Physiological evidence for two sturgeon gonadotrophins in Acipenser transmontanus

Aquaculture ELSEVIER

Aquaculture

135

( 1995) 27-39

Physiological evidence for two sturgeon gonadotrophins in Acipenser transmontanus Gary P. Moberg *, Jack G. Watson ‘, Serge Doroshov, Harold Papkoff, Raymond J. Pavlick, Jr. Department of Animal Science, University of California, Davis, CA 95616, USA

Abstract evidence in white sturgeon Acipenser transmontanus suggests that two gonadotrophins, termed sturgeon gonadotropin I (stGTH 1) and sturgeon gonadotropin II (stGTH II), are involved in regulating reproduction. Pituitary and plasma concentrations of stGTH I were found to be higher than concentrations of stGTH II during vitellogenesis and the early stages of spermatogenesis. Conversely, pituitary and plasma concentrations of stGTH II were greater compared with stGTH I during ovulation and spermiation. The gonadotropin releasing hormone analog D-Ala6-des-Gly”-GnRH ethylamide (GnRHa) was effective in stimulating the release of both gonadotrophins in mature males and preovulatory females, with a maximal response occurring in the spring. Collectively, the data supports our view that sturgeon possess a dual gonadotropin system controlling reproduction. Physiological

Keywords: Acipenser transmonranus; Gonadotrophins;

Gonadotropin-releasing

hormone; Steroids; Reproduction

1. Introduction While initial studies in teleosts (Burzawa-Gerard, 1982) suggested a single pituitary gonadotrophin (GTH) controlling reproduction, more recent reports have proposed a dual GTH system similar to tetrapods. Studies in chum salmon, Oncorhynchus ketu (Suzuki et al., 1988a; Suzuki et al., 1988b; Suzuki et al., 1988~) coho salmon, Oncorhynchus kisutch (Swanson et al., 1989; Swanson et al., 1991), common carp, Cyptinus carpio (Van Der Kraak et al., 1992)) and tuna, Thunnus obesus (Okada et al., 1994) have demonstrated two chemically distinct pituitary GTHs. While the functional differences between tuna gonadotrophins (termed tGTH I and tGTH II) have not been fully determined, much more is

* Corresponding I Deceased.

author.

0044.8486/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDlOO44-8486(95)01004-l

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G.P. Moberg et al. /Aquaculrure I35 (1995) 27-39

known about the biological properties of the salmonid gonadotrophins (termed GTH I and GTH II). GTH I and GTH II are synthesized in separate gonadotropes (Nozaki et al., 1990a), with GTH I cells more numerous during vitellogenesis and spermatogenesis and GTH II cells more abundant during spawning (Nozaki et al., 1990b). Correspondingly, peak plasma concentrations of GTH I and GTH II also occur at different times of reproductive development. Plasma GTH I is found in higher concentrations during vitellogenesis and the early stages of spermatogenesis, while plasma GTH II is greater during ovulation and spermiation (Suzuki et al., 1988d). GTH I and GTH II have been shown to behave similarly in stimulating 17P-estradiol ( E2) production in pre-vitellogenic (Swanson et al., 1989) and mid-vitellogenic (Suzuki et al., 1988~) ovarian follicles, but GTH-II is more potent than GTH I in stimulating 17cu, 20/3-dihydroxy-4-pregnen-3-one (17cu, 20P-DP) synthesis in post-vitellogenic follicles. (Suzuki et al., 1988~). Unlike in modern teleosts (Neoptergii), the presence of a dual GTH system in chondrostean species is just being studied. Initially, it was believed that sturgeon possessed only a single gonadotropin that regulated all aspects of reproductive development (BurzawaGerard, 1975). However, we have recently isolated and developed radioimmunoassays (RIAs) for two sturgeon GTHs which have been designated sturgeon gonadotropin I (stGTH I) and sturgeon gonadotropin II (stGTH II) (Moberg et al., 1991a). This paper presents data collected from several experiments which support our view that sturgeon, like modern teleosts, possess a dual GTH system responsible for the regulation of gametogenesis.

2. Materials and methods 2.1. Preparation

and characterization

of sturgeon gonadotrophins

The methods employed for the fractionation of lyophilized sturgeon pituitaries have been previously described in detail (Farmeret al., 198 1). When applied to a variety of mammalian and non-mammalian species (Papkoff et al., 1965; Papkoff et al., 1967; Papkoff et al., 1982; Licht et al., 1977; Farmer and Papkoff, 1977; Roser et al., 1984) these methods resulted in the isolation of highly purified preparations of the gonadotrophins, follicle stimulating hormone and luteinizing hormone. In the current study, sturgeon (Acipenser guldenstudti) pituitary glands were obtained from pre-spawning females from the delta of the Volga River near the Caspian Sea. Briefly, the initial steps followed those described in a study on the isolation of sturgeon growth hormone (Farmer et al., 198 1) and involved an alkaline (pH 9.5) extraction followed by treatment with metaphosphoric acid at pH 4.0 which precipitated the growth hormone-prolactin fraction. Material in the supernatant fluid was extracted with ammonium acetate-ethanol which resulted in a glycoprotein concentrate. This fraction was chromatographed on a column of sulfoethyl-Sephadex C50 (Pharmacia) in 0.03 M ammonium bicarbonate. The unadsorbed material was saved for purification of the fraction named stGTH I. The adsorbed material was further eluted with 1.O M ammonium bicarbonate and then applied to a column of DEAE-cellulose (Bio-Rad) in 0.03 M ammonium bicarbonate, pH 9.0. This fraction was applied to a gel filtration column of Sephadex-GlOO (Pharmacia) in 0.05 M ammonium bicarbonate. The major peak eluting with a Ve/Vo of about 2.5 was

G.P. Moberg et al. /Aquaculture

135 (1995) 27-39

29

re-run on the same column and eluted as a symmetrical peak with a Ve/Vo of 2.59. This yield was designated stGTH II. The unadsorbed material (stGTH I) from the sulfoethyl-Sephadex column described above was passed over a column of the cation resin Amberlite CG-50 (Fisher) to remove any residual stGTH II-like material and then applied to a column of DEAE-cellulose in 0.03 M ammonium bicarbonate, pH 9.0. This was chromatographed on a column of sulfoethylSephadex C50 in a pH 4.0,0.03 M acetate buffer. After removal of the unadsorbed material, elution was performed with 0.03 M ammonium acetate, pH 6.0. This material was gelfiltered 2 X on a column of Sephadex-GlOO. On the final run, the major symmetrical peak emerged with a Ve/Vo of 2.00. In the current study, chromatographic profiles were very similar to those obtained previously in other species (Licht et al., 1977; Farmer and Papkoff, 1977) The procedures for determining amino acid and carbohydrate compositions of both stGTHs were identical to the methods of Matteri et al. ( 1986). 2.2. Hormone analyses The development of RIAs for the measurement of stGTH I and stGTH II was described previously (Moberg et al., 1991a). Specific polyclonal antibodies to purified stGTH I and stGTH II fractions were raised in rabbits. Each antibody showed a minimum cross-reactivity with the other stGTH preparation. The stGTH I antibody displayed 2.0% cross-reactivity with stGTH II and the stGTH II antibody had 9.3% cross-reactivity with stGTH I. These antibodies were also shown to have minimum cross-reactivity ( < 0.0095%) with purified GTH from several fish species including hake, salmon, tilapia, carp, and gillichthes (Moberg et al., 1991a). The purified fractions of stGTH I and stGTH II also served as a source for radioiodinated gonadotrophins and standards. Iodination of the stGTHs with I’25 was performed using the Iodogen@ ( 1,3,4,5-tetrachloro-3-alpha, 6-alpha-diphenyl glycouril, Pierce Chemical Company) method (Moberg et al., 198 1) . stGTH I and stGTH II (5 pg) were dissolved in 50 ~1 of 0.05 M phosphate buffer (pH, 7.5) and mixed with 1 mCi of ‘251Na (Amersham) for 1 min. This stGTH-1251Na mixture was then added to a glass vial dry-coated with 12.5 pg of Iodogen@. Following a 3 min incubation period at room temperature, the mixture was withdrawn from the reaction vial and added to a column of Sephadex G-50 (Pharmacia) to separate free iodine from stGTH-bound iodine via gel filtration. The ‘*‘stGTH fractions were eluted with assay buffer which consisted of 0.05 M phosphate buffer (pH 7.0) containing 0.15 M NaCl (phosphate buffered saline, PBS), 1% gelatin (Type A from porcine skin, 60 bloom; Sigma Chemical Company), and 0.1% thimerosal (SigmaChemical Company). This buffer was also used to serially dilute standards, plasma, and pituitary extracts. PBS supplemented with 0.05 M EDTA (United States Biochemical Corporation) was used to dilute sera containing antibodies to stGTH I ( 1:25 000) and stGTH II (1:30 000). Triplicate standards and duplicate samples ( 100 ~1 in 400 ~1 assay buffer) were incubated with 200 ~1 of diluted antisera at 4°C for 24 h, followed by addition of 100 ~1 of 125stGTH (30 000-35 000 c.p.m.). After incubation for 48 h at 4°C antibody-bound stGTH was precipitated with 200 ~1 of goat anti-rabbit gamma-globulin (Antibodies, Inc.) which was

30

G.P. Moberg et al, /Aquaculture

slGTHI(ng/ml)

135 (1995) 27-39

slGTHII (@ml)

Fig. 1. Competition binding curves for (a) stGTH I RIA and (b) stGTH II RIA, for stGTH I standards (A), stGTH II standards ( + ), plasma (Cl), male pituitary extracts ( W), and female pituitary extracts (0). Each point represents the mean of duplicate samples.

initially diluted 1: 140 in assay buffer. Following incubation for 48 h at 4”C, 2 ml of double distilled water (4°C) was added to each test tube. The samples were then centrifuged (5000 g, 4°C) for 40 min. The supernatant was poured off and radioactivity in the remaining precipitated bound fraction was analyzed with a lo-well gamma counter (Micromedic). The minimum detectable concentration for the stGTH I RIA was 0.84 ng ml-’ with an inter- and intra-assay variability of 10.2% and 7.3%, respectively. The minimum detectable concentration for the stGTH II RIA was 1.25 ng ml-’ with an inter- and intra-assay variability of 9.7 and 6.1%. Competition curves of plasma and pituitary extracts from both male and female white sturgeon are shown in Fig. 1A (stGTH I) and 1B (stGTH II). For both stGTH I and stGTH I, an analysis of covariance ( ANCOVA) model showed the curves to be parallel. Testosterone (T) (Gay and Kerlan, 1978), E, (England et al., 1974)) and 17a, 20PDP (Scott et al., 1982) were analyzed by RIA procedures that have been previously described. Minimum detectable concentrations for T, E, and 17a, 20/3-DP were 0.52 ng ml-‘, 0.13 ng ml-‘, and 0.10 ng ml-‘, respectively. 2.3. Pituitary concentrations

of stGTHs

White sturgeon (Acipenser transmontanus) raised in captivity were killed at various stages of reproductive development by an overdose of tricaine methane sulfonate (MS-222; 5g 1- ’ ) . Pituitaries were quickly removed, frozen in cryogenic vials over dry ice, and stored at - 70°C. Once thawed, pituitaries were weighed and homogenized on ice with a motordriven mixer (Barnant Company) and pestle for approximately 1 min in 500 ~1 of 0.05 M P04, pH 7.0. The homogenized pituitaries were centrifuged (15 min, 7000 g) and the supernatant was analyzed for both stGTHs by RIA. Total pituitary protein was measured with an assay kit (Bio-Rad) which employed a protein-dye binding method (Bradford,

G.P. Moberg et al. /Aquaculture

1976) and was used to standardize protein.

135 (1995) 27-39

31

pituitary stGTH content as pg stGTH mg- ’ of pituitary

2.4. Histological analysis of gonadal development To determine the stage of reproductive development, gonadal tissue was collected at the time of death and fixed in 10% phosphate-buffered formalin. Following fixation, tissue was dehydrated and embedded in paraffin, cut in 6~ sections, and stained with either haematoxylin/eosin or periodic acid Schiff’s stain for differentiation of ovarian follicle structure (Doroshov et al., 1991). Under light microscopy, female sturgeon oocytes were assigned a score that discriminated between ovarian stages. Scores ranging from O-3 described ovaries that mainly consisted of clusters of gonial cells, early oocytes with undifferentiated follicular envelope, and adipose tissue. Fish with score 4 had initiated the development of egg chorion without any visible yolk deposition. Fish with scores 5-8 had oocytes in different stages of vitellogenesis (continuing 16-18 months in white sturgeon), while fish with score 9 approached the end of vitellogenic growth. At score 9, the oocytes of white sturgeon are already polarized, with germinal vesicle displaced into the animal hemisphere. Further observations on pre-ovulatory germinal vesicle migration are conducted by measurement of Polarization Index (germinal vesicle distance from the animal pole cortex:oocyte diameter at A-V axis) using boiled bisected eggs and computer-assisted digital image analysis. Ovulation is typically induced when Polarization Index reached < 0.01 (Doroshov et al., 1994). Male sturgeon development was assessed by the presence of gonial cells, spermatocytes, and differentiated spermatozoa in testicular cysts. 2.5. Plasma concentrations

of stGTHs

Six months prior to spawning, cultured female sturgeon (n = 11) were bled from caudal vasculature with 5 ml sodium-heparinized vacutainers in November 1992, February 1993, March 1993,36 h prior to spawning (April 1993)) during spawning, and 1 month following spawning (May 1993). Spawning was conducted in April using procedures described by Conte et al. ( 1988). Briefly, the gonadotropin releasing hormone analog D-Ala6-des-Gly”GnRH ethlyamide (GnRHa) was used to stimulate ovulation ( 10 pg kg-‘), followed by the injection of carp pituitary extracts 12 h later. Such empirically derived hormone induction procedures have been proven to be the most efficient in farm spawning management due to accurate timing of ovulatory response (22 f 2 h after second injection, mean f standard deviation, temperature 15-16°C). Ovulation is recognized by the initial discharge of small numbers of cohesive eggs onto the bottom of the tank, and the eggs are removed by caesarian section 1 h after onset of ovulation. The plasma was collected just prior to first injection, at ovulation (surgical egg removal), and 1 month after injection allowing for incision healing. The plasma was frozen at - 20°C until analyzed by RIA for stGTH I and stGTH II, T, E2, and 17q 20P-DP. 2.6. Effects of GnRHa on seasonal plasma stGTH concentrations GnRHa is generally similar in structure to sturgeon GnRH (Sherwood et al., 1991) and induces spawning in sturgeon (Doroshov and Lutes, 1984). To determine if season effects

32

G. P. Moberg et al. /Aquaculture

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GnRHa-induced secretion of stGTHs, a group of ten mature male sturgeon were given an injection ( 10 pg kg- ‘) of GnRHa in December 1990, April 1991, and August 1991. Blood was sampled at 0, 6, 24, and 48 h after GnRHa-injection. The plasma was separated by centrifugation and frozen at - 20°C until analyzed by RIA for stGTH I and stGTH II. 2.7. Statistical analyses Pituitary and plasma stGTH data was analyzed using either the Student’s t-test or one way analysis of variance (ANOVA) with Duncan’s New Multiple Range Test (SuperANOVA, Abacus Concepts). To test for parallelism in the stGTH I and stGTH II competition curves, an analysis of covariance (ANCOVA) model was used which considered both treatment and regression effects (SAS System for Windows@, version 6.08, SAS Institute, Inc., 1992). The accepted significance level for all statistical tests was P < 0.05.

3. Results Carbohydrate analyses showed stGTH I to contain 12.6% carbohydrate (5.5% hexose, 6.8% hexosamine, and 0.2% sialic acid) and stGTH II to contain 13.7% carbohydrate (6.4% hexose, 7.0% hexosamine, and 0.3% sialic acid). N-terminal analyses showed leucine and tyrosine to be the major termini for stGTH I and aspartic acid, tyrosine, and leucine the major termini for stGTH II. Both preparations showed traces of several other amino acids. Preliminary amino acid analysis of the two preparations (Table 1) showed differences between the two with respect to the content of several amino acids (aspartic acid, serine, Table I Amino acid composition

of sturgeon gonadotropina

Amino acid

stGTH I

stGTH II

Aspartic acid Threonine Serine Glutamic acid Profine Half Cystine Glycine Alanine Valine Methionine lsoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine

10.6 7.7 5.9 9.1 5.3 6.7 7.9 4.7 4.7 1.4 3.2 7.0 4.8 5.5 2.8 9.8 4.4

8.8 7.8 8.6 10.0 7.1 9.1 4.0 3.9 5.5 1.8 3.5 8.0 4.4 4.5 2.6 7.1 3.3

“Results are expressed as residues/

100 residues analysed;

uncorrected

for hydrolytic

destruction.

G.P. Moberg et al. /Aquaculture

135 (1995) 27-39

33

140 130 3 120 9 g& 110 8 .-&

‘; 80

p

70

s

60

E

50

;: ‘2

20

b

10 n

GORE0

(n%?Z)

2 (n=3)

3 (n=14)

4 cn=n

$4,

Pre-vitellogenic -+

+Immahm+

7 (r&=1)

Vitellogenic ___t

8 O-4)

9 (“=I)

Ovulatory _I

Fig. 2. The mean ( f SEM) pituitary concentrations (kg stGTH mg-’ pituitary protein) of stGTH I (stipled bars) and stGTH II (diagonal bars) in cultured female sturgeon during the immature (scores (X2). pre-vitellogenic (scores 34). vitellogenic (scores 6-8). and ovulatory (score 9) stages of reproductive development. See text for description of scoring system. Values in parentheses indicate the number of sturgeon per score. Pituitary stGTH I was significantly higher (P < 0.05) than pituitary stGTH II in fish with score 0, 3.4, and 6 oocytes. Pituitary stGTH I and stGTH II in fish with score 8 and 9 oocytes were significantly greater (P< 0.05) than in fish with all other scores. 7x) 700 6M 600 5.50 500 4w 400 350 300 250 200

150 100 50 :::::::::::::::;:i: ... ..\. ..

0-

‘TFre ”

Mature cn=n

Spymgting n

REPRODUCDVE STAGE Fig. 3. The mean ( f SEM) pituitary concentrations (pg stGTH mg-’ pituitary protein) of stGTH I (&pled bars) and stGTH II (diagonal bars) in reproductively immature (genial), mature (meiotic), and spermlating male sturgeon. Values in parentheses indicate the number of sturgeon per reproductive stage. Concentrations of pituitary stGTH I and stGTH II in spermiating fish were significantly greater (PCO.05) than in immature and mature males.

34

G.P. Moberg etal. /Aquaculture

NOV

135 (1995) 27-39

Feb

TIME

Fig. 4. The mean ( f SEM) plasma concentration (ng ml-‘) of stGTH I (open bars), stGTH II (solid bars), oestrogen (diagonal bars), testosterone ( stipled bars), and 17% 20/3-dihydroxykt-pregnen-3-one (vertical bars) in cultured female sturgeon (n = 11) in November, February, march, 36 h prior to spawning (pm-spawn), at spawning and 1 month following spawning (post-spawn). Plasma stGTH I was significantly higher (P < 0.05) than plasma stGTH II in fish sampled in November, February, March and 36 h prior to spawning.

proline, glycine, and lysine) . Both analyses are typical of analyses reported previously for gonadotrophins (Licht et al., 1977). The concentration of pituitary stGTHs during ovarian development in cultured female sturgeon is shown in Fig. 2. In the immature stage, both stGTHs were present in low amounts. As the fish progressed towards a pre-vitellogenic stage, both stGTHs rose in the pituitary, with pituitary stGTH I significantly higher (P < 0.05) than pituitary stGTH II in fish with score 0, 3,4, and 6 oocytes. This same pattern existed throughout the early stages of vitellogenesis. However, during late vitellogenesis the level of stGTH II increased sharply and eventually exceeded the concentration of stGTI-I I at the pre-ovulatory stage. Concentrations of pituitary stGTH II in fish with score 8 and 9 oocytes were significantly higher (P < 0.05) than in fish with all other scores. Pituitary concentrations of stGTHs in developing male sturgeon (Fig. 3) are comparable to the results in female sturgeon. Both stGTHs are low in the immature (gonial) stage, but

G.P. Moberg et al. /Aquaculture

135 (1995) 27-39

35

&li 8 g

i a

30

20 ‘00 40

Summer i

0

24

6 TIME

48

(lus)

Fig. 5. Seasonal effects of GnRHa ( 1Opg kg-‘; administered at 0 h) on the mean ( f SEM) plasma concentrations (ng ml-’ ) of stGTH I (.&pled bars) and stGTH II (diagonal bars) at 0,6,24,48 h post GnRHa injection in adult male sturgeon (n = IO). Bars represent mean f SEM of plasma concentrations of stGTH I at 24 h post GnRHa injection were significantly greater (P < O.OS)thanat 0 h for aI1seasons. Plasma concentrations of stGTH II at 24 h post GnRHa injection were significantly greater (P CO.05) than at 0 h for winter and spring only. Also, plasma concentrations of both stGTH I and sgGTH II at 24 h post GnRHa injection were significantly higher (P < 0.05) in the spring compared with 24 h post GnRHa injection during the winter and summer.

rise as testicular development (meiosis and spermiation) progresses. Similar to ovulation, the pituitary concentration of stGTH II exceeds stGTH I during spermiation. Concentrations of pituitary stGTH I and stGTH II in spermiating fish were significantly greater (P < 0.05) than in immature and mature males. Plasma concentrations of stGTH I in mature female broodstock (Fig. 4) were significantly higher (P < 0.05) than stGTH II during the vitellogenic stages in fish sampled in November, February, March, and 36 h prior to spawning. At spawning, both plasma stGTH I and stGTH II increased, with stGTH II greater, but not statistically higher, than stGTH I. Both T and E2 decreased prior to and during spawning, whereas 17a, 20PDP reached peak levels at ovulation. Plasma concentrations of all steroids declined 1 month following spawning. The effect of GnRHa on the secretion of stGTHs from the pituitaries of mature male sturgeon depended on the time of year (Fig. 5). Plasma concentrations of both stGTH I and stGTH II at 24 h after GnRHa injection were significantly higher (P < 0.05) in the spring (April) compared with 24 h post GnRHa injection during the winter (December) and summer (August). Plasma concentrations of stGTH I at 24 h post GnRHa injection were

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G.P. Moberg et al. /Aquaculture

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significantly greater (P < 0.05) that at zero hours for all three seasons. Plasma concentrations of stGTH II at 24 h post GnRHa injection were significantly greater (P < 0.05) that at zero hours for winter and spring only. Of the two stGTHs, stGTH II showed the greater response to GnRHa stimulation during the spring, while stGTH I had the greater response during the winter and summer.

4. Discussion Based on these data, it appears that sturgeon possess two gonadotrophins which may be functional analogs of salmonid GTH I and GTH II (Kawauchi et al., 1989; Swanson et al., 1989). While plasma levels of both stGTH I and stGTH II are low in reproductively immature or pre-vitellogenic sturgeon, stGTH I is the predomindant gonadotropin found in the pituitary at these stages of development (Fig. 2). At the onset of and throughout vitellogenesis, both gonadotrophins in the pituitary increase, with the ratio of stGTH I to stGTH II remaining relatively constant. However, prior to and during final ovarian maturation and ovulation, stGTH II begins to rise sharply and eventually becomes the more abundant gonadotropin in the pituitary, exceeding stGTH I two-fold. These results are, in general, similar to observations reported in salmonids (Swanson et al., 1989). It is not known if stGTH I and stGTH II are synthesized in separate gonadotropes, as is the case in salmonids (Nozaki et al., 1990a). Elevations of plasma stGTH I that occurred during vitellogenesis (Fig. 4) are similar to reported increases of GTH I at the same stage of development in salmonids (Suzuki et al., 1988d). Coupled with the fact that stGTH I is the predominant gonadotropin in the pituitary at this time, the data suggest that stGTH I may play an important role in regulating the onset of vitellogenesis. Previous in vitro studies in rainbow trout have shown than GTH I causes an increase in vitellogenin uptake by developing oocytes, thereby stimulating their growth (Tyler et al., 199 1) . Although we have been unable to experimentally assess this effect in sturgeon, stGTH I could potentially act in a similar fashion. Current information has shown that reproductive development of cultured female sturgeon often becomes arrested at the pre-vitellogenic stage (score 3 or 4). Even when sturgeon are treated with estradiol implants to increase vitellogenin production by the liver, the developing oocytes fail to sequester circulating vitellogenin (Moberg et al., 1991 b). The stGTH I is not found in the circulation in these animals, and secretion of stGTH I cannot be induced with GnRHa (Moberg et al., 1991b). It is believed that because the pituitary fails to secrete stGTH I during this stage of development, vitellogenesis and further gonadal maturation can not occur. Plasma stGTH II is not found in high concentrations until the time of final ovarian maturation, ovulation, and spawning, in agreement with observations in salmonids (Suzuki et al., 1988d) for stGTH II. Low plasma levels of stGTH II during early development is not surprising based on the fact that there are low amounts of this hormone stored in the pituitary. The functional differences of stGTHs have been previously assessed by an in vitro germinal vesicle breakdown (GVBD) response with ripe ovarian follicles of white sturgeon (Moberg et al., 1991a). Based on these results, stGTH II is much more potent in inducing GVBD than GTH I when administered in equal concentrations. Coupled with the fact that

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135 (1995) 27-39

31

both pituitary and plasma concentrations of stGTH II are greater than stGTH I at the time of final ovarian maturation and ovulation, these results reaffirm that stGTH II is one of the hormones responsible for regulating spawning events. However, whether stGTH II acts through the stimulation of C-21 steroid production is not known at this time. The pattern of pituitary stGTH content in male sturgeon (Fig. 3) was similar to that in the female sturgeon. Mature males in the early meiotic stages of spermatogenesis have greater amounts of pituitary stGTH I compared to stGTH II. However, during spermiation, pituitary concentrations of stGTH II exceed levels of stGTH I. Biological functions of these two hormones in male sturgeon have not been assessed. White sturgeon males in culture complete meiosis and spermiogenesis in winter, develop mature spermatozoa in spring, and undergo mitotic and early meiotic phases of testicular development in summer. The fact that stGTH I is released in greater amounts than stGTH II in response to GnRHa in December (Fig. 5) suggests that stGTH I may be responsible for regulating the meiotic phase of spermatogenesis during the winter. GnRHa-induced secretion of stGTH II, However, is maximal during spermiation in the spring, implying that stGTH II may be more important in controlling this event. In summary, the present set of experiments provides evidence that sturgeon, like salmonids, possess two pituitary gonadotrophins. Pituitary and plasma concentrations of stGTH I are greater that stGTH II in pre-vitellogenic and vitellogenic sturgeon, while pituitary and plasma levels of stGTH II are higher during final ovarian maturation, ovulation, and spermiation. As determined by an in vitro response, stGTH II is a more potent inducer of GVBD that stGTH I. GnRHa was effective in stimulating the release of both gonadotrophins in mature male sturgeon, with a maximal response occurring in the spring. While the present data offer new evidence for the presence of two sturgeon gonadotrophins, a more complete assessment of these stGTHs on steroid production, vitellogenesis, and gonadal development is needed as more purified preparations of stGTHs become available. Additionally, future work must also be directed towards identifying the structures and physicochemical properties of stGTHs which would permit comparisons with teleost GTHs.

Acknowledgements The authors would like to thank Joel Van Eenennaam and Kevin J. Kroll for their technical assistance in handling the fish and collecting pituitaries and blood samples. This work was supported by NOAA grant NA89AA-D-SG138, project R/A-85.

References Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72:248-254. Burzawa-Gerard, E., 1975. L’hormone gonadotrope hypophysaire d’un Poisson chondrosteen, I’esturgeon (Acipenser stellarus Pall.). 1.Purification. Gen. Comp. Endo., 27:289-295. Burzawa-Gerard, E., 1982. Chemical data on pituitary gonadotrophins and their implication to evolution. Can. J. Fish. Aquat. Sci., 39:80-91.

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