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S. R. Milligan and L. Martin

Summary. The hydrostatic pressures generated during controlled flushing of the mouse uterus increased at implantation and under conditions of uterine closure. These pressures may be responsible for inducing tissue damage during flushing. The possibility that samples collected by flushing might be contaminated with interstitial fluid or plasma was studied using intravenously administered51 Cr-labelled EDTA and 125I-labelled human serum albumin as markers. The presence of both tracers was detected in all flushings and was greatest in flushings from uteri with luminal closure and early implantation sites. These observations raise serious doubts about the validity of the flushing technique for analysing uterine luminal constituents in mice.

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S. R. Milligan and C. Edwards

Summary. Changes in the extracellular and blood spaces of the uterus were assessed from the distribution volumes of 51Cr-EDTA and 51Cr-labelled red blood cells during the development and regression of the artificially induced decidual cell reaction in ovariectomized, steroid-treated mice. The normally high values for uterine extra cellular space (0·35–0·40 μl/mg) fell to less than 0·20 μl/mg in association with decidual growth. Uterine blood space increased from around 0·02 μl/mg to 0·03–0·05 μl/mg with decidual development. Induction of decidual regression by removal of s.c. progesterone implants caused a rapid decline in tissue blood volume to reach control values (0·01–0·02 μl/mg) within 24 h and preceded any reduction in uterine weight. Uterine vascular permeability, as determined from the tissue accumulation of 125I-labelled human serum albumin, fell with a similar time course. Tissue extracellular space returned to the higher control values within 48 h of initiating decidual regression.

Keywords: mouse; decidual cell reaction; tissue space

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C. Edwards and S. R. Milligan

Summary. A tissue-sampling paired-tracer method was used to investigate the effect of plasma proteins on uptake by the decidualized endometrium of [3H]progesterone, [3H]oestradiol and [3H]corticosterone. When injected arterially in protein-free Ringer, the extraction of progesterone and oestradiol was 100%, while that of corticosterone was only 60%. The addition of 4% albumin or injection in mouse plasma resulted in significant decreases in progesterone extraction to about 80% and 65% respectively. Injection in pregnant guinea-pig plasma reduced progesterone extraction further (to 33%). While neither 4% albumin nor mouse plasma had any significant effect on the uptake of oestradiol, neonatal rat plasma reduced oestradiol extraction to 40%. These results are consistent with high-affinity binding proteins having a limiting effect on the availability of steroids to target tissues.

Keywords: mouse; uterus; decidua; steroid-uptake; plasma proteins

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S. R. Milligan and C. A. Finn

Summary. The possible role of platelet-activating factor (PAF) in the uterine responses associated with implantation was investigated. Attempts to trigger a decidual cell response in the uteri of hormonally sensitized, ovariectomized mice by instilling PAF-acether (1–1000 ng) intraluminally were unsuccessful. The effect of PAF antagonists on implantation was investigated in females ovariectomized on Day 3 of pregnancy and treated with progesterone. Implantation was induced in these females by injection of 10 ng oestradiol-17β on Day 8. Hourly intraperitoneal injections of three PAF antagonists (WEB 2086, CV 3988 and BN 52021 at doses of 1·2–1·4 mg/kg) given over a 24-h period starting 1 h before the injection of oestradiol-17β had no significant effect on the occurrence of implantation sites. Intraluminal injection of WEB 2086 (15 μg) or BN 52021 (5 μg) either 3 h before or 6 h after the nidatory oestradiol also had no significant inhibitory effect on implantation. SRI 63-441 given once daily over the first 4 days of pregnancy at a dose of 40 μg/30 g body weight had no inhibitory effect on the establishment of pregnancy. These results are not consistent with a critical role for PAF in implantation in mice.

Keywords: PAF; implantation; mouse; decidualization; PAF antagonists

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S. R. Milligan and Pamela C. B. MacKinnon

Department of Agricultural Science and Department of Human Anatomy, University of Oxford, Oxford, U.K.

Ovulation in the vole (Microtus agrestis) occurs about 9-12 hr after mating (Austin, 1957; Breed & Clarke, 1970). Following ovulation, the CL may be `short-lived' and start to degenerate within 48 hr of their formation, or become fully functional when the animal becomes pregnant or pseudopregnant (Milligan, 1974, 1975a). The fate of the CL is determined by whether mating activates a reflex mechanism separate from that controlling ovulation; limited amounts of mating, e.g. a single intromission, often give rise to only short-lived CL and greater amounts of mating stimulate luteal function more consistently (Milligan, 1975a). The hormonal basis of such observations was investigated in the present study by relating the plasma levels of LH and prolactin after mating with the fate of the resulting CL.

Laboratory-bred voles (see Breed, 1969) were used ; females were mature virgins

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S. R. Milligan and Florence M. Mirembe

Summary. Uterine vascular permeability and tissue blood volume during the development of the oil-induced decidual cell reaction (DCR) in ovariectomized steroid-treated rats were assessed by measuring the extravascular accumulation of 125I-labelled human serum albumin and the tissue content of 51Cr-labelled red cells 30 min after intravenous administration. Within 15 min of oil instillation into one uterine horn, the vascular permeability of the horn was significantly elevated. Permeability rose to a sharp peak (10 times control levels) 9 h after oil instillation, but dropped to 5 times control values by 12 h and continued a steady decline over the next 7 days. Although a marked increase in uterine weight was associated with the development of the DCR, there was no significant change in blood volume/g tissue until 4 days after oil instillation.

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S. R. Milligan and Fiona D. C. Lytton

Summary. Intrauterine instillation of oil, but not saline, induced both a decidual cell reaction and a marked elevation in the uterine PGF-α content of suitably sensitized ovariectomized mice. Uterine PGF-α concentrations were elevated within 5 min of the oil instillation, reached maximal levels within 30–60 min and then declined to near baseline levels again by 3 h. A similar increase in uterine PGF-α content in response to oil instillation was seen in non-sensitized females, although no decidual cell reaction developed. No significant changes in PGE or 6-oxo-PGF-1 content were observed. These results suggest that although the increase in uterine PGF-α content is not solely due to the distension of the uterus after intrauterine injection, the increase is not necessarily sufficient to induce a decidual cell reaction.

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P. E. Cohen and S. R. Milligan

Silastic implants containing oestradiol were developed for delivering a range of physiological concentrations of oestradiol to mice over long periods. The implants consisted of discrete lengths of Silastic tubing containing oestradiol in arachis oil, with a small reservoir of the oestradiol solution at either end of the implant. Studies showed that the release of oestradiol in vitro was proportional to the concentration of steroid within the implant. Implants containing oestradiol at concentrations from 1 to 100 μg ml−1 could induce biological responses in ovariectomized mice, ranging from minimal effects on uterine weight and vaginal smears to supraphysiological increases in uterine weight and rapid vaginal cornification. Studies of uterine vascular permeability indicated that significant effects occurred within a few hours of initial placement of the implant. These results suggest that the design of the Silastic implants described in this study provides a useful method for delivering controlled and easily manipulated physiological doses of oestradiol to mice.

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S. R. Milligan and Florence M. Mirembe

Summary. After suitable sensitization of ovariectomized mice with progesterone and oestradiol, the intrauterine instillation of oil produces a massive decidual cell reaction. Vascular permeability, as reflected by the extra-vascular accumulation of 125I-labelled human serum albumin, increased after oil instillation and was maintained at 2–3 times control values for at least the next 3 days. Although oil instillation did not produce a decidual response in females treated with progesterone alone, an increase in vascular permeability (about 2 times control levels) still occurred. This response peaked 8 h after oil instillation and was not maintained. These results indicate that the progesterone-dominated uterus which has not been sensitized with oestradiol cannot be viewed as completely unresponsive to the stimulus of oil and demonstrate that a marked increase in vascular permeability is not itself sufficient to induce decidualization of progesterone-dominated uterine stromal cells. The uterine extravascular accumulation of 125I-labelled albumin was increased both in association with tribromoethanol anaesthesia and after oestradiol treatment of progesterone-primed animals. In pregnant mice, the appearance of Pontamine Sky Blue spots provided an earlier indication of implantation than did determination of total uterine extravascular 125I-labelled albumin accumulation.

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C. A. Finn, M. Pope, and S. R. Milligan

The relationship between mitosis, ovarian hormones, decidual stimuli and decidualization was investigated using progestagen-treated ovariectomized mice. Oestradiol, or the intraluminal instillation of oil or saline, all stimulated stromal mitosis. When oil or saline was instilled following oestradiol, the response depended on the dose of oestradiol, the interval between the oestradiol and the instillation, and the time when the mice were killed. After 20 ng oestradiol, the instillation of oil 7 h later induced large mitotic and decidual responses that were evident within 17 h of instillation and increased with time. Smaller mitotic and decidual responses were obtained when the interval between oestradiol and oil was 24 h; there was no response when the interval was 42 h. When a higher dose (100 ng) of oestradiol was given, oil injected 7 h later initially stimulated mitosis (at 17 h), but this effect was reduced at 24 h and no decidualization occurred. After instilling oil 24 or 42 h after 100 ng oestradiol, the mitotic response was limited, and there was no decidual response. Regardless of the dose of oestradiol, saline induced only a transient mitotic response and no decidualization occurred. It is concluded that there are three stimuli that can cause stromal mitosis in the progestagen-treated mouse uterus: oestrogen, an intraluminal stimulus (blastocyst, oil or saline) and factors associated with decidualization. Oestradiol not only induces mitosis, but also produces a period of heightened sensitivity to the mitotic effects of intraluminal stimuli. In addition, low doses of oestradiol induce a period of sensitivity to decidual stimuli. When these phases of uterine sensitivity to intraluminal stimuli have passed, decidualization and mitosis within the stroma can continue, presumably stimulated by local signals from the decidua in a self-propagating cascade of cellular proliferation and differentiation.