Epididymal Histology Research Articles

Effect Of Obstruction To Sperm Egress On The Male Testis And Epididymis

Objective: The effects of obstructive azoospermia on the testis and epididymis have been studied in vasectomized subjects. However, effects of obstruction to sperm egress in primary obstructive azoospermia on the testes and epididymis have not been extensively reported. The present study on fifty patients of primary obstructive azoospermia focuses on the local immune response and its effect on the testis and epididymis. This was a prospective study in a tertiary hospital setting comparing epididymal histology and epididymal fluid antisperm antibody in men with primary obstructive infertility with those in men with proven fertility. Material and Methods: Fifty males with primary obstructive infertility in the study group and ten subjects with proven fertility as controls were included in the study. While performing vasoepididymostomy on such patients, testicular and epididymal tissues were taken, and epididymal fluid was aspirated for estimation of antisperm antibodies. Testicular and epididymal histologies were studied under light microscopy for evidence of cellular immune response to obstruction. Antisperm antibodies in epididymal fluid were assayed by ELISA. Result(s): No effects of obstruction were evident on spermatogenesis or the testicular histology. Epididymal epithelial flattening with local or diffuse loss of cilia (n=25); pigment in the epithelial cells as a remnant of sperm ingestion (n=20); intraductal macrophages (n=29) with sperm ingestion in 62%; breach of epithelium with sperm extravasation (n=20) were considered as evidence of effects of obstruction leading to proximal ductal dilatation and damage. Presence of plasma cells and lymphocytes (n=31) and macrophages (n=11) in the interstitium were considered effects of antigenic sperm extravasation in the interstitium. These effects could be a local cell mediated immune response. Significant ELISA for antisperm antibodies was found in 29 patients; all patients with inflammatory cell infiltrate in the interstitium had significant ELISA for antisperm antibodies in the epididymal fluid. The cellular immune response as assessed by the presence of interstitial inflammatory cells and macrophages in epididymal histology was found in 12/20 patients. Testicular biopsies in all patients were normal. Humoral immune response as assessed by significant titres of antisperm antibodies in epididymal fluid was found in 14/20 patients. Evidence of local immune response (cellular or humoral or both) was found in 17/20 patients. Conclusion(s): We believe that intermittent exposure to small amounts of extravasated sperms in the epididymal interstitium would be sufficient to produce a local cell mediated immune response with its attendant effects on the epididymis, the primary site of sperm maturation, producing irreversible changes in its structure.

Null Mutation of the Transcription Factor Inhibitor of DNA Binding 3 (Id3) Affects Spermatozoal Motility Parameters and Epididymal Gene Expression in Mice

ID3 is a transcription factor that acts as a dominant-negative regulator of other transcription factors by sequestering them, thus rendering them unable to bind DNA. We have shown previously that ID3 is expressed in a unique, region-specific manner along the epididymis, a highly specialized tissue of the male reproductive tract that functions in the transport and maturation of spermatozoa. The goal of these studies was to test the hypothesis that ID3 plays a role in the epididymis in the region-specific regulation of gene expression that is responsible for establishing the microenvironment required to carry out sperm-related functions. The consequences of ID3 deficiency on epididymal histology and gene expression profiles, as well as spermatozoal motility parameters, were determined. Although ID3 deficiency (Id3(-/-) mice) had no noticeable impact on epididymal histology, the targeted mutation adversely affected sperm motility parameters. Moreover, principal component analysis of microarray data indicated that the gene expression signatures for tissues obtained from Id3(-/-) mice and their genotypic controls were distinct from each other in each epididymal region. The predominant effect of the Id3 null mutation was in the cauda region where the expression of many transcription factors, including Hoxb8 and Bclaf1, was markedly affected. ID3 may play an important role in the molecular circuitry involved in the establishment and maintenance of the region-specific differences in gene expression that are characteristic of the epididymis.

Alterations in the Testis and Epididymis Associated With Loss of Function of the Cystatin-Related Epididymal Spermatogenic (CRES) Protein

Cystatin-related epididymal spermatogenic protein (CRES) or cystatin 8 (Cst8 gene) is a member of the cystatin superfamily of cysteine protease inhibitors. It differs from typical cystatins because it lacks consensus sites for cysteine protease inhibition and exhibits reproductive-specific expression. In the present study, we examined CRES expression within the testes, efferent ducts, and epididymides of normal mice by light microscope immunolocalization. Alterations to these tissues in male mice lacking the Cst8 gene (Cst8(-/-2)) were also characterized by histomorphometry and electron microscopy. In the normal testis, CRES was localized exclusively in mid and late elongating spermatids. In the efferent ducts, CRES was localized to the apical region of the epithelial cells suggestive of localization in the endosomes. In the initial segment of the epididymis, principal cells showed supranuclear and luminal reactions. In the cauda region, CRES was present exclusively as aggregates in the lumen and was detected in clear cells. Compared with wild-type mice (Cst8(+/+)), older (10-12 months) Cst8(-/-) mice had modest but statistically significant reductions in tubular, epithelial, and/or luminal profile areas in the testis and epididymis. By electron microscopy, some Cst8(-/-) tubules in the testis were normal in appearance, but others showed a vacuolated seminiferous epithelium, degenerating germ cells, and alterations to ectoplasmic specializations. In the epididymal lumen, abnormally shaped sperm heads and tails were noted along with immature germ cells. In addition, principal cells contained numerous large irregularly shaped lysosomes suggestive of disrupted lysosomal functions. In both the testis and epididymis, however, these abnormalities were not apparent in younger mice (4 months), only in the older (10-12 months) Cst8(-/-) mice. These findings suggest that the altered testicular and epididymal histology reflects a cumulative effect of the loss of CRES and support a role for CRES in maintaining the normal integrity and function of the testis and epididymis.

Dose-Response Effects of Epichlorohydrin on Male Reproductive Function in Rats.

Present study was conducted to investigate potential effects of epichlorohydrin on testicular and epididymal function in male rats. The test chemical was administered to adult male rats by gavage at dose levels of 0, 3.125, 12.5, and 50 mg/kg/day for 7 days. Testicular and epididymal function were assessed by measurement of reproductive organ weight, testicular spermatid count, epididymal sperm count, motility and morphology, and histopathology in rats. At 50 mg/kg, a decrease in the sperm motility and an increase in the incidence of sperm abnormalities were observed. Histopatho-logical examinations revealed an increase in the incidence of histopathological changes including cell debris in the ducts, vacuolization of the epithelial cells, oligospermia, and epithelial disruption in the proximal caput epididymidis. At 12.5 mg/kg, an increase in the incidence of histopathological changes of the epididymidis was found. There were no treatment-related effects at 3.125 mg/kg. These results show that 7-day repeated oral administration of epichlorohydrin to male rats results in adverse effects on sperm motility, sperm morphology, and epididymal histology at ≥ 12.5 mg/kg/day.

Effects of Chronic Exposure to Octylphenol on the Male Rat Reproductive System

p-tert-Octylphenol (OP) is a degradation product of alkylphenol ethoxylates. OP is an endocrine disruptor known to bind to the estrogen receptor; however, effects on males are controversial. The objective of this study was to evaluate the effects of chronic exposure to OP on male reproduction. Adult Sprague-Dawley rats were administered OP for 60 d, representing 1.5 cycles of spermatogenesis. Experimental groups included a vehicle control, and three doses of OP (25, 50, or 125 mg/kg body weight [bw]) administered daily by gavage. There was a significant decrease in body weight in the 125-mg/kg group after 60 d of treatment. Both testicular and epididymal weights and histology were not altered by treatment with OP at any of the doses administered. There were no marked differences in cauda epididymal sperm counts at any doses; however, total percent sperm motility was significantly lower in rats exposed to the intermediate dose (50 mg/kg bw). There was an increase in percent static sperm cells in all OP-treated groups, with the intermediate dose (50 mg/kg) displaying a significantly higher proportion of static cells relative to untreated controls. Caput epididymal sperm motility was unaltered by OP treatment. Gene expression profiles of testes from control and high-dose-exposed rats indicate that 14 genes were modulated by at least twofold, although these changes were not statistically significant. Taken together, results from this study indicate that OP treatment of adult rats does not appear to exert major effects on male reproductive endpoints at relevant environmental exposure doses.

Seasonal changes in testicular and epididymal histology of the tropical lizard, Tropidurus itambere (Rodrigues, 1987), during its reproductive cycle

The reproductive cycles of lizards, including Tropidurus species, have been widely studied. However, few studies describe in detail the ultrastructure and the epithelial changes in the epididymis. Using histology and trasmission electron microscopy, we show the seasonal changes in the testis and epididymis of the lizard Tropidurus itambere, during its annual reproductive cycle. The reproductive cycle of T. itambere was analysed from June 1988 to June 1989 and from June 2001 to June 2002. While the frequency of reproductive males in the population varied throughout the year, there were reproductive males in most months except for February through April. During this nonreproductive period, there is a reduction in the mean seminiferous tubule volume and few sperm were found in both the testis and the epididymis.

Effect of deprivation of endogenous follicle stimulating hormone on rat epididymis: a histological evaluation

The growth and function of the epididymis are regulated by testosterone produced by Leydig cells in the testes. In the present study it was observed that neutralization of endogenous follicle stimulating hormone (FSH) in immature rats using a highly specific antiserum to ovine FSH resulted in changes in the histology of the epididymis along with a decrease (50–60%) in its weight compared with the normal serum-treated controls. These changes were observed in both rat and monkey epididymis without any decrease in serum testosterone, on which epididymis is known to be dependent. A detailed study was therefore carried out on the effects of deprivation of FSH or testosterone on the histology of epididymis. The changes in epididymal histology following FSH deprivation included a decrease in the size of the tubule lumen in the rat as well as in the adult male bonnet monkey in which the antiserum against ovine FSH was raised. Intensive vacuolization and uneven surface of the luminal epithelium was also observed. In contrast, the effect of deprivation of testosterone support by way of administration of LH antiserum or flutamide resulted in a decrease in the size of the lumen and degenerative changes. These results suggest that cauda epididymidis is a target for FSH action.

Comprehensive assessment of the effect of Sphenocentrum jollyanum root extract on male reproductive activity in albino rats.

Aim: To evaluate the effect of methanol extract from the Sphenocentrum jollyanum root on male reproductive activity. Methods: Male albino rats were treated orally with distilled water (vehicle for the extract; control) and 50, 100 and 150 mgkg-1 body weight of Sphenocentrum jollyanum root extract for 8weeks. Each group had its own recovery. Rats were killed 24h after the last treatment. Caudal epididymal sperm count, motility, viability, morphology and organ weights were determined. Hematological indices, serum proteins, enzymes, testicular superoxide dismutase (SOD) activity, and testicular and epididymal histology were determined. Results: Compared with the control, the extract caused a dose dependent significant (P<0.05) reduction in progressive motility of spermatozoa, viability and total sperm counts. The number of abnormal spermatozoa and epididymal volume were not statistically significant. There was a significant increase (P<0.05) in serum testosterone levels in rats treated with 50 (P<0.01) and 100mgkg-1 (P<0.05) of Sphenocentrum jollyanum. There was a significant (P<0.05) increase in red blood cell count, packed cell volume and hemoglobin concentration, whereas there was no change in white blood cell count, mean total serum protein, albumin and globulin in the sera of Sphenocentrum jollyanum treated rats when compared with the control. The extract caused a significant decrease (P<0.05) in serum aspartate and alanine aminotransferase activities with a significant increase (P<0.05) in testicular SOD activity at a dose of 50mgkg-1 bodyweight. Testicular cytoarchitecture of the extract treated rats showed degeneration of seminiferous tubules, whereas regeneration of germinal epithelium and restructuring of the germinal interstitium occurred in the recovery rats. No lesions were observed in the epididymis of the rats. Conclusion: The results suggest that methanol extract of the Sphenocentrum jollyanum root could produce harmful effects on reproductive functions in male albino rats which can be attributed to poor sperm quantity (epididymal sperm count), quality (sperm motility, viability and morphology) and testicular degeneration. The steroidogenic potential of the plant could explain its use as an aphrodisiac agent. (Reprod Med Biol 2006; 5: 283-292).

Testosterone Replacement Therapy Induces Spermatogenesis and Partially Restores Fertility in Luteinizing Hormone Receptor Knockout Mice

Testosterone (T) is essential for spermatogenesis, fertility, and maintenance of the male phenotype. We analyzed in hypogonadal LH receptor knockout (LuRKO) male mice whether T treatment can restore their phenotype, spermatogenesis, and fertility. In LuRKO mice, spermatogenesis is arrested at round spermatids, adult-type Leydig cells are absent, T production is dramatically decreased, the animals are cryptorchid, and their accessory sex organs are atrophic. T replacement therapy from 21 d of life for 60 or 120 d in LuRKO mice induced a male phenotype macroscopically indistinguishable from that of wild-type littermates as well as full spermatogenesis and testicular descent. Thus, the absence of LH-dependent prepubertal androgen priming is not necessary for subsequent maturation of the male phenotype. Conspicuously, some abnormalities remained in epididymal histology after T treatment despite normal expression of several epididymis-specific genes in caput epididymis. The mice displayed normal mating behavior, although at lower frequency than wild-type controls. The spermatozoa were able to fertilize oocytes, but their impaired passage from epididymis to uterus was apparent. The mice remained subfertile, because only 9% of all breedings resulted in pregnancy, and only two of 13 mice (15%) were fertile. Moreover, inflammation in epididymides and prostate was found in many T-treated LuRKO mice, which probably impaired sperm transport and contributed to their high rate of subfertility. In conclusion, T replacement initiated prepubertally only partially restores the fertility of LuRKO mice, even though most features of the male phenotype recover. Full fertility may require higher and/or earlier postnatal T exposure or production of other Leydig cell factors lacking in this model.

The use of URYX for reversible vasectomy in a rabbit model.

URYX is a biocompatible polymer of ethylene vinyl alcohol dissolved in a dimethyl sulfoxide (DMSO) carrier to allow injection of a very low-viscosity fluid into tissue. Once the material comes into contact with body tissue or fluid, the DMSO rapidly dissipates from the polymer, which results in a precipitate of a coherent solid mass. The purpose of the present study was to determine whether URYX can effectively occlude the vas deferens and whether patency can be restored by redissolving the URYX in vivo using the solvent DMSO. Eight male New Zealand White rabbits (age range, 25-41 weeks; mean age, 33.9 +/- 7.5 weeks; mean weight, 4.0 +/- 0.2 kg) were used in 2 experiments (E1 and E2). In E1, 3 rabbits underwent unilateral vasectomy, and the contralateral vas was injected with either 0.05 or 0.10 mL of URYX, to determine the amount of URYX required to cause obstruction. Two animals underwent bilateral vasectomy, to serve as controls. In E2, 3 animals underwent bilateral URYX injection and were compared with the bilateral vasectomy control rabbits used in E1. After 1 month of initial bilateral URYX treatment, all animals in E2 underwent attempted unilateral reversal with 1.5 mL of DMSO injected into 1 occluded vas deferens. Two end points were evaluated-a clinical end point assessed by semen analyses and a pathological end point assessed by histological analysis of treated tissues, to assess for safety. A 1.5-cm infrapubic incision was made to expose both vasa in anesthetized rabbits. The vasal injection of URYX was performed with a 30-gauge needle. Vasectomy was performed by excision of a 1-cm segment of the vas deferens and subsequent ligation with a 6-0 prolene suture. Semen was collected using an artificial vagina 2-3 times/wk before and 1 month later, after injection treatments and vasectomy. Manual sperm counts were performed. All animals were sacrificed, and tissues (distal vas, injection site, proximal vas, cauda epididymis, caput epididymis, and testis) were harvested and examined for the presence of URYX. The inflammatory response of the wall and adventitia of the vas deferens was given a score (0-15) based on the sum of grades (0 = none, 1 = mild, 2 = moderate, and 3 = severe) for the following categories: foreign body giant cell reaction, granulation tissue, lymphocytes, eosinophils, and scarring, as evaluated by a single pathologist (J.M.). Vasal injection with 0.05 mL of URYX was not sufficient to cause occlusion. Both animals injected with 0.1 mL of URYX were effectively occluded. The injection of occluded vasa with DMSO did not dissolve the URYX plug in the vas lumen. There was no significant difference in vasal inflammatory response scores between vasal units treated with URYX only and vasal units in the vasectomy model. Vasal units subjected to URYX followed by DMSO demonstrated greater inflammatory response scores than vasal units treated with URYX followed by normal saline, URYX alone, or vasectomy. Epididymal and testicular histology remained unaffected in all vasal units in E1. The vasal units in E2 subjected to URYX followed by normal saline showed no histological abnormalities of the epididymis and testis. However, those vasal units subjected to URYX followed by DMSO in E2 showed evidence of adhesions, necrosis, and degenerating cells in the epididymis and a focal foreign body giant cell reaction in the testis. The bilateral vasal injection of URYX can result in azoospermia in the rabbit model. Reversal with subsequent DMSO injection was not achieved. A minimal inflammatory response of the vas deferens was observed with URYX injection alone; however, DMSO following URYX injection resulted in increased vasal inflammation, in addition to epididymal and testicular changes.

126.A repository of ENU mutant mouse lines and their potential for male fertility research

One in 25 western men are infertile and the causal factor is frequently unknown, although it is expected that many are genetic in origin. My project aims to identify genes critical to mouse spermatogenesis using ENU mutagenesis. A further aim was to develop a repository of mutant mice and data on their fertility parameters for use by the reproductive biology community. This research will aid the diagnosis and development of specific treatments for human infertility and the development of contraceptive agents. The potent mutagen N-ethyl-N-nitrosourea (ENU) was utilized to generate libraries of C57BL/6 mice with random point mutations throughout their genomes. A 3 generational breeding program produced mice that were homozygous for a number of mutations. I subsequently performed a number of large scale screens on 3rd generation males, identifying lines carrying recessive mutations specifically affecting male fertility. Thus far we have observed a wide range of abnormal testis phenotypes including Sertoli Cell only, hypospermatogenesis, meiosis arrest, abnormal sperm morphology and abnormal hormone levels. From these analyses a repository including all data and tissues collected from 1200 3rd generation male mice from 122 different lines has been developed and will become publicly available. This includes testis and epididymal histology and serum levels of FSH, LH, activin A and inhibin. Further, I have stored gDNA long term and cryopreserved sperm to enable regeneration of lines in the future. In addition, I have developed a high throughput mutation screening protocol for the detection of mutations within genes of interest using denaturing high performance liquid chromatography (DHPLC). Collectively, our repository and gene screening techniques can be used in conjunction with artificial reproductive technologies to generate mouse models reflective of human conditions and altered specific gene function.

Alpha-Chlorohydrin inhibits glyceraldehyde-3-phosphate dehydrogenase in multiple organs as well as in sperm.

Numerous studies have documented inhibitory effects of alpha-chlorohydrin (ACH) on glyceraldehyde-3-phosphate dehydrogenase (G3PDH) activity in spermatozoa. A sperm-specific G3PDH isoform has been described. The possibility that ACH may inhibit G3PDH in cell types other than sperm was investigated in this work. In addition, the onset of ACH-induced epididymal toxicity was described. Changes to epididymal histology occurred 6 h following a single dose of ACH (50 mg/kg po) and were confined to the proximal initial segment. By 24 h, no epithelial cells lined the basement membrane of that region. Three h after ACH administration (50 mg/kg po), G3PDH activity was significantly decreased in sperm (85%) as well as in kidney (31%), liver (49%), and epididymis (35%). Enzyme activity remained inhibited at 6 and 24 h. G3PDH was immunolocalized in the epididymis and staining was highest in the efferent ducts and initial segment as well as in smooth muscle. Since G3PDH is a microtubule-associated protein and microtubule-dependent endocytosis occurs in the epididymis, beta-tubulin was also immunolocalized. beta-tubulin densely stained the apical region of initial segment and caput epithelial cells. Disruption of beta-tubulin immunostaining correlated with the localization and onset of the lesion. Co-localization of G3PDH and beta-tubulin immunostaining was not observed although both antibodies most densely stained the initial segment. Our data indicate that histologic changes to the proximal initial segment of the epididymis occur rapidly, but subsequent to G3PDH inhibition. Moreover, ACH inhibition of G3PDH is not confined to sperm, although the sperm enzyme is most sensitive to inhibition.

4(R)-5, 10, Seco 19-Norpregna 4,5 Diene 3, 10, 20 Trione (Seco), an Inhibitor of Epididymal 5α-Reductase Arrests Sperm Forward Motility in Wistar Rats

The effect of 4(R) - 5, 10, Seco 19 - norpregna 4,5 diene 3,10, 20 trione (SECO), a potent inhibitor of 5α-reductase, an intracellular enzyme that converts testosterone to dihydrotestosterone (DHT), on epididymal sperm count and motility was studied in Wistar rats. Administration of (i.m.) 250, 500, 750,1000 μg SECO/Kg b.wt/day to mature rats for 45 days impaired sperm forward motility in a dose dependent manner. Epididymal weight and sperm number decreased in rats treated with 750 and 1000 μg SECO, whereas weight of other accessory organs decreased in rats treated with 1000 μg SECO only. The epididymal histology showed degraded sperm and increased number of clear cells indicating high endocytotic activity, to clear dead sperm, whereas no obvious change was observed in the testicular histology. SECO treatment increased testosterone concentration whereas, it decreased estradiol concentration in circulation and in the epididymal tissue. SECO treatment specifically decreased 5α-reductase in epididymis without affecting the same in the liver, indicating its targeted effect on the epididymis. Thus, the preliminary in vivo study points out that SECO impairs the acquisition of sperm forward motility.

Evaluation of apomorphine HCl effects on reproductive endpoints: studies in male rats and dogs.

Apomorphine HCl is currently in phase III clinical trials for the treatment of erectile dysfunction. The potential for reproductive toxicity in males was assessed based on a 13-week rat study--a fertility study in male rats--and a 6-month study in dogs. The subcutaneous (s.c.) route was selected to simulate the sublingual route in humans. Dosages of apomorphine were 0.0 (vehicle), 0.8, 2, and 8 mg/kg/day in the 13-week study in rats (20/group), with 8 mg/kg/day used for only 9 weeks. In the fertility study, 24 males/group were cohabited with females, and doses were 0.0, 0.2, 0.8, and 2 mg/kg/day. Males were treated for 4 weeks prior to cohabitation and for 5 weeks throughout cohabitation. Organ weights, including testis and left epididymis, sperm count and morphology in the epididymis, and sperm motility in the vas deferens were evaluated. Male fertility index, and in females, the numbers of fetuses, implantation sites, and corpora lutea were counted. Male dogs (five/group) were dosed with 0.04, 0.1, or 0.4 mg/kg/day for 6 months. Epididymal and prostate weight, and testicular and epididymal histology were evaluated. Daily morbidity/mortality, weekly clinical observations, body weight, and food consumption were evaluated in all studies. No adverse effect was observed in any of the reproductive parameters in the studies. The NOEL for reproductive toxicity was approximately equal to 0.4 mg/kg/day in dogs and > or = 2 mg/kg/day in rats. These doses in rats and dogs correlated with plasma levels of approximately 240 and 130 ng/mL, and AUCs of 200 and 100 ng.h/mL, respectively. These levels suggest a safety margin for the evaluated male reproductive endpoints of at least 104 times based on the Cmax, and 44 times based on AUC of the clinical dose.

Histologic variations in the epididymis: findings in 167 orchiectomy specimens.

Nonpathologic morphologic variations in the epididymal histology in 167 orchiectomy specimens were analyzed to assess and document the nature, frequency, and possible relation to patient age and underlying testicular pathology. Variations in histology included intranuclear eosinophilic inclusions, lipofuscin pigment, cribriform hyperplasia, Paneth cell-like metaplasia, and nuclear atypia. Intranuclear eosinophilic inclusions were observed in 72.5% of patients, and they appeared to occur at an older age than cribriform hyperplasia and Paneth cell-like metaplasia. Lipofuscin pigment was found in 32.9% of patients; this change was observed predominantly in ductuli efferentes and was more commonly associated with obstructive changes. Cribriform hyperplasia was seen in 41.9% of patients, and it occurred in 1 normal testis and in 33 testes with diverse pathologic alterations. Paneth cell-like metaplasia characterized by bright eosinophilic intracytoplasmic hyaline-like granules and globules, was present in 8.3% of patients and was accompanied by changes of obstruction in almost all instances. The globules were strongly periodic acid-Schiff positive, both before and after diastase digestion, and were negative for chromogranin A, KP-1, and MAC387 immunostains. Nuclear atypia, similar to that seen in seminal vesicles, was focally present in 13.8% of patients and tended to occur at an older age. The authors conclude that variations in epididymal morphology are fairly common and, therefore, surgical pathologists should be aware of these changes. Although exuberant in some patients, in no cases did these variations cause serious diagnostic problems.

Segment-specific morphological changes in aging Brown Norway rat epididymis.

In aging Brown Norway rats, both spermatogenesis and steroidogenesis decrease. Little is known about changes in the epididymis during aging. However, since the two major components entering the epididymis from the testis change, we hypothesized that epididymal histology would be affected by advancing age. The epididymides of Brown Norway rats ranging in age from 3 to 24 mo were prepared for light and electron microscopy. Striking quantitative and qualitative changes were noted. There was an age-dependent increase in the thickness of the basal membrane and in the number of halo cells. There were also major segment-specific changes in the appearance of cells along the epididymis with age. At 12 mo, basal cells in the initial segment emitted pseudopods into the basement membrane. By 18 mo, in the caput epididymidis, clear cells were filled with lysosomes; these cells frequently showed bulging protrusions into the lumen. In the corpus epididymidis, the cytoplasm of principal cells had numerous large lysosomes both below and above the nucleus; apical cells were usually occupied by one giant membranous lysosome. In the proximal cauda, clear cells became filled with dense lysosomes, and principal cells presented large clear vacuoles; debris from spermatozoa was found in the larger vacuoles. In summary, aging of the epididymis was accompanied by the emergence of characteristic features of aging and activation of the immune system. Furthermore, there were many cell- and segment-specific changes. Finally, these changes were not related to the presence of spermatozoa, often preceding their disappearance, thus indicating that there may be an intrinsic mechanism of aging in epididymal epithelial cells.

Changes in luminal fluid protein composition in the rat cauda epididymidis following partial sympathetic denervation.

Sympathetic denervation of the rat cauda epididymidis by surgical removal of the inferior mesenteric ganglion (IMG) results in an excessive accumulation of sperm in the cauda epididymidis as well as significant changes in cauda sperm motility and cauda epididymal gross histology. The objective of the present study was to determine if the cauda-specific changes in sperm storage, sperm motility, and epididymal histology following the loss of sympathetic innervation were accompanied by changes in the protein composition of epididymal fluid. One and 4 weeks after surgical IMG removal or sham operations, luminal fluid obtained from the caput and cauda epididymidis and cauda epididymal sperm-associated proteins were subjected to two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and silver-stained proteins were quantitated. One week after IMG removal, two cauda epididymal fluid (CEF) proteins (2 and 13) had increased 43% and 49%, respectively, whereas four CEF proteins (5, 8, 9, and 19) had decreased between 30% and 73% compared to controls. Four weeks after IMG removal, changes in CEF proteins observed 1 week following surgery were no longer present, but the staining intensities of three additional CEF proteins (11, 12, and 18) were reduced an average of 70% compared to control CEF proteins. By obstructing the cauda epididymidis, we confirmed that the changes in CEF protein composition observed following IMG removal were not the result of sperm accumulation but were due directly to the loss of innervation; the staining intensity of CEF protein 2 increased as a result of excessive sperm accumulation in the cauda epididymidis both in the presence and absence of innervation from the IMG. No significant changes in caput epididymal fluid proteins or cauda epididymal sperm-associated proteins were detected following IMG removal. These data show that the protein composition of rat CEF is significantly affected by the loss of sympathetic innervation and suggest that neuronal input may play an important role in the maintenance of epididymal function.

Preliminary screening for the potential of drinking water disinfection byproducts to alter male reproduction

There is increasing epidemiologic interest in the role drinking water disinfection byproducts (DBPs) may play in adverse reproductive outcomes such as inability to conceive, spontaneous abortion, and low birth weight. Although dozens of DBPs already have been identified, only a few studies have attempted to determine whether DBPs alter male reproductive parameters such as testicular and epididymal histology, testicular and epididymal sperm numbers, and epididymal sperm morphology and motility in laboratory animals. In these studies, alterations in epididymal sperm motility seemed to be predictive of more generalized toxicity of the male reproductive system. Because there is a need to prioritize DBPs for thorough reproductive and developmental toxicity testing, preliminary screening for the potential of DBPs to alter reproductive function seems warranted. Here, we elected to examine only cauda epididymal sperm motion parameters and testicular and epididymal histopathology. The effects of exposure to two commonly occurring DBPs, bromodichloromethane (BDCM) and chloral hydrate (CH), via drinking water were evaluated in F344 rats at an interim (52 week) necropsy during cancer bioassay studies. Exposure to 22 and 39 mg/kg BDCM and 55 and 188 mg/kg CH did not produce any systemic toxicity. Histopathologic evaluation revealed no gross lesions in the reproductive organs, and no tumors were detected in any tissues. In contrast, exposure to 39 mg/kg BDCM significantly decreased the mean straight-line, average path, and curvilinear velocities of sperm recovered from the cauda epididymidis. This BDCM exposure shifted the average path velocity distribution to a lower modal velocity range. Exposure to 188 mg/kg CH significantly decreased both the percentage of motile and progressively motile sperm. This CH exposure shifted the straight-line velocity distribution to a lower modal velocity range. These are the first reproductive toxicity data from exposure to BDCM and CH. The observed effects on sperm motion occurred in the absence of carcinogenesis. Because the effects of BDCM on sperm motility occurred at a lower exposure than that of other DBPs that compromise sperm motility, a thorough reproductive evaluation now is underway.

Assessment of the Reproductive and Developmental Toxicity of Pesticide/Fertilizer Mixtures Based on Confirmed Pesticide Contamination in California and Iowa Groundwater

Assessment of the Reproductive and Developmental Toxicity of Pesticide/Fertilizer Mixtures Based on Confirmed Pesticide Contamination in California and Iowa Groundwater. Heindel, J. J., Chapin, R. E., Gulati, D. K., George, J. D., Price, C. J., Marr, M. C., Myers, C. B., Barnes, L. H., Fail, P. A., Grizzle, T. B., Schwetz, B. A., and Yang, R. S. H. (1994). Fundam. Appl. Toxicol. 22, 605-621.Pesticides and fertilizers, as used in modern agriculture, contribute to the overall low-level contamination of groundwater sources. In order to determine the potential of pesticide and fertilizer mixtures to produce reproductive or developmental toxicity at concentrations up to 100X the median level found in groundwater, we prepared and studied two mixtures of pesticides and a fertilizer (ammonium nitrate). One mixture containing aldicarb, atrazine, dibromochloropropane, 1,2-dichloropropane, ethylene dibromide, and simazine plus ammonium nitrate was considered to be a representative of groundwater contamination in California (CAL). The other, containing alachlor, atrazine, cyanazine, metolachlor, metribuzin, and ammonium nitrate, simulated groundwater contamination in Iowa (IOWA). Each mixture was administered in the drinking water of either Swiss CD-1 mice during a Reproductive Assessment by Continuous Breeding study or pregnant Sprague-Dawley rats (gd 6-20) at three dose levels (1×, 10×, and 100×) where 1× was the median concentration of each pesticide component as determined in the groundwater surveys in California or Iowa. Unlike conventional toxicology studies, the purpose of this study was to evaluate the health effects of realistic human concentrations. Thus, the testing concentrations are probably well below the maximally tolerated dose. Propylene glycol was used as the solubilizer for the pesticides in drinking water formulations in both studies. In the reproductive study, neither mixture caused any clinical signs of toxicity, changes in food or water consumption, or body weight in either F0 or F1 mice at doses up to 100× the median groundwater concentrations. There were no treatment-related effects on fertility or any measures of reproductive performance of either the F0 or the F1 generation mice exposed to either CAL or IOWA at up to 100×. Similarly, measures of spermatogenesis, epididymal sperm concentration, percentage motile sperm, percentage abnormal sperm, and testicular and epididymal histology were normal. In the developmental study, CAL- or IOWA-exposed females did not exhibit any significant treatment-related clinical signs of toxicity. No adverse effects of CAL or IOWA were observed for measures of embryo/fetal toxicity, including resorptions per litter, live litter size, or fetal body weight. CAL or IOWA did not cause an increased incidence of fetal malformations or variations. In summary, administration of these pesticide/fertilizer mixtures at levels up to 100-fold greater than the median concentrations in groundwater supplies in California or Iowa did not cause any detectable reproductive (mice), general, or developmental toxicity (rats).

Diet-induced changes in the male reproductive system of rats.

Either quantitative or qualitative modification of diet interferes markedly with the reproductive functions of the rat. The effect of chick mash or cowpeas (Vigna unquiculata) on body growth, accessory sex organ weights, serum testosterone, and epididymal histology was studied. Body growth was markedly decreased in cowpeas fed group. Binucleated, multinucleated giant cells (MGCs), and lymphocytes were found in large numbers in the lumen of caput and cauda epididymis of the cowpea-fed group. No noticeable effect was seen in chick mash-fed group. Serum testosterone levels were significantly decreased in cowpea-fed group. Presence of MGCs in cowpea-fed animals indicates epididymal dysfunction and probably renders them infertile.