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Little is known about previtellogenic oocyte growth in fish despite its importance in determining age of puberty. This study aimed to reveal ovarian genes expressed during previtellogenic growth in coho salmon (Oncorhynchus kisutch), a semelparous teleost that exhibits synchronous follicle development. Cortical alveolus stage ovaries were enriched for follicle/interstitial cell layers prior to RNA isolation to increase their contribution relative to abundant oocyte transcripts. A cDNA library was constructed and 1783 ESTs were analyzed. Numerous follicle/interstitial cell related genes were identified, including anti-Müllerian hormone, lipoprotein lipase, gonadal-soma derived growth factor, connective tissue growth factor, relaxin-3, basigin, and 3β-HSD. Claudin genes that may relate to cell-cell contact were also observed. Several genes involved in the regulation of apoptosis/atresia were identified, such as caspase-1, TNF receptor, and clusterin. Oocyte related genes identified included zona pellucida proteins, lectins, and cathepsins. This research sheds light on ovarian genes expressed during previtellogenic growth and provides tools for future candidate gene studies.

Captive breeding programs have been established to prevent extinction and aid recovery of imperiled stocks of salmonid fish in North America. Several reproductive problems limit their effectiveness, including early age of male puberty, asynchronous maturation of adults, inadequate development of secondary sex characters and behavior, and poor fertility. Contrary to commercial aquaculture, selective breeding cannot be used to solve these problems since preserving the genetic diversity of the wild stocks is paramount. Studies were conducted to improve broodstock management and mitigate these reproductive problems. Restricting body growth during the fall-winter period in yearling spring Chinook salmon significantly reduced the percent of males maturing at age 2; body fat levels affected maturation rates only in small fish (< 50 g). In females, restricting body growth reduced the rate of previtellogenic oocyte growth, but did not affect fecundity up to the late cortical alveolus stage. Implants containing gonadotropin-releasing hormone analogue were used to advance and synchronize spawning, without impairing fertility of adults spawned in captivity or spawning behavior in an artificial stream. Screening for Y-chromosome specific genetic markers, ultrasound and plasma sex steroid levels were tested as methods to identify gender and state of maturity. Maturing males could be identified 7 months prior to spawning using plasma 11-ketotestosterone levels, while ultrasound could be used 5 months prior to spawning. The GH-pseudo gene served as a genetic marker for male Chinook and coho salmon. Although these techniques improved effectiveness of captive breeding programs, egg fertility problems persist and habitat restoration remains as a significant impediment to success of stock restoration. Thus, new germ cell cryopreservation technology is being tested for stocks most at risk of extinction.

[EN] The luteinizing hormone (LH) is a key regulator of the processes of gonad maturation, ovulation/spermiation and spawning in vertebrates. The present study describes the purification of LH from pituitaries of a teleost fish, the European sea bass (Dicentrarchus labrax), by three-step chromatography (gel filtration, ion-exchange and FPLC). The LH α and β subunits were isolated by rpHPLC. The molecular weight of LH was estimated, on SDS-PAGE, at 31 kD, and for its α and β subunits, at 12 and 22 kD, respectively. Specific antibodies against the sea bass LHβ subunit (AbLHβ) were obtained and used to develop a specific enzyme-linked immunosorbent assay (ELISA), which had a sensitivity of around 0.65 ng mL-1 (Bi/Bo 80%) and intraand inter-assay coefficients of variation of 11.7% (n = 8) and 11% (n = 10), respectively. Validation of the assay showed parallelism between the standard curve and plasma and pituitary samples from sea bass, as well as with pituitary extracts from other perciform fishes. We measured, using ELISA, plasma LH levels in female sea bass given a single injection of different doses of GnRHa ([D-Ala6, Pro9-Net]-LHRH), as a hormonal therapy for spawning induction. All treatments increased plasma LH levels after 90 min of the injection and were maintained elevated during 24 h. In conclusion, the immunoassay developed is sensitive and accurate, useful for LH analysis in biological samples of sea bass, and represents a valuable tool for studies on the reproductive endocrinology of this species., [ES] La hormona luteinizante (LH), secretada por la glándula hipofisaria, regula los procesos de maduración gonadal, ovulación/espermiación y puesta en vertebrados. El presente trabajo describe la purificación de la LH de un pez teleósteo, la lubina europea (Dicentrarchus labrax), mediante triple cromatografía en columna (gel filtración, intercambio iónico y FPLC). Las subunidades α y β de la LH se aislaron mediante rpHPLC. El peso molecular de la LH se estimó, mediante SDS-PAGE, en 31 kD y el de sus subunidades α y β en 12 y 22 kD, respectivamente. Se obtuvieron anticuerpos específicos contra la subunidad LHβ (AbLHβ), que fueron utilizados para desarrollar un inmunoensayo tipo ELISA. La sensibilidad del ELISA fue de unos 0.65 ng mL¯¹ (Bi/Bo 80%) y los coeficientes de variación intra e interensayo, de 11.7% (n = 8) y 11% (n = 10), respectivamente. La validación del ensayo mostró paralelismo entre la curva patrón (LH) y muestras de plasma e hipófisis de lubina, así como con extractos hipofisiarios de otras especies de peces perciformes. Mediante ELISA, se analizaron los niveles plasmáticos de LH en lubinas sometidas a tratamiento de inducción hormonal a la puesta, por inyección simple de diferentes dosis de GnRHa ([D-Ala6, Pro9 - Net]-LHRH). Todos los tratamientos provocaron un marcado incremento de la LH plasmática a los 90 min después de la inyección, manteniéndose elevados durante 24 h. En conclusión, el ELISA puesto a punto es un inmunoensayo sensible y preciso, apropiado para cuantificar LH en muestras biológicas de lubina y representa una valiosa herramienta para realizar estudios sobre la endocrinología de la reproducción de esta especie., Este estudio ha sido financiado por la Comunidad Europea(proyecto FAIR CT97-3785) y el gobierno español (proyectoCICYT MAR1998-1542CE).