1 Introduction

Considerable information is available on the ontogeny and hormonal regulation of Leydig cells (LC) in mammals. Much of this information has been obtained using rodents as a model^[1]. Due to ethical reasons and scarcity of normal testicular tissue from prepubertal boys, our knowledge on hormonal control of LC differentiation in primates is limited to results available from clinical samples and histological analysis.

In the case of studies on LC using rat as a model, the alkylating agent ethane-1,2-dimethyl sulphonate (EDS) has proved to be a valuable tool by its ability to selectively destroy the LC. The reversible destruction of mature androgen secreting LC following the administration of EDS offers a unique opportunity to study hormonal regulation of the development of the adult-type LC population^[2], since the effect of EDS appears to be limited primarily to the LC. Depletion of LC after EDS administration results in a drastic decrease in serum testosterone levels, with a concomitant regression of androgen-dependent tissues^[3]. Within 2-3 weeks after treatment, functional LC regenerate from precursor LC. The regenerated cells in the EDS-treated mature rats are comparable in their histologic and steroidogenic characteristics to LC that develop naturally during puberty^[4]. Thus in recent years, the EDS-treated rat (75 mg/kg body weight, single dose) has become a useful model for studying LC development and function^[5].

Several reports have revealed that LC toxicity is species-specific, a property which has limited its applicability. Thus, while it was reported that EDS acted as a potent toxicant for LC in the  rat, rabbit, guinea pig, hamster, and frog, only substantial systemic toxicity without changes in LC occurred in the mouse, dog, marmoset and quail^[6,7,8].

2 Materials and methods

2.1 Animals

Adult male bonnet monkeys (Macaca radiata, 6-8 kg, 7-8 years) obtained from the Primate Research Laboratory (Sc., Bangalore, India) were housed in galvanized iron cages under conditions of regulated light (12 h light:12 h darkness). The animals were fed with pelleted feed (Mysore Snack Feeds, Bangalore), fresh banana  and water every day. The surgical procedures employed for the studies involving monkeys have been approved by the Institutional Ethical Committee. Testes were removed after anaesthetising the monkeys by administration of ketamine hydrochloride (8-10 mg/kg body weight).

2.2 Chemicals

Ethane-1,2-dimethylsulphonate (EDS, provided by Prof. Rommerts) was dissolved in dimethyl sulfoxide and water (1:3, vol:vol) for use. HEPES, Soyabean trypsin inhibitor (SBTI), DNAse, Bovine Serum Albumin (BSA), Collagenase, were purchased  from Sigma Chemical Company (St. Louis, MO). Dulbecco's Modified Eagle's Medium (DMEM) and Medium 199 were obtained from Hi-media, Bombay. hCG was a gift from Prof. M.R. Sairam, Clinical Research Institute of Montreal, Montreal, Canada. and National Hormone & Pituitary Program, NIDDK, USA.³HTestosterone (3.3 TBq/mmol) was obtained from Amersham International, UK.

2.3 Effect of in vitro addition of EDS on testicular interstitial cells

Since no literature is available on the effect of EDS in monkeys, experiments were carried out to assess the effect  of in vitro addition of EDS on interstitial cells. Testes were flushed via the testicular artery with 5 mL of M-199 twice  and cut into four pieces which were incubated in 10 mL of M-199 containing 1 mg/mL collagenase, 0.1 % BSA, 1000 units of DNAse and  1g/mL SBTI at 34 in a Dubnoff shaking incubator (70 revolutions/min) for 20 min. The colloidal mass of tissue was gently inverted after addition of 30 mL of the medium several times, filtered through a 100 m nylon mesh and centrifuged at 500g for 10 min at room temperature (23). The supernatant was removed, and the cell pellet was resuspended in DMEM supplemented with 0.1% BSA and 12 g/mL gentamycin. The crude interstitial cells were incubated for 4 h in the presence or absence of 100 ng hCG, with or without varying concentration of EDS in DMEM in 96-well plates under an atmosphere of 5% CO₂ and 95% O₂  at 34 up to 4 h.

Testosterone concentration in the incubation medium was quantitated using a sensitive RIA standardized in our laboratory^[9] and expressed as percentage considering the controls as 100%. The testosterone antiserum was used at a final dilution of 1:6000, and the range of the assay was from 3 pg-2000 pg. The minimum detectable quantity of testosterone was 25 pg. The inter-assay variation was 7%, while the intra-assay variation was 3.5%.

2.4 Effect of in vivo administration of EDS on testicular function in the monkey testes

Dose response studies: Adult monkeys were injected a single dose of 5, 10, 20, and 50 mg of EDS/testis in 1:3 Me₂SO:water intratesticularly to both the testes. Six animals were included in the group that was injected 5 mg of EDS and for 10, 20, and 50 mg dose 2 animals were tested per dose. The animals were bled daily for 45 days after EDS treatment from  the femoral vein at 10:00 am, and generally not more than 1-2 mL of blood was drawn at a time. The animals were supplemented with Uniferon B12 injection (Glaxo Laboratories, Bombay) to prevent anaemic conditions. All blood samples were processed within 2-3 h after collection, serum separated, and stored at -20 for further use. After the extraction of the steroids with ether, serum samples were analyzed for testosterone levels as described earlier in a single assay. The extraction efficiency varied from 80%-85% and the values reported are uncorrected for recovery. In order to assess the other parameters of Leydig cell destruction by EDS a second group of monkeys (n=4) were injected 5 mg of EDS into both the testes, and after third day of injection animal was unilaterally castrated to estimate testicular testosterone, ¹²⁵IhCG binding,  and  for histological analysis. Intratesticular testosterone (in the testis homogenate) was assayed by employing the protocol used for serum testosterone estimation. The binding of ¹²⁵IhCG by the testicular homogenate was assessed as described by Morris^[10]. For histological analysis, sections of the testes were fixed in Bouins fluid, and 5 m paraffin sections were prepared and stained with hematoxylin.

Crude interstital cells were  also isolated  to determine  the number of 3--hydroxysteroid dehydrogenase (3-HSD) positive cells and interstitial cell phenyles terase activity. 3-HSD staining was carried out as described by Browning^[11]. Tissue phenylesterase activity  which is reported to be specific for LC was monitored as described^[12].

2.5 Statistical analysis

3 Results

3.1 In vitro studies

In view of the absence of any published reports on the effects of EDS in the bonnet monkeys, a study was undertaken to assess the effect of in vitro exposure of monkey testicular interstitial cells to different concentrations of EDS. Studies were carried out by incubating the interstitial cells suspended in DMEM in 96 well plates under an atmosphere of 5% CO₂ and 95% O₂ up to 4 h at 34.

The effect of addition of different concentrations of EDS ranging from 0.1 mmol/L to 3 mmol/L was examined at 4 h time point on testosterone production. Analysis of the incubation medium for testosterone at 4 h time point in the presence of 1 mmol/L EDS revealed that it decreased by 50%. There was a dose-dependent decrease in testosterone production with increase in EDS concentration. A significant decrease in testosterone level was observed in the presence of 1 mmol/L and 3 mmol/L EDS (Figure 1). The fact that the  cells affected by EDS in the interstitial cell preparation are indeed Leydig cells was ascertained by the observation that these cells responded with increased testosterone in the medium after incubated in the presence of hCG (100 ng) for 4 h (data not shown).

Figure 1. Dose-dependent effect of EDS on  testosterone  production in the  cultured adult  monkey   interstitial    cells. n=3 observations. means. ^bP<0.05 vs control.

The above results obtained following in vitro exposure of monkey testis interstitial cells to EDS suggest that the monkey LC are sensitive to EDS which formed the basis for the in vivo studies. 

3.2 In vivo studies

Following administration of EDS to adult male monkeys by intratesticular route, serum samples were collected for 7 weeks at 24-h intervals  (Figure 2).

Figure 2. Effect of intratesticular administration of different doses (5, 10,15, and 20 mg)of EDS on serum testosterone level in adult male bonnet monkeys. means.

It can also be seen that there was a dose-dependent decrease in serum testosterone level, and a maximum decrease in serum testosterone level was seen in the 50 mg EDS-treated group. The steady decline was seen in all groups up to the 5th day, and from then onwards, the level of serum testosterone increased gradually. In the 5 mg EDS-treated group, a 62% decrease in serum testosterone level was observed on the 3rd day and the levels reached normal values by 45 days. In the remaining groups the serum testosterone level was less than 50% of the pre-injection value on the 3rd day and this did not reach the pre-injection level even on 45th day.

In view of the fact that a maximum decrease in serum testosterone was seen on the 3rd day in the 5 mg EDS treated group, another group of monkeys were injected EDS (5 mg)  intratesticularly into both the testes and  were unilaterally castrated on the 3rd day after EDS injection and isolated interstitial cells were used for various studies. The intratesticular testosterone levels showed a 60% decrease in the experimental animals, compared with the controls (Figure 3A). The binding of [¹²⁵I] hCG, an indicator of LH receptor level, was significantly reduced (55%) in the testes of EDS-treated monkeys (Figure 3B). A 45% reduction in the activity of the LC marker enzyme phenylesterase, indicate the fall in the number of functional LC following EDS exposure (Figure 3C), which is also supported  by the observation that there was a 50% decrease in 3-HSD cells in the EDS treated group (data not shown).

Figure 3. Effect of intratesticular injection of  EDS  on  testicular testosterone (A),  [¹²⁵I] hCG binding (B),  phenylesterase activity (C). n=3 observations. means. ^cP<0.01 vs control.

Photographs of the histological sections of the vehicle and EDS treated monkey testes are presented in Figure 4. While the interstitium of the Me₂SO treated monkey appeared normal with LC population, in the EDS treated group the interstitial space was devoid of LC indicating the destruction of LC following EDS treatment.

References

[1] Lejeune H, Habert R, Saez JM. Origin, proliferation and differentiation of Leydig cells. J Mol Endo 1998; 20: 1-25.
[2] Teerds KJ. Regeneration of Leydig cells after depletion by EDS: a model for postnatal Leydig cell renewal. In: Payne AH, Hardy MP, Russell LD, editors. The Leydig cell. Cache River Press; 1996. p 203-20.
[3] Kerr JB, Bartlett JM, Donachie K. Acute response of testicular interstitial tissue in rats to the cytotoxic drug ethane dimethanesulphonate. An ultrastructural and hormonal assay study. Cell Tissue Res 1986; 243: 405-14.
[4] Myers RB, Abney TO. Testosterone and  androstanediol production by regnerating Leydig cells in the ethylene  dimethanesulfonate-treated mature rat. Int  J Androl 1990; 13: 4-16.
[5] Molenaar R,  Rooij DG, Rommerts FFG, Reuvers PJ, Van der Molen HJ. Specific destruction of  Leydig cells in the mature rats after in vivo administration of ethane dimethylsulfonate. Biol Reprod 1985; 33: 1213-22.
[6] Gray LE, Klinefelter G, Kelce W, Laskey J, Ostby J, Ewing L. Hamster Leydig cells are less sensitive to ethane dimethylsulphonate when compared to rat Leydig cells both in vivo and in vitro. Toxicol Appl Pharmacol 1995; 130 : 248-56.
[7] Kerr JB, Klnell CM, Abott M, Donachie K. Ultrastructural analysis of the effect of ethane  dimethylsulphonate on the testes of the rat, guinea pig, hamster and mouse. Cell Tissue Res 1987; 249: 451-7.
[8] Morris ID. Leydig cell toxicology. In: Payne AH, Hardy MP, Russell LD, editors. The Leydig cell. Cache River Press; 1996. p 573-96.
[9] Rao AJ, Charaborti BR, Kotagi SG, Ravindranath N. Effect of constant infusion of GnRH agonist buserelin and antagonist CDB 2085A using osmotic minipumps on testicular function in adult male bonnet monkeys (Macaca radiata). Andrologia 1990; 20: 567-72.
[10] Morris ND, Phillips DM, Bardin CW. Ethylene dimethanesulfonate destroys Leydig cells in rat testis. Endocrinology 1986; 118: 709-19.
[11] Browning JY, Heindel JJ, Grotjan HE. Primary culture of purified Leydig cells isolated from adult rat testes. Endocrinology 1983; 112: 543-9.
[12] Rommerts FFG, Van Doorn  LG, Galjaard  H, Cooke  BA, Van der  Molen  HJ. Dissection of wet tissue and  freeze-dried sections in the investigation of seminiferous tubules and interstitial tissue from rat testis. J Histochem  Cytochem 1973; 21: 572-9.
[13] Greco TL, Payne AH. Ontogeny of expression of the genes for steroidogenic enzymes P450 side-chain cleavage, 3 beta-hydroxysteroid dehydrogenase, P450 17 alpha-hydroxylase/C17-20 lyase, and P450 aromatase in fetal mouse gonads. Endocrinology 1994; 135: 262-8.