The effect of indomethacin (1M), an inhibitor of prostaglandin (PG) synthesis, on fertilization, early development and hatching of blastocysts was examined using ICR mouse eggs.
(1) When eggs obtained from the oviducts of superovulated females 15 hours after hCG injection were denuded with hyaluronidase and cultured for 2 hours in a modified Krebs-Ringer bicarbonate solution containing 100nM, 1μM, 10μM, 100μM or 200μM of 1M before being inseminated iv vitro, 55.9%, 50.0%, 47.1%, 50.0% and 47.5% of the eggs were fertilized respectively, showing no significant differences from that obtained in an 1M-free (control) medium (55.8%). Fertilization rate was significantly lowered only in the highest (400μM) concentration of 1M.
(2) When 2-cell embryos were cultured for 48 hours in the presence of 100nM, 1μM. 10μm or 100μM of 1M, 79.6%, 81.0%, 81.1% and 85.7% of the embryos respectively were developed into blastocysts, but the percentages showed significant decrease in higher concentrations (200μM) of 1M and no blastocysts were observed in a medium containing 400μM of 1M.
(3) The percentages of hatched blastocysts were significantly decreased when blastocysts obtained 96 hours after hCG were cultured with graded concentrations of 1M higher than 1μM, resulting in only 6.7% of hatched biastocvsts in a medium containlng 400μM of 1M.
These results suggest that PG synthesis plays an important role in early development, especially in the hatching of blastocyst.

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The ability of male pronucleous formation of hamster follicular oocytes matured in vitro was examined by using in vitro fertilization technique of zona-free oocytes.
When hamster oocytes, matured in vitro in a modified Tyrode's solution (mTALP) without amino acids and hormones, were fertilized in vitro, any oocytes could not get the ability to support the development of male pronucleus.
By incorporating 12 amino acids (alanine, arginine, cystidine, glutamine, histidine, isoleucine, leucine, methionine, phenylalanine, proline and valine) and hormones (LH, FSH, PRL and estradiol-17β) in the maturation medium, higher percent (65%) of 75 oocytes gained the ability to support the development of male pronucleus.

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In order to test the effect of Metal-EDTA chelate on the preimplantation development of mouse embryos fertilized in vitro, ICR mouse zygotes were cultured with 10μM Mg-, Ca-, Mn-, Co-, Ni-, Cu-, Zn-, La- or Fe-EDTA in Whitten's medium up to 120hr after insemination. Development to the 4-cell stage of embryos was improved by addition of Mg-, Ca-, La-, or Fe-EDTA in the medium, while Co-, Ni- or Zn-EDTA showed no effect as control medium. At 120hr after imsemination, developmental rates to the blastocyst of the embryos in culture with Mg-(41.7%), Ca-(45.8%) or La-EDTA (68.3%) were not significantly different compared with EDTA control (67.9%). In the presence of Fe-EDTA, ICR embryos overcame the 2-cell block (86.7%) yet most embryos could not developed to the blastocyst stage (10.0%). These results indicated that Ca-, Mg- or La-EDTA was effective to support the preimplantation development in ICR zygotes, and EDTA was effective not only on the second cleavage but also the blastocyst formation.

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A study has been made of the viability of rat ovarian tissue after exposure-196°. The ovaries were plunged into LN₂ directry after soaking in the the vitrification solution (Rall & Fahy, 1985). After thawing and transplantation, such tissue formed functional grafts in the ovarian bursa of the ovariectomised donors, and ovulated.

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Interspecific chimaeric blastocysts (mouse-rat, mouse-hamster, rat-hamster) were produced in the present experiment.Feulgen staining of nuclei and scanning electron microscopic observations of the chimaeric embryos were undertaken to analyse the origin of trophoblast cells, cell distribution and the interaction between cells derived from different species.
In Feulgen nuclear reaction, the cells derived from mouse, rat and hamster were distinguished by observation of its nuclear size, and the shade of stained nuclei in chimaeric embryos.The cells derived from mouse had large nuclei which were homogeneously stained. The cells derived from rat had various size of nuclei which were smaller than those derived from mouse and were unevenly stained.The cells derived from hamster had small nuclei which were homogeneously stained.
Observation with SEM showed the difference among the cells derived from each species embryos in density of microvilli on the surface of trophoblast cells.Trophoblast cells of which surface was covered with few microvilli were derived from mouse embryo. Dense microvilli were found on the surface of the cells derived from rat embryo. The cells derived from hamster were covered with many microvilli which were thicker and shorter than those of mouse or rat.
In chimaeric blastocysts, trophoblast cells derived from each embryos distributed in groups at random regardless of their polarity.

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The localization of fibronectin in mouse embryos in vivo was first found in the inner cell mass of early blastocyst and was later found also massively in the trophectoderm of late blastocyst. In contrast, fibronectin could not be found even in the late blastocyst stage in cultured embryos.
When the blastocysts developed in vivo or in vitro were further cultured for three days in Dulbecco's Modified Eagle Medium (D'MEM) containing 10% fetal calf serum, the in vitroimplantation (trophoblastic outgrowth in vitro) rates were 90%, 37% respectively; the latter showed a significantly low rate. If in vivo blastocysts were cultured in D'MEM without fetal calf serum, the in vitro-implantation rate was as low as 29%. However, if fibronectin at the concentration of 5, 10, 20, or 40μg/ml was added, instead of fetal calf serum, to the D'MEM, the in vitro-implantation rates were improved to 76, 84, 82 and 79%, respectively.

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When mammalian eggs are placed in a highly concentrated solution of neutral salt, the egg cytoplasm shrinks to become a small spherule, whereas the non living zona pellucida remains morphologically unchanged. In this study we examined the function of zona pellucida of salt-stored eggs in about sperm-zona interactions.
(1) Zona penetration by spermatozoa began about same time when salt-stored and living eggs were inseminated with 2hr preincubated (capacitated) spermatozoa in the same dish. Also the time course changes in the ratio of penetration (or fertilization) was almost same.
(2) When 2hr preincubated (capacitated) hamster spermatozoa were mixed with salt-stored and living eggs, almost same number of spermatozoa attached to each type of zona. The number of spermatozoa tightly bound to the two types zona was also about the same.
(3) Acrosome reaction inducing ability of the zona was well mainteined after storage of eggs in salt solution. The major biological and biochemical properties of the zona were retained in the salt solution, therefore salt-stored eggs can be used to study sperm-zona interactions as the substitute of living eggs.

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A possible function of progesterone (P₄) produced by mouse embryo was investigated during preimplantation period. Four cell embryos were obtained from PMS-hCG treated ICR mice 56h after hCG administration and cultured in modified BWW. Hexokinase (HK) activity of the embryos increased 4 fold after 36hr culture period, i. e. from 2. 68±0.11 to 10.5±0.61 pmol NADPH/embryo/h (mean±SEM). Addition of P₄ did not significantly increase HK activity. When P₄ production of the embryo was inhibited by trilostane (TRL), a specific inhibitor of 3β-hydroxysteroid dehydrogenase, the elevation of HK activity was suppressed dose-dependently (TRL 200μM, 4.02±0.18), and recovered by P4 supplement (P₄ 0.5μg/ml, 7.54±0.46). The inhibition was dominant when TRL was added in the first 24h. Actinomycin-D (ACTD) and cycloheximide, which inhibited mRNA transcription and protein translation respectively, both inhibited the elevation of HK activity dose-dependently, and the inhibition by ACTD was also dominant when added in the first half. These results suggest that P₄ play a role in the induction of HK enzyme protein at the transcriptional level perhaps through autocrine fashion.

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We have previously reported the uptake of ¹⁴C-leucine and ¹⁴C -glutamic acid in rat eggs of the preimplantation stage. In this experiment, the simultaneous uptake of ³H-leucine and ¹⁴C-glutamic acid in rat eggs by the double labelling method was studied to compare the single uptake of ³H-leucine and the previously reported single uptake of ¹⁴C-glutamic acid in rat eggs.
(1) Both the single uptake of ³H-leucine and simultaneous uptake of ³H-leucine+¹⁴C-glutamic acid in rat eggs were found to increase until 60 minutes after culture. When glucose, lactate and pyruvate were added, the uptake of these amino acids was augmented.
(2) The uptake of ³H-leucine and ¹⁴C-glutamic acid in rat eggs by the double labelling method tended to be reduced, compared to the single uptake of either ³H-leucine or ¹⁴C-glutamic acid.
(3) Using the uptake of ³H-leucine in rat eggs as an index basis of 1, the uptake of ¹⁴C-glutamic acid in the same eggs showed the same pattern whether singly or doubly labelled. The similarity between the ratios of leucine and glutamic acid to total free aminoacids in the uterine fluid and the ratios of uptake of these aminoacids in the present experiment suggested the possible relationship between the contents of free amino acids in the uterine fluid and the uptake of amino acids.

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[in Japanese]

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