The influence of progestin and androgen on the fine structure of the male reproductive tract of the rat. I. General effects and observations on the testisкод для вставкиСкачать
The lnfluence of Progestin and Androgen on the Fine Structure of the Male Reproductive Tract of the Rat I. GENERAL EFFECTS AND OBSERVATIONS ON THE TESTIS CHARLES J. FLICKINGER D e p a r t m e n t of Anntomy, School of Me dic ine , Uniuersitg of Vir ginin, Cknrlottesuille, Virginia 22901 ABSTRACT The combination of a progestin and androgen has received attention a s a possible male contraceptive. The progestin is thought to reduce gonadotropin release and suppress spermatogenesis, while the sex accessory organs and male characteris tics are maintained by the simultaneous administration of testosterone. In the present study, the histology and ultrastructure of parts of the male reproductive tract of rats treated with medroxyprogesterone (Provera, Upjohn) (1 mg/100 g body weight/day) alone and combined with testosterone (15, 30, o r 100 pg1100 g/day) were studied following treatment for up to 16 weeks. The testes and epididymides of rats administered Provera alone or Provera and testosterone weighed less than those of control rats. The weights of the accessory glands of rats treated with Provera were greatly reduced; it was possible to maintain them at approximately control levels by simultaneously administering sufficient testosterone (100 pg/100 g body weight/day). The fertility of some of the animals was tested by caging them with female rats, and none of the treated rats tested i n this way was fertile. Similar microscopic alterations were present in the testes of animals administered Provera alone or Provera and different levels of testosterone. Spermatogonia, spermatocytes, and early spermatids were abundant in treated rats and did not show ultrastructural changes. However, many degenerating or necrotic spermatids of the cap phase (approximately stages 6-7) and later were present. Late spermatids of the acrosome and maturation phases were rare. Some necrotic spermatids were surrounded by Sertoli cells, and parts of spermatids lay within lysosome-like structures in the cytoplasm of Sertoli cells. Many large lipid droplets were also present in Sertoli cells of treated rats. Leydig cells were smaller i n treated animals than in control rats. The results suggest that germ cells can develop up to cap phase spermatids but then undergo degeneration. These alterations in spermatogenesis may be responsible in large part for the antifertility effect of the progestin and androgen combination. Some rats were permitted to recover following the end of treatment. The microscopic appearance of the testis returned to normal within three to six weeks, although epididymal alterations persisted in some animals six weeks after the end of treatment. By 9 to 12 weeks after the end of treatment the reproductive organs had a normal microscopic appearance in all the rats studied. Many different kinds of compounds have been studied for their antifertility effects in the hope that a suitable chemical contraceptive for men can be found. Recently the combination of a progestin and an androgen has been tested and is believed to show considerable promise as a male contraceptive (Segal, '73), because it is reported to produce reversible infertility while libido and male sex characteristics are maintained (Frick, '73; Coutinho and Melo, ANAT. REC., 1 8 7 : 4 0 5 4 3 0 . '73). The rationale behind the administration of a progestin-androgen combination for male contraception is as follows (Terner and MacLaughlin, '73; Kragt et al., '73). The progestin is thought to diminish gonadotropin release and suppress spermatogenesis. However, the Leydig cells of the cells of the testis are also suppressed, resulting in decreased secretion of testosterReceived Mar. 18, '76. Accepted Oct. 1 , '76. 405 406 CHARLES J . FLICKINGER one. Secondary sex characteristics and libido are then maintained by the simultaneous administration of exogenous testosterone. Stumes in the rat have shown that it is indeed feasible with this combination to abolish fertility and to maintain the weight of the sex accessory glands and sexual behavior (Terner and MacLaughlin, '73; Kragt et al., '73). The influence of this regimen on the structure of the male reproductive tract has not previously been studied, however, and many questions remain concerning its effects on the reproductive organs. Therefore, in the present investigation, the influence of a progestin alone and in combination with testosterone on the histology and ultrastructure of the testis, epididymis, and sex accessory glands of the male rat were studied at intervals up to 16 weeks of treatment. The general aim of the study was to determine the effects of these agents on the structure of the male reproductive tract as a basis for understanding the antifertility action of these compounds. An important characteristic of a potential male contraceptive agent is that its action be reversible. Consequently, observations were also made on the weight and microscopic appearance of the reproductive organs of rats permitted to recover following the cessation of treatment with progestin and testosterone. The present article is concerned with the general effects of the progestin-androgen regimen, including the influence of the agents on the weights of the reproductive organs and on fertility, and with the histology and ultrastructure of the testis. Specific objectives with respect to the study of the testis were to determine which stages of spermatogenesis are affected by the treatment, and the nature of possible cytological changes in germ cells, Sertoli cells,and Leydig cells. The companion paper will consider alterations in different parts of the epididymis and in the prostate and seminal vesicle (Flickinger, '77). MATERIALS A N D METHODS Young adult male rats of the SpragueDawley strain weighing approximately 200 g were obtained from Hilltop Lab Animals, Inc., Scottdale, Pennsylvania. A total of 55 animals was used; of these, 17 were treated with a progestin alone, 23 were administered progestin and testosterone combined, and 15 served as controls. Animals were maintained at 20-22°C and had constant access to food and water. Rats were injected subcutaneously with steroids in 0.1-0.2 ml of sesame oil. Control animals received an equivalent amount of the oil alone. The compounds and the doses administered in the different experiments are listed below. Experiment I . (table 1). Progesterone was administered at a dose of 2 mglratl TABLE 1 The f i n a l w e i g h t s of the reproductiue organs arid t h e f i n a l b o d y w e i g h t s of rats treated w i t h progesterone Testis Time iwk ) 1 2 4 Seminal ve sic Le Prost ate Body weight (average) ~ Treatment (Expt. No. 1) Control (2) Prog 2 rng (1) Prog 1 0 m g (1) Control (2) Prog 2 mg (1) Prog 10 mg (1) Control (2) Prog 2 mg (1) Prog 10 mg (1) 7-8 Epididymis Control (1) Prog 10 m g (2) g/100 g 9% control g/100 g 0.57 - 0.14 0.63 0.46 0.44 0.45 0.42 0.38 0.38 0.33 0.33 111 0.43 75 96 98 90 90 100 8 70 0.10 0.14 0.12 0.11 0.1 1 0.13 0.12 0.10 0.14 0.10 % control g/lOO g control g/100 g control g - 0.13 0.08 - 0.13 0.10 0.08 0.10 0.05 0.09 - 255 315 230 303 340 278 38 8 330 350 485 450 71 100 92 92 0.09 62 69 0.12 0.08 0.11 67 92 - - 0.15 - 92 77 0.09 0.07 0.16 0.05 60 47 71 - 31 0.13 0.04 0.05 0.15 0.05 77 62 - 50 90 31 38 33 The number of animals is shown i n parentheses after the designation for the treatment. The average values for the testis. epididymis, and seminal vesicle include the two organs from each rat. The number of rats is smaller than i n the following experiments, since this w a s a preliminary experiment to test the effects of progesterone on the reproductive organs. The organ weights are shown in g per 100 g body weight and as 8 control. A b b r e ~ ~ i a t i o nProg s: 2 mg, 2 mg progesteronelratlday, Prog 10 mg, 10 mg progesteroneiratlday. PROGESTIN AND ANDROGEN. I day, or 10 mglratlday. Rats were killed a t intervals of 1 , 2, 4, 7, and 8 weeks. In this preliminary experiment, for simplicity a constant dose of progesterone was used. In all the subsequent experiments the dose of steroids was adjusted to the weight of the individual animal. The lower dose of 2 mglratlday was selected because it had been reported to alter spermatogenesis in rats (Kar et al., '67). When this effect was not observed i n our animals, the dose of progesterone was increased to 10 mg/rat/ day in a n effort to influence spermatogenesis. Experiment 2. (table 2). Medroxyprogesterone (Provera, Upjohn) was administered at a dose of 1 mg/100 g body weight/ day. Rats were killed a t intervals of 4, 8, and 16 weeks. This dose of Provera was used because i t is similar to that employed in previous studies on the effects of ProV e r a on the reproductive tract of male rats (Terner and MacLaughlin, '73). This amount of Provera resulted in microscopic alterations in the reproductive tract, and therefore it was continued in the following experiments, i n which the amount of testosterone was varied. Experiment 3 . (table 2). Provera 1 mg/ 100 g/day was given in combination with testosterone propionate 15 pg/lOO g/day. Animals were killed a t intervals of 4, 8, and 16 weeks. This amount of testosterone was used because a similar dose had been reported to prevent or diminish the effects of various progestins (Terner and MacLaughlin, '73). Experiment 4. (table 2). Provera 1 mg/ 100 g/day plus testosterone propionate 30 pgl100 glday were given. Rats were killed at intervals of 4, 8, and 16 weeks. This dose of testosterone was employed because the weight of the sex accessory glands was not maintained a t control levels i n the presence of 15 pg/lOO g/day. Therefore, the dose of testosterone was doubled. Experiment 5 . (table 2). Provera 1 mg/ 100 glday plus testosterone propionate 100 pg/lOO g/day were administered. Animals were killed at intervals of four and eight weeks. Since the weight of the sex accessory glands remained below control levels in the presence of testosterone 30 pg/100 glday, the dose of testosterone was increased. The level of 100 pg1100 glday was 407 used because it was reported that this was sufficient to maintain the sex accessory glands of castrated rats (Ramirez and McCann, '65). Some control and treated animals were not killed during the course of the experiment, but instead were permitted to recover for 3, 6, or 9 weeks after the cessation of treatment as shown i n table 3. Medroxyprogesterone (Provera) was obtained through the courtesy of Millard W. Beucler and Marvin R. Guthaus of the Upjohn Co., Kalamazoo, Michigan. The progesterone and testosterone propionate were purchased from the Sigma Chemical Co.,St. Louis. The reproductive organs, including testes, epididymides, ventral prostate, and seminal vesicles were removed and weighed. Whole testes were immersed for one hour in a glutaraldehyde, formaldehyde, and picric acid fixative (It0 and Karnovsky, '68), 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 the outside of the testes, diced, and fixed for a n additional one hour. After fixation for a total of two hours i n aldehyde, the tissue blocks were rinsed with 0.1 M cacodylate buffer and post-fixed for one hour in 1% OsO4 in 0.1 M cacodylate buffer a t pH 7.3. The tissue was dehydrated in a graded series of ethanols followed by propylene oxide, and was embedded i n Araldite. Sections 1 pm thick for light microscopy were cut with glass knives, mounted on slides, and stained with 0.5% toluidine blue in 0.5 % sodium borate. Thin sections showing silver to pale gold interference colors were cut with a diamond knife, mounted on uncoated copper grids, and stained with lead citrate (Reynolds, '63). The preparations were examined and photographed using a Philips EM-300 electron microscope. The sizes of profiles of Leydig cells of the testis in sections for light microscopy at the 8-week interval were estimated in the following way. Regions of accumulation of interstitial tissue between three or more seminiferous tubules were chosen at random for study. Within these regions, the length and width of all the cells iden- 4 3 2 15pg P v f T 30pg v h + T Control Pv+TlOOpg 4 5 + h. Pv+T 15pg Pv+T 30pg Pv T 100 p g 2 3 P Treatment Control Control Pv P v + T 15pg Pv+T 30pg 2 3 4 5 Expt. No. 0.42 0.16 0.18 0.18 0.25 0.38 0.16 0.16 0.16 0.19 0.36 0.13 0.18 0.16 g/100 g 36 50 44 - 42 42 42 50 - 38 43 43 60 - % control Testis 0.03 0.04 0.05 0.08 0.13 0.04 0.05 0.04 0.08 0.14 0.04 0.06 0.05 o.ii g/100 B 29 43 36 - 31 38 31 62 - 27 36 45 73 - 5% control Epididymis 0.11 0.02 0.05 0.07 0.14 0.14 0.02 0.06 0.06 0.14 0.13 0.03 0.05 0.04 g/100 g 23 38 31 - 14 43 43 00 - 0.01 0.04 0.04 0.06 0.08 0.02 0.04 0.05 0.12 0.07 0.03 0.04 0.06 0 07 18 45 64 27 g/100 g - 43 57 86 - 25 50 63 150 - 14 57 57 86 - % control Seminal vesicle % control Prostate 323 363 328 455 353 345 335 383 52 1 42 1 473 415 41 0 . ._ 45 1 g body weight (average) Each value is the average of the individual organ weights from two rats, except for F'v 16 weeks which is the average from three animals. The values for the testis, epididymis, and seminal vesicles include the two organs from each rat. The weights are shown in g per 100 g hody weight and in % control. Abbreviations: Pv, Rovera 1 mg/100 g/day; P v + T, Provera 1 mg/100g/day plus testosterone 15, 30, or 100 f i g / l O O g/day a s indicated. 16 8 4 Time (wk) The weights of the reproductive organs and t h e f i n a l body weights of rats treated w i t h Prouera or w i t h Provera combined w i t h testosterone for 4, 8, or 1 6 weeks TABLE 2 ?? 9 m 0 z EE 9 CI k 25 409 PROGESTIN AND ANDROGEN. I TABLE 3 The t y p e a n d l e n g t h of t r e a t m e n t a n d t h e mlcroscop?c nppearunce of rats v e r m t t t e d t o recover a f t e r t h e e n d of a t r e a t m e n t p e n o d Time of recovery (wk) Expt. No. Length of treatment (wk) Treatment 3 3 3 6.5 6 Control Pv Pv+T 15pg 16 16 16 16 16 7 7 Pv+T 30pg P v + T 30pg 16 16 8 9 Pv Control T 100 p g 8 8 Pv+T 15pg Pv 9 + Pv + T 100 p g 12 P v + T 15pg + indicates a normal microscopic appearance, while tifiable as Leydig cells were measured using a Zeiss optical micrometer. Assuming the profiles of the cells to be eliptical, the area of a cell represented within a section was estimated by multiplying the length/ 2 X width/2 X T. Twenty-five cells were measured for each of four rats treated with Provera (Experiment 2), two rats treated with Provera and testosterone (Experiment 5), two normal untreated animals, and one control rat. The mean areas of Leydig cells in sections of control and normal rats were compared with those of treated animals using a T test. The fertility of some of the rats was tested by caging each male rat for five days with two female rats and examining the females one week later for the presence of fetuses. Animals tested in this way included seven controls, four rats treated with Provera (Experiment 2) and eight animals treated with Provera and testosterone (Experiments 3 , 5). RESULTS General observations Progestin A preliminary experiment (Experiment 1) was carried out using progesterone as the prototype and representative of the class of progestational agents. Rats received 2 mg or 10 mg of progesterone daily for up to eight weeks. A dose similar to the lower one had been reported to arrest sper- 8 16 Microscopic appearance T - + + testes rest - + + + testes rest - + + + - designates the persistence of alterations matogenesis in male rats (Kar et al., '67). Although the weight of the prostate and seminal vesicle of treated animals was in most cases 30-50% that of control animals after treatment for four weeks or longer (table l), alterations in the height of the epithelium and the cytology of epithelial cells in the prostate and seminal vesicles were minimal, and the histology of the testis and epididymis appeared unchanged. Therefore, we turned to medroxyprogesterone (Provera), because it is a more potent progestational agent and was used in studies on the effects of progestin-testosterone combinations in rats by others (Terner and MacLaughlin, '73). Treatment with Provera 1 mg/lOO g/day (Experiment 2) resulted in a decline in the weight of the prostate and seminal vesicles in most animals to 10-25% control values after treatment for four weeks (table 2). The weight of the testis and epididymis of treated animals decreased to about 3040 % the control. Progestin and testosterone combined In our first experiment in which testosterone was given along with Provera (Experiment 3), rats were administered 15 cLg testosterone/lOO g/day in combination with 1 mg Provera/100 g body weightlday, since it had been reported (Terner and MacLaughlin, '73) that even a small dose of testosterone such as this was capable of preventing or decreasing the effects of pro- 410 CHARLES J. FLICKINGER gestins. However, we found that although the prostate and seminal vesicles of these rats were larger and weighed more than in rats administered Provera alone, they weighed only about one-half those of control animals (table 2). Therefore, in the next experiment (Experiment 4), we maintained the amount of Provera a t 1 mg/100 glday and increased the accompanying testosterone to 30 pg/lOO g/day. However, the weights of the prostate and seminal vesicles of treated animals still did not equal those of controls (table 1). Thus, in the final experiment (Experiment 5), we utilized Provera plus 100 pg testosterone/ 100 g/day. At this level, the weights of the prostate and seminal vesicles of treated animals approximated or exceeded those of control rats. The testes and epididymides of animals treated with Provera and the different doses of testosterone (Experiments 3 , 4, 5) were small, and in the majority of treated rats weighed approximately half those of control animals. Fertility The fertility of some of the treated rats was tested by caging them with two female rats. Since our main interest was in the histology and ultrastructure of the reproductive organs, the number of animals tested in this way is small, but the results are nevertheless of interest in relation to the morphological observations. All but one of the seven controls impregnated a t least one female. In contrast, none of the four rats treated with Provera for eight weeks (Experiment 2) or the eight animals treated with Provera and testosterone for eight weeks (Experiments 3 , 5) impregnated a female. It is uncertain precisely how many of the rats administered Provera and testosterone actually mated, but mating did occur i n a t least some instances since vaginal plugs were observed in females placed with two of the treated males. Since most of our animals were killed for microscopic study during the course of the treatment, we have little data on the recovery of fertility after the end of treatment, except that one of two animals tested eight weeks after the end of treatment with Provera and testosterone (Experiment 5) had regained fertility. Testis The structure of the testis of normal rats resembles that of other mammals (Burgos et al., '70). Seminiferous tubules contain Sertoli cells and combinations of germ cells in different stages of development (fig. 1). The interstitial tissue includes testosterone-secreting Leydig cells, blood vessels, lymphatics, and other connective tissue elements (Fawcett et al., '73). The testes of control rats administered the oil vehicle did not differ from the normal. Alterations in the testes of rats treated with Provera were observed at all the intervals studied, including the earliest samples which were taken after treatment for four weeks. Changes in the microscopic structure of the testes of rats treated with Provera and testosterone closely resembled those observed after treatment with ProVera alone. Furthermore, the structure of the testis did not vary significantly with the amount of testosterone added to the ProVera. Therefore, the microscopic structure of the testes of animals treated with ProVera alone and with Provera and the different levels of testosterone will be described together. Since no consistent progression of the alterations after four weeks of treatment was observed, to reduce redundancy the different intervals are not all sequentially illustrated and described individually. At the light microscope level, spermatogonia, primary spermatocytes, and early spermatids up to the cap phase were readily visible, but later spermatids were rare or appeared to be completely absent i n the light microscope preparations (figs. 2, 3). The testes of treated animals showed numerous large lipid droplets and denselystaining cells that appeared to be degenerating within the seminiferous epithelium. In different profiles of the seminiferous tubules, different types of the early germ cells were present. It was thought that this might reflect the presence of different stages of the cycle of the seminiferous epithelium from which late spermatids were absent or depleted in number, but a systematic comparison of each of the stages of the cycle was not carried out. Study of the seminiferous epithelium with the electron microscope revealed no morphological alterations in spermatogonia, spermatocytes, or Golgi phase spermatids (figs. 4-7). Spermatogonia were recognized as round or oval cells close to or i n contact with the base of the seminiferous epithelium PROGESTIN AND ANDROGEN. I (fig. 5 ) . The cytoplasm contained clusters of free ribosomes but only small amounts of membranous organelles such as endoplasmic reticulum and Golgi apparatus. The round to oval nucleus had a uniform distribution of chromatin in some cells which may represent type A spermatogonia. Other nuclei that had more prominent clumps of chromatin may be those of type B spermatogonia. Many primary spermatocytes were present (fig. 4). These were larger than spermatogonia and in favorable sections their cytoplasm could be seen to contain a larger Golgi apparatus and more abundant profiles of endoplasmic reticulum. The round nuclei of the primary spermatocytes presented a variety of chromatin patterns and nucleolar morphologies, probably reflecting different stages of meiotic prophase. The identification of many synaptinemal complexes (fig. 4) suggested that pairing of homologous chromosomes occurred and that meiosis proceeded through zygotene i n treated animals. Golgi phase spermatids were present (fig. 6). Their most prominent features included the usual round nucleus, a large Golgi apparatus, proacrosomal granules, and the single large acrosomal granule, which was enclosed i n the acrosomal vacuole and apposed to one side of the nucleus (fig. 6). Some normal-appearing spermatids of the early cap phase were present, but as described in more detail below other cap phase spermatids were degenerating. Those of normal appearance (fig. 7) displayed a large Golgi apparatus and the characteristic acrosomal vacuole, which formed a cap over the anterior surface of the round spermatid nucleus. Additional features of these cells included mitochondria located around the margin of the cytoplasm, cytoplasmic vesicles, and, frequently, a chromatoid body. Intercellular bridges were observed between spermatocytes and between spermatids. Although the early spermatogenic stages of spermatogonia, spermatocytes, and early spermatids remained abundant in sections, i t should be noted that changes in their numbers were not ruled out, because a quantitative analysis of the different types was not undertaken. As suggested by the light microscope observations, degenerating cells were numerous i n samples of the testes of treated animals viewed with the electron microscope (fig. 8). These cells displayed in- 411 creased cytoplasmic density, organelles with disrupted membranes, and myelin figures. The severity of the morphological changes suggests that many of the cells were irreversibly damaged and thus may be considered necrotic (Robbins, '74). Many of these cells were identified further as early spermatids i n the latter part of the cap phase (approximately stages 6-7: Leblond and Clermont, '52) because they contained the acrosomal cap that is characteristic of these cells. The presence of some degenerating cells has been described in normal testes (Oakberg, '56; Clermont and Bustos-Obregon, '68), but spermatogonia and spermatocytes are involved. Degenerating spermatids such as those seen in treated animals were not observed in our samples of normal and control rats. The morphological changes that precede those obvious necrotic alterations were not identified with certainty. However, some spermatids displayed increased cytoplasmic density, numerous cytoplasmic vacuoles, and irregularly shaped nuclei, and these features may reflect early degenerative changes. Few later spermatids of the acrosome and maturation stages remained in the seminiferous epithelium, but some could usually be identified with the electron microscope. Many of those present, however, appeared also to be undergoing degeneration and death as indicated by the presence of dense cytoplasm and alterations in the architecture of the acrosome, tail fibers, and other organelles (fig. 9). Sertoli cells in treated rats retained many normal features (fig. 4). They had an irregular shape, with the nucleus and perikaryal cytoplasm being located near the basal lamina, and with long processes extending throughout the seminiferous epithelium in the interstices between the varieties of germ cells. The nuclei of Sertoli cells contained much euchromatin and a large nucleolus. Their cytoplasm displayed multiple stacks of Golgi cisternae, scattered rough endoplasmic reticulum, numerous tubules and vesicles of smooth endoplasmic reticulum, and mitochondria. In the long processes of Sertoli cells, microtubules were abundant. The characteristic junctional specializations between Sertoli cells were conspicuous in treated rats. As previously described (Flickinger and Fawcett, '67), they consisted of cisternae of 412 CHARLES J. FLICKINGER endoplasmic reticulum lying parallel to the plasma membrane and bundles of fine filaments interposed between the endoplasmic reticulum and the cell surface. Since late spermatids were reduced in number, the junctional specializations of Sertoli cells next to these germinal cells were uncommon. When present, however, they had the normal morphology reminiscent of onehalf of one of the Sertoli-Sertoli specializations (Flickinger and Fawcett, '67). Many large lipid droplets were readily visible in the cytoplasm of Sertoli cells (fig. 10). In contrast to the lipid droplets of normal Sertoli cells which are prominent at stage IX of the cycle of the seminiferous epithelium (Ken- and DeKretser, '75), in treated rats the lipid droplets were numerous in almost all of the profiles of seminiferous tubules. In addition, they frequently were so large in treated animals as to indent the nucleus, and sometimes they appeared to displace other organelles. Although most of the lipid droplets in the seminiferous epithelium were found in the basal cytoplasm of Sertoli cells, a few germ cells contained large lipid droplets as well. The cytoplasm of Sertoli cells also contained numerous lysosome-like structures with a polymorphous content of cellular debris (figs. 8, 10). Parts of spermatids were found in these bodies and necrotic cells were surrounded by the cytoplasm of Sertoli cells (fig. 8), suggesting that spermatids were phagocytosed and digested by the Sertoli cells. The Leydig cells of the interstitial tissue appeared smaller in treated rats than in controls at all the intervals studied. They were not measured at all the intervals, but this was done for the 8-week specimens in an attempt to confirm the impression gained from inspection of sections that the size of Leydig cells in treated animals was reduced. The mean area of a Leydig cell represented in sections of normal animals was 96 f i 2 and of the control, 91 p2. For animals treated with Provera for eight weeks (Experiment 2), the corresponding value was only 24 p 2 , and in rats administered ProVera and testosterone (Experiment 5, testosterone 100 Wg/lOO g/day) it was 25 p2. In both experiments, the difference between the treated and the control and between the treated and the normal is highly significant (P < 0.001). Values similar to those found here for normal and control rats were also obtained for Leydig cells measured in a larger number of control animals in a previous study (Flickinger and Loving, '76). Large amounts of smooth endoplasmic reticulum are characteristic of the ultrastructure of normal Leydig cells. In treated animals, smooth tubules and other normal-appearing organelles remained in the cytoplasm of Leydig cells, but as indicated by the measurements performed with the light microscope, the amount of cytoplasm was less than in normal or control rats. Recovery Rats allowed to recover following treatment with Provera or with Provera plus testosterone for 8 or 16 weeks were killed at intervals of up to 12 weeks after the end of the treatment (table 3). Alterations resembling those of treated animals persisted in the structure of the testis and epididymis of two rats killed three weeks after the end of treatment. By six weeks after the end of treatment, the reproductive organs of two of four animals had a normal histology and ultrastructure. In the other two recovery appeared to have begun with a return of abundant late spermatids in the testis, but the epididymis continued to show alterations. Three animals killed 9 or 12 weeks after cessation of treatment had a normal microscopic structure, including a return of late stages of germ cells in all the seminiferous tubules (fig. 11). As shown in the companion paper (Flickinger, '77) the structure of the epididymis of animals permitted to recover for 9 to 12 weeks also returned to normal. DISCUSSION The observation of great diminution or absence of late spermatids of the acrosome and maturation stages, along with the presence of degenerating spermatids of the cap phase and beyond, suggests that in the testes of rats treated with Provera or Provera and testosterone germ cells develop approximately up to the early spermatid stages 6-7 and then undergo degeneration. In accord with this hypothesis, there was a general lack of morphological alteration in the less mature germ cells, including spermatogonia and spermatocytes. However, as pointed out above, PROGESTIN AND ANDROGEN. I changes in the numbers of earlier stages of germ cells might have gone undetected since the morphological methods employed in this study did not include a quantitative analysis of the different cell types. In addition, it was not possible to identify with certainty the stage of all the necrotic cells, and it is conceivable that some of these might have represented earlier germ cells. In any event, the scarcity of late germ cells helps to account for the small size of the testes and their reduced weight in treated rats. Furthermore, the alterations in the development of spermatids seemingly were of sufficient severity to be responsible in large measure for the infertility of the treated animals. The mechanism by which Provera and testosterone produce alterations in spermatogenesis is not definitely known, but some speculations can be based on the present observations. Provera could suppress spermatogenesis by reducing the release of gonadotropins. Although hormone levels were not measured directly, the reduced size of Leydig cells in treated animals seems likely to be the result of decreased stimulation by gonadotropins. In addition, the suggestion that gonadotropins were reduced is supported by the similarity between the testicular changes in the present study and those after hypophysectomy (Clermont and Morgentaler, '55). Spermatids from stage 8 on were found to degenerate after hypophysectomy, while the slightly earlier stages 6-7 appeared to be the first to degenerate in the present study. In the case of Provera administered alone, diminution in LH levels and consequently in testosterone secretion by the Leydig cells would be expected adversely to affect spermatogenesis, since androgen binds to a cytoplasmic receptor protein (CR) in Sertoli cells or early germ cells (Hansson et al., '74, '75) and is necessary for spermatogenesis to proceed (Steinberger, '71). Although the role of FSH in maintaining spermatogenesis has been debated, FSH does appear to influence the seminiferous tubules (Lostroh, '63; Hall, '70), interacting with Sertoli cells and/or early germ cells to influence their synthetic capacities (Means, '75) and specifically stimulating the synthesis of androgen binding protein (ABP) by Sertoli cells (Hansson et al., '74, '75). Thus a reduction in FSH and conse- 413 quently in ABP synthesis might also affect the seminiferous tubules. The observation that the later stages of spermatogenesis were affected in the present study is of interest with regard to the contention that FSH is necessary for maturation of spermatids beyond stage 15 in the rat (Steinberger, '71; Steinberger and Duckett, '67). Changes in FSH and ABP might help to account for the observation that changes in spermatogenesis persisted in the presence of sufficient exogenous testosterone to maintain the sex accessory glands. It is likely that the seminiferous tubules in ProVera and testosterone-treated rats received less than their normal supply of testosterone, despite the administration of exogenous testosterone sufficient to maintain the sex accessory glands, because the tubules normally receive a greater supply of androgen as a consequence of their proximity to the Leydig cells and because testosterone is accumulated in the seminiferous tubules by ABP (Hansson et al., '74). Thus, if ABP were reduced as a result of diminished FSH, accumulation of testosterone in the tubules would be diminished. Then less testosterone would be available for subsequent binding to the cytoplasmic receptor protein (CR) of Sertoli cells or early germ cells, which is thought to mediate the response of spermatogenesis to androgen (Hansson et al., '74). Previous studies on the influence of progestational agents on the testis have yielded varying results. Progestins, including progesterone and a variety of other agents, have been reported to suppress spermatogenesis in several species (Setty and Kar, '67; Kar et al., '67; Patanelli and Nelson, '59; Ericsson et al., '64; Skinner and Adams, '69; Ericsson and Dutt, '65; Skinner and Rowson, '69; Heller et al., '58, '59; MacLeod, '65), although little influence on spermatogenesis has been reported with some compounds (Jackson, '69; Steinberger, '71). A finding similar to the present observations was that spermatogenesis in men proceeded no further than early spermatids in the presence of norethandrolone (Heller and Clermont, '64). In rats administered progesterone and other progestins percutaneously , spermatogenesis was reported to be arrested at the secondary spermatocyte stage (Setty and Kar, '67). 4 14 CHARLES J. FLICKINGER The fate of at least some of the degenerating and necrotic spermatids appeared to be phagocytosis by Sertoli cells, since lysosome-like structures in Sertoli cells were numerous and contained cellular debris that included identifiable spermatid structures. In some cases, entire necrotic spermatids appeared to lie within Sertoli cells. The suggestion that Sertoli cells take up degenerating spermatids is in accord with the activities of Sertoli cells in phagocytosis of injected particulates (Clegg and MacMillan, '65) and damaged germ cells under a number of deleterious conditions (Lacy and Lofts, '65; Villar et al., '67; Hugon and Borgers, '66; Fkddy and Svoboda, '67; Roosen-Runge and Leik, '68; Black, '71) and their retention of residual bodies (Smith and Lacy, '69; Brokelmann, '63; Dietert, '66). It is not known if phagocytosis by Sertoli cells is the fate of all the spermatids. Some altered germ cells may also have been shed from the seminiferous epithelium and transported to the epididymis to accumulate in the distal part of the cauda epididymidis (see the companion paper, Flickinger, '77). The many lipid droplets in Sertoli cells of treated animals could be the result of accumulation of lipid from ingested germ cells, as may be the case in the accumulation of lipid from ingested residual cytoplasm of spermatids at certain stages of the cycle of the normal seminiferous epithelium (Niemi and Kormano, '65; Kerr and DeKretser, '75). The lipid accumulation might also be due to an alteration in the metabolism of the Sertoli cells themselves, since lipid accumulation is seen in a variety of cells under pathological conditions (Robbins, '74). In any event, an increase in lipid in Sertoli cells has been observed in other cases in which spermatogenesis is disrupted (Lacy, '62; Lacy and Lofts, '65; Collins and Lacy, '69; Chung, '74). The weight of the reproductive organs of rats treated with Provera was less than that of control animals at all the intervals studied. In rats administered testosterone along with the Provera, the weight of the testes and epididymides remained less than those of control animals, as might be anticipated since spermatogenesis remained suppressed. It proved possible to maintain the weight of the ventral prostate and seminal vesicles at a level comparable to controls by the addition of testosterone to Provera, but the dose of testosterone required was greater than initially anticipated. The matter of maintenance of the accessory glands in the presence of ProVera and different amounts of testosterone will be considered further in the companion paper, which deals with the sex accessory glands (Flickinger, '77). The number of animals allowed to recover in the present study is relatively small compared to those killed during the course of treatment, and further tests will be required before definite conclusions on the ability of animals to recover from Provera and testosterone treatment are formed. However, the results available from the present study suggest that recovery of a normal microscopic appearance of the testis is attained between three to six weeks after the end of treatment. Taking into account the length of the cycle of the seminiferous epithelium in the rat (Clermont et al., '59; Steinberger and Steinberger, '75), about 12 days, or nearly two weeks, would be required for spermatids to mature once they were able to proceed normally past spermatid stages 6-7. Since the testis did not return to normal in three weeks, but required between three and six weeks to do so, there is apparently a lag period of several weeks after the end of treatment before spermatids begin to proceed normally past the cap phase. Apparently the testis recovers first, followed by reversal of epididymal alterations, and a return to normal of the entire reproductive tract in 6 to 12 weeks. The results suggest that infertility in animals treated with Provera and testosterone is due in large part to an alteration in spermatogenesis. Specifically this involves changes in the development of spermatids, beginning with the cap phase and resulting in the degeneration of many spermatids at this and subsequent stages. However, as described in the following paper (Flickinger, '77), alterations in the epididymis may contribute also to infertility because of microscopic evidence of masses of debris in the distal cauda epididymidis and indications of uptake of luminal material by the epithelium of the proximal cauda epididymidis. ACKNOWLEDGMENTS The author is indebted to Mrs. Jeannette PROGESTIN AND ANDROGEN. I Charlton and Miss Sharon Odum for technical assistance. This research was supported by a grant from the Population Council (M74.82), a contract with the National Institute of Child Health and Human Development (NOl-HD-1-2506), and a grant from N.I.H. (1 RO1 HD10073-01). 415 1973 Comparative observations on intertubular lymphatics and the organization of the interstitial tissue of the mammalian testis. Biol. Reprod., 9: 500-532. Flickinger, C. J . 1977 The influence of pronestin and androgen on the fine structure of the-male reproductive tract of the rat. 11. Epididymis and sex accessory glands. 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Leik 1968 Gonocyte degeneration in the postnatal male rat. Am. J. Anat., 122: 2 7 5 4 0 0 . Segal, S. J. 1973 Male fertility control studies: An editorial comment. Contraception, 8 : 187189. Setty, B. S., and A. B. Kar 1967 Interruption of spermatogenesis by percutaneous application of steroids. Steroids, 10: 687-698. Skinner, J. D., and C. E. Adams 1969 Some effects of a single injection of chlormadinone acetate or methallibure on sexual function in the pubescent male animal. 11. The rabbit. J. Reprod. Fert., 20: 105-110. Skinner, J . D., and L. E. A. Rowson 1969 Some effects of a single injection of chlormadinone acetate or methallibure on sexual function i n the pubescent male animal. I. The ram. J. Reprod. Fertil., 20: 95-104. Smith, B. U., and D. Lacy 1959 Residual bodies of seminiferous tubules of the rat. Nature, 184: 249-251. Steinberger, E. 1971 Hormonal control of mammalian spermatogenesis. Physiol. Rev., 51: 1-22. Steinberger, E., and G. E. Duckett 1967 Hormonal control of spermatogenesis. J. Reprod. Fertil. (Suppl.), 2: 75-87. Steinberger, E., and A. Steinberger 1975 Spermatogenic function of the testis. In: Handbook of Physiology. Vol. V, Endocrinology. Section 7 , Male Reproductive System. R. 0. Greep and E. B. Astwood, eds. American Physiological Society, Washington, pp. 1-19. Terner, C., and J. MacLaughlin 1973 Effects of sex hormones on germinal cells of the rat testis: a rationale for the use of progestin and androgen combinations i n the control of male fertility. J. Reprod. Fertil., 32: 4 5 3 4 6 4 . Vilar, O., A. Steinberger and E. Steinberger 1967 An electron microscopic study of cultured rat testicular fragments. Z. Zellforsch., 78: 221-233. PLATES PLATE 1 E X P L A N A T I O N OF F I G U R E S Figs. 1-3 Light micrographs of the testis of normal and treated rats. Normal testis. Germ cells in different stages of development are present including primary spermatocytes (P), early spermatids with round nuclei (Q), and late spermatids with elongated and condensed nuclei (S). X 300. Testis of a rat treated with Provera for 16 weeks (Experiment 2 ) . Early stages of germ cells are present, including spermatogonia ( G ) , primary spermatocytes (P), and early spermatids ( Q ) ,but there are no late spermatids with condensed or elongated nuclei. The arrows indicate dense, degenerating cells (fig.8). X 300. Testis of a rat treated with Provera and testosterone for eight weeks (Experiment 5). As i n the case of animals administered Provera alone, the early stages of germ cells are present, including primary spermatocytes (P) and early spermatids (Q), but late spermatids i n the acrusome and maturation phases are virtually absent. Lipid droplets (L) are found near the basal lamina of the seminiferous tubules. A dense degenerating cell is indicated by the arrow. The few profiles of tails i n the lumen may belong to early spermatids or to the small number of germ cells that reach later stages i n treated animals. x 300. 418 PROGESTIN A N D ANDROGEN. I Charles J. Rickinger PLATE 1 419 PLATE 2 E X P L A N A T I O N OF FIGURE 4 420 Low power electron micrograph of the seminiferous epithelium of a rat treated with Provera and testosterone for eight weeks (Experiment 5 ) . Sertoli cell cytoplasm (C), nucleus (N), and some of the characteristic junctional specializations (J) between Sertoli cells are visible, a s well as a large primary spermatocyte (P).The organelles of the Sertoli cell and the spermatocyte have a normal appearance. The presence of a synaptinemal complex (arrow) in the nucleus of the spermatocyte suggests that prophase of the first meiotic division proceeded in this specimen. x 11,500. PROGESTIN AND ANDROGEN. I PLATE 2 Charles J. Flickinger 42 1 PLATE 3 EXPLANATION O F FIGURES 422 5 Basal portion of a seminiferous tubule from a rat treated with Provera and testosterone for eight weeks (Experiment 5 ) . A normal-appearing spermatogonium ( G ) is identified as a small, oval cell with a round nucleus. It lies next to the basal lamina (B) of the tubule. Also present i n this field are parts of a Sertoli cell (C) a n d the cytoplasm of a spermatocyte (P). X 10.000, 6 A portion of a late Golgi phase spermatid (Stage 3 ) from a rat treated with Provera for four weeks (Experiment 2 ) . This cell was not altered b y the treatment. It displays a round nucleus (N), large Golgi apparatus (G), and the acrosomal vacuole which contains a dense acrosomal granule (A). x 18,000. PROGESTIN A N D ANDROGEN. I Charles J. Flickinger PLATE 3 423 PLATE 4 EXPLANATION OF FIGURE 7 424 A c a p phase spermatid from a n animal treated with Provera a n d testosterone for 16 weeks (Experiment 3). In treated animals, some cap phase spermatids such as this retained a normal appearance, but others were necrotic (fig. 8 ) . Those with a normal structure, as i n this example, exhibited a round nucleus (N), prominent Golgi apparatus ( G ) , the characteristic acrosomal cap (A), and a rim of mitochondria around the margin of the cell (M). Frequently a chromatoid body (B) was also visible. X 12,000. PROGES'TIN AND ANDROGEN. I Charles J. Flickinger PLATE 4 425 PLATE 5 EXPLANATION O F FIGURE 8 426 Electron micrograph of a degenerating or necrotic cell in the seminiferous epithelium of a rat treated with Provera and testosterone for eight weeks (Fxperiment 4). The cell is abnormally dense and its organelles are distorted. It can be identified a s a cap phase spermatid, possibly stage 6 or 7. by the shape of the remaining acrosomal vacuole (A). The degenerating cell is surrounded by Sertoli cell cytoplasm (C). At the bottom of the field is a membrane-bounded structure that morphologically resembles a lysosome (D) and also lies within the cytoplasm of a Sertoli cell. N. nucleus. X 13,500. PROGESTIN A N D ANDROGEN. I Charles J. Flickinger PLATE 5 42 7 PLATE 6 EXPLANATION 428 O F FIGURES 9 Portion of a seminiferous tubule of a rat treated with Provera and testosterone for four weeks (Experiment 4). Parts of a late spermatid are present within a mass of cytoplasm, but they lack their usual relation to one another. Organelles present include the condensed nucleus (N), coarse tail fibers (T),and a structure that is not identified with certainty but may be a misshapen acrosome (A). X 16,500. 10 Part of a Sertoli cell of a rat administered Provera for four weeks (Experiment 2 ) . Many large lipid droplets (L) are present in treated animals, and in some instances, as here, they are so large as to indent the nucleus (N) and displace other organelles. A structure resembling a lysosome is also present (D). The length of the bar is 1 fim. X 9,700. PROGESTIN AND ANDROGEN. I Charles J. Flickinger PLATE G 429 PROGESTIN AND ANDROGEN. I PLATE 7 Charles J . Flickinger EXPLANATION OF FIGURE 11 430 Light micrograph of the testis of a rat permitted to recover for nine weeks after the end of treatment for eight weeks with Provera and testosterone (Experiment 5). Numerous condensed nuclei and tails of late spermatids ( S ) are present in the seminiferous epithelium. X 295.