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Effects of human chorionic gonadotropin and salmon gonadotropin on testis in hypophysectomized toads (Bufo bufo bufo L.)
GENERAL
AND
COMPARATIVE
36, 371-379 (1978)
ENDOCRINOLOGY
Effects of Human Chorionic Gonadotropin and Salmon Gonadotropin on Testis in Hypophysectomized Toads (Bufo bufo bufo L.) K. KUMAR
GUHA’
AND C. BARKER
JQ~RGENSEN
Zoophysiological Laboratory A, August Krogh Institute, Universitetsparken 13, DK-2100 Copenhagen, Denmark Accepted July 10, 1978 The effects of gonadotropin on spermatogenesis and on histology and function of interstitial tissue were studied in male toads after extirpation of the’ pars distalis of the pituitary gland. Four weeks after hypophysectomy, 5 IU of HCWOO g body weight practically normalized spermatogenesis and stimulated the interstitial tissue as judged from the histological appearance of the tissue and the development of thumb pads. The dosage needed to restore normal histological differentiation of seminiferous tubules, spermatogenesis, and interstitial tissue increased with increasing deterioration of normal testicular structure and function following hypophysectomy. In the highly dedifferentiated testes, 8 weeks after hypophysectomy, it was possible to distinguish between trophic effects of HCG treatment, including restoration of normal testicular histology and spermatogenesis and of the intersritial tissue, and effects on functions such as spermiation and secretion of androgen. Salmon gonadotropin, at a daily dosage of 20 &IO0 g body weight, only slightly stimulated spermatogenesis and the interstitial tissue.
We have shown previously (Guha and Jargensen, 1978) that in adult male toads, Bufo bufo bufo, hypophysectomy initially inhibits normal proliferation and maturation of cells in the secondary spermatogonial cysts, thus interrupting the normal spermatogenetic process. Several weeks after the seminiferous tubules start to disintegrate and become replaced by connective tissue. The pituitary gland thus exerts both special gonadotropic functions and more general trophic functions on the tissue of the testis. The question arises whether these different types of function are due exclusively to gonadotropin(s), or whether other pituitary hormones are active in maintaining the normal structure and function of the toad testis. In the pituitary gland of the frog Rana catesbeiana, two gonadotropins related to i Present address: Department of Anatomy, versity of Kuopio, 70101 Kuopio 10, Finland.
Uni-
mammalian FSH and LH have recently been identified (Licht and Papkoff, 1974; Papkoff et al., 1976). The amphibian hormones are not yet avaiIabie for general use (Muller, 1976). However, during work on the gonadotropic effect of human chorionic gonadotropin (HCG) in female toads it was observed incidentally that in an animal possessing one ovotestis small doses of HCG strongly stimulated spermatogenesis within the male parts of the bisexual gonad (unpublished observation). It was, therefore, found of interest to treat hypophysectomized male toads with HCG in order to see to what extent the hormone was capable of restoring the normal structure and function af regressed testes. Some experiments were also made with salmon gonadotropin. In another teleost, the carp, the gonadotropin that has been isolated seems to be chemically related to the LHs, and thus to HCG (Fontaine and Burzawa-Gerard, 1977). A preliminary report of the results has been published (Guha, 1976). 371
0016~6480/78/0363-0371$OI .00/O Copyright 0 1978 by Academic Press, Inc. All rights 3f reproduction in any form reserved.
372
GUHA AND JORGENSEN
MATERIAL
AND METHODS
Experiments were made on adult male toads kept at 22-23”, and exposed to 12 hr of light alternating with 12 hr of darkness. The toads were placed in groups of up to 10 in plastic boxes (36 x 55 cm) with access to water. They were fed mealworms un libitum for periods of 24 hr three times a week. The pars distalis of the pituitary gland was extirpated as previously described (van Dongen et al., 1966), and these toads are referred to below as “hypophysectomized.” The HCG preparation used was Physex, Leo. The effects of hypophysectomy, and subsequent treatment with gonadotropin, on testicular structure and function were evaluated as previously described (Guha and Jorgensen, 1978). The hypophysectomized toads were injected with 10 IU of ACTH (Orthana) three times a week in order to maintain the animals’ health (Jorgensen and Larsen, 1963). Three experiments were made. One experiment started in March, using 31 toads collected the previous autumn and stored at about 5”. The toads were acclimated for 2 weeks in the laboratory before hypophysectomy, in April. Four weeks after the operation HCG was injected SC daily for 4 weeks in doses of 2, 5, 10, or SO IU/lOO g body weight. The second experiment included 51 toads collected in the breeding ponds in spring and stored afterward in a refrigerator. The toads were acclimated as above for 2 weeks before hypophysectomy in July. Starting 6 weeks after the operation, HCG was injected daily for 4 weeks in doses of 0.5, 1, 2, or 5 IU/lOO g body weight. Salmon gonadotropin was injected daily in a dose of 20 pg/lOO g. In the third experiment 35 toads were collected in spring and acclimated for 5 weeks before hypophysectomy in July. This experiment was designed to study immediate and delayed effects of gonadotropin administered for varying periods of time. Ten international units (IU) of HCGilOO g were injected daily for periods of 5, 10, or 20 days, and the effects on the testes were studied from I day to 5 weeks after the discontinuation of the HCG treatment.
RESULTS
Effects of HCG Treatment 4 Weeks after Hypophysectomy Figures 1 and 2 show the effect, on spermatogenesis, of 4 weeks of treatment with HCG, starting 4 weeks after hypophysectomy. In the untreated hypophysectomized controls secondary spermatogonia had almost disappeared, and later spermatogenetic stages were scarce. Even
the smallest dosage used, 2 IU of HCG, appreciably stimulated spermatogenesis, and maximum effects were reached with daily doses of 5-10 IU. At this dose level the numbers of cysts from primary spermatogonia to spermatids were about the same as in the normal controls, i.e., spermatogenesis had become normalized. The relationship between dose of gonadotropin and number of cells produced per spermatocyst is shown in Fig. 3. It seems that the mean number of cells in the cysts of primary spermatocytes was higher in the groups of toads receiving 5 or 10 IU of HCG, when compared with those receiving 2 or 50 IU. Thus, the highest dose level tended to be less efficient in stimulating spermatogenesis, both with respect to number of spermatocysts formed and of cells produced within the cysts. The effect of the HCG treatment on spermiation increased with increasing dosage, and 50 IU practically depleted the seminiferous tubles of sperm bundles (Fig. 1). The effect of HCG treatment for 4 weeks on the interstitial tissue is summarized in Table 1. It may be seen that extirpation of the pars distalis reduces androgen secretion to insignificant levels, as judged from the absence of the secondary sexual character, the thumb pads; these had completely regressed in the hypophysectomized controls, whereas in the unoperated controls they were well-developed. Two international units of HCG daily stimulated the interstitial tissue and restored the thumb pads; 10 IU of HCG daily produced maximum effects both on the interstitial tissue and the thumb pads. Effects of HCG Treatment 6 Weeks after Hypophysectomy In the previous experiment the threshold dosage of HCG needed to stimulate testes in hypophysectomized toads was below 2 IWO0 g body weight. The effects of a lower range of HCG doses were therefore
EFFECTS
7 61
OF GONADOTROPIN
ON TOAD
TESTIS
373
Hypophysectomized controls
2 i.u. hCG
5 i.u. hCG
FIG. 1. Effect of 4 weeks of treatment with HCG on spermatogenesis in male toads hypophysectomized for 4 weeks prior to treatment. (March-May). 0, primary spermatogonia; I. secondary spermatogonia; II, primary spermatocytes: III, secondary spermatocytes; IV, spermatids; V, sperm bundles attached to Sertoli cells. Figures above or in bars indicate SEM.
studied in toads that had been hypophysectomized for 6 weeks. In addition, the effectiveness of salmon gonadotropin was tested. Figure 4 shows that at the end of the experiment spermatogenesis had stopped completely in the hypophysectomized controls. Even the smallest doses, 0.5 and 1 IU of HCG, seemed to increase the number of primary spermatogonial cysts, but the spermatogenetic effect was small. Spermatogenesis was clearly *stimulated by 2 and 5 IU, but the number of spermatocysts within the seminiferous tubules was far from normalized (Figs. 4 and 5). Also 20 pg
of salmon gonadotropin daily per 100 g body weight exerted only a slight spermatogenetic effect, comparable to that of 1 IU of HGG. The spermiation effect increased with increasing doses of HCG (Fig. 4). At the dose levels of 2 and 5 IU the effects were about the same as in the previous experiment, when expressed as the percentage reduction in number of sperm bundles per tubule section, using the hypophysectomized controls as a reference. Salmon gonadotrop& did not induce spermiation. The effects of the two gonadotropins on the interstitial tissue are shown in Table 2.
GUHA AND JQlRGENSEN
%
11
N
H
2
5 10 50 i.u. hCG
FIG. 2. Effect of 4 weeks of treatment with HCG on spermatogenesis in male toads hypophysectomized 4 weeks prior to treatment. Ordinate: total number of spermatocysts from stage of primary spermatogonia to spermatids. N, normal controls; H, hypophysectomized controls. Vertical lines indicate twice the SEM.
At the time of extirpation of the pars distalis in July, the thumb pads were only moderately developed in most animals. When the hormone treatment began 6 weeks later, the thumb pads were well developed in most intact toads and maximally regressed in the hypophysectomized controls. After a further 4 weeks, the thumb pads were fully developed in the unoperated controls (Table 2). Also, the interstitial tissue was highly developed in most of the intact toads at the end of the experiment, whereas in the hypophysectomized toads the interstital tissue was histologically regressed. Daily doses of 0.5 IU of HCG for 4 weeks had no effect on appearance and function of the interstitial tissue. Response in all animals of a group was only observed with 5 IU, and the effect was smaller than in the previous experiment (Table 2). Twenty micrograms of salmon gonadotropin slight-
60
Normal
controls
LO 20
Numbers
of cells
per sectioned
cyst
FIG. 3. Relationship between dosage of HCG and frequency distribution of numbers of cells in sections of cysts of primary spermatocytes. Hypophysectomized toads treated 4 weeks with HCG (MarchMay).
ly stimulated the interstitial tissue histologically, but did not stimulate thumb pad development. Effects of HCG Treatment Hypophysectomy
8 Weeks after
The two previous experiments showed differences in sensitivity of the testes toward HCG that might be related to differences in the duration of the period that lapsed between extirpation of the pars distalis and the treatment with gonadotropin. It is suggested that the dosage of gonadotropin, or duration of treatment, needed to restore normal spermatogenesis and development of the interstitial tissue increased with progressing deterioration of testis structure and function after hypophysectomy. An experiment was, therefore, made in which HCG treatment was postponed until 8 weeks after extirpation of the pars distalis, and the duration of treatment was varied from 5 to 20 successive days.
EFFECTS
TABLE
OF
GONADOTROPIN
1
EFFECTS OF 4 WEEKS OF HCC TREATMENT ON HISTOLOGICAL APPEARANCE OF INTERSTITIAL TISSUE AND THUMB PAD DEVELOPMENT IN TOADS TREA-CED 4 WEEKS AFTER HYPOPHYSECTOMY (APRIL-MAY)~
Stages Groups Intact controls Hypophysectomized 2 IU of HCG 5 IU of HCG IO IU of HCG 50 IU of HCG
0 1 2 3 1.t. T.p. 1.t. T.p. 1.t. T.p. 1.t. T.p. 1.t. T.p. 1.t. T.p.
5 5 5 5 5 3 2 4 1 3 2 5 1 1 3 6 1 5
u I.t., interstitial tissue; T.p., thumb pads. Staging of development of interstitial tissue: 0, very few cells, with pycnotic nuclei; 1, few cells, with slender and shrunken nuclei; 2, many cells, with round and medium-sized nuclei; 3, numerous cells, with round and large nuclei. Staging of thumb pad development: 0, yellowish thumb pads lacking papillae; 1, yellowish with small papillae; 2, light brown with medium-sized papillae; 3, dark brown with large papillae. Figures indicate numbers of toads.
Eight weeks after hypophysectomy primary spermatogonial cysts were practically the only normal spermatogenetic stage present in the seminiferous tubules (Fig. 6A). Sometimes sperm still remained in bundles, but the Sertoli cells carrying the bundles were disintegrating. Moreover, there was extensive histological dedifferentiation of the tubules, which became replaced by connective tissue. After five injections of 10 IU of HCG/lOO g body weight the number of primary spermatogonial cysts had about doubled (Fig. 6B) and had reached the level typical of intact toads (Figs. 1 and 4), but only few secondary cysts had been produced. Two weeks after the discontinuation of the treatment, the number of primary sper-
ON
TOAD
TESTIS
37s
matogonial cysts was still high, and the secondary spermatogonial cysts were still scarce. No further progress in the spermatogenetic process had been made, al1 subsequent stages being absent. Treatment for 10 days resulted in higher numbers of primary and secondary spermatogonial cysts, with maximum effects 2 weeks after the discontinuation of the treatment (Fig. 6C). Scattered cysts of primary spermatocytes were also observed, indicating that a few cysts of secondary spermatogonia had entered meiosis. However, 5 weeks after the end of the HCG treatment most of the secondary spermatogonial cysts had degenerated, without giving rise to a spermatogenetic wave, and the number of primary spermatogonial cysts was, again, decreasing. Twenty days of treatment with HCG induced a spermatogenetic wave that after 2 weeks had reached the meiotic stages. Again the effect peaked after the discontinuation of the treatment (Fig. 6D). The cysts contained cells in normal numbers. Disorganized seminiferous tubules infiltrated with connective tissue were not observed after 20 days’ treatment with HCG, which thus presumably caused redifferentiation of the deranged parts of the testes. The effect of HCG on the interstitial tissue is shown in Table 3. Five days of treatment stimulated the interstitial tissue, 10 days of treatment resulted in slight to moderate development of the thumb pads in about half of the toads, and 20 days of treatment resulted in thumb-bad development in most of the toads. The response of the interstitial tissue toward the ‘CG treatment tended to increase after the discontinuation of the injections. However, 5 weeks after the 10 days of treatment, the interstitial tissue had once more completely regressed (Table 3). DlSCUSSfQN The testes of the toads in the three experiments differed in degree of dysfunction and
376
GUHA AND JORGENSEN
9 a 7
Normal
controls
, Hypophysectomized controls
1-I
6 5 I
d
d
20 pg salmon gonadotropin
d
3 2 I
i 0
1 11 JJJ IV V
0.5 i.u. hCG
I
0
J
JJ JJJ IV
V
0
J
JJ JJI IV
V
5 i.u. hCG
1 i.u. hCG I
0
J
II JJJ IV
V
0
J
JJ 111 IV
V
0
J
11 IJJ IV
V
0
J
JJ JJJ IV V
FIG. 4. Effect of 4 weeks of treatment with gonadotropin on spertnatogenesis in male toads hypophysectomized 6 weeks prior to treatment (July-September). For further explanation, see legend to Fig. I. TABLE
2
EFFECTS OF 4 WEEKS OF GONADOTROPIN TREATMENT ON HISTOLOGICAL APPEARANCE OF INTERSTITIAL TISSUE (Lt.) AND THUMB PADS (T.p.) IN TOADS TREATED 6 WEEKS AFTER HYPOPHYSECTOMY (JULY-SEPTEMBER)” Stages
Groups Intact controls Hypophysectomized 0.5 IU of HCG
H 20 0.5 1
s%
2
5
i.u. hCG
FIG. 5. Effects of 4 weeks of treatment with gonadotropin on spermatogenesis in male toads hypophysectomized 6 weeks prior to treatment. Ordinate: total number of spermatocysts from stage of primary spermatogonia to spermatids. N, normal controls; H, hypophysectomized controls; S.G., salmon gonadotropin. Vertical lines indicate twice the SEM.
1 IU of HCG 2 IU of HCG 5 IU of HCG 20 pg of salmon gonadotropin
0 I 1.t. T.p. I.K. T.p. 1.t. T.p. 1.t. T.p. 1.t. T.p. 1.t. T.p. 1.t. T.p.
2 3
1 2 7 10 5 5 6 6 1 2 1 2
4 3 2 I 2 1 5 1 5 1 5 6
u For explanation of symbols and staging, see footnote to Table 1.
EFFECTS
lzr-
:, .5
6-
A
OF
GONADOTROPIN
ON
TOAD
TESTIS
377
12 controls
6-
0.42
-
s 0 I 0 I 1 day 2 weeks nfter 5 x 10 IU HCG
1.50
1
1.25
2.40
2.00
6
1 day 2 weeks after 10 x 10 Ill
5 weeks HCG
1 day 2 weeks after 20 x 10 IU HCG
FIG. 6. Effect of HCG treatment for various periods of time on spermatogenesis in long-term hypophysectomized toads (June-November). A, Controls 8 weeks after hypophysectomy. B, Effect of live daily injections of 10 IU of HCG; one testis was excised 1 day after discontinuation of hormone treatment, and the remaining one at sacrifice of the toads after 2 weeks. C, Effect of 10 daily injections; one testis was excised 1 day after the treatment, the remaining one at sacrifice after 2 weeks. In the 5-week sample both testes remained. D, Effect of 20 daily injections; one testis was excised after 1 day, the remaining one at sacrifice after 2 weeks. The toads fin this group had received 10 daily injections of 10 IU of HCG g weeks after hypophysectomy. The series of 20 injections was given more than a month after the 10 injections, when the effect of these on spermatogenesis had vanished (Fig. 6C). For further explanations, see legend to Fig.1.
atrophy resulting from extirpation of the Lofts, 1974), and androgen secretion conpars distalis. It was, therefore, possible to stitutes a well-defined biochemical event. It compare various types of effect exerted by is noteworthy that low doses of HCG, which is chemically closely r&ted to gonadotropin, especially human chorionic gonadotropin. The types of effect may be mammalian LH, were found capable of restoring all aspects of structure and function classified as general or trophic, including differentiation and development of the of the toad testis to normal. It is therefore seminiferous tubules, stimulation of sper- suggested that gonadotropin tilone is capable of maintaining the normal structure and matogenesis, and multiplication of interstifunction of the testis. tial cells, or as special, such as spermiation Salmon gonadotropin was only slightly and secretion of androgen. Like other processes of growth and differentiation the effective but produced some stimulation of trophic actions are camplex and difficult to both spermatogenesis and interstitial cdls. ,It remains to be seen whether the low ‘efdefine compared with the special functions. The spermiation process and its hormonal ficiency of this hormone is due to low sencontrol have been described in detail (see sitivity of the target organs or to fast elimi-
378
GUHA AND J@RGENSEN TABLE
3
differentiation phase. In his classic paper on spermatogenesis in Rana temporaria Witschi (1924) states that spermatogonial cells divide about eight times before they start further differentiation. In Xenopus laevis the number of cell divisions within Stages the secondary spermatogonial cysts is stated to vary between 4 and 8 (Kalt, 1976). Groups 0 I 2 The present experiments, indicate that the 8 weeks, 5 1.t. number of mitoses that precede further difhypophysectomized T.p. 5 ferentiation in the spermatogonial cysts depends upon the level of gonadotropin in the 5 x IO IU of HCG I day 1.t. 2 7 blood. At the lowest dosage of HCG the (one testis excised) T.p. 9 spermatocysts beyond the gonadotropin2 weeksb 1.t. 5 2 dependent stages contained fewer cells than (remaining testis) T.p. 6 1 at the higher dose levels. It is therefore suggested that at this dose level the number 10 x 10 IU of HCG 1.t. 6 4 I day of mitotic divisions in the spermatogenetic (one testis excised) T.p. 8 2 process may have been reduced. 2 weeksb 1.t. 3 4 In order to normalize structure and func(remaining testis) T.p. 3 2 2 tion of regressed testes in long-term 5 weeks 1.t. 5 hypophysectomized toads the treatment T.p. 5 20 x 10 IU of HCG with gonadotropin had to be continued for a 1 day 1.t. 1 4 period of at least 3 weeks. If treatment was 1 1 3 (one testis excised) T.p. discontinued after 5 or 10 days the effects 2 weeksb 1.t. I 4 observed were slight. Treatment for 20 days T.p. I 3 1 (remaining testis) resulted in some spermatocysts reaching n For explanation of symbols and staging, see the gonadotropin-independent stages of the legend to Table 1. spermatogenetic process, but the majority * Missing toads died. of the secondary spermatogonial cysts prenation of the hormone (Roos and Jorgensumably degenerated. Similar findings have sen, 1974). been made in hypophysectomized AmbysSpermatogenesis, and secretion of an- toma tigvinum (Moore, 1975). Guha and Jorgensen (1978) found that drogen as judged by the appearance of the eight daily injections of 100 IU of HCG in thumb pads, declined rapidly after hyintact B&o bufo initiated a spermatogenetic pophysectomy. The disorganization and degeneration of the seminiferous tubules and wave that progressed to the final stages other testicular histological structures pro- even in toads hypophysectomized imceeded more slowly. The sensitivity of the mediately after the last injection. Eight various components of the testis toward days of treatment with HCG may thus inHCG seemed to decrease with time after duce a spermatogenetic wave that proceeds to completion in the differentiated testes, hypophysectomy. Presumably, this reflects but not in the atrophic testes of long-term the degree of posthypophysial degeneration hypophysectomized toads. Redifferentiaand atrophy of the testis. tion of the disorganized testes thus seems to During normal spermatogenesis the secbe a prerequisite for the spermatogenetic ondary spermatogonia undergo a number of mitoses before they start the premeiotic action of gonadotropin. EFFECTS OF HCG TREATMENT FOR VARIOUS PERIODS OF TIME ON HISTOLOGICAL APPEARANCE OF INTERSTITIAL TISSUE AND THUMB PAD DEVELOPMENT IN LONG-TERM HYPOPHYSECTOMIZED TOADS’
EFFECTS
OF
GONADOTROPIN
ACKNOWLEDGMENT Dr E. M. Donaldson, Fisheries Research Board of Canada, Vancouver, B. C., generously supplied the salmon gonadotropin (SC-GlOO).
REFERENCES Fontaine, Y. A., and Burzawa-Gerard, E. (1977). Esquisse de l’evohttion des hormones gonadotropes et thyreotropes des Vertebres. Gen. Camp. Endocrinol. 32, 341-347. Guha, K. K. (1976). Effect of human chorionic gonadotropin (HCG) on spermatogenesis and thumb pad development in pars distalis extirpated toads, Bufu bufo bufo (L.). Gen. Camp. Endocrinol. 29, 278 (Abstr.). Guha, K. K., and Jorgensen, C. B. (1978). Effects of hypophysectomy on structure and function of testis in adult toads, Bufo b&o bufo (L.). Gen. Camp. Endocrinol. 34, 201-210. Jorgensen, C. B., and Larsen, L. 0. (1963). Effect of corticotropin and growth hormone on survival in hypophysectomized toads. Proc. Sot. Exp. Biol. Med. 113, 94-96. Kalt, M. R. (1976). Morphology and kinetics of spermatogenesis in Xenopus laevis. J. Exp. 2001. 195, 393-408.
Licht, P., and Papkoff, H. (1974). Separation of two distinct gonadotropins from the pituitary gland of
ON
TOAD
TESTIS
378
the buLlfrog Rnnn catesbeiana. Endocrinology 94, 1587-1594. Lofts, B. (1974). Reproduction. 01 “Physiology of the Amphibia” (B. Lofts, ed.), Vol. 2, pp. 107-228. Academic Press, New York. Moore, i;. L. (1975). Spermatogenesis in larva1 Ambystoma tigrinum: Positive and negative interactions of FSH and testosterone. Gen. C’orrrp. Endocrinol. 26, 525-533. Muller, C, (1976). “Steroidogenesis and Spermatogenesis in the Male Bullfrog, Rana catesbeiana: Regulation by Purified Bullfrog Gonadotropins,” Ph.D. Thesis, University of California, Berkeley. Papkoff, H., Farmer, S. W., and Licht, P. (1976). Isolation and characterization of luteinizing hormone from amphibian (Rana catesheinna) pituitaries. Life Sci. 18, 245-250. Roos, J., and Jorgensen, C. B. (1974). Rates of disappearence from blood and biological potencies of mammalian gonadotropins (HCG and ovine LR) in the toad Bufo bufo bufo (L.). Gen. Camp. Endocrinol. 23, 423-437. van Dongen, W. J.. Jorgensen, C. B., Larsen, L. 0.. Rosenkilde, P., Lofts, B., and van Oordt, P. G. W. J. (1966). Function and cytology of the normal and autotransplanted pars distalis of the hypophysis in the toad Bufo bufo (L,). Ger7. Cornp. Endocrinol. 6, 491-5 1,s. Witschi, E. (1924). Die Entwicklung der Keimzellen der Rana ternpornria L. Erster Teil: Urkeimzellen und Spermatogenese. Z. Zellen Cewehelehre 2, 523-561.
AND
COMPARATIVE
36, 371-379 (1978)
ENDOCRINOLOGY
Effects of Human Chorionic Gonadotropin and Salmon Gonadotropin on Testis in Hypophysectomized Toads (Bufo bufo bufo L.) K. KUMAR
GUHA’
AND C. BARKER
JQ~RGENSEN
Zoophysiological Laboratory A, August Krogh Institute, Universitetsparken 13, DK-2100 Copenhagen, Denmark Accepted July 10, 1978 The effects of gonadotropin on spermatogenesis and on histology and function of interstitial tissue were studied in male toads after extirpation of the’ pars distalis of the pituitary gland. Four weeks after hypophysectomy, 5 IU of HCWOO g body weight practically normalized spermatogenesis and stimulated the interstitial tissue as judged from the histological appearance of the tissue and the development of thumb pads. The dosage needed to restore normal histological differentiation of seminiferous tubules, spermatogenesis, and interstitial tissue increased with increasing deterioration of normal testicular structure and function following hypophysectomy. In the highly dedifferentiated testes, 8 weeks after hypophysectomy, it was possible to distinguish between trophic effects of HCG treatment, including restoration of normal testicular histology and spermatogenesis and of the intersritial tissue, and effects on functions such as spermiation and secretion of androgen. Salmon gonadotropin, at a daily dosage of 20 &IO0 g body weight, only slightly stimulated spermatogenesis and the interstitial tissue.
We have shown previously (Guha and Jargensen, 1978) that in adult male toads, Bufo bufo bufo, hypophysectomy initially inhibits normal proliferation and maturation of cells in the secondary spermatogonial cysts, thus interrupting the normal spermatogenetic process. Several weeks after the seminiferous tubules start to disintegrate and become replaced by connective tissue. The pituitary gland thus exerts both special gonadotropic functions and more general trophic functions on the tissue of the testis. The question arises whether these different types of function are due exclusively to gonadotropin(s), or whether other pituitary hormones are active in maintaining the normal structure and function of the toad testis. In the pituitary gland of the frog Rana catesbeiana, two gonadotropins related to i Present address: Department of Anatomy, versity of Kuopio, 70101 Kuopio 10, Finland.
Uni-
mammalian FSH and LH have recently been identified (Licht and Papkoff, 1974; Papkoff et al., 1976). The amphibian hormones are not yet avaiIabie for general use (Muller, 1976). However, during work on the gonadotropic effect of human chorionic gonadotropin (HCG) in female toads it was observed incidentally that in an animal possessing one ovotestis small doses of HCG strongly stimulated spermatogenesis within the male parts of the bisexual gonad (unpublished observation). It was, therefore, found of interest to treat hypophysectomized male toads with HCG in order to see to what extent the hormone was capable of restoring the normal structure and function af regressed testes. Some experiments were also made with salmon gonadotropin. In another teleost, the carp, the gonadotropin that has been isolated seems to be chemically related to the LHs, and thus to HCG (Fontaine and Burzawa-Gerard, 1977). A preliminary report of the results has been published (Guha, 1976). 371
0016~6480/78/0363-0371$OI .00/O Copyright 0 1978 by Academic Press, Inc. All rights 3f reproduction in any form reserved.
372
GUHA AND JORGENSEN
MATERIAL
AND METHODS
Experiments were made on adult male toads kept at 22-23”, and exposed to 12 hr of light alternating with 12 hr of darkness. The toads were placed in groups of up to 10 in plastic boxes (36 x 55 cm) with access to water. They were fed mealworms un libitum for periods of 24 hr three times a week. The pars distalis of the pituitary gland was extirpated as previously described (van Dongen et al., 1966), and these toads are referred to below as “hypophysectomized.” The HCG preparation used was Physex, Leo. The effects of hypophysectomy, and subsequent treatment with gonadotropin, on testicular structure and function were evaluated as previously described (Guha and Jorgensen, 1978). The hypophysectomized toads were injected with 10 IU of ACTH (Orthana) three times a week in order to maintain the animals’ health (Jorgensen and Larsen, 1963). Three experiments were made. One experiment started in March, using 31 toads collected the previous autumn and stored at about 5”. The toads were acclimated for 2 weeks in the laboratory before hypophysectomy, in April. Four weeks after the operation HCG was injected SC daily for 4 weeks in doses of 2, 5, 10, or SO IU/lOO g body weight. The second experiment included 51 toads collected in the breeding ponds in spring and stored afterward in a refrigerator. The toads were acclimated as above for 2 weeks before hypophysectomy in July. Starting 6 weeks after the operation, HCG was injected daily for 4 weeks in doses of 0.5, 1, 2, or 5 IU/lOO g body weight. Salmon gonadotropin was injected daily in a dose of 20 pg/lOO g. In the third experiment 35 toads were collected in spring and acclimated for 5 weeks before hypophysectomy in July. This experiment was designed to study immediate and delayed effects of gonadotropin administered for varying periods of time. Ten international units (IU) of HCGilOO g were injected daily for periods of 5, 10, or 20 days, and the effects on the testes were studied from I day to 5 weeks after the discontinuation of the HCG treatment.
RESULTS
Effects of HCG Treatment 4 Weeks after Hypophysectomy Figures 1 and 2 show the effect, on spermatogenesis, of 4 weeks of treatment with HCG, starting 4 weeks after hypophysectomy. In the untreated hypophysectomized controls secondary spermatogonia had almost disappeared, and later spermatogenetic stages were scarce. Even
the smallest dosage used, 2 IU of HCG, appreciably stimulated spermatogenesis, and maximum effects were reached with daily doses of 5-10 IU. At this dose level the numbers of cysts from primary spermatogonia to spermatids were about the same as in the normal controls, i.e., spermatogenesis had become normalized. The relationship between dose of gonadotropin and number of cells produced per spermatocyst is shown in Fig. 3. It seems that the mean number of cells in the cysts of primary spermatocytes was higher in the groups of toads receiving 5 or 10 IU of HCG, when compared with those receiving 2 or 50 IU. Thus, the highest dose level tended to be less efficient in stimulating spermatogenesis, both with respect to number of spermatocysts formed and of cells produced within the cysts. The effect of the HCG treatment on spermiation increased with increasing dosage, and 50 IU practically depleted the seminiferous tubles of sperm bundles (Fig. 1). The effect of HCG treatment for 4 weeks on the interstitial tissue is summarized in Table 1. It may be seen that extirpation of the pars distalis reduces androgen secretion to insignificant levels, as judged from the absence of the secondary sexual character, the thumb pads; these had completely regressed in the hypophysectomized controls, whereas in the unoperated controls they were well-developed. Two international units of HCG daily stimulated the interstitial tissue and restored the thumb pads; 10 IU of HCG daily produced maximum effects both on the interstitial tissue and the thumb pads. Effects of HCG Treatment 6 Weeks after Hypophysectomy In the previous experiment the threshold dosage of HCG needed to stimulate testes in hypophysectomized toads was below 2 IWO0 g body weight. The effects of a lower range of HCG doses were therefore
EFFECTS
7 61
OF GONADOTROPIN
ON TOAD
TESTIS
373
Hypophysectomized controls
2 i.u. hCG
5 i.u. hCG
FIG. 1. Effect of 4 weeks of treatment with HCG on spermatogenesis in male toads hypophysectomized for 4 weeks prior to treatment. (March-May). 0, primary spermatogonia; I. secondary spermatogonia; II, primary spermatocytes: III, secondary spermatocytes; IV, spermatids; V, sperm bundles attached to Sertoli cells. Figures above or in bars indicate SEM.
studied in toads that had been hypophysectomized for 6 weeks. In addition, the effectiveness of salmon gonadotropin was tested. Figure 4 shows that at the end of the experiment spermatogenesis had stopped completely in the hypophysectomized controls. Even the smallest doses, 0.5 and 1 IU of HCG, seemed to increase the number of primary spermatogonial cysts, but the spermatogenetic effect was small. Spermatogenesis was clearly *stimulated by 2 and 5 IU, but the number of spermatocysts within the seminiferous tubules was far from normalized (Figs. 4 and 5). Also 20 pg
of salmon gonadotropin daily per 100 g body weight exerted only a slight spermatogenetic effect, comparable to that of 1 IU of HGG. The spermiation effect increased with increasing doses of HCG (Fig. 4). At the dose levels of 2 and 5 IU the effects were about the same as in the previous experiment, when expressed as the percentage reduction in number of sperm bundles per tubule section, using the hypophysectomized controls as a reference. Salmon gonadotrop& did not induce spermiation. The effects of the two gonadotropins on the interstitial tissue are shown in Table 2.
GUHA AND JQlRGENSEN
%
11
N
H
2
5 10 50 i.u. hCG
FIG. 2. Effect of 4 weeks of treatment with HCG on spermatogenesis in male toads hypophysectomized 4 weeks prior to treatment. Ordinate: total number of spermatocysts from stage of primary spermatogonia to spermatids. N, normal controls; H, hypophysectomized controls. Vertical lines indicate twice the SEM.
At the time of extirpation of the pars distalis in July, the thumb pads were only moderately developed in most animals. When the hormone treatment began 6 weeks later, the thumb pads were well developed in most intact toads and maximally regressed in the hypophysectomized controls. After a further 4 weeks, the thumb pads were fully developed in the unoperated controls (Table 2). Also, the interstitial tissue was highly developed in most of the intact toads at the end of the experiment, whereas in the hypophysectomized toads the interstital tissue was histologically regressed. Daily doses of 0.5 IU of HCG for 4 weeks had no effect on appearance and function of the interstitial tissue. Response in all animals of a group was only observed with 5 IU, and the effect was smaller than in the previous experiment (Table 2). Twenty micrograms of salmon gonadotropin slight-
60
Normal
controls
LO 20
Numbers
of cells
per sectioned
cyst
FIG. 3. Relationship between dosage of HCG and frequency distribution of numbers of cells in sections of cysts of primary spermatocytes. Hypophysectomized toads treated 4 weeks with HCG (MarchMay).
ly stimulated the interstitial tissue histologically, but did not stimulate thumb pad development. Effects of HCG Treatment Hypophysectomy
8 Weeks after
The two previous experiments showed differences in sensitivity of the testes toward HCG that might be related to differences in the duration of the period that lapsed between extirpation of the pars distalis and the treatment with gonadotropin. It is suggested that the dosage of gonadotropin, or duration of treatment, needed to restore normal spermatogenesis and development of the interstitial tissue increased with progressing deterioration of testis structure and function after hypophysectomy. An experiment was, therefore, made in which HCG treatment was postponed until 8 weeks after extirpation of the pars distalis, and the duration of treatment was varied from 5 to 20 successive days.
EFFECTS
TABLE
OF
GONADOTROPIN
1
EFFECTS OF 4 WEEKS OF HCC TREATMENT ON HISTOLOGICAL APPEARANCE OF INTERSTITIAL TISSUE AND THUMB PAD DEVELOPMENT IN TOADS TREA-CED 4 WEEKS AFTER HYPOPHYSECTOMY (APRIL-MAY)~
Stages Groups Intact controls Hypophysectomized 2 IU of HCG 5 IU of HCG IO IU of HCG 50 IU of HCG
0 1 2 3 1.t. T.p. 1.t. T.p. 1.t. T.p. 1.t. T.p. 1.t. T.p. 1.t. T.p.
5 5 5 5 5 3 2 4 1 3 2 5 1 1 3 6 1 5
u I.t., interstitial tissue; T.p., thumb pads. Staging of development of interstitial tissue: 0, very few cells, with pycnotic nuclei; 1, few cells, with slender and shrunken nuclei; 2, many cells, with round and medium-sized nuclei; 3, numerous cells, with round and large nuclei. Staging of thumb pad development: 0, yellowish thumb pads lacking papillae; 1, yellowish with small papillae; 2, light brown with medium-sized papillae; 3, dark brown with large papillae. Figures indicate numbers of toads.
Eight weeks after hypophysectomy primary spermatogonial cysts were practically the only normal spermatogenetic stage present in the seminiferous tubules (Fig. 6A). Sometimes sperm still remained in bundles, but the Sertoli cells carrying the bundles were disintegrating. Moreover, there was extensive histological dedifferentiation of the tubules, which became replaced by connective tissue. After five injections of 10 IU of HCG/lOO g body weight the number of primary spermatogonial cysts had about doubled (Fig. 6B) and had reached the level typical of intact toads (Figs. 1 and 4), but only few secondary cysts had been produced. Two weeks after the discontinuation of the treatment, the number of primary sper-
ON
TOAD
TESTIS
37s
matogonial cysts was still high, and the secondary spermatogonial cysts were still scarce. No further progress in the spermatogenetic process had been made, al1 subsequent stages being absent. Treatment for 10 days resulted in higher numbers of primary and secondary spermatogonial cysts, with maximum effects 2 weeks after the discontinuation of the treatment (Fig. 6C). Scattered cysts of primary spermatocytes were also observed, indicating that a few cysts of secondary spermatogonia had entered meiosis. However, 5 weeks after the end of the HCG treatment most of the secondary spermatogonial cysts had degenerated, without giving rise to a spermatogenetic wave, and the number of primary spermatogonial cysts was, again, decreasing. Twenty days of treatment with HCG induced a spermatogenetic wave that after 2 weeks had reached the meiotic stages. Again the effect peaked after the discontinuation of the treatment (Fig. 6D). The cysts contained cells in normal numbers. Disorganized seminiferous tubules infiltrated with connective tissue were not observed after 20 days’ treatment with HCG, which thus presumably caused redifferentiation of the deranged parts of the testes. The effect of HCG on the interstitial tissue is shown in Table 3. Five days of treatment stimulated the interstitial tissue, 10 days of treatment resulted in slight to moderate development of the thumb pads in about half of the toads, and 20 days of treatment resulted in thumb-bad development in most of the toads. The response of the interstitial tissue toward the ‘CG treatment tended to increase after the discontinuation of the injections. However, 5 weeks after the 10 days of treatment, the interstitial tissue had once more completely regressed (Table 3). DlSCUSSfQN The testes of the toads in the three experiments differed in degree of dysfunction and
376
GUHA AND JORGENSEN
9 a 7
Normal
controls
, Hypophysectomized controls
1-I
6 5 I
d
d
20 pg salmon gonadotropin
d
3 2 I
i 0
1 11 JJJ IV V
0.5 i.u. hCG
I
0
J
JJ JJJ IV
V
0
J
JJ JJI IV
V
5 i.u. hCG
1 i.u. hCG I
0
J
II JJJ IV
V
0
J
JJ 111 IV
V
0
J
11 IJJ IV
V
0
J
JJ JJJ IV V
FIG. 4. Effect of 4 weeks of treatment with gonadotropin on spertnatogenesis in male toads hypophysectomized 6 weeks prior to treatment (July-September). For further explanation, see legend to Fig. I. TABLE
2
EFFECTS OF 4 WEEKS OF GONADOTROPIN TREATMENT ON HISTOLOGICAL APPEARANCE OF INTERSTITIAL TISSUE (Lt.) AND THUMB PADS (T.p.) IN TOADS TREATED 6 WEEKS AFTER HYPOPHYSECTOMY (JULY-SEPTEMBER)” Stages
Groups Intact controls Hypophysectomized 0.5 IU of HCG
H 20 0.5 1
s%
2
5
i.u. hCG
FIG. 5. Effects of 4 weeks of treatment with gonadotropin on spermatogenesis in male toads hypophysectomized 6 weeks prior to treatment. Ordinate: total number of spermatocysts from stage of primary spermatogonia to spermatids. N, normal controls; H, hypophysectomized controls; S.G., salmon gonadotropin. Vertical lines indicate twice the SEM.
1 IU of HCG 2 IU of HCG 5 IU of HCG 20 pg of salmon gonadotropin
0 I 1.t. T.p. I.K. T.p. 1.t. T.p. 1.t. T.p. 1.t. T.p. 1.t. T.p. 1.t. T.p.
2 3
1 2 7 10 5 5 6 6 1 2 1 2
4 3 2 I 2 1 5 1 5 1 5 6
u For explanation of symbols and staging, see footnote to Table 1.
EFFECTS
lzr-
:, .5
6-
A
OF
GONADOTROPIN
ON
TOAD
TESTIS
377
12 controls
6-
0.42
-
s 0 I 0 I 1 day 2 weeks nfter 5 x 10 IU HCG
1.50
1
1.25
2.40
2.00
6
1 day 2 weeks after 10 x 10 Ill
5 weeks HCG
1 day 2 weeks after 20 x 10 IU HCG
FIG. 6. Effect of HCG treatment for various periods of time on spermatogenesis in long-term hypophysectomized toads (June-November). A, Controls 8 weeks after hypophysectomy. B, Effect of live daily injections of 10 IU of HCG; one testis was excised 1 day after discontinuation of hormone treatment, and the remaining one at sacrifice of the toads after 2 weeks. C, Effect of 10 daily injections; one testis was excised 1 day after the treatment, the remaining one at sacrifice after 2 weeks. In the 5-week sample both testes remained. D, Effect of 20 daily injections; one testis was excised after 1 day, the remaining one at sacrifice after 2 weeks. The toads fin this group had received 10 daily injections of 10 IU of HCG g weeks after hypophysectomy. The series of 20 injections was given more than a month after the 10 injections, when the effect of these on spermatogenesis had vanished (Fig. 6C). For further explanations, see legend to Fig.1.
atrophy resulting from extirpation of the Lofts, 1974), and androgen secretion conpars distalis. It was, therefore, possible to stitutes a well-defined biochemical event. It compare various types of effect exerted by is noteworthy that low doses of HCG, which is chemically closely r&ted to gonadotropin, especially human chorionic gonadotropin. The types of effect may be mammalian LH, were found capable of restoring all aspects of structure and function classified as general or trophic, including differentiation and development of the of the toad testis to normal. It is therefore seminiferous tubules, stimulation of sper- suggested that gonadotropin tilone is capable of maintaining the normal structure and matogenesis, and multiplication of interstifunction of the testis. tial cells, or as special, such as spermiation Salmon gonadotropin was only slightly and secretion of androgen. Like other processes of growth and differentiation the effective but produced some stimulation of trophic actions are camplex and difficult to both spermatogenesis and interstitial cdls. ,It remains to be seen whether the low ‘efdefine compared with the special functions. The spermiation process and its hormonal ficiency of this hormone is due to low sencontrol have been described in detail (see sitivity of the target organs or to fast elimi-
378
GUHA AND J@RGENSEN TABLE
3
differentiation phase. In his classic paper on spermatogenesis in Rana temporaria Witschi (1924) states that spermatogonial cells divide about eight times before they start further differentiation. In Xenopus laevis the number of cell divisions within Stages the secondary spermatogonial cysts is stated to vary between 4 and 8 (Kalt, 1976). Groups 0 I 2 The present experiments, indicate that the 8 weeks, 5 1.t. number of mitoses that precede further difhypophysectomized T.p. 5 ferentiation in the spermatogonial cysts depends upon the level of gonadotropin in the 5 x IO IU of HCG I day 1.t. 2 7 blood. At the lowest dosage of HCG the (one testis excised) T.p. 9 spermatocysts beyond the gonadotropin2 weeksb 1.t. 5 2 dependent stages contained fewer cells than (remaining testis) T.p. 6 1 at the higher dose levels. It is therefore suggested that at this dose level the number 10 x 10 IU of HCG 1.t. 6 4 I day of mitotic divisions in the spermatogenetic (one testis excised) T.p. 8 2 process may have been reduced. 2 weeksb 1.t. 3 4 In order to normalize structure and func(remaining testis) T.p. 3 2 2 tion of regressed testes in long-term 5 weeks 1.t. 5 hypophysectomized toads the treatment T.p. 5 20 x 10 IU of HCG with gonadotropin had to be continued for a 1 day 1.t. 1 4 period of at least 3 weeks. If treatment was 1 1 3 (one testis excised) T.p. discontinued after 5 or 10 days the effects 2 weeksb 1.t. I 4 observed were slight. Treatment for 20 days T.p. I 3 1 (remaining testis) resulted in some spermatocysts reaching n For explanation of symbols and staging, see the gonadotropin-independent stages of the legend to Table 1. spermatogenetic process, but the majority * Missing toads died. of the secondary spermatogonial cysts prenation of the hormone (Roos and Jorgensumably degenerated. Similar findings have sen, 1974). been made in hypophysectomized AmbysSpermatogenesis, and secretion of an- toma tigvinum (Moore, 1975). Guha and Jorgensen (1978) found that drogen as judged by the appearance of the eight daily injections of 100 IU of HCG in thumb pads, declined rapidly after hyintact B&o bufo initiated a spermatogenetic pophysectomy. The disorganization and degeneration of the seminiferous tubules and wave that progressed to the final stages other testicular histological structures pro- even in toads hypophysectomized imceeded more slowly. The sensitivity of the mediately after the last injection. Eight various components of the testis toward days of treatment with HCG may thus inHCG seemed to decrease with time after duce a spermatogenetic wave that proceeds to completion in the differentiated testes, hypophysectomy. Presumably, this reflects but not in the atrophic testes of long-term the degree of posthypophysial degeneration hypophysectomized toads. Redifferentiaand atrophy of the testis. tion of the disorganized testes thus seems to During normal spermatogenesis the secbe a prerequisite for the spermatogenetic ondary spermatogonia undergo a number of mitoses before they start the premeiotic action of gonadotropin. EFFECTS OF HCG TREATMENT FOR VARIOUS PERIODS OF TIME ON HISTOLOGICAL APPEARANCE OF INTERSTITIAL TISSUE AND THUMB PAD DEVELOPMENT IN LONG-TERM HYPOPHYSECTOMIZED TOADS’
EFFECTS
OF
GONADOTROPIN
ACKNOWLEDGMENT Dr E. M. Donaldson, Fisheries Research Board of Canada, Vancouver, B. C., generously supplied the salmon gonadotropin (SC-GlOO).
REFERENCES Fontaine, Y. A., and Burzawa-Gerard, E. (1977). Esquisse de l’evohttion des hormones gonadotropes et thyreotropes des Vertebres. Gen. Camp. Endocrinol. 32, 341-347. Guha, K. K. (1976). Effect of human chorionic gonadotropin (HCG) on spermatogenesis and thumb pad development in pars distalis extirpated toads, Bufu bufo bufo (L.). Gen. Camp. Endocrinol. 29, 278 (Abstr.). Guha, K. K., and Jorgensen, C. B. (1978). Effects of hypophysectomy on structure and function of testis in adult toads, Bufo b&o bufo (L.). Gen. Camp. Endocrinol. 34, 201-210. Jorgensen, C. B., and Larsen, L. 0. (1963). Effect of corticotropin and growth hormone on survival in hypophysectomized toads. Proc. Sot. Exp. Biol. Med. 113, 94-96. Kalt, M. R. (1976). Morphology and kinetics of spermatogenesis in Xenopus laevis. J. Exp. 2001. 195, 393-408.
Licht, P., and Papkoff, H. (1974). Separation of two distinct gonadotropins from the pituitary gland of
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TESTIS
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the buLlfrog Rnnn catesbeiana. Endocrinology 94, 1587-1594. Lofts, B. (1974). Reproduction. 01 “Physiology of the Amphibia” (B. Lofts, ed.), Vol. 2, pp. 107-228. Academic Press, New York. Moore, i;. L. (1975). Spermatogenesis in larva1 Ambystoma tigrinum: Positive and negative interactions of FSH and testosterone. Gen. C’orrrp. Endocrinol. 26, 525-533. Muller, C, (1976). “Steroidogenesis and Spermatogenesis in the Male Bullfrog, Rana catesbeiana: Regulation by Purified Bullfrog Gonadotropins,” Ph.D. Thesis, University of California, Berkeley. Papkoff, H., Farmer, S. W., and Licht, P. (1976). Isolation and characterization of luteinizing hormone from amphibian (Rana catesheinna) pituitaries. Life Sci. 18, 245-250. Roos, J., and Jorgensen, C. B. (1974). Rates of disappearence from blood and biological potencies of mammalian gonadotropins (HCG and ovine LR) in the toad Bufo bufo bufo (L.). Gen. Camp. Endocrinol. 23, 423-437. van Dongen, W. J.. Jorgensen, C. B., Larsen, L. 0.. Rosenkilde, P., Lofts, B., and van Oordt, P. G. W. J. (1966). Function and cytology of the normal and autotransplanted pars distalis of the hypophysis in the toad Bufo bufo (L,). Ger7. Cornp. Endocrinol. 6, 491-5 1,s. Witschi, E. (1924). Die Entwicklung der Keimzellen der Rana ternpornria L. Erster Teil: Urkeimzellen und Spermatogenese. Z. Zellen Cewehelehre 2, 523-561.