Hormonal and local factors control the immunohistochemical distribution of immunocytes in the rat uterus before conceptus implantation: effects of ovariectomy, fallopian tube section, and injection.

The distribution of immunocytes in the rat uterus undergoes profound changes during early pregnancy. This study was designed to evaluate the respective contributions of hormonal and local factors to regulation of the distribution and number of MCA341+ monocyte-macrophage antigen-bearing cells and T-lymphocyte-polymorphonuclear leukocyte (PMN) antigen-bearing cells before and during implantation of the fertilized ovum. Immunohistological data in normal rat pregnancy were compared to those found in cycling rats, ovariectomized rats, pseudopregnant rats (the oviducts of which had been sectioned on Day 0.5 of pregnancy), and pregnant rats injected with the antiprogesterone RU-486 on Day 0.5 of pregnancy. Four major events were observed: (1) transient accumulation of T-lymphocyte-PMN antigen-bearing cells in the endometrium close to the lumen and occurring only in the pregnant state 12 h after mating; (2) accumulation of an MCA341+ antigen-bearing monocyte-macrophage subset in the uterus, especially the luminal endometrium, 12 h after ovulation in pregnant as well as cycling rats; (3) progressive disappearance of these labeled cells starting 1 day after ovulation in the pregnant and nonpregnant states and influenced by RU-486 injection; (4) relative persistence of labeled cells in the deep endometrium before the implantation of the conceptus--which requires the presence of fertilized ovum in the genital tract. In conclusion, a complex multifactorial and sequential control of the distribution and number of cells bearing MCA341+ monocyte-macrophage or T-lymphocyte antigens appears to be at work before and during implantation of the rat conceptus, and may involve hormonal factors as well as local factors produced by the embryo or trophoblastic cells.     

Nucleic acid metabolism of cells of the luminal epithelium and stroma of the rat uterus during early pregnancy.

The uptake of [5-3H]uridine into RNA and DNA of the cells of the uterine luminal epithelium, stroma and myometrium of the rat has been studied in early pregnancy using a technique for separation of the cell fractions before quantitative analysis. Comparisons of the metabolism between the pregnant and pseudopregnant horn of the unilaterally ovariectomized rat has shown that RNA and DNA synthesis are markedly increased by 04.00 hours on the morning of Day 5 in the pregnant horn. This increased metabolism occurs in all cell fractions and before the zona pellucida is shed. The results are discussed in relation to the onset of the decidual response.     

Differential expression of prostaglandin E receptor subtype EP2 in rat uterus during early pregnancy

PGE2 is essential for mammalian female reproduction. This study was to examine the expression of EP2 gene in the rat uterus during early pregnancy, delayed implantation and artificial decidualization by in situ hybridization and immunohistochemistry. There was no detectable EP2 mRNA expression in the uterus from days 1 to 4 of pregnancy (day 1 = day of vaginal sperm). A low level of EP2 immunostaining was observed in the luminal and glandular epithelium from days 1 to 4 of pregnancy. Both EP2 mRNA and protein expression were highly detected in the luminal epithelium at implantation sites on day 6 of pregnancy. EP2 expression decreased from day 7 of pregnancy and was undetectable on days 8 and 9 of pregnancy. After delayed implantation was terminated by estrogen treatment and the embryo implanted, both EP2 mRNA and protein expression were strongly observed in the luminal epithelium at the implantation site. There was no detectable EP2 expression in both control and decidualized uteri. In conclusion, these data suggest that EP2 expression at implantation site may play an important role during embryo implantation in rats.     

Catecholoestrogen synthesis and metabolism in the rabbit uterus during the periimplantation period

Microsomal oestradiol-2/4-hydroxylase (OE-2/4-H) and cytosolic catechol-O-methyltransferase (COMT) (EC 2.1.1.6) activity in the uteri of pregnant and pseudopregnant rabbits during the periimplantation period were studied. The apparent Km for the 4-hydroxylation of oestradiol (3.18 microM) was considerably less than for the 2-hydroxylation reaction (13.36 microM), whereas the Vmax were almost equal. This suggests that 4-hydroxyoestradiol (4-OH-OE2) is the predominant product of OE-2/4-H in the rabbit uterus. These reactions were inhibited by SKF-525A, indicating the involvement of cytochrome P450 dependent monooxygenases. Uterine cytosolic COMT utilized 2-hydroxyestradiol (2-OH-OE2) as the preferred substrate as compared to 4-hydroxyoestradiol (4-OH-OE2). Since the rabbit uterus has a considerable capacity to synthesize 4-OH-OE2 and a lower capacity to metabolize it, it could be suggested that more 4-OH-OE2 than 2-OH-OE2 could be available to the uterus for its physiological activities. Furthermore, an increase in OE-2/4-H in Day 6 pseudopregnant and pregnant uteri with a concomitant decrease in COMT suggests the involvement of catecholoestrogens in the implantation process in the rabbit.     

Immunohistochemical localization of prostaglandin synthase in the rat uterus and embryo during the peri-implantation period

Prostaglandins (PGs) appear to have a role in the appearance of the increased uterine vascular permeability and subsequent decidualization observed at implantation in many species. However, the sites of production of these PGs have not been clearly established. To clarify the PG synthetic capacity of the blastocyst and the various types of cells in the uterus at implantation, we have studied the immunohistochemical localization of PG synthase in the rat blastocyst on Days 5 to 7 and uterus on Days 1, 4, 5, 6, and 7 of pregnancy. Labeling of PG synthase was negligible in the uterus on Day 1 of pregnancy. On Day 4, there was increased labeling in the luminal and glandular epithelium, in stromal cells adjacent to the luminal epithelium, and in blood vessels and some leukocytes. PG synthase was detected in the blastocysts on Days 5 to 7, but there was a gradual loss of label in the luminal and glandular epithelial cells during this period. Early differentiating stromal cells adjacent to the luminal epithelium in the implantation site on Day 5 showed bright labeling, whereas peripheral stromal cells were only slightly labeled. By Day 7, the differentiated cells of the primary decidual zone showed little or no label, but cells in the secondary decidual zone were brightly labeled. These results indicate that PG synthase is present in the rat blastocyst and in several kinds of uterine cells, and that its localization in uterine cells changed markedly during the implantation process.     

The glycocalyx of the mouse uterine luminal epithelium during estrus, early pregnancy, the peri-implantation period, and delayed implantation. I. Acquisition of Ricinus communis I binding sites during pregnancy

Mouse uteri were examined during estrus, early pregnancy, the peri-implantation period, and delayed implantation to determine whether changes in the surface coat of the luminal epithelium could be associated with receptivity of the uterus to the presence of blastocyst-stage embryos or blastocyst adhesion. By using alkaline bismuth subnitrate to label periodate-oxidized glycols within the glycocalyx we were able to measure the thickness and examine the morphology of the glycocalyx by electron microscopy. Ferritin-conjugated Ricinus communis agglutinin (RCA-I) demonstrated the presence of D-galactose at terminal, nonreducing positions within the glycocalyx. A relatively thick (0.06-0.1-micron) surface coat was present during estrus, but contained almost no RCA-I binding sites. During Day 3 of pregnancy the surface coat remained up to 0.1 micron thick and RCA-I binding sites were present. At Day 4 and during delay the glycocalyx had a fibrillar appearance, contained RCA-I binding sites, and was reduced to 0.06-0.08 micron in thickness. During Day 5 of pregnancy the thickness of the surface coat was greatly reduced, but there remained uniform lectin binding adjacent to the plasma membrane both at sites of blastocyst attachment and between implantation sites. The results indicate that the luminal epithelium of the mouse uterus acquired RCA-I binding sites during pregnancy and that the thickness of the surface coat was greatly reduced at the time of implantation.     

Conceptus-mediated integrity of endometrial epithelial cells and maintenance of relaxin synthesis in pregnant rabbits: effects of unilateral oviduct ligation

A previous study indicated rabbit endometrial relaxin synthesis is stimulated by blastocyst (Lee VH, Fields PA, Biol Reprod 1990; 40:737-745). To evaluate this hypothesis, unilateral oviduct ligations were placed (A) at the oviduct isthmus on Day 1 post-copulation and (B), in a separate group of rabbits, at the infundibulum before copulation. Blastocysts migrate into and implant in the uterine horn contralateral to the ligated oviduct only (conceptus-bearing uterus). The uterine horn ipsilateral to the ligated oviduct will be referred to as the non-conceptus-bearing uterus. Uteri and ovaries were removed on Days 4-28 of pregnancy and were evaluated for relaxin using guinea pig anti-porcine relaxin serum and avidin-biotin light microscopy immunohistochemistry. Results were identical for both models. Blastocysts first attach to the antimesometrial uterine surface by Day 7 post-copulation. Implantation on the mesometrial surface occurs on Days 8-11. Relaxin was observed in antimesometrial endometrial glands of both conceptus and non-conceptus-bearing uteri on Days 4-7 of pregnancy. Beyond Day 7, relaxin was observed in antimesometrial and mesometrial endometrial glandular and luminal epithelial cells at implantation sites of the conceptus-bearing uterus only. Relaxin was not found between implantation sites. Endometrial epithelial cells of the non-conceptus-bearing uterus were regressing by Day 9. These data indicate a conceptus-mediated maintenance of endometrial epithelial cells. Furthermore, the data suggest a paracrine maintenance of epithelial cell integrity and relaxin synthesis since these parameters are preserved only in the conceptus-bearing uterus. Cell-cell communication between conceptus and endometrium appears to be specific since endometrium between implantation sites does not contain relaxin. Uterine tissue from pseudopregnant rabbits (Days 1-16) was evaluated. Relaxin was observed in the antimesometrial glands on Day 7 only. Like the endometrium in the ligation model, endometrial epithelial cells of the pseudopregnant rabbit uterus were regressing by Day 9. These results indicate that pregnancy is not required for, but may enhance, relaxin synthesis. In addition, endometrial epithelial cells regress in the absence of pregnancy. Regression of endometrial epithelial cells on Day 9 suggests that maternal recognition of pregnancy occurs during the preimplantation period (Days 4-8).     

Effect of decidual tissue on the uterine production of prostaglandins in pseudopregnant rats.

The concentration of prostaglandin F (PGF) in uterine vein plasma of non-traumatized pseudopregnant rats and pseudopregnant rats with deciduomata were not significantly different from each other at any of the times of pseudopregnancy studied (Days 7, 10 and 12). There was a significant increase in PGF levels on Day 10 in both groups of pseudopregnant animals (P less than 0-05) compared to the Day 4 values, and PGE values were significantly greater on Day 10 in the decidual tissue-bearing rats (P less than 0-01). A slight but not significant elevation in PGE concentration was observed on Days 7 and 12 in rats with deciduomata, but there was no significant difference in the control rats on Days 4, 7, 10 or 12. The results indicate that the prolongation of pseudopregnancy in rats with deciduomata is not due to a decreased production of uterine PGs and lend support to the recent suggestion of a luteotrophic effect of decidual tissue in the rat.     

Arachidonic acid in uterine phospholipid during early pregnancy and following hormone treatment

Evidence that prostaglandins are involved in intercellular communication during blastocyst implantation suggested that development and loss of uterine sensitivity to deciduogenic stimuli during early pregnancy might depend upon changes in uterine capacity to mobilize arachidonic acid from phospholipid. We measured levels of arachidonic acid in lipid fractions on Day 6 of pregnancy in uterine segments containing implantation sites, in uterine segments between implantation sites, and in luminal epithelial cells after a deciduogenic stimulus. Arachidonic acid in uterine phospholipid was depleted at implantation sites. With an intrauterine deciduogenic stimulus of hormonally primed ovariectomized rat uteri, the arachidonic acid content of the luminal epithelium decreased. When the fatty acid composition of the luminal epithelium was examined during pseudopregnancy and after progestin-estrogen treatment, however, no changes in arachidonic acid composition and content were observed. These data suggest that during blastocyst implantation, luminal epithelial cells at implantation sites mobilize arachidonic acid from phospholipid for prostaglandin synthesis, but that uterine sensitivity and the capacity to synthesize prostaglandins in response to the blastocyst does not depend upon changes in arachidonic acid levels in uterine phospholipid.     

Abnormal expression of matrix metalloproteinase-2 and -9 in interspecific pregnancy of rat embryos in mouse recipients

The high failure rate of interspecific pregnancy is a major obstacle to the successful interspecific cloning of mammals. Embryo transfer between rats and mice provides a unique model for studying the causes of such failures. Previous research has shown that the upper time limit for the survival of rat embryos in mouse uteri was the seventh day of pregnancy (Day 7). To study the reasons for the failure of interspecific pregnancy between rats and mice, we transferred rat blastocysts into mouse uteri on the third day of pseudopregnancy. Unexpectedly, intact rat embryos could still be observed in mouse uteri on Day 9 and the implantation rate was as high as 30.6%. However, compared with mouse embryos, the further development of transferred rat embryos in mouse uteri was retarded. On Day 10, transferred rat embryos shrank with much blood. From Day 11 on, they lost their intact structure and the recipient uteri developed dropsy. On Day 12, the embryos shrank further and completely separated from the mouse uteri. By Day 13, they had been absorbed without any remains. In an in vitro co-culture (CT) system, the attachment rate of rat embryos on a monolayer of mouse uterine epithelial cells was similar to that of mouse embryos, but the outgrowth rate of rat embryos was significantly lower. Further investigation by gelatin zymography showed that matrix metalloproteinase-2 (MMP-2) and metalloproteinase-9 (MMP-9) activities in transferred rat embryos was significantly less than in mouse embryos. The same result was obtained in the in vitro CT assay. These results suggest that rat embryos can complete adhesion but not the invasion when transferred into mouse uteri. The reduced invasive ability, and especially, the associated reduction of MMP-2 and -9 activity, is one of the reasons for the failure of interspecific pregnancy.     

Dihydrotestosterone secretion in pregnant and pseudopregnant rats

The objective of this study was to determine levels of dihydrotestosterone in the peripheral circulation and the source of its secretion throughout pregnancy and pseudopregnancy. Rats were bled from the jugular vein and ovarian or uterine vein from Day 8 through Day 22 of pregnancy and from Day 8 through the end of pseudopregnancy in various types of pseudopregnant rats. Jugular vein samples alone were obtained on the day of parturition and every fourth day thereafter during the lactational period. Our results indicate that the levels of dihydrotestosterone in the peripheral circulation were high throughout pregnancy and pseudopregnancy then declined during the lactational period. The sources of dihydrotestosterone were primarily the ovary in pseudopregnant rats and the ovary plus uterus in pregnant rats.     

Steroid regulation of cell specific secreted phosphoprotein 1 (osteopontin) expression in the pregnant porcine uterus

Secreted phosphoprotein 1 (SPP1, commonly referred to as osteopontin and formerly known as bone sialoprotein 1, early T-lymphocyte activation 1) is an extracellular matrix/adhesion molecule that is upregulated in the pregnant uterus of all mammals examined to date. This study focused on the pig, which has true epitheliochorial placentation and exhibits induction of SPP1 mRNA in luminal epithelium (LE) just before conceptus attachment and in glandular epithelium (GE) after Day 30 of pregnancy. The objective of this study was to determine steroid regulation of SPP1 mRNA and protein in porcine uterine epithelium. To examine the effect of estrogen, cyclic gilts were treated daily (Days 11-14) with 5 mg estradiol benzoate (i.m.) and hysterectomized on Day 15. To evaluate the long-term effect of pseudopregnancy, cyclic gilts were given daily injections (Days 11-15) with steroid as above and hysterectomized on Day 90. In situ hybridization showed high expression of SPP1 mRNA only in LE contiguous with apposing conceptus tissue on Day 15 of pregnancy. In contrast, estrogen injection resulted in moderate but uniform SPP1 mRNA in all LE of Day 15 nonpregnant gilts, with expression maintained through Day 90 of pseudopregnancy. SPP1 mRNA also localized to the GE of Day 90 pseudopregnant gilts, similar to expression in late gestation. Consistent with in situ hybridization results, SPP1 protein localized to the apical surface of LE in all estrogen-treated gilts and in the GE on Day 90 of pseudopregnancy. We conclude that, in pregnant pigs, SPP1 is induced by conceptus estrogen in uterine LE and is regulated in GE in a manner coincident with CL/placental progesterone production.     

Expression of cellCAM-105 in the apical surface of rat uterine epithelium is controlled by ovarian steroid hormones

Affinity-purified antibodies to cellCAM-105, an adhesive cell surface glycoprotein, were used in immunohistochemical investigations of rat uteri at various functional stages: (i) the oestrous, pro-oestrous, metoestrous, and dioestrous stages of the oestrous cycle, (ii) Days 1-8 of normal pregnancy, (iii) delayed implantation, (iv) 18 h after oestrogen reactivation from delay of implantation, and (v) juvenile rats, and normal ovariectomized adults, respectively, before and after experimental injection of progesterone and/or oestrogen. CellCAM-105 was present in the apical zones of the luminal and glandular epithelium cells in a stage-specific and hormone-dependent manner. The results indicate that: (1) steroid hormones are essential for the expression of cellCAM-105 in the uterine epithelial cells; (2) progesterone induces cellCAM-105 expression in the glandular epithelium, and oestrogen induces cellCAM-105 expression in the luminal epithelium; (3) progesterone induces down-regulation of cellCAM-105 from the surface of the uterine luminal epithelium of juvenile rats; (4) cellCAM-105 is absent in the luminal epithelial cells but present in the glandular epithelial cells of the rat uterus at the time of blastocyst implantation.     

Pseudopregnancy and the decidual response in the obese Zucker rat: a reexamination

The ability to induce the pseudopregnant (psp) state and the decidual response (DR) in the obese Zucker rat was reexamined. Lean and obese rats were cervically stimulated on the evening of proestrus or morning of estrus. This procedure lengthened the period of vaginal diestrus in both groups to approximately 13 days. The percentage of obese rats (70.0%) that became psp was not significantly different from that of lean rats (72.2%). To test sensitivity to a decidual stimulus, sesame oil was injected into the lumen of uteri of psp rats on Day 4 of vaginal diestrus. Treated uterine horns of all rats decidualized. The mean percentage increases in weights of Day 9 decidualized horns were not significantly different between obese and lean rats (752 +/- 85% SE and 875 +/- 133%, respectively). These data contradict previous reports that obese rats do not become psp following cervical stimulation, and that their uteri do not develop a typical DR. Although it is recognized that the reproductive capacity of the obese female Zucker rat is severely limited, this study describes aspects of reproductive function that are less abnormal than previously reported. The ability of the obese Zucker rat to become psp following cervical stimulation suggests that the progestational state of pregnancy might be induced reflexly. Furthermore, the ability of its uterus to respond to a decidual stimulus suggests that the obese Zucker rat may be able to support implantation.     

Differential responses to inducers of delta-aminolaevulinate synthase and haem oxygenase during pregnancy.

The responses of hepatic delta-aminolaevulinate synthase and microsomal haem oxygenase to inducers were examined in pregnant rats. 2-Allyl-2-isopropylacetamide-mediated induction of delta-aminolaevulinate synthase was greatly decreased during pregnancy and in the early post-partum period. Administration of allylisopropylacetamide to pseudopregnant rats induced delta-aminolaevulinate synthase normally. Treatment of pregnant rats with cortisol failed to restore the drug-mediated induction of delta-aminolaevulinate synthase. Microsomal cytochrome P-450 content and the activities of drug-metabolizing enzymes such as aniline hydroxylase and ethylmorphine. N-demethylase were significantly lowered during pregnancy. In contrast with the greatly impaired induction of delta-aminolaevulinate synthase, the induction of haem oxygenase in response to CoCl2 remained unaltered in pregnant rats. The normal perturbations of delta-aminolaevulinate synthase, consisting of an initial inhibition followed by a rebound increase in the enzyme activity associated with CoCL2 treatment, were observed during pregnancy. These findings indicate that hormones and metabolic factors associated with gestation exert significant but differential controls on the induction patterns of delta-aminolaevulinate synthase and haem oxygenase.     

Viability and development of 'tube-locked' mouse embryos

'Tube-locked' morulae and blastocysts were recovered from the ampulla of the oviduct of centchroman-treated mice between Days 4 and 12 post coitum and transferred to the uteri of pseudopregnant female mice. Pregnancy and implantation rates were lower and the post-implantation resorption rate was higher in the treated than in the control group. There was little difference in the pregnancy or implantation rates between embryos recovered on Days 4 or 12 post coitum, but the resorption rate increased with increasing duration of embryos in the oviducts and was 100% for the Day-12 embryos. The resorption rate was similar even when these embryos were transferred to the sterile uterine horn of unilaterally pregnant mice. Centchroman did not produce any deleterious effect on embryos which survived until Day 19 of pregnancy in foster mothers. The average fetal weight was also comparable to those of control fetuses.     

Localization of prostaglandin F in the ovine uterus during early pregnancy

As part of a study on the anti-luteolytic action of the sheep conceptus, the distribution of prostaglandin F (PGF) within the uteri of 19 pregnant and 14 non-pregnant ewes was studied using three antisera to PGF_2α and fluorescent antibody tracing. In the uteri of seven non-pregnant ewes up to Day 11 after estrus (Day 0) and in uteri of $\text{18}\text{19}$ pregnant ewes up to Day 50, PGF was localized mainly in the lamina propria, with very little on epithelial cells lining the uterine lumen. After Day 11, in the uteri of seven non-pregnant ewes, PGF was localized on the surface of luminal epithelial cells and throughout their cytoplasm, and to a lesser extent in the lamina propria. The distribution of PGF on Days 14 and 17 in the gravid and non-gravid horns of the uteri of 13 ewes made unilaterally pregnant (conceptus confined to one uterine horn) was similar to that described above for normal pregnant and non-pregnant ewes after Day 11 respectively.These results are interpreted to indicate a change in the distribution of PGF in sheep uteri due to the presence of a conceptus.