Transcriptional control of the MUC16 promoter facilitates follicle-stimulating hormone peptide-conjugated shRNA nanoparticle-mediated inhibition of ovarian carcinoma in vivo.

Ovarian cancer is the leading cause of cancer death among gynecological malignancies. The high mortality rate has not been significantly reduced despite advances in surgery and chemotherapy. Gene therapy shows therapeutic potential, but several key issues must be resolved before clinical application. To minimize toxicity in noncancerous tissues, tumor-specific ligands are conjugated to vectors to increase the selectivity of drug delivery. The expression pattern of follicle-stimulating hormone (FSH) receptor in normal and cancer tissues provides an opportunity for highly selective drug delivery in ovarian cancer. Furthermore, tumor-specific promoters can conditionally regulate therapeutic gene expression in tumor or normal tissues. The mucin 16 (MUC16) promoter might be a potential tool to drive ovarian cancer-localized gene expression since MUC16/CA125 is overexpressed in most ovarian carcinomas. Here, we screened the possible MUC16 promoter sequences and constructed MUC16 promoter-driven gro-α shRNA plasmid vectors. The vectors were specifically delivered into ovarian cancer cells via FSH peptide-conjugated nanoparticles. The predicted promoter sequence with TAAA repeats showed high transcriptional activity. The nanoparticle complex containing MUC16 promoter-driven gro-α shRNA and FSH peptides had the ability to decrease gro-α protein secretion in ovarian cancer cells and block tumor growth without obvious toxic effects in a nude mouse model bearing ovarian cancer. Our study provides a novel gene delivery system using a MUC16 promoter trigger and FSH peptide-mediated active targeting in ovarian cancer, and this system may be a promising strategy for specific genetic therapeutic delivery.

Molecular cloning and functional analysis of the goose FSHß gene.

The objective of this investigation was to clone goose FSHß-subunit cDNA and to construct a FSH fusion gene to identify the function of FSHß mRNA during stages of the breeding cycle. The FSHß gene was obtained by reverse transcription-PCR, and the full-length FSHß mRNA sequence was amplified by rapid-amplification of cDNA ends. FSHß mRNA expression was detected in reproductive tissues at different stages (pre-laying, laying period, and broody period). Additionally, the expression of 4 genes known to be involved in reproduction (FSHß, GnRH, GH, and BMP) were evaluated in COS-7 cells expressing the fusion gene (pVITRO2-FSHαß-CTP). The results show that the FSHß gene consists of a 16 base pair (bp) 5'-untranslated region (UTR), 396 bp open reading frame, and alternative 3'-UTRs at 518 bp and 780 bp, respectively. qPCR analyses revealed that FSHß mRNA is highly transcribed in reproductive tissues, including the pituitary, hypothalamus, ovaries, and oviduct. FSHß mRNA expression increased and subsequently decreased in the pituitary, ovaries, and oviduct during the reproductive stages. Stable FSH expression was confirmed using enzyme-linked immunosorbent assays after transfection with the pVITRO2-FSHαß-CTP plasmid. FSHß, GnRH, and BMP expression increased significantly 36 h and 48 h after transfection with the fusion gene in COS-7 cells. The results demonstrate that the FSHß subunit functions in the goose reproductive cycle and provides a theoretical basis for future breeding work.

Molecular characterization of the Chinese alligator follicle-stimulating hormone ß subunit (FSHß) and its expression during the female reproductive cycle.

The Chinese alligator Alligator sinensis is an endangered species endemic to China, it has a highly specialized reproductive pattern with low fecundity. Up to date, little is known about the regulation of its female reproductive cycle. Follicle-stimulating hormone (FSH), a glycoprotein hormone, plays a key role in stimulating and regulating ovarian follicular development and egg production. In this study, the complete FSHß cDNA from the ovary of the Chinese alligator was obtained for the first time, it consists of 843-bp nucleotides, including 120-bp nucleotides of the 5'-untranslated region (UTR), 396-bp of the open reading frame, and 3'-UTR of 327-bp nucleotides. It encodes a 131-amino acid precursor molecule of FSHß with a signal peptide of 18 amino acids followed by a mature protein of 113 amino acids. Its deduced amino acid sequence shares high identities with the American alligator (100%) and birds (89-92%). Phylogenetic tree analysis of the FSHß amino acid sequence indicated that alligators cluster into the bird branch. Tissue distribution analyses indicated that FSHß mRNA is expressed in ovary, intestine and liver with the highest level in the ovary, while not in stomach, pancreas, heart, thymus and thyroid. Expression of FSHß in ovary increases in May (breeding prophase) and peaks in July (breeding period), it is maintained at high levels through September, then decreases significantly in November (post-reproductive period) and remains relatively low from January to March (hibernating period). These temporal changes of FSHß expression implicated that it might play an important role in promoting ovarian development during the female reproductive cycle.

Targeted paclitaxel nanoparticles modified with follicle-stimulating hormone ß 81-95 peptide show effective antitumor activity against ovarian carcinoma.

The majority of patients with advanced ovarian cancer will experience a relapse and ultimately die from refractory diseases. Targeted therapy shows promise for these patients. Novel therapeutic strategies should be developed on the basis of the molecular mechanisms involved in ovarian cancer and the steroid hormone environment of ovaries. The ovary is the main target organ of follicle-stimulating hormone (FSH), which bind to its receptor with high affinity. In this study a FSH receptor-targeting ligand, FSH ß 81-95 peptide, was used as a targeting moiety to synthesize an FSH receptor-mediated drug delivery system. FSH ß 81-95 peptide-conjugated nanoparticles (FSH81-NPs) and paclitaxel-loaded FSH81-NPs (FSH81-NP-PTXs) were synthesized. In vitro studies showed that FSH ß 81-95 peptide enabled the specific uptake of cytotoxic drugs and increased the intracellular paclitaxel concentration in FSH receptor-expressing cancer cells, resulting in enhanced cytotoxic effects. In vivo studies showed that FSH81-NP-PTXs possessed higher antitumor efficacy against FSH receptor-expressing tumors without any clinical signs of adverse side effects or body weight loss due to modification with FSH ß 81-95 peptide. Therefore, FSH binding peptide-targeted drug delivery system exhibited high potential in the treatment of ovarian cancer, and tumor targeting via reproductive hormone receptors might improve the outcome of diseases.

Molecular cloning and sequence analysis of follicle-stimulating hormone beta polypeptide precursor cDNA from the bovine pituitary gland.

Follicle-stimulating hormone (FSH) plays an essential role in mammalian spermatogenesis and follicular development. In a previous study, we demonstrated that some bulls carry numerous linked mutations in the FSH beta-subunit (FSHB) gene, and that these bulls have poor-quality semen, low fertility, and slightly lower serum FSH concentration compared to those without such mutations. Here, we identified the different FSHB mRNA transcripts in such individuals and analyzed the evolutionary pattern of the FSHB open reading frame (ORF) in different species. Two different lengths of FSHB mRNA transcripts corresponding to two different polyadenylation sites in the 3'-UTR were detected in wild-type bull pituitary glands, and four different mRNA transcripts resulting from the different polyadenylation sites and linked mutations were identified in mutation-bearing bull pituitaries. All transcripts had almost the same putative FSHB precursor molecule. When the ORF sequences of wild-type and mutation-bearing genes were compared with those of other tetrapod species, the leopard frog had the lowest level of homology (57.8 and 58.1%) and the buffalo had the highest level (95.9 and 96.7%), respectively. These results indicated that the bovine FSHB gene transcribes at least two classes of mRNA in the wild-type and four classes of mRNA in the mutation-bearing individuals, which provides a new insight into the bovine FSHB evolutionary pattern. In addition, these findings lay a foundation for further study of gene expression regulation and the effects of mutations on male fertility traits in cattle.

Up-regulation of FSHR expression during gonadal sex determination in the frog Rana rugosa.

In vertebrates, gonadal production of steroid hormones is regulated by follicle-stimulating hormone (FSH) and luteinizing hormone (LH) via their receptors designated FSHR and LHR, respectively. We have shown recently that steroid hormones are synthesized in the differentiating gonad of tadpoles during sex determination in the frog Rana rugosa. To elucidate the role of gonadotropins (GTHs) and their receptors in the production of gonadal steroid hormones during sex determination, we isolated the full-length FSHß, LHß, FSHR and LHR cDNAs from R. rugosa and determined gonadal expression of FSHR (FSH receptor) and LHR (LH receptor) as well as brain expression of FSHß and LHß during sex determination in this species. The molecular structures of these four glycoproteins are conserved among different classes of vertebrates. FSHß expression was observed at similar levels in the whole brain (including the pituitary) of tadpoles, but it showed no sexual dimorphism during gonadal sex determination. By contrast, LHß mRNA was undetectable in the whole brain of tadpoles. FSHß-immunopositive cells were observed in the pituitary of female tadpoles with a differentiating gonad. Furthermore, FSHR expression was significantly higher in the gonad of female tadpoles during sex determination than in that of males, whereas LHR was expressed at similar levels in males and females. The results collectively suggest that FSHR, probably in conjunction with FSH, is involved in the steroid-hormone production during female-sex determination in R. rugosa.

Expression of bovine follicle-stimulating hormone subunits in a Hansenula polymorpha expression system increases the secretion and bioactivity in vivo.

Bovine follicle-stimulating hormone (bFSH), a pituitary gonadotropin, is a heterodimer hormone that consists of a common alpha-subunit non-covalently associated with the hormone-specific beta-subunit. Unfortunately, expression levels of recombinant bFSH or its subunits are invariably low. We report here the secretory expression of biologically active bFSHalpha and bFSHbeta subunit in the methylotrophic yeast Hansenula polymorpha. A slightly higher level of expression of recombinant bFSH subunits was achieved by using the Saccharomyces cerevisiae-derived calnexin (ScCne1) as a chaperone in engineered H. polymorpha strains. The preliminary data also suggested that bFSH subunits expressed in H. polymorpha appeared to be less-glycosylated. This isoform had been shown to be 80% increase in in vivo bioactivity compared with the hyperglycosylated Pichia pastoris-derived recombinant bFSHalpha/beta. More sophisticated applications of bFSH would profit from the assembled less-glycosylated heterodimer.

Toward fully synthetic homogeneous beta-human follicle-stimulating hormone (beta-hFSH) with a biantennary N-linked dodecasaccharide. synthesis of beta-hFSH with chitobiose units at the natural linkage sites.

A highly convergent synthesis of the sialic acid-rich biantennary N-linked glycan found in human glycoprotein hormones and its use in the synthesis of a fragment derived from the beta-domain of human Follicle-Stimulating Hormone (hFSH) are described. The synthesis highlights the use of the Sinay radical glycosidation protocol for the simultaneous installation of both biantennary side-chains of the dodecasaccharide as well as the use of glycal chemistry to construct the tetrasaccharide core in an efficient manner. The synthetic glycan was used to prepare the glycosylated 20-27aa domain of the beta-subunit of hFSH under a Lansbury aspartylation protocol. The proposed strategy for incorporating the prepared N-linked dodecasaccharide-containing 20-27aa domain into beta-hFSH subunit was validated in the context of a model system, providing protected beta-hFSH subunit functionalized with chitobiose at positions 7 and 24.

Expression of three gonadotropin subunits in Southern catfish gonad and their possible roles during early gonadal development.

The three gonadotropin (GtH) subunit cDNAs, GtHalpha, FSHbeta and LHbeta, which contain complete open reading frames were isolated from Southern catfish (Silurus meridionalis Chen) ovary. RT-PCR revealed that GtHalpha, FSHbeta and LHbeta mRNA were expressed in ovary, female and male pituitaries, but not in testis. Ontogeny study showed that GtHalpha and FSHbeta expressed in ovary from 25 dah (days after hatching) and LHbeta expressed from 40 dah onwards. The expression levels of these genes in all-female Southern catfish gonad were down-regulated after treatment with tamoxifen from 5 to 25 dah when measured at 65 dah. These results indicated the involvement of the three subunits in gonadal development and sexual differentiation.

Molecular characterization of gonadotropin subunits and gonadotropin receptors in black porgy, Acanthopagrus schlegeli: effects of estradiol-17beta on mRNA expression profiles.

The cDNAs of three gonadotropin (GTH) subunits (GTHalpha, FSHbeta, and LHbeta) and two GTH receptors (FSHR and LHR) from pituitary and gonads of black porgy were cloned. The nucleotide sequences of the GTHalpha, FSHbeta, and LHbeta cDNA were 354, 363, and 414 base pairs (bps) in length with open reading frames (ORF) encoding peptides of 117, 120, and 137 amino acids, respectively. The FSHR and LHR cDNA was 2118 and 2076 bps in length with ORFs encoding peptides of 705 and 691 amino acids, respectively. To study the mechanism of the estradiol-17beta (E(2)) action, we examined the expression pattern of GTH subunit mRNAs in pituitary and GTH-receptor mRNAs in gonads, and the changes of plasma E(2) level when E(2) treatment was applied to immature black porgy. E(2) treatment increased mRNA expression levels of the genes and plasma E(2) levels, indicating that E(2) stimulated the increases in GTH subunit and GTH-receptor mRNAs. These data indicate that E(2) plays an important regulatory role in the brain-pituitary-gonad axis of immature black porgy. We provide the molecular characterization and expression of the GTH subunits and GTH receptors during sex change in the protandrous black porgy.

Highly purified FSH is as efficacious as recombinant FSH for ovulation induction in women with WHO Group II anovulatory infertility: a randomized controlled non-inferiority trial.

BACKGROUND: The objective of this study was to demonstrate non-inferiority of a highly purified urinary follicle stimulating hormone (HP-FSH) preparation compared with a recombinant (rFSH) preparation with respect to ovulation rate (primary end-point). METHODS: This was a randomized, open-label, assessor-blind, multinational study. Women with anovulatory infertility WHO Group II and resistant to clomiphene citrate were randomized (computer-generated list) to stimulation with HP-FSH (n=73) or rFSH (n=78) using a low-dose step-up protocol. The non-inferiority limit was prespecified at -20%. RESULTS: The ovulation rate was 85.2% (51/62) with HP-FSH and 90.9% (60/66) with rFSH (per-protocol population), and non-inferiority was demonstrated [95% confidence interval: -16.9; 5.6]. No differences were noted between groups in number of follicles>or=12 mm, >or=15 mm or >or=18 mm, mono-follicular development, pregnancy rates, endometrial thickness, number of ovarian stimulation syndrome cases or frequency of injection site reactions/pain. The singleton live birth rate was 15% in both groups (11/73 with HP-FSH and 12/78 with rFSH). CONCLUSIONS: This urinary HP-FSH preparation is non-inferior compared with a rFSH preparation with respect to ovulation rate in anovulatory WHO Group II women failing to ovulate or conceive on clomiphene citrate.

Assembly and structural characterization of an authentic complex between human follicle stimulating hormone and a hormone-binding ectodomain of its receptor.

Follicle stimulating hormone (FSH) is secreted from the pituitary gland to regulate reproduction in vertebrates. FSH signals through a G-protein coupled receptor (FSHR) on the target cell surface. We describe here the strategy to produce a soluble FSH-FSHR complex that involves the co-secretion of a truncated FSHR ectodomain (FSHR(HB)) and a covalently linked FSHalphabeta heterodimer from baculovirus-infected insect cells. FSH binds to FSHR(HB) with a high affinity comparable to that for the full-length receptor. The crystal structure of the FSH-FSHR(HB) complex provides explanations for the high affinity and specificity of FSH interaction with FSHR, and it shows an unexpected dimerization of these complexes. Here we also compare the crystal structure with theoretical models of the FSH-FSHR-binding mode. We conclude that the FSH-FSHR(HB) structure gives an authentic representation of FSH binding to intact FSHR.

All-or-none N-glycosylation in primate follicle-stimulating hormone beta-subunits.

Human FSH exists as two major glycoforms designated, tetra-glycosylated and di-glycosylated hFSH. The former possesses both alpha- and beta-subunit carbohydrates while the latter possesses only alpha-subunit carbohydrate. Western blotting differentiated the glycosylated, 24,000 M(r) hFSHbeta band from the non-glycosylated 21,000 M(r) FSHbeta band. Postmenopausal urinary hFSH preparations possessed 75-95% 24,000 M(r) hFSHbeta, while pituitary hFSH immunopurified from 21- to 43-year-old females and 21-43-year-old males possessed only 35-40% 24,000 M(r) hFSHbeta. The pituitary hFSH from a postmenopausal woman on estrogen replacement was 75% 21,000 M(r) hFSHbeta. Other immunopurified postmenopausal pituitary hFSH preparations possessed 50-60% 21,000 M(r) hFSHbeta. Gel filtration removed predominantly 21,000 M(r) free hFSHbeta and reduced its abundance to 13-22% in postmenopausal pituitary hFSH heterodimer preparations. A major regulatory mechanism for FSH glycosylation involves control of beta-subunit N-glycosylation, possibly by inhibition of oligosaccharyl transferase. Two primate species exhibited the same all-or-none pattern of pituitary FSHbeta glycosylation.

[PCR-SSCP polymorphism of FSHbeta gene and its relationship with prolificacy of Jining grey goats].

The follicle-stimulating hormone beta-subunit (FSHbeta) gene was studied as a candidate gene for the prolificacy in Jining Grey goats. According to the sequence of ovine FSHb gene, nine pairs of primers were designed to detect single nucleotide polymorphisms of 5' regulatory region, exon 1 and exon 2 of FSHbeta gene in both high fecundity breed (Jining Grey goat) and low fecundity breeds (Liaoning Cashmere goat, Boer goat and Angora goat) by PCR-SSCP. The results indicated that the homology of nucleotide sequence of this fragment between goat and sheep was 98 percent. Only the products amplified by primer P9 displayed polymorphism. Three genotypes (AA, AB and AC) were detected in Jining Grey goats and Liaoning Cashmere goats. Three genotypes (AA, CC and AC) were detected in Boer goats. Six genotypes (AA, BB, CC, AB, AC and BC) were detected in Angora goats. Sequencing revealed a G-->A mutation at 94 bp of exon 2 of FSHbeta gene in the BB genotype in comparison to the AA genotype and a C-->T mutation at 174 bp of exon 2 of FSHbeta gene in the CC genotype in comparison to the AA genotype. The former mutation resulted in an amino acid change: alanine-->threonine, and the latter mutation did not cause any amino acid change. Genotype frequency of AA, AB and AC was 0.686, 0.137 and 0.177 in Jining Grey goats, respectively. The does with genotype AA had 0.78 (P<0.05) or 0.64 (P<0.05) kids more than those with genotype AB or AC in Jining Grey goats, respectively.

Comparative glycomics of the glycoprotein follicle stimulating hormone: glycopeptide analysis of isolates from two mammalian species.

Follicle stimulating hormone (FSH) is one of the important hormones that regulate gonadal functions. This hormone is glycosylated, and the glycans greatly influence the biological properties. In the present study the negatively charged glycopeptides of equine and human pituitary follicle stimulating hormone (eFSH and hFSH) have been characterized in a glycosylation site-specific manner using FT-ICR-MS and Edman sequencing. The characteristic pattern of glycan distribution at each glycosylation site has been deduced and compared between horse and human FSH preparations. The data suggest that site-specific differences exist between glycoforms of human and equine FSH. For instance, except for one site in the beta subunit (Asn7) of hFSH all other sites in both species have sulfated glycoforms. Also, glycoforms at Asn52 of hFSH are all complex type, whereas in eFSH, both complex and hybrid structures exist at this site. There is also a higher percentage of sulfated glycans in the latter site compared to the former. This is the first study that characterizes the glycans from this hormone in a glycosylation site-specific manner, and these data can be used to begin correlative studies between glycosylation structure and hormone function.

Biological properties of a novel follicle-stimulating hormone/human chorionic gonadotropin chimeric gonadotropin.

A chimeric recombinant human gonadotropin, termed C3, demonstrates both follitropic and lutropic bioactivities. The alpha-subunit construct for C3 is comprised of the recombinant wild-type human glycoprotein hormone alpha-subunit. The beta-subunit DNA construct for C3 encodes residues 1-145 from human chorionic gonadotropin (hCG)-beta with the exceptions that FSH beta amino acid 88 (D) is substituted for hCG beta amino acid 94 (R) and FSH beta amino acids 95-108 (TVRGLGPSYCSFGE) are substituted for hCG beta amino acids 101-114 (GGPKDHPLTCDDPR). C3 is a potent FSH and LH agonist able to bind and to signal through FSH and LH receptors in vitro. In in vivo bioassays optimized to quantify each type of activity, C3 was found to have lutropin and follitropin potencies at levels similar to those of recombinant human LH and recombinant human FSH, respectively. In immature rats, C3 was sufficient to support the maturation of normal ovarian follicles. Moreover, a significant portion of follicles matured by C3 ruptured in response to an ovulatory hCG stimulus and gave rise to morphologically normal oocytes. Furthermore, a low dose of C3 promoted weight gain in the rodent uterus, suggesting it also supported preparation for implantation without histological evidence of excessive luteinization of the ovary. In summary, the biological properties of C3 indicate that its chimeric nature has resulted in a fully functional, dual-acting human gonadotropin.

Single-chain, triple-domain gonadotropin analogs with disulfide bond mutations in the alpha-subunit elicit dual follitropin and lutropin activities in vivo.

The human glycoprotein hormones chorionic gonadotropin (CG), TSH, LH, and FSH are heterodimers composed of a common alpha-subunit and a hormone-specific beta-subunit. The subunits assemble noncovalently early in the secretory pathway. LH and FSH are synthesized in the same cell (pituitary gonadotrophs), and several of the alpha-subunit sequences required for association with either beta-subunit are different. Nevertheless, no ternary complexes are observed for LH and FSH in vivo, i.e. both beta-subunits assembled with a single alpha-subunit. To address whether the alpha-subunit can interact with more than one beta-subunit simultaneously, we genetically linked the FSHbeta- and CGbeta-subunit genes to the common alpha-subunit, resulting in a single-chain protein that exhibited both activities in vitro. These studies also indicated that the bifunctional triple-domain variant (FSHbeta-CGbeta-alpha), is secreted as two distinct bioactive populations each corresponding to a single activity, and each bearing the heterodimer-like contacts. Although the data are consistent with the known secretion events of gonadotropins from the pituitary, we could not exclude the possibility whether transient intermediates are generated in vivo in which the alpha-subunit shuttles between the two beta-subunits during early stages of accumulation in the endoplasmic reticulum. Therefore, constructs were engineered that would direct the synthesis of single-chain proteins completely devoid of heterodimer-like interactions but elicit both LH and FSH actions. These triple-domain, single-chain chimeras contain the FSHbeta- and CGbeta-subunits and an alpha-subunit with cystine bond mutations (cys10-60 or cys32-84), which are known to prevent heterodimer formation. Here we show that, despite disrupting the intersubunit interactions between the alpha- and both CGbeta- and FSHbeta-subunits, these mutated analogs exhibit both activities in vivo comparable to nonmutated triple-domain single chain. Such responses occurred despite the absence of quaternary contacts due to the disrupted bonds in the alpha-subunit. Thus, gonadotropin heterodimer assembly is critical for intracellular events, e.g. hormone-specific posttranslational modifications, but when heterodimers are present in the circulation, the alpha/beta-contacts are not a prerequisite for receptor recognition.

Cloning of the elk common glycoprotein alpha-subunit and the FSH and LH beta-subunit cDNAs.

We report the nucleotide and deduced amino acid sequences of the pituitary glycoprotein hormone common alpha-subunit, and the FSH and LH beta-subunits of the elk (Cervus elaphus). These sequences were obtained by RT-PCR of pituitary gland polyadenylated RNA. A partial genomic fragment of the elk follicle stimulating hormone beta-subunit was also amplified from genomic DNA and sequenced. These sequences show high similarity to other related domestic species, including sika deer, ovine, bovine, porcine and equine. The presently reported cDNAs were used as probes for Northern analysis of pituitary gland and muscle samples from elk and sheep. Mature mRNA transcripts of approximately 700, 1700 and 550 bases were expressed in the pituitary gland but not in muscle tissue, for the glycoprotein hormone common alpha-subunit and FSH and LH beta-subunits, respectively. These observations are consistent with reports for other related species. The genomic fragment of elk FSH beta-subunit revealed an intronic microsatellite of 6 CT repeats, which differs from the 19 CT repeats documented for the sheep genomic sequence. We propose that differences in the CT repeats of the elk FSH beta-subunit gene may be used as a marker for elk parentage testing. The elk DNA sequences presently reported represent a unique addition to the growing collection of the pituitary hormone genes for various vertebrates, which will facilitate evolutionary and phylogenetic studies of the gonadotropin genes. In addition, this information and the cloned cDNAs will be useful for studying seasonal expression of the elk pituitary glycoprotein genes.

Molecular cloning of cDNAs and structural model analysis of two gonadotropin beta-subunits of snakehead fish (Channa maculata).

The cDNAs encoding beta-subunits of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) have been cloned from the pituitary of snakehead fish, Channa maculata, and the three-dimensional structural models of the encoded FSH and LH were investigated. The cloned cDNAs, including 5'-untranslated region (UTR), open-reading frame, and 3'-UTR followed by a poly(A) tail, were obtained by reverse transcription-polymerase chain reaction and rapid amplification of cDNA end methods. The open-reading frames of FSH-beta cDNA encodes a 120-amino acid protein with a signal peptide of 18 amino acids and a mature protein of 102 amino acids; while LH-beta cDNA encodes a 140-amino acid protein with a signal peptide of 33 amino acids and a mature protein of 115 amino acids. The amino acid sequence identities of snakehead fish FSH-beta and LH-beta in comparison with other fish are 27.8-81.9% and 45.2-88.8%, respectively; while in comparison with tetrapods are 26.2-28.9% and 37.5-51.2%, respectively. Both FSH-beta and LH-beta of snakehead fish resemble most to those of Perciformes, implying their closer phylogenetic relationship. All 12 cysteine residues are conserved in snakehead fish LH-beta; while 11 cysteine residues are conserved in its FSH-beta. The third cysteine is absent in snakehead fish FSH-beta; instead, a positionally shifted cysteine residue is present at the N-terminus, as found in some phylogenetic related fish. The structure models of snakehead fish FSH and LH, constructed by using the crystal structures of human FSH and human chorionic gonadotropin as respective template, showed that the positionally shifted N-terminal cysteine residue of snakehead fish FSH-beta likely can substitute the third cysteine to form a disulfide bond with the 12th cysteine.

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.