EGR1 Promotes Ovarian Hyperstimulation Syndrome Through Upregulation of SOX9 Expression.

Angiogenesis is strongly associated with ovarian hyperstimulation syndrome (OHSS) progression. Early growth response protein 1 (EGR1) plays an important role in angiogenesis. This study aimed to investigate the function and mechanism of EGR1 involved in OHSS progression. RNA-sequencing was used to identify differentially expressed genes. In vitro OHSS cell model was induced by treating KGN cells with human chorionic gonadotropin (hCG). In vivo OHSS model was established in mice. The expression levels of EGR1, SOX1, and VEGF were determined by Quantitative Real-Time polymerase chain reaction (qRT-PCR), Western blot, immunofluorescence staining, and immunochemistry assay. The content of VEGF in the culture medium of human granulosa-like tumor cell line (KGN) cells was accessed by the ELISA assay. The regulatory effect of EGR1 on SRY-box transcription factor 9 (SOX9) was addressed by luciferase reporter assay and chromatin immunoprecipitation. The ERG1 and SOX9 levels were significantly upregulated in granulosa cells from OHSS patients and there was a positive association between EGR1 and SOX9 expression. In the ovarian tissues of OHSS mice, the levels of EGR1 and SOX9 were also remarkedly increased. Treatment with hCG elevated the levels of vascular endothelial growth factor (VEGF), EGR1, and SOX9 in KGN cells. Silencing of EGR1 reversed the promoting effect of hCG on VEGF and SOX9 expression in KGN cells. EGR1 transcriptionally regulated SOX9 expression through binding to its promoter. In addition, administration of dopamine decreased hCG-induced VEGF in KGN cells and ameliorated the progression of OHSS in mice, which were companied with decreased EGR1 and SOX9 expression. EGR1 has a promoting effect on OHSS progression and dopamine protects against OHSS through suppression of EGR1/SOX9 cascade. Our findings may provide new targets for the treatment of OHSS.

Recombinant human chorionic gonadotropin induces signaling pathways towards cancer prevention in the breast of BRCA1/2 mutation carriers.

BACKGROUND: Strategies for breast cancer prevention in women with germline BRCA1/2 mutations are limited. We previously showed that recombinant human chorionic gonadotropin (r-hCG) induces mammary gland differentiation and inhibits mammary tumorigenesis in rats. The present study investigated hCG-induced signaling pathways in the breast of young nulliparous women carrying germline BRCA1/2 mutations. METHODS: We performed RNA-sequencing on breast tissues from 25 BRCA1/2 mutation carriers who received r-hCG treatment for 3 months in a phase II clinical trial, we analyzed the biological processes, reactome pathways, canonical pathways, and upstream regulators associated with genes differentially expressed after r-hCG treatment, and validated genes of interest. RESULTS: We observed that r-hCG induces remarkable transcriptomic changes in the breast of BRCA1/2 carriers, especially in genes related to cell development, cell differentiation, cell cycle, apoptosis, DNA repair, chromatin remodeling, and G protein-coupled receptor signaling. We revealed that r-hCG inhibits Wnt/ß-catenin signaling, MYC, HMGA1 , and HOTAIR , whereas activates TGFB/TGFBR-SMAD2/3/4, BRCA1, TP53, and upregulates BRCA1 protein. CONCLUSION: Our data suggest that the use of r-hCG at young age may reduce the risk of breast cancer in BRCA1/2 carriers by inhibiting pathways associated with stem/progenitor cell maintenance and neoplastic transformation, whereas activating genes crucial for breast epithelial differentiation and lineage commitment, and DNA repair.

AAV-mediated gene therapy produces fertile offspring in the Lhcgr-deficient mouse model of Leydig cell failure.

Leydig cell failure (LCF) caused by gene mutation results in testosterone deficiency and infertility. Serum testosterone levels can be recovered via testosterone replacement; however, established therapies have shown limited success in restoring fertility. Here, we use a luteinizing hormone/choriogonadotrophin receptor (Lhcgr)-deficient mouse model of LCF to investigate the feasibility of gene therapy for restoring testosterone production and fertility. We screen several adeno-associated virus (AAV) serotypes and identify AAV8 as an efficient vector to drive exogenous Lhcgr expression in progenitor Leydig cells through interstitial injection. We observe considerable testosterone recovery and Leydig cell maturation after AAV8-Lhcgr treatment in pubertal Lhcgr-/- mice. Of note, this gene therapy partially recovers sexual development, substantially restores spermatogenesis, and effectively produces fertile offspring. Furthermore, these favorable effects can be reproduced in adult Lhcgr-/- mice. Our proof-of-concept experiments in the mouse model demonstrate that AAV-mediated gene therapy may represent a promising therapeutic approach for patients with LCF.

Genetic variants of gonadotrophins and their receptors: Impact on the diagnosis and management of the infertile patient.

This narrative review is concerned with genetic variants of the genes encoding gonadotrophin subunits and their receptors, as well as their implications into the diagnosis and treatment of infertility. We first review briefly the basics of molecular biology and biochemistry of gonadotrophin and gonadotrophin receptor structure and function, then describe the phenotypic effects of polymorphisms and mutations of these genes, followed by diagnostic aspects. We will then summarise the information that inactivating gonadotrophin receptor mutations have provided about the controversial topic of extragonadal gonadotrophin action. Finally, we will close with the current and future therapeutic approaches on patients with gonadotrophin and their receptor mutations.

ERß Regulation of Gonadotropin Responses during Folliculogenesis.

Gonadotropins are essential for regulating ovarian development, steroidogenesis, and gametogenesis. While follicle stimulating hormone (FSH) promotes the development of ovarian follicles, luteinizing hormone (LH) regulates preovulatory maturation of oocytes, ovulation, and formation of corpus luteum. Cognate receptors of FSH and LH are G-protein coupled receptors that predominantly signal through cAMP-dependent and cAMP-independent mechanisms that activate protein kinases. Subsequent vital steps in response to gonadotropins are mediated through activation or inhibition of transcription factors required for follicular gene expression. Estrogen receptors, classical ligand-activated transcriptional regulators, play crucial roles in regulating gonadotropin secretion from the hypothalamic-pituitary axis as well as gonadotropin function in the target organs. In this review, we discuss the role of estrogen receptor ß (ERß) regulating gonadotropin response during folliculogenesis. Ovarian follicles in Erß knockout (ErßKO) mutant female mice and rats cannot develop beyond the antral state, lack oocyte maturation, and fail to ovulate. Theca cells (TCs) in ovarian follicles express LH receptor, whereas granulosa cells (GCs) express both FSH receptor (FSHR) and LH receptor (LHCGR). As oocytes do not express the gonadotropin receptors, the somatic cells play a crucial role during gonadotropin induced oocyte maturation. Somatic cells also express high levels of estrogen receptors; while TCs express ERα and are involved in steroidogenesis, GCs express ERß and are involved in both steroidogenesis and folliculogenesis. GCs are the primary site of ERß-regulated gene expression. We observed that a subset of gonadotropin-induced genes in GCs, which are essential for ovarian follicle development, oocyte maturation and ovulation, are dependent on ERß. Thus, ERß plays a vital role in regulating the gonadotropin responses in ovary.

Maturation conditions, post-ovulatory age, medium pH, and ER stress affect [Ca2+]i oscillation patterns in mouse oocytes.

Insufficiency of oocyte activation impairs the subsequent embryo development in assisted reproductive technology (ART). Intracellular Ca2+ concentration ([Ca2+]i) oscillations switch the oocytes to resume the second meiosis and initiate embryonic development. However, the [Ca2+]i oscillation patterns in oocytes are poorly characterized. In this study, we investigated the effects of various factors, such as the oocytes age, pH, cumulus cells, in vitro or in vivo maturation, and ER stress on [Ca2+]i oscillation patterns and pronuclear formation after parthenogenetic activation of mouse oocytes. Our results showed that the oocytes released to the oviduct at 17 h post-human chorionic gonadotrophin (hCG) displayed a significantly stronger [Ca2+]i oscillation, including higher frequency, shorter cycle, and higher peak, compared with oocytes collected at earlier or later time points. [Ca2+]i oscillations in acidic conditions (pH 6.4 and 6.6) were significantly weaker than those in neutral and mildly alkaline conditions (pH from 6.8 to 7.6). In vitro-matured oocytes showed reduced frequency and peak of [Ca2+]i oscillations compared with those matured in vivo. In vitro-matured oocytes from the cumulus-oocyte complexes (COCs) showed a significantly higher frequency, shorter cycle, and higher peak compared with the denuded oocytes (DOs). Finally, endoplasmic reticulum stress (ER stress) severely affected the parameters of [Ca2+]i oscillations, including elongated cycles and lower frequency. The pronuclear (PN) rate of oocytes after parthenogenetic activation was correlated with [Ca2+]i oscillation pattern, decreasing with oocyte aging, cumulus removal, acidic pH, and increasing ER stress. These results provide fundamental but critical information for the mechanism of how these factors affect oocyte activation.

Oocyte competence is independent of the ovulation trigger adopted: a large observational study in a setting that entails vitrified-warmed single euploid blastocyst transfer.

PURPOSE: To assess whether the GnRH-agonist or urinary-hCG ovulation triggers affect oocyte competence in a setting entailing vitrified-warmed euploid blastocyst transfer. METHODS: Observational study (April 2013-July 2018) including 2104 patients (1015 and 1089 in the GnRH-a and u-hCG group, respectively) collecting ≥1 cumulus-oocyte-complex (COC) and undergoing ICSI with ejaculated sperm, blastocyst culture, trophectoderm biopsy, comprehensive-chromosome-testing, and vitrified-warmed transfers at a private clinic. The primary outcome measure was the euploid-blastocyst-rate per inseminated oocytes. The secondary outcome measure was the maturation-rate per COCs. Also, the live-birth-rate (LBR) per transfer and the cumulative-live-birth-delivery-rate (CLBdR) among completed cycles were investigated. All data were adjusted for confounders. RESULTS: The generalized-linear-model adjusted for maternal age highlighted no difference in the mean euploid-blastocyst-rate per inseminated oocytes in either group. The LBR per transfer was similar: 44% (n=403/915) and 46% (n=280/608) in GnRH-a and hCG, respectively. On the other hand, a difference was reported regarding the CLBdR per oocyte retrieval among completed cycles, with 42% (n=374/898) and 25% (n=258/1034) in the GnRh-a and u-hCG groups, respectively. Nevertheless, this variance was due to a lower maternal age and higher number of inseminated oocytes in the GnRH-a group, and not imputable to the ovulation trigger itself (multivariate-OR=1.3, 95%CI: 0.9-1.6, adjusted p-value=0.1). CONCLUSION: GnRH-a trigger is a valid alternative to u-hCG in freeze-all cycles, not only for patients at high risk for OHSS. Such strategy might increase the safety and flexibility of controlled-ovarian-stimulation with no impact on oocyte competence and IVF efficacy.

A matched propensity score study of embryo morphokinetics following gonadotropin-releasing hormone agonist versus human chorionic gonadotropin trigger.

PURPOSE: To compare morphokinetic parameters and quality of embryos derived from GnRH antagonist ICSI cycles triggered either with GnRH agonist or standard hCG between matched groups of patients. METHODS: Morphokinetic parameters of embryos derived from matched first GnRH antagonist ICSI cycles triggered by GnRH agonist or standard hCG between 2013 and 2016 were compared. Matching was performed for maternal age, peak estradiol levels, and number of oocytes retrieved. Outcome measures were: time to pronucleus fading (tPNf), cleavage timings (t2-t8), synchrony of the second and third cycles (S2 and S3), duration of the second and third cycle (CC2 and CC3), optimal cell cycle division parameters, and known implantation data (KID) scoring for embryo quality. Multivariate linear and logistic regression analyses were performed for confounding factors. RESULTS: We analyzed 824 embryos from 84 GnRH agonist trigger cycles and 746 embryos from 84 matched hCG trigger cycles. Embryos derived from the cycles triggered with hCG triggering cleaved faster than those deriving from GnRH agonist trigger. The differences were significant throughout most stages of embryo development (t3-t6), and a shorter second cell cycle duration of the hCG trigger embryos was observed. There was no difference in synchrony of the second and third cell cycles and the optimal cell cycle division parameters between the two groups, but there was a higher percentage of embryos without multinucleation in the hCG trigger group (27.8% vs. 21.6%, p < 0.001). CONCLUSION: The type of trigger in matched antagonist ICSI cycles was found to affect early embryo cleavage times but not embryo quality.

Rare genetic variants suggest dysregulation of signaling pathways in low- and high-risk patients developing severe ovarian hyperstimulation syndrome.

PURPOSE: To investigate if rare gene variants in women with severe ovarian hyperstimulation syndrome (OHSS) provide clues to the mechanisms involved in the syndrome. METHODS: Among participants in a prospective randomized study (Toftager et al. 2016), six women with predicted low and six women with predicted high risk of OHSS developing severe OHSS (grades 4 and 5, Golan classification) were selected. In the same cohort, six plus six matched controls developing no signs of OHSS (Golan grade 0) were selected. Whole-exome sequencing was performed. Analysis using a predefined in silico OHSS gene panel, variant filtering, and pathway analyses was done. RESULTS: We found no convincing monogenetic association with the development of OHSS using the in silico gene panel. Pathway analysis of OHSS variant lists showed substantial overlap in highly enriched top pathways (p value range p < 0.0001 and p > 9.8E-17) between the low- and high-risk group developing severe OHSS, i.e., "the integrin-linked kinase (ILK) signaling pathway" and the "axonal guidance signaling pathway," both being connected to vasoactive endothelial growth factor (VEGF) and endothelial function. CONCLUSION: Rare variants in OHSS cases with two distinct risk profiles enrich the same signaling pathways linked to VEGF and endothelial function. Clarification of the mechanism as well as potentially defining genetic predisposition of the high vascular permeability is important for future targeted treatment and prevention of OHSS; the potential roles of ILK signaling and the axonal guidance signaling need to be validated by functional studies.

N- and O-glycosylation patterns and functional testing of CGB7 versus CGB3/5/8 variants of the human chorionic gonadotropin (hCG) beta subunit.

The classical function of human chorionic gonadotropin (hCG) is its role in supporting pregnancy. hCG is a dimer consisting of two highly glycosylated subunits, alpha (CGA) and beta (CGB). The beta-hCG protein is encoded by CGB3, CGB5, CGB7 and CGB8 genes. CGB3, 5 and 8 code for an identical protein, CGB3/5/8, whereas CGB7 differs in three amino acids from CGB3/5/8. We had observed earlier that CGB7 and CGB3/5/8 display very distinct tissue expression patterns and that the tumor suppressor and transcription factor p53 can activate expression of CGB7 but not of CGB3/5/8 genes. Here, we investigate the glycan structures and possible functional differences of the two CGB variants. To this end, we established a system to produce and isolate recombinant CGA, CGB7 and CGB3/5/8 proteins. We found that N- and O-glycosylation patterns of CGB7 and CGB3/5/8 are quite similar. Functional assays were performed by testing activation of the ERK1/2 pathway and demonstrated that CGB7 and CGB5/5/8 appear to be functionally redundant isoforms, although a slight difference in the kinetics of ERK1/2 pathway activation was observed. This is the first time that biological activity of CGB7 is shown. In summary, the results lead to the hypothesis that CGB7 and CGB3/5/8 do not hold significant functional differences but that timing and cell type of their expression is the key for understanding their divergent evolution.

Human chorionic gonadotropin improves endometrial receptivity by increasing the expression of homeobox A10.

Homeobox A10 (HOXA10) is a characterized marker of endometrial receptivity. The mechanism by which hCG intrauterine infusion promotes embryo implantation is still unclear. This study seeks to investigate whether hCG improves endometrial receptivity by increasing expression of HOXA10. HOXA10 expression with human chorionic gonadotropin stimulation was analyzed in vitro and in vivo. Our results demonstrate that HOXA10 was decreased in the endometria of recurrent implantation failure patients compared to that in the healthy control fertile group, also we observed that hCG intrauterine infusion increased endometrial HOXA10 expression. HOXA10, blastocyst-like spheroid expansion area was increased, whereas DNA (cytosine-5-)-methyltransferase 1 was decreased when human endometrial stromal cells (hESCs) were treated with 0.2 IU/ml of hCG for 48 h. HOXA10 promoter methylation was also reduced after hCG treatment. Collagen XV (ColXV) can repress the expression of DNA (cytosine-5-)-methyltransferase 1, and hCG treatment increased the expression of ColXV. However, when the hESCs were treated with LH/hCG receptor small interfering RNA to knock down LH/hCG receptor, hCG treatment failed to repress DNA (cytosine-5-)-methyltransferase 1 expression or to increase ColXV expression. Our findings suggest that hCG may promote embryo implantation by increasing the expression of HOXA10.

Unbalanced human embryos secrete more hyperglycosylated human chorionic gonadotrophin (hCG-H) than balanced ones.

PURPOSE: The aim of this study was to compare the levels of hyperglycosylated human chorionic gonadotropin (hCG-H) secreted from balanced and unbalanced human embryos. METHODS: Single-step culture media samples from 155 good quality embryos, derived from 90 good prognosis patients undergoing intracytoplasmic sperm injection (ICSI), were collected on the fifth day of embryo cultivation. All embryos were tested by next-generation sequencing (NGS) technique. The hCG-H levels in the culture media were evaluated by ELISA kit (Cusabio Biotech, CBS-E15803h) according to the manufacturer's instructions. Statistical analysis was performed using SPSS v.21 (IBM Corp., Armonk, NY, USA). RESULTS: The NGS analysis revealed that 36% of the embryos (n = 56) were balanced, and 64% of the embryos were unbalanced (n = 99). The presence of hCG-H was confirmed in all embryo culture media samples but was absent in the negative control. In addition, hCG-H concentration was significantly higher in the culture media from unbalanced embryos compared with the balanced ones (0.72 ± 0.30 mIU/ml vs. 0.62 ± 0.12 mIU/ml, p = 0.02, respectively). Furthermore, the mean levels of hCG-H were significantly increased in the samples from embryos with multiple abnormalities. Finally, the highest levels of hCG-H were expressed from embryos with monosomy of chromosome 11 (1.28 ± 0.04 mIU/ml) and those with trisomies of chromosomes 21 (2.23 mIU/ml) and 4 (1.02 ± 0.35 mIU/ml). CONCLUSION: Our results suggest that chromosomal aberrations in human embryos are associated with an increased secretion of hCG-H. However, hCG-H concentration in embryo culture media as a single biomarker is not sufficient for an accurate selection of balanced embryos.

Vitamin D and sex steroid production in men with normal or impaired Leydig cell function.

Production of testosterone is under tight control by human chorion gonadotropin (hCG) during fetal life and luteinizing hormone (LH) in adulthood. Several animal and human studies have linked vitamin D status with sex steroid production although it is not clear whether there exist a direct or indirect involvement in androgen production. Few studies have investigated this crosslink in young healthy men and putative direct or synergistic effect of activated vitamin D (1,25(OH)2D3) and LH/hCG on sex steroid production in vitro. Here, we present cross-sectional data from 300 young men and 41 hCG-stimulated men with impaired Leydig cell function combined with data from an ex vivo culture of human testicular tissue exposed to 1,25(OH)2D3 alone or in combination with hCG. Serum 25-OHD was positively associated with SHBG (ß:0.002; p = 0.023) and testosterone/estradiol-ratio (ß:0.001; p = 0.039), and inversely associated with free testosterone (%) (free testosterone/total testosterone) (ß:-0.002; p = 0.016) in young men. Vitamin D deficient men had higher total and free estradiol concentrations than men with higher vitamin D status (19% and 18%, respectively; p < 0.01). Interestingly, men with impaired Leydig cell function and vitamin D deficiency had a significantly lower hCG-mediated increase in total and free testosterone compared with vitamin D sufficient men (p < 0.05). Accordingly, testicular tissue exposed to 100 nM 1,25(OH)2D3 had a 15% higher testosterone release into the media compared with vehicle treated specimens (p = 0.030). In conclusion, vitamin D deficiency is associated with lower testosterone/estradiol ratio in young men and lower Leydig cell sensitivity after hCG-stimulation in men with impaired gonadal function. The significant effect of 1,25(OH)2D3 on testosterone production in a human testis model supports that the stimulatory effect at least in part may be direct. Larger placebo-controlled studies are needed to determine whether vitamin D supplementation can influence testosterone production.

A study on enhanced O-glycosylation strategy for improved production of recombinant human chorionic gonadotropin in Chinese hamster ovary cells.

Human chorionic gonadotropin (hCG) is a glycoprotein hormone that exists as a heterodimer comprised of an α subunit and ß subunit linked with disulfide bridges. The ß subunit contains four O-glycosylation sites. Previous studies have found that the translation of mRNA to polypeptides of the ß subunit was a severely limiting step for the expression of recombinant hCG protein in Chinese hamster ovary (CHO) cells. The effects of O-glycosylation on recombinant hCG protein expression were assessed by adding O-glycan precursors and overexpressing and knocking down key regulatory genes of O-glycan precursor synthesis and O-glycan sugar chain synthesis or hydrolases. The results indicated that O-glycosylation was indeed limiting in the expression of recombinant hCG protein, and N-acetylgalactosamine (GalNAc) was the major limiting precursor. Glutamine-fructose-6-phosphate transaminase 2 (Gfat2) and Uridine diphosphate-glucose pyrophosphorylase 2 (Ugp2), key regulatory genes of O-glycan precursor synthesis, were overexpressed. Ugp2 overexpression significantly increased the recombinant hCG protein level by 1.92 times compared to that of the control. The LC-MS/MS analysis and Phaseolus vulgaris leucoagglutinin (PHA-L) lectin blot analysis showed that Ugp2 overexpression significantly increased the total galactosylation levels of intracellular proteins and the O-glycosylation of recombinant hCG protein. The stability of the hCG protein to trypsin digestion was also enhanced. Ugp2 is the major limiting enzyme of the O-glycan precursor synthesis in recombinant hCG protein production. Furthermore, the effects and mechanisms of the key genes of O-glycan sugar chain synthesis and hydrolases such as polypeptide N-acetylgalactosaminyltransferase1 (Galnt1), Core 1 synthase, glycoprotein-N-acetylgalactosamine 3-beta-galactosyltransferase (C1galt1), O-linked N-acetylglucosamine transferase (Ogt) and Hexosaminidase (Hex), were evaluated. The results indicated that Galnt1 overexpression increased the recombinant hCG protein level by 1.57 times and improved the total galactosylation of intracellular proteins, O-glycosylation and the stability of recombinant hCG protein. Galnt1 is the major limiting enzyme of O-glycan sugar chain synthesis. Overexpression of Ugp2 and Galnt1 simultaneously improved the recombinant hCG protein level by 2.44 times, and both had synergistic effects. Based on the results of overexpression of Galnt1, the major limiting gene of O-Glycan chain synthesis, the precursors GalNAc and Gal were added and increased the recombinant hCG protein level by 3.68 times. This study revealed the major limiting factors of O-glycosylation of recombinant hCG protein in CHO cells and proposed an effective expression regulation strategy.

Characterization of tethered equine chorionic gonadotropin and its deglycosylated mutants by ovulation stimulation in mice.

BACKGROUND: To directly assess the biological role of oligosaccharides in recombinant equine chorionic gonadotropin (rec-eCG) functioning, cDNA encoding the full-length eCGß-subunit was fused with the mature protein part of the α-subunit, and we examined the expression levels of deglycosylated eCG mutants, the ovulation rate for deglycosylated mutants in C57BL/6 mice. RESULTS: The characterizations of heterodimeric and tethered mutants were studied following their respective secretions in culture medium, molecular weight and ovulation in vivo. Rec-eCG variants containing mutations at glycosylation sites at Asn82 of the α-subunit (eCGß/αΔ82) and Asn13 of the ß-subunit (eCGßΔ13/α) were not efficiently secreted into the culture medium from transfected cells. Western blot analysis revealed that the rec-eCGß/α proteins have an approximate broad range of molecular weights of 40-46 kDa. Three rec-eCG mutants-a deglycosylated site at Asn56 of the α-subunit (eCGß/αΔ56), a deletion of the C-terminal region of the ß-subunit (eCGß-D/α), and the double mutant (eCGß-D/αΔ56)-turned out to have clearly lower (approximately 4-23 kDa) molecular weights. Protein N-glycosydase F (PNGase F) treatment markedly decreased the molecular weight to approximately 2-10 kDa. Normal oocytes were significantly more abundant in the natural eCG-treated group than in mutant rec-eCG-treated groups. In particular, numbers of nonfuntional oocytes were remarkably lower in all rec-eCG groups. CONCLUSIONS: Our results indicate that the ovulation rates of oocytes are not affected by the deglycosylated rec-eCGß/α mutant proteins. There are around 20% non-functional oocytes with natural eCG and only 2% with the rec-eCGs tested. These results provide insight into the molecular mechanisms underlying the production of rec-eCG hormones with excellent bioactivity in vivo.

Induction of Gonadal Development in Protogynous Grouper with Orally Delivered FSH DNA.

The availability of sexually mature fish often dictates the success of its captive breeding. In this study, we induced reproductive development in juvenile protogynous tiger grouper through oral administration of a plasmid (p) containing an engineered follicle-stimulating hormone (FSH). An expression construct (pcDNA3.1) was designed to express a single-chain FSH consisting of giant grouper FSH ß-subunit and glycoprotein subunit-α (CGα), linked by the carboxy-terminal peptide (CTP) sequence from the human chorionic gonadotropin (hCG). Single oral delivery of pFSH encapsulated in liposome and chitosan to tiger grouper yielded a significant increase in plasma FSH protein level after 4 days. Weekly pFSH feeding of juvenile tiger groupers for 8 weeks stimulated ovarian development as indicated by a significant increase in oocyte diameter and progression of oocytes to cortical alveolar stage. As the pFSH treatment progressed from 20 to 38 weeks, female to male sex change was initiated, characterized by oocyte regression, proliferation of spermatogonial cells, and occurrence of spermatogenic cysts. It was also associated with significantly lower mRNA expression of steroidogenic genes (cyp11b, cyp19a1a, and foxl2) and basal plasma levels of sex steroid hormones 17ß-estradiol (E2), testosterone (T), and 11-ketotestosterone (11KT). Results suggest that pFSH stimulates ovarian development up to cortical alveolar stage and then initiates sex change in tiger grouper. These findings significantly contribute to our knowledge on the role of FSH in the development of protogynous hermaphroditic fish. This study is the first to demonstrate induction of reproductive development in fish through oral delivery of plasmid gonadotropin.

ß-hCG-induced mutant BRCA1 ignites drug resistance in susceptible breast tissue.

ß-hCG expression in breast cancer is highly controversial with reports supporting both protective and tumorigenic effects. It has also been reported that risk of breast cancer at an early age is increased with full-term pregnancies if a woman is a BRCA1 mutation carrier. We have already demonstrated that BRCA1-defective cells express high levels of ß-hCG and that when BRCA1 is restored, ß-hCG level is reduced. Also, BRCA1 can bind to the promoter and reduce the levels of ß-hCG. ß-hCG induces tumorigenicity in BRCA1-defective cells by directly binding to TGFBRII and induces TGFBRII-mediated cell proliferation. In this study, we analyzed the mechanism of action of ß-hCG on BRCA1 expression and its influence on drug sensitivity in breast cancer cells. We demonstrate that ß-hCG induces mutant BRCA1 protein expression in BRCA1 mutant cells; however, in BRCA1 wild-type cells, ß-hCG reduced wild-type BRCA1 protein expression. Transcriptionally, ß-hCG could induce Slug/LSD1-mediated repression of wild-type and mutant BRCA1 messenger RNA levels. However, ß-hCG induces HSP90-mediated stabilization of mutant BRCA1 and hence the overexpression of mutant BRCA1 protein, resulting in partial restoration of homologous recombination repair of damaged DNA. This contributes to drug resistance to HSP90 inhibitor 17AAG in BRCA1-defective cancer cells. A combination of HSP90 inhibitor and TGFBRII inhibitor has shown to sensitize ß-hCG expressing BRCA1-defective breast cancers to cell death. Targeting the ß-hCG-HSP90-TGFBRII axis could prove an effective treatment strategy for BRCA1-mutated breast tumors.

Association of hCG and LHCGR expression patterns with clinicopathological parameters in ovarian cancer.

In addition to its critical role during pregnancy, human chorionic gonadotropin (hCG) has been shown to be expressed by various tumor types. Recent studies have similarly documented the presence of the luteinizing hormone (LH)/hCG receptor (LHCGR) in a variety of nongonadal organs; however, its clinicopathological significance in ovarian cancer remains unclear. The present study used a combination of immunohistochemical, real-time PCR, and western blot analyses to examine hCG and LHCGR expression in normal and cancerous tissues collected from patients with epithelial ovarian cancer (EOC). hCG and LHCGR expression levels were resultantly shown to be significantly increased and decreased in cancerous versus normal (or benign) ovarian tissues, respectively (P < 0.05), and both expression pattern changes were associated with more advanced tumor stages and a higher rate of metastasis. Furthermore, patients with tumors with high or low levels of hCG and LHCGR, respectively, experienced a worse overall survival (OS) rate than those with low hCG or high LHCGR expression levels (P < 0.05). In fact, hCG and LHCGR expression levels were independent prognostic factors of patient OS (P < 0.05) for EOC. Collectively, these findings indicate that hCG and LHCGR expression pattern changes are associated with EOC occurrence and progression. Thus, hCG and LHCGR represent promising potential targets to improve the diagnosis, treatment, and prognosis of patients with EOC.

Perfluorooctanoic acid (PFOA) and perfluooctane sulfonate (PFOS) induce different modes of action in reproduction to Japanese medaka (Oryzias latipes).

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) have widely and frequently used in many industrial sectors, and thus have been frequently found in the environment. These chemicals may act as endocrine disrupting chemicals (EDCs), although the molecular mechanisms are still debated. In this study, Japanese medaka (Oryzias latipes) were exposed to 10 mg/l PFOA and 1 mg/l PFOS for 21 days, and the reproductive responses, such as the fecundity, secondary sexual characteristics and transcriptional levels of vitellogenin (vtg1 and vtg2) and choriogenin (chgh, chghm and chgl), were time-dependently evaluated (day 7, 14 and 21). PFOA and PFOS significantly reduced fecundity, and caused expression changes in the genes with time, although the patterns were different for each chemical and each sex. Different transcriptional regulations of vitellogenin and choriogenin in male suggest that PFOA and PFOS have different mode of actions in reproductive effects despite their similar chemical structure.

Therapeutic potential of adenovirus-mediated TFF2-CTP-Flag peptide for treatment of colorectal cancer.

TFF2 is a small, secreted protein with anti-inflammatory properties. We previously have shown that TFF2 gene delivery via adenovirus (Ad-Tff2) suppresses colon tumor growth in colitis associated cancer. Therefore, systemic administration of TFF2 peptide could potentially provide a similar benefit. Because TFF2 shows a poor pharmacokinetic, we sought to modify the TFF2 peptide in a manner that would lower its clearance rate but retain bioactivity. Given the absence of a sequence-based prediction of TFF2 functionality, we chose to genetically fuse the C-terminus of TFF2 with the carboxyl-terminal peptide of human chorionic gonadotropin ß subunit, and inserted into adenoviral vector that expresses Flag. The resulting Ad-Tff2-CTP-Flag construct translates into a TFF2 fused with two CTP and three Flag motifs. Administered Ad-Tff2-CTP-Flag decreased tumorigenesis and suppressed the expansion of myeloid cells in vivo. The fusion peptide TFF2-CTP-Flag delivered by adenovirus Ad-Tff2-CTP-Flag as well purified recombinant fusion TFF2-CTP-Flag was retained in the blood longer compared with wild-type TFF2 delivered by Ad-Tff2 or recombinant TFF2. Consistently, purified recombinant fusion TFF2-CTP-Flag suppressed expansion of myeloid cells by down-regulating cyclin D1 mRNA in vitro. Here, we demonstrate for the very first time the retained bioactivity and possible pharmacokinetic advantages of TFF2 with a modified C-terminus.