Summary. Preimplantation mouse embryos were recovered by flushing the oviducts on Day 3 at 09:30–10:00 h, 15:30–16:00 h and 21:30–22:00 h: When placed in culture for 48 h, 79% of the 4–8 cell embryos recovered at 09:30–10:00 h developed into blastocysts, but a large number of these embryos failed to form blastocysts when exposed to trifluoperazine, a calmodulin antagonist, at 0·5 or 0·6 μm in culture. About 45% of the embryos recovered at 15:30–16:00 h were compacted and blastocyst formation was again markedly depressed in the presence of the drug. Advanced compacted embryos recovered at 21:30–22:00 h showed normal development into blastocysts in the presence of 0·6 μm-trifluoperazine. Trifluoperazine sulphoxide (the inactive form of trifluoperazine) at 0·6 or 1·2 μm concentration had no effect on blastocyst formation of uncompacted embryos recovered at 09:30–10:00 h. These embryos and those recovered at 21:30–22:00 h and developed into blastocysts in the presence of trifluoperazine were transferred to Day-4 pseudopregnant mice and healthy young were born. When exposed to calcium-free medium or medium containing trifluoperazine all compacted embryos recovered at 18:30 h became decompacted; development to the blastocyst stage was normal in medium alone but reduced when trifluoperazine was added. Compacted embryos recovered at 23:00 h showed 100% decompaction in the calcium-free medium but completely failed to decompact in the presence of 0·6 μm-trifluoperazine. We suggest that extracellular calcium is essential for the continuance of compaction, while intracellular calcium is required only during the initial phase of this process.
P. L. Pakrasi and S. K. Dey
S. K. Dey and D. C. Johnson
Summary. Mouse embryos recovered on the 4th day of pregnancy produced histamine, as evidenced by the14CO2 produced from carboxy labelled l-histidine, at the rate of 1·5 ± 0·3 (s.e.m.) pmol/embryo per hour. Most (83·2 ± 4·6%) of the embryos flushed from the oviducts on Day 3 of pregnancy (4–8-cell stage) developed into blastocysts within 48 h after being placed in culture. Inclusion of l-histidine hydrochloride (4·7 × 10−4 m) in the culture medium did not alter this development but dl-α-methylhistidine (3·8 × 10−4 m), an inhibitor of histidine decarboxylase, reduced the number of embryos developing into blastocysts to only 10·8 ± 6·8%. A combination of l-histidine and dl-α-methylhistidine in the medium prevented the growth-retarding effect of the latter compound. The results indicate that mouse embryos can produce histamine and suggest that this is necessary for normal development.
Y. M. Huet and S. K. Dey
Summary. Oestrogen action in the uterus is expressed in an early phase (Phase I) and a late phase (Phase II). The role of this biphasic oestrogen action in implantation is not clear. To determine the relative importance of Phase I and II responses, triphenylethylene compounds (CI-628, LY-117018, nafoxidine, clomiphene citrate and tamoxifen) and oestrogens (oestriol and oestradiol-17β) were used in a physiologically relevant experimental system for studying implantation. All compounds elicited uterine water imbibition to various degrees in ovariectomized–progesterone-treated mice at 6 h (Phase I response) and their effectiveness in inducing implantation in delayed implanting mice correlated with their respective potency to increase uterine wet weight. This suggests that Phase I might be an essential component of oestrogen action in implantation and that the efficiency of a compound to elicit a Phase I response might serve as a predictive indicator of its potential action on implantation.
S. K. Dey and D. C. Johnson
Summary. The effect of adrenal steroids upon implantation was evaluated by examining the efficacy of oestradiol-17β on the initiation of implantation in ovariectomized, ovariectomized plus adrenalectomized or hypophysectomized pregnant rats treated with progesterone. More oestrogen (× 5) was required in ovariectomized animals to obtain results equivalent to those obtained with the other animal models.
M. T. McMaster, S. K. Dey, and G. K. Andrews
The distribution and activation of monocytes and neutrophils in the mouse uterus were examined early in the process of blastocyst implantation. Implantation regions, detected by increased capillary permeability at sites of blastocyst apposition to the uterine luminal epithelium, could be distinguished at about 21:00 h on day 4 of pregnancy (day 1 = day of vaginal plug), and were also examined at 01:00, 05:00 and 09:00 h on day 5. Serial sections containing the implanting blastocyst (implantation sites), and random sections of interimplantation regions were examined by immunohistochemistry using interleukin-1β as a marker for monocytes–macrophages and lactoferrin as a marker for neutrophils in the uterine stroma. At implantation sites, interleukin-1β-positive cells were transiently abundant within endometrial capillaries. In interimplantation regions only a few interleukin-1β-positive cells were dispersed at the myometrial–stromal junction. Northern blot hybridization to RNAs from implantation and interimplantation regions showed that the abundance of interleukin-1β and interleukin-1α mRNAs was much lower than that found in the uterus during acute inflammation. However, these cytokine mRNAs were more abundant in implantation regions. On the evening of day 4 and the early morning of day 5, lactoferrin-positive neutrophils were detected juxtaposed to the basolateral surface of the antimesometrial epithelial cells surrounding the implanting blastocyst. They were primarily at the myometrial–stromal junction in interimplantation regions. Metallothionein gene expression was examined as a marker for uterine responses to inflammatory reactions. In situ hybridization showed high metallothionein mRNA specifically in antimesometrial epithelial cells underlying the implanting blastocyst and in deeper stromal cells in the implantation region at 05:00 h on day 5. These data suggest that monocytes and neutrophils are transiently abundant near implantation sites, and neutrophils become closely associated with the luminal epithelium. Although monocytes are not highly active in cytokine gene expression, neutrophils and monocytes may play a role in a localized inflammatory response during the initiation of blastocyst implantation.
S. Long, S. K. Dey, and D. C. Johnson
Summary. An intravenous injection of 2-fluoro-oestradiol simultaneously with an implantation-inducing dose of oestradiol reduced the number of implantation sites in delayed implanting hypophysectomized rats maintained with progesterone. Administration of 2-fluoro-oestradiol 1 h before or after oestradiol had no effect. Furthermore, injection of as much as 500 ng 2-fluoro-oestradiol 48 h before administration of oestradiol failed to have any effect upon implantation, i.e. failure to block implantation was correlated with failure to induce the uterine refractory state. These results suggest that conversion of primary oestrogens to catechol oestrogens could be important for implantation as well as for the induction of the oestrogen refractory state in the uterus.
S. K. Dey, F. Kimura, A. Mukherjee, and Z. Dickmann
Summary. Concentrations of both nucleotides were significantly higher in Day-6 than in Day-5 blastocysts but the ratio of cAMP to cGMP changed from 0·5 to 1·5.
R. C. Hoversland, S. K. Dey, and D. C. Johnson
Summary. Rabbit blastocysts were homogenized by sonication, and centrifuged at 105 000 g for 60 min. The pellet was resuspended and incubated in phosphate buffer containing [1β-3H]testosterone and a NADPH generating system. The amount of 3H2O produced was determined by liquid scintillation counting. Enzyme activity was calculated, after subtracting blank values obtained with boiled embryos, and expressed as pg testosterone aromatized per embryo per hour. Aromatase activity was undetectable to low on Day 5 and increased on Day 6 of pregnancy. There was a 10-fold increase in activity in Day-6 embryos cultured for 24 h, with a further 6-fold increase in activity in Day-6 embryos cultured for 48 h. The enzyme had an apparent K m of 0·77 μm and was completely inhibited by an aromatase inhibitor. The results clearly indicate that the rabbit blastocyst has an increasing capacity for aromatization of testosterone at about the time of implantation.
S. K. Dey, C. Villanueva, S. M. Chien, and R. D. Crist
Summary. When disodium cromoglycate, an inhibitor of histamine release, was instilled into the uterine lumen on Days 5 or 6 of pregnancy, the number of blastocysts implanting was significantly (P < 0·002) reduced. Simultaneous instillation of histamine and disodium cromoglycate prevented the effect.
Amal Mukherjee, S. K. Dey, Jayasree Sen Gupta, C. S. Ramadoss, and Z. Dickmann
Summary. The activities of phosphofructokinase, pyruvate kinase, citrate synthase and creatine kinase were determined in blastocysts from rabbits at 144 h post coitum and in similar blastocysts cultured for 24 h with or without oestradiol-17β (1 μg/ml). There was a significant increase in all the enzymes during the 24-h culture period but oestradiol had no effect.