S. PANDEY2. Received April 10, 1969

Transcription

1 BIOLOGY OF REPRODUCTION 1, (1969) The Role of Pituitary and Gonadal Hormones in the Differentiation of Testis and Secondary Sex Characters of the Juvenile Guppy Poecilia reticulata Peters1 S. PANDEY2 Department of Zoology, University of British Columbia, Vancouver, British Columbia Received April 10, 1969 Hypophysectomy of the juvenile guppy prevents the development of both gametogenetic and endocrine tissues of testis as well as the differentiation of secondary sex characters. Administration of methyl testosterone to hypophysectomized juveniles for 8 weeks stimulates the differentiation of the sperm ducts and secondary sex characters but has no effect on either spermatogenesis or the differentiation of interstitial cells. Two doses of methyl testosterone (1:2 X 10 and 1:10) were used but no differences in results were evident. Although these results are based on only five hypophysectomi.zed male juveniles, they are highly consistent and significant when compared with the controls. Details of the technique for the hypophysectomy of the juvenile guppy are described. Vivien (1941) seems to have been the only investigator to use juvenile teleosts (Gobius paganellus) in an attempt to analyze the role of the pituitary in development of sex. This study, however, only demonstrated that hypophysectomy prevented the development of the gonad but provided no details of the regulation of cytogenesis in the testis or the control of secondary sex characters. Yet, juvenile fishes prior to the differentiation of the endocrine system offer unique advantages in attempts to analyze the hormonal regulation of reproductive functions. Tissues at this stage have never been stimulated by the hormones and there are no possibilities of the residual hormones in the organism which may interfere with the analysis of the relationship between the hormone-producing and the target organs. If the technical difficulties of hypophysectomy can be solved, the hypophysectomized 1 Based on a section of a Ph.D. thesis accepted by the Department of Zoology, University of British Columbia; with financial assistance from the National Research Council of Canada through a grantin-aid of research to W. S. Hoar. 2 Present address: Department of Zoology, Patna University, Patna 5, India. juvenile should provide a valuable preparation for analysis of relative roles of pituitary and gonadal hormones in sex differentiation. The technique of hypophysectomy was found to be relatively successful with the juvenile guppies. The present study describes the effects of hypophysectomy and subsequent methyl testosterone treatment on the testis and secondary sex characters of the juvenile guppy Poecilia reticulata Peters. MATERIALS AND METHODS Maintenance of Juvenile Fish Juvenile guppies of both sexes (2-3 weeks old, 9-11 mm in standard length, mg), prior to the differentiation of gonads and secondary sex characters, were used. One week before the operation, the fish were taken from the breeding aquaria and put in aquaria containing 16 liters of continuously aerated fish saline (Young, 1933). All the fish were maintained at a temperature of 25 ± 0.5 C and daily light period of 16 hours and were fed daily with finely ground commercial trout fish food (J. R. Clark Co). A group of intact juveniles was killed as initial controls at the time another group was hypophysectomized. In Exp. 1 the juveniles were hypophysectomized and in Exp. 2 the hypophysectomized juveniles were treated with methyl testosterone. These two experiments were terminated after 6 and 8 weeks, respectively; by this time, the control 272

2 PITUITARY AND GONADAL HORMONES 273 sham-operated juveniles had differentiated into adults. Procedure of Hypophysectomy Juvenile guppies were anesthetized with 1:800 MS222 (tricaine methane sulphonate, Sandoz). The fish was placed with the ventral surface upwards on a small piece of soft plastic sponge fixed in the middle of a wax-filled rectangular plastic tray. The fish was immersed in fish saline and secured in place by a thin strip of soft plastic sponge stretched across the ventral surface just posterior to the gills. All subsequent procedures were carried out under a dissecting microscope (x 10). A transverse incision just behind the lower lip was extended on both right and left sides to the branchiostegal membrane. The whole gular area connecting the lower jaw was deflected back. After removing the mucus membrane covering the palate, the parasphenoid bone was broken with a pair of blunt forceps. The pituitary was then picked up with a pair of fine forceps. It was always ascertained under the microscope that all three lobes of pituitary were completely removed. Sham-operated Fsh received the same treatment without pituitary removal. After the operation, the fish was left in fish saline of the operation tray until it started swimming actively. It was then transferred to an aquarium containing fish saline. The wound healed in days and no infection was ever noticed. Mortality amounted to 30-35% during the operation; a further mortality of 25-30% occurred in the 2 weeks following the operation. Methyl Testosterone Treatment The treatment of hypophysectomized juvenile guppies with methyl testosterone was begun 1 week after the operation. Two different concentrations of methyl testosterone (California Corporation for Biochemical Research, Los Angeles) were used: 1:2 X 106 (8 mgm of testosterone in 16 liters of fish saline every week) and 1:10 (1.6 mgm of testosterone in 16 liters of fish saline every week). Methyl testosterone was added as a suspension to the aquaria. Four liters of fish saline were renewed in each aquarium every week. The testosterone treatment was continued for 8 weeks. Hypophysectomized juvenile guppies without testosterone treatment and sham-operated juveniles were the two types of controls. Histology Fish were killed by anesthesia. The testes of the sham-operated juveniles (mature by the end of the experiment) were removed and fixed in Bouin s fluid. The hypophysectomized and testosteronetreated juveniles, because of their small size, were fixed entire in formic acid-bouin solution and left for 7 days in fixative to decalcify. Testes of shamoperated and whole bodies of hvpophysectomi.zed fish were processed in the usual way; longitudinal sections cut at 5.t were stained with Ehrlich s hematoxylin and eosin. Measurements To compare the histological picture of the testes cf different experimental groups, the total number of cysts or acini of each spermatogenetic stage in the median sagittal section was counted and percentage of each spermatogenetic stage calculated. The total number of efferent ducts in the median sagittal section was also counted. The total width and lumina of five efferent ducts just before they join to form the main sperm duct were measured in one section of each testis of different experimental groups. The height of the epithelial cells and the width of lumen of the main sperm duct in the median sagittal section of each testis were measured at five different places along the entire length of the main sperm duct. Statistical Method The data were analyzed bs analysis of variance. The meats for each column (see Table 1) were compared bs Tukey s %V (Steel and Torrie, 1960). RESULTS Experiment 1 Sham-Operated Control Fish By the end of the experiment the shamcperated juveniles had grown to adult size and differentiated into males and females. The structure of the testis and secondary sex characters of adult guppy have already been described (Pandey, 1969a: see also Figs. 1, 2b, 3, 4, 5; Table 1). Juvenile Control Fish (Sacrificed at the Time Other Juveniles Were Hypophvsectomized) The juvenile testis consists of two lobes connected by a bridge of stroma cells. The testis is surrounded by a thin, squamous peritoneal membrane and is situated in the posterior part of the body cavity, ventral to the airbladder and dorsal to the pancreas and the coils of intestine.

3 274 PANDEY TABLE 1 EFFECTS OF HYPOPHYSECTOMY AND TESTOSTERONE TREATMENT ON EFFERENT Ducrs AND MAn, SPERM DUCT OF THE JUVENILE GUPPY Efferent Main sperm duct No. 1 2 Juvenile Treatment Number1 ducts Width (s)2 Lumen ()2 Epithelial Experiment shamoperated 18.8 ± ± ± ± ± 9.55 Juvenile* Juvenile control 7.8 ± 0.58b ± 0.74b 1.22 ± 0.lOb 2.70 ± 0.l5b 1.66 ± 0.lSb hypophysectomized 7.2 ± 0.37b ± 0.63b 1.33 ± 0.llb 2.52 ± O.12b 1.76 ± O.13b Juvenile shamoperated 19 ± 0.71C ± 445t ± 354e ± 0.94C ± 6.49c Juvenile hypophysectomized 8 ± ± ± 0.13d 2.92 ± O.17d 1.58 ± 0.13d Juvenile and hypophysectomized testosteronetreated 33 ± ± ± 107t ± 0.85e ± 1.68e 1 Mean of five counts ±Sx (one in each of five fish). 2Mean of 25 counts ±Sx (five in each of five fish). icantly different (p <.01) except dtgroups (lumen (p <.05). Only spermatogonia are found in the testis (Figs. 1, 6). These occur in small cysts at the periphery of testis; the center of the testis is filled with stroma cells (Fig. 7). Mitotic divisions were not observed in the spermatogonial cysts. Spermatogonia are oval with distinct cellular membranes and homogenous cytoplasm except for the presence of occasional fine basophilia. There is usually a single nucleolus. Stroma cells, on the other hand, have no distinct cellular membranes. Their nuclei are elliptical and each contains an irregular and coarse reticulum. Interstitial cells and Sertoli cells are not evident in these testes. The efferent ducts, which are formed during development by the rearrangement of stroma cells, are small and have narrow lumina (Fig. 7; Table 1). The efferent ducts from the two lobes of testis open at the posterior end into cells ()2 Lumen ()2 * Killed at the time other juveniles were hypophysectomized. Experiment 1. abgroups with different superscripts are significantly different (p <.01) ; Experiment 2. CdefGroups with different superscripts are signifof efferent ducts) which are significantly different a main sperm duct with a narow lumen (Fig. 8; Table 1); the latter empties into the urogenital sinus. The epithelial cells lining the main sperm duct are squamous. No secondary sex characters are present in these juveniles. The anal fin is in the sexually indifferent condition (Fig. 2a). Hypophysectomized Fish No change is evident in the histology of the testis 6 weeks after hypophysectomy (Figs. 1, 6, 7, 8; Table 1); secondary sex characters also remain undifferentiated (Fig. 2a). Experiment 2 Hypophysectomized Control and Sham- Operated Control Fish The structures of the testes and secondary sex characters of hypophysectomized control

4 PITUITARY AND GONADAL HORMONES CONTROL #{149} 80 SHAM-OPERATED U 0 I- z Lu U a LI a SPG SPC SPD FIG. 1. Percentage of different stages of spermatogenesis in juvenile control, juvenile hypophysectomized and juvenile sham-operated guppies. The values given are means of five observations ± standard error. Test of significance was based on total number of cysts containing each stage of spermatogenesis (row X column contingency table) p. <.001-between juvenile control and juvenile sham-operated; juvenile hypophysectosnized and juvenile sham-operated. Keys: SPG-Spermatogonia; SPC-Spermatocytes; SPD-Spermatids; SPM-Sperm; SPR-Spermatophores. and sham-operated control fish are similar to those described in Exp. 1 (see Figs. 2a, 2b, 3,4, 5,6, 7,8, 11; Table 1). Testosterone-Treated Fish Treatment of undifferentiated gonads of hypophysectomized fish with methyl testosterone neither stimulates mitotic division in the spermatogonial cysts nor induces development of later spermatogenetic stages such as spermatocytes (Fig. 11), nor do the interstitial cells and the Sertoli cells become evident. The most significant change is the marked differentiation of the sperm ducts. The total number, width and the lumina of the efferent ducts increase significantly (Fig. 9; Table 1). The lumen of the main sperm duct becomes very wide and the epithelial cells lining sperm duct become tall and columnar (Fig. 10; Table 1). SPM SPR T Changes associated with the transformation of the anal fin into a gonopodium are initiated but the sexual modifications of the fin are never complete. Ray 3 becomes thick (bone deposition) and develops a small hood which does not extend beyond the tip of Ray 3 (as in a normal gonopodium). The segments of Rays 3 and 4 do not develop spines, which are characteristically well developed in a normal gonopodium. The small hook at the tip of Ray S is considerably smaller than the hook of a normal gonopodium (compare Figs. 2b and 2c). The lipophores appear as two narrow bands, one on the upper margin and the other on the lower margin of the caudal fin. These two bands join at the posterior margin of the caudal peduncle and extend onto the caudal peduncle as a single band. However, the development of this secondary sex character, like the gonopodium, is less complete than that of normal males. The

5 (a) L I- FIG. 2. Anal fin of the guppy. (a) anal fin of juvenile control; fin-rays are numbered from cranial (ventral) to caudal (dorsal) border of fin, the line below the figure represents 1 mm; (b) modified anal fin (gonopodium) of adult male which was sham-operated as juvenile; (c) anal fin of hypophysectomized juvenile treated with testosterone (Drawings by Mrs. J. Fenwick). 276

6 rt.:ii FIG. 3. Sagittal section of testis of adult guppy which was sham-operated as juvenile showing different stages of spermatogenesis and efferent ducts (ED). Hemotoxylin and eosin. X200 (see Fig. 1 legend for Key to lettering). Fin. 4. Sagittal section of testis of adult guppy which was sham-operated as juvenile showing spermatophores in efferent ducts (ED). Note tall epithelial cells (EC) of efferent ducts. Hemotoxylin and eosin. X200. Fin. 5. Sagittal section of testis of adult guppy which was sham-operated as juvenile showing spermatophores in main sperm duct (MD). Hemotoxylin and eosin. X200. Fin. 6. Sagittal section of testis of juvenile guppy showing only spermatogonia (SPG). Hemotoxylin and eosin. X I

7 278 PANDEY brightness of the lipophores was assigned structure of the testes or secondary sex char- ++ in the arbitrary scale described before acters of hypophysectomized fish exposed to (Pandey, 1969a). two different concentrations of methyl testos- No differences were evident either in the terone (1:2 X 10 and 1: 10). FIG. 7. Sagittal section of testis of juvenile guppy showing spermatogonia (SPG), efferent ducts (ED) and stroma cells (ST). Hemotoxyline and eosin. X900. FIG. 8. Sagittal section of testis of juvenile guppy showing main sperm duct (MD) lined by squamous epithelial cells. Note reduced lumen of main sperm duct. Hemotoxylin and eosin. X900. Fin. 9. Sagittal section of testis of hypophysectomized juvenile treated with testosterone showing efferent ducts (ED). Note large lumen of efferent duct. Hemotoxylin and eosin. x900. Fin. 10. Sagittal section of testis of hypophysectomi.zed juvenile treated with testosterone showing main sperm duct (MD). Note large lumen and columnar epithelial cells of main sperm duct. Hemotoxylin and eosin. X900.

8 PITUITARY AND GONADAL HORMONES 279 HYPOPHYSECTOMIZED #{149}TESTOSTERONE 80 SHAM-OPERATED LI (9 4 z LI 0 a LI a nfl SPG SPC SPD SPM SPR FIn. 11. Percentage of different stages of spermatogenesis in juvenile hypophysectomized, juvenile hypophysectomized and testosterone treated; juvenile sham-operated guppies. The values given are means of five observations ± standard error. Test of significance was based on total number of cysts containing each stage of spermatogenesis (row x column contingency table); p <.001-between juvenile sham-operated and juvenile hypophysectomized; juvenile sham-operated and juvenile hypophysectomized + testosterone treated (see Fig. 1 legend for Key to lettering). DISCUSSION This study provides definite evidence concerning the respective roles of the pituitary hormones and the gonadal steroids in spermatogenesis, the differentiation of the sperm ducts and the appearance of the secondary sex characters. These aspects will now be considered Spermato separately. genesis Hypophysectomy of the juvenile guppy prevents the development of both gametogenetic (spermatogenesis) and endocrine tissues (interstitial cells) of the testis. The testes of juveniles 6 weeks after hypophysectomy contain only spermatogonia and no mitotic divisions are apparent. Hypophysectomized larval amphibians, Triturus, Rana, and Bufo have shown similar results (Chang and Witschi, 1955). Likewise, in the adult guppy, hypophysectomy prevents the mitotic divisions in the spermatogonia and their transformation into spermatocytes (Pandey, 1969a). T Administration of methyl testosterone to the hypophysectomized juvenile guppy does not appear to stimulate spermatogenesis or cause the differentiation of interstitial cells. Since the pituitary was removed 3 or 4 weeks after birth before gonadotrophs had become granulated (presumably before gonadotropins had been secreted) (Sokol, 1961), the findings suggest that the initial development of the testis in the guppy is under the direct control of the pituitary gonadotropins; androgen is not required. In intact juvenile guppies and also in embryos (within pregnant guppies), however, treatment with testosterone propionate or methyl testosterone produces a higher male: female sex ratio and stimulates the development of the testes (Eversole, 1941; Miyamori,

9 280 PANDEY 1961; Dzwillo, 1962; Clemens et al., 1966). The mechanism of action of testosterone in theee ca!es is not clear. In the present work, administration of methyl testosterone to the hypophysectomized juveniles has not altered the sex ratio nor, as mentioned above, stimulated the development of testes. Thus, it seems possible that testosterone acted in the intact juveniles and embryos via the pituitary causing an early release of gonadotropins of the male type. A similar action of testosterone has also been suggested in rats (J#{216}rgensen, 1968). In the adult guppy, on the other hand, the same dose and duration of methyl testosterone treatment as used in the present work induce spermatogenesis in the absence of the pituitary (Pandey, 1969b). Similar results have been obtained in other teleosts (Lofts et al., 1966; Sundararaj and Nayyar, 1967). Thus, it appears that while in adult teleosts, testosterone alone can induce spermatogenesis, this role is played only by the pituitary in the juvenile guppy. Perhaps, the initiation of spermatogenesis (during early testis development) is under the direct control of the pituitary, whereas the subsequent maintenance of spermatogenesis is under the control of androgen produced by the interstitial cells. Hypophysectomy of adult teleosts stops the androgen production since interstitial cells become regressed; spermatogenesis, therefore, is not maintained. Testosterone treatment may then compensate for this loss in androgen production following hypophysectomy and thus induce spermatogenesis. Differentiation of Sperm Ducts Hypophysectomy prevents the normal development of the sperm ducts. Methyl testosterone treatment of the hypophysectomized juvenile brings about their complete differentiation. Likewise, the regressed sperm ducts of the hypophysectomized adult guppy and Fundulus heteroclitus are restored to normal following methyl testosterone administration (Pandey, 1969b; Lofts et al., 1966). Thus, it seems likely that the differentiation and maintenance of the sperm ducts, unlike the gametogenetic and endocrine tissues of the juvenile testis, depend solely on androgens; gonadotropin secretion is not required. On the other hand, the Sertoli cells are neither differentiated in hypophysectomized juveniles nor in methyl testosterone-treated hypophysectomized juveniles. The differentiation of Sertoli cells may therefore be under the direct control of pituitary gonadotropins. Secondary Sex Characters Secondary sex characters including both the lipophore pigments and the gonopodium, are absent in the juvenile guppy; the juvenile appearance is maintained in the hypophysectomized juveniles until the end of experimental period (6 weeks). During this period, the sham-operated juveniles developed into adult males with distinct secondary sex characters. When the hypophysectomized juveniles are treated with testosterone, the secondary sex characters (gonopodium and lipophores) become differentiated but the differentiation is not complete. Turner (1960) showed that increasing concentrations of steroid hormones are required for the complete development of secondary sex characters in cyprinodont fishes. Thus, it seems likely that the incomplete differentiation of secondary sex characters following methyl testosterone treatment in the present experiment is due to the fact that a continuing uniform dose was used. Alternatively, the incomplete differentiation may be due to degree of dissimilarity between the synthetic exogenous androgen and the naturally occurring endogenous androgen or that the exogenous androgen cannot lead to complete differentiation of secondary sex characters in the absence of pituitary gonadotropins. ACKNOWLEDGMENTS The author thanks Dr. W. S. Hoar for his guidance, support and encouragement and for his help in the preparation of the manuscript. Thanks are also due to Drs. T. J. Lam and J. F. Leatherland for criticizing the manuscript. The author is grateful

10 PITUITARY AND GONADAL HORMONES 281 to the authorities of Patna University for granting study leave. REFERENCES CHANG, C., AND WITSCHI, E. (1955). Independence of adrenal hyperplasia and gonadal masculinization in the experimental adrenogenital syndrome of frogs. Endocrinology 56, CLEMENS, H. P., MCDER1VIITT, C., AND INSLEE, T. (1966). The effects of feeding methyl testosterone to guppies for sixty days after birth. Copeia 1966, DZWILLO, V. M. (1962). Uber k#{252}nstliche Erzeugung funktioneller M#{228}nnchen weiblichen Genotype bei Lebistes reticulatus. Biol. Zentralbl. 81, EVER5OLE, W. J. (1941). The effects of pregneninolone and related steroids on sexual development in fish (Lebistes reticukitus). Endocrinology 28, J#{216}RGENSEN, C. B. (1968). Chemical correlation and control. In Animal Function: Principles and Adaptations (M. S. Gordon, ed). Macmillan, New York. LOFTs, B., PICKFORD, G. E., AND ATZ, J. W. (1966). Effects of methyl testosterone on the testes of a hypophysectomized cyprinodont fish, Fundulus heteroclitus. Gen. Corn p. Endocrinol. 6, MIYAM0RI, H. (1961). Sex modification of Lebistes reticulatus induced by androgen administration. Zool. Mag. 70, PANDEY, S. (1969a). Effects of hypophysectomy on the testis and secondary sex characters of the adult guppy Poecilia reticulate Peters. Can. I. Zool. 47 (in press). PANDEY, S. (1969b). Effects of methyl testosterone on the testis and secondary sex characters of the hypophysectomized adult guppy Poecilia reticulata Peters. Can. J. Zool. 47 (in press). SOKOL, H. W. (1961). Cytological changes in the teleost pituitary gland associated with the reproduction cycle. J. Morph. 109, STEEL, R. G. D., AND TORRIE, J. H. (1960). Principles and procedures of statistics. McGraw-Hill, New York. SUNDARARAJ, B. I., AND NAYYAR, S. K. (1967). Effects of exogenous gonadotrophins and gonadal hormones on the testes and seminal vesicles of bypophysectomized catfish, Heteropneustes fossiis (Bloch). Gen. Comp. Endocrinol. 8, TURNER, C. L. (1960). The effects of steroid hormones on the development of some secondary sexual characters in cyprinodont fishes. Trans. Amer. Micro. Soc. 79, VIVIEN, J. H. (1941). Contribution a l #{233}tude de Ia pbysiologie hypophysaire dans ses relations avec l appareil genital, Ia thyroide et les crops suprar#{233}naux chez les poissons S#{233}laciens et T#{233}l#{233}ost#{233}ens Scyliorhinus canicula et Gobius paganellus. Bull. Biol. Fr. BeIg. 75, YOUNG, J. Z. (1933). The preparation of isotonic solutions for use in experiments in fish. Publ. Staz. Zool. Napoli. 12,