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Effects of acut and chronic doses of methoxy acetic acid on hamster sperm fertilising ability

L.D.C. Peiris1, H.D.M. Moore2

1Dept. of Zoology, University of Colombo, Colombo 03, Sri Lanka
2Dept. of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK

Asian J Androl  2001 Sep; 3: 185-191

Keywords:  methoxy acetic acid; fertilisation in vitro; testis; spermatozoa; fertility
Abstract

Aim: To evaluate the effects of acute and chronic doses of methoxy acetic acid (MAA) on in vitro fertilisation by hamster sperm and to correlate the data with the testicular damage. Methods: Adult male hamsters were gavaged with 3 single doses (0, 80, 160 and 650 mg/kg) and 3 chronic doses (0, 8, 32 and 64 mg/kg daily for 5 weeks) of MAA in distilled water. After treatment hamsters were killed at weekly intervals and spermatozoa recovered from the distal cauda epididymides were used to assess the fertilising capacity in vitro. The testes were processed for histological examination. Results: Acute doses showed a significant reduction in sperm fertilising ability from week 3 and 4 after treatment and with the chronic doses, the effects were more extensive and persistent.The results were in correpondence with the testicular damages observed. Conclusion: It is evident that both acute and chronic doses of MAA can impair the sperm function by damaging one or more cell populations in the testis.

1 Introduction

Fertilisation in vivo requires adequate numbers of spermatozoa to be ejaculated with normal morphology and motility[1]. Since spermatogenesis involves a complex process of cellular development, the impairment to any of the stages may lead to a reduction in fertility. Spermatogenesis has been shown to be susceptible to the damaging effect of a variety of chemical agents[2].

The relationship between the chemical damage to the testis and the male fertility is poorly understood in mammals. Specific damage to germ cells within the testis may take several weeks to become apparent and may not affect the fertilising capacity of all the sperm so that residual fertility remains. Furthermore, at low exposure of toxicant, the effects on sperm development may be subtle and involve changes to sperm function that are not apparent by standard testing such as animal mating trials. In this respect, rodent animal models have been useful to assess the effects of testicular toxicants[3,4].

Methoxy acetic acid (MAA) is widely used as a solvent in the manufacture of protective coatings such as lacquers, enamels, phenolic varnishes and alkyl resins. MAA is also used as an intermediate chemical for the production of ethylene glycolmonomethyl ether (EGME), a common plasticiser used by paint, printing, textile and leather industries[5,6].

Studies have shown that administration of single oral doses of MAA to rats caused selective and stagedependent destruction of late pachytene and diplotene spermatocytes[6-9]. Later the degeneration extended to early and mid-stages of spermatocytes and to round spermatids[9]. At lower dose levels, the effects were less pronounced or confined to fewer stages of spermatogenesis. Ratnasooriya and Sharpe[10] treated rats with a single dose of 650 mg/kg of MAA and found similar results to earlier toxicological studies. In addition, they observed a reduction in sperm motility at 4-7 weeks post-treatment but with no evidence of infertility. Chronic doses of MAA resulted in the reduction in testicular weights[11] in addition to the above mentioned effects.

Although there have been a considerable number of investigations on the effect of reproductive toxicants on the histology of the testis, it is less known about how damage is reflected in the fertilising ability of sperm. Further, the effects of MAA on fertility and testicular damages of hamsters have not been studied. The in vitro fertilisation (IVF) assay is sensitive enough to detect subtle changes in the fertilising capacity of spermatozoa[12]. Therefore the present study was undertaken to examine the sperm fertilising ability in vitro in male hamsters treated with acute and chronic doses of MAA and to compare the fertility data with the results on testicular damage.
2 Materials and methods

2.1  Hamster colony

Adult (6 weeks old) Syrian (Golden) hamsters (Mesocricetus auratus)  were purchased from Harlan (UK). Animals were maintained on 13 h light and 11 h dark cycle in a room that was lit by artificial light from 06:00 to 19:00. The animal room was kept at (202) and a relative humidity of 45 %-55 %. Animals were fed on a pellet diet with free access to water and animals were allowed to acclimatise for at least 2 weeks before use.

Toxicant was given orally by gavage using smooth-ended flexible stainless steel gavage needles connected to a 1 mL syringe. 

2.2 Chemicals

MAA, >98 % pure, was purchased from Aldrich(UK), while all other chemicals were purchased from Sigma (UK) unless stated otherwise. Solutions of MAA were prepared with distilled water just prior to use.

2.3 Treatment of hamsters

Experiment 1 (acute dose): 36 male hamsters were given a single dose of MAA at either 650, 160 or 80 mg/kg. The total dosing solution was 1 mL. Control animals (n=12) received the same amount of distilled water.

Experiment 2 (chronic dose): 36 animals were dosed daily for 5 weeks by oral gavage with 8, 32 or 64 mg/kg MAA. Control animals were treated as above.

2.4 In vitro fertilisation

The method described by Bavister[13] was used. The medium used for in vitro fertilisation was a modified Krebs-Ringers solution (BWW) developed by Biggers et al[14]. This medium was supplemented with 4 mg/mL crystalline bovine serum albumin. The final solution was filter sterilised (0.2 m, Gelman Sciences, UK) and 100 l aliquots of medium were placed in the tissue culture Petri dishes (3510 mm, Corning Glass Wear, USA) and immediately covered with warm liquid paraffin (Boots Co., Nottingham, UK). Petri dishes with medium were equilibrated overnight in an atmosphere of 5 % carbon dioxide in air at 37 in a humidified incubator. The final pH of the medium was 7.4. The osmotic pressure was between 280-300 mOsm as determined by freezing point depression osmometer (Camlab, UK).

2.5 Collection of cauda epididymal sperm

The hamsters were killed by sodium pentobarbitone (J.M. Loveridge, Southampton, UK) given intraperitoneally. For the IVF procedure, the left distal cauda epididymidis was dissected out and placed in 2 mL of equilibrated BWW medium in a tissue culture dish. The epididymal tubules were teased apart to allow sperm to swim out into the medium.

2.6  Staining of hamster spermatozoa with Hoechst 33342

Sperm released from the distal cauda epididymidis were incubated in BWW medium with 2 g/mL Hoechst 33342 (bisbenzimide) for 30 min and centrifuged (Beckman, UK) for 4 min at 600 g. The sperm pellet was resuspended in BWW medium and washed again by centrifugation. To induce capacitation, sperm (~107/mL) were incubated in the medium in a humidified atmosphere of 5 % carbon dioxide in air at 37 for 3 h.

2.7  Collection of oocytes

Immature, female Syrian hamsters were superovulated with 40 IU of PMSG (Pregnant Mare's Serum Gonadotropin). The PMSG was administered at 11.00 h intraperitoneally (i.p.). Two days later, 40 IU of hCG (Human Chrionic Gonadotropin) was given i.p. to each female at 17:00. Animals were killed at 11:00 the next d. Ovaries and oviducts were dissected out in BWW medium and oviducts were ruptured with a fine needle to release the cumulus mass.

The cumulus was dissolved by 0.1 % bovine serum testicular hyaluronidase in the medium for 10 min. Cumulus-free eggs were washed twice by pipette in the BWW medium and were kept at 37 under oil until insemination.

To obtain zona-free hamster oocytes, cumulus free eggs were treated with 1 % trypsin in BWW for 20 s. The eggs were washed thoroughly and stored as above.In both instances, about 25-30 eggs were kept in a single drop.

2.8 Insemination of oocytes

After 3 h of incubation, the stock sperm suspension was adjusted so that the final concentration of sperm used for inseminating oocytes was 5105/mL-1106/mL. The oocytes were transferred to the sperm suspension and the culture dishes were returned to the humidified incubator at 37 in 5 % CO2 in air.

2.9 Evaluation of fertilisation

Four hours after insemination, eggs were pipetted out, washed with BWW medium thoroughly to remove the sperm attached to the zona surface and mounted between a slide and a coverslip with Vaseline spots for support. The eggs were moderately compressed under the coverslip and examined with a phase-contrast microscope (BH2, Olympus, Japan) and ultraviolet light with a wavelength of 395-440 nm filter for evidence of a decondensing sperm head or the presence of pronuclei. Fertilisation was normally confirmed by the presence of the sperm tail in the egg vitellus under phase-contrast.

2.10 Histological studies

The right testis was dissected out, blotted dry to remove any blood,  cut into small slices and fixed in Bouin's fluid for 24 h. The tissues were washed free of Bouin's fluid and stored in 70 % alcohol until embedding.

The tissues were dehydrated in alcohol series and embedded in melted wax. Tissue sections of 4 m thickness were made and placed on glass slides.

The sections were stained with heamotoxylin and eosin and mounted in DPX mounting medium. Slides were examined under a light microscope (BH-2. Olympus Ltd, Japan). For each testis several crosssections composing of 20-50 tubule sections was examined for signs of damage to the germinal epithelium.

2.11 Statistical analysis

Statistical analysis was performed with one way analysis of variance (ANOVA) using Minitab software for the main effect of toxicants. Where a significant treatment effect was found, differences among individual group means were tested by the least significant test. The null hypothesis for each testing was that there was no significant difference between the fertilisation rates or sperm counts ofthe control and treated animals. Additionally, the relationship between dose and fertilisation rates was examined by linear regression analysis. Values were considered statistically significant at P<0.05. The data are expressed as meanSD.

3 Results  

3.1 Effect of fluorescent staining of hamster sperm on IVF

Staining of hamster sperm with Hoechst 33342 did not effect their fertilising ability since it was able to obtain highly consistent (>90 %) fertilisation rates for the controls.

With stained sperm, all stages of sperm head decondensation (Figure 1A-B) and the presence of pronuclei (Figure 1C) in fertilised oocytes were easily and quickly identified by distinct blue fluorescence. Decondensed sperm heads were of either elliptical (Figure 1A) or round (Figure 1B) shape of different sizes. Care was taken when scoring oocytes to confirm the presence of the sperm tail in the egg vitellus.

Figure 1. Micrographs of fluorescence of DNA stained at different stages of fertilisation. Penetrated spermatozoon head within the egg appeared initially elliptical (A) & later became large and diffuse (B). Both are indicated by arrows  (C). fertilised oocyte showing male & female pronuclei. Polar body is also fluorescent. Magnification  200.

3.2  General  toxicity of MAA

At acute and chronic dose levels no overt signs of general toxicity were observed in the treated animals and the body weights were comparable to those of the control hamsters.

3.3 Effects of MAA on fertilisation in vitro

Similar patterns of results were obtained with both zona-intact and zona-free eggs.

3.3.1 Effects of acute doses of MAA

The fertilisation rates in the control group were consistently greater than 90 % throughout the experiment (Figure 2).

Males dosed with 80 mg/kg showed no significant reduction in sperm fertilising ability. Males given 160 mg/kg had significantly reduced sperm fertility at weeks 4 and 6 after exposure. Males treated with 650 mg/kg had significantly lower sperm fertility at week 1, which  reached a nadir from week 3 with zona-intact eggs. With zona-free eggs, there was a similar trend in fertilisation rates but were slightly higher at week 5. At week 6 post-treatment, males had vestigial cauda epididymides and hence, no viable sperm were obtained to carry out IVF. These data are summarised in Figures 2A and 2B.

3.3.2  Effects of chronic doses of MAA

Males treated with 8 mg/kg MAA had significantly reduced sperm fertility from week 1 to week 4 after exposure. Males given 32 mg/kg MAA showed a reduction in sperm fertility throughout the experimental period except at week 5.

At week 1, males treated with 64 mg/kg had a small cauda epididymidis and the sperm reserves were not adequate to carry out IVF. These males exhibited a significant reduction in sperm fertilising capacity throughout the observation period with a gradual recovery. Data are presented in Figures 2C and 2D.

Figure 2. Fertilisation rates of male hamsters treated with three acute doses (A, zona intact eggs & B, zona free eggs) and three chronic doses (C, zona intact eggs & D, zona free eggs) of MAA. Spermatogenic stages are given with the graphs. Ed s'zoa=epididymal spermatozoa, S'tids=spermatids; P=pachytene spermatocytes, Z=zygotene spermatocytes, L=leptotene spermatocytes, PL=preleptotene spermatocytes and S'gonia=spermatogonia. *P<0.05 & **P<0.01,  compared with controls.

3.4 Histology

Testes sections from control males displayed consistently good histological preservation indicating that the fixation method and tissue processing was optimal. A series of representative micrographs are shown in Figure 3 (A-B) with increasing order of magnification. The germinal epithelium is well preserved with all the cell types present. There are no vacuoles in the epithelium and the lamina of the tubules do not contain sloughed immature cell types.

3.4.1 Acute exposure to MAA

At the lowest acute dose, testicular damage was only evident at the first week of treatment and was restricted to individual tubule sections. Sloughed clusters of spermatocytes were observed in certain sections (Figure 3C). At later stages after treatment there was no obvious differences between the control and treated animals. At the higher dose levels, the effects of toxicant became more apparent at the first two weeks after treatment. Tubule sections contained many sloughed cells, the majority of which were pachytene spermatocytes, although some other cell types (i.e., spermatids) were also present. This effect was most pronounced at the first week after treatment. Representative micrographs of damage are shown in Figures 3D and 3E. By 3 and 4 weeks after treatment, the lamina of tubule sections were free of debris but the germinal epithelium exhibited a diminished population of elongatated spermatids (Figure 3F).

3.4.2 Chronic exposure to MAA

The lowest chronic dose had little effect on testis histology at any time after treatment. At the first two weeks sporadic tubule sections contained sloughed cells of mixed cell types but many tubule sections looked normal. At the high dose levels there were noticeable effects on the testis. Two weeks after treatment many tubules contained sloughed cells similar to the high acute dose after week 1 (Figure 3G). However by week 4 of treatment a proportion of tubules was depleted of later stages cells, which were not recovered at the end of the observation period (Figure 3H).

Figure 3. Micrographs of testicular sections. 
A: Control animals at different stages, (120, normal spermatogeneis).  
B: Control animals at Stages IX* and XII**, (240, normal spermatogeneis). 
C: Animals one week after MAA 80 mg/kg (Stage XI, (400), showing sloughing off (arrow) of spsermatocytes.
D: One week after MAA 160 mg/kg (Stages VIII\|XII, 120), showing sloughed cells, mainly spermatocytes and some spermatids, in the lumen.
E: One week after MAA 650 mg/kg (Stages VIII\|XII, 240), showing sloughed cells, mainly spermatocytes and some spermatids, in the lumen.
F: Four weeks after the highest acute dose of MAA (Stages IX\|XV, 120), showing depletion of elongated spermatids and spermatozoa.
G: Two weeks after MAA 32 mg/kg (Stage XI, 240), showing sloughed cells in the lumen.
H: Animals given high chronic dose of MAA (Stage IX), showing depletion of later stages of dell development. 

4 Discussion

It is well documented that MAA damages the germinal epithelium in the rat. A number of toxicity trials has been carried out using mating protocols and only a few studies[4,12,15] used in vitro fertilisation procedures.

The number of sperm inseminated is a crucial factor to obtain successful fertilisation rates. Talbot et al[16] failed to achieve high fertilisation rates at sperm number higher or below 1106/0.5 mL. They were able to obtain the highest fertilisation rates when they inseminated a sperm concentration of 1106/0.5 mL. A drop in the percentage of eggs penetrated at sperm concentration above 1106/0.5 mL were observed and this may be due to exhaustion of certain metabolites, accumulation of toxic products, or inhibition of capacitation.

A fall in fertilisation rates with low sperm concentration could be due to diminishing probability of egg and capacitated sperm collision. Holloway et al[12] found that the best concentration of sperm to obtain optimal fertilisation rates was between 3105-1106/mL per 20-30 eggs. Hence, in the present study, the sperm concentration for insemination was always kept between 5105 and 1106 /mL. A consistently high percentage of oocyte fertilised in the controls (90 %-100 %) indicated that this sperm concentration gave optimal fertilisation rates. The results were similar to those reported by Stein and Schnieden (90 %-100 %)[17] and  Yanagimachi (100 %)[18].

Removal of the zona pellucida provides an indication of whether a chemical can effect sperm penetration or sperm fusion[19]. There was no marked difference between the fertilisation rates of zona-free and zona-intact eggs in both MAA and mDNB treated hamsters, suggesting that the toxicants had a major effect on the sperm fertilising capacity irrespective of the presence or absence of zona.

The spermatogenic cycle of the hamster is about 35 d and epididymal sperm transit varies from 7 to 10 d[20]. Therefore in the case of acute treatment, there is a lag period from the time of initial exposure to a testicular toxicant to the time when the cauda epididymidis sperm may be affected. For example, if at week 1, sperm fertilising capacity is already affected, the toxicant may have penetrated the cauda epididymidis to directly compromise sperm. If sperm in the cauda epididymidis are affected only after 3 and 4 weeks, it would indicate that the round spermatids and pachytene spermatocytes were compromised at the time of the treatment.

As the acute dose of MAA was increased, the number of stages of germ cells affected and the severity of toxicity were also increased. At the lowest dose of 80 mg/kg,  the fertilising capacity of sperm was reduced only slightly at week 3. At the 160 mg/kg dose, the decline in sperm fertilising capacity was in keeping with pachytene spermatocytes and possibly also spermatogonia damage, while at the 650 mg/kg, the results were consistent with round spermatids and epididymal spermatozoa damage as well. Histological studies also revealed that spermtocytes are the most sensitive stage.

Previous in vivo and in vitro studies in the rat identified meiotic pachytene spermatocytes as an early target for MAA toxicity[21]. Later studies with the related compound EGME also indicated that spermatids were affected[12]. In keeping with the studies in other rodents, the present histological data also revealed that the spermatocytes are the most sensitive cell type affected by MAA. In addition, early spermatids were also sensitive to the toxicant. Stages 8-12 being particularly prone to damage after exposure. Pachytene spermatocytes make up a greater complement of the germinal epithelium in these stages.

A significant observation made in the present study was the inhibition of fertility of epididymal sperm with 650 mg/kg of MAA after week 1. This has not been recorded in previous studies and may be due to species differences. The result would indicate that epididymal sperm or very late spermatids can be directly affected by MAA.

In serial mating trials in rats with MAA using comparable doses to the present study, Ratnasooriya and Sharpe[10] did not observe any significant reduction in fertility, although they observed histological depletion in selective germ cells. This may indicate species difference or perhaps the sensitivity of the IVF model used in this study[4,12,15].

Compared with the males treated with an acute dose of MAA, those given chronic doses had lower sperm fertilising capacity throughout the observation period, thus indicating that chronic doses may be more toxic than acute doses. Compared with the  controls, there was a significant decline in sperm fertility of males exposed to 8 mg/kg or 32 mg/kg of MAA, although this was not as great as in case of 64 mg/kg. At the lowest dose, the sperm fertilising ability was fully recovered by week 5, whereas for males treated with 32 mg/kg or 64 mg/kg, recovery was incomplete even at week 6. The results show that 64 mg/kg of MAA must have caused extensive damage even to the spermatid population which is evident by very low fertilisation rates.

Histological studies also revealed that chronic doses of MAA caused similar effects on the testis as the acute dose, but were effective at a lower level and caused a more persistent damage to the seminiferous tubules. Spermatocytes were the first to be affected, but with the progress of time more cell types were damaged. Beyond week 4, the fertilisation rates began to increase and this may be due to a partial recovery of the pachytene and preleptotene spermatocyte populations.

Immediately after the end of the treatment period, males given 64 mg/kg of MAA had no sperm in the cauda epididymidis. This observation indicates that spermatogonia are affected when a chronic dose is given over a long time. Histological studies carried out by Foster et al[8] revealed that repeated treatment of rats for 11 d with EGME resulted in continuous spermatocyte degeneration and depletion of spermatid population, leaving only the Sertoli cells. The present study also showed extensive damage to the germ cells, yet the effects are more marked in the spermatid population. Since no studies have been conducted previously on hamsters exposed to such a prolonged dosing, it is difficult to compare the present results with previously recorded results.

From the present study it is evident that both acute and chronic doses of MAA can impair sperm fertilising capacity and the testicular effects indicated that this decline in fertility is mainly due to depletion of pachytene spermatocytes and to alesser extent, other cell populations. The reduction in sperm fertilising ability was in correspondence with the damages observed in the testis.

Acknowledgements

The authors thank the Commonwealth Scholarship Commission,  UK for the financial support and Mr. Nick Jenkins for gavaging the hamsters.

References

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Correspondence to: Dr. L.D.C. Peiris, Dept. of Zoology, University of Colombo, Colombo 03, Sri Lanka.
Tel: +94-1-503-399    
E-mail: dinithsamay@eureka.lk  
Received 2001-08-22    Accepted 2001-09-05