The results of this study demonstrated the viability of embryos obtained from in-vitro maturation of bovine oocytes followed by in-vitro fertilization and culture to the blastocyst stage in vitro.

Ca 2+ -regulated exocytosis is enhanced by an autocrine mechanism via the PGE

In the prespermatogenesis period, male germ cells (gonocytes) begin to reproliferate and move to the basement membrane of the seminiferous tubule. Although these two events-reproliferation and relocation-are important for establishment of spermatogenesis, they have not been greatly analyzed both in a mechanical and in an endocrine or paracrine aspect. In this study, the relationship between reproliferation and relocation of gonocytes was examined, using the thymidine analog bromodeoxyuridine (BrdU) labeling method and transmission electron microscopy (TEM). BrdU was injected into the fetuses [day 13.5 post coitus (dpc) to 18.5 dpc] and pups [day 0. 5 post partum (dpp) to 6.5 dpp] of C57BL/6J mice. Two hours later, BrdU positive gonocytes were examined immunohistochemically and these data were analyzed. TEM and LM observation was carried out as well. Gonocytes began to relocate on the basement membrane from 18.5 dpc (1.4%) while BrdU-labeled gonocytes were first detected on 1.5 dpp (13.6%). Relocated BrdU-negative gonocytes were recognized from 18.5 dpc (1.4%), and relocated BrdU-labeled gonocytes were recognized from 1.5 dpp (8.4%). On the other hand, non-relocated BrdU-labeled gonocytes were detected from 1.5 dpp (5.2%). Gonocyte relocation began 2 days earlier than reproliferation during the late fetal period. After birth, the two events occurred at random. These results indicate that the reproliferation of the gonocyte does not correlate with relocation. The two events may be regulated by different mechanisms.

We have evaluated the effects of embryo density and the co-culture of unfertilized (degenerating) oocytes on the development of in-vitro fertilized (IVF) mouse embryos. In experiment 1, groups of one, five, 10 or 20 zygotes were cultured in 20 microliter drops of modified human tubal fluid (HTF) medium for 168 h at 38.7 degrees C in 5% CO2 and 95% air. As the embryo density increased, significantly (P < 0.05) higher rates of embryos reached hatched blastocyst stage. In addition, the time required for hatching after IVF was significantly (P < 0.05) shortened by the increase in embryo density. In experiment 2, 10 IVF zygotes were cultured with or without 10 unfertilized (degenerating) oocytes in 20 microliter drops of HTF medium. The rates of IVF embryos that developed to morula, blastocyst, expanded blastocyst and hatched blastocyst stages were decreased significantly (P < 0.01) by culturing embryos with unfertilized oocytes compared with culturing embryos alone. In experiment 3, groups of one or 10 IVF zygotes or 10 IVF zygotes plus 10 unfertilized oocytes were cultured in 20 microliter drops of HTF medium and the number of cells per blastocyst was examined at 120 h after IVF. Increasing embryo density resulted in a significant (P < 0.05) increase in the number of cells per blastocyst. In contrast, the cell number of IVF embryos that developed to blastocyst decreased significantly (P < 0.05) when they were cultured with unfertilized oocytes. The results suggest that in-vitro development of IVF mouse embryos is enhanced by increasing embryo density and is impaired by co-culture with unfertilized (degenerating) oocytes.

Prostaglandin E 2 (PGE 2 ), which is generated by two isoforms of cyclo-oxygenase (COX 1 and COX 2 ), is a key mediator in gastric mucosal defense. In the present study, antral mucosa of guineapigs was incubated with various agonists or antagonists in a medium, the PGE 2 concentration of which was measured using a PGE 2 EIA kit. Prostaglandin E 2 was released from the antral mucosa spontaneously (basal PGE 2 release) and acetylcholine (ACh, 10 μM) enhanced the PGE 2 release (ACh-stimulated PGE 2 release) was mediated via intracellular Ca 2+ concentration ([Ca 2+ ] i ). Arachidonic acid enhanced both forms of PGE 2 release, and a phospholipase A 2 inhibitor (amylcinnamoyl anthranilic acid) and COX inhibitors (acetylsalicylic acid and indomethacin) decreased them. 5-(4-Chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazol (SC560, 100 nM, a COX 1 -selective inhibitor) inhibited ACh-stimulated PGE 2 release without any decrease in basal PGE 2 release. N -(2-Cyclohexyloxy-4-nitrophenyl) methanesulphonamide (NS398, 20 μM, a COX 2 -selective inhibitor) decreased basal PGE 2 release without any reduction of AChstimulated PGE 2 release. However, ionomycin (a Ca 2+ ionophore) increased PGE 2 release from antral mucosa in the presence of SC560 or NS398, suggesting that COX 1 and COX 2 are regulated by [Ca 2+ ] i . These findings indicate that COX 1 -containing cells have ACh receptors but COX 2 -containing cells do not. Moreover, in isolated antral epithelial cells, SC560 decreased basal and ACh-stimulated PGE 2 release, but NS398 did not. In conclusion, in antral mucosa, basal PGE 2 release is mainly maintained by COX 2 of non-epithelial cells, and ACh-stimulated PGE 2 release is maintained by COX 1 of epithelial cells.