After conception and during early pregnancy in pigs, uterine motility plays a keyrole in sperm transport and embryo spacing prior to placentation. This process is modulated by gonadal steroids. Early pig embryos secrete estradiol with a peak d. 12-13 and estradiol elicits part of its influence on target cells in the genital tract via the insulin-like growth factor-I (IGF-I). Therefore, in the present study, the levels of the estrogen receptor (ER) and progesterone receptor (PR), as well as their mRNAs and the IGF-I mRNA, were measured in the myometrium of early pregnant pigs (days 8-30 after onset of estrus) by EIA and solution hybridization, in order to establish relationships among the expressions of IGF-I, ER and PR during early pregnancy. The ER level was low on days 8-10 and decreased even further to very low levels on days 25-30 while the level of ER mRNA was unchanged during the same period. The PR level was decreasing during the days monitored, although PR mRNA showed a tendency to increase from days 12-13 to days 25-30. The level of myometrial IGF-I mRNA was higher on days 25-30 as compared to days 8-18 indicating that myometrial IGF-I mRNA expression is not affected by the production of conceptus estradiol on days 12-13. Significant correlations were found between the expressions of IGF-I mRNA and PR mRNA and between the levels of ER and PR. The results suggest that in the porcine myometrium the expressions of IGF-I and PR genes, respectively ER and PR, may have common regulating factors.

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In order to clarify the individual follicular development during superovulation treatment in Japanese black cows, consecutive ultrasonography was performed. A total of 5 cows was superovulated in mid-luteal phase. Ovaries were monitored with ultrasonography at 12 h intervals from the initial FSH injection (=0 h) until 24 h after the first ovulations. All follicles more than 5 mm in diameter were determined in freezing ultrasonography images with built-in calipers. Blood samples were collected at 12 h intervals for 168 h and the concentrations of progesterone and estradiol-17β were determined. Few follicles more than 5 mm in diameter emerged at 12 h after FSH injection, but most follicles more than 5 mm in diameter emerged at 36 h. Then, these emergence tended to decrease gradually. Every ovulatory follicles were retrospectively identified more than 5 mm in diameter at 84 h. The follicles that emerged later tended to grow faster than those emerged earlier within 84 h after treatment. The progesterone profile after prostaglandin F_2α (PG) injection in superovulated cows was similar to those in normal cycling cows synchronized with PG. The estradiol-17β profile showed a slight increase between 0 and 72 h during the follicular growth, then the levels reached peaks in accordance with estrous behavior prior to a sharp decrease before ovulation.

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The present study was undertaken to determine whether insulin-like growth factor-I (IGF-I) is involved in mediating the stimulatory effects of growth hormone (GH) on nuclear maturation of bovine oocytes in vitro and subsequent embryonic development after in vitro fertilization. When cumulus-oocyte complexes (COCs) or cumulus-free oocytes were cultured in modified Medium 199 supplemented with 0-100 ng/ml recombinant human IGF-I (rhIGF-I), the proportion of COCs with slight cumulus expansion was increased with increasing concentration of rhIGF-I after 12 and 24 h of culture. Nuclear maturation of oocytes to the metaphase II (M-II) stage was promoted by rhIGF-I only in COCs, with higher proportions of M-II oocytes at 10-100 ng/ml (74 ± 3-76 ± 3%) than at 0-1 ng/ml (53 ± 4-61 ± 1%) after 24 h of culture. When COCs were cultured in the presence of 100 ng/ml rhIGF-I or 10 ng/ml recombinant bovine GH (rbGH) and/or anti-IGF-I antibody (Ab) for 24 h, the proportion of oocytes reaching the M-II stage was higher with rhIGF-I (73 ± 2%) or rbGH (74 ± 1%) than in the absence of these additives (54 ± 3%), in Ab alone (54 ± 3%), or with IGF-I or GH in combination with Ab (54 ± 3-58 ± 1%). The inhibition of the stimulatory effects of rbGH on nuclear maturation by Ab was dose-dependent. After in vitro maturation and fertilization, no differences were observed in the penetration rate (75 ± 3-77 ± 6%), pronuclear formation (56 ± 3-64 ± 10%) and polyspermy (28 ± 4-36 ± 5%) at 8 h post-insemination among cumulus enclosed oocytes cultured in the presence of different additives. When the oocytes were cultured in a chemically defined medium starting from 8 h after insemination, 20 ± 1-26 ± 2% of oocytes developed to the ≥ morula stage 144 h post-insemination without difference among different additives during maturation. However, at 192 h post-insemination, higher proportions of oocytes developed to the blastocyst stage when COCs were matured in the presence of rhIGF-I (16 ± 1%) or rbGH (15 ± 2%) than in the absence of the additives (4 ± 2%) or when IGF-I or GH was combined with Ab (4 ± 2-5 ± 3%). These results suggest that the stimulatory effects of GH on bovine oocyte maturation may not be direct, but mediated via IGF-I probably produced by the cumulus cells.

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The sno (ski-related novel) gene, was originally identified as a homologue of cellular proto-oncogene, c-ski. Ski and SnoN are localized in the nucleus and probably act as transcriptional factors, although their functions have not yet been fully elucidated. In an attempt to identify a role of snoN in vivo, we produced 4 transgenic mice, harboring human snoN (hsnoN) cDNA driven by the simian virus 40 (SV40) enhancer/promoter. One of the transgenic mice (#11-4) exhibited retarded growth, and the hsnoN expression was confirmed in heart, stomach, skeletal muscles and kidney. Another transgenic mouse (#9-3), on the other hand, did not show any obvious phenotypic alteration except sterility, although hsnoN expression was detected in the soleus and the plantaris muscles, where the endogenous snoN expression was not observed. While the soleus of normal mice contains much higher proportion of type I fibers (high ATPase activity) than type II fibers (low ATPase activity), in the #9-3 mouse the soleus was smaller, and contained reduced number of type I fibers, compared to normal mice. It is suggested that snoN might play a role in the differentiation of type I muscle fibers.

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To investigate whether the fertilizing ability is changed during transit of spermatozoa in the cauda epididymis in BALB/c mouse which is known as a low in vitro fertilization (IVF) rate, we examined the capacitation rates of spermatozoa collected from 3 regions of the cauda epididymis by chlortetracycline (CTC) fluorescence assay. In addition, their fertilizing ability was examined by IVF with cumulus-intact and cumulus-free mouse eggs. Spermatozoa immediately collected from the cauda epididymis showed no significant differences in capacitation rates (the proportion of spermatozoa having a fluorescence-free dark band in the postacrosomal region; B pattern) estimated by CTC test among regions. Nevertheless, the capacitation rates in the proximal region were significantly (P<0.05) higher than that in distal region at 60 min of incubation and the middle region at 120 min of incubation. Non-preincubated spermatozoa regardless of the collection regions in the cauda epididymis showed signs of low fertilizing ability in cumulus-intact (9-10%) and cumulus-free (0-4%) mouse eggs at 6 h after insemination. After 2 h sperm preincubation, fertilization rates of spermatozoa collected from the proximal and distal regions were only slightly increased (26-44%) but that of middle region was greatly increased in both cumulus-intact (68%) and cumulus-free (61%) mouse eggs. These results suggest that the fertilizing ability of BALB/c mouse spermatozoa is changed during transit of the cauda epididymis, and the spermatozoa in the middle region of the cauda epididymis could be utilized efficiently for IVF in the BALB/c mouse strain.

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The present study was undertaken to evaluate ovulation time after superovulation treatment in ewes during the non-breeding season. Ewes (n=17) were treated with vaginal sponges impregnated with fluorogesterone acetate (FGA sponge) for 12 days. To the superovulation group (n=9), an intramuscular injection of 20 mg porcine follicle stimulating hormone (pFSH) was given at 48 h before sponge removal, and 500 IU equine chorionic gonadotropin (eCG) was given 24 h later after the pFSH injection. Ewes in the control group (n=8), were given 500 IU eCG alone 24 h before sponge removal. The time of ovulation was assessed by repeated observation of the ovaries in both groups of ewes using a laparoscope. Observations were commenced 24 h after sponge removal and were conducted every 6 h for a total of 60 h. The intervals between sponge removal and the onset of estrus, and ovulations were shorter in the superovulation group than the control group (P<0.02). There were significant differences in the ovulation rate between superovulated and control ewes. In 9 superovulated ewes, 12%, 53% and 27% of the total 99 ovulations occurred within 0-24 h, 36-42 h and 42-48 h after sponge removal, respectively. The present study indicates that a single pFSH injection combined with 500 IU eCG induces a high synchrony (80%) of ovulation at 36-48 h after sponge removal in the ewes treated during the non-breeding season.

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To assess the direct effects of growth hormone (GH) on the release of progesterone (P) in the microenvironment within the ovine corpus luteum (CL) in vivo, a microdialysis system (MDS) was implanted into the CL during the breeding season. In experiment 1 (single CL model), on Day 7 after estrus, three microcapillary dialysis membranes (cut off Mr=1000 kDa; transfer rate, 1% for P and 0.1% for LH and GH) were surgically implanted into the cyclic CL paralleled to each other with a distance of at least a 5 mm between capillaries (n=5 ewes). Each MDS line was continuously perfused with Ringer's solution during the experiment. Serial fractions were collected every 30 min for 35 h on Days 8-9 following a pre-perfusion period of 10-12 h. A 16-h perfusion with GH (10 μg/ml) induced an acute increase of P release to 300% of the baseline throughout the infusion (P<0.001). A 3-h perfusion with oxytocin (OT: 2 × 10^-5 M) also stimulated the P release to approximately 200% of the baseline (P<0.01). When OT was infused following the GH infusion, no additional effect was observed. In experiment 2 (a multiple CL model), four ewes were treated for superovulation, and multiple CLs formed thereafter were implanted with the MDS (1 line/CL) on Day 7 after GnRH injection. Infusions with GH or LH on Days 9-10 at doses of 2.5, 5 or 10 μg/ml for 4 h twice with an 8-h interval similarly induced a clear increase of P release. The 2nd GH infusion had a greater stimulative effect than the 2nd LH infusion at all the doses tested (P<0.05). Furthermore, the 2nd infusion with GH at 5 and 10 μg/ml induced a higher increase of P release than did the 1st infusion (P<0.05), while LH, at any of the tested doses, stimulated P release to a similar extent during the 1st and 2nd infusions. The finding that GH, like LH, directly stimulates the local release of P within the CL in cyclic and superovulated ewes in vivo strongly suggest that GH, like LH, is an essential luteotropic pituitary hormone in ewes.

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To determine whether phosphatidylinositol 3-kinase (PI 3-kinase) cascade plays any role in mammalian oocyte meiotic progression, porcine oocytes were matured in vitro with PI 3-kinase specific inhibitors, wortmannin and LY294002. In cumulus-oocyte complexes (COCs), the addition of 10^-8, 10^-7 or 10^-6 M wortmannin or 0.5, 5.0 or 7.5 ×10^-5 M LY294002 to the medium significantly reduced the proportion of oocytes exhibiting germinal vesicle breakdown (GVBD) compared with the inhibitor-free control. In denuded oocytes (DOs), no significant differences in the incidence of GVBD were found between 10^-8 M wortmannin treatment and controls, and between 10^-8 and 10^-7 M treatments, respectively. LY294002 treatment at all concentrations tested did not significantly affect GVBD in DOs. When COCs were cultured in the presence of wortmannin or LY294002, the proportions of oocytes arrested at GVII were significantly higher than the control. The degree of cumulus cell expansion was significantly decreased with wortmannin or LY294002 treatment compared to the control. We concluded that activation of PI 3-kinase in cumulus cells is closely associated with meiotic resumption of the porcine oocytes and the cumulus cell expansion.

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Secretion of prostaglandins (PG) E₂ and PGF_2α by the bovine endometrium and the effects of oxytocin (OT) on these secretions during the estrous cycle were investigated. Bovine uteri were classified into five luteal stages (early I: Days 2-3; early II: Days 5-7; mid-: Days 8-12; late: Days 15-17; regressed: Days 19-21). After 1 h of pre-incubation, endometrial tissues (20-30 mg) were exposed to 0 or 100 nM OT for 4 h. PGE₂ concentration in the medium was higher than that of PGF_2α during the luteal phase, and declined at the regressed luteal stage (P<0.05). In contrast, PGF_2α secretion reached the highest value at the regressed luteal stage (P<0.05). OT significantly stimulated both PGE₂ and PGF_2α secretion at the early luteal stage (P<0.05). Although OT enhanced PGF_2α secretion at the late and regressed luteal stages (P<0.05), PGE₂ secretion was reduced by OT at the same time (P<0.05). These results indicate that there are significant differences in the pattern of PGE₂ and PGF_2α secretion by the bovine endometrium during the estrous cycle and suggest that OT may play a role in modulating the secretion of PGE₂ as well as PGF_2α.

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Inner cell masses (ICM) were isolated immunosurgically from 110 bovine blastocysts derived from in vitro produced day-8 embryos and subsequently cultured in Dulbecco's modified Eagle's medium containing 20% fetal bovine serum, 10^-4 M mercaptoethanol, nonessential amino acids and nucleosides on mitomycin C-treated mouse embryonic fibroblast feeder cells. Approximately 5 days after plating, growing ICMs were dissociated by trypsinization and the colonies composed of cells with ES cell-like morphology were selected for further passage at 4 to 6 days intervals. Sixty seven percent of the ICMs were clumped and 7.8% of the ICMs brought into culture produced ES cell-like cell lines. Histochemical staining revealed that all cell lines after five passages were positive for the alkaline phosphatase activity. Immunohistochemical localization of the carbohydrate antigen by 4C9 monoclonal antibody reactivity also gave a positive result similar to that seen immediately following immunodissection. Cytogenetic analysis revealed a karyotype with an euploid number of chromosomes in 48.1 to 71.0% of cells. In all cell lines, dissociated cells formed embryoid bodies 8 to 10 days after culture in the absence of feeder cells. These results suggest that the bovine ES cell-like cell lines established were pluripotent, at least in vitro.

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A model of induced follicular atresia consists of hypophysectomizing hamsters on the morning of estrus (day 1 of cycle) followed by injecting 30 IU equine chorionic gonadotropin (eCG). On the morning of day 4 (0 h) atresia is induced by administration of an antiserum to eCG (eCG-AS). One hour after eCG-AS, plasma estradiol and testosterone decline to 15.8% and 25.9%, respectively, of 0 h values with progressive decreases until at 12 h after eCG-AS, circulating estradiol is no longer detectable although baseline levels of testosterone (10.7%) are still present. The question raised was whether the simultaneous drop in both steroids at 1 h post eCG-AS is attributable to the fall in both hormones or to a more drastic decline in thecal androgens, thus depriving the granulosa of the precursors needed to synthesize estrogens. To answer this question, antral follicles were dissected 2 and 4 h after eCG-AS and incubated for 5 h, with or without the addition of 10 ng androstenedione. Addition of androgen at either time led to an increase in estradiol accumulation in the medium after the first hour which was sustained over the next 4 h. These experiments indicate that the immediate effect of eCG-AS is to sharply reduce thecal androgen production thus depriving granulosal aromatase of the necessary steroid substrate. In previous histological studies, we have shown that the earliest time when morphological signs of atresia are first discernable is by 4 h post eCG-AS as judged by pyknotic granulosa nuclei in the cumulus oophorus. In this study, using gel electrophoresis and autoradiography of 3’ labeled DNA followed by quantitating low and high molecular weight DNA, the earliest consistent onset of atresia was also at 4 h after eCG-AS.

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The effect of melatonin on testosterone release was investigated in Sprague Dawley rats in vivo and in vitro. When plasma testosterone levels were determined in rats 2 weeks after pinealectomy (Px) or in rats given daily melatonin injections (MI), no significant differences were observed compared with those in intact or sham-operated rats (Sham). Although luteinizing hormone (LH) stimulated testosterone release by Leydig cells from intact, Px and MI rats in a dose-dependent manner, there was no significant difference in the magnitude of the response to LH. In addition, neither basal nor LH (5 mIU/ml) -stimulated testosterone release were affected by co-culture of various doses of melatonin (1 nM-10 μM) for 4 h and 12 h. When various doses of LH (10 μIU-100 mIU/ml) were co-administered with 0.5 or 5 μM melatonin, no significant change was observed in testosterone release compared with that in the presence of LH alone. Although forskolin, an adenylate cyclase activator, stimulated testosterone release by Leydig cells from intact rats in a dose-dependent manner, melatonin did not reduce forskolin-stimulated testosterone release in 4 h and 12 h cultures. These results do not support the hypothesis that melatonin directly suppresses LH-stimulated testosterone release by rat Leydig cells.

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