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


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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A Foghi, K J Teerds, H van der Donk and J Dorrington


In each estrous cycle dominant follicles are selected from a growing pool to develop to the preovulatory stage and to ovulate. Those follicles that do not ovulate must be eliminated in order to maintain the constant mass and homeostasis of the ovary. Granulosa cells are lost by apoptosis at the onset of follicular atresia, whereas apoptotic thecal cells are identified at later stages of atresia. Since transforming growth factor (TGF) α and TGFβ1 have been implicated in the regulation of thecal cell physiology we have localized these growth factors by immunohistochemistry in sections of ovaries from 25-day-old rats, an age at which the ovary exhibits a wave of atresia of preantral follicles. Thecal cells contained TGFα and TGFβ1 throughout the entire process of follicular atresia. To determine if these growth factors could influence thecal cell death, thecal/interstitial cells were isolated from 25-day-old rats, and maintained in culture with growth factors. Subconfluent cultures treated with TGFα or TGFβ1 alone remained healthy whereas in the presence of both TGFα and TGFβ1 there was light microscopical evidence of rounding up of cells and detachment from the monolayer. Chromatin condensation and internucleosomal fragmentation, characteristic of apoptosis, were observed by nucleic acid staining and fluorescence microscopy of thecal/interstitial cells treated with TGFα plus TGFβ1. Further evidence that these cells were undergoing apoptosis came from DNA analysis and the demonstration of DNA laddering. This response of thecal/interstitial cells to TGFα plus TGFβ1 was density dependent; confluent cultures were protected from the induction of apoptosis under these conditions. We conclude that thecal cells are eliminated from atretic follicles by the active and strictly regulated process of apoptosis involving the combined actions of TGFα and TGFβ1.

Journal of Endocrinology (1997) 153, 169–178

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


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


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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K A Slot, M de Boer-Brouwer, M Houweling, A B Vaandrager, J H Dorrington and K J Teerds

Gonadotrophins including LH have been suggested to play an important role in the etiology of epithelial ovarian cancers. The goal of the present study was to obtain more insight in the mechanism of gonadotrophin action on ovarian surface epithelium (OSE) cells. As the Fas system is known to be a major player in the regulation of the process of apoptosis in the ovary, we investigated whether LH interfered with Fas-induced apoptosis in the human OSE cancer cell lines HEY and Caov-3. Activation of Fas receptor by an agonistic anti-Fas receptor antibody induced apoptosis, as was evaluated by caspase-3 activation, poly(ADP-ribose) polymerase fragmentation, phosphatidylserine externalization and morphological changes characteristic of apoptosis. Co-treatment with LH reduced the number of apoptotic cells following activation of Fas in a transient manner, while LH by itself did not affect apoptosis or cell proliferation. The anti-apoptotic effect of LH could be mimicked by the membrane-permeable cAMP analog 8-(4-chlorophenylthio) cAMP (8-CPT-cAMP), and blocked by H89, a specific inhibitor of protein kinase A (PKA). In conclusion, these findings suggest that LH protects HEY cells against Fas-induced apoptosis through a signaling cascade involving PKA. Although it is plausible that in vivo LH might also enhance OSE tumor growth through inhibition of apoptosis, further research is necessary to confirm this hypothesis.

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K. J. Teerds, D. G. de Rooij, F. F. G. Rommerts, R. van den Hurk and C. J. G. Wensing


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

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K. J. Teerds, J. Closset, F. F. G. Rommerts, D. G. de Rooij, D. M. Stocco, B. Colenbrander, C. J. G. Wensing and G. Hennen


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