Production of recombinant channel catfish (Ictalurus punctatus) FSH and LH in S2 Drosophila cell line and an indication of their different actions.

Due to the lack of purified, native gonadotropins (GtH) for almost all species of fish, we designed a system for the production of recombinant bioactive luteinizing hormone (LH) and follicle stimulating hormone (FSH) using the channel catfish (Ictalurus punctatus) as a model animal. The strategy was to produce the three subunits composing FSH and LH, i.e. the common alpha-subunit (alpha-glycoprotein hormone (alpha-GP)), beta-FSH, and beta-LH subunit, individually in stable recombinant insect cells (S2) with C-terminal His-tag. This expression system was also used to co-express the alpha-subunit without the His-tag with each of the His-tagged beta-subunits. The recombinant S2 cells were capable of secreting FSH and LH heterodimers and alpha-GP in abundance; however, expression of the individual beta-subunits was much less successful. The recombinant GtHs were partially purified from the cell medium by immobilized metal affinity chromatography to ~15% purity with a yield of 7 and 4 mg per liter of medium for FSH and LH respectively. These recombinant GtHs activated their receptors in vitro, enhanced estrogen secretion, up-regulated several steroidogenic enzyme genes in channel catfish ovarian follicles, and increased androgen secretion from African catfish testis. Interestingly, the FSH and LH dose-response curves for each of these biological activities clearly demonstrate differences in their cellular action and physiological roles. This expression system may be an important development for the production of species-specific GtHs so that FSH- and LH-specific mechanisms of actions within the reproductive endocrine processes can finally be examined with homologous, albeit recombinant, hormones.

Cyclooxygenases in rat Leydig cells: effects of luteinizing hormone and aging.

Previous studies suggested that increased Leydig cell cyclooxygenase (COX)2 expression may be involved in the reduced testosterone production that characterizes aged Leydig cells. Our objective herein was to further elucidate the relationships among LH stimulation, Leydig cell COX2 and COX1 expression, aging, and testosterone production. Incubation of Leydig cells from young or aged rats with LH or dibutyryl cAMP resulted in increases in both intracellular COX2 protein expression and testosterone production. COX1 expression did not respond to LH or dibutyryl cAMP. Incubation of adult cells with a protein kinase A inhibitor suppressed the stimulatory effects of LH on COX2 and testosterone production. Short-term incubation of Leydig cells with TGF-alpha or IL-1beta also increased COX2 protein levels; IGF-I had no effect. In vivo, LH also was found to stimulate both COX2 and testosterone, but not COX1. As reported previously, COX2 expression was greater in old than in young cells, and old Leydig cells responded to inhibition of COX2 in vitro with increased testosterone production. However, the effects of the COX2 inhibitors were not restricted to old cells; young Leydig cells also responded to COX2 inhibition with increased testosterone production. This and the observation that the incubation of young or old cells with LH resulted in increased COX2 and testosterone production in both cases suggests that the relationship between COX2 and testosterone production is not unique to aged Leydig cells. Moreover, the close correlation between increases in COX2 and testosterone in LH-stimulated young and aged Leydig cells is difficult to reconcile with the contention that the increased expression of COX2 in aged cells is responsible for age-related suppression of Leydig cell testosterone production.

Receptor-selective determinants in catfish gonadotropin seat-belt loops.

Mammalian gonadotropins are highly selective. Charge differences between the Cys(10-11) sequence of FSHbeta and LHbeta/CGbeta seat-belt loops determine the ability of these hormones to interact with the LH-R. Selective FSH-R binding is mainly dependent on the presence of an FSHbeta-specific sequence between Cys(11-12) of the seat-belt loop. Intriguingly, African catfish LHbeta (cfLHbeta) lacks a positively charged Cys(10-11) region and stimulates both catfish LH-R and FSH-R with comparable potencies. Our studies on the promiscuous behaviour of cfLH using chimeric gonadotropins revealed that the Cys(10-11) region of cfLHbeta contains cfLH-R-selective determinants, whereas the Cys(11-12) region of cfLHbeta confers FSH-R-stimulating activity to cfLH. Hence, the location of receptor-selective determinants appeared to be fairly well conserved throughout evolution, despite the low sequence identity between mammalian and catfish seat-belt loops. Moreover, various structure-function differences between gonadotropins are discussed in the context of the different (female) reproductive strategies between mammalian and non-mammalian species that required the divergence to a more specific LH-R-stimulating activity of one of the gonadotropins in mammals.

betaFSH, betaLH and growth hormone gene expression in blue gourami (Trichogaster trichopterus, Pallas 1770) during spermatogenesis and male sexual behavior.

The relationship between gonadal development (histological evidence for spermiogenesis and/or spermatogenesis), sexual behavior (nest-building) and mRNA levels of gonadotropins (betaFSH and betaLH) and growth hormone (GH) in the male pituitary was investigated. Amplification of betaFSH cDNA showed a significantly higher mRNA level in mature males (whether sexually active or not) than in juveniles. However, following PCR amplification of betaLH cDNA, a significantly higher mRNA level was found in the sexually active group compared to the sexually inactive group. These results suggest that FSH may participate in spermatogenesis, whereas LH is more involved in spermiogenesis. The GH mRNA level increased slightly during the maturation process but no significant differences were found between the groups studied.

Functional study of a recombinant form of human LHbeta-subunit variant carrying the Gly(102)Ser mutation found in Asian populations.

Genetic variants of human LH caused by amino acid replacements in the beta-subunit have been demonstrated to affect reproductive function. Occurrence of a G(1502)A substitution in the LHbeta gene leading to Gly(102)Ser replacement of the LHbeta protein has been found to be associated with infertility in the Singapore Chinese population. In the present study, a search for this LHbeta allele from 383 DNA samples from different continents, using a PCR-based strategy, demonstrated its total absence in these populations. Functional properties of the variant (V) (Gly(102)Ser substitution) LHbeta subunit were assessed using a recombinant (r) form of V-LH produced in HEK293 cells, in comparison with wild-type (WT) LH or hCG. The synthesized V-LH was purified by a single step of immunoaffinity chromatography, and it had a molecular weight of 30 kDa as determined by SDS-PAGE. The affinities of the WT-hCG and rV-LH in mouse Leydig tumour (mLT-1) cell LH receptor binding were similar, with K(d) values of 0.140 +/- 0.03 and 0.156 +/- 0.01 nmol/l respectively. Likewise, the effects of WT- and V-rLH preparations on mLT-1 cell cAMP and progesterone production were concentration-dependent and with similar biopotencies. In addition, HEK293 cells expressing the human LH receptor documented similar dose-dependent increases in inositol phosphate production by the two rLH forms. In conclusion, these findings demonstrate that Gly(102)Ser mutation of the LHbeta gene does not affect receptor binding and bioactivity of the hormone, when tested in vitro.