The reporter gene encodes enzymes whose activities are visable by staining with a chromogenic substrate. The reporter gene is developmentally neutral as proved by the normal developmental potential of LacZ positive embryos. In order to clarify the early expression of the myogenin-LacZ gene during mouse embryogenesis, transgenic mice harboring the reporter gene linked to the myogenin flanking region have been developed. Stereoscopic visualization of LacZ expression of these transgenic mouse F1 embryos revealed that the LacZ gene expression in rostral somites appeared to correspond to myogenin which was first detected in mesodermal tissues at high levels at 8.0 days p.c. (post coitum), Which is the furthermore, LacZ expression in the somites and in the limb bud was observed at 10.5 days p.c. and 11.5 days p.c. respectively. At 12.5 days p.c., LacZ gene expression in cells started to migrate to the ventral region of the embryo, where eventually LacZ gene-expressed cells formed the abdominal musculature. This suggested that the role of the myogenin gene is crucial for muscle formation and for myogenic progenitors that migrate from the somite into the developing limb bud. The results also indicated that the myogenin gene is potentially involved in the early stages of muscle determination.

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Forty-two ewes were treated with intravaginal insertion of progesterone sponge and single injection of pregnant mare serum gonadotropin (PMSG) either in May (mid-anestrus; Group 1, n=14), in July (late-anestrus; Group 2, n=13) or between October and December (breeding season; Group 3, n=15). The sequence of estrus, LH surge and ovulation, thus induced in each group was compared with that in untreated cycling ewes during the breeding season (Group 4, n=10). Although intervals from the sponge removal to the onset of estrus (22.3-25.5 h) and to the LH peak (23.3-28.5 h) were not different among groups, the duration of estrus in Group 1 (19.3 ± 4.4 h) was shorter (P<0.05) than Groups 3 and 4. The intervals between the onset of estrus and the peak of cervical mucus secretion were also shorter in Groups 1 and 2 as compared with Group 4. The number of ewes ovulated during the period between 40-50 h after sponge removal was less (P<0.05) in Group 1 than Groups 2 and 3 . The conception rates were 62.5, 83.3, 100 and 100 % in Groups 1, 2, 3 and 4, respectively. The present results suggest that synchrony among timing of estrous behavior, LH surge and ovulation following the progesterone and PMSG treatments become less coordinated during anestrous season, which could be one of the primary factors responsible for low conception rate in these animals.

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Immature bovine oocytes aspirated from small follicles (2-5 mm) are not all developmentally competent following maturation and fertilization. It is suspected that the follicles provide the proper signal to the oocyte during late folliculogenesis. To assess the effects of follicular components, steroids, and gonadotropins on subsequent development, it is important to maintain the oocyte in a state of maximum receptivity, at the germinal vesicle (GV) stage. This study evaluates the inhibitory effects of co-culture with hemi-sections follicles on the meiotic resumption and subsequent development of cumulus-enclosed primary oocytes in vitro. One or 2 follicular hemi-sections prepared by careful dissection of follicles (2-5 mm) were co-cultured for 15 h with groups of 10 oocytes in the presence of FSH, estradiol or both. Oocytes were fixed after incubation for 15 h or incubated for an additional 24 h in a conventional maturation medium (FSH-LH and estradiol) or a medium containing progesterone and LH to stimulate meiotic resumption. Co-culture with 1 or 2 hemi-sections resulted in 37 to 76% GV rates. Inhibition of spontaneous oocyte maturation due to the co-culture was influenced by neither estradiol nor FSH, and 57 to 97% of oocytes reached the metaphase II stage in the subsequent 24 h culture, but at a lower rate with estradiol alone. Successful embryonic development of these oocytes after successive treatments of in vitro maturation (24 h), fertilization (10 h) and culturing with oviduct cells (96 h) indicates that viability is maintained after co-incubation with hemi-sections.

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The development of rat embryos after exposure to a medium that contained phosphate was examined. After exposure to a medium that contained phosphate, embryos were washed 10 times in watch glasses with 1 ml of modified HECM-1. Then they were rinsed 8 times with 100-μl droplets of the same medium in an attempt to bring the concentration of phosphate after washing below 1.0 fM. After exposure for 0, 10 and 20 min to Ca²⁺-Mg²⁺ and substrate-free Dulbecco's Phosphate Buffered Saline (D-PBS (-)), the development rates of 1-cell embryos to the blastocyst stage were 44%, 39% and 52%, respectively, and the mean numbers of blastomeres in the blastocysts were 20.0 17.7 and 19.4, respectively. After exposure for 0, 30 and 60 min to mHECM-1 supplemented with 1.19 mM KH₂PO₄, the development rates to the blastocyst stage from the 1-cell stage were 47%, 60% and 55%, respectively, and mean numbers of blastomeres in the blastocysts were 16.3, 16.6 and 15.5, respectively. By contrast, 93% to 100% of 2-cell embryos developed to the blastocyst stage. In each six experiments, the development of embryos after breif exposure to phosphate was not significantly different from the group witout exposure. When 2-cell embryos were exposed to phosphate for 15 h and 18 h, the rate of development to blastocysts decreased markedly to 60% and 22%, respectively. Embryonic development was completely inhibited by exposure to phosphate for more than 21 h.

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Oocyte maturation is triggered by the activation of maturation promoting factor (MPF). In activation of MPF during meiotic maturation of porcine oocytes, a possible involvement of phosphorylation and subsequent dephosphorylation of a inhibitory tyrosine residue in p34^cdc2 was examined using vanadate, a potent protein tyrosine phosphatase inhibitor. Porcine oocytes were cultured in porcine follicular fluid supplemented with pregnant mare's serum gonadotrophin with or without vanadate for 48 h. Vanadate blocked germinal vesicle breakdown (GVBD) almost completely at the concentration of 250 and 500 μM. The addition of an effective dose of vanadate into the maturation medium at 24 or 30 h after the start of maturation culture revealed that the porcine oocytes before GVBD were sensitive to vanadate and had their GVBD blocked, but those that had undergone GVBD were not-sensitive to vanadate and reached to the second metaphase normally. When vanadate was removed after 24 h treatment, GVBD occurred in 64% of oocytes but most of them were arrested at the first metaphase with dispersed chromosomes and abnormal spindles. These results suggest that tyrosine dephosphorylation in p34^cdc2 takes place during the first meiosis but not in the second meiosis and that a prolonged exposure to vanadate might deteriorate microtubules function of the oocytes.

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The aim of the present study was to examine the survival rate of embryos after bilateral nonsurgical transfer in recipient cows given a superovulatory treatment of low-dose FSH. In total, 54 parous Holstein or Japanese Black breed cows were used as recipients. These recipients were assigned to the three groups based on hormonal treatments during the preceding estrous cycle. The treatments included A) FSH (10 or 15 mg) and PGF_2α (n=18), B) PGF_2α (n=14), and C) no treatment (n=22). Morula to blastocyst-staged embryos that were transferred were derived either from in vitro maturation & in vitro fertilization or they were collected from superovulated Japanese Black heifers. Some embryos collected from donors were routinely frozen and thawed. Most of the cows (83%, 15/18) that were given the combination of FSH and PGF₂α formed corpora lutea (CLs) on the ovaries (mean number CLs ± s.e. per cow=3.1 ± 0.48). Pregnancy rates on day 30 in groups A, B and C were 67% (12/18), 64% (9/14) and 55% (12/22), respectively (P>0.05). The rates of twin pregnancies to all pregnancies were 50% (6/12), 22% (2/9) and 42% (5/12), respectively (P>0.05). Consequently, 19 cows carried their pregnancies to term, producing 25 calves, including six pairs of twins. Calving rates in the three groups were 67% (8/12), 33% (3/9), and 67% (8/12), respectively (P>0.05). Calf crop rates were 125% (10/8), 133% (4/3), and 138% (11/8), respectively (P>0.05). The embryonic survival rate at Day 30 in group A was likely to be higher than those of cows in the other two groups (50% [18/36] vs 39% [28/72]). No difference between the origins of embryos was observed in the embryonic survival rates. These results suggested that superovulatory treatment with low-dose FSH to recipients bilaterally receiving embryos would be beneficial for increasing embryonic survival, although the results were not statistically significant.

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Changes in MMP-11 (stromelysin-3) and TIMP-1 gene expression in the uterus and the placenta of the mouse during different reproductive stages were examined by quantitative RT-PCR. MMP-11 expression rose steeply from baseline levels in early estrus, and reached a peak at estrus, and then declined rapidly on diestrus. During pregnancy, high levels of MMP-11 mRNA were detected in the uterus on Days 1 and 19 of gestation, while it was minimal on Day 7. A reciprocal relationship was found between MMP-11 and TIMP-1 expression both in the uterus and the placenta. These observations suggest a role for MMP-11 in the remodeling of uterine extracellular matrix during the estrous cycle and the pregnancy.

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This study was designed to determine whether preimplantation mouse embryos carrying the MT-neo/MT-β-gal transgene, which contains a neomycin resistance gene (neo) and E. coli β-galactosidase (β-gal) gene, can be selected by incubation in the presence of G-418 prior to transfer to recipients. Mouse 1-cell eggs were injected with MT-neo/MT-β-gal DNA, allowed to develop to the 4-cell stage and then treated for 3 days with 200 μg/ml of G-418, a concentration known to completely inhibit development of normal embryos in vitro. Twenty-eight (15.1%) of 186 4-cell embryos that had been microinjected developed to the blastocyst stage after culture with G-418. Histochemical staining of the resulting blastocysts for β-gal activity revealed that 28.6% (8 of 28) of G-418-resistant embryos were positive for β-gal activity. Of 158 control 4-cell eggs that had been injected with MT-neo/MT-β-gal DNA but not treated with G-418, 55 (34.8%) developed to the blastocyst stage. Four (7.3%) of these 55 blastocysts expressed the β-gal gene. These findings suggest that mouse preimplantation embryos with exogenous DNA after injection can be concentrated approximately 4-fold after selection in the presence of G-418.

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It is known that a four-day estrous cycle is frequently shortened to 3 days in IV CS female mice in the presence of males. This phenomenon is generally attributed to functions of the female's olfactory system. This olfactory system consists of two systems: 1) the olfactory epithelium-main olfactory bulb (main olfactory) system and 2) the vomeronasal organ-accessory olfactory bulb (accessory olfactory) system. In this study we examined the effects of the removal of the vomeronasal organ on the shortening of the estrous cycle. Eight of 18 sham operated females underwent a 3-day estrous cycle (44.4%), while all of 15 females with their vomeronasal organs excised underwent a regular 4-day estrous cycle. These results suggest that the accessory olfactory system plays an important role in the shortening of the estrous cycle.

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Ultrasound-guided transvaginal follicular oocyte aspiration combined with in vitro maturation/in vitro fertilization/in vitro culture (IVM/IVF/IVC) was used to obtain bovine embryos. The effects of aspiration frequency and FSH stimulation on follicular development, oocyte recovery and embryo development following IVM/IVF/IVC were evaluated. In Experiment 1, transvaginal follicular aspiration was performed once or twice weekly with or without FSH stimulation in the same cows (2 cows). During the 1st session (5 aspirations/2.5 weeks), follicular aspiration was performed twice a week following the administration of FSH for 2 days (a total of 18 mg FSH). During the 2nd session (5 aspirations/2.5 weeks), follicular aspiration was performed twice a week without FSH treatment. During the 3rd session (5 aspirations/5 weeks), follicular aspiration was performed once a week without FSH treatment. During the 4th session (5 aspirations/5 weeks), follicular aspiration was performed once a week following the administration of FSH for 2 days. The follicular fluid containing aspirated oocytes was kept warm (36 C) in the 2nd, 3rd and 4th sessions but it was kept at air temperature (2-8 C, winter) in the 1st session. There were no significant differences in the mean number of follicles ≥ 2 mm in diameter during the 4 treatment sessions. The mean number of oocytes collected from each cow remained constant during the 4 treatment sessions except in the 1st session with one cow. In vitro development of oocytes collected from each cow showed similar rates of development to the morula/blastocyst stages among the last 3 treatment sessions. The lower development rate of oocytes after IVM/IVF/IVC in the 1st session may be due to the cooling of oocytes after aspiration from the ovarian follicles. This was confirmed in Experiment 2 showing that cooling (5 C, 2 h) of oocytes before IVM decreased significantly (P<0.05) the proportions of embryos that cleaved and developed to transferable stage after IVM/IVF/IVC.

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Changes in plasma concentrations of immunoreactive (ir-) inhibin, steroid hormones and gonadotropins were determined in relation to follicular growth throughout the bovine estrous cycle. Three waves of follicular development occurred; the first, second and third waves were observed during the early-luteal, mid-luteal and follicular phases, respectively. A dominant follicle was identified in each wave. During the growth of the dominant follicle, the other follicles ceased their growth, while the emergence of follicular wave was observed after the dominant follicle ceased to grow or ovulated. Plasma concentrations of ir-inhibin increased (p<0.05), concomitantly with the emergence of each wave. A high concentration of ir-inhibin was noted in the growing phase of each dominant follicle. Concentrations of estradiol in the plasma increased (p<0.01) coincidentally with the growth of the first and third dominant follicles. However, during the second wave, plasma estradiol levels did not show any significant rise, suggesting that the second dominant follicle has a very low activity to produce estradiol. Progesterone levels declined (p<0.01) sharply due to the spontaneous luteolysis and increased again during the early luteal phase, reached the high levels during the mid-luteal phase. Concentrations of plasma FSH were high before the emergence of each follicular wave and began to decrease in accordance with the emergence of the wave, then remained low until the dominant follicle regressed or ovulated. These results suggest that the dominant follicle, during its growing phase, lowers the plasma FSH concentration by enhancing the secretion of inhibin alone and or estradiol, and suppresses the recruitment and growth of the other follicles. In addition, progesterone also probably affects FSH secretion during the early-luteal phase in combination with estradiol.

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To characterize the local release of progesterone (P) and oxytocin (OT) within the corpus luteum (CL) formed after superovulation during the non-breeding season in ewes, a microdialysis system (MDS) was implanted in corpora lutea (CcL). Superovulation was induced with a combination of PMSG, FSH and GnRH injections in ewes (n=5) that had been pretreated with P using an intravaginal sponge. On Day 5 after GnRH injection, the microdialysis capillary membrane (cut off Mr=1000 kDa) was surgically implanted into the individual CL of both ovaries, and continuously perfused with a Ringer's solution up to Day 17. Serial fractions were collected every 30 min on Days 6-7, Days 9-10, Days 12-13 and Day 15, to observe in detail the release patterns of P and OT at different ages of the CL. Both P and OT were released into the MDS in a pulsatile manner throughout the experiment. The frequency of P pulses changed by the age of the CL (P<0.05); it decreased temporally on Days 12-13, but gradually increased on Day 15. The percentages of concomitant P release (coincident rate of pulses) decreased on Days 12-13, but extremely increased to 52.3% on Day 15 (P<0.01). On the other hand, the profiles of OT pulses were constant throughout the experiment. The absolute P concentrations gradually decreased as the CL age progressed, while the absolute OT concentrations remained constant. A high concomitant rate of OT pulses with a P pulse within individual lines of the MDS was observed on Days 6-7 when plasma P concentrations were still rapidly increasing, but such a close relationship between OT pulses and a P pulse disappeared thereafter (P<0.05). Furthermore, a 3 h-infusion with LH on Day 10 stimulated P release during the infusion, whereas a PGF_2α infusion induced an inhibition of P release after the infusion (P<0.05). These observations characterized the profiles of a local release of P and OT within an individual CL formed after superovulation during the non-breeding season. The characteristics of P release, but not those of OT release, change with the CL age. Collectively, the data appear to be well comparable to those in the cyclic CL during the breeding season. Thus, a local secretory function of the CL formed after superovulation during the non-breeding season is likely to have a close similarity to those of the cyclic CL.

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The prolactin (PRL)-responsive cell line, HC11, derived from normal mammary epithelial cell line COMMA-1D, expressed the long form of prolactin receptor (L-PRLR) mRNA, though the level was considerably lower than that in the mammary gland of the normal lactating mouse. HC11 cells expressed whey acidic protein (WAP) mRNA, when they were cultured to confluence and 'in vitro lactogenesis' was induced by the addition of PRL, insulin and dexamethasone. The levels of PRL-induced WAP expression, as measured by reverse transcriptase mediated polymerase chain reaction (RT-PCR), increased in a dose-dependent manner, as PRL concentrations were raised from 0.05 μg/ml to 50 μg/ml. Treatment of HC11 cells with human growth hormone (hGH) caused increase in levels of WAP mRNA. In contrast to PRL, hGH showed its maximum effect at 0.5 μg/ml. Beyond this concentration, the effect of hGH was greatly reduced. These results suggest that hGH has a narrow optimal concentration range with regard to the stimulation of WAP expression in HC11 cells.

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Syrian (Golden) hamster zygotes (pronuclear eggs) were collected from oviducts of donors and transferred to either oviducts or ovarian bursae of recipient females. Zygotes could develop into live young regardless of the post-ovulatory age of the zygotes and of the recipient females tested. The best results were obtained when the zygotes were transferred to ovarian bursae shortly before their first cleavage. Intra-ovarian bursal transfer of the zygotes is easy to perform. It may be applied to assessment of developmental potential of microsurgically operated hamster oocytes/zygotes.

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An attempt was made to use the zygotes arrested at the M phase of the cell cycle as the recipient cytoplasm for nuclear transfer. i) Zygotes which were collected 24, 28 and 32 h after hCG injection were incubated in M16 + nocodazole (3 μl/ml) for 10 to 14 h. After elimination of the drug, they were cultured in vitro for 4 days. ii) Blastocysts were cultured with nocodazole for 5 to 12 h and the mitotic index was examined. iii)M phase and G₁/S phase inner cell mass (ICM) cells synchronized with nocodazole and aphidicolin treatment, and fetal germ cells isolated from fetuses on days 11.5 (in mitosis) and 15.5 (G₀ phase) of pregnancy were fused with enucleated M phase zygotes. The results were as follows. i) Zygotes obtained 24 h after hCG injection and treated with nocodazole for 10 to 14 h were at the M phase. After release from the drug, they developed to blastocysts at a rate similar to that of non-treated zygotes (83∼87% vs 100%). ii) Mitotic index of blastocysts treated with nocodazole for 11 to 12 h was 94%. iii) Nuclear transfer into M phase zygotes did not improve the developmental ability of reconstituted embryos into blastocysts. In the present study, it was clear that mouse zygotes arrested at the M phase with nocodazole treatment, and after washing they developed to the blastocysts at the similar rate to non-treated zygotes. But, the developmental ability of reconstituted zygotes at the M phase did not support in vitro development.

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One of the target tissues for prolactin (PRL) family members is the central nervous sytem (CNS) in adult animals. In this study, we tried to detect PRL receptor (PRL-R) mRNAs in fetal rat brains. mRNAs extracted from various tissues, including the brain, on embryonic days 12 (E12), 14, 16, 18 and 20 were subjected to reverse transcriptional polymerase chain reaction (RT-PCR) using two sets of primers specific to the short and long forms of the PRL-R. The short form cDNA band (330 bp) was detected in the fetal brain from E12 to 20, whereas the long band (420 bp) was not apparently observed, suggesting that the developing fetal brain is a target for PRL family members. Short form PRL-R mRNA was also expressed in the liver (E20), heart (E12 to 20), intestine (E16 to 20) and forelimb (E20), and long form PRL-R was observed in the heart on E20. Semi-quantitative PCR analysis revealed that short form PRL-R mRNA expression in the fetal brain was as biphasic: constant on E12 and 14, and increased progressively on E18 and 20. Thus, PRL-R gene expression is initiated very early in developing fetal tissues, including the CNS.

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Immature bovine oocytes were equilibrated with a mixture of 1.6 M 1, 2-propanediol and various concentrations of sucrose (0, 0.01, 0.05, 0.1, 0.2 or 0.3 M) by the three-step method. Subsequently, the oocytes were cooled from 0 C to -5.5 C at a rate of 1 C/min. They were seeded at -5.5 C, cooled at a rate of 0.3 C/min to -30 C and plunged into liquid nitrogen. After being thawed and having their cyroprotectants diluted by the three-step method, the oocyte survival rate was assessed by examination of morphology and development following in vitro maturation/in vitro fertilization and culture. The rate of development of morphologically normal oocytes and of development to the two-cell stage in immature oocytes frozen in the presence of 1, 2-propanediol containing 0.3 M sucrose were significantly lower (P<0.05) than those observed at lower concentrations of sucrose (0 to 0.2 M). Of the oocytes frozen in the presence of 1, 2-propanediol containing sucrose at concentrations less than 0.2 M, very few frozen-thawed oocytes (0 M, 1.7%; 0.01 M, 1.3%; 0.05 M, 1.6%; 0.1 M, 0.6%; 0.2 M, 2.2%) developed to the blastocyst stage. Five blastocysts derived from immature oocytes frozen in the presence of 1, 2-propanediol containing 0.2 M sucrose were transferred nonsurgically to three recipients. Consequently, one recipient was diagnosed as pregnant with a single fetus by an ultrasonographer after 60 days of estrus.

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The ability of equine blastocysts to produce embryonic capsule was investigated in vitro. Blastocysts were nonsurgically recovered from Day-6 donor mares (Day-0; detection of ovulation), and were subjected to 5 days of culture at 37 C in 5% CO₂ in air. The blastocysts cultured in TCM199 supplemented with 0.1% polyvinylalcohol (M199/PVA; n=8) had poor developmental abilities; only one blastocyst initiated hatching on the fifth day of culture. In TCM199 with 10% fetal equine serum (M199/FES; n=7), all blastocysts initiated their hatching until the third day of culture. Five of them continued spherical development after hatching. The embryonic capsule was shed together with the zona pellucida throughout the hatching process. Pattern of blastocyst development in TCM199 which had been repeatedly used for uterine infusion/recovery/filtration (M199/IRF; n=6) was similar to that in M199/PVA; hatching was initiated in one blastocyst on the first day of culture, but was not completed. In vitro development of blastocysts (n=5) in uterine fluid recovered from mares 3-4 days post ovulation was, in part, comparable with that in M199/FES. Hatching was initiated in 3 blastocysts on the second or third day of culture, and one blastocyst completed its hatch within 24 h. However, the hatched blastocyst did not possess a capsule. These results suggest that mare uterine fluid as well as fetal equine serum support the in vitro development of equine blastocysts, but fail to support the maintenance of embryonic capsule under the culture conditions employed here.

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Corpus luteum formed in cows after ovulation induced by the use of prostaglandin (PGF_2α) and gonadotropin releasing hormone (GnRH) showed hypoplasia. In the present study, peripheral blood levels of estradiol-17β (E₂) were determined in the cow in which ovulation leading to luteal hypoplasia was induced. Ovulation was induced in 6 cows by injecting GnRH 32 h after the injection of PGF_2α analogue (PGF_2α-A) at 8 to 11 days after spontaneous ovulation. The animals were divided into 2 groups of 3 each. In one group, FSH was injected twice at 16 h intervals, starting 0 h after PGF_2α treatment. Cows in the other group were not injucted. Progesterone (P₄) values in blood remained low even after the induced ovulation in all 3 cows of the group not injected with FSH. Blood P₄ levels increased gradually and remained at a high level after induced ovulation in all 3 cows treated with FSH. E₂ values in the FSH injection group increased and peaked at 8 h after GnRH injection. At this time, average levels of E₂ were 9.1 and 4.7 pg/ml in FSH treated and non-treated group, respectively, and the difference was significant (P<0.01). These results suggest that the ability of the follicle to secrete estrogen before ovulation, which causes luteal hypoplasia, is low.

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Liposome-encapsulated plasmid pMT-neo/MT-β-gal DNA containing a neomycin resistance gene and a E. coli β-galactosidase gene was injected through a needle into mature mouse testes bilaterally 3 times at 4-day intervals. Eighty percent of blastocysts derived from eggs fertilized by spermatozoa of male mice injected with the DNA exhibited expression of β-galactosidase gene and the presence of pMT-neo/MT-β-gal DNA. Thus, direct injection of foreign DNA into the testis will provide a novel and convenient tool for production of transgenic animals.

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