Luteotrophic effect of decidual tissue was examined in Holtzman rats by observing length ofpseudopregnancy or by measuring progesterone and 20α-hydroxyprengn-4-en-3-one (20a-OHP) in>ovarian effluent blood plasma. The length of pseudopregnancy was significantly longer in traumatized rats than in hysterecto->mized-pseudopregnant animals (23.0±0.97 υs 19.3±0.93; mean±SE). When deciduomectomy (removalof uterus bearing deciduomata) was performed on days 7 and 9, pseudopregnancy lasted only 17.4±0.92 and 19.4±1.25 days, respectively, which were similar to the value observed for hysterecto-mized-pseudopregnant animals. Progestin levels in both the traumatized and the hysterectomized-pseudopregnant rats were>similar to each other on day 7. An increase in progestin levels, however, occured in traumatizedanimals from days 7 to 9 of pseudopregnancy and the levels of progesterone and 20α-OHP on day >9 were significantly higher in the animals with deciduomata by 1.6- and 2.2-fold, respectively, ascompared with those in hysterectomized-pseudopregnant ones. Following deciduomectomy on day 7, the increase in each progestin level from days 7 to 9 was not seen. On the other hand, hypophy->sectomy performed on day 7 in traumatized rats resulted in a dramatic decrease in progesteronelevel coincident with an increase in 20α-OHP level on day 9 without changing total progestin level(sum of progesterone and 20α-OHP). Thus, the luteotrophic action of decidual tissue was suggested by the prolongation of pseudo-pregnancy and the elevation of ovarian progesterone secretion, and it was not seen in the absence of pituitary.
The patterns of TSH, LH and FSH levels after TRH injection were studied in female rats in order to know if TRH induces the release of gonadotropin concurrently with TSH. TRH were subcutaneously injected to immature (2729 days of age, 6080 g b. w.) and adult (180250 g b. w. in the late diestrus state with the correct 4 days cycle) female rats of Wistar strain at the different dose as shown in Table 13. Three to 5 animals consist of each group. Rats were killed by bleeding at different times (Tables 13) after injection, and serum levels of TSH, LH and FSH were measured by radioimmunoassay. TSH release was evoked 5 min after injection of more than 2 μg TRH in immature rats. In adults, TSH release was shown 30 min after injection of 0.2 and 2 μg TRH and 5 min after injection of more than 20 μg TRH. The time of recovery of TSH levels to 0 min had some relation with TRH dose in both female groups. LH and FSH levels were not affected by TRH treatment. It is concluded that higher blood TSH levels are not necessarily followed by higher gonadotropin levels although UMEZU et al.9) observed higher LH levels with elevated TSH in immature female hypothyroid rats. The authors acknowledge the gift of rat TSH, LH and FSH radioimmunoassay materials from the Rat Pituitary Hormone Distribution Program of the National Institute of Arthritis, Metabolism and Digestive Diseases.
Preovulatory changes and the time of ovulation were studied in adult rats treated with various doses (0 to 50 i. u.) of gonadotrophins. A total of 235 rats were raised under artificial lighting condition (darkness from 1 a. m. to 11 a. m.). They were injected with pregnant mare serum gonadotrophin (PMS) at 3 p. m. in early diestrus and human chorionic gonadotrophin (HCG) at 9 p. m. in proestrus (at 54 hours' interval, or 4 hours before the beginning of darkness). These rats were killed 12, 16, and 20 hours after HCG injection (or 8, 12, and 16 hours after the beginning of darkness). At autopsy, ovulated ova were counted. Then the ovaries of each rat were fixed in Bouin's solution and stained with Heidenhain iron hematoxylin. In them, oocytes in follicles of 300μ or over in diameter were examined for the stage of maturation division. The results obtained are as follows. 1. In untreated rats ovulation was almost completed 8 hours after the beginning of darkness.It was delayed, however, in superovulation-treated rats even when an appropriate dose of HCG (more than 1/2 or 1/3 of the dose of PMS) had been injected. The time of complete ovulation in relation to the HCG injection (time in hours after the beginning of darkness is shown in parenthesis) was estimated as follows: a dose of 10 i. u. of PMS, 16 (12) hours; a dose of 20 i. u. of PMS, 16 (12) hours or later up to 20 (16) hours; a dose of 30 to 50 i. u. of PMS, about 20 (16) hours. 2. The percentage of a total of mature and ovulated oocytes to a total of oocytes in follicles of 300 μ or over in diameter was 48.2, 35.6, 28.1, and 19.2% in rats injected with 50, 30, 20, and 10 i. u. of PMS, respectively, together with the same dose of HCG. It was 15.8% in the untreated control rats. In those treated rats, the percentage was reduced with decrease in the dose of HCG. It was reduced slightly, however, when rats had been treated with such a dose of HCG as cor-responding to more than 1/2 of the dose of PMS. (It was 92.5 to 97.8, as expressed by index to 100 which represented the percentage in the case of injection with th same doses of PMS and HCG.) 3. The correlation (γ) between a sum total of mature plus ovulated oocytes and a total of oocytes contained in follicles of 300 μ or over in diameter was calculated to be 0.787, 0.555, and 0.288 in rats treated with 50, 30, and 20 i. u. of PMS, respectively, together with more than 1/2 of the respective dose of HCG. From these results, it seems that the number of ovulated ova may depend on the number of oocytes contained in large follicles of the ovaries in superovulation-treated rats. At present, however, it is difficult to draw any conclusion. Further studies are needed to verify this presumption.
Present study was designed to isolate immunoglobulins from serum and colostrum whey, and determine immunoglobulins in uterine fluid of proestrous rat. Results obtained were as follows: 1. Subclass of IgG and IgA in serum and colostrum whey were obtained by DEAE cellulose, sephadex-G 200 and sepharose-6B column chromatography (Figs. 13). 2. Physicochemical analysis of immunoglobulins were performed by SDS acrylamide gel electro-phoresis and density gradient ultracentrifugation methods (Figs. 4, 5). Molecular weight of isolated IgG and IgA was about 16 × 104 and 50 × 104, respectively. 3. Immunoglobulins in uterine fluid were determined by immunoelectrophoresis. Although high molecular IgA in uterine fluid determined by physicochemical analysis was very small amount, uterine fluid contained high level of the protein component reacted to serum IgA, of which molecular weight is smaller than 20 × 104. Immunoglobulin G was not appeared in the rat uterine fluid at 22:00 of proestrus.
In previous papers15), an alteration of protein in rat uterine fluid, and physicochemical and immunological analysis of protein components in uterine fluid and serum were studied. In the present report, it was designed to compare uterine fluid with serum other external secretions for immunoglobulins and other protein components. Results obtained were as follows: 1. Protein components in serum and colostrum whey fractionated by sephadex-G200 column chromatography, were analyzed by disc electrophoresis and immunoelectrophoresis (Figs. 13). Uterine fluid and intestinal fluid (Figs. 4 & 6) were resolved into only two peaks although the fractionation of the uterine fluid protein by the use of sepharose-6B column chromatography obtained several peaks (Fig. 5). Their protein patterns represented the immunoglobulins and specific protein components in each secretions. 2. Physicochemical properties of proteins in serum, uterine fluid, colostrum whey and intestinal fluid were furthermore analyzed by means of SDS gel electrophoresis. Specificity and community of protein components in each secretions were showed in Fig. 7. 3. It was confirmed by immunochemical analysis that some of the specific protein in uterine fluid appeared in other external secretions (Fig. 8).
Prepuberal gilts about 180 days of age were injected with 750 iu pregnant mare serum gonadotrophin (PMSG) and 500 iu human chorionic gonadotrophin (HCG) or 0.1 mg synthetic luteinizing hormone releasing hormone analog (LHRH-A) at 72 h interval (method A or C), or with mixed 400 iu PMSG and 200 iu HCG (method B) to compare the effect of methods for the induction of estrus, ovulation and subsequent fertilization after an artificial insemination at 98 h from the initiation of treatment.The effect of 10 mg estriol (ET) injection to each treatment above mentioned, given at 88 h from the initiation of treatment was also investigated (method A', B' and C'). All the treated gilts, 27 in total, showed red colouring and swelling of vulva which reached its maximum about 92 h to 100 h from the initiation of treatment. Standing reflex was not induced effectively, induced in only 30% of the gilts, and a pars of semen was frequently discharged from the vulva at the time of insemination. By ET injection the vulval reaction was augmented but retained longer and not so effective for the induction of standing reflex. When the gilts were killed at 141 h from the initiation of treatment, ovulation was induced in all the gilts except for two gilts by method B' where the ovulation appeared to be delayed by ET injection. Number of follicles ovulated was comparatively moderate (mean no. 8.6) but variable and did not differ significantly by the methods. Eggs were recovered quite efficiently from the oviducts of gilts treated by method A, B and C (96% to 100%), but less efficiently from the gilts after ET injection (50% to 62.5%). Fertilized eggs were recovered from 80% of gilts ovulated. The failure of fertilization in the others was considered not to be due to the methods used, but mainly attributable to the delayed ovulation from unknown reason. The fertilization rate of recovered eggs did not differ significantly by methods (overall mean 64.5%) except for those of method A where the rate was significantly higher (82%). However, the stage of development of fertilized eggs was significantly different by the methods used; the most advanced stage (61.4% were of cleaved) by method C and C', the middle stage (35.4% were of cleaved) by method A and A', and the delayed stage (all at pronuclear) by method B. This difference suggests that there may be subtle difference of ovulation time by induction methods used and careful choice of the timing of insemination may be necessary. Whether normal estrus can be cycled after the induced ovulation and whether high conception rate and normal range of litter size can be obtained are now under investigation.