The markers that have been frequently used in many researches are basically based on their enzymatic activities. Therefore, prior to the detection of the markers, we needed to add substrate to the system. In case of the observation of thin section of the organs, the addition of the substrate will not be a big obstacle in the research. In order to detect the expression of the reporter gene, newly designed substrates were developed and available in the market to allow us to stain the live cells or organs. However, the loading the substrate into cell makes it a difficult procedure to stain the live cells by these enzyme markers. A green fluorescent protein (GFP) was reported as a new type of marker especially in marking the live cells by Chalfie in 1994. The GFP emits the green fluorescent light in response to ex citation light without any pretreatment. This nature will enable us to observe the marked object in real time and in continuous manner. From this idealistic nature of GFP, many researchers are now trying to use the GFP in versatile systems as a vital marker. In the present review, we introduce the basics of GFP and our examples of using GFP in transgenic mouse lines together with a newly discovered developing usage of GFP.

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Using a progesterone agent [Controlled Internal Drug Releasing-Bovine (CIDR-B)] made of a silicone resin containing 1.9 g of natural progesterone that is inserted into the vagina, continuous superovulation (I course) treatment was intentionally performed twice at an interval of 35 days in Japanese black multiparous cows regardless of the estrous cycle. It was then investigated whether this continuous treatment could be further repeated twice at an interval of 42 days (total of 3 repetitions=approximately 1 year). The test animals were 9 Japanese Black cows. CIDR-B was inserted into the vagina regardless of the estrous cycle and superovulation treatment was started after Day 10. The method of treatment consisted of either a decreasing dose administration over a period of 3 days of FSH (total amount=20 AU), or simultaneous and separate subcutaneous administration of FSH 25 AU dissolved in 30% PVP (polyvinyl pyrodine) and FSH 5 AU in physiological saline, in the neck. Ovum collection was non-surgical methods on Day 7. Estrous was confirmed in all the animals (total of 54) that were treated and ovum collection was performed. The results of the superovulation treatment were as follows: mean estimated number of luteal bodies,10.5 ± 0.8 (S.E); mean number of residual ovarian follicles, 9.3 ± 0.9; mean number of normal embryos, 6.7 ± 0.3; and mean percentage of normal embryos, 71.7%. In addition, the results by course (I, II, III course), with 18 animals in each course, were as follows: mean estimated number of luteal bodies, 9.2, 9.8, and 12.5, respectively; mean number of recovered ovaries, 8.2, 8.7, and 11.0, respectively; and mean number of normal embryos, 6.0, 5.8, and 8.2, respectively. There were no statistically significant differences in the results and they were similar to those obtained using the conventional FSH decreasing dose administration method. From the above results, it was evident that continuous superovulation can be done intentionally over 1 year in cows using CIDR-B, regardless of the estrous cycle.

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In Experimental 1, when in vitro matured (IVM) bovine oocytes were inseminated with frozen-thawed spermatozoa in in-vitro fertilization (IVF) medium (BO solution containing 5 μg/ml heparin) (Control) and IVF medium supplemented with 5 mM caffeine (Caf) or 10 mM hypotaurine (HT) for 1, 3 and 5 h, significantly higher percentages of oocytes were penetrated in Caf and HT groups than Control group after 3 h of insemination. Then the rates showed no difference between groups after 5 h of insemination. However, a significantly lower percentage of monospermic oocytes was obtained in Caf group than Control and HT groups. In Experimental 2, IVM oocytes were inseminated with spermatozoa in IVF medium supplememted with 0, 1, 5 and 10 mM HT, and then co-cultured with cumulus cells in TCM 199 with 5% calf serum. There was no difference in the ability of spermatozoa to penetrate oocytes between groups but significantly higher rates of fusion of male and female pro nuclei in monospermic oocytes were obtained in the medium supplemented with 10 mM HT than no addition.The addition of HT in IVF medium significantly improved percentages of embryos developed to the stage of blastocyst. In Experimental 3, IVM oocytes were inseminated in IVF medium supplemented with 10 mM HT or 5 mM Caf and then co-cultured as described in Exp.2. significantly higher percentages of monospermic oocytes and embryos developed to the stage of blastocyst were obtained with HT groups than Caf group. In monospermic oocytes significantly higher rates of fusion of male and female pro nuclei were obtained in HT group than Caf group. These results indicate that addition of HT in the ferti lization medium promotes monospermic fertilization compared with Caf and shortens the duration of insemination needed for spermatozoa to penetrate oocytes resulting in higher percentages of IVM/IVF oocytes develop to the stage of blastocyst than those of no addition.

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Nulliparous Holstein heifers were administered 20 mg of xylazine for tranquilization before embryo transfer (ET). A frozen-thawed compacted morula embryo from Japanese Black cows was transferred to each heifer, and pregnancy rates were investigated. Division 1 recipients were kept in stanchion stalls, while Division 2 recipients were kept in treatment stalls. The pregnancy rate in the group to which xylazine was administered before transfer (xylazine administration group) tended to be higher than in the control group to which embryos were transferred without xylazine administration in both Divisions 1 and 2 (55.0% vs 48.6% and 50.0% vs 46.2%, respectively). The proportion of Division 1 and 2 recipients requiring 5 or more minutes for ET completion was lower for xylazine treated heifers than for control heifers (10.0% vs 19.0%, respectively). The proportion of Division 1 and 2 recipients leaving blood adhered to the tip of the ET gun was lower for xylazine treated heifers than for control heifers (12.8% vs 22.2%, respectively). These results suggest that administration of xylazine during embryo transfer is effective for improvement in pregnancy rate when nulliparous Holstein heifer recipients are kept in stanchion stalls before transfer.

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In the present study, the pluripotency of embryonic cells on the germ line, especially fetal germ cells, was examined. The results obtained are as follows; when donor cells on the germ line were aggregated with precompacted stage embryos or injected into blastocysts, fertile chimeric mice or fetuses were obtained from 3.5, 4.5 and 5.5 days cells and ES-cells, respectively. However, further stage cells never contributed to the conceptuses even though analyzed at 10.5 days of pregnancy. When fetal germ cells on days 14.5-16.5 were reprogrammed by fusion with enucleated oocytes and then treated with fertilized embryos, they can form chimeric fetuses and extraembryonic tissues. It was suggested that differentiated cells on the germ line such as fetal germ cells can also develop to chimeric fetuses after reprogramming. However, it was also clear that they died at the midgestation probably due to the inappropriate imprinting.

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The present study was carried out to clarify the morphological and functional characteristics of the corpus luteum during the estrous cycle in cattle. The major findings are summarized as follows: 1) Morphological and endocrinological characteristics of corpora lutea corresponded to each other in the different luteal stages. During the endocrinologically active stage, the corpus luteum consists mainly of luteal tissue, in which active luteal cell types I, II and III dominate. Total progesterone concentration of the corpus luteum correlates with the appearance rate of the luteal cell type I, II and III (r=0.69, r=0.85, r=0.67, respectively). 2) A transrectal real-time B mode linear scanner was used to deterime whether the appearance of a cavity in the corpus luteum in cows causes in fertility. The mean period for return to estrus, mean serum progesterone concentration and mean pregnancy rate were not significantly different for cows that had a corpus luteum with or without a cavity. These results suggest that the cavity does not cause in fertility. 3) Specific receptors for oxytocin (OT) on intact luteal cells are demonstrated. Significant binding of OT was observed at all luteal stages. The receptors were localized on both large and small luteal cells by immunohistochemistry. OT binding was maximal at the mid-luteal stage. Furthermore, the specific binding in cultured bovine luteal cells increased dose-dependently with PGF_{2 α}. When PGF_2α synthesis in the cultured cells was inhibited by indomethacin, the specific binding of OT decreased significantly. The overall results indicate that a direct action of OT on the bovine corpus luteum is mediated by the OT receptor in a paracrine and autocrine fashion. In addition, the results suggest that PGF_2α may be one of the potent regulators for luteal OT receptors in a paracrine and/or autocrine manner.

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It is well documented that the corpus luteum is required for a successful pregnancy. The primary function of the corpus luteum is to secrete progesterone, which has several biological effects on target tissues in the reproductive system to prepare them for support of pregnancy or to provide nourishment to the conceptus. In most domestic species including cattle, three organ systems regulate the function of the corpus luteum. The anterior pituitary gland secretes LH, which is the primary hormone responsible for stimulating the secretion of progesterone. The uterus has a luteolytic effect during the late luteal phase of the estrous cycle. This luteolytic effect of the uterus is mediated via secretion of prostaglandin F_2α. Finally, the conceptus has either direct luteotropic or antiluteolytic effects. However, there is increasing evidence for the existence of substances in luteal tissue which are able to act locally, either alone or by modulating the action of the gonadotropins, thus modifying the functions of corpus luteum. The intra-luteal regulators consist of peptides, eicosanoids, growth factors, cytokines, and neurotransmitters. Available data suggest an important role for these substances in the control of luteal function during not only pregnancy but the estrous cycle as well.

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The corpus luteum (CL) of the estrous cycle in the cow is a dynamic organ with a lifetime of approximately 17-18 days. In other words, the CL regresses within a few days when pregnancy does not occur successfully. There is universal agreement that prostaglandin F_2α (PGF_{2 α}) is a physiological luteolysin in the cow. Nevertheless, cellular and molecular mechanisms involved in local actions of PGF_2α in the CL have not been fully elucidated. Recent observations suggest that the endothelial cell-derived vasoconstrictive peptide endothelin-l (ET-l) interacts with PGF_2α, and that luteal ET-l participates in the rapid cascade of functional luteolysis in vivo. In the bovine CL, ET-l inhibits P secretion in luteal cells in culture and in the in vitro microdialysis system (MDS) of luteal explants. Moreover, PGF_2α stimulates ET-l secretion in this MDS. Further in vivo observation confirmed that PGF_2α injection rapidly increased ET-l release within the regressing CL as well as into the ovarian venous blood in the cow. All these findings strongly suggest a physiological impact of PGF_2α and ET-l in the acute depletion of blood flow into the CL and the rapid cascade of functional luteolysis in vivo, and thus a possible interaction between endothelial cells and luteal cells during luteolysis. Overall results strongly support the hypothesis that luteal ET-l is a local luteolytic mediator/promotor in the regressing bovine CL.

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Pig is a polytocous animal and average number of newborn piglets is around ten. In pig, it is a interesting point that how many number of littermate embryos (conceptuses) in the early pregnant uterus is needed for the establishment of pregnancy. Frontier research showed that four embryos are necessary for establishment of pregnancy in pig. On the contrary, embryo transfer study provided that one embryo can not maintain a pregnancy, however two embryos establish it, and more than two embryos in the uterus improve a pregnancy rate. These facts support the evidence that plural growing embryos in the reproductive tracts of early pregnant pig synthesize some signal substances above the threshold level for the recognition of pregnancy to their maternal system. In pig, the most important substance from conceptus for the recognition of pregnancy is estradiol-17 β (E₂). When conceptus starts an elongation in the uterus at during week 2 of pregnancy, concomitant E₂ synthesized and secreted from trophoblast is a first signal to their mother for a recognition of pregnancy. This signal from conceptus is necessary for maintenance of corpora lutea during week 2 of pregnancy, and conceptus has to spread over both uterine lumens to play an effective role for maintenance of corpora lutea. In pig, as same as other domestic animals, prostaglandin (PG) F_2α causes a luteolysis at the estrous cycle. An exocrine direction of synthesized and secreted PGF_2α in the endometrium is critical at the time of recognition of pregnancy. Conversely, PGE₂ probably plays a luteotropic role for corpora lutea. The recognition and establishment of pregnancy is the results of many successive reactions between sow and conceptuses in the maternal system. What kinds of substances and mechanisms are related to the recognition of pregnancy in pig, is not completely resolved. The relationship between conceptus and their maternal system, and signals for maintenance of corpora lutea may have still unknown parts. Therefore, in this article, following subjects are discussed, 1) E₂ synthesis by conceptus, 2) Retinal binding protein synthesis by conceptus and uterine endometrium, 3) Secretion and its direction of PGE₂ and PGF_2α from endometrium, 4) proteins synthesized by conceptus.

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[in Japanese]

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Unique expression of HLA class I antigen on human trophoblast might be one of the mechanisms by which the fetus escapes recognition by maternal immune system. The regulation of classical class I, HLA-A, gene and nonclassical class I, HLA-G, gene in cultured trophoblastic cell lines was investigated by nuclear proteins binding to HLA class I regulatory elements using gel mobility shift assay. The results indicated that nuclear protein binding to the class I regulatory element (CRE) was detected in the trophoblastic cell lines expressed classical HLA class I. In the cell lines expressed HLA-G, nuclear protein bound to the 5' upstream region (–190 bp- –120 bp).

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Once fertilized, the embryos begin to develop by undergoing a series of mitotic divisions before differentiating into specific cells, resulting in the organization of tissues and organs. This developmental programme ensures the successful transition from oocyte to offspring. In general, once the developmental program of embryos is started, it cannot be reversed. After the first one or two cleavage divisions, individual cells of embryos cannot form a new individual presumably because changes have taken place in the DNA that alter gene expression during development. However, using nuclear transfer techniques to expose the chromatin (nuclei) to ooplasm, it is possible to reprogram the DNA. These techniques provide a method of embryo cloning since the nuclei of early (and perhaps advanced) stage embryos can be transferred into the ooplast to produce several new individuals of identical genotype. When we reconstitute eggs by nuclear transfer the combination of cell cycles between the donor nuclei and the recipient cytoplasm should be synchronized for successful development, which requires both reconstitution of eggs containing diploid chromosomes and the nuclear reprogramming. A new approach to synchronize the donor nuclei at M phase of the cell cycle appears to be very successful, perhaps owing to the precision of synchrony, and has led to the procedure of identical sextuplet mice from the donor four-cell stage embryo. The molecular mechanisms involved in reprogramming donor nuclei, however, remain unknown. The principles of nuclear transfer are outlined with respect to nuclear remodelling, nucleocytoplasmic interactions and cell cycle effects.

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The present study was carried out to investigate the effects of different donor cell types (in vivo-fresh, cloning, recloning, in vivo-frozen or in vitro embryos by ultrasound guided transvaginal aspiration) on development into compacted morulae and pregnancy rates of reconstituted embryos by nuclear transfer. The fusion rate of in vivo frozen donor cells and resipient oocytes (82%) was significantly (P<0.05) lower than in the other donor cells (92-96%). The mean number of compacted morulae dirived from a in vivo-fresh embryo (9.0) was significantly (P<0.05) higher than in the other donor cells. Two of 13 recipient that received the reconstituted embryos dirived from recloning donor cells. However, this pregnancy rate (15%) was significantly (p-0.05) lower than those of the other groups (50-56%).

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Animal cloning from embryo-derived cultured cell line by nuclear transfer would be of considerable value for mass production and transgenic technology in domestic animals. Two kind of embryo-derived stem cell lines, embryonic stem (ES) cells and embryonic germ (EG) cells, are established in the mouse and have been extensively used for functional genetic analysis by gene targeting approach. The application of these cell lines by nuclear transfer and transgenic technology to genetic modification in domestic species has been hampered due to the unavailability of cell lines with pluripotent characteristics. In contrast to these undifferentiated cell lines, it has been recently reported that the differentiated cell lines from embryo disc or fetus in sheep can be successfully contributed to full term development of embryo by nuclear transfer. This provides us an alternative method for the efficient production of transgenic animal in domestic species, in place of microinjection of gene into fertilized eggs. However this method can not overcome the limitation of genetic control of transgene due to undesirable gene insertion into chromosome. In view of this point, the establishment of embryo-derived stem cell line and their use in the generation of transgenic animal seem to have considerable significant for precise control of transgene expression. In this review, recent progress of research on establishment of pluripotential stem cell lines and their in vivo developmental capacity in domestic species is discussed.

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[in Japanese]

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