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K. Hashizume and K. Ōhashi

Summary. In about 60% of rats that were paired with fertile males before vaginal opening, vaginal opening occurred between 15:00 and 21:00 h under controlled lighting conditions (lights on 06:00–18:00 h). No rats mated before 12:00 h or after 03:00 h. Most of the rats (52/82) mated within 3 h after vaginal opening. About 90% of the rats that were paired with fertile males before vaginal opening mated and most conceived. Serum LH and FSH levels rose from 14:00 h to a peak at 18:00 h, whereas hypothalamic LHRH content suddenly decreased by 18:00 h.

This study shows that the timing of sexual receptivity, ovulation and the release of gonadotrophins during puberty are similar to those at pro-oestrus in adult rats and suggest that the diurnal rhythm of hormonal changes plays an important role in timing of the first oestrus.

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K. Hashizume, S. Sugawara and S. Takeuchi

Laboratory of Animal Reproduction, Faculty of Agriculture, Tohoku University, Sendai, Japan

We have previously described studies of parturition and subsequent reproductive phenomena in rats which showed that the time of the LH surge and post-partum ovulation are related to the time of parturition (Hashizume, Sugawara & Takeuchi, 1973a, b, 1975). In an attempt to clarify further the factors governing the timing of the post-partum ovulation in rats, we have examined the effects of an oestrogen antagonist, clomiphene (see Labhsetwar, 1970), on the time of parturition and the post-partum ovulation.

Rats of the Wistar strain, bred in our own laboratory and kept in controlled lighting (lights on 06.00-18.00 hours), were used. Rats which had experienced three consecutive 4-day cycles were caged at pro-oestrus with fertile males and mating was verified by finding spermatozoa in the vaginal smear the following morning. This day was designated Day 1 of pregnancy.

Clomiphene citrate (Clomid

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H Nakano, A Shimada, K Imai, T Takahashi and K Hashizume

Binucleate cells in ruminant placenta are differentiated from fetal mononucleate trophoblast cells and secrete many glycoproteins. This study characterized bovine placental binucleate cells in primary culture and a bovine trophoblastic cell line (BT-1) with an N-acetyl-galactosamine-binding lectin, Dolichos biflorus agglutinin (DBA). DBA specifically bound to the surface membrane and the cytoplasm of binucleate cells. Mononucleate epithelial cells and fibroblasts were free of DBA. DBA-positive binucleate cells corresponded to the fully matured cells, producing placental lactogen, and the cytoplasm was devoid of cytokeratin. Binucleate cells assumed a flattened shape on a collagen substratum in an extended culture, and entered a dedifferentiated state with a degranulation of placental lactogen. In these flattened cells, DBA reactions were attenuated in the cytoplasm. DBA binding in BT-1 was subsequently examined. BT-1 was derived from blastocysts produced in vitro and is trophectodermal as shown by the expression of cytokeratin. BT-1 was able to differentiate into placental lactogen-producing binucleate cells on a collagen gel substratum. Cytokeratin expression in BT-1 was downregulated with the differentiation into binucleate cells. However, DBA bound to neither mononucleate nor the differentiated binucleate cells in BT-1. These results indicate that binucleate cells in vivo but not binucleate cells derived from BT-1, specifically developed glycoconjugates recognized by DBA. The glycoconjugate expression was associated with fully differentiated cells. The onset of DBA binding in binucleate cells coincides with placental development, and binucleate cells differentiated from BT-1 cell cultures may reflect those cells at earlier stages of gestation.

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K Kizaki, H Nakano, H Nakano, T Takahashi, K Imai and K Hashizume

This study reports the identification and sequence of a partial cDNA for bovine heparanase and the expression of its mRNA in the placenta during gestation. The 364 amino acid residues deduced from the 1092 bp cDNA fragment share 81.9% and 80.5% identity with amino acid sequences of human and rat heparanase, respectively. Northern blot hybridization showed that two mRNAs (2.0 and 3.5 kb) are strongly expressed in placenta, and weakly expressed in the kidney, lung, spleen and non-pregnant uterus. In the placenta, these transcripts were detected in the cotyledon at all stages of gestation examined, and in the intercotyledonary fetal membrane and caruncle on day 60, day 120 and day 260. Quantitative real-time RT-PCR analysis showed very low expression of heparanase mRNA in the conceptus before implantation (day 17), but high expression in the cotyledon-containing fetal membrane (days 27-34) after implantation. Furthermore, heparanase mRNA was detected in the cotyledon, intercotyledonary fetal membrane and caruncle after days 60-64 of gestation. However, no significant expression of heparanase mRNA was observed in intercaruncular endometrium at all stages of gestation examined. These results demonstrate that heparanase mRNA is expressed in the placentome, indicating that heparanase may play a role in implantation, and in placental development and function.

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O Yamada, J Todoroki, K Kizaki, T Takahashi, K Imai, OV Patel, LA Schuler and K Hashizume

The bovine placenta secretes multiple molecules during implantation and placentation, many of which are produced by binucleate cells. In this study, production of prolactin-related protein I (PRP-I), a member of the non-classical prolactin-related family, was investigated during the implantation period in cows. Expression of bovine PRP-I (bPRP-I) in the placentome was examined during the preimplantation (days 17-19), implantation (days 20-25) and post-implantation (days 30-60) periods by immunohistochemistry, immunofluorescence and in situ hybridization. During the preimplantation period, both bPRP-I and bovine placental lactogen (bPL) were undetectable in trophoblastic cells. Both bPRP-I mRNA and protein appeared first at day 20 of gestation in trophoblastic binucleate cells and multinuclear cells that might migrate into the endometrium and fuse to epithelium; however, no bPL was detected in binucleate cells at this time. After implantation, on day 30, both bPRP-I and bPL were detected in binucleate cells and were co-expressed in the same cells. These data indicate that bPRP-I may play a role before implantation and that bPRP-I may be an excellent marker for trophoblastic cell differentiation, as well as a candidate for pregnancy diagnosis.