GENERALANDCOMPARATIVEENDOCRINOLOGY 56, 111-120(1984)

Serum Levels of 11 Gxotestosterone in Male and 17P-Estradiol in Female Rainbow Trout (Salmo gairdneri) during the First

Reproductive Cycle

RUDIGER SCHULZ

Ruhr-Universitdt Bochum Arbeitsgruppe fir vergleichende Endokrinologie ND 5127, UniversitSitsstrasse 150,04630 Bochum 1, West Germany

Accepted December 15, 1983

From September 1980 to August 1981 the serum levels of 1 I-oxotestosterone (11-O-T) in 231 male and of 17B-estradiol (Ez) in 207 female rainbow trout were measured radioimmuno- logically, and compared with the developmental stages of the gonads. Using histological criteria, testicular development was divided into seven stages, and ovarian development into four stages. In males, 11-0-T levels remained below 6 &ml until the appearance of spermatozoa. Maximum values (126 &ml) were observed in spermiating males. In females, E, levels were below 2 &ml during the period of slow oocyte growth. Maximum values (average of 31 &ml) were observed during exogenous vitellogenesis.

The aim of the present study was to de- termine quantitatively the sex steroids ll- oxotestosterone and 17@estradiol during the first reproductive cycle in rainbow trout. 11-Oxotestosterone is the major an- drogen in male salmonids (Idler et al., 1960, 1971; Scott et al., 1980; Scott and Baynes, 1982; Fostier et al., 1982), and 17P-estradiol the main estrogen in female salmonids (Bil- lard et al., 1978; van Bohemen and Lam- bert, 1981; Scott et al., 1982).

The serum levels of both steroids have already been measured in Salmo gairdneri (Schreck et al., 1973; Fostier et al., 1978, 1982; Scott et al., 1980, 1982; Campbell et al., 1980; van Bohemen and Lambert, 1981; Bromage et al., 1982) using radioimmu- noassay and double isotope derivative assay methods. However, measurements with frequent blood sampling and a con- comitant correlation of the endocrine data with the histological status of the gonads are rare. This is the case especially with the early stages of gonadal development in both sexes. Therefore we correlated histological and endocrine data from a large number of individual rainbow trout which had not

been subjected to any experimental manip- ulation.

MATERIAL AND METHODS

Experimental animals. Twice a month between Sep- tember 1980 and August 1981, 20 rainbow trout were caught in open air ponds (Forellenzucht N. Nigge- mann, Hattingen-Elfringhausen) near the Ruhr- Universitat Bochum. The trout hatched in April 1978. Their body weight ranged between 180 and 488 g, and standard length between 21.5 and 30.5 cm. They were exposed to natural temperature and photoperiod re- gimes. The water temperature (monthly averages) ranged from 2” in January to 18°C in August. The rab- bits used in the immunization procedure were derived from a randomly bred population.

Blood and gonad sampling. Blood (3-6 ml) was taken between 10 and 11 AM by heart puncture. It was allowed to clot at 4” for 4 hr. Serum was obtained by centrifugation and stored at - 35” until RIA determi- nation. After measuring the total gonad weight, a tissue sample from the cranial part of the organ or a complete gonad was fixed in Bouin’s solution and em- bedded in Paraplast (Shandon). Gonad sections (7 pm thick) were stained with a combination of Goldner’s (1938) and Gomori’s (1950) methods to monitor the stage of gonadal development. The gonadosomatic index (GSI) was calculated using the equation GSI = (gonad weightibody weight) x 100.

Serum extraction. Serum (50 to 400 ~1) was mixed with 50 pl phosphate-buffered saline (PBS) containing 1500 cpm of recovery tracer. The volume was brought

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112 RijDIGER SCHULZ

to 0.5 ml with PBS. The samples were incubated for 30 min at room temperature and then extracted twice with 3 ml of cold diethyl ether. The combined organic phases were evaporated by a gentle stream of air. The residue was solubilized in 2 x 100 (~1 ethanol and transferred to methanol-washed thin-layer chromatog- raphy plates (E. Merck, Darmstadt, West Germany; TLC-plates 60 F-254), which were developed subse- quently with the solvent systems cyclohexaneibenzol (l/l; v/v) and chloroform/methanol/Hz0 (9011011; v/v/v). Sample areas which were isopolar with coch- romatographed 11-0-T and E, were scraped off and eluted with ethanol. After evaporation, the dry residue was solubilized in 500 pl of the assay buffer described by Simpson and Wright (1977).

Preparation of immunogens and antisera. Nonra- dioactive steroids were purchased from Sigma Chem- ical Company (St. Louis, MO.). 1 I-Oxotestosterone and estradiol were conjugated to bovine serum al- bumin using the mixed anhydride procedure (for ex- ample, Erlanger, 1973). The androgen was coupled through the 3-position carbonyl group. 6-Oxoestradiol was prepared according to Marrian and Sneddon (1960) and then conjugated through the 6-position ac- cording to Lindner et al. (1971). Conjugate in PBS (500 ug/ml) was emulsified with an equal volume of Freund’s complete adjuvant and injected intrader- mally at multiple sites. Five injections were made over a period of 8 weeks, immunizing male rabbits with the estrogen conjugate and female rabbits with the an- drogen conjugate. Arterial blood was taken from the ear after intraperitoneal booster injections of conju- gate emulsified with incomplete adjuvant. The serum was diluted 1:5 with PBS containing 0.05% (w/v) so- dium azide, 10% glycerol (v/v), and 5 ml/liter Trasylol (Bayer). It was stored in l-ml aliquots at -20”. The results presented here were obtained using antisera from a single bleeding date. They were diluted 1:8000 (1 I-oxotestosterone) and 1: 12,000 (estradiol) just be- fore running the assays using the phosphate buffer de- scribed by Simpson and Wright (1977). With these di- lutions the antisera bound 40% of the label (10,000 cpm) in the absence of nonradioactive steroids.

Assay procedure. [6,7-3H]Estradiol (1969 GBqi mmol) was purchased from Amersham Buchler. [ 1,2-‘HI 1 I-oxotestosterone was prepared from [l ,2-3H]cortisone (1592 GBq/mmol, New England Nu- clear Corp., Boston, Mass.) according to Simpson and Wright (1977). Antiserum (0.2 ml). sample aliquots (0.1 ml, in duplicate) or standard solutions (0.1 ml, in trip- licate), and 0.05 ml of assay label containing 10,000 cpm were mixed and incubated in sealed tubes over- night at 4”. All solutions were made using assay buffer. Unbound steroids were absorbed with 0.5 ml of a chilled Dextran-coated charcoal solution (1 g charcoal and 0.1 g Dextran T-70 in 100 ml assay buffer), which was sedimented after 5 min by centrifugation (5000 r-pm for 5 min at 4”). The supernatant (0.6 ml) was

transferred into counting vials containing 5 ml of scin- tillator (Unisolve I Zinsser). A 0.2-ml sample aliquots were directly pipetted into scintillation vials for the determination of steroid recovery. Using a semiloga- rithmic plot, the standard curve in the ll-oxotestos- terone assay showed linearity in the range of 1000 to 31.25 pg. the estradiol assay in the range of 1600 to 50 pg of the respective unlabeled tracers.

Quality control. The interassay coefficients of vari- ation were calculated using the data obtained from eleven 1 I-oxotestosterone and nine estradiol standard curves. The intraassay coefficients of variation were calculated using 500 pg 1 I-oxotestosteroneil00 (~1 assay buffer and 800 pg estradiolilO0 ~1 assay buffer respectively (n = 6) which had been processed like sample aliquots as described under Assay procedure. The antibodies’ specificities were determined as fol- lows: in the presence of radioactive and nonradioac- tive antigen (62.5 pg 1 I-oxotestosterone; 100 pg estra- diol), lo-, 50-, and loo-fold amounts of selected ste- roids were incubated with antiserum. The degree of cross-reactivity was calculated using the equation

measured 11-0-T or Ez minus 62.5

or 100 (pg) % cross-reactivity = - x 100

actual amount of cross-reacting

steroid (pg)

Statistics. The steroid hormone values of the dif- ferent developmental stages were compared by the Student t test.

RESULTS

Assay Characterization

The interassay coefficients of variation ranged between 4.6 and 9.1% for six ll- oxotestosterone concentrations. They varied from 5.5 to 10.3% for six estradiol concentrations. The intraassay coefficients of variation in the 1 1-oxotestosterone assay showed an average of 8.5%, the one in the estradiol assay an average of 7.9%. The cross-reaction rates of both antisera with selected steroids are listed in Table 1. None of the selected compounds exceeded a cross-reaction rate of 5%.

Testicular Development

The testicular development of the rainbow trout and of other bony fish has been described repeatedly (Billard et al.,

SEX STEROIDS IN RAINBOW TROUT 113

TABLE 1 CROSS-REACTION OF THE 11-0-T AND E, ANTISERA

WITH SELECTED STEROIDS

% Cross-reactivity”

11-0-T E2 Steroid antiserum antiserum

Androstentrion 4.7 <O.l Testosterone 3.4 <O.l Dihydrotestosterone 2.25 co.1 11 P-Hydroxy-

testosterone 0.16 co. 1 1 I P-Hydroxyandro-

stendione co. 1 <o. 1 Androstendione co. 1 co.1 Aldosterone co.1 <o. 1 Cortisone co. 1 <o. 1 Cortisol co. I <O.l Progesterone co.1 co.1 17a-Hydroxypro-

gesterone co. 1 co. 1 Estriol co.1 2.25 Estron co. 1 3.3

a Values are means of cross-reaction rates obtained with three different steroid concentrations (see Quality Controls).

1978; Billard, 1983; van den Hurk ei af., 1978; review, Grier, 1981). In the present study, the developmental stages (Fig. 1) have been defined mainly according to Grier (1981).

I. Spermatogonia and Sertoli cells as intratubular cell types; GSI = 0.08 * 0.07, all months except October.

II. Spermatogonia and -spermatocytes present; GSI = 0.19 +- 0.11, April-Sep- tember, accumulation July/August.

III. Spermatogonia, spermatocytes, and spermatids present; GSI = 0.54 & 0.42, July-November, accumulation Au- gust/September.

IV. All types of germ cells, many sper- matogenetic cysts, no spermiation; GSI = 4.82 t 1.53, July- January, accumulation October-December.

V. No or few spermatogenetic cysts with small numbers of cells, tubuli filled with spermatozoa, 18% of spermiating males found in this state; GSI = 3.63 2

1.54, December-April, accumulation Jan- uary-March.

Va. mbulus lumen lined by a layer of Sertoli cells, no spermatogenetic cysts, tubuli filled with spermatozoa, 82% of sper- miating males found in this stage; GSI = 2.04 & 0.99, January-June, accumulation March-May.

VI. Small and enlarged tubuli, enlarged tubuli similar to those in stage Va, some- times with spermatozoa apparently under- going phagocytosis, small tubuli similar to those in stage I or II; GSI = 0.49 t 0.25, May-August, accumulation June/July.

Expressible milk (“spermiation”) was first recorded at the 18th of December. Twenty-seven males found throughout the study showed incomplete tubulus forma- tion, a low number of germ cells, and very low GSI values. Hence they were consid- ered as juvenile and their data were omitted from this study.

Ovarian Development

The preovulatory development of mono- estrous teleostean species has been de- scribed repeatedly (van den Hurk and Peute, 1979; Khoo, 1979; Billard et al., 1978; review: Wallace and Selman, 1981). In the present study the developmental stages were defined according to staining properties of the oocytes and histological changes in the follicular envelope (Fig. 2).

I. Vesicle-free oocytes with Aldehyd- fuchsin (a@positive cytoplasm, squamous follicle cells, oocyte diameter up to 0.27 mm; GSI = 0.12 ? 0.06, all months except March, and August-October.

II. Peripheral chromophobe vesicles, af-positive cytoplasm, squamous follicle cells, oocyte diameter = 0.19-0.36 mm; GSI = 0.14 ? 0.05, all months’.

III. Cytoplasm randomly vesiculated with partly chromophobe, partly light green-staining vesicles, af-negative cyto- plasm, cubical follicle cells, oocyte diam-

RUDIGER SCHULZ

FIG. 1. Rainbow trout testes sections from different developmental stages: Cl, spermatogonia; S, Sertoli cells; C, spermatocytes; 2, spermatozoa. (A) Stage I (X 100); (B) stage II (X 100); (C) stage V (x 16). small cysts (arrows) with a small number of germ cells; (D) stage Va, tubulus lumen lined by a layer of Sertoli cells (arrows) ( x 100); (E) stage VI, sperm-filled tubulus (left) next to an involuted tubulus (right) containing younger germ cells (x 100).

eter = 0.33-0.78 mm; GSI = 0.23 * 0.1, The classifications were made according December to August, accumulation April- to the most mature oocytes present in three July. well separated ovary sections. Eight ani-

IV. Cytoplasm (af-negative) randomly mals showed oocytes with a homogenous vesiculated with chromophobe and light mass of Ponceau-S-staining yolk and a pe- green-staining vesicles as well as Ponceau- ripheral ring of cortical alveoli. Another 13 S-staining yolk globules, fully developed females had ovulated eggs in the body Zona radiata, cubical follicle cells, oocyte cavity. The sampling technique did not diameter = 0.31-3.7 mm; GSI = 3.0 + allow us to monitor when the processes 2.5, July to March. leading up to ovulation commenced and

SEX STEROIDS IN RAINBOW TROUT 115

FIG. 2. Rainbow trout ovary sections from different developmental stages. (A) Stage III ovary with far differently developed oocytes: stage I oocyte (1); stage II oocyte (2); stage III oocyte (3) (x 12.8); (B) detail of a stage III ovary showing light green-staining yolk vesicles (arrows), and peripheral chromophobe vesicles of an adjacent stage 11 oocyte (points), and nucleus (N) (x 40); (C) oocyte at the beginning of exogenous yolk incorporation ( x 12.8); (D) oocytes during full vitellogenesis (x 40).

when the ovulation itself took place. Since these processes are correlated with pro- found alternations of the endocrine status, the data of these animals were omitted, so that only animals before the end of the vi- tellogenic process constituted stage IV. The high standard deviation of the GSI in stage IV is due to females with considerably higher gonad weights (maximal value: 98.46 g with a GSI of 23.39) during late vitello-

genesis. The first female with ovulated eggs in the body cavity was found on the 8th of January.

11-Oxotestosterone in Male Trouts

The recovery value for both steroids and all developmental stages if 68.8 ? 7.5%. The data have been corrected for proce- dural losses.

Stage I males show an average 11-0-T

116 RUDIGER SCHULZ

FIG. 3. Average amounts of 1 I-oxotestosterone/ml serum of male rainbow trout in developmental stages I-VI. Vertical bars represent standard deviations. Number of fish in parentheses. Levels of significance: * , P -s 0.05; * ’ , P < 0.025, *. . ) P < 0.0125, * . . . , P < 0.01.

level of 3.4 t 1.3 rig/ml (Fig. 3). Until the first appearance of spermatozoa, the con- centration of this androgen hardly exceeds 5 nglml, except for a small but significant increase in stage II (P < 0.05 for both I- II, and II-III). Concomitantly with the in- crease of the GSI in stage IV the serum 1 l- O-T levels increase sixfold to reach an av- erage of 20.1 t 5.1 &ml. In stage V a threefold, and again in stage Va (where the majority of spermiating males were found) a further twofold increase up the maximal value of 126.4 + 37.9 rig/ml has been de- termined. In stage VI the ll-oxotestos- terone concentration falls to a low value (3.7 -t 1.1 nglml), comparable with the values obtained in the early stages of tes- ticular development.

Estradiol in Female Trout

During the course of ovarian develop- ment, the serum levels of estradiol augment with progressing oocyte growth (Fig. 4) If the individual E, values are plotted on a linear ordinate against the individual oocyte diameters on a linear abscissa, the equation derived from linear regression analysis is y = 18.31x - 5.38, the correlation coeffi- cient being 0.92. In stages I and II, low and

FIG. 4. Average amounts of 17lSestradioVml serum of female trout in developmental stages I-IV. Vertical bars represent standard deviations. Number of fish in parentheses. Levels of significance: . , P < 0.05; . . * * ( P < 0.005.

relatively constant levels of estradiol have been found (0.48 + 0.05 and 0.82 t 0.14, respectively). The average E, level doubles to 1.61 + 0.57 @ml in stage III. Between July and September, eight females in Stage IV showed E, levels in the range of 5 to 14 nglml. This corresponded to oocytes showing few and small Ponceau-S-positive yolk globules apparently soon after the be- ginning of exogenous yolk incorporation. All other stage IV females showed E, levels above 20 r&ml resulting in an average value of 31.5 + 6.6 rig/ml.

DISCUSSION

In the present study alterations of sex steroid concentrations were measured in the serum during the first reproductive cycle of rainbow trout. In another circan- nual study on l-year-old trout of the same population (Seifert et al., 1983), all females were immature (N = 207), whereas 53 out of 267 males were found in different phases of spermatogenesis and spermiation. How- ever, residual sperm was not found in ma- turing testes of 2+ males, and the GSI values during early spermatogenesis were lower than in males which were about to enter the next reproductive cycle (stage VI). This indicates that the fish investigated here were previously immature.

There seems to exist considerable intra- species variation in the timing of the repro- ductive cycle in rainbow trout. Exogenous vitellogenesis is reported to start in April (van den Hurk and Peute, 1979), or July/ August (Lambert et al., 1978; van Bohemen et al., 1981), and Billard et al., (1981) noted that the time when spermatogenesis is ini- tiated varies. It may therefore be mis- leading to subdivide the cycle on the basis of a time scale. This study is based upon the supposition that given gametogentic steps (e.g., exogenous vitellogenesis) are regulated by comparable endocrine events regardless of their timing. This seems a rea- sonable basis for a comparison of results obtained from studies on different popula- tions of rainbow trout.

(Campbell and Idler, 1980; Schulz et al., 1981; van Bohemen and Lambert, 1981). The highest serum E, levels were found in this stage and concentrations exceeding 5 rig/ml were restricted to females with oo- cytes showing Ponceau-S-positive yolk globules. With an average of 3 1.5 nglml, the present results are closer to those of van Bohemen and Lambert (1981) and Scott et al., (1982) who found maximal values above 40 rig/ml, than to those of Bromage et al., (1982) with maximal values of 5 rig/ml.

The preovulatory development observed in this study is in agreement with observa- tions made in the same and other species showing the group-synchronous type of oocyte recruitment (Wallace and Selman, 1981).

A linear relationship between ovarian morphometric parameters and serum E, levels before the end of vitellogenesis is known from the rainbow trout and other species (Wingtield and Grimm, 1977; Lam- bert et al., 1978; Crim and Idler, 1978; Breton et al., 1983). Shortly after gonadal sex differentiation the rainbow trout ovary is capable of aromatizing androgens (van den Hurk et al., 1982). In vitellogenic fe- males the ovarian estrogen secretion is under the control of the maturational GtH (Kagawa et al., 1982; Billard et al., 1978). The situation is less clear in previtellogenic females. Although Billard et al., (1978) and Bromage et al., (1982) reported a small and transient serum GtH increase during early recrudescence, significant changes of con- comitantly measured serum E, levels were not recorded, which is in agreement with the present results. The significance of es- trogens during stage IV is seen in context with the heuatic vitelloeenin svnthesis

The testicuiar development observed in this study is typical for teleosts showing the unrestricted spermatogonial-type testes (Grier, 1981). It corresponds for the most part with the observations of van den Hurk et al. (1978), except for the timing of the cycle which seems to be initiated earlier in the present population. In contrast to the other developmental stages, stage Va has been defined according to a change of the intratubular somatic Sertoli cells which led to the distribution of spermiating males into two developmental stages. Although it has not yet been investigated systematically, the present results indicate that Sertoli cells do not undergo this change synchronously, since tubuli of the mesorchial part seem to be favored in this respect. The simulta- neous presence of residual sperm, sper- matogonia, and spermatocytes in stage VI suggests that these males were entering the next cycle, indicating that testicular recru- descence is not delayed until the complete elimination of residual sperm. During early testicular development (stages I-III) the 11-0-T concentration remains below 6 ng/ ml. Comparable results have been pub- lished by Scott et al. (1980) and Scott and Baynes (1982). The transient 11-0-T con- centration rise in stage II could be consid- ered a reflection of early gonadotropic stim- ulation initiating spermatogenesis, since the steroidogenetic system of the testes ap- pears to be sensitive to GtH at that partic-

1 ” , ~~~-~- ular time (Ng and Idler, 1980). The appear-

SEX STEROIDS IN RAINBOW TROUT 117

118 RUDIGER SCHULZ

ante of spermatozoa and a substantial augmentation of the serum 11-0-T con- centration are observed simultaneously in stage IV. These results are consistent with the findings of Idler et al. (1971), who re- ported that the final stages of spermatogen- esis are dependent on and accelerated by this androgen, and with observations of Scott and Baynes (1982), who noted that androgen levels rise sharply in correlation with the GSI values. The coincidence of maximal androgen levels and the Sertoli cell alteration in stage Va bears the question whether this change is a consequence of or a prerequisite for the high serum 11-0-T concentrations. Sertoli cells have often been implicated with steroidogenesis (O’Halloran and Idler, 1970; Grier, 1981), but ultrastructural data argue against a ste- roid formation starting from cholesterol (van den Hurk et al., 1978). This does not exclude, however, the possible participa- tion in certain steps of steroidogenesis, and it is tempting to speculate that Sertoli cells are a part of the adrenal-hepatic-gonadal axis which Kime (1978) proposed to con- tribute to androgen synthesis in mature sal- monids. On the other hand, it is possible that Sertoli cells simply undergo a sponta- neous alteration after the disappearance of spermatogenetic cysts. Males in stage VI showed serum 11-0-T levels below 5 rig/ml. Therefore, precipitating androgen levels could be regarded as a factor involved in the termination of sperm storage and the reinitiation of spermatogenesis.

The present results obtained from male rainbow trout are closer to those of Scott et al. (1980) than to those of Fostier et al.

(1982). In another study, however, Scott and Baynes (1982) showed that, beginning with spermiation, the serum androgen levels start to decline. The difference con- cerning androgen levels during spermiation may possibly be explained by the fact that in contrast to their earlier study the males have been stripped during the period of

spermiation, a feature shared by the study of Fostier et al. (1982) but not by Scott et uE. (1980) or the present study. This implies that the loss of considerable amounts of spermatozoa has an impact on the endo- crine system. Nevertheless, intraspecies differences of serum GtH and sex steroid concentrations have been reported by Leatherland et al. (1982) who worked on Oncorhynchus ketu obtained from different habitats. Thus, the above mentioned differ- ences in both sexes might also be related to the use of rainbow trout obtained from dif- ferent strains and/or habitats.

ACKNOWLEDGMENTS

I thank R. Oberstebrink-Scholl, who provided ex- pert technical assistance. This study was supported in part by DFG Grants Bl 62/14-l and 2.

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