Yutaka Sendai

Growth, Antrum Formation, and Estradiol Production of Bovine Preantral Follicles Cultured in a Serum-Free Medium

Abstract The objective of this study was to identify factors that would allow the establishment of a serum-free culture system that could support follicular and oocyte growth, antrum formation, and estradiol-17β (E2) production in preantral follicles of bovine ovaries. Large preantral follicles (145–170 μm in diameter) were microsurgically dissected from ovaries, embedded in 0.15% type I collagen gels, and maintained in a serum-free medium for up to 13 days at 38.5°C in 5% CO2 in air. This culture environment allowed most preantral follicles to maintain a three-dimensional structure with the presence of a thecal layer and basement membrane surrounding the granulosa cells throughout the entire culture period. The effects of insulin, insulin-like growth factor (IGF)-I, IGF-II, FSH, and LH on preantral follicle growth were investigated in serum-free medium. Follicular diameters were significantly larger in the presence of insulin, IGF-I, IGF-II, or FSH after 13 days in culture. Oocyte diameters were also significantly larger in the presence of all hormones tested. The single addition of insulin, IGF-I, or FSH induced antrum formation between Days 7 and 13 of culture. Insulin progressively induced E2 secretion by follicles after antrum formation, but IGF-I and FSH had no apparent effect. FSH and LH caused an increase in oocyte diameter in the presence of insulin. The addition of three hormones (insulin, FSH, and LH) initiated antrum formation and E2 production earlier than insulin-containing medium alone. Furthermore, maximal E2 secretion was maintained steadily between 7 and 13 days in this culture condition. From these results, we have demonstrated that insulin, FSH, and LH play substantial roles in the growth and development of bovine large preantral follicles in a serum-free medium.

Establishment of a Porcine Cell Line from In Vitro-Produced Blastocysts and Transfer of the Cells into Enucleated Oocytes

Abstract The present study was conducted to establish a porcine cell line from blastocysts produced in vitro and to examine the developmental ability of nuclear transfer embryos reconstituted with the cells and enucleated mature oocytes. When hatched blastocysts were cultured in Dulbeccos modified Eagles medium with supplements, no colonies of embryo-derived cells were observed. In contrast, 56% of embryos that were attached to feeder layers of STO cells formed colonies in NCSU-23 with supplements. When the colonies were subcultured in the absence of feeder cells, a cell line with an epithelial-like cell morphology was obtained. This cell morphology was stable up to at least passage 30. Although no fused embryos were observed when a pulse of 100 V/mm was applied, the fusion rate increased significantly at 150 V/mm (28%) and 200 V/mm (64%). At 200 V/mm, 39% of fused embryos cleaved, but no embryos developed beyond the 3-cell stage. When cocultured with electro-activated oocytes, percentages of reconstructed embryos cleaved (65%) and developed to the 4-cell stage (23%) were significantly higher than percentages for those (cleavage: 38%; 4-cell stage: 3%) in the absence of activated oocytes. At 7 days after culture, one reconstructed embryo successfully developed to the blastocyst stage in the presence of activated oocytes. When green fluorescent protein-expressing cells and enucleated oocytes were fused and the fused embryos were cultured with electro-activated oocytes, 3 of 102 reconstructed embryos developed to the blastocyst stage. All of the blastocysts were positive for fluorescent green under ultraviolet light. The results of the present study indicate that a porcine cell line can be established from the hatched blastocyst and maintained in vitro for a long period, and that reconstructed embryos obtained by transferring the blastocyst-derived cells into enucleated oocytes have the ability to develop to the blastocyst stage in vitro.

Effect of a null mutation of the oviduct-specific glycoprotein gene on mouse fertilization.

The mammalian fertilization process takes place in a complex microenvironment within the female genital tract. A member of the chitinase protein family, oviduct-specific glycoprotein (OGP), has been identified in oviductal fluid from various mammalian species, including humans. Although OGP is widely believed to be involved in the process of mammalian fertilization, including spermatozoon function and gamete interactions, based on experimental results obtained in vitro, its physiological significance remains controversial. The present study established OGP gene-null ( ogp (-/-)) mice, and primarily characterized their reproductive properties to study the physiological function(s) of OGP. Results obtained from studies using an in vivo or in vitro system showed that the fertility of ogp (-/-) females was within normal limits. These results indicate that OGP is not essential for the process of in vivo fertilization, at least in mice.

Fibroblast growth factor 7 stimulates in vitro growth of oocytes originating from bovine early antral follicles

Essential factors required for growing oocytes derived from bovine early antral follicles and their mechanisms of action are poorly understood. Fibroblast growth factor 7 (FGF7) is a member of the heparin‐binding FGF family with a distinctive pattern of target‐cell specificity. The effect of FGF7 on the stimulation of oocyte growth in a culture of cumulus–oocyte complexes with granulosa cells (COCGs, oocyte diameter; 90–100 µm) was investigated. The oocyte diameter of COCGs was increased significantly in the FGF7‐containing medium (10 ng/ml; 117.2 ± 3.2 µm, 50 ng/ml; 116.5 ± 3.5 µm) compared to the control (0 ng/ml; 110.5 ± 2.8 µm) after 16 days. However, there was no stimulatory effect of FGF7 on the proliferation of cumulus‐granulosa cells. The FGF7 receptor, fibroblast growth factor receptor 2IIIb (FGFR2IIIb), was detected in cumulus‐granulosa cells from COCGs. Messenger RNA expression of FGFR2IIIb was induced to cumulus‐granulosa cells by FGF7. The mRNA expression levels of KIT ligand (KITLG), KIT (KIT), growth differentiation factor 9 (GDF9), and bone morphogenetic protein 15 (BMP15) in the cultured COCGs were determined in FGF7‐treated (10 ng/ml) cultures using real time RT‐PCR analysis. The levels of KITLG and KIT, but not GDF9 and BMP15 mRNA expression were stimulated by FGF7. Furthermore, neutralizing antibody for KIT attenuated the stimulatory action of FGF7 on the oocyte growth. These results strongly suggest that FGF7 may be an important regulator for oocyte growth and its action is mediated via the KIT/KITLG signaling pathway. Mol. Reprod. Dev. 75: 1736–1743, 2008.

Molecular Cloning of a cDNA Encoding a Bovine Growth Differentiation Factor-9 (GDF-9) and Expression of GDF-9 in Bovine Ovarian Oocytes and In Vitro-Produced Embryos

Growth differentiation factor-9 (GDF-9), a member of the transforming growth factor-beta superfamily, is known to be expressed specifically in ovaries of various mammalian species and to be important for normal follicular development in mice. In the present study, a cDNA encoding for bovine GDF-9 was isolated and characterized, and expression of GDF-9 mRNA in ovarian oocytes and in-vitro-derived embryos was examined. Isolation of bovine GDF-9 was achieved using the polymerase chain reaction (PCR) with primers based on an ovine GDF-9 cDNA sequence. A 1385 bp cDNA encodes a deduced 453-amino acid sequence which contains the proregion (318 residues) and the mature protein (135 residues) portion. The deduced amino acid sequence of bovine GDF-9 is 98 and 93% identical to ovine GDF-9 and human GDF-9 in the mature portion of the molecule, respectively. Results from reverse transcription (RT)-PCR analysis detected bovine GDF-9 mRNA in preantral follicles (150-200 microm in diameter), early antral follicles (400-800 microm in diameter), and immature oocytes, whereas no detectable PCR signal was observed in cumulus/granulosa cells. In addition, bovine GDF-9 mRNA continued to be expressed in developing embryos up to the 8-cell stage, but was undetectable at the blastocyst stage. These findings give rise to new possibilities regarding an additional physiological role of GDF-9 in early embryonic development as well as in the development of follicles.

Mouse Oviduct-Specific Glycoprotein Gene: Genomic Organization and Structure of the 5′-Flanking Regulatory Region

Abstract A member of the chitinase protein family, oviduct-specific glycoprotein (OGP), can directly associate with gametes or with the early embryo in the oviduct. Although the glycoprotein is widely distributed among mammalian species and there is indirect evidence concerning the involvement of the molecule in the fertilization process, its physiological functions are far from completely understood. To understand the fundamental mechanisms that direct gene expression as well as to know the physiological significance of OGP, we have isolated and characterized a mouse OGP gene (mogp-1). The gene was found to span 13.4 kilobases (kb) including 11 exons and 10 introns. The genomic organization of mogp-1 is well conserved compared to the other members of the chitinase family. Two transcription initiation sites were found at positions 18 and 14 upstream from the first ATG codon. Fluorescence in situ hybridization analysis demonstrated that the mogp-1 was located on the R-positive F3 band of mouse chromosome 3. Although the putative promoter region of mogp-1 lacked typical TATA, CAAT, or GC box sequences, the region contained several motif sequences of transcription factor binding sites including 10 half-palindromic estrogen responsive elements (ERE) and an imperfect ERE. Transient transfection experiments demonstrated that promoter activity could be modulated by various sequences within the 2.2 kb of the 5′-flanking region, and that the mogp-1 promoter was transactivated in an estrogen receptor-positive cell line, MCF-7, by the addition of estradiol-17β (E2). In addition, relevant promoter activity for E2 responsiveness resides within the first 270 base pairs upstream of the mogp-1. These findings should facilitate our understanding of the regulation of OGP gene expression, and they may be helpful for designing experiments to unravel the role of OGP in the process of mammalian fertilization.

α1,3-Galactosyltransferase-gene knockout in cattle using a single targeting vector with loxP sequences and Cre-expressing adenovirus

Background. Gene targeting in large animals has the potential to be useful in medicine as well as in agriculture. Previously, we reported the first successful targeting of the bovine &agr;1,3-galactosyltransferase (&agr;1,3GT) gene and establishment of a heterozygous knockout cell line. In this report, we generated both heterozygous and homozygous knockout bovine cell lines, and &agr;1,3GT-gene knockout cattle. Methods. &agr;1,3GT gene-disruption was accomplished using primary fetal fibroblasts with a single targeting vector, a promoter-less positive selection vector containing IRES (internal ribosome entry site)-antibiotic-resistance gene (neo) cassette and loxP sequences. At each step in establishing heterozygous and homozygous knockout cell lines, the antibiotic-resistance gene cassette in the targeted allele was removed by a Cre-loxP recombination system that utilizes an adenovirus with transient Cre recombinase expression. A nuclear transfer was performed using &agr;1,3GT−/− fetal fibroblasts, and one &agr;1,3GT knockout calf was generated but died shortly after birth (day 287). Results. Necropsy revealed normal morphology in all organs. The calf weighed 22.3 kg at birth and this value is within the normal range. Conclusion. The &agr;1,3GT knockout- and antibiotic-resistance gene free (&agr;1,3GT−/−neo-) cells could be cloned normally. Thus, cloned cattle from &agr;1,3GT −/−neo- cells are potentially safer for human use. Additionally, our strategy is faster and more economical than backcrossing to produce homozygous knockouts. This method should be useful for future production of knockouts of multiple genes in livestock.

Fibroblast growth factor stimulates the gene expression and production of tissue inhibitor of metalloproteinase-1 in bovine granulosa cells.

SummaryThe hormonal control of tissue inhibitor of metalloproteinase-1 (TIMP-1) gene expression and production by growth factors, gonadotrophins, and serum factors in cultured bovine granulosa cells (BGC) were investigated. Confluent cultures of BGC were exposed to various factors in a defined medium and levels of TIMP-1 in the conditioned medium were determined by enzyme immunoassay. Basic fibroblast growth factor (bFGF) and acidic fibroblast growth factor (aFGF) showed potent stimulation of cell proliferation and TIMP-1 production by BGC, while insulin stimulated growth but not TIMP-1 production. Basic FGF stimulated TIMP-1 production and BGC cell proliferation in a dose-dependent manner. A time course of TIMP-1 production showed substantially increased levels between 18 and 24 h in both control and bFGF-stimulated BGC cultures with bFGF-stimulated cultures having markedly higher TIMP-1 production at all time points. Consistent with the TIMP-1 production data, bFGF and aFGF increased the expression of TIMP-1 mRNA as determined by northern blot analysis, while insulin, inhibited TIMP-1 mRNA levels. These results indicate that FGF-induced TIMP-1 production by BGC may support bovine embryo development in vitro.

Heterozygous disruption of the α1,3-galactosyltransferase gene in cattle

Background. Animal cloning techniques have enabled gene disruption in several species. Here, we report the first successful disruption of the &agr;1,3-galactosyltransferase (&agr;1,3-GT) gene in cattle. Methods. The &agr;1,3-GT gene of the Japanese Black cow (JBC) was used to construct pGT-6, a targeting vector for the bovine &agr;1,3-GT gene, and pGT-6 was introduced into the fetal fibroblast cell line JBC906 by the lipofection method. Four polymerase chain reaction (PCR)-positive colonies were obtained from 797 G418-resistant colonies, and Southern blot analysis revealed successful homologous recombination at the &agr;1,3-GT locus in one of the four colonies. Nuclear transfer was performed, and the four embryos were transferred to a heifer. Results. To establish fetal fibroblasts that were heterozygously disrupted at the &agr;1,3-GT locus, one of the fetuses was recovered at 5 weeks of pregnancy, and PCR and Southern blot analysis of the fetal fibroblasts established from it showed definite homologous recombination of the &agr;1,3-GT gene. Conclusions. Heterozygous knockout of the &agr;1,3-GT gene was performed in JBC, and production of a homozygous &agr;1,3-GT knockout JBC by a second round of targeting 906htGT is currently in progress. The technique described here can be applied to disruption of other genes in cattle.

Generation of exogenous germ cells in the ovaries of sterile NANOS3-null beef cattle.

Blastocyst complementation (BC) systems have enabled in vivo generation of organs from allogeneic pluripotent cells, compensating for an empty germ cell niche in gene knockout (KO) animals. Here, we succeeded in producing chimeric beef cattle (Wagyu) by transferring allogenic germ cells into ovaries using somatic cell nuclear transfer and BC technology. The KO of NANOS3 (NANOS3−/−) in Wagyu bovine ovaries produced a complete loss of germ cells. Holstein blastomeres (NANOS3+/+) were injected into NANOS3−/− Wagyu embryos. Subsequently, exogenous germ cells (NANOS3+/+) were identified in the NANOS3−/− ovary. These results clearly indicate that allogeneic germ cells can be generated in recipient germ cell-free gonads using cloning and BC technologies.