Estrous Uterus Research Articles

Female reproductive abnormalities in mouse adolescent pregnancy.

There are around 300 million adolescent pregnancies worldwide, accounting for 11% of all births worldwide. Accumulating evidence demonstrates that many adverse perinatal outcomes are associated with adolescent pregnancies. However, how and why these abnormalities occur remain to be defined. In this study, pregnancy at different stages was compared between 25- and 30- day-old and mature female mice. We found that the litter size of adolescent pregnancy is significantly decreased from F1 to F3 generations compared to mature pregnancy. On days 8 and 12 of pregnancy, multiple abnormalities in decidual and placental development appear in F3 adolescent pregnancy. On days 5 and 8, uterine endoplasmic reticulum stress is dysregulated in F3 adolescent pregnancy. Embryo implantation and decidualization are also compromised in adolescent pregnancy. Many genes are abnormally expressed in adolescent estrous uteri. The abnormal endocrine environment and abnormal implantation from uterine immaturity may result in multiple pregnancy failures in adolescent pregnancy. The aim of this study is to shed light on human adolescent pregnancy.

Expression and Hormonal Regulation of Hoxa10 in Canine Uterus During the Peri‐implantation Period

Hoxa10, a homeobox gene, is necessary for endometrial receptivity to blastocyst implantation. The aim of this study was to investigate the differential expression of Hoxa10 in canine uterus during early pregnancy and its regulation under different conditions by in situ hybridization. Hoxa10 mRNA was mainly localized in glandular epithelium and myometrium in canine uterus. There was a low level of Hoxa10 expression in the glandular epithelium on days 6, 12 and 17 of pregnancy. On day 20 of pregnancy when embryo implanted, Hoxa10 mRNA was highly expressed in the glandular epithelium surrounding the embryo, but not in the luminal epithelium. The expression of Hoxa10 mRNA gradually declined from day 23 and reached a low level on day 28. In the myometrium, a low level of Hoxa10 mRNA signal was seen on days 6, 12 and 17 of pregnancy and reached a high level on day 20 of pregnancy. During the estrous cycle, a high level of Hoxa10 mRNA expression was seen in the estrous uterus. Either estrogen or progesterone significantly induced the expression of Hoxa10 mRNA in the ovariectomized canine uterus. These results suggest that Hoxa10 expression is closely related to canine embryo implantation and upregulated by estrogen and progesterone.

Murine SPAM1 is secreted by the estrous uterus and oviduct in a form that can bind to sperm during capacitation: acquisition enhances hyaluronic acid-binding ability and cumulus dispersal efficiency

Sperm uptake of epididymal sperm adhesion molecule 1 (SPAM1) in vitro has recently been shown to be a marker of sperm maturation, since acquisition of this surface hyaluronidase increases cumulus dispersal efficiency. Here, we demonstrate that this glycosyl phosphatidylinositol-linked sperm antigen, previously shown to be expressed during estrous in the female reproductive tract, is secreted in the uterine and oviductal fluids (ULF and OF respectively) in a 67 kDa form, which can bind to sperm. We show that it can be acquired by caudal sperm from Spam1 null, Spam1-deficient mutant, and wild-type (WT) mice in vitro during incubation in ULF or OF at 37 degrees C, as detected by immunocytochemistry and flow cytometry. SPAM1 binding after ULF incubation was localized predominantly to the acrosome and the mid-piece of the flagella of Spam1 null sperm in a pattern identical to that of WT sperm. After ULF incubation, WT sperm demonstrated a significantly (P<0.001) enhanced hyaluronic acid-binding ability, and the involvement of SPAM1 in this activity was shown by a significant (P<0.001) decrease in binding when sperm were exposed to SPAM1 antiserum-inhibited ULF. Importantly, when Spam1 null sperm were exposed to ULF with SPAM1 accessible (in the presence of pre-immune serum) or inaccessible (in the presence of SPAM1 antiserum) for uptake, there was a significant difference in cumulus dispersal efficiency. Taken together, these results suggest that in the sperm surface remodeling that occurs prior to and during capacitation, the fertilizing competence of sperm is increased via acquisition of SPAM1, and likely other hyaluronidases, from the female tract.

Gene expression pattern and hormonal regulation of Small Proline-Rich Protein 2 family members in the female mouse reproductive system during the estrous cycle and pregnancy

Small proline-rich proteins (SPRR) are known to construct the cornified cell envelope (CE) in the stratified squamous epithelial cell. Their functions in the simple epithelium such as the uterine epithelium are not clear hitherto. In the present study, the mRNA expression patterns of sprr2 family members in the mouse uterus and vagina during the estrous cycle and pregnancy as well as their regulation by steroids were investigated. Using semi-quantitative RT-PCR, it was revealed that the transcripts of sprr2b, 2e and 2g genes were up-regulated in the proestrous and estrous uteri, and sprr2d was up-regulated only in the estrous uterus. In the vagina, transcription of sprr2a, 2b, 2d, 2e and 2k genes were up-regulated at the metestrous stage. Northern blot analysis demonstrated that the overall expression of sprr2 was highly up-regulated in the estrous uterus and the metestrous vagina. During pregnancy, the sprr2 mRNA in the uterus was sharply repressed from day 3 postcoitus on, and began to be induced around labor time. In situ hybridization showed that the sprr2 transcripts were localized in uterine luminal and glandular epithelial cells as well as vaginal stratified epithelial cells. In ovariectomized mice, the expression of sprr2a, 2d, 2e and 2f genes in the uterus were induced by estrogen, and the effect of estrogen on sprr2d and 2e expression could be partly abolished by progesterone. The data indicate that the sprr2 genes have unique regulation patterns in different reproductive tissues under different physiological conditions, and the encoded proteins might play diverse functions in the female reproductive system.

Specific expression of haptoglobin mRNA in implantation-stage rabbit uterine epithelium.

A glycoprotein, termed GP42, was previously identified in uterine fluid obtained from peri-implantation-stage rabbits. N-terminus amino acid sequencing of purified GP42 demonstrated identity through the first 13 amino acids with the beta subunit of liver haptoglobin. The present study was undertaken to determine if GP42 is indeed identical to haptoglobin and, if so, to determine whether it is expressed in the uterus as opposed to being present as a transudate from plasma. Reverse transcription-PCR amplification of poly(A)+ RNA prepared from implantation-stage rabbit endometrium with GP42- and haptoglobin-specific primers yielded a predicted 667 bp cDNA product. Sequence analysis of the cloned cDNA confirmed the identity of GP42 with beta-haptoglobin. Northern blot analysis demonstrated the specific expression of haptoglobin mRNA in the peri-implantation-stage endometrium and the absence of its expression in the estrous or day 4 pseudopregnant endometrium. Non-isotopic in situ hybridization revealed that the haptoglobin mRNA was restricted to the epithelium lining the luminal surface and mucosal folds of day 6(3/4) pregnant or pseudo-pregnant uteri and that no haptoglobin mRNA was detectable in the epithelium of the deep glands or cells of the stroma or myometrium. Similarly, in situ hybridization revealed no expression of haptoglobin mRNA in any cell types of the estrous uterus. These data establish the identity of GP42 with beta-haptoglobin and demonstrate that it is expressed in a stage-specific manner just prior to implantation, correlating with uterine receptivity to blastocyst implantation. Endometrial GP42 mRNA expression is not dependent on the presence of blastocysts.

Prostaglandin output from and the spontaneous inotropism of uterine horns isolated from underfed rats at different stages of the sex cycle. smooth muscle contractile influences of indomethacin and of methoxamine

The influences of a period of 15 days of restricted diet (50% of the normal food intake) in rats sacrificed at different stages of the sex cycle (proestrus, estrus, metestrus and diestrus), were explored on: (1) the magnitude of uterine spontaneous phasic contractions (isometric developed tension= IDT); (2) the release of prostaglandins (PGs) F 2α and E 2; from the uterus and (3) the myometrial inotropic responses evoked by methoxamine and indomethacin. At estrus and at proestrus, preparations from restricted diet rats exhibited greater initial (postisolation) IDT and better contractile constancy during 60 min than did uteri from normal fed rats. This enhanced contractile constancy, but not that of preparations from control fed rats, was prevented by incubation “in vitro” with indomethacin (10 −6M). At metestrus and at diestrus, uteri from normal fed rats presented higher initial levels of IDT and even more sustained contractile constancy than at estrus or at proestrus. Moreover, contractile profiles remained unaltered following the dietary restriction and the presence of indomethacin evoked similar negative inotropic actions in both experimental groups (fed and underfed). Dose-response curves for methoxamine documented its possitive, but different, inotropic actions in the two groups and at the four periods of the estrous cycle. Indeed, in the underfed group at estrus and at proestrous, dose-response curves for methoxamine were shifted to the left of those in fed controls,a situation prevented by indomethacin (10-6M); whereas at metestrus and at diestrus, no differences in the inotropic reactivity towards methoxamine between the two experimental groups, were detected. On the other hand, indomethacin shifted to the right dose-response curves for the agonist, both in preparations from normal fed and from restricted diet rats. The generation and release of PGF 2α and of PGE 2 were explored under normal and restricted diet conditions, both at estrus and at diestrus. Following the dietary restriction, the output of PGE 2 from estrous uteri was augmented in comparison to controls, whilst the release of PGF 2α was not affected. At diestrus, dietary restriction failed to alter the uterine output of either one of these PGs. Results suggest that a greater generation and release of PGE 2, following underfeeding, appears to subserve the increased spontaneous motility and the greater sensitivity of the rat uterus for — adrenoreceptor agonists.

Prostaglandin synthesis by rat uterine homogenates in the presence of copper ions, and the effects of indomethacin and salicylic acid

While no significant effects on the in vitro production of PGF 2α by homogenates of rat estrous uteri were observed in the presence of 10 −3 and 10 −6M Cu 2+, the presence of Cu 2+ at 10 −4 and 10 −5M was found to stimulate production with maximal synthesis of PGF 2α occurring with 10 −4M Cu 2+. By contrast, the synthesis of PGE 2 and PGI 2 (determined as 6-keto PGF 1α) were unaffected at all of the different Cu 2+ concentrations used. When indomethacin and salicylic acid were tested for their effects on the Cu 2+-mediated levels of PG synthesis by the homogenates, indomethacin (at 20μM) was found to cause similar pronounced decreases in PGF 2α, PGE 2 and 6-keto PGF 1α whereas salicylic acid (400μM) showed preference towards suppressing PGE2 and 6-keto PGF 1α production.

Effects of epinephrine and oxytocin on the release of prostaglandin F from the rat uterus in vitro

Isolated uteri from rats with regular 4-day cycles were incubated in Krebs-Ringer bicarbonate buffer and the release of PGF into the medium was measured by radio-immunoassay after extraction of the incubation medium with ethyl acetate at pH 3.0–3.5. PGF was produced from endogenous precursors and accumulated in equal amounts in the medium during two successive 60 min periods on each day of cycle, but the magnitude of the production varied significantly during the cycle, being greatest in estrus. Oxytocin in doses up to 500 mU/ml had no effect on PGF accumulation in the incubation period at any stage of the cycle, while epinephrine (10 −3) greatly stimulated PGF release from the estrous uterus but had no effect on PGF release from the diestrous uterus. Phentolamine, an α-blocking agent, had no effect on the epinephrine-induced release of PGF, while propranolol, a β-blocking agent, not only prevented the increase in PGF production induced by epinephrine but also reduced the basal release of PGF by the estrous uterus. Since oxytocin contracts and epinephrine relaxes the nonpregnant rat uterus both in vivo and in vitro, it is unlikely that the effects of these two compounds on uterine contractility are mediated by the release of PGF 2α.

The accumulation of malachite green stainable phospholipid in rabbit spermatozoa during maturation in the epididymis, and its possible role in capacitation.

Up to 70% of live, ejaculated rabbit spermatozoa contain a phospholipid in the postacrosomal region of the head, which is specifically stabilized by fixation in glutaraldehyde containing malachite green. This MGA-material has been shown to consist predominantly of choline plasmalogen. It accumulates within the spermatozoa during the final stages of maturation in the epididymis. It does not appear to be essential to the spermatozoa for initial motility, nor for survival under aerobic, in vivo culture conditions; however, it disappears from live spermatozoa after 12 h in the estrous uterus, which suggests that it may be involved in capacitation. While the precise role of the phospholipid in sperm function remains to be elucidated, it is evident that malachite green staining adds an important new technique to the assessment of semen quality.

Effect of uterine environment and uterine extract on guinea pig sperm metabolism.

It was demonstrated that rabbit spermatozoa was acquired the fertilizing capacity in estrous uterus. And this capacitated spermatozoa was inhibited to penetrate into the ova treating with seminal plasma. In the present report, to clear these phenomena from the biochemical aspect, it was studied that how respiration and anaerobic fructolysis of guinea pig spermatozoa were influenced by the uterine environment and uterine extract. 1) Guinea pig spermatozoa incubated in estrogen dominated uterus shows much greater respiration and anaerobic fructolysis activity than that of incubated in progesterone dominated uterus. 2) Treatment with seminal plasma caused great inhibition of respiratory activity of spermatozoa that was incubated in estrogen dominated uterus. 3) Respiration and anaerobic fructolysis stimulating factor of guinea pig spermatozoa was extracted from the estrogen treated guinea pig uterus. And from bovine uterus the same stimulating factor (a protein of approximately 50000 molecular weight estimated from the behavior on Sephadex G-75 gel filtration). 4) This bovine uterine extract recovered or accelerate the low respiratory activity of the spermatozoa for the existence of seminal plasma. From these results, it is suggested that the extract from the guinea pig and bovine uterus might play a major role in sperm respiration and anaerobic fructolysis, and further be related to capacitation process in estrous uterus.

Phlogistic activity of secalonic acid A.

The phlogistic activity of secalonic acid A (S.A.A) was examined, isolation of which has recently been completed from Aspergillus ochraceus. S.A.A. gave rise to a biphasic increase of vascular permeability in the abdominal cavity of mice by intraperitoneal application in the dye-leakage test. The peak of this biphasic increase was at about 1 hr and about 18 hr respectively. Exudation of leucocyte in the abdominal fluid was remarkable in the delayed phase. S.A.A also induced intensive sustained edema in a rat paw by a single local application, making the edema last more than 10 days. Pus-like thick fluid and a scab developed around the injected position 3-4 days after the injection of S.A.A and lasted as similarly as edema did. The increased vascular permeability and the rat paw edema were inhibited by aspirin, hydrocortisone and indomethacin in appropriate doses. The phlogistic activity of S.A.A considerably decreased when its chemical structure was changed. The effect of the abdominal fluid exuded by intraperitoneal application of S.A.A was examined on the isolated preparation of guinea pig ileum, rat duodenum and rat estrous uterus, respectively. The fluid contracted the ileum but this activity was supposed not to be due to S.A.A itself.

SPERM CAPACITATION BY UTERINE FLUID OR BETA-AMYLASE IN VITRO

Rabbit sperm developed the capacity to fertilize ova when incubated in utero or in vitro in fluids from estrous uteri. Incubation of sperm with beta-amylase in phosphate-buffered Locke's solution also resulted in capacitation. The data suggest that sperm capacitation involves enzymatic alteration of carbo-hydrate-containing seminal macromolecules which coat sperm and inhibit fertilization.

Sperm capacitation by uterine fluid or beta-amylase in vitro.

Rabbit sperm developed the capacity to fertilize ova when incubated in utero or in vitro in fluids from estrous uteri. Incubation of sperm with beta-amylase in phosphate-buffered Locke's solution also resulted in capacitation. The data suggest that sperm capacitation involves enzymatic alteration of carbo-hydrate-containing seminal macromolecules which coat sperm and inhibit fertilization.

Effects of Diethylstilbestrol and Magnesium on the Rat Oxytocic Potencies of Some Neurohypophysial Hormones and Analogues

Precise in vitro oxytocic potencies of synthetic oxytocin, arginine vasotocin, arginine vasopressin and deamino-oxytocin were obtained from metestrous or diestrous rats and from diethylstilbestroltreated rats. In each of the 2 endocrine conditions, van Dyke-Hastings solution with 0.5 mM Mg or no magnesium was used to bathe the uterus. Four oxytocic potencies were therefore obtained for each peptide. Both estrogen and magnesium usually influenced potency significantly. Alterations of potency by these 2 factors appeared to vary independently from one another. The most consistently useful and sensitive rat uterus is one treated with diethylstilbestrol and bathed in a solution containing magnesium ion. {Endocrinology 76: 90, 1965) T IN VITRO rat oxytocic potencies of several highly purified neurohypophysial hormones and analogues were previously determined with 0.0 and 0.5 mM Mg++ in van Dyke-Hastings solution (1). The ratio, Potency, USP U/mg in 0.5 HIM Mg++ Potency, USP U/mg in 0.0 HIM Mg++' RMK++, was greater than 1.0 for the following peptides: 8-arginine vasopressin (1.6), 8-arginine oxytocin (arginine vasotocin) (1.9), 3-phenylalanine oxytocin (oxypressin) (1.4), 2-phenylalanine oxytocin (2.9), 8-lysine vasopressin (2.6), and 3-valine oxytocin (3.6). Oxytocin was the sole substance assayed whose potency was not significantly augmented by the presence of magnesium. Although responses to oxytocin were increased by magnesium, they were less potentiated than the responses to other peptides. Arginine vasopressin and oxytocin are both present in the USP Posterior Pituitary Reference Standard. Magnesium Received June 23, 1964. Supported in part by grants in aid from the National Institutes of Health (HD-00589); Fluid Research Fund, University of Colorado Medical Center; and Sandoz Pharmaceuticals, Inc., San Francisco, California. potentiates responses to the vasopressin far more than to the oxytocin. Therefore, in assaying against this mixed standard, which is more potentiated than oxytocin because of its vasopressin content, pure oxytocin appears to have relatively less potency (RMK++=0.86). Rats used for the above study were not treated with an estrogen, nor were they examined to determine whether or not they were cycling. Some were probably very immature and anestrous, but others were cycling, as judged by the occasional appearance of typical estrous uteri. Practical use was made of the apparently unique RMg++ of oxytocin by comparing it to the RjigH of natural peptides which resembled oxytocin in their chromatographic and pharmacological characteristics. Munsick et al. (2) thus tentatively identified oxytocin in eluates of paper chromatograms of extracts of chicken posterior pituitary glands. They based this identification partially upon an Rjtfg++ of less than 1.0 for the distal, oxytocin-like principle in paper chromatograms. Presence of oxytocin in the chicken posterior lobe was later proved chemically by Acher et al. (3). Recently, Chan et al. (4) stated that

Oxytocic action of arachidonic acid under different hormonal environments.

The minimum effective doses of oxytocin and arachidonic acid to produce contractions of pregnant and estrous rat uteri were determined. The pregnant rat uteri were significantly less sensitive to oxytocin and more sensitive to arachidonic acid than the estrous uteri. This mode of action of arachidonic acid on the uterine musculature indicates that the fatty acid may serve as an oxytocic substance for parturition.

Isolation of Enteramine from Extracts of Posterior Salivary Glands of Octopus Vulgaris and of Discoglossus Pictus Skin

The present paper describes the main characteristics of natural enteramine as well as a procedure for its isolation. In another paper the preparation and the characteristics of synthetic enteramine have been considered (1). Vertebrates and some groups of invertebrates possess a particular system of cells, known as the enterochromafin cell system, which contains specific granules characterized by several well defined and distinctive chemical and physicochemical features (chromaffinity, argentaffinity, fluorescence in Wood’s light, ability to couple with diazonium salts, and fixation by formolcontaining histological fixatives). The cells of this system may be distributed within the gastrointestinal mucosa (2-4) or skin (or both) (5) or else massed in well defined glandular formations (6-9). The specific secretion or storage product of the enterochromaffin system has been termed enteramine (10, 11). A number of color tests and biological reactions suitable for the detection and quantitative determination of this substance have been described (12-14). The pharmacological effects exerted by enteramine are (a) a powerful stimulating action on the estrous uterus and the duodenum of the rat, the small intestine of the rabbit, the urinary bladder of the dog, both isolated and in situ, and the heart of mollusks (15) ; (b) a moderate action on the systemic blood pressure, which may be hypotensive, hypertensive, or biphasic, according to the animal species, the dose, and the state of the circulatory system (16) ; (c) an intense antidiuretic action, especially evident in hydrated rats. Diuresis may be reduced in these animals even after a subcutaneous injection of as little as 5 y of enteramine per kilo of body weight. This dose is at least 50 to 100 times smaller than that needed to affect the systemic blood pressure and at least 10,000 times smaller than the median lethal dose by the subcutaneous route. Furthermore, enteramine acts selectively on the contractile structures of the afferent vascular bed of the glomerulus, causing an increase in tonus, or spasm (17). With high single doses or with repeated doses, the vascular spasm leads to a severe and lasting ischemia, resulting in more or less extensive degeneration and necrosis of large sections of the cortex of the kidney.’