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BOR - Papers in Press, published online ahead of print August 20, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.020560
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BIOLOGY OF REPRODUCTION 69, 2109–2117 (2003)
DOI: 10.1095/biolreprod.103.020560
© 2003 by the Society for the Study of Reproduction, Inc.

Testis

Endocrine Changes During Onset of Puberty in Male Spring Chinook Salmon, Oncorhynchus tshawytscha1

B. Campbell3, J.T. Dickey3, and P. Swanson2,4,5

School of Aquatic and Fishery Sciences,3 University of Washington, Seattle, Washington 98195 Integrative Fish Biology Program,4 Resource Enhancement Utilization Technology Division, Northwest Fisheries Science Center, Seattle, Washington 98112 Center of Reproductive Biology,5 Washington State University, Pullman, Washington 99164


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In male salmonids, the age of maturation varies from 1 to 6 years and is influenced by growth during critical periods of the life cycle. The endocrine mechanisms controlling spermatogenesis and how growth affects this process are poorly understood. Recent research has indicated that gonadotropins, 11-ketotestosterone, and insulin-like growth factor I play roles in spermatogenesis in fish. To expand our understanding of the roles of these endocrine factors in onset of puberty, male spring chinook salmon (Oncorhynchus tshawytscha) were sampled at monthly intervals 14 mo prior to spermiation. This sampling regime encompassed two hypothesized critical periods when growth influences the initiation and completion of puberty for this species. Approximately 80% of the males matured during the experimental period, at age 2 in September 1999. An initial decline in the ratio of primary A to transitional spermatogonia was observed from July to December 1998, and during this period plasma levels of 11-ketotestosterone and pituitary levels of FSH increased. From January 1999 onward, males with low plasma 11-ketotestosterone levels (<1 ng/ml) had low pituitary and plasma FSH levels and no advanced development of germ cells. Conversely, from January through September 1999, males with high plasma 11-ketotestosterone levels (>1 ng/ml) had testes with progressively more advanced germ cell stages along with elevated pituitary and plasma FSH. Plasma levels of insulin-like growth factor I increased during maturation. These data provide the first physiological evidence for activation of the pituitary-testis axis during the fall critical period when maturation is initiated for the following year.

follicle-stimulating hormone, puberty, spermatogenesis, steroid hormones, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Male Pacific Coast salmon show a large variation in age of sexual maturation both within and between populations [1]. Both genetic and environmental factors influence the age of sexual maturation, but the underlying controlling mechanisms are poorly understood. Most Pacific salmon species are semelparous, and thus the onset and completion of puberty constitute the only reproductive period these animals will experience in their lifetime. Several studies have suggested that the physiological commitment or "decision" to mature occurs up to a year prior to spawning [2–4] and that proximate (predictive) cues are used to control the timing of maturation to optimize gamete production and survival of offspring in the wild. One proposed predictor of future reproductive success is body growth and/or fat stores. Thus, for a species that spawns in the fall, growth during the late winter and early spring of the year of maturation affects the proportion of males maturing [2, 5]. In addition, there is evidence to suggest that growth during the fall the year prior to spawning affects subsequent maturation [3, 5, 6]. Based on these data, it has been hypothesized that there is a decision period for initiation of maturation in the fall followed by a decision period (permissive) for continuing maturation in the spring [5]. However, there is very little physiological evidence to support the existence of these two decision periods and no data pertaining to a mechanism whereby growth directly affects the reproductive endocrine axis. The present study was designed to provide evidence for physiological changes in the pituitary-testis axis associated with these hypothetical decision periods.

Spermatogenesis in vertebrates can be divided into four distinct sequential phases: mitotic stem cell renewal, mitotic proliferation of spermatogonia and supporting cells (e.g., Sertoli and Leydig cells), meiosis (the meiotic division of germ cells), and spermiogenesis (the maturation of germ cells into fully functional spermatozoa) [7]. Although the specific mechanisms for the regulation of spermatogenesis are poorly understood, studies of the Japanese eel (Anguilla japonica) and trout have indicated that pituitary gonadotropins, the testicular steroid 11-ketotestosterone (11-KT), and insulin-like growth factor I (IGF-I) are important regulating factors [8, 9]. IGF-I also influences gonadotropin production at the level of the pituitary [10–13]. Plasma IGF-I is a principal component of the growth axis and is highly correlated with growth rate in chinook salmon (Oncorhynchus tshawytscha) [14–16]. This growth factor may therefore act to signal growth and nutritional status to the reproductive axis.

We measured the above hormones known to be involved with spermatogenesis to determine their temporal relationship with testis development. Changes in these hormones and testis development were related to the hypothesized decision periods of maturation in the fall and spring to indicate physiological mechanisms by which maturation is controlled in spring chinook salmon.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Fish, Rearing Conditions, Diets, and Feeding

Eyed embryos from the Willamette River stock of spring chinook salmon were obtained from the Willamette Hatchery (Oregon Department of Fish and Wildlife) and incubated at the Northwest Fisheries Science Center (Seattle, WA). After hatching (20 January 1998), fish were placed into a single circular fiberglass tank 4 feet (1.2 m) in diameter supplied with water from a recirculation system (8°C). Fry were fed a commercial salmon starter feed (BioOregon-Starter, Bio-Oregon, Inc., Warrenton, OR; 23.1 kJ/g energy, 50% protein, 22% lipid) for 1 mo. Fish (0.5 g) were then randomly assigned (700/tank) into four 4-foot-diameter circular fiberglass tanks and fed a low-fat diet at 100% satiation. The experimental diet was formulated to contain 54% protein, 7% fat, and 19.7 kJ/g energy. Fish were fed a single meal, by hand, each morning. The fish were fed 6 days/wk for the first 16 wk and 5 days/wk thereafter. A natural photoperiod was used, and water temperature varied seasonally between 8°C and 12.5°C. After 18 wk, 1400 fish were distributed equally into four circular fiberglass tanks 6 feet (1.8 m) in diameter (350 fish/tank). Prior to the move, all fish were treated with a potassium permanganate bath (5 ppm) for 1 h to control a Costia necatrix infestation. On 9 November, all fish were killed because of a severe outbreak of bacterial kidney disease (BKD, caused by Renibacterium salmoninarum). A new group of fish of the same age, stock, and rearing history was distributed into four 4-foot-diameter tanks (130 fish/tank). Because of the limited number of fish available, the sampling period was extended to 4- to 7-wk intervals. Fish in these tanks were fed at an 88% ration because of loading constraints of the recirculation system. Erythromycin (100 mg kg body mass-1 day-1 for 21 days) was added to the feed on two occasions (January–February and April–May 1999) to control BKD. The final major sample was taken on 28 June, when mature and immature males were clearly distinguishable by gonadosomatic indices (GSIs) and testis histology. A small number of fish were maintained until 29 September, when mature males were spermiating, and the experiment was terminated at this stage.

Animal use procedures followed the policies and guidelines of the University of Washington Institutional Animal Care and Use Committee (Argent, Redmond, WA).

Sample Collection and Hormone Analyses

A minimum of 10 males were sampled from each tank (at least 40 males for each time point). Fish were killed in a lethal dose of buffered tricaine methanesulfonate (MS-222, 0.05%), and body weight and length were recorded. Blood was collected via the caudal vein, using heparinized capillary tubes after severing the caudal peduncle in small fish and then by venipuncture using heparinized syringes and 21-ga needles once fish were >30 g body weight. Pituitaries were frozen in liquid nitrogen and stored at -70°C. Testes were weighed and fixed in Bouin fixative for 24 h prior to storage in 70% ethanol. Blood was centrifuged at 1000 x g for 5 min, and plasma was stored at -70°C. Plasma 11-ketotestosterone (11-KT) was measured by RIA according to Schulz [17], using a methylene chloride extraction method described by Planas and Swanson [18]. Pituitary FSH and plasma FSH and LH were measured by RIAs according to the method of Swanson et al. [19] as described by Shearer and Swanson [4]. Plasma IGF-I was measured by RIA [20] using commercially available salmon IGF-I as standard and label, and anti-barramundi IGF-I serum (GroPep, Adelaide, Australia). Pituitary FSH and plasma 11-KT levels were measured in all males. Because of the small volume of plasma recoverable from fish sampled in the initial months, plasma IGF-I levels were measured in a randomly selected number of males from the July–October 1998 samplings and in all subsequent samples from males. Plasma FSH was measured in all samples from November 1998 onward, and plasma LH was only measured from May 1999 onward because levels were assumed to be below the detection limits of the assay (0.2 ng/ml) prior to that point [4].

Testis Histology

Fixed testes were dehydrated through ethanol and imbedded in paraplast, sectioned (4 µm), and stained with hematoxylin and eosin. Stages of spermatogenesis were determined by light microscopy using the methods of Loir [21] and Schulz [17] as a guide (Fig. 1). For quantification of germ cell types, three fields of view were selected at random from a section taken from the middle portion of the testis (for small testes, the whole testis was contained on one section). Using an eyepiece with a 10 x 10 grid, each germ cell type that fell on an intersection was identified and counted. The proportion of each germ cell type in the testis was expressed as a percentage of the total number of germ cells counted. Germ cell staging was as follows (see Fig. 1): primary A spermatogonium, transitional spermatogonium, late (secondary) B spermatogonium, primary spermatocyte, secondary spermatocyte, spermatid, and spermatozoon. All proliferation stages between primary A spermatogonia and late B spermatogonia were categorized as transitional spermatogonia; this category includes the A2 to B5 spermatogonia described by Loir [21].



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  		FIG. 1. Chinook salmon testes showing stages of germ cell development (arrowhead): primary A spermatogonia (A), transitional spermatogonia (B), late B spermatogonia (C), primary spermatocytes (D), secondary spermatocytes (E), and spermatids (arrow) and spermatozoa (arrowhead) (F). Bar = 20 µm

From January to September 1999, comparisons of reproductive parameters and maturation stage were performed by grouping individuals into seven stages of spermatogenesis according to the most advanced germ cell type in the testis as follows: stage 1, primary A spermatogonia and transitional spermatogonia; stage 2, late B spermatogonia; stage 3, primary spermatocytes; stage 4, secondary spermatocytes; stage 5, spermatids; stage 6, spermatozoa; stage 7, milt (spermiation).

Statistics

Initial comparisons of parameters among the four replicate tanks within each sample date, using one-way ANOVA, indicated no significant differences among tanks (P > 0.05). Thus, data were pooled within sample date for subsequent analyses. Comparisons of parameters according to sample date or testis stage were done by one-way ANOVA. The Scheffé test was used to conduct multiple mean comparisons. Body weight, pituitary FSH, plasma 11-KT, and plasma FSH were log transformed and percentage data were arcsine transformed prior to ANOVA to meet test criteria. Correlation analyses were performed on untransformed data. All analyses were performed using Statview (Abacus Concepts, Berkeley, CA). Data in the text are given as mean ± SEM (with sample size in parentheses). Differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Appearance of Germ Cells in the Testis

From the initial sampling in July through December 1998, histological analyses of the germ cell stages present in the testes showed only early stages of spermatogenesis (Fig. 2). From January 1999 onward, the testes of some individual fish had more advanced germ cell stages, whereas testes of other fish remained in the first stage of spermatogenesis. Late B spermatogonia were first observed in two individuals in January 1999 (Fig. 2). The number of individuals with testes containing late B spermatogonia as the most advanced stage of germ cell development (stage 2) increased in February 1999, but in some individuals the testes had advanced to stage 3 (primary spermatocytes). By April 1999, the testes of several individuals had advanced to the spermatid stage (stage 5), but the majority of fish had progressed to the late B spermatogonia stage (stage 2). In May 1999, the testes of a small number of individuals had spermatozoa (stage 6), but the majority of individuals had germ cells developed as far as the spermatid stage. At this time, the testes of only one male had late B spermatogonia as the most advanced stage of germ cell development. By June 1999, the testes of the majority of males were in stage 6, with only a small number at earlier stages of development, and none were at the late B spermatogonia stage. Thus, by April 1999 the testes of the majority of individuals were committed to maturation.



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  		FIG. 2. Proportion of chinook salmon males at the seven designated stages of spermatogenesis, July 1998 to June 1999

Relative Changes in Germ Cell Numbers

During the initial months from July to December 1998, only two categories of germ cells were identified, primary A spermatogonia and transitional spermatogonia. The proportion of these two germ cell types changed from July to December: primary A spermatogonia decreased from 14% in July to 6.58% in December, with an accompanying increase in transitional spermatogonia (Fig. 3).



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  		FIG. 3. Proportion of primary A spermatogonia in chinook salmon testes containing only primary A and transitional spermatogonia. From July to December 1998, data were collected mainly from testes of fish that were expected to mature based on the final maturation rate of 80%. From January to February, a small number of fish had reached stage 2 of testis development and were not included in this figure. From April to June 1999, data were collected mainly from testes of fish that would be expected not to mature because 70%–80% of the fish sampled per date had reached stage 2 or beyond and were therefore definitively identified as maturing and were not included in this figure. For each date, the percentage of the sampled fish that were at stage 1, and therefore included in the analysis, are shown in parentheses. Bars with similar letters are not significantly different. Data are presented as mean ± SEM (n = 8–40 fishes/mo)

During January and February 1999, the majority of males were on the verge of producing late B spermatogonia (as indicated by the large increase in the incidence of late B spermatogonia in the subsequent months). These individuals had a low ratio of primary A spermatogonia to transitional spermatogonia during this period (Fig. 3). By April, the majority of males had developed to the late B spermatogonia stages or beyond. The testes of the remaining undeveloped males from April onward had a proportion of primary A spermatogonia similar to that in the testes from males sampled at the beginning of the experiment in July 1998 (Fig. 3).

To compare reproductive parameters with testis development from January onward, males were grouped into stages of maturation based on the most advanced germ cell type identified in their testes. The percentages of germ cell types present in stages 1–6 are shown in Figure 4 (testes from spawning males in September were not processed for histology). The percentage of transitional spermatogonia declined from stage 2 to stage 6. The percentage of late B spermatogonia increased from stage 2 to a peak at stage 4 and then declined in stages 5 and 6. The percentage of primary spermatocytes increased from stage 3 to stage 6. Secondary spermatocytes remained relatively rare from stage 4 to stage 6. The percentage of spermatids increased from stage 5 to stage 6, and spermatozoa constituted 18.2% of the germ cell types present in stage 6.



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  		FIG. 4. Relative proportion of germ cell types in the designated 7 stages of chinook salmon spermatogenesis (based on the most advanced germ cell type identified in their testis): stage 1, primary A and transitional spermatogonia; stage 2, late B spermatogonia; stage 3, primary spermatocytes; stage 4, secondary spermatocytes; stage 5, spermatids; stage 6, spermatozoa. Data from spermiating males (stage 7) were not available

Body Size

Males grew from an average weight of 12 g in July 1998 to 268 g in June 1999 and an average spawning size of 291 g in September 1999 (Fig. 5a). During January and February 1999, when histological evidence of maturation began to appear in the testes of some fish, there was no obvious relationship between size and maturation. Body weights of maturing fish were distributed throughout the range of body weights measured in all fish. By April 1999, the majority of maturing fish weighed >100 g, with only two fish weighing <50 g. The body weights of nonmaturing fish ranged from 35 g to 250 g. By May and June 1999, maturing fish were generally larger than the nonmaturing fish, although the difference was not significant. When individuals were grouped according to testis stage, there was a general increase in body weight from stage 1 to stage 7. This increase in body weight became significant from stage 4 onward (Fig. 6a).



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  		FIG. 5. Changes in body weight (A), GSI (B), pituitary FSH (C), 11-KT (D), plasma FSH (E), and plasma IGF-I (F) for individual males over the sample period from July 1998 (J) to September 1999 (S). Circles indicate males with testes developed to the transitional spermatogonia stage (stage 1). Diamonds indicate males with testes containing late B spermatogonia or more advanced germ cells (stages 2–7)



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  		FIG. 6. Changes in body weight (A), GSI (B), pituitary FSH (C), plasma 11-KT (D), plasma FSH (E), and plasma IGF-I (F) according to the seven stages of spermatogenesis from January through June 1999. Data are mean ± SEM (n = 5–80). Bars with same letter are not significantly different

For the initial summer and fall sampling periods (July 1998–December 1998) the relationship between body size and pituitary FSH, plasma 11-KT, plasma FSH, and plasma IGF-I are shown in Table 1. Pituitary FSH levels were positively correlated with body weight from July to December. Plasma 11-KT levels were not correlated with body weight during any of the months except November, when a positive correlation was observed. Plasma IGF-I levels were positively correlated with body weight in December.


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  		TABLE 1. Correlation coefficients (sample size in parentheses) between body size and pituitary FSH, plasma 11-KT, plasma FSH, and plasma IGF-I for the initial summer and fall sample periods: July 1998–December 1998

Gonadosomatic Index

The overall mean GSI (Fig. 5b) gradually increased from 0.028% ± 0.001% in July 1998 to 0.246% ± 0.019% in April 1999 (P < 0.01). From May to June 1999, GSI increased rapidly from 0.518% ± 0.068% to 3.102% ± 0.394% (P < 0.0001). During January and February 1999, late B spermatogonia first appeared in the testes of some individuals, indicating their commitment to maturation, and the highest GSI values were consistently associated with these maturing males (Fig. 5b). By April, there was a clear difference in GSI values between nonmaturing (0.098% ± 0.039%, n = 8) and maturing (0.29% ± 0.016%, n = 28) males. In April, two individuals had relatively high GSIs and high plasma 11-KT levels, but only primary A and transitional spermatogonia were observed in the testes. The average GSI remained low for nonmaturing males in May (0.046% ± 0.004%, n = 9) and June (0.064% ± 0.018%, n = 7), whereas GSI increased six-fold in maturing males from 0.725% ± 0.054% (n = 21) in May to 4.216% ± 0.413% (n = 28) in June. When individuals were grouped according to testis stage, there was a significant increase in GSI between stage 1 and stage 3 (P < 0.05, Fig. 6b), a gradual increase to stage 5, and a large, significant increase to stage 6 (P < 0.0001).

Pituitary and Plasma FSH

Pituitary FSH content increased significantly from 7.3 ± 0.689 ng/pituitary in July 1998 to 75.15 ± 8.558 ng/pituitary in September 1998 (P < 0.0001). Pituitary FSH gradually increased in maturing males, with levels reaching 3.153 ± 0.244 µg/pituitary (n = 24) in June 1999 and 16.579 ± 2.096 µg/pituitary (n = 6) in spermiating males during September 1999 (Fig. 5c). During January and February, when histological evidence of maturation began to appear in the testes of some fish, maturing males tended to have higher pituitary FSH levels. However, relatively high levels of pituitary FSH were also seen in individuals that did not exhibit any germ cell development beyond transitional spermatogonia. By May, there was a clear association between pituitary FSH content and maturity, with nonmaturing males having low pituitary FSH levels. This difference became more evident from June onward.

Plasma FSH levels gradually rose from November 1998 (0.56 ± 0.022 ng/ml) to peak in June 1999 (1.48 ± 0.14 ng/ml; P < 0.0001) and declined to initial levels by September 1999 (Fig. 5e). From January to April, maturing males tended to have high plasma FSH levels, and there was a clear division between nonmaturing males (<0.5 ng/ml) and maturing males from May to June. However, plasma FSH levels in spermiating males declined and approached the values seen in nonmaturing males.

When individuals were grouped according to testis stage, there was a significant increase in pituitary FSH levels between stages 1 and 2 (P < 0.0001, Fig. 6c). Pituitary FSH levels gradually increased between stages 2 and 5, with a large, significant increase between stages 6 and 7 (P < 0.0001). Plasma FSH levels also increased significantly between stages 1 and 2 (P < 0.01), followed by a gradual increase to peak at stage 6 and then a significant decline at stage 7 (P < 0.001, Fig. 6e).

11-Ketotestosterone

Plasma 11-KT levels (Fig. 5d) increased significantly (P < 0.0001) from July (0.165 ± 0.012 ng/ml) to September 1998 (1.004 ± 0.116 ng/ml). A gradual increase occurred from September 1998 onward, reaching mean levels of 20.7 ± 2.11 ng/ml in June 1999 and 161.8 ng/ml in spermiating males during September 1999. The distribution of 11-KT levels in males first appeared to be bimodal in January 1999, when a small proportion of males fell into a low group (<1 ng/ml). The proportion of males that fell into this low group from January onward ranged between 11% and 28%, similar to the proportion of nonmaturing males observed at the end of the experiment when all other males were spermiating. Males with plasma levels of 11-KT above this apparent threshold (1 ng/ml) were observed as early as September 1998. The percentage of males that had plasma 11-KT levels >1 ng/ml increased from 30% in September 1998 to 85% in January 1999 and 81% in June 1999 (Fig. 5d).

During January and February 1999, a large number of males had plasma 11-KT levels >1 ng/ml but did not show any germ cell development beyond transitional spermatogonia. However, by April 1999 all but two males in this upper mode of plasma 11-KT levels had testes containing late B spermatogonia or more advanced germ cell stages. By May and June 1999, there was a relationship between stage of spermatogenesis and 11 KT levels: nonmaturing males had significantly lower plasma 11-KT (May, 0.31 ± 0.036 ng/ml, n = 8; June, 0.44 ± 0.05 ng/ml, n = 6) than did maturing males (May, 17.7 ± 1.17 ng/ml, n = 21; June, 26.4 ± 1.6 ng/ml, n = 27) (P < 0.0001).

When individuals were grouped according to testis stage there was a significant increase in plasma 11-KT levels between stages 1 and 2 (P < 0.0001; Fig. 6d). Subsequently, levels gradually increased between stages 2 and 5, and a large, significant increase occurred between stages 6 and 7 (P < 0.05).

For the initial summer and fall sampling periods (July 1998–December 1998), the relationships between plasma 11-KT and GSI, pituitary FSH, plasma FSH, and plasma IGF-I are shown in Table 2. Pituitary FSH levels were positively correlated with plasma 11-KT during all months except August. There was a positive correlation of GSI with plasma 11-KT levels during all months except July and October. There was a high positive correlation of plasma FSH with plasma 11-KT in both November and December. Plasma IGF-I was positively correlated with 11-KT levels only in December.


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  		TABLE 2. Correlation coefficients (sample size in parentheses) between plasma 11-KT and pituitary FSH, GSI, plasma IGF-I, and plasma FSH for the initial summer and fall sample periods: July 1998–December 1998

Insulin-Like Growth Factor I

Initially, in July 1998, plasma IGF-I levels were at a minimum of 13.7 ± 1.2 ng/ml and subsequently increased during the summer to peak in September 1998 (35.8 ± 2.9 ng/ml, P < 0.0001). Over the winter, levels declined to a low of 21.3 ± 0.97 ng/ml in February 1999 (P < 0.01) and increased to a maximum mean of 49.7 ± 5.5 ng/ml in spermiating males in September 1999 (Fig. 5f).

During January and February, when histological evidence of maturation appeared in the testis, there was no obvious relationship between plasma IGF-I levels and maturation (Fig. 5f). By April, maturing males tended to have higher plasma IGF-I levels, and this association became stronger in May and June, with a complete separation in plasma IGF-I levels between spermiating (high levels) and nonmaturing (low levels) males evident in September 1999.

When individuals were grouped according to testis stage there was a general increase in plasma IGF-I levels from stage 1 to stage 6; however, this increase was only significant at stages 5–7 (Fig. 6f, P < 0.01). A large, significant increase in plasma IGF-I levels occurred in spermiating males (stage 7; P < 0.0001).

Luteinizing Hormone

Plasma LH levels were measured in samples collected from May 1999 onward. Levels of LH were similar in nonmaturing and maturing males during May (0.413 ± 0.012 ng/ml, n = 29) and June (0.396 ± 0.012ng/ml, n = 37) and increased significantly (P < 0.0001) in September only in maturing males (0.844 ± 0.11 ng/ml, n = 8). When samples were grouped according to testis stage, a significant increase in LH was observed only between stage 7 (spermiating males) and the other six stages (P < 0.0001).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The goal of this study was to describe changes in the pituitary-testis axis during the onset of puberty in male chinook salmon and during seasonal periods when growth affects the proportion of fish maturing in a given year. Significant changes associated with the onset of puberty were detected in the testis and plasma of male spring chinook salmon over a year prior to when the fish produced viable sperm (spermiation). A decrease in the ratio of primary A spermatogonia to transitional spermatogonia and an increase in plasma 11-KT levels occurred during the fall and early winter prior to the appearance of late B spermatogonia (the hallmark for the last mitotic division prior to the onset of meiosis). These data provide the first physiological evidence for the hypothesized fall decision period for initiation of maturation in salmonid fish [5]. In addition, the timing of entry into meiosis in the testis coincided with the hypothesized spring decision period for continuing maturation.

Spermatogenesis has been classed into four distinct development stages: mitotic stem cell renewal, mitotic proliferation of spermatogonia, meiotic production of haploid spermatids, and final differentiation into fully functional spermatozoa [7]. In the rainbow trout (Oncorhynchus mykiss), detailed investigation of the early mitotic stages of spermatogenesis has indicated that the immature testis contains a self-renewing population of large primary A spermatogonia [21, 22]. Loir [21] suggested that spermatogenesis commences with the mitotic division of these primary A spermatogonia to eventually form primary (early) B spermatogonia, which are connected by cytoplasmic bridges and gathered into a cyst surrounded by Sertoli cells. These early spermatogonia undergo a predetermined number of divisions (possibly five) and give rise to a last generation of secondary (late) B spermatogonia, which then divide to form primary spermatocytes. This process is followed by meiosis and differentiation of spermatids into spermatozoa [21]. Studies of spermatogenesis in fish have generally used the appearance of late B spermatogonia or primary spermatocytes as the hallmark for the initiation of maturation and have not assessed changes in the earlier stages of primary A and transitional spermatogonia [17, 23, 24].

In the present study in chinook salmon, late B spermatogonia were first identified in a small number of males during January, 8 mo prior to spermiation. However, quantification of earlier germ cell types (prior to the production of late B spermatogonia) demonstrated a significant decrease in the ratio of primary A spermatogonia to transitional spermatogonia from July to December 1998 (during the year prior to spermiation). It is hypothesized that this change in the relative numbers of these early germ cell types reflects the initiation of spermatogenesis (as suggested by Loir [21]) during the fall prior to spermiation, with primary A spermatogonia undergoing mitotic proliferation prior to the production of late B spermatogonia. This hypothesis is supported by the observation that immature males at the beginning of the study in July had a high proportion of primary A spermatogonia, similar to the nonmature males examined the following spring when maturing males had more advanced stages of germ cells.

In addition to the changes in types of spermatogonia, plasma 11-KT increased significantly during the first fall of the study. September 1998 was the first time that some yearling males had plasma 11-KT levels greater than levels in nonmature males in the following May, June, and September (1999) (<1 ng/ml). Other studies have shown elevations in plasma 11-KT levels prior to the appearance of late B spermatogonia in rainbow trout [23]. Previous researchers working with individually marked Atlantic salmon (Salmo salar) also reported early increases in plasma 11-KT levels in males that subsequently matured 10 mo later [25, 26]. 11-KT probably plays a physiologically important role in initiating spermatogenesis at this time, because studies in the Japanese eel and African catfish (Clarias gariepinus) have shown that 11-KT acts directly on the testis to stimulate early mitotic divisions of spermatogonia and of surrounding somatic cells [27, 28]. Furthermore, the capacity of the testis to produce androgens at this early stage of development has been confirmed. Male rainbow trout and African catfish release high levels of 11-KT or 11ß-hydroxyandrostenedione (a precursor of 11-KT in the African catfish) in vitro, and steroid-producing Leydig cells are active [29, 30].

Data from the present study suggests that from September 1998 onward, some yearling male spring chinook salmon commenced early stages of spermatogenesis, as indicated by the increase in the proportion of transitional spermatogonia and the increase in plasma 11-KT levels above those of immature males. The proportion of males with elevated levels of plasma 11-KT increased from September 1998 onward and was similar to the final maturation rate for this group from January 1999 onward. During February 1999, a large proportion of males had elevated levels of plasma 11-KT, but no late B spermatogonia were found in their testes. By April 1999, only two such individuals were found, and the remaining males with elevated plasma 11-KT levels had germ cells developed to the late B spermatogonia stage or beyond. This finding supports our hypothesis that plasma 11-KT is elevated prior to the appearance of late B spermatogonia and plays a role in the early mitotic proliferation of germ and somatic cells. Furthermore, these data demonstrate that 11-KT levels can be used as an early indicator of maturation.

Although 11-KT has an established role in spermatogenesis in fish, it is centrally regulated by pituitary gonadotropins [18]. In this study, pituitary and plasma levels of FSH also increased during the first fall and were positively correlated with plasma 11-KT during this period. In salmonid fishes, FSH directly stimulates the production of 11-KT by the testis [18, 31] and induces spermatogonial proliferation [8]. Human chorionic gonadotropin can induce complete spermatogenesis in the Japanese eel [32, 33], but this stimulation probably occurs through its effect on 11-KT production because exogenous 11-KT can induce complete spermatogenesis in vitro in the Japanese eel in the absence of hCG [27]. In salmon, it has previously been suggested that FSH, but not LH, is involved in regulating early stages of gonad development and spermatogenesis, because it is detectable in the pituitary and plasma and can increase total androgen levels in juvenile salmonids [19, 34–37], whereas LH is not detectable in the plasma until the months just prior to spermiation. In mammals, FSH plays a major role in early spermatogonial development [38]. The results of the present study support the hypothesis that FSH is also involved in early testis maturation in fishes, possibly acting via the stimulation of testis 11-KT production and/or other mechanisms yet to be defined.

In addition to FSH and 11-KT, IGF-I also has a stimulatory effect on the early mitotic stages of spermatogenesis in salmonids in vitro [8, 39–41] and stimulates pituitary gonadotropin production [13]. Because IGF-I is produced by a number of fish tissues, including the testis, it is not known whether the actions of IGF-I on spermatogenesis are endocrine or are paracrine and autocrine as reported in mammals [42]. However, because peripheral levels of IGF-I are correlated with growth rate in salmonids [14–16, 43], we hypothesized that the mechanism whereby growth affects the proportion of fish maturing involves an endocrine effect of IGF-I on the testis and/or pituitary. Therefore, we expected elevations in plasma IGF-I during early stages of spermatogenesis. In the present study, plasma IGF-I changed seasonally, with highest levels occurring during September and lowest levels occurring during February, as has been reported in other studies of juvenile spring chinook salmon [44, 45]. Plasma levels of IGF-I increased from July to September 1998, and this increase coincided with a significant decrease in the proportion of primary A spermatogonia in the testes during that period. Plasma levels of 11-KT and pituitary FSH also increased during this period of early spermatogonial proliferation. This finding is consistent with a stimulatory role for IGF-I in the early mitotic stages of spermatogenesis in salmonids. In the Japanese eel, IGF-I and 11-KT act synergistically, and both are required for spermatogonial proliferation [41]. However, plasma levels of IGF-I in the chinook salmon subsequently fell during the fall period as spermatogonial proliferation proceeded and plasma levels of 11-KT and pituitary FSH continued to rise. In addition, from January onward the gradual increase in plasma IGF-I with the progression of spermatogenesis only became significant with the appearance of spermatids and tended to follow the general increase in body size. Thus, the specific actions of plasma IGF-I in spermatogenesis remain unclear, although the significant increase in plasma IGF-I at spermiation suggests a role during the final stages of spermatogenesis. Further work is required to determine the roles of intratesticular and plasma IGF-I on spermatogenesis in this species.

There are several additional growth factors and steroid hormones that have been implicated in the control of spermatogenesis in fishes, especially during early mitotic proliferation, including estradiol-17ß, 17{alpha}, 20ß-dihydroxy-4-prenen-3-one, fibroblast growth factor, and activin B [8, 46–49]. The importance of these factors at the different stages of spermatogenesis remains to be determined.

By January, several males had testes containing late B spermatogonia, an indication that these males were about to enter the meiotic stage of spermatogenesis. The appearance of late B spermatogonia was associated with a significant increase in pituitary and plasma FSH and plasma 11-KT levels. Increases in plasma 11-KT and/or FSH have previously been associated with this stage of spermatogenesis in eels and salmonids [2, 32, 49–51]. In the present study, as spermatogenesis proceeded through meiosis and spermiogenesis, a general increase in GSI, pituitary FSH, plasma FSH, and 11-KT occurred. Peak levels of plasma FSH and GSI coincided with the appearance of spermatozoa, whereas pituitary FSH and plasma 11-KT reached peak levels at spermiation. Plasma LH levels increased significantly only at spermiation. Similar changes in these parameters associated with the later stages of spermatogenesis have been reported for a variety of fish species [17, 23, 24, 36, 51–54]. These changes are similar to those seen in mammals, where testosterone, FSH, and LH are important for successful sperm production [38]. In fishes, both FSH and LH stimulate 11-KT, testosterone, and 17{alpha}, 20ß-dihydroxy-4-prenen-3-one production by the testis of maturing salmon [18, 31], and 17{alpha}, 20ß-dihydroxy-4-prenen-3-one is involved with final sperm maturation in the Japanese eel [55]. However, the specific roles of these and other factors in the later stages of spermatogenesis remain to be determined [56].

The present study provides physiological evidence for the initiation of spermatogenesis and activation of the pituitary-testis axis during the fall, 12 mo prior to spawning. This process coincides with the hypothesized fall decision period for initiation of maturation in salmonids [5]. Our data suggest that as fall proceeds the number of males commencing the initial stages of spermatogenesis increases and approaches the final rate of maturation of the population by January. It appears that by January the intent to mature or remain immature has been made, and maturing males proceed through the final stages of mitotic proliferation and differentiation of spermatogonia from late winter onward. The onset of meiosis coincides with the hypothesized spring decision period for the continuation of maturation. Two blocks in spermatogenesis have been described in the cultured eel model. An initial block to spermatogenesis prior to mitotic proliferation is overridden by injection of hCG and acts via the stimulation of 11-KT [32, 50, 57] and the reduction of an inhibitory factor similar to mammalian Mullerian-inhibiting substance [58]. A second block in spermatogenesis, which has been described in the Japanese eel and a newt (Cynops pyrrhogaster), occurs at a mitotic division prior to the appearance of late B spermatogonia, therefore controlling entry into meiosis [59, 60]. These animal models provide mechanisms whereby spermatogenesis may be controlled at specific stages of development. Similar blocks to spermatogenesis could be present in male chinook salmon represented by the fall and spring maturation decision periods described for salmonids. The present study was not specifically designed to test for these blocks to spermatogenesis; however, the results provide physiological evidence for the activation of the pituitary-testis axis during the fall critical period. Further work is required to determine whether the two critical maturation decision periods in spring chinook salmon are controlled by specific physiological blocks in spermatogenesis.


   ACKNOWLEDGMENTS
 
We thank Dr. Brian Beckman, Brad Gadberry, Paul Parkins, and Dr. Karl Shearer (Northwest Fisheries Science Center, Seattle, WA) for care of experimental fish. We also thank Kathy Cooper and Nicholas Hodges (Northwest Fisheries Science Center) for assistance with immunoassays and histology, respectively. Comments from Drs. Brian Beckman and Walt Dickhoff (Northwest Fisheries Science Center) during the preparation of this manuscript are also appreciated.


   FOOTNOTES
 
1 This work was supported by Bonneville Power Administration Project grant 93-056 to P.S. Back

2 Correspondence: Penny Swanson, Northwest Fisheries Science Center, 2725 Montlake Blvd. East, Seattle, WA 98110. FAX: 206 860 3467; penny.swanson{at}noaa.gov Back

Received: 25 June 2003.

First decision: 14 July 2003.

Accepted: 31 July 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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