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K. R. STEVENS, H. D. HAFS and K. T. KIRTON

Summary.

The pH, protein concentration, and amount of oestrous uterine fluid were determined in mature Dutch type rabbits from 1 to 20 weeks after ligation of the uteri. Uterine fluid accumulated at the rate of 2·10 to 4·48 ml per cornu per week. These fluids were serous, slightly turbid, and colourless or slightly yellow. The protein concentration decreased from 5·13 mg/ml after the first week to 1·82 mg/ml after the 20th week of accumulation. The high initial protein concentration may have been due to contamination of the uterine fluid with blood at the time the uterus was ligated. The in-utero pH of fluids was 7·64. A rapid increase in pH with time and handling after removal from the uterus indicated the importance of carbon dioxide in buffering uterine fluids.

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K. R. STEVENS, H. D. HAFS and A. G. HUNTER

Summary.

Fluids were obtained from 185 rabbit uteri 1 to 20 weeks after uterine ligation. The uterine fluid proteins were concentrated with an ultrafilter and characterized by diffusion in agar gel, moving boundary electrophoresis, and immunoelectrophoresis. Eight electrophoretic components were identified by means of moving boundary electrophoresis. One which migrated as a pre-albumin and another which migrated as an alpha-globulin were not found in blood sera. Agar-gel diffusion tests revealed thirteen antigenic components in uterine fluid. Three precipitin lines appeared to be specific to uterine fluid after absorption of guinea-pig antisera to rabbit uterine fluid with rabbit blood sera. However, at least five antigens which could not be found in blood sera were identified in uterine fluid by means of immunoelectrophoresis. The mobilities of two were similar to prealbumins and the mobilities of the remaining three were similar to beta-globulins. The results indicated that at least two classes of proteins, which do not exist in rabbit blood serum, may be found in uterine fluids obtained by ligation.

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CAROLE A. SAMUEL, W. R. ALLEN and D. H. STEVEN

Summary.

By the 60th day of gestation, the microcotyledons of the equine placenta can be recognized as a simple folding of the trophoblast and adjacent uterine epithelium. Secondary folds are evident by Day 100. By 150 days, further complex folding has resulted in the formation of mature multibranched villous structures each surrounded by a maternal connective tissue capsule.

There is no evidence to suggest that the microcotyledons are formed by a grouping of chorionic villi around the mouths of the uterine glands. Each microcotyledon develops from a number of primary folds which are related to small aglandular areas of the endometrium.

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Carole A. Samuel, W. R. Allen and D. H. Steven

Summary. In early pregnancy the equine placenta consists of a simple apposition of fetal and maternal epithelia, but it becomes more complex with the formation of microcotyledons between 75 and 100 days of gestation. Although the placental barrier maintains an epitheliochorial arrangement throughout the course of pregnancy, a thinning of the maternal epithelium and a progressive indentation of the chorionic epithelium by fetal capillaries shortens the length of the diffusion pathway and reduces the amount of placental tissue between fetal and maternal bloodstreams. These structural modifications may reflect the changing requirements of the fetus for O2 and other metabolites as gestation proceeds.

During the first 200 days of pregnancy there is evidence of intense pinocytotic activity by the cells of the trophoblast. From the 100th day of pregnancy there is a pronounced development of smooth endoplasmic reticulum, while rough endoplasmic reticulum and irregular, dense, membrane-bound bodies are a prominent feature of the paranuclear cytoplasm from Day 200. These changes suggest that the cells of the trophoblast become more highly involved in synthetic processes with increasing gestational age.

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Carole A. Samuel, W. R. Allen and D. H. Steven

Summary. Ultrastructural studies of the uterine glands at intervals during pregnancy in the mare show that secretory activity continues after formation of the placental exchange units. The nature of the glandular secretion appeared initially to be proteinaceous, but cellular debris was also present during the last third of gestation. These secretions were absorbed by the trophoblast overlying the mouths of the glands. The fate of the secretions and their significance for the fetus and placenta are unknown.

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R. J. AITKEN, J. BURTON, J. HAWKINS, R. KERR-WILSON, R. V. SHORT and D. H. STEVEN

Summary.

The ultrastructure of four roe-deer blastocysts at different stages of embryonic development were studied. During delayed implantation, the outer surface of the trophoblast possessed numerous microvilli and periodic invaginations or caveolae. There was a marked lack of organelles such as mitochondria or endoplasmic reticulum in the cytoplasm of the trophoblast cells, though many lipid droplets, granular inclusions and a lamina of fine fibrillae were present. Elongation of the blastocyst was associated with a decrease in the size and number of the microvilli, the disappearance of lipid droplets and granular inclusions, a reduction in the amount of fibrillar material and a dramatic increase in the development of mitochondria, granular endoplasmic reticulum, ribosomes and Golgi apparatus.

The histology of the ovaries and uterus was studied in thirty-one roe deer. No prominent changes occurred in the ovaries at any stage of development; all ovaries possessed active CL and showed signs of follicular growth and atresia. Changes in the degree of mitotic activity, epithelial cell height, endometrial vascularity and stromal oedema were observed in the uterus throughout the period of delayed implantation and during the phase of rapid embryonic growth. Elongation of the embryo was associated with a marked decline in the height of the glandular epithelium and an increase in endometrial vascularity.

The most important ultrastructural changes in the uteri of six roe deer were observed in the endometrial glands. Delayed implantation was associated with the accumulation of numerous supranuclear vesicles derived from the Golgi apparatus, while the resumption of embryonic growth was correlated with their sudden disappearance. When elongation had been completed, there was a sudden decrease in the cellular activity of these endometrial glands.

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Byeong-Gyun Jeon, Gianfranco Coppola, Steven D Perrault, Gyu-Jin Rho, Dean H Betts and W Allan King

The poor outcome of somatic cell nuclear transfer (SCNT) is thought to be a consequence of incomplete reprogramming of the donor cell. The objective of this study was to investigate the effects of treatment with S-adenosylhomocysteine (SAH) a DNA demethylation agent, on DNA methylation levels and X-chromosome inactivation status of bovine female fibroblast donor cells and the subsequent impact on developmental potential after SCNT. Compared with non-treated controls, the cells treated with SAH revealed (i) significantly (P<0.05) reduced global DNA methylation, (ii) significantly (∼1.5-fold) increased telomerase activity, (iii) diminished distribution signals of methylated histones H3-3mK9 and H3-3mK27 on the presumptive inactive X-chromosome (Xi), (iv) alteration in the replication pattern of the Xi, and (v) elevation of transcript levels for X-chromosome linked genes, ANT3, MECP2, XIAP, XIST, and HPRT. SCNT embryos produced with SAH-treated donor cells compared with those derived from untreated donor cells revealed (i) similar cleavage frequencies, (ii) significant elevation in the frequencies of development of cleaved embryos to hatched blastocyst stage, and (iii) 1.5-fold increase in telomerase activity. We concluded that SAH induces global DNA demethylation that partially reactivates the Xi, and that a hypomethylated genome may facilitate the nuclear reprogramming process.