Epididymis-Specific Pathologic Disorders in Rats Exposed to Gossypol from Weaning Through Puberty

Abstract

Previous work in our laboratory revealed that the pubertal period of reproductive development in the male rat was particularly vulnerable to gossypol exposure, with a higher frequency of round structures in the lumen of the cauda epididymidis in the treated rats. Herein, we utilized hemicastration and electron microscopy to confirm that the epididymis is a definitive target of gossypol. Although exposure to gossypol from weaning through puberty caused a significant decrease in daily sperm production, as well as in the concentration of sperm in the epididymis, serum testosterone levels and reproductive organ weights were not altered. In gossypol treated rats, sperm morphology was compromised severely, but the epithelium in testis and epididymis appeared morphologically normal. Ultrastructural examination revealed that round structures, present only in gossypol exposed males, represented: (1) principal cells exfoliated from the epididymal epithelium; (2) epididymal epithelial cell cytoplasm containing degenerating sperm; and (3) degenerating epithelial cells, consisting of vesicles and particles of different sizes, forms and densities. Taken together, the data confirm that gossypol targets the epididymis, disturbing both the structure and function of this organ, and presumably disrupts sperm maturation.

Introduction

Almost 40 years after the introduction of the oral birth-control pill, a male contraceptive that is effective, safe and reversible, as is the pill for women, remains to be developed (Coutinho et al., 2000). In general, men have not been taking the same responsibility as women in family planning. This fact probably can be explained by the lack of options, as few usable methods for males are available, notably the condom and the vasectomy (Mazaro et al., 2000).

In search of a male contraceptive, several plant-derived substances have been tested, including gossypol. Gossypol is a polifenolic yellow pigment, found in the cotton plant (Gossypium sp.), especially in the seeds. For a long time gossypol was just considered a toxic residue in the processing of products derived from cotton (Abou-Donia, 1976). In spite of the controversies in relation to its collateral effects and mechanism of action (Waites et al., 1998), gossypol has been considered a candidate to become the base for the production of an oral male contraceptive (Udoh et al., 1992). The use of gossypol as a contraceptive was suggested initially in the 1950s, when high rates of infertility were registered in several areas of China. Epidemiologic studies associated this fact with the use of raw oil, extracted from cottonseeds, as a new culinary method (Bender et al., 1988). After that observation several studies have been done to evaluate the male contraceptive property of gossypol. However, despite considerable research, the exact mechanism responsible for the contraceptive effect of gossypol has not been clarified sufficiently. Several studies suggest the testis as the main target organ (Qian and Wang, 1984; Arshami and Ruttle, 1989; Monsees et al., 1998), while others support the notion that gossypol acts directly on the epididymis (Srivastava et al., 1989; Swan et al., 1990), promoting alterations in the maturation of sperm, thereby rendering them unable to fertilize, whereas a third group believe that gossypol alters the function of both the testis and the epididymis (Srivastava et al., 1989). These discrepancies can be attributed, at least in part, to fundamental differences in experimental design including: the purity and dosage(s) of gossypol, the duration and route of gossypol administration, and the species of the test animal.

In a study performed previously in our laboratory to evaluate the effects of gossypol on sexual development of the male rat, the pubertal phase was revealed to be especially vulnerable, with the presence of round structures in the lumen of the cauda epididymidis in a higher frequency in the treated rats (Romualdo et al., 2002). The objective of the present work was to evaluate the origin of gossypol-associated appearance of round structures in the lumen of the epididymis, as well as changes in epididymal sperm transit/storage of the epididymis following exposure to gossypol from weaning through puberty.

Material and Methods

Animals and Gossypol Exposure

Thirty Wistar rats (21 days old, around 60 g) were supplied by Central Biotery of the State University of São Paulo. The animals were maintained under controlled temperature (21–23°C) and light (12 L:12 D), and were provided with lab chow and water ad libitum. The experimental protocol followed the ethical principles in animal research adopted by the Brazilian College of Animal Experimentation.

Male rats were weaned at 21 days of age and divided into 2 experiments, hemicastrated and intact rats, as described later. Each experiment had 2 groups: control and treated with gossypol. Treated rats received gossypol (Sigma Chemical Co., St. Louis, Mo.) diluted in sunflower oil (7.5 mg/ml), administered daily by oral intubation at a dosage of 15 mg/kg body weight. Control animals received vehicle only. Dosing began at weaning (21 days) and continued through puberty (61 days). During the experiment, body weight gain was recorded on a daily basis.

Experimental Design

Rationale

To control experimentally for a testicular origin of gossypol-associated appearance of round structures in the lumen of the epididymis at 60 days of age, rats were hemicastrated 7 days before the sacrifice, approximate time that is required for normal sperm transit from testis to distal cauda. So, in 7 days, the round structures should not be observed in regions proximal to distal cauda epididymidis, unless they originate in the epididymis.

Hemicastrated Rats

This study consisted of 14 animals (7 controls, 7 treated) that were hemicastrated while under a surgical plan of pentobarbital (Nembutal, 30 mg/kg) anesthesia. The left testis and epididymis were exposed via an abdominal incision, and while taking care to avoid trauma and bleeding, the artery coursing toward the testis was isolated. A ligature of 4-0 silk was tightly drawn around the testicular artery and efferent ducts, and when the fascia between the ligature and testis was cut, the testis was removed and the epididymis was returned to the scrotum. The abdominal musculature and skin were sutured with 3-0 chromic gut. At the end of gossypol treatment, animals were slightly anesthetized with ether and soon after were decapitated, with blood collected for the determination of testosterone levels. The right testis and epididymis, and the left epididymis were removed and weighed.

Small segments (1 mm³) of the testis and each region of the epididymis (i.e., caput, corpus, proximal cauda) were fixed in Karnovsky’s fixative (25% glutaraldehyde, 8% paraformaldehyde, 0.2 M phosphate buffer, pH 7.2) and after 24 hours were processed for light microscopy (embedding in methyl methacrylate, cuts of 3μm, hematoxilin-eosin) and electron microscopy. Seminal vesicles and ventral prostate were removed and weighed with the prostate frozen for determination of fructose concentration. The left (hemicastrated side) and right (intact side) vas deferens were weighed and used for the semen collection for sperm morphology evaluation.

Intact Rats

This study consisted of 16 animals (8 controls, 8 treated). The animals were sacrificed and blood was collected as described previously. The left testis and epididymis were removed and frozen for determination of the number of homogenization-resistant spermatids and sperm, respectively. The seminal vesicles and ventral prostate were removed and weighed with the prostate frozen for determination of fructose concentration. Small segments (1 mm³) of the right testis were fixed in Karnovsky’s fixative and processed for histology as described previously.

Hormone and Fructose Assays

Plasma was obtained by centrifugation (2500 rpm, 30 minutes, 4°C) and determination of the testosterone concentration made through double-antibody radioimmunoassay (Rosa-Silva, 1999). It is known that the functional activity of the accessory sex glands is correlated with plasma levels of androgens (Mann and Lutwak-Mann, 1981; Coffey, 1988). Fructose levels were determined in the ventral portion of the prostate as described previously (Kempinas et al., 1988) in order to have and additional parameter to evaluate the androgen status of the rats.

Electron Microscopy

Pieces (1mm³) of the testis and epididymis were immersed in Karnovsky’s fixative later were postfixed in 1% osmium tetroxide. The samples were dehydrated with acetone and embedded in araldite. Thin sections were stained with uranyl acetate and lead citrate and examined with a Phillips 300 electron microscope.

Morphometry

The mean height of principal cells in the cauda epididymidis of the hemicastrated rats was estimated using a computer image analysis system, KS-300 (Kontron Elektronik, Release 2.0, 1995, Germany). Fifty cells per animal were chosen from the proximal cauda of the intact side and measured. This region corresponds to subzone 6A as classified by Reid and Cleland (1957). In this region there are principal cells and abundant and very active clear cells.

Sperm Morphology

Sperm morphology was evaluated by sperm smears made from both vas deferens contents of the hemicastrated rats. Two hundred spermatozoa were analyzed per animal and abnormalities of head and tail were evaluated (Linder et al., 1992).

Daily Sperm Production, Epididymis Sperm Number and Transit Time

Homogenization-resistant testicular spermatids (step 19 spermatids) and sperm in both the caput/corpus epididymis and cauda epididymis were enumerated as described previously (Robb et al., 1978). Daily sperm production (DSP) was derived by dividing the total number of homogenization-resistant spermatids per testis by 6.1 days, the number of days of a seminiferous cycle in which these spermatids are present. Transit time through the caput/corpus epididymis or cauda epididymidis was calculated by dividing the number of sperm within each of these regions by the DSP.

Statistical Analysis

For comparison of the results between control and treated groups, data were analyzed by the nonparametric Mann–Whitney test. Percentages were compared using the Fisher test. Data were considered significant when p < 0.05.

Results

Body Weight and Organ Weights

Treatment with gossypol caused a decrease in body weight gain of the intact rats, starting in the middle of the treatment, i.e., 20 days after the beginning of gossypol exposure (data not shown), but not in the hemicastrated animals, when compared to the control group. Alterations were not observed in the weight of the reproductive organs (Table 1).

Testosterone and Fructose Measurements

No differences were observed in the plasma testosterone levels or in the fructose content in the prostate (Table 2).

Light Microscopic Changes

Treatment with gossypol did not perturb the histology of the testis or the epididymal epithelium. A carefully examination of the seminiferous epithelium did not reveal signals of inhibited spermiation or Stage VII spermatocyte and spermatid death in the testis, reassuring that there was no functional lowering of testosterone, as stated before. On the other hand, there was a significant increase in sperm with abnormal morphology in the vas deferens of treated animals (hemicastrated side: Control = 9%; Treated = 54%; Intact side: Control = 14%, Treated = 52%, p < 0.05), with the most frequent abnormality being the presence of isolated sperm heads.

As we observed in our original experiment, there was an increase in round structures in the lumen of the epididymis of males exposed to gossypol, but not in control males. These bodies were observed throughout the epididymis, and in the epididymides on both intact and hemicastrated sides (Figures 1 and 2).

Morphometric data showed that there was a significant increase in the height of the principal cells in region 6A of the cauda epididimydis of the hemicastrated treated rats (Control = 17.0 ± 0.16; Treated = 21.2 ± 0.18, mean ± SEM, p < 0.05). Finally, there was an apparent increase in both the size and number of clear cells in the cauda epididymidis (Figure 3).

Ultrastructural Changes

The histopathologic results of this study revealed that neither gossypol exposure nor the hemicastration perturbed the integrity of the seminiferous epithelium in the testis. However, elongate spermatids (steps 18 and 19 of spermiogenesis) were found in the lumen of the seminiferous tubules. These spermatids were found to have degenerating plasma membranes and segmentary aplasia of the mitochondrial sheet in the midpiece indicating that this particular lesion, which was also observed in sperm in the lumen of the epididymis, originated in the testis (Figure 4).

Based on ultrastructural examination and the fact that the round structures were present in the lumen of the epididymis even after 7 days of removal of the testis, they appeared to represent: (1) principal cells that were exfoliated from the epididymal epithelium; (2) epididymal epithelial cell cytoplasm containing degenerating sperm; and (3) degenerating cells consisting of vesicles and particles of different sizes, forms and densities (Figures 5 and 6).

Sperm Counts

There was a decrease in number of homogenization-resistant spermatids in the testis as well as a decrease in the number of sperm in both the caput/corpus and cauda regions of the epididymis. Thus, exposure to gossypol from weaning through puberty resulted in a decrease in daily sperm production and an apparent decrease in cauda epididymal transit time (Table 3).

Discussion

At the end of the treatment it was observed that gossypol administration provoked a significant decrease in the body weight gain of the animals of the intact group. This did not happen with the hemicastrated rats, and we do not know how to explain this fact, based on the parameters studied. Several authors reported reduction in the body weight of animals treated with gossypol in doses, treatment periods and variable ages (Hadley et al., 1981; Srivastava et al., 1989; Swan et al., 1990). On the other hand, Wang et al. (1984); Giridharam et al. (1986) and Bhiwgade and Nair (1989), did not find alterations in the body weight of rats treated with gossypol, even in doses and periods that exhibit antispermatogenic action.

The weights of the reproductive organs were not altered by gossypol treatment in the present work, indicating that gossypol did not cause systemic effects, even in the intact group, that lost body weight. Other studies report no effect of gossypol on testis weight (Wang et al., 1984; Soufir et al., 1989; Chang et al., 1980; Kaur et al., 1988) while some observed a decrease in testis weight (Srivastava et al., 1989). Changes in epididymal weight following gossypol exposure are equally conflicting, with a decrease observed in some studies (Srivastava et al., 1989; Bhiwgade and Nair, 1989; Soufir et al., 1989; Taylor et al., 1991) and no decrease observed in other studies (Swan et al., 1990; Wang et al., 1984; Chang et al., 1980; Kaur et al., 1988). Similar disagreement exists in the literature for reported effects of gossypol on the accessory sexual glands. Taylor et. al. (1991) and Chang et al. (1982) reported a decrease in the weight of the seminal vesicle of rats treated with gossypol. Singh and Rath (1990), observed the same in mice, and Chang et al. (1982) observed a decrease in the weight of the prostate in rats. On the other hand, some authors reported no alterations in the weight of these organs in treated animals (Chang et al., 1980; Kalla et al., 1982; Wang et al., 1984; Kaur et al., 1988; Swan et al., 1990). The failure of gossypol to decrease organ weights in the present study is consistent with our observation that androgen status was not compromised, as discussed as follows.

Neither testosterone levels nor fructose levels changed following gossypol administration either in hemicastrated or intact rats, indicating the normal function of the Leydig cells and prostate (Mann, 1948; Mann and Lutwak-Mann, 1981; Coffey, 1988). The effects of gossypol on the endocrine system, especially with respect to the effects on testosterone levels is controversial. Several studies using animals showed that gossypol reduced fertility without alteration in testosterone, other androgens, or luteinizing hormone (Shandilya et al., 1982; Wang et al., 1984; Soufir et al., 1989). Studies on men confirm this and show that gossypol does not decrease sexual potency or libido (Zatuchni and Osborn, 1981; Coutinho et al., 1984; Coutinho et al., 2000). However, some studies suggest otherwise, indicating that gossypol has an inhibitory effect on testosterone production by the Leydig cell via a lesion subsequent to pregnenolone formation. In these studies, the antifertility effect of gossypol appears secondary to the decrease of testosterone synthesis (Hadley et al., 1981; Saksena et al., 1981; Peyster and Srebnik, 1988; Dabrowisky et al., 2000). It is very likely that the great contradiction on the endocrinal effects of gossypol can be due to the use of different animal species, different doses and times of treatment, or on account of different administration routes. However, it is clear that infertility can be induced by gossypol without altered androgen status.

The histopathologic results of this study revealed the presence of damaged sperm in the lumen of the seminiferous tubules and epididymis duct. This is consistent with previous ultrastructural evaluations of testicular and epididymal sperm in rats (Oko and Hrudka, 1982; Hoffer et al., 1987), hamsters (Hoffer et al., 1987; Hoffer 1985), and monkeys (Shandilya et al., 1982) treated with gossypol. Aplasia within the mitochondrial sheath in the midpiece seems to be specific to gossypol and is observed initially in late spermatids, and then later as these sperm appear in the epididymis. This lesion in the sperm midpiece undoubtedly accounts for the non-motile sperm found in rodents and primates treated with gossypol (Randel et al., 1992).

Light microscope analysis revealed structural alterations in the epididymal epithelium of the treated animals. The detachment of cells of the epididymis epithelium, both in the caput and in the cauda of both intact and hemicastrated rats, is indicative of a direct action of gossypol on the epididymis as there is no evidence that epididymal epithelial participate in a process of epithelial renewal or halocrine secretion (Clermont and Flannery, 1970). As mentioned previously, this exfoliation had to occur after castration and was observed on both intact and castrate sides of the treated animals. Furthermore, round structures with different morphologic characteristics were observed in the lumen of the caput, corpus, and proximal cauda epididymis on both intact and castrated sides of the gossypol exposed animals. Based on the observed epididymal sperm transit times in this study (4.3 days for control, 3 days following gossypol treatment), one can assume that the presence of these structures in the epididymis 7 days after castration indicates that they were formed in the epididymis, not the testis. If these structures were formed in the testis, were present in the caput epididymis at the time of castration, and moved along the duct in a manner similar to that of sperm, they would only be observed beyond the proximal cauda region in the course of 7 days. Electron microscopy revealed that the round structures were of 3 types: (1) principal cells exfoliated from the epididymal epithelium; (2) epididymal epithelial cell cytoplasm containing degenerating sperm; and (3) degenerating cells consisting of vesicles and particles of different sizes, forms and density, present only in the animals treated with gossypol.

We do not know the origin of the cytoplasmatic material consisting of vesicles and particles of different sizes, forms and densities, only observed in the epididymal lumen of animals treated with gossypol. Swan et al. (1990) observed the presence of spherical masses (similar to the structures observed in the present work) bounded by membrane in the epididymal lumen of rats treated with gossypol; they suggested that these structures represented apical portions of principal cells that would detach due to the toxic effect of gossypol. While we found that the height of principal cells was increased in treated animals, it is difficult to imagine how these cells might become attenuated to the point of rupture. Alternatively, these luminal structures represent various stages degeneration in sloughed clear cells that had undergone excessive phagocytosis.

One of the most common alterations as a consequence of gossypol treatment is the presence of morphologically abnormal sperm (Srivastava et al., 1989; Swan et al, 1990; Bhiwgade and Nair, 1989; Soufir et al., 1989; Chang et al., 1980; Chang et al., 1982). In this study, the morphologic analysis of the sperm in the vas deferens revealed a significant increase in the percentage of abnormal sperm in the rats treated with gossypol, with the most common defect being the presence of isolated heads (sperm without tail). There were no differences between proportions of normal and abnormal sperm collected in the hemicastrated or the intact side, suggesting that the surgery did not influence this parameter.

The action of gossypol on the epididymis epithelium, like its effects on organ weight and testosterone production, is controversial. Srivastava et al. (1989), described normal histology and a reduction in the diameter of the duct in the cauda, accompanied by an increase in the intertubular conective tissue and, in some animals, exfoliated germ cells in the lumen. Bhiwgade and Nair (1989) did not observe alterations in the architecture of the epithelium of the epididymis. On the other hand, degenerative changes in the epididymis epithelium, with ciliary loss and cytoplasmatic vacuolization as well as a reduction in the sperm density, necrotic spermatid nuclei and sperm heads broken into fragments in the epididymidis lumen were described by Kaur et al. (1988), in rats treated with gossypol.

In the present study, the daily sperm production (DSP) was significantly reduced in the gossypol treated animals. In rats, it is known that gossypol provokes specific ultrastructural damages in the intermediary piece of spermatids, including degeneration of the mitochondrial sheet (Hoffer et al., 1983). Consequently, gossypol causes a decrease in DSP (Cerelli and Johnson, 1999). As expected, there was also a decrease in the sperm concentration in both the caput/corpus and cauda epididymidis. Besides, there was an apparent acceleration of sperm transit in the cauda epididymidis (or, in other words, decrease in sperm transit time, that means, less days were necessary for the sperm be transported through the organ). As described before, transit time through the caput/corpus or cauda epididymidis was calculated by dividing the number of sperm within each of these regions by the DSP. Most of the related literature agrees with our results, showing a decrease in the sperm density in the epididymis of gossypol treated rats (Chang et al., 1980; Kaur et al., 1988; Srivastava et al., 1989; Swan et al., 1990). However, the decrease of sperm density in the cauda epididymidis might also reflect phagocytosis of abnormal and degenerated sperm by epithelial cells in the cauda. This would account for the apparent decrease in sperm transit time. There is morphological evidence that spermiophagy occurs, under normal conditions, by cells lining the excurrent ducts (Robaire and Hermo, 1988). Indeed, our observation that gossypol treatment resulted in hypertrophy of clear cells, and perhaps an increased number of clear cells, is consistent with increased phagocytosis within the epithelium.

We conclude that treatment of rats with gossypol from weaning throughout puberty provoked alterations in the male reproductive system that were not mediated by alterations in the androgen levels. Taken together the results following hemicastration indicate that gossypol has a direct effect on the epididymis, provoking exfoliation of epididymal epithelial cells, increased phagocytosis of sperm in the cauda, and/or accelerated transit through the cauda.