To elucidate whether or not the promoting effects on embryo development is derived from the characteristic factors which the oviduct possesses, we cultured mouse embryos in the oviduct, the ureter and the vein, all of which have "ductal structures", and we compared the culture efficacy among 3 organs. Furthermore, we measured oxygen tensions within the 3 organs and performed an ultrastructural analysis of the three ductal organs after the culture. Embryos cultured in the oviduct developed to blastocysts at a significantly higher rate (13.5%) than those in the ureter (1.7%) and the vein (0%). The oxygen tensions of the inner lumen of all the ductal organs were relatively low, 36-45 mmHg, and no remarkable difference was observed among the 3 organs. These results suggest that the oviduct may have embryo growth promoting effect besides low oxygen tension. Furthermore, the ultrastructural results suggest that not only the normality of structural components but the inherent character of the oviduct may play an important role in the promotion of embryo development.
Neurosecretion of hypothalamic GnRH was monitored during the LH surge in ovariecto-mized goats given estradiol using microdialysis sampling. The microdialysis probe with a polycarbon-ate dialysis membrane was stereotaxically implanted in the median eminence, and was perfused with the artificial cerebrospinal fluid at a flow rate of 5μl/min under conscious and unrestrained conditions. The LH surge was induced by means of an intravenous infusion of estradiol (3 μg/h, i.v.) for 16 h and hourly fractions of microdialysis perfusate were collected for GnRH measurement. The perfusate GnRH showed a marked increase to peak levels of 25.3±6.9 μg/ml over basal levels of 2.6±1.1μg/ml at the onset of the LH surge, and then gradually decreased so that the GnRH surge lasted longer than the LH surge. These results suggest that the LH surge is induced by a surge release of GnRH from the hypothalamus but ends despite a continued supply of GnRH.
In mouse blastocysts cultured in vitro, trophoblast cells undergo extensive spreading which represents the invasion stage of implantation in vivo. The trophoblast spreading is accompanied by striking increase of cellular volume and transformation of the cells into the giant trophoblast cells. Since, Na+/H+ exchanger system (or Na+/H+ antiporter) on the plasma membrane of vertebrate cells plays a crucial role in regulating cellular volume in mammalian cells, the carrier mediated Na+/H+ exchanger system might be involved in the process of trophoblast spreading. To obtain clues to approach the problem, we examined effects of amiloride which is a potent inhibitor of Na+/H+ exchanger and other amiloride sensitive Na+ channels, upon mouse blastocysts cultured in vitro. Amiloride inhibited, in dose-dependent manner, hatching as well as the trophoblast spreading in mouse blastocysts cultured in vitro. Our findings strongly indicated that the amiloride sensitive Na+/H+ exchanger system might be operating, in particular, during the initial phase of trophoblast spreading: the system appeared less active in the fully spread cells. To corroborate the view, experiments with more specific inhibitors of Na+/H+ exchanger are currently in progress in our laboratory. In addition, while amiloride and its derivatives have been used as diuretic drugs, little has been known concerning their effects upon the peri-implantation embryos. Our data clearly indicates their potentially adverse effects upon the conceptuses during early phase of gestation.
The blood supply of the hypophysis in the dog was examined by light microscopy and scanning electron microscopy. The anterior hypophyseal arteries converged toward the infundibular stalk, subdivided in the stalk, and entered the primary capillary net and capillary loops in the median eminence lying at the base of the third ventriculus. The portal vessels originated from the capillary net and loops, and were continuous with the capillary sinusoids in the pars distalis. Some of the anterior hypophyseal arteries supplied the pars distalis directly and were also continuous with capillary sinusoids. In its caudal pole, the pars nervosa received the posterior hypophyseal arteries, which formed a capillary plexus in the pars nervosa. The vessels of the pars nervosa broke up into a primary capillary net at the caudal areas in the median eminence. The vasculature in the dog hypophysis is compared with that in the rat.
Superovulated eggs in (BALB/c×C57BL/6) F1 and ICR (outbred in a closed colony) female mice were fertilized in vitro with spermatozoa obtained from caudal epididymides of ICR males. Air-dried chromosome preparations were made from colcemid-primed 1-cell eggs and stained by the C-banding method. The fertilization rate was lower in F1 eggs than in ICR eggs (88 vs. 92%, P<0.02). The incidence of metaphase ("syngamy") eggs was higher in F1 eggs, and the incidence of pronuclear and late prometaphase ("pre-syngamy") eggs was higher in ICR eggs (P<0.001, P<0.005), showing delayed progress of the first cleavage in the ICR eggs. Developmental rates from 2-cell to 4-cell stage and from morula to blastocyst stage were significantly lower in ICR eggs (P<0.001) than in F1 eggs. These results show that a delay of embryo development had already appeared before male and female genomes fused (pre-syngamy). The incidence of triploidy, which might be caused by dispermy, was higher in F1 eggs, in both the pronuclear and mitotic stages (P<0.001). A little aneuploidy and structural aberration of chromosomes occurred in both F1 and ICR eggs, and no significant difference was observed between F1 and ICR eggs. The sex ratio at the first cleavage stage in both F1 and ICR eggs showed no significant deviation from equality.
Superovulation was induced in intact and acutely hypophysectomized mature rats by a successive eCG-hCG treatment. In the rats hypophysectomized after the onset of GTH surges, increment in eCG dosages maximized the number of ova shed by the following hCG treatment, and the maximum number was similar to those in non-hypophysectomized rats. In the rats hypophysecto-mized before GTH surges, however, the number of ova shed was significantly less, but this decrease was restored by an hCG injection immediately after the hypophysectomy. These results indicate that the maximum number of ova shed is determined by the limited number of eCG-responsive follicles that are generated during exposure to an antecedent LH surge. In intact rats, excess doses of eCG elicited enhanced progesterone secretion which, in turn, established PRL surges. These PRL surges appears to cause atretic changes in growing follicles and decreased the number of ova shed by the following hCG treatment. Thus, the upper limit of the number of ova shed in mature cycling rats subjected to the eCG-hCG treatment was estimated to be approximately 80.
The objective of the present investigation was to purify the early pregnancy factor (EPF) from swine pregnancy serum using diafiltration, ion-exchange chromatography, HPLC-gel permeation, and affinity chromatography. EPF activity was determined by using RIT after the pregnancy serum obtained from a mature swine 25 days after mating was divided into 2 parts (below M.W. 100KD and above M.W. 100KD) by the diafiltration. Using RIT, EPF activity was found in the fraction below M.W. 100KD. When this fraction was applied onto the DEAE-Shepharose column, the EPF activity was only found in the unadsorbed fraction. Next, this fraction was applied onto the CM-Sepharose column and was divided into 3 parts. A fraction which was eluted by 50 mM ammonium acetate buffer containing 50 mM NaCl had the EPF activity and the other fractions did not have this activity. The fraction which was eluted with 50 mM NaCl was analysed by SDS-PAGE and contained 6 bands between M.W. 30KD and 20KD. When the pattern of this SDS-PAGE was compared with the bands obtained from non-pregnant swine serum (day 0 of cycle, estrus), the 3 bands were not confirmed in the non-pregnancy serum. The molecular weight of these bands were 21KD, 23.7KD, and 26KD, respectively. On the HPLC analysis, EPF activity was found between M.W. 14 and 21KD. Therefore, it was thought that the swine EPF active material of M.W. 21KD might be the major active form of the swine EPF in this gestation period. In addition, the affinity gel which was coupled with Rabbit anti nonpregnancy swine whole serum lgG was effective for the purification of EPF.
The aim of the work is to determine the susceptibility of mouse preimplantation embryos to vitrification at different developmental stages. The experiment was carried out in embryos at 1-cell, morula and blastocyst stages. As a vitrification solution, a mixture of 2.5 M dimethylsulphoxide + 2.5 M propylene glycol (DP), DP + 1.0 M sucrose (DPS) or DP + 0.16 M raffinose (DPR), was used. The in vitro survival, evaluated by the development into a late blastocyst stage in vitro, of control and vitrified mouse embryos was examined. A high survival rate (80%) after vitrification was obtained for 1-cell embryos after a 15 sec-exposure to DPS. High survival rates for morulae (91-96%) and relatively low survival rates for blastocysts (30-55%) were observed after vitrification in either DPS or DPR after short exposures (within 30 sec). In vivo survival rate for each embryos vitrified in DPS by a short exposure (within 30 sec) at 1-cell, morula or blastocyst stage was 56, 53 or 40%. Though the rates for 1-cell and molura stages were comparable with those in control embryos (55 and 58%), the rate for blastocyst stage was significantly lower (P<0.05) than that in control embryos (56%). Thus, the proposed vitrification procedure can be useful in the cryopreservation of mouse preimplantation embryos at 1-cell and morula stages.
We have generated 2 transgenic mouse lineages expressing a mWAP/hGH fusion gene where 5' sequence from the mouse whey acidic protein (mWAP) gene is linked to the coding region for human growth hormone (hGH). In the present study, we investigated whether hGH was synthesized in the mammary gland of the transgenic mice and secreted into milk, and whether physiological functions of the transgenic mice were affected by the expression of the transgenes or not. Milk taken from female mice of the transgenic lineages contained about 4.77±0.1 μg/ml (n=4) hGH in average. The hGH was also detected in plasma of the transgenic mice expressing the transgene. The plasma hGH was probably derived from several organs, including submandibular glands, pancreases and tongues as well as mammary glands. The results suggested that expression of the transgenes were probably higher in organs with exocrine functions. In addition, hGH was produced in the brain. Transgenic females derived from one of the founder males showed the sterility accompanied by obesity, insulinemia and hyperglycemia. Histological examination confirmed that the number of azocarmine-positive cells which represent GH secreting-cells in the anterior hypophysis was dramatically reduced in the sterile transgenic females compared to the control mice. The ovary showed signs of pronounced disorders accompanied by failure of follicular growth and by inability of the corpus luteum formation.
The lactose content of the mammary gland and plasma and the ion concentrations of the plasma were investigated in primiparous lactating rats after oxytocin (OT) administration. OT was given sc or iv at 1500 h on day 12 of lactation to mothers whose pups were removed 6 h earlier. At this time a large amount of milk was accumulated in the mammary glands. Mammary gland lactose content of animals injected sc with 10IU of OT was markedly reduced within 30 min and continued to decline to 120 min, whereas saline-injected controls maintained high levels throughout the experimental period. The value at 240 min in the OT-injected animals was 4.8% of that of the control. In contrast, the lactose concentration in plasma rose sharply up to 60 min and then returned to the baseline level at 240 min after OT injection. Potassium and calcium concentrations in plasma increased and plasma sodium decreased after OT injection. The iv administration of OT at graded doses (0.01, 0.1 and 1IU) resulted, at the end of 120 min, in a dose-dependent decrease in mammary gland lactose content with the higher 2 doses being statistically significant. These results indicate that OT administration to lactating rats shifts milk components from the mammary gland to the plasma.
To identify an in vitro culture procedure which can be used to assess porcine in vitro fertilized embryos, three culture systems were examined. A total of 103 in vivo fertilized zygotes were co-cultured in modified BMOC-3 medium with granulosa cells, or in modified BMOC-2 with oviduct cells, and 35 were cultured in Whitten's medium supplemented with 1.5% BSA. After 8 days in culture, 66.0 and 73.6% of the co-cultured zygotes, and 51.4% of the zygotes cultured in the modified Whitten's medium developed to blastocyst stage. In contrast, only 20.5% (18/88) and 1.4% (9/627) of in vitro fertilized zygotes derived from in vivo and in vitro matured oocytes developed to blastocysts in the co-culture with modified BMOC-3 and granulosa cells. It is concluded that the culture systems examined are competent to support development of porcine zygotes to blastocysts and evaluate in vitro fertilized porcine embryos.
We have previously purified the 20α-hydroxysteroid dehydrogenase (20α-HSD) molecule from normal mature rat ovarian cytosol [Biochim. Biophys. Acta, 1991, 1079: 112-118]. In order to further characterize the 20α-HSD and to obtain information for molecular cloning of the enzyme, we determined its N-terminal amino acid sequence. The sequence was shown to be Ser-Lys-lle-Gln-LysMet-Glu-Leu-Asn-Asp-Gly-His-Ser-Ile-Pro-Val-Leu-Gly-Phe-Xaa-Thr. A search of the SWISS-PROT protein sequence database for N-terminal amino acid sequence similarities revealed 6 highly homologous proteins, including bovine prostaglandin F synthase, rat liver 3α-hydroxysteroid dehydrogenase, bovine lens aldose reductase, human aldose reductase, frog ε-crystallin and human liver chlordecone reductase, suggesting that rat ovarian 20α-HSD belongs to the aldo-keto reductase gene superfamily.