Spermiating Fish Research Articles

Seasonal variation of pituitary gonadotropin subunit, brain-type aromatase and sex steroid receptor mRNAs, and plasma steroids during gametogenesis in wild sablefish

Pituitary-hormone signaling plays critical roles in the onset and progression of gametogenesis in vertebrates. This study characterized expression patterns of pituitary gonadotropin beta-subunits (fshb and lhb), brain-type aromatase (cyp19a1b), androgen (ar1, ar2) and estrogen receptors (esr1, esr2a, esr2b), and changes in plasma steroid levels by liquid chromatography/tandem mass spectrometry in wild sablefish (Anoplopoma fimbria, order Scorpaeniformes) during a complete reproductive cycle. Transcripts for fshb increased during early gametogenesis and peaked in late vitellogenic females and late recrudescent males, while expression of lhb reached maximum levels in periovulatory and spermiating fish. Pituitary levels of cyp19a1b and ar1 were strongly correlated with those of lhb in females and males, increasing during gametogenesis and reaching maximum levels prior to spawning. By contrast, expression of ar2, and the three estrogen receptors differed between female and male sablefish. 17β-estradiol (E2) was the dominant steroid in females during vitellogenesis, while a range of at least 6 steroids (11β-hydroxyandrostenedione, testosterone [T], E2, 11-ketotestosterone [11KT], 11-deoxycortisol, and 17α,20β,21-trihydroxyprogesterone) were detected at similar levels in males during testicular development. Prior to spawning, a marked increase in 4-androstenedione, T, 11KT and E2 was found in both periovulatory females and spermiating males. In conclusion, the concomitant changes in plasma androgen levels and pituitary ar1 expression during gametogenesis suggest a specific role for androgens in pituitary hormone regulation of reproduction in sablefish. Further, our data highlight the importance of E2 during final stages of maturation in this species, which may regulate the transcription of pituitary lhb in a paracrine fashion.

Development of a flatfish-specific enzyme-linked immunosorbent assay for Fsh using a recombinant chimeric gonadotropin

In flatfishes with asynchronous and semicystic spermatogenesis, such as the Senegalese sole (Solea senegalensis), the specific roles of the pituitary gonadotropins during germ cell development, particularly of the follicle-stimulating hormone (Fsh), are still largely unknown in part due to the lack of homologous immunoassays for this hormone. In this study, an enzyme-linked immunosorbent assay (ELISA) for Senegalese sole Fsh was developed by generating a rabbit antiserum against a recombinant chimeric single-chain Fsh molecule (rFsh-C) produced by the yeast Pichia pastoris. The rFsh-C N- and C-termini were formed by the mature sole Fsh β subunit (Fshβ) and the chicken glycoprotein hormone common α subunit (CGA), respectively. Depletion of the antiserum to remove anti-CGA antibodies further enriched the sole Fshβ-specific antibodies, which were used to develop the ELISA using the rFsh-C for the standard curve. The sensitivity of the assay was 10 and 50pg/ml for Fsh measurement in plasma and pituitary, respectively, and the cross-reactivity with a homologous recombinant single-chain luteinizing hormone was 1%. The standard curve for rFsh-C paralleled those of serially diluted plasma and pituitary extracts of other flatfishes, such as the Atlantic halibut, common sole and turbot. In Senegalese sole males, the highest plasma Fsh levels were found during early spermatogenesis but declined during enhanced spermiation, as found in teleosts with cystic spermatogenesis. In pubertal males, however, the circulating Fsh levels were as high as in adult spermiating fish, but interestingly the Fsh receptor in the developing testis containing only spermatogonia was expressed in Leydig cells but not in the primordial Sertoli cells. These results indicate that a recombinant chimeric Fsh can be used to generate specific antibodies against the Fshβ subunit and to develop a highly sensitive ELISA for Fsh measurements in diverse flatfishes.

Effects of Gonadectomy on Plasma Gonadotropins I and II in Coho Salmon,Oncorhynchus kisutch

Studies of both salmon and trout have indicated that the levels of gonadotropins, GTH I and GTH II, in the pituitary and peripheral circulation vary during the reproductive cycle. To evaluate the possible feedback relationship between the gonads and pituitary GTH secretion, we studied the effects of bilateral gonadectomy on plasma levels of GTH I and GTH II in coho salmon,Oncorhynchus kisutch.During late spermatogenesis in males and late vitellogenesis in females, plasma GTH I levels increased significantly after gonadectomy, approximately 6- and 5-fold over presurgery levels at 3 and 14 days after surgery, respectively, and then declined to near presurgery levels by Day 17. No change in GTH I levels occurred in sham-operated fish. In all groups, GTH II levels were nondetectable and did not change significantly up to 17 days postsurgery. In males gonadectomized during spermiation, plasma GTH I levels increased significantly, approximately 10-fold over presurgery levels by 7 days postsurgery, and remained elevated thereafter. In contrast to the males in late spermatogenesis, the spermiating fish had detectable levels of GTH II (2–3 ng/ml), and significant elevations in plasma GTH II levels (approximately 60-fold) were observed 7 days after gonadectomy. These experiments demonstrate that the gonads exert negative feedback effects on secretion of both GTH I and GTH II, but the effect varies seasonally and the nature of the specific factor(s) from the gonads that inhibits and/or stimulates GTH production and secretion remains to be clarified.

Binding characteristics and regulation of the 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S) receptor on testicular and sperm plasma membranes of spotted seatrout (Cynoscion nebulosus)

The binding characteristics of 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S) to plasma membranes prepared from the testes and sperm of spotted seatrout (Cynoscion nebulosus) were investigated using a filtration method to retain the bound 20β-S. A single class of high affinity (Kd = 17.9 nM), low capacity (Bmax = 0.072 nM g-1 testes) binding sites was identified by saturation and Scatchard analyses on testicular membranes of spermiating spotted seatrout. A corresponding receptor (Kd = 22.17 nM, Bmax = 0.00261 nM ml-1 milt) was also detected in spermatozoan membrane preparations. The rates of 20β-S association and dissociation were rapid, both had Thalfs of less than 1 min. Competition studies indicated that the receptor was highly specific for 20β-S. 17,20β-dihydroxy-4-pregnen-3-one, which had the highest affinity of the other steroids tested, had a relative binding affinity (RBA) of 14.3%. Progesterone, 11-deoxycortisol and testosterone competed with an order of magnitude less affinity (RBA's of 7.4, 1.8 and 1.1%, respectively). Estradiol displayed low affinity for the receptor (RBA = 0.4%) and cortisol did not cause any displacement at 1000-fold excess concentration. Specific 20β-S receptor binding was detected in plasma membranes from testes of both spermiating and non-spermiating seatrout and on spermatozoa. Prolonged incubation of testicular fragments from a spermiating fish with gonadotropin (15 IU ml-1 human chorionic gonadotropin) or forskolin (10 µM) caused a 2–3 fold increase in membrane receptor binding. Previous studies have shown that gonadotropin-induced upregulation of the 20β-S plasma membrane receptor in seatrout ovaries is required for the oocytes to become responsive to 20β-S and undergo final maturation. The existence of a 20β-S membrane receptor on sperm and its upregulation in the testes by gonadotropin raises the possibility that final maturation of spermatozoa in male seatrout may be regulated by a similar mechanism.

Estradiol-17? suppresses testicular development and stimulates sex reversal in protandrous black porgy, Acanthopagrus schlegeli

Two year old black porgy (Acanthopagrus schlegeli) fed a diet containing 4.0 mg kg(-1) of estradiol-17β (E2) for 5 months had significantly lower GSI than the control group during the spawning season. E2 suppressed testicular development, spermiation and plasma testosterone (T) and 11-ketotestosterone (11-KT) and stimulated ovarian development, vitellogenesis and sex reversal. Spermiation in the control group occurred in January and February with the concentrations of 1.08-1.36 × 10(10) sperm ml(-1) of milt. Higher plasma T and 11-KT, but lower E2 levels were detected in the spermiating fish (control group). Higher plasma E2 levels were detected in the sex reversing black porgy during the pre-spawning season. A sharp rise in plasma 11-KT and a drop in T levels were detected in spermiating fish (control group) from January to February. Plasma 11-KT levels correlated with the testicular development and spermiation. The data suggest that E2 plays an important role in controlling the sex reversal of black porgy.

Localization of two gonadotropin receptors in the salmon gonad by in vitro ligand autoradiography.

Receptors for two salmon gonadotropins, GTH I and GTH II, were localized by use of in vitro ligand autoradiography of coho salmon gonads at various stages of sexual maturation. The results in both sexes revealed the presence of two types of GTH receptors: type I (GTH-RI), which interacts with both GTHs, and type II (GTH-RII), which interacts specifically with GTH II. GTH-RI was found at all stages of spermatogenesis examined and was localized on cells that were most likely Sertoli cells; however, it could not be determined whether GTH-RI was also localized on Leydig cells. In contrast, GTH-RII was found only in Leydig cells in the testis from a spermiating fish. In the vitellogenic ovary, GTH-RI was localized in the thecal layer and intensely on granulosa cells; in the preovulatory follicle, in contrast, GTH-RI was found in the thecal layer and in interstitial connective tissue, but not in the granulosa layer. Among all the stages of oogenesis examined, only granulosa cells of the preovulatory follicle exhibited GTH-RII. The appearance of GTH-RII coincides well with the increase in plasma levels of GTH II that occurs during final oocyte maturation and spermiation in coho salmon. The nature, distribution, and timing of appearance of these two receptors can explain, at least in part, the results of previous studies on steroidogenic activities of the two GTHs. The present study also suggests the functional homology of salmon GTH I and GTH II to mammalian FSH and LH, respectively, during gonadal development.

Plasma levels of sex steroids during sexual maturation of snapper, Pagrus auratus (Sparidae), caught from the wild

Snapper, Pagrus auratus, were caught by baited hook from the Hauraki Gulf, northeast New Zealand, from April (autumn) 1990 to January (summer) 1991. Fish were blood-sampled immediately upon being taken aboard the vessel. From April to August, all female fish caught were sexually immature, the first ovulated fish was caught in late September and, thereafter, they occurred through to December. Male fish caught from April to July were sexually immature, the first spermiating fish was caught in October and they occurred through to January. In females, plasma estradiol (E 2) increased from low levels in sexually immature fish, to peak (0.93 ng ml −1) in fish undergoing ovarian recrudescence and ovulation. Plasma testosterone (T) levels followed a similar pattern to E 2, with a maximum mean of 0.63 ng ml −1. There was evidence that levels of both of these steroids changed according to time of day during the spawning season. Levels of 17 α,20 β-dihydroxy-4-pregnen-3-one (17,20 β-P) were low (mean level 0.3 ng ml −1) and did not change significantly with either month or time of day. In males, plasma T level increased from a minimum in sexually immature fish, to peak at 1.1 ng ml −1 in fish undergoing spermiation. Plasma 11-ketotestosterone levels followed a very similar pattern to T levels, but the range of values was greater (max. 2.8 ng ml −1). Levels of both androgens varied according to time of day in spermiating fish. Plasma levels of 17,20 β-P changed significantly with month, and were highest (0.63 ng ml −1) during the period of spermiation, but no change with time of day was apparent. Plasma levels of 17,20 β-P sulphate were determined in selected fish, and were low ( < 1 ng ml −1) at all times. The absence of changes in plasma 17,20 β-P level during sexual maturation in females is at variance with its suggested role in final oocyte maturation in snapper.

Sperm production and steroidogenesis in testes of the common carp, Cyprinus carpio L., at different stages of maturation

Testes from carp, Cyprinus carpio L., at five different maturational stages from immature through to spermiation and regression were incubated with or without addition of carp hypophysial homogenate (chh) for 8 or 20 h. Concentrations of steroids and spermatozoa were measured in the medium and the residual tissue examined histologically. There was an increase in the area of the germinal cysts containing spermatozoa, the percentage of the testis which they occupied and in the production of spermatozoa as the gonadosomatic index (GSI) increased, but this was unaffected either by incubation or by pretreatment with chh. The major steroid in plasma and in in vitro testicular cultures from all of the maturing fish captured in winter was 1 I‐ketotestosterone. The production rate of this steroid in virro was unaffected by GSI, while plasma levels tended to increase with GSI. 17.20β‐Dihydroxy‐4‐pregnen‐3‐one was detectable in significant amounts in only a few spermiating fish in summer, but was stimulated more in incubations with chh in maturing winter than in summer spermiating or post‐spawning fish. 17,20a‐Dihydroxy‐4‐pregnen‐3‐one was not detectable in incubations, but plasma concentrations tended to increase towards spermiation and were positively correlated with the size of the cyst. After spawning, fish had low plasma steroid levels and failed to respond in vitro to pituitary extract, indicating a testicular post‐spawning refractoriness.