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1 Exp. An i m. 34 (3), , 1985 Studies on Implantation Traces in Rats II. Staining of Cleared Uteri, Formation and Distribution of Implantation Traces Takashi YAMADA, Mariko HARA, Yuka OHBA, Tadahiro INOUE and Hiroshi OHNO Research Center of Taisho Pharmaceutical Co., Ltd., Yoshino-cho Ohmiya-shi Saitama 330, Japan. (Received 12 October 1984 / Accepted 3 April 1985) We made an investigation of implantation traces in delivered rats. 1. Nonfixed rat uteri were immersed in 2 %-NaOH solution for over one hour. The uteri were then cleared and the implantation traces were seen to be stained yellowish-brown. This staining method was convenient for observation of the implantation traces. 2. Stillbirth was induced by stabbing or crushing, either in some embryos on one day between the 6 th and 12 th day of gestation, or in all embryos after the 13 th day of gestation. Also, abortion was induced by stabbing or crushing all embryos before the 10 th day of gestation. 3. When live embryos existed in the uterine horns, abortion traces were not detected. 4. In cleared uteri stained with 2%-NaOH solution, abortion traces were observed as small globes, reddish-brown. Normal delivery traces were observed as large globes, yellowish-brown, covered with yellowish-white of agglomerate cells, while stillbirth traces appeared as middle-sized, orange or yellowish-brown masses. 5. Though the implantation traces in rats which had been delivered four times were arranged like beads, they were recognizable by NaOH stain as either old or new traces, the former appearing smaller without agglomerate cells and a little more brown in comparison with the latter. 6. As the implantation traces increased in the uterine horns they became distributed from the central region to the cervical end, and the mean space between them narrowed. The implantation traces occupying the ovarian end or central region of the uterine horns trendes smallest size or largest size. 7. The implantation traces were composed of cicatrical tissue, and the area they occupied did not show adhesion of the placenta. On the other hand, placenta adhering to the uterine wall proliferated, and formed new implantation traces which did not overlap the old traces after decollement of the placenta.

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3 Table 1. Experimental abortion induced by fetal stabbing or crushing in rats No. of frequency / No. of rats observed 1). Treatment : Stabbing was performed once during the period from day 6 to day 15 of gestation and crushing was performed on either day 6 or day 7 of gestation. 2). Fetuses were treated alternately from the cervical side. 3). Abortion (dead fetuses expulsed before day 19 of gestation) was confirmed by autopsy on day 20 of gestation. 4). Delivery included stillbirth (dead fetuses expulsed between day 20 to full term) was confirmed expulsion or autopsy after term.

4 252 Table 2. Intrauterine position of implantation traces in first delivered rats No. of rats observed : 31 (SLc : Wistar strain) 58 (Wistar-Imamichi strain) Trace position : Distance between cervical canal and center of implantation trace/length of uterine horn ~10 } standard deviation (cm) \ First delivery \: Second delivery \: Third delivery No. of rats observed : 31 (first), 6 (second) and 8 (third) [SLc : Wistar strain] O : Ovarian end M : Middle C : Cervical end Fig. 1. Distribution of implantation traces in rats delivered

5 253 Table 3. Mean spacing between implantation traces Distance : Mean (mm) } standard deviation No. of rats observed : 58 (Wistar-Imamichi) 31 (SLc : Wistar) Table 4. Mean spacing between implantation traces in different uterine positions

6 O : Ovarian end, OP: Ovarian penultimate, OI : Ovarian intermediate, M : Middle, CI: Cervical intermediate, CP : Cervical penultimate, C : Cervical end No. of observations : 499 traces, 68 horns, 36 rats (Wistar-Imamichi) Fig. 2. Relative frequency of largest or smallest implantation traces in intrauterine positions

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8 [1] Barr, MJ., and Brent, R. L. (1978). Uterine vascular interruption and combined radiation and surgical procedures. In Handbook of teratology, 4. Research procedures and data analysis. pp , Wilson, J. G. and Fraser, F. Clarke. (edit.). Plenum Press, New York and London. [2] Barr, M. JR., Jensh, R. P., and Brent, R. L. (1970). Placental growth in the albino rat : effects of number, intrauterine position and resorptions. Am. J. Anat., 128, [3] Barr, M. JR., Jensh, R. P., and Brent, R. L. (1969). Fetal weight and intrauterine position in rats. Teratofgy. 2, [4] Brent, R. L. (1965). Uterine vascular clamping and other surical techniques in experimental mammalian embryology. In Teratology-principles and techniques. pp , Wilson, J. G. and Warkany, J. (edit.). University of Chicago press, Chicago. [5] Davis, J., and Glassen, S. R. (1965). Morphological changes in the rat placenta after fetectomy. Anat. Rec., 151, 341. [6] Frazer, J. F. D. (1955). The site of implantation of ova in the rat. J. Embryol. Exp. Morph., 3, [7] Gibori, G., Antzak, E., and Rothchild, I. (1977). The role of estrogen in the regulation of luteal progesterone secretion in the rat after day 12 of pregnancy. Endo., 100, [10] Huggert, A. St. G., and Pritchard, J. J. (1945). Experimental foetal death, the surviving placenta. Proc. R. Soc. Med., 38, [11] Mclaren, A. (1965). Placental weight loss in late pregnancy. J. Reprod. Fertil., 9, [15] Norman, N. A., and Bruce, N. W. (1979). Fetal and placental weight relationships in the albino rat near term. Teratology, 19, [16] Shintani, S., Glass, L., and Page, E., (1966). Studies of induced malignant tumors of placental and uterine origin in the rat. I. Survival of placental tissue following fetectomy. Am. J. Obst. and Gynec., 95, [17] Shintani, S., Glass, L., and Page, E. (1966). Studies of induced malignant tumors of placental and uterine origin in the rat. II. Induced tumors and their pathogenesis with special reference to chorioarcinoma. Am. J. Obst. and Gynec., 95,

9 257 Explanation of Figures Fig. 3-1 Implantation traces stained with 0.2 (left) or 2 % (right) sodium hydroxide on the 21 st day after delivery I : Implantation trace 0: Ovary Fig. 3-2 Implantation traces in normal delivery (left), stillbirth (middle) or abortion (right) stained with 2 % sodium hydroxide on 21 st day after delivery I : Implantation trace 0 : Ovary Fig. 3-3 Implantation traces from first (left), second (middle) or third (right)-time deliveries with 2 % sodium hydroxide on the 21 st day after final delivery I : Implantation trace 0 : Ovary Fig. 3-4 Magnified view implantation traces from a third time deliveries F : First trace S : Second trace T : Third trace Fig. 3-5 Implantation traces (first delivery) and embryos on the 7 th day of gestation E : Live embryo I : Implantion trace Fig. 3-6 New (second delivery) and old (first delivery) implantation traces on the 4 th day after delivery N : New trace O : old trace

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