Hiroyoshi Hoshi

In vitro production of bovine embryos and their application for embryo transfer.

This review introduces newly developed serum-free media (IVD101 and IVMD101), that are effective for producing high yields of transferable embryos of good quality from in vitro-matured and -fertilized oocytes. Both serum-free media produced better results than serum-containing medium, including increased rates of blastocyst formation, post-thaw embryo viability, and pregnancy after transfer. In addition, reduced risks of calf mortality and large calf syndrome were also observed for the serum-free-derived embryos. Serum-derived embryos contained a large number of lipid droplets and immature mitochondria in their cytoplasm that may account for the lower production of transferable embryos and poor embryo quality. A non-invasive technique using scanning electrochemical microscopy was successful in quantitatively measuring oxygen consumption of single embryos. This technique may prove to be reliable for predicting embryo viability and subsequent developmental ability.

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.

Ultrastructural differences in bovine morulae classified as high and low qualities by morphological evaluation

Bovine morulae collected from superovulated cows were classified into four groups (excellent, good, fair, poor) using morphological evaluation under a phase contrast microscope. The number of blastomeres in morulae classified as excellent and good (high quality) was significantly higher than the number in fair and poor (low quality) morulae. The ultrastructure of morulae in each group was compared. In morulae classified as excellent and good, two characteristic cell types could be identified by the electron density of their cytoplasm and by their ultrastructural features. One type (presumptive trophoblast cells) was located in the peripheral layer of the embryo mass, was generally characterized by low electron density of cytoplasm, and possessed numerous cellular organelles such as mitochondria and Golgi apparatus and had numerous microvilli projecting into the perivitelline space. The other type (presumptive embryonic cells) was located in the interior of the embryo mass and distinguished by cytoplasm that stained more densely than that of the lighter-appearing cells. The darker-appearing cells generally possessed fewer organelles than the lighter cells, but many lysosome-like structures were present in the cytoplasm. All high quality morulae contained well-developed mature nucleoli. Many embryos classified as fair contained both types of cells, while cells in some fair and most poor quality morulae could not be distinguished by the electron density of their cytoplasm and ultrastructural features. The morulae classified as fair and poor showed less well-developed junctional complexes between cells, less well-developed apical microvilli on the blastomeres, and contained large numbers of lipid droplets, vesicles, immature mitochondria, and nucleoli with low transcriptional activity. This study demonstrates conspicuous differences in the ultrastructural features between the high quality and the low quality morulae, suggesting that these ultrastructural features may reflect the various physiological anomalies observed in previous studies.

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; 117.2u2009±u20093.2 µm, 50 ng/ml; 116.5u2009±u20093.5 µm) compared to the control (0 ng/ml; 110.5u2009±u20092.8 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.

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.

Glucose and glycine synergistically enhance the in vitro development of porcine blastocysts in a chemically defined medium

In the present study, the effects of glucose and/or glycine on the in vitro development of Day 5 (Day 0=IVF) porcine blastocysts were determined. The addition of 2.5-10 mM glucose to the chemically defined culture medium porcine zygote medium (PZM)-5 significantly increased blastocyst survival rates compared with those of blastocysts cultured in the absence of glucose. The addition of 5 and 10 mM glycine to PZM-5 containing 5 mM glucose significantly enhanced the development to hatching and the number of hatched blastocysts compared with no addition of glycine. However, the addition of glycine to PZM-5 with no glucose did not improve blastocyst development. The ATP content of Day 6 blastocysts cultured with glucose was significantly higher than that of blastocysts cultured in the absence of glucose, regardless of glycine supplementation. The diameter and total cell numbers were significantly greater, and the apoptotic index was significantly lower, in Day 6 blastocysts cultured with both glucose and glycine. These results indicate that glucose is an important energy source for the porcine blastocyst and that glucose and glycine act synergistically to enhance development to the hatching and hatched blastocyst stage in vitro.

Transforming growth factor-α in a defined medium during in vitro maturation of porcine oocytes improves their developmental competence and intracellular ultrastructure

We examined the effects of transforming growth factor-alpha (TGF-alpha) to develop a defined medium for in vitro maturation (IVM) of porcine (Sus scrofa domesticus) oocytes. Cumulus-oocyte complexes (COCs) were matured in porcine oocyte medium containing 3mg/mL polyvinyl alcohol (POM) and TGF-alpha (0, 1, 10, or 100 ng/mL) in the presence or absence of the gonadotropins equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). In the absence of gonadotropins, adding 10 ng/mL TGF-alpha increased (P<0.05) the percentage of oocytes that reached metaphase II (24.2%) compared with that of the control (no TGF-alpha addition; 5.6%). In the presence of gonadotropins, although maturation rate did not differ among TGF-alpha treatments (75.4% to 84.8%), the rate of blastocyst formation (28.1%) was higher (P<0.05) in the TGF-alpha group (28.1%) than that in the control group (15.9%) after in vitro fertilization and embryo culture. An electron microscope study revealed that TGF-alpha-treated oocytes contained more homogenous lipid droplets than did control oocytes. Moreover, mitochondria surrounded by the endoplasmic reticulum were observed only in the TGF-alpha-treated oocytes. In blastocysts derived from the latter oocytes, mitochondria with numerous cristae were frequently observed compared with that in blastocysts from control oocytes. When the Day-5 blastocysts obtained from oocytes matured with TGF-alpha were surgically transferred into four recipients, a total of 29 piglets were farrowed. We concluded that the addition of TGF-alpha to the defined IVM medium of porcine oocytes improved the subsequent blastocyst formation and that the blastocysts produced by the defined in vitro production system have developmental competence to full term after embryo transfer.

Recombinant human follicle‐stimulating hormone and transforming growth factor‐alpha enhance in vitro maturation of porcine oocytes

The biological functions of recombinant human follicle‐stimulating hormone (FSH) and transforming growth factor‐α (TGF‐α) on in vitro maturation of porcine oocytes were investigated. Cumulus–oocyte complexes were matured in defined porcine oocyte medium containing 0–0.1u2009IU/ml FSH in the presence or absence of 10u2009ng/ml TGF‐α. The percentage of oocytes reaching metaphase II was significantly higher with the addition of 0.01–0.1u2009IU/ml FSH compared with no addition, and was further enhanced in the presence of TGF‐α. The rates of sperm penetration and blastocyst formation were significantly higher with the addition of 0.05–0.1u2009IU/ml FSH compared with no addition after in vitro fertilization and embryo culture. There was no beneficial effect of FSH and TGF‐α on nuclear maturation of denuded oocytes. The specific EGF receptor inhibitor, AG1478, completely inhibited TGF‐α‐induced meiotic resumption, but did not completely prevent the stimulatory effect of FSH. Addition of both FSH and TGF‐α significantly enhanced cumulus expansion compared with no addition. When cumulus expansion‐related genes (HAS2, HAPLN1, and VCAN) mRNA expression in COCs was measured during in vitro maturaiton, addition of both of FSH and TGF‐α upregulated the expression of HAS2 mRNA after 20u2009hr culture and HAPLN1 mRNA after 44u2009hr culture compared with no addition. Expression of VCAN mRNA was significantly higher in the presence of FSH compared with addition of TGF‐α alone. These results suggest that FSH and TGF‐α synergistically enhance porcine oocyte maturation via cumulus cells, and act through different signaling pathways. Mol. Reprod. Dev. 80: 549–560, 2013.

Birth of piglets from in vitro–produced porcine blastocysts vitrified and warmed in a chemically defined medium

We examined the effect of different embryo developmental stages, culture periods, and media on the cryotolerance of inxa0vitro-produced porcine blastocysts. All media used for inxa0vitro production, vitrification, and warming were chemically defined. When inxa0vitro-produced embryos at the blastocyst and expanded blastocyst stages were vitrified using the Cryotop method on Day 5 or 6 (Day 0xa0=xa0the day of IVF), the survival rate and hatching rate of expanded blastocysts after warming were higher than those of blastocysts. The viability after vitrification and warming of Day-6 embryos cultured in porcine blastocyst medium from Day 5 were higher than that of embryos cultured in porcine zygote medium 5. On the other hand, there were no significant differences on the cryotolerance between Day-5 embryos cultured in porcine zygote medium 5 and those replaced with porcine blastocyst medium on Day 4. There were no significant differences in viability between the embryonic ages of 5 and 6xa0days after vitrification and warming. When expanded blastocysts vitrified on Day 5 or 6 were surgically transferred to recipient gilts, all three recipients became pregnant in the Day-5 group, whereas only one out of three recipients became pregnant in the Day-6 group. These results indicate that the cryotolerance of porcine inxa0vitro-produced blastocysts after vitrification appears to depend on the embryonic stage, culture period, and medium.

Successful Production of Piglets Derived from Expanded Blastocysts Vitrified Using a Micro Volume Air Cooling Method without Direct Exposure to Liquid Nitrogen

Abstract This study was conducted to clarify the feasibility of newly developed vitrification techniques for porcine embryos using the micro volume air cooling (MVAC) method without direct contact with liquid nitrogen (LN2). Expanded blastocysts were vitrified in a solution containing 6 M ethylene glycol, 0.6 M trehalose and 2% (wt/vol) polyethylene glycol in 10% HEPES-buffered PZM-5. The blastocysts were collected from gilts and vitrified using the new device (MVAC) or a Cryotop (CT). Blastocysts were stored in LN2 for at least 1 month. After warming, cryoprotective agents were removed using a single step. Survival of the embryos was assessed by in vitro culture (Experiment 1) and by embryo transfer to recipients (Experiment 2). In Experiment 1, the embryos vitrified by the MVAC or CT and fresh embryos without vitrification (Control) were used. The survival rates of embryos in the MVAC, CT and Control groups were 88.9% (32/36), 91.7% (33/36) and 100% (34/34), respectively, after 48 h culture, and the hatching rates of embryos after 48 h incubation were 69.4% (25/36), 63.9% (23/36) and 94.1% (32/34), respectively. In Experiment 2, 64 vitrified embryos were transferred to 5 recipient gilts, and 8 healthy piglets were produced from 3 recipients in the MVAC group. Similarly, 66 vitrified embryos were transferred to 5 recipient gilts, and 9 healthy piglets were produced from 2 recipients in the CT group. These results indicated that porcine expanded blastocysts can be cryopreserved using the MVAC method without potential pathogen contamination from LN2.