T. Nagai

Birth of piglets derived from in vitro fertilization of pig oocytes matured in vitro

Abstract Follicular oocytes collected from prepubertal gilts at a local slaughterhouse were matured for 36 hours in mTLP-PVA medium with pig follicular fluid (pFF) fraction and cysteine, and were fertilized (mTALP-PVA) in vitro. After 12 hours of insemination, a total of 78% ( 14 18 ) of the oocytes was fertilized in vitro and the normal fertilization rate was 43% ( 6 14 ). Of the 356 embryos developed (mKRB) in vitro, 84 (24%) cleaved to the 2- to 4-cell stage by 36 hours after insemination. Of 84 the embryos, 75 were subsequently transferred into the oviducts of a recipient gilt. The recipient farrowed 3 healthy piglets 117 days after embryo transfer. The results indicate that pig embryos derived from in vitro fertilization of oocytes matured in vitro in medium with pFF fraction and cysteine can develop to term.

Full-term development of in vitro-matured, vitrified and fertilized bovine oocytes

In vitro-matured bovine oocytes were vitrified in a mixture of 2 M-dimethyl sulphoxide (DMSO), 1 M-acetamide and 3 M-propylene glycol dissolved in mTCM199. After vitrification and thawing, the oocytes were exposed to 2-0.1M-sucrose solution in 1 or 12 steps to remove the cryoprotectants. Then the oocytes were fertilized in vitro and co-cultured with a monolayer of cumulus cells for 7 days. Nine of 88 inseminated oocytes developed to the blastocyst stage. Three blastocysts were transferred to 3 recipients, resulting in 2 pregnancies.

Promoting Effect of β-Mercaptoethanol on In Vitro Development under Oxidative Stress and Cystine Uptake of Bovine Embryos

Abstract The effects of β-mercaptoethanol (β-ME) on in vitro development under oxidative stress and cystine uptake of bovine embryos were investigated. Bovine 1-cell embryos obtained by in vitro fertilization were cultured in TCM-199 or synthetic oviductal fluid (SOF) in 20% O2 supplemented with β-ME. Addition of β-ME significantly (P < 0.01) promoted embryo development when cultured in both TCM-199 and SOF under high levels of O2, to almost the same rates when they were cultured in 5% O2. To investigate whether the growth-promoting effect of β-ME was related to cystine uptake, which is an important amino acid for intracellular glutathione (GSH) synthesis, 1-cell, 8-cell, morula, and blastocyst stage embryos were incubated in cystine, cysteine-free TCM-199 containing radioisotope-labeled cystine supplemented with or without β-ME. It was found that cystine uptake was consistently low in each embryo stage incubated without β-ME. In contrast, addition of β-ME significantly (P < 0.05 to 0.0001) promoted cystine uptake in each stage of embryo development. This increase of cystine uptake by β-ME was significantly inhibited by supplementation of buthionine sulfoximine, a specific inhibitor of GSH biosynthesis (P < 0.0001). High-performance liquid chromatography (HPLC) analysis clearly revealed a decrease of cystine in culture medium after supplementation by β-ME, thereby forming another peak. HPLC analysis also showed the incorporated cystine by supplementation of β-ME was possibly metabolized for GSH synthesis in the embryos. These results indicate that β-ME has a protective effect in embryo development against oxidative stress and that the effect of β-ME is associated with the promotion of cystine uptake of low availability in embryos.

EFFECT OF FOLLICLE CELLS ON IN VITRO FERTILIZATION OF PIG FOLLICULAR OOCYTES

We investigated the effect of cumulus and granulosa cells (follicle cells) on in vitro fertilization of pig follicular oocytes matured in vitro. Oocytes surrounded by cumulus and connected with a piece of parietal granulosa cells (complexes) were matured in vitro for 46hours and were then divided into 4 groups: Group I oocytes were surrounded by expanded cumulus and granulosa cells; Group II oocytes were surrounded by expanded cumulus cells; Group III were denuded oocytes; and Group IV were denuded oocytes with cumulus cells from other complexes. After incubation for 4 hours and 40 minutes with frozen, thawed and preincubated pig epididymal spermatozoa, the oocytes were cultured for 5 hours and 20 minutes. When oocytes were inseminated in the presence of cumulus cells, the penetration rates were higher (92.5% for Group II and 89.5% for Group IV) than when cumulus cells were not used for insemination (Group III, 66.8%) or when oocytes with follicle cells were inseminated (Group I, 72.3%). Denudation of follicle cells before insemination (Group III) decreased the percentage of male pronuclear formation (50.8%) compared with that of oocytes surrounded by follicle cells (66.7% for Group II and 80.2% for Group I). These results support the ability of a moderate number of follicle cells to facilitate sperm penetration of pig follicular oocytes and male pronuclear formation.

Current status and perspectives in IVM-IVF of porcine oocytes

Abstract Boar spermatozoa have been shown to be capacitated without preincubation if the fertilization medium contains caffeine and a high concentration of Ca 2+ , but the incidence of polyspermy in IVM-IVF oocytes is still high (60%–100%). To prevent polyspermy, co-culture with oviductal cells, preincubation with porcine follicular fluid, or the addition of oligopeptides in the fertilization medium, have been tested with significant effects but still remarkably high rates of polyspermy (32%–58%). On the other hand, techniques for in-vitro maturation of porcine oocytes have progressed such that the problem of the low rate of pronucleus formation with in-vitro matured oocytes after IVF has been nearly solved. Irrespective of the collection method porcine follicular oocytes are morphologically selected based on the morphology of cytoplasm and cumulus cells and can be matured in vitro in a static system or in the non-static shaking culture system with addition of follicle cells and a high percentage of oocytes with both male and female pronuclei is observed. Beneficial effects of gonadotrophins, estradiol-17β, porcine follicular fluid and/or cysteine on maturation of oocytes have been reported. However, the number of piglets obtained from IVM-IVF oocytes is low. Possible experimental strategies to improve success rates of porcine IVM-IVF are discussed.

Forskolin stimulates porcine sperm capacitation by increasing calcium uptake

Using the fluorescent calcium indicator fura‐2, forskolin was found to dose‐dependently cause an immediate increase in the concentration of intracellular free calcium of porcine cauda epididymal sperm. This stimulatory effect of forskolin is due to the enhancement of Ca2+ uptake by the verapamil‐sensitive transporter on the sperm plasma membrane and results in the promotion of the sperm capacitation and subsequent acrosome reaction.

Effect of b-mercaptoethanol on the viability of IVM/IVF/IVC bovine embryos during long-distance transportation in plastic straws

Experiments were conducted to assess the effect of beta-mercaptoethanol (beta-ME) on the quality and viability of bovine blastocysts derived from in-vitro culture (IVC) of in-vitro matured and fertilized (TVM-IVF) oocytes during their transport between 2 distant places. Follicular oocytes were collected from ovaries obtained at a slaughterhouse and were cultured for 20 to 21 h in modified TCM-199. The IVM oocytes were fertilized in vitro with frozen-thawed spermatozoa. Fertilized oocytes were cultured for 7 d, and embryos that developed to the blastocyst stage were used for the experiments. The blastocysts, packed in straws with transportation medium that consisted of modified TCM-199 with HEPES equilibrated in air and supplemented with 20 % calf serum and 0, 10, 50, 100 or 150 microM beta-ME, were transported at 37 degrees C from Tokyo to Sapporo by air (18.3 h). The quality of blastocysts was assessed and ranked as excellent (A), good (B), fair (C) or poor (D) after transportation. The percentages of blastocysts ranked as A or B were significantly higher (P < 0.05) when the embryos were transported in beta-ME supplemented medium (80 to 100%) than when transported without beta-ME (54 %). Blastocysts ranked as A or B after transportation in medium with or without 150 microM beta-ME were nonsurgically transferred to synchronous recipients; 60 d after embryo transfer, 21/36 and 19/35 cows, respectively, were diagnosed as pregnant by palpation per rectum. These results indicate that beta-ME maintains the quality of bovine blastocysts in plastic straws for several hours without control of CO2 and that the concentration of beta-ME used in this experiment is not detrimental to the blastocysts.

Changes in the nature of calcium transport systems on the porcine sperm plasma membrane during epididymal maturation

Comparative studies of 45Ca(2+)-transport across the plasma membrane were performed using porcine caput, corpus and cauda epididymal sperm. The Ca(2+)-uptake is dependent on the presence of the substrates for respiration and is sensitive to verapamil. The Ca(2+)-efflux is mediated by both Na(+)-dependent and -independent systems. In the immature sperm in caput epididymis, Na(+)-independent efflux is predominant, but it is gradually replaced by Na(+)-dependent efflux during the epididymal transit. The net activity of Ca2+ accumulation into sperm increases with the epididymal maturation.

Native zona pellucida structure is required for completion of sperm acrosome reaction in porcine fertilization

In fertilization in vitro, the penetration rate of zona-intact porcine oocytes by cryopreserved epididymal spermatozoa was about 100% while that of zona-free oocytes was only 30%. Spermatozoa treated with calcium ionophore A23187 penetrated both zona-intact and zona-free oocytes at the rate of more than 90%. Treatment of spermatozoa with solubilized procine zonae pellucidae hardly induced acrosome reaction and did not increase the penetration rate. These results suggest that the structure of the zona is necessary for completion of acrosome reaction.

Forskolin promotes sperm penetration of pig oocytes in vitro without increasing cyclic AMP levels in sperm

Abstract Experiments were conducted to assess the effects of forskolin on the ability of cryopreserved boar epididymal spermatozoa to penetrate in-vitro matured oocytes and on the levels of cyclic AMP in the spermatozoa. When preincubated spermatozoa were introduced into medium containing forskolin (0, 0.5, 5 and 50 μM) and in-vitro matured oocytes, the penetration rates were found to increase with increase in the concentration of forskolin. However, when spermatozoa were incubated in forskolin-containing (50 μM) medium for 2 h before insemination, only 8% of oocytes in forskolin-free medium were penetrated by the spermatozoa, whereas similarly preincubated spermatozoa penetrated 43% of oocytes in forskolin-containing medium. When oocytes were incubated with spermatozoa for 1, 2 and 3 h in forskolin-containing (50 μM) medium, and then either freed from the spermatozoa by pipetting (− sperm) or washed gently to avoid removal of attaching spermatozoa (+sperm), and finally transferred to forskolin-free medium, their penetration rates increased with increasing duration of incubation with sperm (−sperm: 0%, 5% and 37% and +sperm: 21%, 61% and 82%, respectively). When matured oocytes were further incubated for 3 h with forskolin and then inseminated with spermatozoa in the medium with or without forskolin, only 14% of oocytes were penetrated in the absence of forskolin. The addition of forskolin (50 μM) to the fertilization medium had no effect on the levels of cyclic AMP in sperm. These results indicate that during 2–3 h of incubation with oocytes in forskolin-containing medium, the boar spermatozoa gain considerably in fertilizing ability without a concomitant rise in their cyclic AMP levels.