Purification of Mummichog (Fundulus heteroclitus) Gonadotropins and Their Subunits, Using an Immunochemical Assay with Antisera Raised against Synthetic Peptides

Shimizu, Akio; Yamashita, Michiaki

doi:10.1006/gcen.2001.7741

Cited by 50 publications

(40 citation statements)

References 41 publications

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“…The antisera used for this study were obtained from the mummichog F. heteroclitus (Cyprinodontidae; Cyprinodontiforms, Acantopterygii). The synthetic peptides used as antigens, carried the conserved sequences of teleost FSH and LH (Shimizu and Yamashita 2002;Shimizu et al 2003a, b). These antisera were successful on detecting gonadotrops of several acantopterygian species (Shimizu et al 2003a, b).…”

Section: Discussionmentioning

confidence: 99%

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Identification of immunoreactive FSH and LH cells in the cichlid fish Cichlasoma dimerus during the ontogeny and sexual differentiation

et al. 2006

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Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) expressing cells were detected in pituitary, brain and ovary of the Perciform cichlid fish Cichlasoma dimerus. This detection was carried out by immunohistochemistry (IHC) and Western blot techniques using antisera of the Cyprinodontiform Fundulus heteroclitus raised against the conservative region of the teleost betaFSH and the betaLH subunits. The estimated molecular weights were 24 kDa for LH and 19 and 15 kDa for FSH. In the adult pituitary, both cell types were distributed along mid and ventral zones of the proximal pars distalis (PPD, mid-immunoreactive cells), and along the ventral and dorsal external border of the pars intermedia (PI, high-immunoreactive cells). Double IHC showed that FSH and LH are mainly expressed in different pituitary cells. FSH cells were detected in the pituitary around day 21 after hatching (ah) (prior to sex differentiation), while LH cells were detected by day 60 ah (during the sexual differentiation period). A correlation between gonadal sex differentiation and FSH was demonstrated in a 15 days organ culture system. FSH and LH neurons were localized in the nucleus lateralis tuberis and their fibers project through the ventral hypothalamus, preoptic area and neurohypophysis. FSH neurons differentiated on day 21 ah, while LH neurons appeared on day 15 ah. In the ovary, the immunoreactivity for both FSH and LH was restricted to the cytoplasm of previtellogenic and early vitellogenic oocytes.

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Section: Discussionmentioning

confidence: 99%

“…The generation and characterization of these antisera were previously described (Shimizu and Yamashita 2002). These antisera were specifically raised against conservative sites of teleost GtHs.…”

Section: Antiseramentioning

confidence: 99%

Identification of immunoreactive FSH and LH cells in the cichlid fish Cichlasoma dimerus during the ontogeny and sexual differentiation

et al. 2006

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“…The polypeptide was synthesized with a peptide synthesizer (model 432A; Applied Biosystems, Tokyo, Japan). The region was selected based on the criteria as described elsewhere (Shimizu and Yamashita, 2002). For immunization, 100 mg of the hemocyanin-coupled peptide of Vasa was dissolved in 100 mL saline, emulsified with an equal volume of Freund's complete adjuvant (COSMO BIO, Tokyo, Japan) and injected into the lymph node of a rabbit (Goudie et al,'96).…”

Section: Production and Validation Of An Antibody Against Vasa Proteinmentioning

confidence: 99%

Isolation and characterization of Vasa in the frog Rana rugosa

et al. 2010

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We cloned a cDNA encoding Vasa, a member of the DEAD (Asp-Glu-Ala-Asp) family of proteins, from the ovary of the frog Rana rugosa. Comparative alignment of amino acid sequences with known Vasa from several species of vertebrate showed that the R. rugosa orthologue shares eight conserved regions with Vasa from other vertebrates. Vasa gene expression was restricted to the testis and ovary among ten different tissues examined. Vasa expression showed no sexual dimorphism during sex determination in R. rugosa, but became higher in the ovary thereafter. By Western blot analysis, a single Vasa band with a molecular weight of 80.9 kDa was detected. The same antibody immunohistochemically detected Vasa in a few cells in the embryonic endoderm at stage 15; the beginning of closure of neural folds, and in the cytoplasm of spermatogonia in the testis, and oocytes in the ovary of tadpoles at stage XX; marked by one or both forelegs protruded. Together, these results suggest that Vasa is a highly specific marker of germ cells and hence useful for studies of germ cell specification and function in amphibians as it already is in other species of both invertebrates and vertebrates such as Drosophila and zebrafish.

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“…The GtHs doses used were 0.1, 0.5 and 1 µg/ml. These doses were chosen based on some published works related to the well-known effect of in vitro administrated GtHs in the onset of meiosis and gonadal steroidogenesis [12,13,14]. After 24 h, incubation culture media was removed and frozen (day 2) and pituitaries were fixed in Bouin’s solution and included in Paraplast in order to confirm their viability and cytoarchitecture by topographic colorations and by IHQ (fig.…”

Section: Methodsmentioning

confidence: 99%

“…Four control pituitaries and 4 pituitaries per treatment were used in each experiment (n = 32). During the purification of mummichog GtHs [13], it was shown that there were no detectable levels of FSH contamination in the obtained LH native hormone and vice versa. To demonstrate the biological effect of mummichog LH and FSH in C. dimerus , we performed a bioassay of steroidogenic activity in a vitellogenic ovary.…”

Section: Methodsmentioning

confidence: 99%

Presence of β-Follicle-Stimulating Hormone and β-Luteinizing Hormone Transcripts in the Brain of <i>Cichlasoma dimerus</i> (Perciformes: Cichlidae)

Pandolfi

Ag²,

Canepa³

et al. 2008

Neuroendocrinology

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Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play key roles in vertebrate gametogenesis and steroidogenesis. They are mainly synthesized in the pituitary gland. While investigating the ontogeny of FSH and LH cells in the cichlid fish Cichlasoma dimerus by immunohistochemistry (IHC), we unexpectedly found immunoreactive neurons in the preoptic area, sending their projections through different brain areas and neurohypophysis. Our previous work using Western blot and IHC techniques applied to the adult brain confirmed these findings. To further demonstrate the extrapituitary expression of these hormones, we performed RT-PCR detecting sequences coding for β-FSH and β-LH subunits in the C. dimerus pituitary and brain (preoptic-hypothalamic area). The expression of these transcripts in both organs was consistent with their peptide expression showing a high sequence homology when compared with other phylogenetically related fish. An individual pituitary in vitro culture system was utilized to study the possible modulatory effect of brain-derived gonadotropins on pituitary hormone secretion. Pituitary explants were cultured with different concentrations of LH or FSH, and the culture media were analyzed by Western blot. Exogenous LH produced a dose-dependent increase in pituitary β-LH, β-FSH and somatolactin (SL) releases. No effect was observed on growth hormone (GH). The effect on prolactin (PRL) was not consistent among treatments. Exogenous FSH produced an inhibition in β-LH release, dose-dependent increases in β-FSH and SL releases, and no effect on PRL and GH releases. These findings support the concept of regulation of pituitary trophic hormones by brain-derived gonadotropins.

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Purification of Mummichog (Fundulus heteroclitus) Gonadotropins and Their Subunits, Using an Immunochemical Assay with Antisera Raised against Synthetic Peptides

Cited by 50 publications

References 41 publications

Identification of immunoreactive FSH and LH cells in the cichlid fish Cichlasoma dimerus during the ontogeny and sexual differentiation

Identification of immunoreactive FSH and LH cells in the cichlid fish Cichlasoma dimerus during the ontogeny and sexual differentiation

Isolation and characterization of Vasa in the frog Rana rugosa

Presence of β-Follicle-Stimulating Hormone and β-Luteinizing Hormone Transcripts in the Brain of <i>Cichlasoma dimerus</i> (Perciformes: Cichlidae)

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