Involvement of gonadotropin and steroid hormones in the spermiation of the goldfish (Carassius auratus)

GEliERAL

AND

COMPARATIVE

ESDOC’RISOLOGY

12, 491-497

(1969)

Involvement of Gonadotropin and Spermiation of the Goldfish FUMIO Fisheries

Research

YAMAZAKI’ Board

of Drive,

E’DWARD

AND

Cn~uIn. I’nncoutw

Received

Steroid Hormones in the (Carassius aurafus) M. DONALDSOh6640

Vancouver Laboratory, 8, B. C., Canatln

August

X.

IiT.

Marit~e

8, 1968

3,8-hydroxysteroid dehydrogenasc activity has been located in the interstitial cells of the goldfish testis. This activity falls rapidly after hypophysxtomy rcaching a very low or undetectable level within 25 postoperative days. Rrplaccmcnt therapy using salmon gonadotropin showed that this enzymic activity and spermiation are dependent on pituitary gonadotropin. However, the cnzymic activity did not increase significantly at spermiation and spermiation could bc induced even when there is a very low or undctcctable level of 3 p-01 dehydrogenase. A single injection of salmon gonadotropin induces spcrmiation and hypertrophy of the interstitial cells. Androgen was also found to be effective for the induction of spermiation. These facts suggest that despite the lack of a direct correlation between 3 p-01 dchydrogenase and sprrmiation, the pituitary gonadotropin may act on the interstitial cells and induce the secretion of androgens which in turn hring about spermintion.

Spermiation in fish is defined as the release of spermatozoa int’o the sperm ‘ducts by thinning of the semen and leads to the state of functional maturity. At spermiation, the water content of testes increases dramatically. Clemens and his co-workers st,udied this increase in connection with pituitary factors using goldfish and carp and concluded that the pituitary gland secretes a hormone which causes the gonadal hydration iClemens and Grant,, 1964; Clemens et al.,, 1964; Clemens and Johnson, 1964; Clemens and Grant, 1965). Other evidence showing that spermiation in fish is dependent on the pituitary hormone has been obtained by Yamamoto and Yamazaki (1967 1, and Yamazaki and Donaldson (1968a). They showed that hypophysectomy in goldfish inhibits spermiation and administration of HCG (Human Chorionic Gonadot,ropin) or salmon

gonadotropin induces it in hypophysectomized goldfish. Furthermore, Yamazaki and Donaldson (1968a) have found that interstitial cells in the testis show hypertrophy at’ spermiation which was induced by HCG or salmon gonadotropin. Further, the interstitial cells in the testis of teleost fish have been found to have 3 p-01 dehydrogenase, an enzyme which is known t’o be involved in steroid hormone biosynthesis (Stanley et u/., 196.5; Yaron, 1966). This raises the question whether or not 3 p-01 dchydrogenase is concerned with spermiation or whether steroid hormone is involved directly in the mechanisms of spermiation. The present investigat’ion was carried out to study the activity of 3 p-01 clehydrogenase at spermiat,ion, and the effect of androgens on spermiation to elucidate the involvement of steroid hormones in spermiat.ion in fish.

’ Postdoctoral Fellow, Fisheries Research Board of Canada. Present address: Faculty of Fishrrirs, Hokkaido University, Hakodate, Japan.

In the first dehydrogenase

MATERIALS

491

experiment, activity

AND

METHODS the changes in at spermiation

3 p-01 aft,cr

hypophysectomy were examined to elucidate the relationship between spermiation and the enzymic activity in the testes. Seventy sexually mature male goldfish ranging from 7.0 to 9.6 cm in standard body length were hypophysectomized by the opercular approach (Yamazki, 1961) and divided into two groups of 35. They were kept at 20” in 025% NaCl solution and killed in groups of five-5, 10, 15, 20, 25, and 50 days after hypophysectomy. One group was injected intraprritoneally with saline (0.6% KaCl. 0.15 ml per fish) and the other was injected with 0.15 mg partially purified salmon gonadotropin (Yamazaki and Donaldson, 1968a.b; Donaldson and Yamazaki, 1968; Dona!dson el al., 1968) 24 hr before being killed, to induce spermiation. Two groups of five normal sexually mature male fish served as controls. Spermiation was induced in one control group but not, in thr othrr. St autopsy, both the body weight nntl the w-eight of testes were detrrmined. h portion of testis was frozrn immediately on Dry Ice foi historhrmical examination of 3 p-01 dehydrogenas activity. The activity was determined according to Bara (1965). Dehydroepiandrosterone was used as substrate. The frozen sections of 8 p thicknes.5 wrre obtained by Cryostat maintained at -20”. were air dried and incubated for 1 hr at 37”. Snother portion of testis was fixed with Bouin’s solution, cut at 6 F and stained with Delafield’s hematoxylin and eosin for histological observation of interstitial cells. In the second experiment, replacement therapy using salmon gonadotropin was carried out in hypophysectomized fish which had been operated 2 months previously to examine the involvement of gonadotropin in both 3 p-01 dehydrogenasr Twenty hypophyactivity and spermiation. sectomized fish were used for this experiment. Five fish served as controls and were killed at the beginning of the experiment. Salmon gonadotropin 0.2 mg per fish per day was injected three times per week. Three groups of five fish were killed 1, 2, and 3 weeks after the first injection. respectively. The 3 p-01 dehydrogenase activit? was determined as described above. Spermiation was checked 1 and 3 weeks after the first, injrction and was evaluated as a percentage of the mnximal method of Yamazaki and response by thr Donaldson (1968a). In the microscopial observation of 3 p-01 dehydrogenase activity, the intensity of the rcaction was graded into four categories fro.m to +++. The negative symbol designates no visible reaction ; +++ designates maximal reaction. Thr activity in each group was then calculated as a percentagc of the mnsimnl reaction. Mark + was given half the value of

+f and +f was given half the value of +++, respectively. In the third experiment, methyl testosterone, dehydroepiandrosterone and 11 ketotestosterone were injected into hypophysectomized goldfish to examine the effect of androgens on spermintion. The fish used in this experiment were sexually mature males, ranging from 18 to 30g in body weight, which had been hypophyscctomized 3-7 days previously. The androgens were dissolved in peanut oil and injected intraperitoncally at various doses per 10 g body weight as shown in Table 1. Peanut oil at a dosage of 0.2 ml per log hody weight was injected into a group of five control fish. Spermiation responses were checked 24 hr after injection and were evaluated according to the method of Yamazaki and Donaldson (1968a). RESULTS 3 p-01 Dehydyogenase Spermifltion

Activity

at

The results arc presented in Fig. 1. In nonspermiatecl control groups, a strongly positive reaction was found along the seminiferous lobules especially where interstitial cells were present in intact fish (Fig. 3). Five days after hypophysectomy, the activity fell rapidly and was evaluated at 55% of the maximal reaction found in intact fish. Further decline in activity was found 10 days after the operation (Fig. 4). On the postoperative days 25 and 50 no visible reaction was found in the interstitial cells (Fig. 5), except in one 25-day fish which gave a very slight reaction. There was no detectable difference between the responses of spermiated and nonspermiated intact fish (Fig. 6‘1. After hypophysectomy, the same postoperative decline in activity which was observed in the nonspermiated group was also found in this group, although the evaluation of the activity was a little higher than that in the former group (Fig. 7 1. No reaction was visible in two fish on 20 days and in three fish on 25 days after the operat’ion, even though they showed spermiation (Fig. 81. This result indicates that there is no significant increase in 3 p-01 dehydrogenase activity in the testes of goldfish at epermiation. Fifty days after the operation, spermiation TYRSnot induced by a single injection

HORMOSES

SPERMIATION

IN

193

GOLDFISH

‘\

w s: 5

‘\ .

$ E G x

AND

‘\ \

l X\ 40-

‘\

\

.

‘\

\

\

20-

\

.-*=--:..-,

d k: 2 0

DAYS

I:IG. 1. Comparison after hypophyaectomy.

20

IO

of 3 p-01 dehydrogenase Solid line: Nonspermiated

AFTER

HYPOPHYSECTOMY

activities of nonspermiated and spermiated-groups group; broken line: spermiated group.

of gonadotropin because the testes were reduced in size and at,rophied by the operation. Changes in Interstitial Cells at Spermiation The interstitial cells in the nonspermiated group showed very rapid shrinking after hypophysectomy. The cells lose a large part of the cytoplasm and the nucleus decreases in size even in 5 days after the operation. A number of the cells were still observed 20 days after the operation, but some cells were below half the size of the normal cells (Fig. 9)) On the contrary, after hypophysectomy in the spermiated group injected once with salmon gonado-

60

30

with

time

tropin, the cells showed dramatic increases, especially in cytoplasm. They were easily identified from the other cells, even in the fish killed 20 or 25 days after hypophysectomy, since most of the spermatocytes had disappeared making the septum of the seminiferous lobule clear and the other cells of connective tissue were small compared with the interst,iCal cells (Fig. 10). Effects of Salmon G’onadotropin on 3 p-01 Dehydrogenase and Spermiation The effect, of repeated injections of salmon gonadotropin on testicular 3 p-01 dehydrogenase activity and spermiation in hypophysertomized goldfish is shown in

t

DAYS

OF

GONADOTROPIN

INJECTION

PIG. 2. lMect,s of iujectioll of 0.2 mg salmon gonadotropin, three times dehydrogenase activity of the test,es and spermiation in hypophysectomized activit,y; (A), spermiation response.

per week, fish. (a),

on the st,eroid 3 p-01 3 p-01 dehydrogenase

494

YAMAZAKI

AND

DONALDSON

FIG. 3. Testis of nonspermiated intact fish showing a strong reaction for 3 8-01 dehydrogenase along the seminiferous lobule where the interstitial cells are present. X 100. FIG. 4. Testis of hypophysectomized nonspermiated fish killed 10 days after the operation showing a weak reaction for 3 p-01 dehydrogenase. X 100. FIG. 5. Testis of hypophysectomized non-spermiated fish killed 25 days after the operation showing no visible reaction for 3 p-01 dehydrogenase. X 100. FIG. 6. Testis of spermiated intact, fish showing a strong reaction for 3 8-01 dehydrogenase similar to t,hat observed in the nonspermiated int,act fish (Fig. 3). X 100. FIG. 7. Testis of hypophysectomized spermiated fish killed 10 days after the operaCon showing a low level of 3 p-01 dehydrogenase activity. X 100. FIG. 8. Testis of hypophysectomized spermiated fish killed 25 days after the operation showing no visible 3 p-01 dehydrogenase reaction, as in the nonspermiated fish (Fig. 5). X 100.

HORMONES

AND

FIG. 9. Photograph showing the shrinkage fish killed 20 days after the operation. X420. FIG. 10. Photograph showing the hypertrophy killed 20 days after the operation. X420.

SPERMIATION

of the interstitial of interstitial

Fig. 2. Seven days after the first injection, the 3 p-01 dehydrogcnase activity had returned to 40% of the maximal reaction. The activity had increased further at 14 days and a maximal reaction was observed 21 days after the first injection. At the end of the first week of the experiment, there was a slight recovery of spermiation, and after 21 clays, a11 fish showed a maximal spcrmiation response. Effects

of Andmgens

on Spemiation

The effect of a single injection of three clifferent androgcnic et,eroids on spermiation TABLE EFFECT

OF ASDROGESS

ox

IN

SPERYIATION

0.1 0.5 1 0 3.0

cells of hypophysectomized cells of hypophysectomized

Dehydroepiandrosterolle

ll-ketotestosterone

Peanut

oil

spermiated

1 OF HYPOPHYSECTOAIIZED

GOLDFISF~

Response -

+

5 3

.i 0

10.0 0 1 1 0 .i 0 10 0 0.1 0 .i I .o 2 0 0 2 rc

nonspermiated fish

is shown in Table 1. Methyl testosterone or dehydroepiandrosterone induced 100% spermiation response at a ,close of 10 mg per 10 g body weight; no spermiation was observed at a dose of 0.1 mg per log body weight. There is a linear relationship between the logarithm of the dose of both androgens and the spermiation response between the 0.1 mg dose and the 10 mg dose (Table 1). Over the dosage range tested, this was also true for ll-ketotestosterone. The peanut oil injection vehicle did not induce any sign of spermiation at 0.2 ml per 10 g body weight.

No. of fish used

17 methyltesitosterone

495

GOLDFISH

5

1

4 2

3 4 4

++

Evaluation

496

YhMhZhKl

AND

DISCUSSIOX

The present study indicates that in goldfish, steroid 3 p-01 dehydrogenase is located in the interstitial cells in the testes, and that this enzyme activity falls rapidly after hypophysectomy, reaching a very slight or undetectable level within 25 postoperative ,days. Three injections of sahnon gonadotropin into hypophysectomized goldfish with a minimal level of 3 p-01 dehydrogenase activity resulted in a marked increase in cnzymic activity. These finding are comparable to those found in rat by Samuels and Helmreich (1956). They stated that the same enzymic activity of the testes in rat dropped rapidly during the first 5-6 days, and t,hen at a decreasing rate over a 58-day period. They also found that more than two daily injections of 100 IU HCG were nccessarv to obtain a significant increase in enzyrliic activity in the testes of the hypophysectomized rat. This indicates t’hat the 3 p-01 dehydrogenasc activity of the interstitial cells in goldfish testes is depend& on pituit,ary gonadotropin, as in mammals. The present study also showed that there is no significant increase in 3 p-01 dehydrogenase activity in the testes at spermiation and that spermiation can be induced even when there is a very low or undetectable level of 3 p-01 dehydrogenase activity in the testes after hypophysectomy. Furthermore, the present, study shows that spermiation does not occur, even if the 3 p-01 dehydrogenase activity is at a high level in the control group of intact fish or recently hypophysertomized fish. These farts suggest that 3 p-01 dchydrogenase activity is not essential for induction of spermiation. However, this suggestion does not discount the involvement of st,eroid hormones in spcrmiation, since many other enz,vmes are involved in steroidogenesis in addition to 3 ,p-01 dehydrogenase (Baillie et al.. 1966; Arai and Tamaoki, 1967). Furthermore, the morphological changes in interstitial cells at sprrmiation and the effects of inject’ed androgens on the spermiation of hypophysectomized fish suggest an in-

DOh-ALDSOS

volvement of steroid hormones in spernliation. A IO-mg dose of methyl tcstostc,rone or dehydroepiandrosterone per 10 g body weight was necessary for induction of 100% spermiation. This dose appears t’o be far beyond the physiological level of androgens, since it is reported that the concentration of testosterone in male skate ranges from 2.2 to 20.8 pg/lOO ml plasma (Idler and Truscott, 1966)) and 11 -ketotestosterone ranges from 0.0 to 67.5 pg/lOO ml plasma in various states of maturation of male salmon (Schmidt and Idl(lr, 1962). Two factors, however, should be consitlerc,(l in discussing this enormous dose. One is t#he t’opographical relation between the Leyclig cells and the androgen target ccllb. The Leydig cells which secrete androgcns are only a few microns away from the seminiferous lobules which arc connected with the sperm duct cells in the posterior part. of the testes. The lat’ter cells show a hypertrophy at spermiation (Yamazaki and Donaldson, 1968a) similar to that, occurring in amphibian Sertoli cells which are known to be the main gonadotropin target at spermiation in the toad (Burgos and Vitale-Calpe, 1967). Therefore, t’he steroidogenic activity of the Leydig cells may produce much higher conccntrntions of androgen in this location than in the blood. or at a distance from the tsstis. The second factor to be consirlcrrrl is the absorpt’ion rate of androgcn from the injection solvent. It may reasonably be considered that this heavy dose is only absorbed gradually from the solvent, since some oil still remained in t’hc ahrlornen several days after inject’ion. This is prohably the reason why such hear\- doses are required to reach the appropriate (~ncentration of andropen at the seminiferous lobular level to induce sprrmiation with 24 hr after injection. In amphibians, spermiation, which is mediated by swelling of the cytoplasm and unfolding of the apical recesse? of the Sertoli cells, is controlled by T,H ilutcinizing hormone) (Burgos and Vitale-Calpe, 1967; Lofts, 1961). In goldfish, mammalian T,H or FSH do not induce spcrmiation, hut

HORMONES

AIiD

SPEII

HCG and salmon gonadotropin are effective (Yamazaki and Donaldson, 1968a). Furthermore, the gonadotropes of the pituitary gland show morphological changes just before spermiation (Yamamoto and Yamazaki, 1967). These facts indicate that bpcrmiation in goldfish is controlled by the pituitary gonadotropin, as in the amphibia. On the basis of the experiments reported here, the gonadotropin in goldfish may act on the interstitial cells and induce the secretion of androgens, which in turn bring about spermiation.

K-t, cxlw’ss thanks to Dr. W. S. Hoar, who read and commented on the manuscript. We are grateful to the stati of thr Spring Creek and Little White, Salmon National Fish Hatchcrips for the use of facilitiw for thr collrction of pituitar: glands, REFERENCES .%1i.41.

H..

synt.htk Saltn~t 305-313.

.4x~

B. (1967). Steroid by testes of rainbow Gen. Cotnp. Endocrinol.

TAMAOKI,

in vitro gnridneri.

biotrout, 8,

A. H., FERGUSOK, M. M., AND HART, D. R,I. (1966). “Developments in Steroid Histochemistry.” Academic Press. Nrw York. B \I(.\. ( ;. (1965). Histochemicxl localization of A’-3 /%hydrosysteroid dehydrogenase in the ovaries of a tcleost fish, Somber scombpr L. Gett. C’omp. Endocri,nol. 5, 284-296. Bcncos. M. H.. .4ND ~‘ITdLI+CL.4PIS, R. (1967). The mechanism of spermiation in the toad. 81~. J. =Itint. 120, 227-252. B.AILLIE.

cLmmP*‘S.

H.

P..

.4ND

GRANT,

F.

B.

(1964).

<:onatlal hydration of carp (Cuprinus carpio) md goldfish (Camssiw nuratus) after injection of ljituitary extracts. Zoologica 49, 193-210. CLK.:.\IES~. H. P., CIERRSZKO, I,. S.. SHOEMAKER, J. D., .IXD GRANT. F. B. (1964). Partial charnrtrrization of the gonndal hgdra.tion principh ilk thr pituitaries of carp (Cyprintcs corpio). Getc. Camp. Enrbocrinof. 4, 503-507. CLEWZNP,

H.

Specificity tlw pituitary 2. 389-394. CLI:UISNS.

H.

P.,

of

AND

JOTTYSON.

thr gonadal of some fresh

I’.,

.4ND

CiR.1R.T.

wminal thinning responw cctr/,io) and rainbow trout after injrctions of pituitary 174-177.

MT.

\I-.

(1%X).

hydration factor in water fishrs. Co[witr F.

1s.

of carp (Snlmo extracts.

(1965).

Tllr

(C~~witLw gairdnerii) Copei 2,

.MIATION

IX

GOLDFISH

d9i

E. M.. .4s1) k7~\~~.t~.4~~, F. (1968). Preparation of gonadotropic hormone from salmon pituitary glands. Annual Confrrence Chemirnl Institutcx of Canada, 51st, p. 64. (Abstr.)

DOSALDSOS.

D. R.. ..~I\‘I) ‘I’M-s:c-OL’T, 13. (1966). Identification and (11!3nl ifitxtion of testosterone in Getz. Cotnp. ~tdoperillheral plasma of skate. critd. 7, 375-383. Low>. I%. (1961). Thcx rffccts of follicle stimulating hormone and lutcinizing hormone on the t.c~tIs of h~l~~~l)ll?.s:~cton~izrd frogs (Rwc~ lcttL/t~t~~ff~~ia) Getl. (“otnp. Btcdocritwl. 1, 179lS9. ~.~MuEI.~. I,. T., AND HELMREICH, M. L. (1956). The influence of chorionic gonadotropin on the 3 p-01 dehydropc,naw activity of testes and adrenals. Endorritrology 58, 435-442. ~CIIMIDT. P. J.. .xsu IIILIZR, D. R. (1962). Strroid hormones in the plasma of salmon at various statf-s of matur:ttion. GP)?. Ct>tt?p. Enrlocrinol. 2, 204-214 ST.~NI,~. H.. ~HXEFFI. C;.. END BOTTE, Jr. (1965). Histological anIt histochtmical observations 011 thta testis of Gohirrz paganellus. Z. Zelljorsch. 65, 350-362. ‘1-.4>1.4.210~0. K.. .\SD ~*.\MAzAKI, F. (1967). Hormonal c,ontrol of ovulation and spermiation in goldlish. (~UUVW Sgtnp. Etdocrit~ol. 4, 131-145. le\nf.\z.ik;l. F. (19613. Thr c%ffwts of hypophywc.to~Ily WI the ovary of thr goldfish, Carassius uurlr/u.s. B~rll. For. b’is/rrri!,.y Hokkairlo I:tlil,. 12, 167-FO. Y4lrr.4~4~1. 1;. (1!)65), Endocrinological studies on tllc, rqxoduction of thr fcmalc goldfish, Caras.siuh rc~~rnlas I,.. with sprciai reference to the func.tion of tllr, l)it,uitary gland. Alem. Fnc. Fish. Hokl;rri,/o t7ttii,. 13, l-64. \~.w.~%.IIcI. F., AND L)oNAI~DBON, E. M. (1968a). Thr 5permiation of goldfish (Carnssiu.~ auratus) :,s :I l,ioasaa\for salmon (Oncorhynchus /sl,rrf~,!/f.sclro) gonadot:.ol)in. Ge,l. Comp. Endorbtt-cd. 10, 383-391. YAMAZ.~KI. I-.. .irn Dos~~usos. E. M. (196812). The rffrctr of purtiall~ purified salmon pituitar>gonadotropin on spermatogenesis, vitellogenesis and ovulation in hypophysectomized goldfish (Ctrrrtssius auratus) . Gen. Comp. Endocrirlol. 11, 292-299. YARON, Z. (1966). Demonstration of 3 P-hydrospstc%roid dr~hydrogc~na,w in the testis of T&pin rttossrrttnhicn ((‘ic~hlida~, Trlrwtri), J. E,xtZr,c!Ytcr,l. 34, IZi-12s. IDLKH,