689–696