Inhibition of activin signalling reduces the growth of LßT2 gonadotroph pituitary tumours in mouse.

Pituitary tumours are benign neoplasms that derive from hormone-producing cells of the pituitary gland. While medical treatments have emerged for most subtypes, gonadotroph tumours that express follicle-stimulating hormone (FSH) and/or luteinizing hormone still lack therapeutic options apart from surgery and radiotherapy. Activin ligands are physiological regulators of production and secretion of FSH by gonadotroph cells, but their role in gonadotroph tumourigenesis remains little explored. Using the LßT2 mouse gonadotroph cell line which produces FSH under activin stimulation, we first tested whether subcutaneous xenografts of LßT2 cells resulted in tumour formation in Rag2KO mice. Histological analysis confirmed the presence of LßT2 tumours with endothelial cells and macrophages in their microenvironment. FSH expression was found in a subset of clusters of LßT2 cells in the tumours. We subsequently addressed the consequences of targeting activin signalling via injection of a soluble activin decoy receptor (sActRIIB-Fc). sActRIIB-Fc treatment resulted in significantly decreased LßT2 tumour volume. Reduced Smad2 phosphorylation as well as inhibition of tumour-induced FSH production confirmed the efficient targeting of activin-downstream signalling in treated tumours. More interestingly, treated tumours showed significantly fewer endothelial cells associated with reduced Vegfa expression. In vitro treatment of LßT2 cells with sActRIIB-Fc had no effect on cell proliferation or apoptosis, but Vegfa expression was inhibited, pointing to a likely paracrine effect of LßT2 cells on endothelial cells through activin-mediated Vegfa regulation. Further in vitro and in vivo studies are now needed to pinpoint the exact roles of activin signalling in these processes prior to translating these observations to the clinic.

Development and characterization of in vitro self-assembled recombinant human follicle stimulating hormone originated from goat mammary epithelial cells.

Follicle stimulating hormone (FSH), composed of FSHα and FSHß subunits, is essential for female follicle development and male spermatogenesis. The recombinant human FSH (rhFSH) products on the market are mainly generated from mammalian cells and are expensive. Large animal mammary gland bioreactors are urgently needed to produce large amounts of rhFSH. However, there are currently no effective methods to prepare rhFSH by large animals mainly due to the fact that excessive accumulation of FSH might cause many adverse effects in animals. We herein report the development and characterization of functional self-assembled rhFSH produced in goat mammary epithelial cells (GMECs). FSHα and FSHß stably expressed in Chinese hamster ovary (CHO) cell lines were secreted into culture medium and well glycosylated. Importantly, FSHα and FSHß expressed apart were able to assemble into functional FSH. We next inserted human FSHα or FSHß gene separately into goat ß-Lactoglobulin locus in GMECs by CRISPR/Cas9. Inactive FSHα and FSHß subunits expressed from GMECs assembled into rhFSH as analyzed by His-tag pull down assay. Functional assessment of rhFSH by cAMP induction assay, mouse ovulation induction and rat ovarian weight gain experiments showed that the bioactivity of self-assembled rhFSH expressed by GMECs was comparable to that of Gonal-F both in vitro and in vivo. Our study demonstrated that FSHα and FSHß can be separately expressed and assembled into functional rhFSH, and provided the basis for future preparing FSH by goat mammary gland bioreactor with less health problems on the producing animals.

Follicle-Stimulating Hormone ß-Subunit Potentiates Bone Morphogenetic Protein 9-Induced Osteogenic Differentiation in Mouse Embryonic Fibroblasts.

Postmenopausal osteoporosis (PMOP)-related fractures usually result in morbidity and mortality in aging women, so it remains a global public health concern, and new effective safe treatments are urgently needed recently. Efficient osteogenesis from mesenchymal stem cells (MSCs) would have the clinical application potential in treating multiple osteal disorders. Follicle-stimulating hormone (FSH), a pituitary glycoprotein hormone highly associated with menopausal bone turnover, whose peculiar part of receptor binding is follicle-stimulating hormone ß-subunit (FSHß). Bone morphogenetic protein 9 (BMP9), a potent osteogenic factor, can up-regulate FSHß in mouse embryonic fibroblasts (MEFs). However, it is unclear, whether extrapituitary FSHß affects BMP9-induced osteogenesis in MEFs. In this study, we investigated the role of FSHß in BMP9-induced osteogenesis in MEFs. We found that exogenous expression of FSHß significantly increased BMP9-induced alkaline phosphatase activity (ALP), the expression of osteogenic transcriptional factors, Runx2 and Osx, and the established late osteogenic markers, osteopontin (OPN) and osteocalcin (OCN), so does the ectopic bone formation. Mechanistically, FSHß dramatically enhanced BMP9-induced BMP/Smad signal transduction, presenting the augment phosphorylation of Smad1/5/8, whereas treatment with anti-FSHß antibodies suppressed these effects. An adenylate cyclase inhibitor obviously suppressed ALP and BMP/Smad signal transduction induced by BMP9 or the combination of BMP9 and FSHß in MEFs. Collectively, our findings suggested that FSHß may promote BMP9-induced activation of BMP/Smad signaling through a FSH/FSH receptor (FSHR)/cAMP dependent pathway in MEFs partly. J. Cell. Biochem. 118: 1792-1802, 2017. © 2016 Wiley Periodicals, Inc.

Effects of α and ß recombinant FSH (Gonal-F, Puregon) and progesterone upon human endometrial cell proliferation in-vitro: a preliminary study.

Endometrial proliferation or regeneration during menstrual cycle is regulated by sexual hormones. However, the effect of gonadotrophins on the endometrial cell growth remains obscure. Herein, we aimed to investigate the effects of r-FSH (Gonal-F, Puregon) and progesterone on the proliferation of human endometrial cells in-vitro. According as gonadotrophin concentrations, the follicular-phase endometrial cells were divided into six groups: (1) 0 (controls), (2) 1; (3) 10; (4) 100; (5) 1000; (6) 100,000 µIU/ml. The cell countings with microscopy and cell proliferation kit assay were used to assess the endometrial cell proliferations. In Gonal-F groups, the cell absorptions (%) after 24/48 h culture were: (1) 100/100; (2) 103.8/102.3; (3) 104.8/102.8; (4) 102.3/101.3; (5) 96.3/94.2; (6) 86.8/84.3. In Puregon groups, the cell absorptions were: (1) 100/100; (2) 102.8/101.9; (3) 103/102.3; (4) 103.9/103.5; (5) 102.9/102.4; (6) 103.7/103.2 (non-different). In progesterone groups, the cell absorptions were: (1) 100/100; (2) 99.1/101.9; (3) 83.5/80.4; (4) 80.7/82.4. Higher dosage of Gonal-F (100,000 µIU/ml) and progesterone (10, 100 µg/ml) appeared the significant inhibition upon endometrium. We conclude that lower dosages of Gonal-F, Puregon, and progesterone appear the non-significant influence upon endometrium. Higher dosage of Gonal-F (10,000 µIU/ml) and progesterone (10, 100 µg/ml), but not Puregon, might interfere with the endometrial proliferation during follicular phase.

Follicle-stimulating hormone peptide can facilitate paclitaxel nanoparticles to target ovarian carcinoma in vivo.

Chemotherapy is an important treatment for ovarian cancer. However, conventional chemotherapy has inevitable drawbacks due to side effects from nonspecific biodistribution of the chemotherapeutic drugs. To solve such problem, targeted delivery approaches were developed. The targeted delivery approaches combine drug carriers with the targeting system and can preferentially bring drugs to the targeted sites. Follicle-stimulating hormone receptor (FSHR) is an ovarian cancer-specific receptor. By using a peptide derived from FSH (amino acids 33-53 of the FSH beta chain, named as FSH33), we developed a conjugated nanoparticle, FSH33-NP, to target FSHR in ovarian cancer. FSH33-NP was tested for recognition specificity and uptake efficiency on FSHR-expressing cells. Then, the antitumor efficiency of paclitaxel (PTX)-loaded FSH33-NP (FSH33-NP-PTX) was determined. FSH33-NP-PTX displayed stronger antiproliferation and antitumor effects compared with free PTX or naked PTX-loaded nanoparticles (NP-PTX) both in vitro and in vivo. In summary, this novel FSH33-NP delivery system showed very high selectivity and efficacy for FSHR-expressing tumor tissues. Therefore, it has good potential to become a new therapeutic approach for patients with ovarian cancer.

[Preparation and in vitro targeting of follicle stimulating hormone polypeptide modified nanoparticles].

OBJECTIVE: To prepare follicle stimulating hormone (FSH) polypeptide modified nanoparticles (NP) in order to achieve specific ovarian tumor targeting. METHODS: Expression of FSH receptor protein in human liver cancer and ovarian cancer cell lines BEL-7402, SKOV-3 and Caov-3 was detected by immunocytochemistry. The polypeptide fragment of FSH beta 81-95 amino acids (FSHL81-95) was synthesized and covalently coupled to NP. The specific binding of FSHL81-95 and FSHL81-95-NP was examined by fluorescence microscopy and flow cytometry. RESULTS: BEL-7402 and SKOV-3 cells were negative for FSH receptor staining, while Caov-3 cells were positive. The diameters of NP were about 100 nm, with a Zeta potential of -25 mV or so. Caov-3 cells showed a more specific interaction with FSHL81-95-NP than SKOV-3 cells (4.17 +/- 0.86 and 2.30 +/- 0.21; P < 0.05). The uptake of FSHL81-95-NP was more than NP in Caov-3 cells (4.17 +/- 0.86 and 0.41 +/- 0.32; P < 0.05). FSHL81-95-NP showed a selective targeting at Caov-3 cells compared with control NP. CONCLUSION: FSH polypeptide modified NP could selectively target ovarian cancer cells expressing FSH receptor, which might contribute to specific endocytosis mediated by FSH receptor.

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.

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 Smad2, Smad3, Smad4, and Smad7 from the goldfish pituitary and evidence for their involvement in activin regulation of goldfish FSHbeta promoter activity.

Follicle-stimulating hormone (FSH), a glycoprotein consisting of an alpha subunit and a unique beta subunit, is essential for gonadal development and function in vertebrates including teleosts. FSH is regulated by a variety of neuroendocrine and endocrine factors, and its biosynthesis is primarily determined by the expression of the beta subunit. Although the regulation of FSH biosynthesis has been well documented in mammals, the molecular mechanisms underlying the regulation are poorly understood. Our previous studies demonstrated that activin stimulated goldfish FSHbeta expression in the primary pituitary cell culture and enhanced its promoter activity in the mouse gonadotrope cell line LbetaT-2 cells. However, little is known about the signal transduction pathway involved in the transcriptional activation of this gene by activin. To assess the involvement of intracellular signaling protein Smads in regulating goldfish FSHbeta promoter, we first cloned full-length cDNAs for goldfish Smad2, Smad3, Smad4, and Smad7 from the pituitary. All Smads cloned show high sequence conservation with their mammalian counterparts. The spatial expression of these Smads overlapped with that of activin subunits and its receptors in various tissues examined. In addition, we demonstrated that activin induced Smad3 and Smad7 expression, but not Smad2 and Smad4. Co-transfection of Smad2 or Smad3 cDNA into the LbetaT-2 cells with the reporter construct of goldfish FSHbeta promoter significantly enhanced basal and activin-stimulated reporter (SEAP, secreted alkaline phosphatase) expression, while Smad7 completely blocked basal and Smad2/3-stimulated FSHbeta activity. Interestingly, the effect of Smad3 was much higher than that of Smad2, suggesting that Smad3 is likely the principal signal transducing molecule involved in activin stimulation of FSHbeta expression in the goldfish. This work lays a foundation for further analysis of goldfish FSHbeta promoter for the cis-regulatory elements involved in activin signaling.

Synergy between activin A and gonadotropin-releasing hormone in transcriptional activation of the rat follicle-stimulating hormone-beta gene.

Both activin and GnRH can independently stimulate expression of the FSHbeta subunit gene. In this study, we used the gonadotrope-derived LbetaT2 cell line to investigate the potential interaction between activin and GnRH in regulating the transcriptional activity of the rat FSHbeta gene promoter. Activin A and GnRH synergistically enhanced rat FSHbeta transcriptional activity. Overexpression of SMAD3 (mediator of decapentaplegic-related protein 3), but not of SMAD2, increased transcriptional activation of the rat (r) FSHbeta gene promoter, which was further enhanced by the combined overexpression of SMAD3 and 4 (3+4). The stimulatory effects of SMAD3 overexpression were localized to -472/-256 of the rFSHbeta gene promoter, and activin- and GnRH-responsive proteins were shown to bind to region -284/-252. Sequence analysis identified a consensus palindromic SMAD-binding site at -266/-259 of the rFSHbeta gene promoter. Mutation of two bases located in the center of this palindrome effectively abrogated SMAD4 binding, markedly reduced SMAD3 and 3+4 stimulation of the rFSHbeta gene promoter, and significantly decreased the synergistic enhancement of promoter activity by both activin A and GnRH, and SMAD3 and GnRH. Blockade of the MAPK-signaling pathway did not significantly affect the response to combined stimulation with activin and GnRH. In contrast, interference with SMAD3 signaling caused a significant reduction in activin and GnRH synergy. The results indicate that SMAD3 plays an important role in the synergistic effects of activin and GnRH and demonstrate that this synergy is mediated by a palindromic cis-element located at -266/-259 of the rFSHbeta gene promoter.

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.