Effects of testosterone enanthate on the structure of the male reproductive tract of the rat.
код для вставкиСкачатьEffects of Testosterone Enanthate on the Structure of the Male Reproductive Tract of the Rat CHARLES J. FLICKINGER Department ofAnatomy, University of Virginia School of Medicine, Charlottesville, Virginia 22908 ABSTRACT The histology and fine structure of the testis, epididymis and sex accessory glands were studied in young adult male rats administered testosterone enanthate, 120 pg/lOO g body weight, three times weekly for 4, 8, or 12 weeks. The weights of the testis and epididymis decreased, and animals treated for 11 weeks were infertile. Alterations were found in the seminiferous tubules of all rats treated for 8 or 12 weeks, including the presence of many degenerating germ cells and a large decrease or absence of late spermatids. Study of different stages of the cycle of the seminiferous epithelium showed that the greatest number of degenerating germ cells, step 7 spermatids and pachytene primary spermatocytes, occurred a t stages VII-VIII of the cycle. Some normal appearing spermatogonia, primary spermatocytes and early spermatids remained in most seminiferous tubules. Sertoli cells contained many lipid droplets and lysosome-like bodies, and degenerating cells were surrounded by Sertoli cell cytoplasm. The Leydig cells of treated animals were greatly reduced in size. Sperm progressively disappeared from the lumen of the middle segment and proximal part of the terminal segment of the epididymis after treatment for 8 or 1 2 weeks. Changes in the middle segment also included the appearance of intraepithelial cavities containing debris, and the presence within the epithelium of phagocytic cells that resembled leukocytes. The lumen of the proximal part of the terminal segment was often collapsed, while in the distal part of the terminal segment, the lumen was filled with cellular debris and degenerating sperm. Organelles of the principal cells of the epididymal epithelium appeared to be qualitatively unaltered. The weight of the sex accessory glands remained close t o normal, and the presence of normal ultrastructural features suggested that production of secretions continued. Testosterone is among the hormones and other compounds considered a s possible chemical contraceptives because i t is capable of suppressing spermatogenesis in men (Hotchkiss, '44; Heller et al., '50, '70; Reddy and Rao, '72; Steinberger and Smith, '77). The basis of its antifertility action is a biphasic action on the seminiferous tubules of several mammalian species (Albert, '61; Ludwig, '50; Steinberger, '71; Desjardins et al., '73; Walsh and Swerdloff, '73; Ewing et al., '73a; Berndtson et al., '74). In relatively low doses i t suppresses spermatogenesis, apparently by decreasing the secretion of gonadotropins by the pituitary gland (Ludwig, '50; Desjardins et al., '73; Ewing et al., '73a, '73b; Heller et al., '70; Berndtson et al., '741, thus greatly lowerANAT. REC. (1978)192: 555-584. ing the secretion of endogenous testosterone by the Leydig cells of the testis. At relatively high dose levels, however, the exogenous testosterone is itself sufficient for spermatogenesis to continue even though pituitary gonadotropin secretion is depressed (Nelson and Merckel, '37; Ludwig, '50; Clermont and Harvey, '65; Walsh and Swerdloff, '73; Desjardins et al., '73; Berndtson et al., '74). The reason that low doses of testosterone, adequate t o maintain the sex accessory glands and libido, are insufficient to maintain spermatogenesis probably lies in the anatomical location of the seminiferous tubules close t o the source of endogenous testosterone in the Received Dec. 7, '77. Accepted July 11, '78. 555 556 CHARLES J . FLICKINGER Leydig cells. Thus t h e seminiferous epithelium normally is exposed t o a much greater concentration of testosterone than t h e rest of the body, and this is not replaced by administration of amounts of hormone sufficient for other androgen-dependent tissues (Heller e t al., '70; Walsh and Swerdloff, '73). Therefore, testosterone in proper amount potentially can result in suppression of spermatogenesis with maintenance of other male characteristics. Various testosterone derivatives have been considered for use in suppressing sperm production, and t h e testosterone ester, testosterone enanthate, has attracted attention because i t has a relatively long action and needs to be administered less frequently than many other androgens such as testosterone or testosterone propionate (Murad and Gilman, '75; Steinberger and Smith, '77). In t h e present study, the histology and fine structure of t h e male reproductive tract of rats administered testosterone enanthate were studied. An objective was t o extend morphological observations on t h e testis t o determine in detail t h e cell types and stages of t h e cycle of t h e seminiferous epithelium t h a t a r e affected, as well as to define ultrastructural aspects of t h e alterations. It is also important to consider t h e sex accessory organs in studies of antifertility agents. First, under some conditions sperm production may be decreased but not abolished, and t h e sex accessory organs, especially t h e epididymis, may affect t h e fate of t h e sperm t h a t continue t o be produced and t h u s contribute to t h e antifertility action of t h e drug. Second, t h e maintenance of t h e sex accessory glands, secondary sex characteristics, and libido a r e important objectives in t h e eventual development of practical chemical contraceptives for men. Therefore, further objectives were t o determine if there are changes in several segments of t h e epididymis and to investigate whether t h e structure of t h e sex accessory glands is maintained or altered. MATERIALS A N D METHODS Young adult male Sprague-Dawley rats weighing -200 g were obtained from Hilltop Lab Animals, Scottdale, Pennsylvania. They were maintained in t h e University Vivarium in a n air conditioned room and were furnished food and water ad libitum. Experimental animals were administered testosterone enanthate (Delatestryl, Squibb) 120 pg/lOO g body weight in 0.1-0.3 ml sesame oil three times weekly by subcutaneous injection. Control rats were administered t h e oil vehicle alone. Three treated and two control animals were studied after treatment for 4, 8, or 12 weeks. Three rats were permitted to recover for 8 weeks following treatment for 12 weeks. Tissue was also obtained from several normal adult male rats for comparison with treated and control animals. Sprague-Dawley rats were used so t h a t t h e results could be compared readily with our previous studies of other antifertility agents (Flickinger and Loving, '76; Loving and Flickinger, '76; Flickinger, '77a,b,c) which were carried out on this strain. The rats were killed by cervical dislocation, and t h e testes, epididymides, ventral prostate a n d seminal vesicles were removed a n d weighed. Whole testes were immersed for one hour in a glutaraldehyde, formaldehyde, and picric acid fixative (It0 and Karnovsky, '681, prepared by mixing 8.5 ml of Karnovsky's fixative (Karnovsky, '65) with 1.5 ml of a saturated solution of picric acid and 10 ml of 0.1 M cacodylate buffer, pH 7.3. After one hour, pieces were cut from t h e testes, diced, and fixed for a n additional one hour. Portions of t h e initial, middle, and terminal segments of t h e epididymis (Glover and Nicander, '71) were dissected. The terminal segment was sub-divided into two parts, one corresponding to t h e proximal part of t h e cauda which has t h e smaller diameter (regions 5B and 6A of Reid and Cleland, '57) and t h e other comprising t h e distal part of t h e cauda (region 6B of Reid and Cleland). The epididymal segments were immersed in Karnovsky's fixative (Karnovsky, '651, and pieces of t h e ventral prostate were placed in t h e glutaraldehyde, formaldehyde, and picric acid fixative. After one hour t h e samples of epididymis and prostate were diced and fixed for a n additional one hour. The seminal vesicles were diced and placed for two hours in Karnovsky's fixative. After fixation for a total of two hours in aldehyde, all t h e tissue blocks were rinsed with 0.1 M cacodylate buffer and post-fixed for one hour in 1%OsO, in 0.1 M cacodylate buffer at pH 7.3. The tissue was dehydrated in a graded series of ethanols followed by propylene oxide, and was embedded in Araldite. Sections 1 p m thick for light microscopy were cut with glass knives, mounted on slides, and stained with 0.5% toluidine blue in 0.5% sodium borate. Light micrographs were obtained with a n Olympus photomicroscope. 557 EFFECTS OF TESTOSTERONE Thin sections showing silver t o pale gold interference colors were cut with a diamond knife, mounted on uncoated grids, and stained with lead citrate (Reynolds, '63). The preparations were examined and photographed using a Philips EM-300 or an AEI EM-801 electron microscope. To supplement visual impressions of changes in the size of Leydig cells of the testis, the following semiquantitative method was employed t o estimate the sizes of profiles of Leydig cells in sections for light microscopy (Flickinger and Loving, '76; Flickinger, '77a). Regions of accumulation of interstitial tissue between three or more seminiferous tubules were chosen a t random for study. Within such a region, the length and width of all the cells identifiable as Leydig cells were measured using a Zeiss optical micrometer. Although the shape of the cells varied, a rough estimate of the area of a cell represented within a section was obtained by assuming the profiles t o be elliptical and multiplying the length/2 x width12 x T.Twenty-five cells were measured for each of the two rats treated for 12 weeks and for two control animals. The mean areas of Leydig cells in sections of treated and control rats were compared using a T test. The fertility of three animals treated for 11 weeks with testosterone enanthate and two accompanying control rats was tested by caging each male rat for five days with two female rats. During this time, the females were checked daily for a vaginal plug, and one week later they were examined for the presence of fetuses. RESULTS Testis The microscopic structure of the testis of control rats did not differ from normal. Semi- niferous tubules contained Sertoli cells and germ cells in various stages of development (fig. 1).Degenerating germ cells were infrequently encountered in normal or control rats. As reported by Russell and Clermont ('77), those present were usually in stages VII-VIII or I of the cycle, and generally no more than one was observed in a cross section of a seminiferous tubule. The weight of the testis declined to less than half of control values after treatment with testosterone enanthate for 8 or 12 weeks (table 1).Microscopic alterations were slight or were undetected in the testes of rats treated for 4 weeks. All rats showed microscopic changes after 8 or 12 weeks, although the severity of the testicular alterations varied somewhat from one animal to another. Testicular alterations visible in the light microscope (figs. 2, 3) included the presence of many degenerating germ cells, a large decrease in number or absence of late spermatids, especially those of the maturation phase, and a large decrease in the size of the Leydig cells of the interstitial tissue. Degenerating cells Degenerating cells were identified in light microscopic sections (figs. 2, 3) by their intense staining with toluidine blue, granular appearance, and in some instances, separation from surrounding cells by a clear space. As many as 10 t o 15 degenerating cells were present in some profiles of the seminiferous tubules. Some of the degenerating cells were identified as early spermatids because of their round shape, location among other early spermatids, and a remnant of the acrosomal cap. Others appeared to be primary spermatocytes because of their large size, round shape, and location in the basal part of the seminiferous TABLE 1 The weights of reproductive organs and the final body weights of rats treated with testosterone enanthate. The average weights are shown in g/IOOg body weight and in percent of the corresponding control. The values for the testis, epididymis and seminal vesicle include the two organs from each of three treated animals and two control rats at each interval Time weeks Treated Control 8 Treated Control 12 Treated Control 4 Testis Epididymis Seminal vesicle gI100 g % control gI100 g 80 0.09 75 0.05 37 0.12 0.08 0.13 62 47 0.07 58 g/100 g % control 0.36 0.45 0.16 0.43 0.16 0.34 0.12 Y, g/lOO g %, control 71 0.11 0.10 0.11 0.11 0.17 0.13 110 0.07 0.07 0.06 0.08 0.10 Ventral prostate control 117 80 100 131 Body weight g 427 423 48 1 509 538 564 558 CHARLES J . FLICKINGER TABLE 2 epithelium alongside normal-appearing primary spermatocytes. Degenerating germ cells A comparison o f the number of degenerating cells at were much more numerous in some profiles of different stages of the cycle o f t h e seminiferous epithelium in a rat treated with testosterone seminiferous tubules than others, so an a t enanthate f o r 12 weeks tempt was made to relate the presence of degenerating cells to stages of the cycle of the Degenerating Degenerating cells per Number of seminiferous epithelium. Stage cells per cross 100 Sertoli Sertoli Various combinations of cells in sections of section nuclei nuclei the seminiferous tubules of treated animals A 1 4.1 345 made it evident that different stages of the cyB 1.2 6.2 291 cle of the seminiferous epithelium were repreC 8.3 38.8 299 sented. Due to the scarcity of late spermatids D (total) 3.5 16.2 214 D, 7.5 32.6 and their absence from many profiles of D2 1.1 4.4 tubules we were not able precisely t o identify all of the stages of the cycle. However, in a study on the development of the seminiferous epithelium in immature rats, Clermont and tially underwent degeneration in rats treated Perey ('57) described a way to divide the cycle with testosterone enanthate. At the electron microscope level, cells were of the seminiferous epithelium into four broad stages based on the appearance of sper- judged to be degenerating if they had an matogonia and spermatocytes. In this scheme unusually dense cytoplasm, distorted and disthe four broad stages, here designated stages rupted membranous organelles, and an abnorA through D, correspond t o the conventional mal degree of clumping and high density of 14 stages (Leblond and Clermont, '52) as fol- nuclear chromatin. Many of the degenerating lows: Stage A = stages I - IV; Stage B = cells were identified as spermatids of approxstages IV - VI; Stage C = stages VII - VIII; imately stages 6-8 because of the persistence of the characteristically shaped acrosome (fig. Stage D = stages IX - XIV. Degenerating cells appeared particularly 4).Other degenerating cells, which lay deep in numerous in Stage C (VII - VIII) with some the seminiferous epithelium close to the basal also present in Stages B and D. To confirm this lamina, were more difficult to identify on the impression, sections from one animal treated basis of their ultrastructure, but probably corfor 12 weeks were used for a further semi- responded to the degenerating primary sperquantitative analysis. The numbers of degen- matocytes observed with the light microscope. erating spermatocytes and early spermatids The scarcity or absence of late spermatids sugwere counted in cross or slightly oblique sec- gested that degeneration of these cells might tions of seminiferous tubules in the four main have occurred also. Some dense elongated instages of the cycle. The persistence of some clusions in the cytoplasm of Sertoli cells (fig. acrosome phase spermatids in this animal 5) resembled the condensed nuclei of late spermade it possible in most cases to subdivide matids and thus may have been derived from Stage D further into two parts designated D, degenerating spermatids. (stages IX t o XI) and D, (stages XI1 to XIV). As shown in table 2, by far the largest number Other components of the seminiferous epithelium of degenerating cells was present in stage C, which includes the conventional stages VII Alterations were not detected in the sperand VIII. The next highest number of degen- matogonia of treated animals. Primary spererating cells was found in Stage D, and sub- matocytes in different stages of meiotic prodivision of this stage showed that most were phase were abundant in all the specimens and present in the early portion of the stage (D,) many had normal ultrastructural features inwhich immediately follows Stage C. Germ cluding a large nucleus containing synapcells present in Stage C of the cycle in addition tonemal complexes. Although numerous det o spermatogonia include pachytene primary generating early spermatids were identified, spermatocytes, steps 7-8 early spermatids, and large numbers of Golgi and cap phase sperstages 18-19 late spermatids. Thus light mi- matids (fig. 5) remained and had a normal croscopic observations suggested that germ morphology. As described above, later spercells a t these stages of development preferen- matids were scarce, and those present usually ~~ ~~ ~ ~~ 559 EFFECTS OF TESTOSTERONE occurred singly rather than in the usual groups. Large lipid droplets were present in the cytoplasm of Sertoli cells (fig. 5). These occurred in most tubules regardless of the stage of the cycle, in contrast to those that are normally found a t stage IX (Niemi and Kormano, '65; Kerr and deKretser, '75). Degenerating cells usually were found in a space bounded by processes of Sertoli cytoplasm (fig. 41, and the Sertoli cells contained prominent polymorphous lysosome-like structures which sometimes enclosed remnants of degenerating cells (fig. 5). The remaining ultrastructural features of the Sertoli cells did not appear to be altered. In some specimens in which the spermatid population was severely depleted, processes of Sertoli cells occupied much of the epithelium near the lumen of the seminiferous tubules. Leydig cells Upon inspection of light microscope sections, it was evident that the size of Leydig cells in the interstitial tissue of the testis was greatly reduced in all the treated animals. The mean area of Leydig cell profiles in animals treated for 1 2 weeks was 22.8 pm2,only 33%of the control value, which was 70.1 pm'. This difference was highly significant (p < 0.001). At the ultrastructural level, cytoplasmic organelles of Leydig cells were greatly reduced in abundance in treated animals (fig. 7). For example, the smooth endoplasmic reticulum, which is known to be involved in testosterone production (Christensen, '751, occupied much of the remaining cytoplasm but did not approach the large fields containing this organelle in sections of normal cells (fig. 6). Fertility The objectives of this study were t o define the structural changes of the male reproductive tract in the presence of testosterone enanthate. However, some information on fertility was also obtained and, although limited, i t is reported here because i t is of interest in relation to the morphological alterations. When the rats subsequently killed a t 1 2 weeks were tested during the eleventh week of treatment, all of the treated males mated, as indicated by the presence of vaginal plugs in each of the females with which they were caged, but none impregnated any females. In contrast, each of the control rats impregnated both females with which they were caged. His- tological observations on the reproductive organs of animals allowed to recover for 8 weeks following 12 weeks treatment showed a return to normal appearance of the reproductive organs, including the presence of many late spermatids in the testes. Epididymis The structure of the epididymis of control rats did not differ from observations and previous descriptions of this organ in normal rats (e.g., Hamilton, '75; Flickinger, '77b,c). Prominent features of the columnar principal cells included apical microvilli, vesicles, vacuoles and lysosomes, a large supranuclear Golgi apparatus, and both smooth and rough endoplasmic reticulum. The principal cells were accompanied throughout the duct by small basal and halo cells. In the initial segment apical cells were present, while light cells appeared more distally. The epithelium decreased in height along the length of the epididymis. Some additional features of the different segments of the normal epididymis will be noted when pertinent to the descriptions of treated animals. The weight of the epididymis decreased to 58% that of the control following treatment with testosterone enanthate for 8 or 1 2 weeks (table 1). Microscopic alterations were small or undetectable in the epididymides of animals treated with testosterone enanthate for 4 weeks, but those of rats treated for 8 or 12 weeks all showed changes. Initial segment The initial segment of the epididymis is normally lined by a tall pseudostratified columnar epithelium. The small lumen contains few sperm compared with more distal parts (Glover and Nicander, '71). Consistent changes from this pattern were not detected in the initial segment of testosterone enanthate-treated rats a t either the histological or ultrastructural level. Middle segment In the middle segment a pseudostratified columnar epithelium normally surrounds a large lumen containing many sperm (fig. 8 ) . The first change in the middle segment of the epididymis in treated rats was the reduction and disappearance of the luminal sperm (fig. 9). In the earlier stages of this change, a t 8 weeks, the sperm were seemingly supplanted 560 CHARLES J. FLICKINGER in the lumen by a variable number of small round cells which had features reminiscent of spermatocytes and early spermatids and did not contain phagocytosed sperm. A conspicuous alteration in t h e middle segment after treatment for 12 weeks was t h e appearance within t h e epithelium of large circular or oval cavities many p m in diameter (fig. 9), which were located extracellularly and were bounded by several principal cells (fig. 10). Microvilli on t h e surfaces of t h e principal cells projected into t h e space. At t h e periphery, a fine flocculent content was common, while t h e center contained solid material, including small membrane-bound pieces of cytoplasm, granules, and dense fibers similar t o those of sperm tails. The intraepithelial cavities appeared to be separate from the epididymal lumen but their morphology afforded no clues as to how they developed. Some cells resembling leukocytes are normally present in all segments of the r a t epididymis and have long been referred t o as "halo" cells (Reid and Cleland, '57; Hoffer et al., '73). I t has been reported t h a t most of t h e halo cells in t h e epithelium of t h e r a t male excurrent duct system a r e small lymphocytes (Dym and Romrell, ' 7 5 ) .The epithelium of t h e middle segment of t h e epididymis of rats t r e a t e d with testosterone e n a n t h a t e contained m a n y p a l e - s t a i n i n g cells t h a t r e sembled leukocytes (figs. 11, 12), but differed from halo cells seen in normal and control rats in two main ways. First, they occurred not only singly, but also in aggregates of up t o about 10 to 12 cells (fig. 131, whereas halo cells normally occur singly. Second, t h e cells of treated animals contained many structures with the polymorphous content of phagocytic vacuoles (figs. 11, 12). The vacuoles varied in size from a few tenths of a micrometer up to large spherical bodies several micrometers in diameter which occupied most of the cytoplasm of a cell within a plane of section. No obvious relationship between the pale-staining cells and t h e intraepithelial cavities was noted, and these cells were not observed in t h e lumen. In some cases, pale-staining cells, lacking phagocytic vacuoles, were found in t h e connective tissue and in t h e smooth muscle coat surrounding t h e epididymal epithelium. The principal cells of t h e middle segment had a normal complement of organelles and did not appear altered, with t h e exception of their unusual arrangement around t h e intraepithelial cavities described above. Terminal segment, proximal part (proximal cauda epididymidis) The proximal cauda epididymidis is normally lined by a pseudostratified columnar epithelium of medium height, which is composed mainly of principal cells and light cells. The smoothly-contoured epithelium surrounds a lumen containing many sperm (fig. 14). Beginning in rats treated for 8 weeks, sperm progressively disappeared from t h e lumen of the proximal cauda epididymidis. Concomitant with t h e loss of sperm, t h e lumen of t h e proximal cauda collapsed and t h e epithelium assumed a n irregular, undulating contour (fig. 15). As in t h e case of t h e middle segment, the epithelium of the proximal cauda of animals treated for 12 weeks contained abundant cells provisionally identified as leukocytes, including aggregates of these pale-staining cells. In many specimens, particularly those from rats treated for 8 weeks, t h e light cells of t h e epithelium were very conspicuous (figs. 15) with their content of apical vesicles and vacuoles and large numbers of membrane-bound dense bodies believed to be lysosomes (Nicander, '70; Flickinger, '72). The principal cells of t h e proximal cauda epididymidis did not appear to differ from those of control or normal rats. Terminal segment, distal part (distal cauda epididymidis) The distal part of t h e terminal segment of t h e normal rat epididymis has a large lumen containing many sperm, surrounded by t h e circular profile of a smoothly contoured, low columnar epithelium (fig. 16). Changes were observed in t h e luminal content of the distal cauda epididymidis in one rat treated for 8 weeks and in all those studied after 12 weeks treatment. In these specimens, t h e entire lumen was filled with a mass of flocculent material, spheres of cytoplasm, cellular debris, and some sperm (figs. 17, 18). Most of the sperm were judged t o be disintegrating by t h e absence of plasma membranes, frequent distortion of t h e cell organelles, and changes in t h e relations of organelles t o one another. There were no regions within t h e lumen t h a t contained groups of normal sperm as in the lumen of normal and control rats. Alterations were not detected in t h e epithelium of t h e distal cauda epididymidis. Sex accessory glands The weights of t h e seminal vesicles and ven- EFFECTS OF TESTOSTERONE tral prostate gland of animals treated with testosterone were maintained a t or slightly above control values (table 1). In accord with maintenance of t h e weights of t h e organs, t h e height of t h e glandular epithelium of treated animals was similar to t h a t of normal and control rats, and histological features of t h e glands were normal (figs. 19, 20). At t h e ultrastructural level, both glands displayed abundant cisternae of rough endoplasmic reticulum, a large Golgi apparatus, and many secretory vacuoles (figs. 21, 22). Thus the organelles known to be involved in t h e synthesis and transport of secretory proteins in both t h e r a t prostate and seminal vesicles (Flickinger, '74a,b) were present and had a normal morphology, suggesting t h a t there was a n active secretory process in these glands. DISCUSSION Testis The results showed t h a t in rats administered testosterone enanthate testis weight declined and spermatogenesis was greatly suppressed. This confirmed earlier reports, based on light microscopic studies, t h a t testosterone in various forms and with different routes of administration can suppress the seminiferous epithelium of t h e r a t (Abdi and Hasan, '73; Berndtson e t al., '74; Walsh and Swerdloff, '73). In t h e present study, observations of t h e effects of testosterone enanthate on t h e testis were extended to show t h a t certain stages of germ cells were ereferentially affected; many degenerating spermatids of stages 6-7 were definitely identified, and pachytene primary spermatocytes probably degenerated as well. Accordingly, degenerating germ cells were most prevalent in seminiferous tubules corresponding to stages VII-VIII (Leblond and Clermont, '52) of t h e cycle of t h e seminiferous epithelium. The stages of degenerating germ cells observed in testosterone enanthate-treated rats closely resemble those t h a t degenerated in increased numbers after hypophysectomy in t h e r a t (Clermont and Morgentaler, '55; Russell and Clermont, '77). Suppression of spermatogenesis by testosterone treatment has usually been attributed t o decreased gonadotropin secretion (Ludwig, '50; Desjardins e t al., '73; Ewing et al., '73a,b; Heller e t al., '70; Berndtson e t al., '74). The present observation t h a t Leydig cells were greatly reduced in size and in content of smooth endoplasmic reticulum is 561 in accord with this, and suggests t h a t decreased gonadotropin and endogenous testosterone secretion form t h e basis for t h e similarity between t h e effects of testosterone enanthate and hypophysectomy on t h e seminiferous tubules. The effects of testosterone enanthate on t h e testis also closely resemble t h e alterations observed following treatment with other antifertility compounds, at least some of which probably act by suppressing gonadotropin stimulation of t h e testis (Flickinger, '77a,c). In a recent study, Dym and Madhwa Raj ('77) described changes in t h e testes of rats administered anti-LH serum. There was a decrease in serum and rete testis fluid testosterone and in t h e smooth endoplasmic reticulum of Leydig cells. Changes in the seminiferous tubules similar to those in t h e present study comprised degeneration of midpachytene spermatocytes, phagocytosis of germ cells by Sertoli cells, and a n increase in Sertoli cell lipid droplets. The rapidity of alterations in Sertoli cells, including the retention of late spermatids and changes in smooth endoplasmic reticulum, nuclei, and mitochondria, suggested t h a t alterations in germ cells were secondary to those in Sertoli cells (Dym and Madhwa Raj, '77). Although not directly comparable t o t h e present study because of differences in dosage and in t h e strain of animals used, it is pertinent t o note t h a t physiological observations on testosterone enanthate-treated rats (Walsh and Swerdloff, '73) revealed decreased serum LH levels while serum FSH levels were not altered. In addition, clinical studies have shown t h a t testosterone enanthate results in suppression of Leydig cells and spermatogenesis in men (Heller e t al., '70; Steinberger and Smith, '77), and i t has been proposed t h a t these effects are mediated through suppression of LH (Heller et al., '70). The situation may be more complicated in primates than in rats and rabbits, however, since testosterone administration via subdermal implantation in Rhesus monkeys reduced sperm production while serum LH levels remained normal (Ewing et al., '76). The seminiferous tubules were altered in the presence of exogenous testosterone enanthate sufficient to maintain t h e weight and fine structure of t h e sex accessory glands. As noted in t h e introductory remarks, i t seems likely t h a t this is explained by t h e location of t h e seminiferous tubules near t h e source of endogenous testosterone in t h e Leydig cells. Thus t h e 562 CHARLES J. FLICKINGER seminiferous epithelium normally receives a greater amount of testosterone t h a n most other organs and i t s supply is not adequately replaced by administration of testosterone sufficient to maintain other androgen-dependent organs in a normal state (Heller et al., '70; Walsh and Swerdloff, '73). Alterations in testosterone e n a n t h a t e treated rats differed from ultrastructural changes in t h e testis of men administered testosterone propionate (Barham and Berlin, '74) with respect to alterations in spermatogonia and spermatocytes. In both cases, however, decreased numbers of spermatids and changes in those t h a t remained were reported, and Sertoli cells had similar features, including apparent uptake of degenerating germ cells. The enclosure of degenerating cells by Sertoli cytoplasm, accompanied by a n increase in t h e size and abundance of lipid droplets in t h e cytoplasm of t h e Sertoli cells, has been observed in many other instances in which germ cells degenerate (for references see Flickinger, '77a,c), and t h e lipid may be derived from t h e breakdown of parts of t h e germ cells. Epididymis Some of t h e changes in t h e epididymis in testosterone enanthate-treated rats, such a s decreased epididymal weight and t h e progressive disappearance of sperm from t h e lumen of most parts of the epididymis, can be accounted for by t h e diminished production of sperm by t h e testis. Similarly, changes in t h e contour of t h e epithelium in t h e proximal part of t h e terminal segment may have resulted from collapse of the lumen in t h e absence of sperm. The presence of very large amounts of cellular debris and degenerating sperm filling t h e distal part of t h e terminal segment seem likely t o be due to t h e failure of sperm t o survive in this region where they are normally stored. Several possible explanations for this can be suggested. First, t h e epididymis receives a dual supply of androgen, both from t h e blood and in t h e luminal fluid from t h e testis (White and Hudson, '68; Waites and Setchell, '69; Hanssori e t al., '75). Since t h e Leydig cells of the testis were very severely suppressed, i t seems likely t h a t t h e normal supply of androgen in t h e luminal fluid was not adequately replaced by th*e administration of exogenous testosterone, with possible deleterious effects on t h e luminal sperm or t h e epididymal epithelium. Second, if sperm were not transported normally beyond t h e distal cauda epi- didymis, they may have aged there, died, and begun to disintegrate. Finally, under the influence of t h e treatment t h e germ cells t h a t reached t h e cauda may have been immature or defective. It is not possible to choose between these alternatives, but the first two imply some changes in t h e interaction between sperm and epididymal epithelium in the distal cauda epididymidis. Such a change could contribute to t h e antifertility action of testosterone enanthate by providing a n additional barrier t o t h e escape of normal sperm from the male reproductive tract. It is to be emphasized t h a t t h e degenerating sperm and cellular debris present in t h e distal cauda epididymidis of treated rats occupied almost t h e entire lumen; no areas with normal sperm were found. Cooper and Hamilton ('77) have described t h e presence of some degenerating sperm in t h e distal epididymis and vas deferens of normal mammals and have questioned t h e significance of reports of degenerating sperm in t h e cauda epididymidis of rats receiving antifertility agents. However, t h e difference between t h e normal and the situation described here and in previous publications (Flickinger and Loving, '76; Flickinger, '77b,c) is dramatic. In t h e normal the masses of degenerating sperm occupied only a fraction of t h e lumen and t h e proportion of degenerating sperm remained small, but in treated animals most of t h e lumen contained debris and almost all t h e sperm appeared t o be degenerating. An interesting feature not previously observed in our studies of various antifertility agents was t h e appearance of many apparently phagocytic cells in t h e epithelium of the middle segment and proximal part of t h e terminal segment of testosterone enanthatetreated rats. The nature of these cells has not definitely been determined, but they have many features characteristic of monocytes, including their nuclear morphology, large Golgi apparatus, rough endoplasmic reticulum and dense cytoplasmic granules (Bloom and Fawcett, '75). The presence of phagocytic vacuoles makes i t seem unlikely t h a t the cells represent lymphocytes, which are not normally phagocytic, even though other cytological features may be consistent with those of the unusual lymphocytes said t o occur in t h e r a t (Hoffer et al., '73). The presence of phagocytosed material may make i t appropriate to consider these cells as macrophages, which is in accord with their aggregation in large EFFECTS OF TESTOSTERONE groups in the epididymal epithelium. Whatever their identity, the functional significance of phagocytic cells in the epididymal epithelium of treated animals and the origin of the ingested material remain uncertain. The phagocytes were never observed in the lumen of the epididymis. Phagocytes in the epithelium might provide a means for disposal of disintegrating germ cells located within the intraepithelial cavities observed in some specimens. However, no preferential association between the phagocytic cells and the epithelial cavities was detected. Sex accessory glands The sex accessory glands, represented by the ventral prostate and the seminal vesicles, were maintained a t close to normal weight, and the ultrastructure of the epithelium suggested that normal secretory activities continued in the presence of testosterone enanthate. Other organs were not investigated, but if other androgen-dependent organs and male sexual characteristics fared as well as the prostate and seminal vesicles it is likely that male characteristics were generally well maintained, even though spermatogenesis was severely suppressed. ACKNOWLEDGMENTS The author is indebted to Miss Mary Stuart for technical assistance and to Mr. Eric Sun for injecting the animals. This research was supported by a grant (1 R01 HD 10073) from the National Institute of Child Health and Human Development. LITERATURE CITED Abdi, S. H. M., and W. Hasan 1973 Effect of testosterone on t h e histological structure of the testis of adult albino rats. Ind. J. Med. 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Mortimore 1950 Improvement in spermatogenesis following depression of the human testis with testosterone. Fertil. Steril., 1: 415-422. Hoffer, A. P., D. W. Hamilton and D. W. Fawcett 1973 The ultrastructure of the principal cells and intraepithelial leukocytes in the initial segment of the r a t epididymis. Anat. Rec., 175: 169-202. Hotchkiss, R. S. 1944 Effects of massive doses of testosterone propionate on spermatogenesis. J . Clin. Endocrinol., 4: 117-120. Ito, S., and M. J . Karnovsky 1968 Formaldehyde-glutaraldehyde fixatives containing trinitro compounds. J. Cell Biol., 39: 168A. Karnovsky, M. J. 1965 A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol., 27: 137A. Kerr, J. B., and D. M. deKretser 1975 Cyclic variations in Sertoli cell lipid content throughout the spermatogenic cycle in t h e rat. J . Reprod. Fertil., 43: 1-8. Leblond, C. P., and Y. Clermont 1952 Definition of the stages of the cycle of the seminiferous epithelium of t h e rat. Ann. N. Y. Acad. Sci., 55: 548-573. Loving, C. K., and C. J. Flickinger 1976 Alterations in the fine structure of the prostate and seminal vesicle of rats treated with cyproterone acetate. Anat. Rec., 185: 13-30. Ludwig, D. J. 1950 The effect of androgen on spermatogenesis. Endocrinology, 46: 453-481. Murad, F., and A. G. Gilman 1975 Androgens and anabolic steroids. In: The Pharmacological Basis of Therapeutics, Fifth ed. L. S. Goodman and A. Gilman, eds. MacMillan Publishing Co., New York, pp. 1451-1471. Nelson, W. O., and C. Merckel 1937 Maintenance of spermatogenesis in testis of the hypophysectomized rat with sterol derivatives. Proc. SOC.Exp. Biol. Med., 36: 825-828. Nicander, L. 1970 Morphological evidence of secretion and absorption in the epididymis. In: Morphological aspects of Andrology. Vol. I. A. F. Holstein, and E. Horstman, eds. Grosse Verlag, Berlin, pp. 121~124. Niemi, M., and M. Kormano 1965 Cyclical changes in and significance of lipids and acid phosphatase activity in the seminiferous tubules of t h e r a t testis. Anat. Rec., 151: 159-170. Reddy, P. R. K., and J. M. Rao 1972 Reversible antifertility action of testosterone propionate in human males. Contraception, 5: 295-301. Reid, B. L., and K. W. Cleland 1957 The structure and function of the epididymis. I. The histology of the rat epididymis. Austral. J . Zool., 5: 223-246. Reynolds, E. S. 1963 The use of lead citrate a t high pH an electron-opaque stain in electron microscopy. J. Cell Biol., 17: 208-212. Russell, L. D., and Y. Clermont 1977 Degeneration of germ cells in normal, hypophysectomized and hormone treated hypophysectomiaed rats. Anat. Rec., 187: 347-366. Steinberger, E. 1971 Hormonal control of mammalian spermatogenesis. Physiol. Rev., 51: 1-22. Steinberger, E., and K. D. Smith 1977 Testosterone enanthate: A possible reversible male contraceptive. Contraception, 16: 261-268. Waites, G. M. H., and B. P. Setchell 1969 Physiology of the testis, epididymis, and scrotum. Adv. Reprod. Physiol., 4: 1-63. Walsh, P. C., and R. S. Swerdloff 1973 Biphasic effect of testosterone on spermatogenesis in the rat. Invest. Urol., 11: 190-193. White, I. G., and B. Hudson 1968 The testosterone and dehydroepiandrosterone concentration in fluids of the mammalian male reproductive tract. J. Endocrinol., 41: 291-292. PLATES PLATE I EXPLANATION OF FIGURES 1 Light micrograph of normal rat testis. The seminiferous tubules contain Sertoli cells and germ cells in different stages of development. Present are spermatogonia next to t h e basal lamina, spermatocytes (P),and many acrosome phase spermatids (T). Leydig cells (L) lie in the interstitial tissue between the tubules. N, Sertoli cell nuclei. x 260. 2 , 3 Light micrographs of the testis of a rat treated with testosterone enanthate for 12 weeks. In the seminiferous tubules, Sertoli cells, spermatogonia, spermatocytes (P), and early spermatids (S) are present, but late spermatids are virtually absent. Degenerating germ cells are numerous and are identified by their dense granular appearance. Many are apparently early spermatids (DS), while others are primary spermatocytes (DP) as indicated by their size and location. Leydig cells in the interstitial tissue are inconspicuous due to their small size. Figure 2 X 290; figure 3 X 250. 566 EFFECTS OF TESTOSTERONE Charles J. Flickinger f’LATE 1 567 EFFECTS OF TESTOSTERONE Charles J Fhckrnger PLATE 2 EXPLANATION OF FIGURES 568 4 Electron micrograph of a degenerating cell in the seminiferous epithelium of an animal treated for 8 weeks with testosterone enanthate. The cell is judged to be degenerating by its extreme density and the disruption of the normal architecture of its organelles. I t is identified as a n early spermatid of the cap phase because of the persistence of an acrosomal cap (A). The degenerating cell is surrounded by Sertoli cell cytoplasm (C). x 11,000. 5 Low power electron micrograph of the seminiferous epithelium in a rat administered testosterone enanthate for 8 weeks. Constituents of Sertoli cells include part of the nucleus (N), a cytoplasmic lipid droplet (L), dense elongate inclusions (I), and degenerating cellular material (D). Some normal appearing germ cells are represented, including a cap phase spermatid 6)and some preleptotene spermatocytes (P). X 6,400. EFFECTS OF TESTOSTERONE Charles J. Flickinger PLATE 3 569 PLATE 4 EXPLANATION OF FIGURES 6 Part of a Leydig cell of a normal rat. Much of t h e cytoplasm of the large cell is occupied by smooth endoplasmic reticulum (El and mitochondria (M). R, rough endoplasmic reticulum; N, nucleus. X 17,000. 7 Leydig cell from a rat treated for 8 weeks. Cytoplasmic organelles including smooth endoplasmic reticulum (E) and mitochondria (M) are not abundant because the cell is very small compared to normal (cf. fig. 6 ) . The nucleus (N) contains prominent clumps of heterochromatin. Leydig cells are frequently dense in testes fixed with the aldehyde mixture used in this study. X 17,000. 570 EFFECTS OF TESTOSTERONE Charles J. Flickinger PLATE 4 571 PLATE 5 EFFECTS OF TESTOSTERONE Charles J Flickinger EXPLANATION OF FIGURES 8 Light micrograph of the middle segment of the epididymis in a control rat. The epididymis is lined by a columnar epithelium of moderate height, and t h e lumen (L) contains many sperm. X 220. 9 Middle segment in a rat administered testosterone enanthate for 12 weeks. Sperm are virtually absent from the lumen (L) which contains only a few round cells. Large cavities (C) are conspicuous within the epithelium. x 220. 10 Low power electron micrograph of the middle segment of t h e epididymis in a rat treated for 12 weeks. A portion of an intra-epithelial cavity (C) is surrounded by several principal cells. Microvilli project from the surface of the cells into the cavity, which contains a light granular material and a central mass of debris consist. ing of small spherical objects and dense fibers. A portion of the epididymal lumen (L) appears in the upper left hand corner of the field. Present within the epithelium is a dense polymorphous mass of material (D)surrounded by a thin rim of pale-staining cytoplasm. X 4,500. 572 EFFECTS OF TESTOSTERONE Charles J. Flickinger PLATE 6 PLATE I EXPLANATION O F FIGURES 11, 12 Examples of the pale-staining cells that resemble leukocytes in t h e epithelium of the middle segment in a rat administered testosterone enanthate for 12 weeks. Prominent in the cytoplasm are dense membrane-bound structures (D) t h a t contain a variety of materials, including cellular organelles. N, nucleus. In figure 11 note the large Golgi apparatus (GI and the cytocentrum (HI. Figure 11 X 15,000; figure 12 X 11,000. 5 74 EFFECTS OF TESTOSTERONE Charles J. Flickinger PLATE 7 PLATE 8 EXPLANATION OF FIGURE 13 Low magnification electron micrograph of a mass of pale-staining cells in the epithelium of the middle segment of a rat treated for 12 weeks. The cells have irregular interdigitated outlines, and their cytoplasm contains dense structures (D) with a polymorphous content, which resemble lysosomes. X 8,500. 576 EFFECTS OF TESTOSTERONE Charles J Flickinger PLATE 8 577 PLATE 9 EXPLANATION OF FIGURES 14 Light micrograph of the proximal part of the terminal segment of a control rat. The lumen (L) contains many sperm and is lined by a columnar epithelium. X 285. 15 Proximal part of the terminal segment in a rat treated for 12 weeks. Sperm are ab- sent from t h e small lumen (L). The epithelium is tall and has an irregular undulating outline, perhaps as a result of collapse of the lumen. Some vacuolated light cells with dense cytoplasmic granules are evident (arrow). X 285. 16 Light micrograph of the distal part of the terminal segment in a control rat. The large lumen (L) is surrounded by a low columnar epithelium and a smooth muscle coat (M). x 285. 17 Distal part of the terminal segment of a r a t treated for 12 weeks. The epithelium is a low columnar type as in the normal, but the luminal contents are altered. Among clumps of sperm (S) are round cells, spherical masses of cytoplasm (0,and debris. X 285. 578 PLATE 9 EFFECTS OF TESTOSTERONE Charles J. Flickinger 579 PLATE 10 EXPLANATION OF FIGURES 18 Lumen of the distal part of t h e terminal segment in a rat treated for 12 weeks. The luminal material includes spherical masses of cytoplasm (C) some of which contain a nucleus (N), a few sperm (S), a flocculent material (F), and scattered debris. X 4,000. 19 Light micrograph of seminal vesicle of a r a t treated with testosterone enanthate for 12 weeks. The epithelium (El contains secretory granules visible as dense spots surrounded by a clear rim. Secretory material (A) is present in the lumen between the folds of epithelium. X 230. 20 580 Light micrograph of ventral prostate of a rat treated with testosterone enanthate for 12 weeks. The prostatic acini are lined by a medium to tall columnar epithelium (E). X 230. EFFECTS OF TESTOSTERONE Charles J. Flickinger PLATE 10 581 PLATE 11 EXPLANATION OF FIGURES 582 21 Seminal vesicle of a rat treated for 12 weeks. The epithelial cells contain abundant organelles associated with the formation of secretions: rough endoplasmic reticulum (R), Golgi apparatus (GI, and t h e characteristic secretory vacuoles (S) that contain a dense secretory granule surrounded by a light rim. L, lumen of the gland. X 15,500. 22 Ventral prostate of a rat treated for 12 weeks. The prostatic epithelial cells display normal organelles, including abundant rough endoplasmic reticulum (R),t h e Golgi x 9,000. apparatus, and secretory vacuoles 6). EFFECTS OF TESTOSTERONE Charles J . Flickinger PLATE 11 583
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