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.

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.

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.

Where to fix in rejuvenation surgeries?: tensile strength of the periosteum.

The aim of this study is to elucidate the tensile strength of the periosteum relating to facial rejuvenation surgeries.Twelve hemifaces of 6 formalin-fixed Korean adult cadavers were used. Two horizontal incision lines were made 3 cm above the supraorbital rim and 1 cm below the infraorbital rim. Another 2 vertical incisions were on the medial orbital rim and 2 cm lateral to the lateral orbital rim. Elevated flaps were turned over, and the undersurfaces of the periosteum were exposed. A silk string was passed below the periosteum with a 3-mm bite and wound. A 3-cm loop was made, and this was pulled away using the tensiometer. The breaking strength was measured.The breaking strengths of the periosteum were different according to the location (P = 0.000, analysis of variation). The strongest point was 2 cm above the supraorbital rim at the medial one third of the orbit (14.05 [2.50] N) followed by 1 cm above the frontozygomatic suture (13.35 [4.70] N). The weakest point was the infraorbital rim at the lateral one third of the orbit (6.93 [3.76] N) followed by the lateral orbital rim at the level of the lateral canthus (7.60 [3.49] N). Breaking strengths of the periosteum of the medial side (11.44 [3.83] N) were significantly greater (P = 0.021, t-test) than the lateral side (9.32 [3.76] N). In the supraorbital area, the breaking strengths of the periosteum of the upper points (12.91 [3.00] N) were significantly greater (P = 0.000, t-test) than the lower points (9.36 [2.76] N).The results of this study can be of use when choosing a fixation point in rejuvenation surgeries.

The effect of horse serum on in vitro development of porcine parthenogenetic embryos.

The objective of this study was to examine the effect of different sera and serum-like substances on the preimplantation development of porcine parthenogenetic embryos. Chemically activated (calcium ionophore A23187+cytochalasin B) pig oocytes were pre-cultured for five days. On day 5, the parthenogenetic embryos were treated with porcine follicular fluid (PFF), fetal bovine serum (FBS), horse serum (HS) or porcine serum albumin (PSA), and were cultured two more days. Horse serum was found to be the most effective protein source in enhancing parthenote development judging by blastocyst formation and hatching. Next, three different concentrations of HS (10, 20 and 30%) were used to determine the optima HS concentration needed to improve the development of porcine parthenogenetic embryos. All HS concentrations increased the blastocyst cell number and decreased the incidence of blastocyst apoptotic cells with 20% being the most effective. In conclusion, horse serum enhanced parthenogenetic embryo development and the quality of porcine parthenogenetic embryos.

Aberrant phenotypes of transgenic mice expressing dimeric human erythropoietin.

BACKGROUND: Dimeric human erythropoietin (dHuEPO) peptides are reported to exhibit significantly higher biological activity than the monomeric form of recombinant EPO. The objective of this study was to produce transgenic (tg) mice expressing dHuEPO and to investigate the characteristics of these mice. METHODS: A dHuEPO-expressing vector under the control of the goat beta-casein promoter, which produced a dimer of human EPO molecules linked by a 2-amino acid peptide linker (Asp-Ile), was constructed and injected into 1-cell fertilized embryos by microinjection. Mice were screened using genomic DNA samples obtained from tail biopsies. Blood samples were obtained by heart puncture using heparinized tubes, and hematologic parameters were assessed. Using the microarray analysis tool, we analyzed differences in gene expression in the spleens of tg and control mice. RESULTS: A high rate of spontaneous abortion or death of the offspring was observed in the recipients of dHuEPO embryos. We obtained 3 founder lines (#4, #11, and #47) of tg mice expressing the dHuEPO gene. However, only one founder line showed stable germline integration and transmission, subsequently establishing the only transgenic line (#11). We obtained 2 F1 mice and 3 F2 mice from line #11. The dHuEPO protein could not be obtained because of repeated spontaneous abortions in the tg mice. Tg mice exhibited symptoms such as short lifespan and abnormal blood composition. The red blood cell count, white blood cell count, and hematocrit levels in the tg mice were remarkably higher than those in the control mice. The spleens of the tg mice (F1 and F2 females) were 11- and -21-fold larger than those of the control mice. Microarray analysis revealed 2,672 spleen-derived candidate genes; more genes were downregulated than upregulated (849/764). Reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qRT-PCR) were used for validating the results of the microarray analysis of mRNA expression. CONCLUSIONS: In conclusion, dHuEPO tg mice caused excessive erythrocytosis that led to abnormal blood composition, short lifespan, and abnormal splenomegaly. Further, we identified 2,672 genes associated with splenomegaly by microarray analysis. These results could be useful in the development of dHuEPO-producing tg animals.

Expression of aldo-keto reductase family 1 member C1 (AKR1C1) gene in porcine ovary and uterine endometrium during the estrous cycle and pregnancy.

BACKGROUND: The aldo-keto reductase family 1 member C1 (AKR1C1) belongs to a superfamily of NADPH-dependent reductases that convert a wide range of substrates, including carbohydrates, steroid hormones, and endogenous prostaglandins. The 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) is a member of AKR family. The aims of this study were to determine its expression in the ovary and uterus endometrium during the estrous cycle and pregnancy. METHODS: Rapid amplification of cDNA ends (RACE) experiments were performed to obtain the 5' and 3' ends of the porcine 20 alpha-HSD cDNA. Reverse-transcriptase-PCR (RT-PCR), real-time PCR, northern blot analysis, and western blot analysis were performed to examine the expression of porcine 20 alpha-HSD. Immunohistochemical analysis was also performed to determine the localization in the ovary. RESULTS: The porcine 20 alpha-HSD cDNA is 957 bp in length and encodes a protein of 319 amino acids. The cloned cDNA was virtually the same as the porcine AKR1C1 gene (337 amino acids) reported recently, and only differed in the C-terminal region (the AKR1C1 gene has a longer C-terminal region than our sequence). The 20 alpha-HSD gene (from now on referred to as AKR1C1) cloned in this paper encodes a deletion of 4 amino acids, compared with the C-terminal region of AKR1C1 genes from other animals. Porcine AKR1C1 mRNA was expressed on day 5, 10, 12, 15 of the cycle and 0-60 of pregnancy in the ovary. The mRNA was also specifically detected in the uterine endometrium on day 30 of pregnancy. Western blot analysis indicated that the pattern of AKR1C1 protein in the ovary during the estrous cycle and uterus during early pregnancy was similar to that of AKR1C1 mRNA expression. The recombinant protein produced in CHO cells was detected at approximately 37 kDa. Immunohistochemical analysis also revealed that pig AKR1C1 protein was localized in the large luteal cells in the early stages of the estrous cycle and before parturition. CONCLUSIONS: Our study demonstrated that AKR1C1 mRNA and protein are coordinately expressed in the luteal cell of ovary throughout the estrous cycle and in the uterus on day 30 of pregnancy. Thus, the porcine AKR1C1 gene might control important mechanisms during the estrous cycle.

Molecular characterization of bovine placental and ovarian 20α-hydroxysteroid dehydrogenase.

The enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD) catalyzes the conversion of progesterone to its inactive form, 20α-hydroxyprogesterone. This enzyme plays a critical role in the regulation of luteal function in female mammals. In this study, we conducted the characterization and functional analyses of bovine 20α-HSD from placental and ovarian tissues. The nucleotide sequence of bovine 20α-HSD showed significant homology to that of goats (96%), humans (84%), rabbits (83%), and mice (81%). The mRNA levels increased gradually throughout the estrous cycle, the highest being in the corpus luteum (CL) 1 stage. Northern blot analysis revealed a 1.2  kb mRNA in the bovine placental and ovarian tissues. An antibody specific to bovine 20α-HSD was generated in a rabbit immunized with the purified, recombinant protein. Recombinant 20α-HSD protein produced in mammalian cells had a molecular weight of ∼37  kDa. Bacterially expressed bovine 20α-HSD protein showed enzymatic activity. The expression pattern of the 20α-HSD protein in the pre-parturition placenta and the CL1 stage of the estrous cycle was similar to the level of 20α-HSD mRNA expression. Immunohistochemical analysis also revealed that bovine 20α-HSD protein was intensively localized in the large luteal cells during the late estrous cycle.

Expression and localization of the 20α-hydroxysteroid dehydrogenase (HSD) enzyme in the reproductive tissues of the cynomolgus monkey Macaca fascicularis.

This study was conducted to characterize and functionally analyze the monkey 20α-hydroxysteroid dehydrogenase (20α-HSD) in the ovary, placenta, and oviduct. We focused on 20α-HSD mRNA expression and protein localization in monkey reproductive tissues and the molecular characterization of the promoter region. Reverse transcription-polymerase chain reaction (RT-PCR) monkey 20α-HSD mRNA was more strongly detected in the ovary at pre-ovulation than in the placenta and oviduct at pre-parturition. The mRNA was approximately 1.2kb in size and the expression was high in the ovary, which was the same as the RT-PCR result. We also produced His tagged 20α-HSD proteins by using an Escherichia coli expression system. In a western blot for the 20α-HSD protein, only 1 band of approximately 37-kDa was detected in the ovary, oviduct tissue, and recombinant protein produced in the Chinese hamster ovary (CHO) cell line. However, in the placenta, additional 2 bands (35 and 39 kDa) were detected. Immunohistochemical analyses suggested that the monkey 20α-HSD protein was localized mainly in the syncytiotrophoblast of the placenta and the isthmus cells of the oviduct. According to promoter analyses with the enhanced green fluorescent protein (EGFP) gene, the monkey 20α-HSD promoter was efficiently expressed in the CHO-K1 cell line; however, the promoter was not expressed in bovine fetal fibroblast (bFF) cell. Taken together, our study showed that the 20α-HSD mRNA and protein are coordinately expressed in the ovary at pre-ovulation and in the placenta and oviduct at pre-parturition. Therefore, monkey 20α-HSD in the placenta, ovary and oviduct plays an important role in the estrous cycle, pregnancy, and parturition.

Soft x-ray microscope constructed with a PMMA phase-reversal zone plate.

A soft x-ray microscope based on a phase-reversal zone plate was constructed and tested using high harmonic radiation as a coherent light source. The 61st harmonic centered at 13.3 nm was optimized in spectral sharpness and intensity by controlling the incident laser energy and chirp. A phase-reversal zone plate made of polymethyl methacrylate more than doubled the first-order efficiency. The nano patterns, imaged on an x-ray CCD with a magnification of 650, showed that the measured resolution of the microscope was better than 100 nm.