More than half of unfertilized oocytes after ICSI failed to activate in human subjects. We investigated whether the fertilization rate after ICSI can be increased by electrical stimulation. At first, the effects of electrical stimulation on the parthenogenesis of hamster oocytes were investigated. Delivering a single 375 V/cm DC pulse for 100 μsec was effective in inducing parthenogenesis. The pattern of Ca²⁺ transient increase in hamster oocytes induced by electrical stimulation was recorded with Indo-1/AM. It seemed that a single sharp increase in Ca²⁺ initiated parthenogenetic activation. Secondly, a 375 V/cm pulse was applied to human oocytes after ICSI. In the case of immotile sperm, the fertilization rate and cleavage rate were increased significantly by electrical stimulation. On the other hand, in the case of motile sperm, no increase in the fertilization rate was observed. This study demonstrated that electrical stimulation combined with ICSI may be expected to contribute to increasing the fertilization rate in the case of immotile sperm.

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The objective of this experiment was to compare the effectiveness of two bulls and two different semen treatments on rates of in-vitro fertilization of in-vitro matured bovine oocytes, cleavage and development of embryos to the stage of morula and blastocyst by using a 2 × 2 factorial design. Oocytes (n=946) aspirated from follicles 2-6 mm in diameter were incubated for 20-22 h in TCM-199 supplemented with 5% calf serum, and then inseminated with frozen-thawed semen from two single ejaculates of two Holstein bulls of proven fertility. For the conventional washing method (C), semen was washed twice by centrifugation at 500 g for 5 min with modified BO medium (BO: without glucose or bovine serum albumin (BSA)) supplemented with 10 mM caffeine and 2.5 IU/ml heparin (BO-wash), and then diluted with the same amount of BO containing 20 mg/ml BSA (BO-dilute). For the Percoll washing method (P) semen was layered on a 45 to 90% percoll gradient in a 15 ml centrifugation tube. After 30 min of centrifugation at 700 g, the sperm pellet was recovered from the bottom of the tube, and then resuspended in BO-wash and double diluted with BO-dilute. With this sperm suspension insemination drops (100 μl) were prepared and the sperm was cultured with oocytes for 5 h. Then the oocytes were transferred to CR1aa supplemented with 5% calf serum for 7 days. The rates of cleavage and morula or blastocyst development of oocytes fertilized in vitro were similar in P (14.2% and 7.9%, respectively) and C (17.7% and 7.7%, respectively) methods for bull A, but these rates were significantly higher (P<0.005) with C (72.0% and 46.9%, respectively) method than with P (58.7% and 28.2%, respectively) method for bull B. In total rates of fertilization, cleavage and morula or blastocyst (combined data for C and P methods) were significantly higher (P<0.05) for bull B (83.8%, 65.8% and 33.9%, respectively) than those for bull A (42.4%, 16.0% and 7.9%, respectively). These results indicate that the Percoll washing method is not superior to the conventional washing method for in-vitro production of bovine embryos independ-ently of the significant differences between bulls.

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This study was conducted to evaluate the effect of supplementation of human or murine leukemia inhibitory factor (hLIF or mLIF) on in vitro maturation medium (TCM-199) containing FCS or BSA, and mLIF on semi-defined maturation medium containing BSA with or without hormones and in two different culture systems (single or group) on in vitro maturation, fertilization and development of bovine oocytes. Immature bovine oocytes were matured in vitro in TCM-199 supplemented with hLIF or mLIF (0 or 1,000 U/ml) containing FCS or BSA (Experiment 1), and mLIF as a supplement (0 or 1,000 U/ml) in TCM-199 containing BSA with or without hormones (Experiment 2) for 24 h. In Experiment 1, hLIF and mLIF added to a maturation medium have no positive effect on maturation, fertilization or development rates. In Experiment 2, the proportion of oocytes matured in TCM-199 with hormones showed significantly higher normal and total fertilization rates (P<0.01) and cleavage rates (P<0.05) than without hormones. The proportion of oocytes matured in a group showed significantly (P<0.01) lower normal and total fertilization rates, and higher polyspermy rates than those matured singly. There was no positive effect of mLIF added to the maturation medium on in vitro maturation, fertilization or development of bovine oocytes. These results indicate that there was no stimulating effect of hLIF or mLIF added to in vitro maturation medium on IVM, IVF or development of bovine oocytes.

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In order to establish a strategy to construct a DNA library from a specific region of a chromosome, bovine Y-chromosome DNA was amplified by the microdissection of whole chromosomes and polymerase chain reaction (PCR). Intact Y-chromosomes were microdissected from metaphase lymphocyte chromosomes and were digested with Proteinase K, followed by DNA extraction with phenol/chloroform. Purified DNA was cleaved with the restriction enzyme Sau3AI and the fragments were ligated to a synthetic linker-primer into which EcoRI site composite was introduced. The resultant Y-chromosome nucleotide sequences were amplified by PCR and cloned with vector DNA (EcoRI-digested pUC19 DNA). Sau3AI-digested pUC19 DNA served as a positive control and could be amplified by PCR using at least 0.2 pg of DNA. The Y-chromosome was successfully microdissected under an inverted microscope (400 ×), DNA was amplified by the PCR method and a smear of DNA (0.15-1.0 kb) was obtained. Two of sixty-five clones (0.05-1.2 kb) were male-specific by dot hybridization to male and female genomic DNA but no clone was found to be Y-chromosome specific by Southern hybridization.

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To evaluate the function of protein kinase C (PKC) during oocyte activation following fertilization, the effects of activators and inhibitors of PKC on the second polar body emission, pronuclear formation and protein phosphorylation of mouse oocytes were investigated during in vitro fertilization and during artificial oocyte activation with Ca ionophore (A23187). One ng/ml of 12-O-tetradecanoyl phorbol 13-acetate (TPA), a PKC activator, accelerated PF without accelerating the second polar body emission. 5-20 ng/ml of TPA suppressed the second polar body emission significantly but did not inhibit female chromosome separation of second meiotic division. Staurosporine, a PKC inhibitor, at 1-5 nM, suppressed the second polar body emission. Female chromosome separation was also suppressed by staurosporine and H7, an another PKC inhibitor. One diploid female pronucleus was formed in an egg or an oocyte without the second polar body emission. TPA itself caused female chromosome separation even in the presence of 20 μM of BAPTA-AM, a selective membrane-permeable calcium chelator. These results show that PKC has supportive effect on pronuclear formation. The activation of PKC is indispensable for female chromosome separation during fertilization.

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The present series of experiments were conducted to examine the effect of Japanese Kampo Medicines (JKMs), namely Tokishakuyakusan (TJ23), Keishibukuryogan (TJ25), Shakuyakukansouto (TJ68) and Unkeito (TJ106) on the physiological function of frozen-thawed bovine spermatozoa in in vitro fertilization. In the first experiment (experiment 1), the most effective JKM was determined by adding JKMs to the BSA-free in vitro fertilization medium (m-Hepes-BO) and examining the developmental capacity of oocytes after in vitro fertilization. In the second experiment (experiment 2), a comparison of the effect of the most effective JKM on in vitro fertilization with that of BSA was conducted in the same manner as in experiment 1. The result of experiment 1 showed that Unkeito (TJ106) was the most effective JKM when added to BSA-free m-Hepes-BO medium, although very few blastocysts were obtained. Furthermore, recording the survivability of the frozen-thawed spermatozoaincubated in fertilization medium supplemented with TJ106 and/or BSA showed that the survivability of the spermatozoa was improved by TJ106. In experiment 2, when TJ106 was added to m-Hepes-BO medium together with BSA, the rates of morulae and blastocysts were better than in the case of TJ106 alone. Moreover, the morphological characteristics of oocytes after 6 h of insemination in fertilization medium with BSA and/or TJ106 as the supplements, also indicated that BSA was indispensable in in vitro fertilization of bovine oocytes, althought the physiological functions of the spermatozoa were somewhat improved by JKM in the present work.

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Changes in the distribution of endoplasmic reticulum (ER) in porcine oocytes during meiotic maturation were examined. In the Germinal Vesicle (GV) stage oocyte, there were many bright clusters of ER present in the peripheral cytoplasm, and some of them formed a mass. This mass was absent in the Prometaphase I (Pro-MI) and Metaphase I (MI) stage oocytes, and the clusters became fewer than in the GV stage oocyte. ER accumulations that appear to be generated by scattering of the cluster or the mass were uniformly distributed in the peripheral cytoplasm and formed a thin layer beneath the plasma membrane. In the Metaphase II (MII) stage oocyte, the layer became more evident and wider, and a space with a relatively sparser distribution of ER was observed beneath the layer. From these results, it is clear that the distribution in the ER of porcine oocytes is changing during miotic maturation.

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Progesterone plays an important role in sperm capacitation and acrosome reaction. In this experiment we examined the effect of a progesterone antagonist, RU486, on the penetration of zona-free hamster eggs by human sperm. Media containing 10, 15, 20, 25 or 30 μM RU486 were prepared. In the control medium without RU486, the sperm penetration rate was 80.7%. In the medium with 10 μM RU486, the sperm penetration rate was 67.0%. The rate further decreased as the level of RU486 increased. In the medium with 30 μM RU486, the sperm penetration rate was reduced to 4.8%. This study demonstrates that RU486 inhibits penetration of zona-free hamster eggs by human sperm in a dose-dependent manner.

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Oocyte-cumulus cells connected to some of the parietal granulosa cells (OCG complexes) were mechanically obtained from pig early antral follicles 0.5-0.7 mm in diameter and were cultured for 8 days in collagen gels. The effects of FSH, dbcAMP, estradiol-17β (E₂) and EGF on antrum formation in the OCG complexes were studied. After 1 day of culture, every OCG complex formed a spherical compact structure. The OCG complexes which had undergone FSH and dbcAMP treatment then formed antra, resulting in antral follicle-like structures, but no antra were formed in the E₂, EGF-treated and control groups. Instead, in these groups the granulosa cells spread away from the oocytes. Proteins similar to those in pig follicular fluid were accumulated in the antral follicle-like structures of the OCG complexes induced by FSH and dbcAMP stimulation. These results indicate that mediated by cAMP rather than estrogen, FSH induces antrum formation in pig granulosa cells, and that EGF itself does not induce antrum formation in vitro.

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Hamster cumulus-oocyte complexes (COCs) were recovered by dissection of antral follicles of ovaries for the germinal vesicle (GV) or metaphase I (MI) stages, and by flushing the oviduct for the metaphase II (MII) stage. The surface morphological characteristics of the oocyte and its investments at each time point were evaluated by scanning electron microscopy (SEM). Oocyte diameter was also measured during the SEM analysis. The cumulus cells had a compact structure with minimal intercellular spaces among them in the GV-stage oocytes. They became greatly expanded with increased intercellular spaces at the MI and MII stages. The zona pellucida (ZP) had an open mesh-like structure, and the size and number of mesh holes increased at the MII stage. The vitelline surface of oocytes at the GV stage was characterized by relatively sparse distribution of microvilli (MV). After maturation, the MV increased in density. The diameter of oocytes decreased significantly during maturation from 82 to 77 μm (P<0.01). Thus the hamster oocyte maturation processes were found to involve the expansion of cumulus cells, an increase in the mesh holes of the ZP and development of MV on the vitelline membrane.

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Pig follicular oocytes with cumulus cells were cultured in different maturation media, modified tissue culture medium 199 (TCM-199B) and BSA-free Whitten's medium (mWM), containing gonadotropins (10 IU eCG/ml + 10 IU hCG/ml) and/or 10% (v/v) fetal calf serum (FCS) or 10% (v/v) pig follicular fluid (PFF). After 48 h of culture, complete (degree +3) cumulus expansion was observed in oocytes cultured in both kinds of media supplemented with gonadotropins together with FCS or PFF. However, when oocytes were cultured in media with gonadotropins only, moderate (degree +2) cumulus expansion was observed in TCM-199B, while no (degree 0) cumulus expansion was observed in mWM. The proportions of metaphase II (M-II) oocytes were significantly (P<0.01) higher in both media in the presence of gonadotropins (61-95%) than in their absence (22-35%), irrespective of FCS or PFF supplementation. The addition of FCS or PFF to the media containing gonadotropins increased the proportion of oocytes reaching M-II in mWM but not in TCM-199B. When gonadotropin-containing media were used, the rate of male pronuclear formation in oocytes fertilized in vitro was significantly higher (P<0.05) in TCM-199B with PFF (70%) than with FCS (30%) although the value in PFF did not differ statistically from that (50%) in unsupplemented medium. In mWM, FCS did not support penetration of oocytes by spermatozoa, while PFF supported high rates of penetration (72%) but not male pronuclear formation. These results indicate that gonadotropins and/or FCS and/or PFF affect nuclear and cytoplasmic maturation of pig oocytes in different ways.

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Freezing of epididymal spermatozoa from red-bellied tamarins (Saguinus labiatus) was achieved by using a semen-diluting medium called TTE (Tes, Tris, egg-yolk base) containing 5% glycerol. When the frozen spermatozoa were suspended and incubated in m-TYH medium (TYH medium supplemented with 1mM caffeine and 1mM dibutyryl cyclic AMP), the frozen-thawed spermatozoa showed a good sperm survival rate and hyperactive movement after 30 min of incubation. A total of 59 germinal vesicle (GV) stage oocytes were collected from 4 ovaries of 4 female red-bellied tamarins regardless of their menstrual cycles. At 24, 48, and 72 h after incubation in TCM-199 medium containing 10% fetal calf serum (FCS), 30%, 53%, and 71% oocytes, respectively, either showed germinal vesicle breakdown (GVBD) or had extruded the first polar body. Although the oocytes matured in vitro were inseminated by frozen-thawed spermatozoa, no eggs became fertilized.

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