Stable Production of a Tethered Recombinant Eel Luteinizing Hormone Analog with High Potency in CHO DG44 Cells.

We produced a recombinant eel luteinizing hormone (rec-eel LH) analog with high potency in Chinese hamster ovary DG44 (CHO DG44) cells. The tethered eel LH mutant (LH-M), which had a linker comprising the equine chorionic gonadotropin (eLH/CG) ß-subunit carboxyl-terminal peptide (CTP) region (amino acids 115 to 149), was inserted between the ß-subunit and α-subunit of wild-type tethered eel LH (LH-wt). Monoclonal cells transfected with the tethered eel LH-wt and eel LH-M plasmids were isolated from five to nine clones of CHO DG44 cells, respectively. The secreted quantities abruptly increased on day 3, with peak levels of 5000-7500 ng/mL on day 9. The molecular weight of tethered rec-eel LH-wt was 32-36 kDa, while that of tethered rec-eel LH-M increased to approximately 38-44 kDa, indicating the detection of two bands. Treatment with the peptide N-glycanase F decreased the molecular weight by approximately 8 kDa. The oligosaccharides at the eCG ß-subunit O-linked glycosylation sites were appropriately modified post-translation. The EC50 value and maximal responsiveness of eel LH-M increased by approximately 2.90- and 1.29-fold, respectively, indicating that the mutant exhibited more potent biological activity than eel LH-wt. Phosphorylated extracellular regulated kinase (pERK1/2) activation resulted in a sharp peak 5 min after agonist treatment, with a rapid decrease thereafter. These results indicate that the new tethered rec-eel LH analog had more potent activity in cAMP response than the tethered eel LH-wt in vitro. Taken together, this new eel LH analog can be produced in large quantities using a stable CHO DG44 cell system.

Production of Recombinant Single-Chain Eel Luteinizing Hormone and Follicle-Stimulating Hormone Analogs in Chinese Hamster Ovary Suspension Cell Culture.

We produced rec-single chain eel luteinizing (rec-eel LH) and follicle-stimulating (rec- eel FSH) hormones displaying high biological activity in Chinese hamster ovary suspension (CHO-S) cells. We constructed several mutants, in which a linker, including an O-linked glycosylated carboxyl-terminal peptide (CTP) of an equine chorionic gonadotropin (eCG) ß-subunit, was attached between the ß- and α-subunit (LH-M and FSH-M) or in the N-terminal (C-LH and C-FSH) or C-terminal (LH-C and FSH-C) regions. The plasmids were transfected into CHO-S cells, and culture supernatants were collected. The secretion of mutants from the CHO-S cells was faster than that of eel LHß/α-wt and FSHß/α-wt proteins. The molecular weight of eel LHß/α-wt and eel FSHß/α-wt was 32-34 and 34-36 kDa, respectively, and that of LH-M and FSH-M was 40-43 and 42-45 kDa, respectively. Peptide-N-glycanase F-treatment markedly decreased the molecular weight by approximately 8-10 kDa. The EC50 value and the maximal responsiveness of the eel LH-M and eel FSH-M increased compared with the wild-type proteins. These results show that the CTP region plays a pivotal role in early secretion and signal transduction. We suggest that novel rec-eel LH and FSH proteins, exhibiting potent activity, could be produced in large quantities using a stable CHO cell system.

Specific Signal Transduction of Constitutively Activating (D576G) and Inactivating (R476H) Mutants of Agonist-Stimulated Luteinizing Hormone Receptor in Eel.

We investigated the mechanism of signal transduction using inactivating (R476H) and activating (D576G) mutants of luteinizing hormone receptor (LHR) of eel at the conserved regions of intracellular loops II and III, respectively, naturally occurring in mammalian LHR. The expression of D576G and R476H mutants was approximately 58% and 59%, respectively, on the cell surface compared to those of eel LHR-wild type (wt). In eel LHR-wt, cAMP production increased upon agonist stimulation. Cells expressing eel LHR-D576G, a highly conserved aspartic acid residue, exhibited a 5.8-fold increase in basal cAMP response; however, the maximal cAMP response by high-agonist stimulation was approximately 0.62-fold. Mutation of a highly conserved arginine residue in the second intracellular loop of eel LHR (LHR-R476H) completely impaired the cAMP response. The rate of loss in cell-surface expression of eel LHR-wt and D576G mutant was similar to the agonist recombinant (rec)-eel LH after 30 min. However, the mutants presented rates of loss higher than eel LHR-wt did upon rec-eCG treatment. Therefore, the activating mutant constitutively induced cAMP signaling. The inactivating mutation resulted in the loss of LHR expression on the cell surface and no cAMP signaling. These data provide valuable information regarding the structure-function relationship of LHR-LH complexes.

The N-Linked Glycosylation Site N191 Is Necessary for PKA Signal Transduction in Eel Follicle-Stimulating Hormone Receptor.

The follicle-stimulating hormone receptor (FSHR) contains several N-linked glycosylation sites in its extracellular region. We conducted the present study to determine whether conserved glycosylated sites in eel FSHR are necessary for cyclic adenosine monophosphate (cAMP) signal transduction. We used site-directed mutagenesis to induce four mutations (N120Q, N191Q, N272Q, and N288Q) in the N-linked glycosylation sites of eel FSHR. In the eel FSHR wild-type (wt), the cAMP response was gradually increased in a dose-dependent manner (0.01-1500 ng/mL), displaying a high response (approximately 57.5 nM/104 cells) at the Rmax level. Three mutants (N120Q, N272Q, and N288Q) showed a considerably decreased signal transduction as a result of high-ligand treatment, whereas one mutant (N191Q) exhibited a completely impaired signal transduction. The expression level of the N191Q mutant was only 9.2% relative to that of the eel FSHR-wt, indicating a negligible expression level. The expression levels of the N120Q and N272Q mutants were approximately 35.9% and 24% of the FSHG-wt, respectively. The N288Q mutant had an expression level similar to that of the eel FSHR-wt, despite the mostly impaired cAMP responsiveness. The loss of the cell surface agonist-receptor complexes was very rapid in the cells expressing eel FSHR-wt and FSHR-N288Q mutants. Specifically, the N191Q mutant was completely impaired by the loss of cell surface receptors, despite treatment with a high concentration of the agonist. Therefore, we suggest that the N191 site is necessary for cAMP signal transduction. This finding implies that the cAMP response, mediated by G proteins, is directly related to the loss of cell surface receptors as a result of high-agonist treatment.

Functional characterization of naturally-occurring constitutively activating/inactivating mutations in equine follicle-stimulating hormone receptor.

OBJECTIVE: Follicle-stimulating hormone (FSH) is the central hormone involved in mammalian reproduction, maturation at puberty, and gamete production that mediates its function by control of follicle growth and function. The present study investigated the mutations involved in the regulation of FSH receptor (FSHR) activation. METHODS: We analyzed seven naturally-occurring mutations that were previously reported in human FSHR (hFSHR), in the context of equine FSHR (eFSHR); these include one constitutively activation variant, one allelic variant, and five inactivating variants. These mutations were introduced into wild-type eFSHR (eFSHR-wt) sequence to generate mutants that were designated as eFSHR-D566G, -A306T, -A189V, -N191I, -R572C, -A574V, and -R633H. Mutants were transfected into PathHunter EA-parental CHO-K1 cells expressing ß-arrestin. The biological function of mutants was analyzed by quantitating cAMP accumulation in cells incubated with increasing concentrations of FSH. RESULTS: Cells expressing eFSHR-D566G exhibited an 8.6-fold increase in basal cAMP response, as compared to that in eFSHR-wt. The allelic variation mutant eFSHR-A306T was not found to affect the basal cAMP response or half maximal effective concentration (EC50) levels. On the other hand, eFSHR-D566G and eFSHR-A306T displayed a 1.5- and 1.4-fold increase in the maximal response, respectively. Signal transduction was found to be completely impaired in case of the inactivating mutants eFSHR-A189V, -R572C, and -A574V. When compared with eFSHR-wt, eFSHR-N191I displayed a 5.4-fold decrease in the EC50 levels (3,910 ng/mL) and a 2.3-fold decrease in the maximal response. In contrast, cells expressing eFSHR-R633H displayed in a similar manner to that of the cells expressing the eFSHR-wt on signal transduction and maximal response. CONCLUSION: The activating mutant eFSHR-D566G greatly enhanced the signal transduction in response to FSH, in the absence of agonist treatment. We suggest that the state of activation of the eFSHR can modulate its basal cAMP accumulation.

Constitutively Activating Mutants of Equine LH/CGR Constitutively Induce Signal Transduction and Inactivating Mutations Impair Biological Activity and Cell-Surface Receptor Loss In Vitro.

The signal transduction of the equine lutropin/choriogonadotropin receptor (eLH/CGR) is unclear in naturally occurring activating/inactivating mutants of this receptor, which plays an important role in reproductive physiology. We undertook the present study to determine whether conserved structurally related mutations in eLH/CGR exhibit similar mechanisms of signal transduction. We constructed four constitutively activating mutants (M398T, L457R, D564G, and D578Y) and three inactivating mutants (D405N, R464H, and Y546F); measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary cells; and investigated cell-surface receptor loss using an enzyme-linked immunosorbent assay in human embryonic kidney 293 cells. The eLH/CGR-L457R-, -D564G-, and -D578Y-expressing cells exhibited 16.9-, 16.4-, and 11.2-fold increases in basal cAMP response, respectively. The eLH/CGR-D405N- and R464H-expressing cells presented a completely impaired signal transduction, whereas the Y546F-expressing cells exhibited a small increase in cAMP response. The cell-surface receptor loss was 1.4- to 2.4-fold greater in the activating-mutant-expressing cells than in wild-type eLH/CGR-expressing cells, but was completely impaired in the D405N- and Y546F-expressing cells, despite treatment with a high concentration of agonist. In summary, the state of activation of eLH/CGR influenced agonist-induced cell-surface receptor loss, which was directly related to the signal transduction of constitutively activating mutants.

Roles of N-linked and O-linked glycosylation sites in the activity of equine chorionic gonadotropin in cells expressing rat luteinizing hormone/chorionic gonadotropin receptor and follicle-stimulating hormone receptor.

BACKGROUND: Equine chorionic gonadotropin (eCG), which comprises highly glycosylated α-subunit and ß-subunit, is a unique member of the glycoprotein hormone family as it elicits both follicle-stimulating hormone (FSH)-like and luteinizing hormone (LH)-like responses in non-equid species. To examine the biological function of glycosylated sites in eCG, the following glycosylation site mutants were constructed: eCGß/αΔ56, substitution of Asn56 of α-subunit with Gln; eCGß-D/α, deletion of the O-linked glycosylation site at the carboxyl-terminal peptide (CTP) region of the ß-subunit; eCGß-D/αΔ56, double mutant. The recombinant eCG (rec-eCG) mutants were expressed in Chinese hamster ovary suspension (CHO-S) cells. The FSH-like and LH-like activities of the mutants were examined using CHO-K1 cells expressing rat lutropin/CG receptor (rLH/CGR) and rat FSH receptor (rFSHR). RESULTS: Both rec-eCGß/α and rec-eCGß/αΔ56 were efficiently secreted into the CHO-S cell culture medium on day 1 post-transfection. However, the secretion of eCGß-D/α and eCGß-D/αΔ56, which lack approximately 12 O-linked glycosylation sites, was slightly delayed. The expression levels of all mutants were similar (200-250 mIU/mL) from days 3 to 7 post-transfection. The molecular weight of rec-eCGß/α, rec-eCGß/αΔ56 and rec-eCG ß-D/α were in the ranges of 40-45, 37-42, and 34-36 kDa, respectively. Treatment with peptide-N-glycanase F markedly decreased the molecular weight to approximately 5-10 kDa. Rec-eCGß/αΔ56 exhibited markedly downregulated LH-like activity. The signal transduction activity of both double mutants was completely impaired. This indicated that the glycosylation site at Asn56 of the α-subunit plays a pivotal role in the LH-like activity of eCG. Similarly, the FSH-like activity of the mutants was markedly downregulated. eCGß-D/α exhibited markedly downregulated LH-like and FSH-like activities. CONCLUSIONS: Rec-eCGß/α exhibits potent biological activity in cells expressing rLH/CGR and rFSHR. The findings of this study suggest that the LH-like and FSH-like activities of eCG are regulated by the N-linked glycosylation site at Asn56 of the eCG α-subunit and/or by the O-linked glycosylation sites of the eCG ß-subunit. These findings improved our understanding of the mechanisms underlying both LH-like and FSH-like activities of eCG.

Luteinizing hormone-like and follicle-stimulating hormone-like activities of equine chorionic gonadotropin ß-subunit mutants in cells expressing rat luteinizing hormone/chorionic gonadotropin receptor and rat follicle-stimulating hormone receptor.

To identify the specific region of eCG involved in FSH-like activity, the following mutant expression vectors were constructed targeting the amino acid residues 102-104 of the eCG ß-subunit: single mutants, eCGßV102G/α, eCGßF103P/α, and eCGßR104K/α; double mutants, eCGßV102G;F103P/α, eCGßV102G;R104K/α, and eCGßF103P;R104K/α; triple mutant, eCGßV102G;F103P;R104K/α. The LH-like and FSH-like activities of eCG mutants were examined in CHO-K1 cells expressing rat LH/CG receptor and rat FSH receptor. The levels of eCGßV102G/α, eCGßR104K/α, and eCGßV102G;R104K/α in the culture supernatant were markedly lower than those of eCGß/α-wt. The other mutants and rec-eCGß/α-wt were efficiently secreted into the culture supernatant. The LH-like activities of eCGV104G/α, eCGßV102G;R104K/α, and eCGßF103P;R104K/α were approximately 61%, 52%, and 54%, respectively, of those of eCG-wt. The Rmax values of the mutants were 58.9%-78.8% those of eCG-wt with eCGßR104K/α exhibiting the lowest value. The FSH-like activities of single mutants were only 16%-20% of those of eCG-wt. Additionally, the FSH-like activity of double mutants was less than 10% of that of eCG-wt. In particular, the FSH-like activities of ßV102G;R104K/α and ßF103P;R104K/α were 2.5-2.9% of that of eCG-wt. These results suggest that the amino acid residues 102-104 of the eCG ß-subunit are dispensable and that the residue 104 of the eCG ß-subunit plays a pivotal role in signal transduction through the rat FSH receptor. Thus, these mutants may aid future studies on eCG interactions with mammalian FSH receptors in vitro and in vivo.

Specific Biological Activity of Equine Chorionic Gonadotropin (eCG) Glycosylation Sites in Cells Expressing Equine Luteinizing Hormone/CG (eLH/CG) Receptor.

Equine chorionic gonadotropin (eCG), produced by the endometrial cups of the placenta after the first trimester, is a specific glycoprotein that displays dual luteinizing hormone (LH)-like and follicle-stimulating hormone (FSH)-like effects in non-equid species. However, in equidaes, eCG exhibits only LH-like activity. To identify the specific biological functions of glycosylated sites in eCG, we constructed the following site mutants of N- and O-linked glycosylation: eCGß/αΔ56, substitution of α-subunit56 N-linked glycosylation site; eCGß-D/α, deletion of the O-linked glycosylation sites at the ß-subunit, and eCGß-D/αΔ56, double mutant. We produced recombinant eCG (rec-eCG) proteins in Chinese hamster ovary suspension (CHO-S) cells. We examined the biological activity of rec-eCG proteins in CHO-K1 cells expressing the eLH/CG receptor and found that signal transduction activities of deglycosylated mutants remarkably decreased. The EC50 levels of eCGß/αΔ56, eCGß-D/α, and eCGß-D/αΔ56 mutants decreased by 2.1-, 5.6-, and 3.4-fold, respectively, compared to that of wild-type eCG. The Rmax values of the mutants were 56%-80% those of wild-type eCG (141.9 nmol/104 cells). Our results indicate that the biological activity of eCG is greatly affected by the removal of N- and O-linked glycosylation sites in cells expressing eLH/CGR. These results provide important information on rec-eCG in the regulation of specific glycosylation sites and improve our understanding of the specific biological activity of rec-eCG glycosylation sites in equidaes.

Comparative gene expression profiling of mouse ovaries upon stimulation with natural equine chorionic gonadotropin (N-eCG) and tethered recombinant-eCG (R-eCG).

BACKGROUND: Equine chorionic gonadotropin (eCG) induces super-ovulation in laboratory animals. Notwithstanding its extensive usage, limited information is available regarding the differences between the in vivo effects of natural eCG (N-eCG) and recombinant eCG (R-eCG). This study aimed to investigate the gene expression profiles of mouse ovaries upon stimulation with N-eCG and R-eCG produced from CHO-suspension (CHO-S) cells. R-eCG gene was constructed and transfected into CHO-S cells and quantified. Subsequently, we determined the metabolic clearance rate (MCR) of N-eCG and R-eCG up to 24 h after intravenous administration through the mice tail vein and identified differentially expressed genes in both ovarian tissues, via quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC). RESULTS: R-eCG was markedly expressed initially after transfection and maintained until recovery on day 9. Glycan chains were substantially modified in R-eCG protein produced from CHO-S cells and eliminated through PNGase F treatment. The MCR was higher for R-eCG than for N-eCG, and no significant difference was observed after 60 min. Notwithstanding their low concentrations, R-eCG and N-eCG were detected in the blood at 24 h post-injection. Microarray analysis of ovarian tissue revealed that 20 of 12,816 genes assessed therein were significantly up-regulated and 43 genes were down-regulated by > 2-fold in the group that received R-eCG (63 [0.49%] differentially regulated genes in total). The microarray results were concurrent with and hence validated by those of RT-PCR, qRT-PCR, and IHC analyses. CONCLUSIONS: The present results indicate that R-eCG can be adequately produced through a cell-based expression system through post-translational modification of eCG and can induce ovulation in vivo. These results provide novel insights into the molecular mechanisms underlying the up- or down-regulation of specific ovarian genes and the production of R-eCG with enhanced biological activity in vivo.

Constitutive Activation and Inactivation of Mutations Inducing Cell Surface Loss of Receptor and Impairing of Signal Transduction of Agonist-Stimulated Eel Follicle-Stimulating Hormone Receptor.

In the present study, we investigated the signal transduction of mutants of the eel follicle-stimulating hormone receptor (eelFSHR). Specifically, we examined the constitutively activating mutant D540G in the third intracellular loop, and four inactivating mutants (A193V, N195I, R546C, and A548V). To directly assess functional effects, we conducted site-directed mutagenesis to generate mutant receptors. We measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary (CHO-K1) cells and investigated cell surface receptor loss using an enzyme-linked immunosorbent assay in human embryonic kidney (HEK) 293 cells. The cells expressing eelFSHR-D540G exhibited a 23-fold increase in the basal cAMP response without agonist treatment. The cells expressing A193V, N195I, and A548V mutants had completely impaired signal transduction, whereas those expressing the R546C mutant exhibited little increase in cAMP responsiveness and a small increase in signal transduction. Cell surface receptor loss in the cells expressing inactivating mutants A193V, R546C, and A548V was clearly slower than in the cell expressing the wild-type eelFSHR. However, cell surface receptor loss in the cells expressing inactivating mutant N195I decreased in a similar manner to that of the cells expressing the wild-type eelFSHR or the activating mutant D540G, despite the completely impaired cAMP response. These results provide important information regarding the structure-function relationships of G protein-coupled receptors during signal transduction.

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.

Site-specific roles of N-linked oligosaccharides in recombinant eel follicle-stimulating hormone for secretion and signal transduction.

Eel follicle-stimulating hormone (eelFSH) is composed of a common α-subunit and a hormone specific ß-subunit, both of which contain two N-linked carbohydrate residues. We characterized the biologically active single chains by fusing the α-subunit to the carboxyl terminal region of the eelFSH ß-subunit. Expression vectors were constructed and the biological activity of the recombinant hormones (rec-hormones) was characterized using Chinese hamster ovary (CHO) K1 cells expressing the eelFSH receptor gene. Mutagenesis of the individual and double glycosylated sites was performed to determine the functions of the oligosaccharide chains on signal transduction. The absence of the Asn22 (eelFSHßΔ22/α) and Asn5.22 (eelFSHßΔ5.22/α) N-linked oligosaccharide chain in the eelFSH ß-subunit completely reduced the secretion level in the medium and cell lysate of CHO-K1 cells. The expression levels of eelFSHß/α wild-type in CHO suspension (CHO-S) cells was approximately 4-fold higher in CHO-k1 cells. The molecular weight of rec-eelFSHß/α wild-type by western blotting analysis was found to be 34 kDa. Mutants (ß/αΔ56, ß/αΔ79, and ßΔ5/α) lacking single oligosaccharide sites showed molecular weights that were reduced by approximately 10%. The digestion of N-linked oligosaccharides using PNGaseF treatment showed that the molecular weights of all mutants were reduced to 27-kDa. The oligosaccharide chains in rec-eelFSHß/α wild-type were modified to a molecular weight of approximately 7-10 kDa in CHO-K1 and CHO-S cells. Oligosaccharide site deletions at positions Asn56 and Asn79 on the α-subunit and Asn5 on the ß-subunit were found to play an essential role in cAMP signal transduction through the eelFSH receptor. The EC50 values of Asn56 and Asn5 resulted in a significant decrease in potency to 64% and 53% of the wild type, respectively. Specifically, the removal of the carbohydrates at Asn79 of the α-subunit (ß/αΔ79) was drastically reduced to 53.8% of the wild-type levels in maximum response. These results have allowed for the identification of the site-specific roles of carbohydrate residues in eel FSH. Our data suggest that N-linked oligosaccharide chains play a pivotal role in biological activity through the eelFSH receptor as suggested in similar studies of other mammalian FSH hormones.

The C-terminal Phosphorylation Sites of eel Follicle-Stimulating Hormone Receptor are Important Role in the Signal Transduction.

The large extracellular domain of glycoprotein hormone receptors is a unique feature within the G protein-coupled receptors (GPCRs) family. After interaction with the hormone, the receptor becomes coupled to Gs, which, in turn stimulates adenylyl cyclase and the production of cAMP. Potential phosphorylation sites exist in the C-terminal region of GPCRs. The experiments described herein represent attempts to determine the functions of the eel follicle-stimulating hormone receptor (eelFSHR). We constructed a mutant of eelFSHR, in which the C-terminal cytoplasmic tail was truncated at residue 614 (eelFSHR-t614). The eelFSHR-t614 lacked all potential phosphorylation sites present in the C-terminal region of eelFSHR. In order to obtain the eelFSHR ligand, we produced recombinant follicle-stimulating hormone (rec-eelFSHß/α) in the CHO-suspension cells. The expression level was 2-3 times higher than that of the transient expression of eelFSH in attached CHO-K1 cells. The molecular weight of the rec-eelFSHß/α protein was identified to be approximately 34 kDa. The cells expressing eelFSHR-t614 showed an increase in agonist-induced cAMP responsiveness. The maximal cAMP responses of cells expressing eelFSHR-t614 were lower than those of cells expressing eelFSHR-wild type (eelFSHR-WT). The EC50 following C-terminal deletion in CHO-K1 cells was approximately 60.4% of that of eelFSHR-WT. The maximal response in eelFSHR-t614 cells was also drastically lower than that of eelFSHR-WT. We also found similar results in PathHunter Parental cells expressing ß-arrestin. Thus, these data provide evidence that the truncation of the C-terminal cytoplasmic tail phosphorylation sites in the eelFSHR greatly decreased cAMP responsiveness and maximal response in both CHO-K1 cells and PathHunter Parental cells expressing ß-arrestin.

Site specificity of eel luteinizing hormone N-linked oligosaccharides in signal transduction.

Eel luteinizing hormone (eelLH) is composed of a common α-subunit and hormone specific ß-subunit, both of which contain asparagine-linked carbohydrate residues, located at positions 56 and 79 on the α-subunit and position 10 on the ß-subunit. The specific roles of the individual carbohydrate chains are poorly defined in eel. Thus, we characterized the biologically active single chains by fusing the α-subunit to the carboxyl terminal region of the eelLH ß-subunit. Site-directed mutagenesis of the three N-linked glycosylation sites was performed to examine the function of individual glycosylation sites in secretion and signal transduction. The absence of the Asn79N-linked sugar chain slightly reduced secretion in Chinese hamster ovary (CHO) cells. The expression of eelLHß/α (wild-type) in CHO suspension cells was increased by approximately 2-fold higher than that of attached CHO cells. By western blotting analysis, the molecular weight of wild-type was found to be 32 kDa. Mutants (ß/α△56, ß/α△79, and ß△10/α) of the oligosaccharide chain at a single site showed molecular weights that were reduced by approximately 10%. However, the double mutant (ß/α△56.79) molecular weight was decreased by more than 20% compared to the wild-type. Enzymatic digestion of oligosaccharides using PNGaseF treatment showed that the molecular weights of all mutants, including wild-type, were reduced to 25 kDa. The results obtained in the absence of carbohydrates at Asn56 of the α-subunit and at Asn10 of the ß-subunit revealed their roles in signal transduction through the eelLH receptor. The EC50 value of the cAMP response at Asn79 of the α-subunit was increased by 5-fold, whereas the maximum response was dramatically reduced to 17.8% of wild-type levels. Specifically, removal of the carbohydrates at double mutant (ß/α△56.79) is approximately 85% to wild-type levels in biopotency. These results revealed the site-specific roles of eelLH carbohydrate residues. Our data suggest that N-linked oligosaccharide chains play a pivotal role in biological activity through the eelLH receptor.

Data on the localization of EGFP and 20α-hydroxysteroid dehydrogenase (20α-HSD) in the placenta and testes of transgenic mice.

In order to investigate the function of monkey 20α-hydroxysteroid dehydrogenase (20α-HSD), transgenic mice (tg) were produced, expressing enhanced green fluorescent protein (EGFP) under the control of the monkey 20α-HSD promoter. The expression levels and localization of EGFP and 20α-HSD were analyzed in immature testis and in placenta. In support of our recent publication, "Characterization of transgenic mice expressing EGFP under control of monkey 20α-hydroxysteroid dehydrogenase promoter" (Park et al., 2018) [1], it was important to characterize the function of EGFP and 20α-HSD in the ovarian luteal cells of tg mice. Here, the expression of EGFP and 20α-HSD in immature testis and placenta are presented. The expression level of EGFP and 20α-HSD were detected in the testes 1 week after birth, and increased dramatically at 8 weeks. Both of proteins strongly detected in the placenta on days 14, 16, and 18 of pregnancy. Immunohistochemical analysis revealed that EGFP was detected in the seminiferous epithelium and 20α-HSD was specifically localized in the seminiferous tubule at 8 weeks.

Characterization of transgenic mice expressing EGFP under the control of the monkey 20α-hydroxysteroid dehydrogenase promoter.

To directly assess the molecular function of the monkey 20α-hydroxysteroid dehydrogenase (20α-HSD) promoter, we generated transgenic mice (tg) expressing enhanced green fluorescent protein (EGFP) under control of this promoter. We demonstrated that prostaglandin F2α induced 20α-HSD promoter activity in CHO cells in a dose-dependent manner. Furthermore, forskolin treatment markedly reduced 20α-HSD promoter activity, and prolactin exhibited weak inhibitory activity. The transgenic mouse obtained one positive founder male. The transgene was propagated in 10 successive generations without any notable defects to the progeny. EGFP and 20α-HSD in the tg mice were colocalized in the luteal cells of the ovary during late pregnancy. Strong EGFP and 20α-HSD protein signals were also detected in the adult testis. Immunohistochemical analysis revealed high EGFP levels in the seminiferous epithelium, whereas 20α-HSD was expressed in the seminiferous tubules. Our data suggest that the ovaries in monkey and mouse exhibit similar expression patterns of 20α-HSD during pregnancy. However, the expression pattern of EGFP in tg mice testis slightly differed from that of the endogenous 20α-HSD. Further investigation is required to elucidate the functional mechanisms underlying regulation of the monkey 20α-HSD promoter in the tg mice.

Signal Transduction of Eel Luteinizing Hormone Receptor (eelLHR) and Follicle Stimulating Hormone Receptor (eelFSHR) by Recombinant Equine Chorionic Gonadotropin (rec-eCG) and Native eCG.

Previous studies showed that recombinant equine chorionic gonadotropin (rec-eCGß/α) exhibits both follicle-stimulating hormone (FSH) and luteinizing hormone (LH)-like activities in rat LHR- and FSHR-expressing cells. In this study, we analyzed signal transduction by eelFSHR and eelLHR upon stimulation with rec-eCGß/α and native eCG. The cyclic adenosine monophosphate (cAMP) stimulation in CHO-K1 cells expressing eelLHR was determined upon exposure to different doses (0-1,450 ng/mL) of rec-eCGß/α and native eCG. The EC50 values of rec-eCGß/α and native eCG were 172.4 and 786.6 ng/mL, respectively. The activity of rec-eCGß/α was higher than that of native eCG. However, signal transduction in the CHO PathHunter Parental cells expressing eelFSHR was not enhanced by stimulation with both agonist rec-eCGß/α and native eCG. We concluded that rec-eCGß/α and native eCG were completely active in cells expressing eelLHR, similar to the activity in the mammalian cells expressing LHRs. However, rec-eCGß/α and native eCG did not invoke any signaling response in the cells expressing eelFSHR. These results suggest that eCG has a potent activity in cells expressing eelLHR. Thus, we also suggest that rec-eCGß/α can induce eel maturation by administering gonadotropic reagents (LH), such as salmon pituitary extract.

Internalization of Rat FSH and LH/CG Receptors by rec-eCG in CHO-K1 Cells.

Equine chorionic gonadotropin (eCG) is a unique molecule that elicits the response characteristics of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in other species. Previous studies from this laboratory had demonstrated that recombinant eCG (rec-eCG) from Chinese hamster ovary (CHO-K1) cells exhibited both FSH- and LH-like activity in rat granulosa and Leydig cells. In this study, we analyzed receptor internalization through rec-eCGs, wild type eCG (eCGß/α) and mutant eCG (eCGß/αΔ56) with an N-linked oligosaccharide at Asn56 of the α-subunit. Both the rec-eCGs were obtained from CHO-K1 cells. The agonist activation of receptors was analyzed by measuring stimulation time and concentrations of rec-eCGs. Internalization values in the stably selected rat follicle-stimulating hormone receptor (rFSHR) and rat luteinizing/chorionic gonadotropin receptor (rLH/CGR) were highest at 50 min after stimulation with 10 ng of rec-eCGß/α. The dose-dependent response was highest when 10 ng of rec-eCGß/α was used. The deglycosylated eCGß/αΔ56 mutant did not enhance the agonist-stimulated internalization. We concluded that the state of activation of rFSHR and rLH/CGR could be modulated through agonist-stimulated internalization. Our results suggested that the eLH/CGRs are mostly internalized within 60 min by agonist-stimulation by rec-eCG. We also suggested that the lack of responsiveness of the deglycosylated eCGß/ αΔ56 was likely because the site of glycosylation played a pivotal role in agonist-stimulated internalization in cells expressing rFSHR and rLH/CGR.

Abnormal gene expression in regular and aggregated somatic cell nuclear transfer placentas.

BACKGROUND: Placental defects in somatic cell nuclear transfer (SCNT) are a major cause of complications during pregnancy. One of the most critical factors for the success of SCNT is the successful epigenetic reprogramming of donor cells. Recently, it was reported that the placental weight in mice cloned with the aggregated SCNT method was significantly reduced. Here, we examine the profile of abnormal gene expression using microarray technology in both regular SCNT and aggregated SCNT placentas as well as in vivo fertilization placentas. One SCNT embryo was aggregated with two 2 to 4 -cell stage tetraploid embryos from B6D2F1 mice (C57BL/6 × DBA/2). RESULTS: In SCNT placentas, 206 (1.6%) of the 12,816 genes probed were either up-regulated or down-regulated by more than two-fold. However, 52 genes (0.4%) showed differential expression in aggregated SCNT placentas compared to that in controls. In comparison of both types of SCNT placentas with the controls, 33 (92%) out of 36 genes were found to be up-regulated (>2-fold) in SCNT placentas. Among 36 genes, 13 (36%) genes were up-regulated in the aggregated SCNT placentas. Eighty-five genes were down-regulated in SCNT placentas compared with the controls. However, only 9 (about 10.5%) genes were down-regulated in the aggregated SCNT placentas. Of the 34 genes known as imprinted genes, expression was lower in SCNT placentas than that in the controls. Thus, these genes may be the cause of placentomegaly in mice produced post SCNT. CONCLUSIONS: These results suggest that placentomegaly in the SCNT placentas was probably caused by abnormal expression of multiple genes. Taken together, these results suggest that abnormal gene expression in cloned placentas was reduced in a genome-wide manner using the aggregation method with tetraploid embryos.