Search

Combining cryopreservation of germline stem cells (GSCs) with their subsequent transplantation into recipient fish is a powerful tool for long-term preservation of genetic resources of endangered fishes. However, application of this technique has been limited because endangered species sometimes have small gonads and do not supply enough GSCs to be used for transplantation. This limitation could be overcome by expanding GSCs in vitro, though this has been difficult due to the complexity of reconstructing the gonadal microenvironment that surrounds GSCs. Here, we describe a novel method of in vitro expansion of rainbow trout GSCs using a feeder layer derived from Sertoli cells and a culture medium containing trout plasma. A transplantation assay demonstrated that the in vitro-expanded GSCs exhibited stem cell activity and potency to produce functional eggs, sperm, and eventually healthy offspring. In vitro expansion of GSCs can aid in rescuing fishes that are on the verge of extinction., Yoshiko Iwasaki-Takahashi et al. report the production of fertilization-capable eggs and sperm from rainbow trout spermatagonia, for application to endangered species preservation. Their method avoids the need for limited numbers of germline stem cells for transplantation, instead allowing in vitro expansion of these cells using a Sertoli cell-derived feeder layer.

A novel immunologic method using antigen-antibody reactions and electrochemical measurement to monitor the oocyte maturation-inducing hormone 17, 20β-dihydroxy-4-pregnen-3-one (DHP) in fish was dev...

Recombinant gonadotropins (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) of goldfish Carassius auratus were previously produced by baculovirus in silkworm larvae Bombyx mori. These GTHs are heterodimeric hormones consisting of a common α subunit and an FSHβ or LHβ subunit; the GTH subunits were produced separately in silkworm larvae. In order to simplify the production process of the hormones and increase production efficiency, we produced single-chain FSH (scFSH) and LH (scLH) in which the FSHβ or LHβ subunit was fused to the α subunit. Biological activities of these single-chain GTHs produced by baculovirus–silkworm larvae system were analyzed in vivo using male goldfish, female bitterling Rhodeus ocellatus ocellatus, and male Japanese eel Anguilla japonica. Injection of hemolymph containing scFSH or scLH induced milt production in male goldfish. scLH induced ovulation in bitterling but scFSH showed lower potency. Both scLH and scFSH induced the initiation of spermatogenesis in the testes of sexually immature male eels after 5–6 administrations of the recombinant GTHs at seven day intervals. Spermatozoa were observed in scLH-administered eels and spermatids in scFSH-administered eels whereas the testes of eels administered with hemolymph of untreated silkworm larvae remained at the spermatogonia stage. Thus, single-chain GTHs of goldfish produced by baculovirus in silkworm larvae showed in vivo biological potency not only in the final phase of gametogenesis (milt production and ovulation) but also in the early phase of gametogenesis (spermatogenesis). We suggest that single-chain fish GTHs could be used for the induction of gonadal development in aquaculture fishes.

Recombinant follicle-stimulating hormone (reFSH) and luteinizing hormone (reLH) of the Japanese eel Anguilla japonica were produced by baculovirus in silkworm Bombyx mori larvae. cDNAs encoding Japanese eel gonadotropin subunits (i.e., FSHβ, LHβ, and common α) were introduced into the baculovirus, which was infected into silkworm larvae after propagation of the recombinant virus in B. mori culture cells. A 100 ml solution of pooled hemolymph from silkworm larvae containing reFSH or reLH were obtained from approximately 250 infected larvae. Ten milliliters of hemolymph were applied to Ni-affinity choromatography, and 5.6 and 3.5 mg of partially purified reFSH and reLH were obtained, respectively. Using Western blot analysis concentrations of reFSH and reLH in the original hemolymph was estimated to be 2.2 and 1.1 mg/ml, respectively. Biological activities of reFSH and reLH were assessed in vitro and in vivo . Purified reFSH and reLH induced eel oocyte maturation in vitro , and administration of hemolymph containing reFSH or reLH induced spermatogenesis in vivo in sexually immature Japanese eel. The present study indicates that a baculovirus–silkworm system could produce large amounts of biologically active recombinant fish gonadotropins for use in investigations in reproductive endocrinology and/or aquaculture of fish.