Hedgehog signaling plays roles in epithelial cell proliferation in neonatal mouse uterus and vagina

Both the uterus and vagina develop from the Müllerian duct but are quite distinct in morphology and function. To investigate factors controlling epithelial differentiation and cell proliferation in neonatal uterus and vagina, we focused on Hedgehog (HH) signaling. In neonatal mice, Sonic hh (Shh) was localized in the vaginal epithelium and Indian hh (Ihh) was slightly expressed in the uterus and vagina, whereas all Glioma-associated oncogene homolog (Gli) genes were mainly expressed in the stroma. The expression of target genes of HH signaling was high in the neonatal vagina and in the uterus, it increased with growth. Thus, in neonatal mice, Shh in the vaginal epithelium and Ihh in the uterus and vagina activated HH signaling in the stroma. Tissue recombinants showed that vaginal Shh expression was inhibited by the vaginal stroma and uterine Ihh expression was stimulated by the uterine stroma. Addition of a HH signaling inhibitor decreased epithelial cell proliferation in organ-cultured uterus and vagina and increased stromal cell proliferation in organ-cultured uterus. However, it did not affect epithelial differentiation or the expression of growth factors in organ-cultured uterus and vagina. Thus, activated HH signaling stimulates epithelial cell proliferation in neonatal uterus and vagina but inhibits stromal cell proliferation in neonatal uterus.     

Physiological studies on nonpregnant and pregnant uterus in experimentally diabetic rats]

Spontaneous intraluminal pressure waves of diabetic nonpregnant uterus and contractile responses to oxytocin and prostaglandin F2 alpha (PGF 2 alpha) of both diabetic nonpregnant and diabetic pregnant uterus were investigated in vitro. Diabetes was induced by streptozotocin (STZ), 60 mg/kg for nonpregnant and 50 mg/kg for pregnant rats. Frequency of spontaneous intraluminal pressure waves of nonpregnant uterus was reduced in diabetic rats when compared with normal, but amplitude was slightly larger in diabetic than in normal uterus. Pressure-volume curves revealed that the compliance of nonpregnant diabetic uterus was remarkably reduced. Normal tubal side-circular muscle was significantly more sensitive to oxytocin and PGF 2 alpha than cervical one in contractile responses. This tendency was lost in diabetic nonpregnant uterus. Contractile responses of both tubal and cervical circular muscles to oxytocin were lower in nonpregnant diabetic than in normal rats, but those of longitudinal muscles were higher in diabetic nonpregnant than in normal rats. Cervical circular muscle of pregnant diabetic rats was more sensitive to both agents than those of normal. However, contractile responses of diabetic longitudinal muscle to both agents were higher than those of normal as in the case of nonpregnant uterus. The mechanism of diabetic changes of the nonpregnant and pregnant uterus was discussed.     

Postnatal developmental expression and localization of apelin and apelin receptor protein in the ovary and uterus of mice

Postnatal ovarian and uterine development is crucial to accomplished female fertility. Thus, the investigations of factors that present in pre-pubertal stages are important as it might be responsible for the regulation of ovarian and uterine function. Apelin, an adipokine and its receptor (APJ) are present in female reproductive organs. However, no study has reported its postnatal expression in uterus and ovary. Thus, we investigated the postnatal developmental changes in expression and localization of apelin and APJ in the ovary and uterus of mice. Postnatal ovary and uterus were collected from postnatal day (PND) 1, 7, 14, 21, 42, 65 and performed western blot analysis and immunohistochemistry. Uterine APJ is elevated in PND14 and PND65, whereas, ovarian APJ elevated in PND7, PND14, and PND65. Apelin expression in both ovary and uterus showed intense staining at PND65 and PND14. Our results showed that apelin and APJ abundance was lower at PND21 in uterus and ovary. In conclusion, apelin and APJ are developmentally regulated in the ovary and uterus, and its localization in the different compartments of ovary and uterus suggest its distribution specific physiological role in the uterus and ovary.     

Sperm-uterine interactions: a review.

The uterus of domestic animals, including the horse, has a dual role in the interaction of the uterus and sperm. On one hand, uterine contractions carry sperm toward the oviduct, and on the other hand the uterus eliminates excessive sperm. The selection of sperm for the small numbers of "good" cells that gain access to the oviduct and for the majority of sperm that will be destroyed takes place in the uterus. The sperm-uterine interaction works both ways; sperm and seminal plasma also have several effects on the uterus. Sperm and seminal plasma probably provoke uterine contractions. Sperm induce leukocytosis in the equine uterus by activating complement. Seminal plasma has immuno-suppressive effects in the uterus. The sperm-uterine interaction can be modified by the type of the inseminate: concentration, numbers, motility/viability of sperm, volume, and absence or presence of seminal plasma. In mares, relaxation of the cervix, myometrial contractions, lymphatic drainage, and stallion contact affect elimination of sperm.     

Analysis of intra-uterine fluid motion induced by uterine contractions

Evaluation of the fluid flow pattern in a non-pregnant uterus is important for understanding embryo transport in the uterus. Fertilization occurs in the fallopian tube and the embryo (fertilized ovum) enters the uterine cavity within 3 days of ovulation. In the uterus, the embryo is conveyed by the uterine fluid for another 3 to 4 days to a successful implantation site at the upper part of the uterus. Fluid movements within the uterus may be induced by several mechanisms, but they seem to be dominated by myometrial contractions. Intra-uterine fluid transport in a sagittal cross-section of the uterus was simulated by a model of wall-induced fluid motion within a two-dimensional channel. The time-dependent fluid pattern was studied by employing the lubrication theory. A comprehensive analysis of peristaltic transport resulting from symmetric and asymmetric contractions is presented for various displacement waves on the channel walls. The results provide information on the flow field and possible trajectories by which an embryo may be transported before implantation at the uterine wall.     

Monosodium glutamate impairs the contraction of uterine visceral smooth muscle ex vivo of rat through augmentation of acetylcholine and nitric oxide signaling pathways

The aim of the study was to examine the toxic effects of Monosodium glutamate (MSG), an extensively used food additive, on the contraction of uterine visceral smooth muscle (UVSM) in rat and to elucidate the probable neurocrine mechanism involved in it. MSG produced significant potentiation of the force and inhibition of frequency of uterus recorded ex vivo in chronic MSG exposure and in single dose acute experiments. MSG also produced significant potentiation of force of acetylcholine induced contraction and no alterations in atropine induced contraction of uterus. Further, MSG produced significant increase in force and frequency of contraction of neostigmine incubated uterus. We have found significant potentiation of the post pause force of contraction of uterus when MSG was applied in adrenaline incubated uterus. MSG also produced significant decrease in frequency of contraction of sodium nitroprusside incubated uterus; increase in frequency of N-ω-Nitro-l-Arginine Methyl Ester incubated uterus and no significant changes in frequency of contraction of methylene blue incubated uterus. These results indicate that MSG potentiates the force of contraction of UVSM predominantly by augmenting the activity of cholinergic intrinsic efferents and inhibits the frequency of contraction probably by augmenting the activity of nitrergic efferents. In conclusion, MSG potentiates the force and inhibits the frequency of contraction of UVSM, and the MSG induced effect is probably mediated through the augmentation of acetylcholine and nitric oxide signaling pathways.     

Studies of the innervation of rabbit myometrium and cervix

We characterized the innervation of isolated circular and longitudinal-oriented muscle strips from the nulliparous rabbit uterus and cervix by field stimulation (FS). FS with increasing frequency (2.5-50 pps) and voltage (2.5-70 V) caused graded increases in isometric contraction with no relaxation or inhibition of spontaneous activity. Tetrodotoxin (TTX, 3.1 X 10(-6) M) significantly reduced the FS response by 75% in all strips at higher stimulus frequencies. Contractile responses to FS were also significantly inhibited by atropine (3.5 X 10(-6) M) in circular uterus and in longitudinal cervix. Guanethidine (5 X 10(-6) M) reduced the response in all strips, as did phentolamine (3.6 X 10(-6) M) in longitudinal uterus and circular cervix. Propranolol (3.9 X 10(-6) M) did not significantly change the response in longitudinal uterus or circular cervix. In longitudinal uterus, combined guanethidine and atropine produced significant inhibition, but not statistically different from either drug alone. Similar results were seen in circular uterus. Electron microscopy and glyoxylic acid histofluorescence indicate that both blood vessels and smooth muscle in rabbit uterus are supplied with adrenergic nerves. The results suggest the presence of TTX-sensitive adrenergic and cholinergic excitatory innervation of rabbit uterus and cervix.     

Metabolic process in uterine structures after different damages to the nervous system

Metabolic processes in the uterus damaged in different parts of its innervation were studied. The specificity of neurodystrophic processes in the uterus depends on the nature of the injury to its innervation. Combination of different methods of modern morphological analysis made it possible to reveal hypoplastic processes in all the layers of the uterus.     

The expression of genes coding for positive acute-phase proteins in the reproductive tract of the female rat. High levels of ceruloplasmin mRNA in the uterus

High levels of ceruloplasmin mRNA were measured in the uterus of both pregnant and non-pregnant rats. No mRNA for alpha 2-macroglobulin and alpha 1-acid glycoprotein could be detected in the uterus in contrast to the high levels of those two mRNAs found in the decidua in the mid-gestation period. Synthesis of plasma proteins with a protective function in the decidua or uterus may be important in maintaining homeostasis at different stages of reproduction. In addition, ceruloplasmin synthesis by the uterus may be part of a system transporting copper to the fetus.     

Multiphasic regulatory system mediating the effect of estradiol in rat uterus]

Estradiol is shown to induce histidine decarboxylase and histamine to activate adenylate cyclase in the rat uterus. Cyclic AMP like histamine simulates the effect of estradiol, intensifying RNA synthesis and inducing glycolytic enzymes and uterus inhibition. It was found by autoradiography that 3H-estradiol is accepted by the nuclei of some myometrium cells, 3H-histamine by their cytoplasm and 3H-cAMP is selectively bound by endothelium cells of the uterus capillaries. The estradiol messengers (histamine and cAMP) seem to mediate hormonal effect of some uterus heterofunctional cells forming a kind of multicellular functional system.     

Changes of progesterone content of rat uterine flushings in relation to serum concentrations of progesterone during the oestrous cycle.

Uterine fluid was collected from four-day cyclic rats at each stage of the oestrous cycle and assayed for progesterone and protein content. Progesterone was determined by radioimmunoassay either after ethanol (or 2.5% NaOH) denaturation of proteins from uterine flushings ('total' progesterone) or without protein denaturation ('ether-extractable' progesterone). The amount of 'ether-extractable' progesterone in the lumen was constant from metoestrus to pro-oestrus (340 pg per uterus) but lower in oestrus (200 pg per uterus). However, 'total' progesterone content of uterine fluid was subject to cyclic variations and was highest in dioestrus (890 pg per uterus) and lowest in oestrus (350 pg per uterus), in contrast to serum progesterone which is lowest in dioestrus and highest in oestrus. Protein content of uterine flushings peaked to 780 micrograms per uterus in pro-oestrus then fell to about 140 micrograms per uterus until the end of the oestrous cycle. Changes in protein content of the lumen were followed by qualitative variations since the mean amount of 'bound' progesterone ('total' progesterone minus 'ether-extractable' progesterone) released per milligram of denatured lumen protein rose from 1.8 pmol in pro-oestrus to 18.2 pmol in dioestrus. The changes of luminal 'bound' progesterone during the oestrous cycle suggest that progesterone binding to luminal proteins could be an important modulator of progesterone action in rat uterus. Moreover, the variations in progesterone content of the lumen, irrespective of serum progesterone concentrations, are consistent with the hypothesis that progesterone synthesis occurs in the uterus.     

Neutral metalloendopeptidase associated with the smooth muscle cells of pregnant rat uterus

The pregnant rat uterus contains a membrane-bound metalloendopeptidase that is biochemically and immunologically similar to kidney enkephalinase (E.C.3.4.24.11). The uterus enzyme readily cleaved specific neutral endopeptidase substrates and oxytocin as well as the synthetic elastase substrate, Suc(Ala)3-pNA, yet did not digest native elastin. Using specific inhibitors, the uterus endopeptidase was identified as a metallopeptidase and not a serine protease, having an absolute requirement for zinc and perhaps calcium for maximal activity. The uterus endopeptidase cross-reacted with polyclonal antiserum to kidney microvillar endopeptidase and a monoclonal antibody to common acute lymphocytic leukemia antigen. Immunohistochemical localization of the enzyme in a 17 day pregnant uterus indicated that the enzyme was localized on the smooth muscle bundles of the myometrium and the endometrial epithelium. Total enzyme activity was 25 times higher in the late-term pregnant uterus (17th day of pregnancy) than in the nonpregnant uterus. Enzyme levels dropped rapidly prior to parturition and within 4 days after delivery the enzyme activity had returned to control levels. Inhibition of NEP in uterine strips with phosphoramidon resulted in a marked potentiation of oxytocin-induced contractions. Our results suggest that the uterine endopeptidase may have an important role in regulating uterine smooth muscle cell contraction during the later stages of pregnancy through its action on oxytocin and perhaps other biologically active peptides.     

Prostaglandin release from the guinea-pig uterus superfused in vitro. Effect of stage of estrous cycle, progesterone, estradiol, oxytocin and A23187

Prostaglandin (PG) E2 was the major PG released from the superfused guinea-pig uterus on Day 7, followed by in descending order 6-oxo-PGF1 alpha, thromboxane (TX) B2 and PGF2 alpha. However, the outputs of all four substances were low and were very similar. By Day 15, PGF2 alpha output from the superfused uterus had increased 21.9-fold, whereas the outputs of PGE2, 6-oxo-PGF1 alpha and TXB2 had increased only 1.8-, 2.9- and 1.2-fold, respectively. A mechanism is apparently "switched on" between Days 7 and 15 which causes a fairly specific increase in the release of PGF2 alpha from the uterus. Progesterone and/or estradiol had no effect on PG or TX release when superfused over the uterus on Day 7, nor did they have any effect on PG and TX release from the Day 15 uterus when administered separately. When administered together, however, they significantly inhibited PGF2 alpha, PGE2 and 6-oxo-PGF1 alpha, but not TXB2, release from the Day 15 uterus. Oxytocin had no effect on PG release from the Day 7 or Day 15 uterus, while A23187 stimulated PGF2 alpha, 6-oxo-PGF1 alpha and, to a lesser extent, PGE2 release from the uterus on both Days 7 and 15. Oxytocin is apparently not important for stimulating PGF2 alpha release from the guinea-pig uterus in relation to luteolysis, whereas increasing intracellular free Ca++ levels may be part of the mechanism for "switching on" uterine PG synthesis. Furthermore, changes in intracellular free Ca++ levels in the endometrium may be responsible for the pulsatile nature of PGF2 alpha release from the uterus.     

A histochemical study of the secretions of reproductive glands and of the egg envelopes of Achatina fulica (Pulmonata: Stylommatophora)

Histochemical investigations of the secretions of reproductive glands—albumen gland, apical uterus, basal uterus and prostate gland—indicate the presence of galactogen in the albumen gland, acid mucopolysaccharide in the apical uterus, and lipoprotein in the basal uterus and prostate gland of A. fulica. The proteinaceous secretions produced by the glands do differ in their terminal reactive sites. Intense alkaline phosphatase reaction is found in albumen gland and apical uterus; carbonic anhydrase activity could be detected mainly in the uterine glands. The cyclical secretory activity of the reproductive glands has been studied preparatory to egg-laying and in the spent phase. Histochemical characteristics of the egg envelopes—albumen, shell membrane and egg-shell—suggest a possible sequential deposition of glandular products during the descent of eggs through the repv. ductive tract. The factors contributing to the stability and resistant nature of the egg envelopes, and the possible role of nutritive materials contained in reproductive gland secretions in the development of the embryo (even while the eggs are inside the uterus), are discussed.     

Effect of median raphe nucleus electrocoagulation and serotonin synthesis blockade on oestrogen receptor content of rat uterus

The number of oestrogen receptors (OR) in rat uterus changes in relation to the cyclic function of the ovary. In the dioestrus phase the OR content of rat uterus is significantly greater than in the oestrus phase. Electrocoagulation of the median raphe nucleus causing in rats increased release of gonadotrophins and consequent disturbances in the cyclic ovarian function is associated also with changes in OR content of the uterus. These changes are, however, not analogous to those observed in the normal sexual cycle. In the oestrus phase a very high accumulation of OR is found in the uterus, while in the dioestrus phase OR are barely detectable. Serotonin synthesis blockade with p-CPA, which is associated with an increase in the number of maturating and mature follicles in the ovary and dioestrus phase prolongation leads to a striking increase in the number of OR in the uterus.     

In vitro study of rat uterus after chronic formaldehyde exposure.

To measure cholinergic, adrenergic and tryptaminergic receptor activity of formaldehyde (HCHO) in rat uterus, albino rats were treated with 5 and 10 mg/kg, ip HCHO for 30 days. Acetylcholine (ACh) in doses 1.33, 2 and 3 micrograms/ml produced mild to moderate contraction of isolated rat uterus in control group. HCHO had no effect on isolated rat uterus per se, however it reduced ACh and carbachol induced contraction and presence of adrenaline influences in respect of ACh and carbachol activity. Adrenaline per se had no effect in control preparations, but reduced carbachol induced contraction. Propranolol had no effect on rat uterus; but its presence in the bathing medium increased activity of adrenaline. 5-Hydroxytryptamine (5-HT) had no effect of its own on isolated rat uterus but its presence in the bathing medium enhanced contractions of carbachol and oxytocin.     

Interleukin-18 (IL-18) mRNA expression and localization of IL-18 mRNA-expressing cells in the mouse uterus

Interleukin-18 (IL-18) belongs to the interleukin-1 family and was identified as an interferon-gamma inducing factor. We investigated IL-18 mRNA-expressing cells in the mouse uterus. By RNase protection assay, IL-18 mRNA and alpha subunit of IL-18 receptor mRNA were detected in the uterus. In the uterus, IL-18 mRNA levels increased during sexual maturation. In situ hybridization analysis demonstrated IL-18 mRNA-expressing cells in the mouse uterus of different ages. At 21 days of age, IL-18 mRNA-expressing cells were detected in the luminal epithelial cells and stromal cells although the IL-18 mRNA signal was weak. At 42 days of age, IL-18 mRNA signal was mainly detected in the stromal cells located near the myometrium, and in some of the luminal and glandular epithelial cells. In the uterus of 63-day-old adult mice, a strong hybridization signal for IL-18 mRNA was detected at estrus, but was weak at diestrus. IL-18 mRNA was mainly detected in the glandular epithelial cells and stromal cells. The effect of estradiol-17beta (E(2)) on IL-18 mRNA-expressing cells in the uterus was examined in ovariectomized mice. In oil-treated mice IL-18 mRNA signal was localized in luminal epithelial cells and stromal cells, while in E(2)-treated mice IL-18 mRNA signal was localized in stromal cells alone. These results suggest that the mouse uterus has an IL-18 system, and IL-18 exerts a physiological role within the uterus in a paracrine manner, and that IL-18 gene expression is regulated by estrogen.     

Estradiol and progesterone regulate the migration of mast cells from the periphery to the uterus and induce their maturation and degranulation

Background

Mast cells (MCs) have long been suspected as important players for implantation based on the fact that their degranulation causes the release of pivotal factors, e.g., histamine, MMPs, tryptase and VEGF, which are known to be involved in the attachment and posterior invasion of the embryo into the uterus. Moreover, MC degranulation correlates with angiogenesis during pregnancy. The number of MCs in the uterus has been shown to fluctuate during menstrual cycle in human and estrus cycle in rat and mouse indicating a hormonal influence on their recruitment from the periphery to the uterus. However, the mechanisms behind MC migration to the uterus are still unknown.

Methodology/Principal Findings

We first utilized migration assays to show that MCs are able to migrate to the uterus and to the fetal-maternal interface upon up-regulation of the expression of chemokine receptors by hormonal changes. By using a model of ovariectomized animals, we provide clear evidences that also in vivo, estradiol and progesterone attract MC to the uterus and further provoke their maturation and degranulation.

Conclusion/Significance

We propose that estradiol and progesterone modulate the migration of MCs from the periphery to the uterus and their degranulation, which may prepare the uterus for implantation.     

Hoxa11 regulates stromal cell death and proliferation during neonatal uterine development

Increasing evidence indicates that the Hoxa11 gene plays a critical role in the proper development of the uterus. In this report, we describe potential altered cellular processes in the developing uterus of Hoxa11 mutants. Histologic analysis demonstrates normal uterine morphology in Hoxa11 mutants as compared with controls at the newborn stage and d 7 after birth. Stromal tissue was moderately reduced in the Hoxa11 mutant uterus by d 14 after birth and was absent by d 21 after birth. There is decreased cellular proliferation in the Hoxa11 mutant uterus both at 7 and 14 d after birth. Terminal deoxyribonucleotide transferase-mediated deoxyuridine triphosphate nick-end labeling analysis demonstrates that apoptosis was markedly increased in the Hoxa11 mutant uterus at d 14 after birth. p27 is decreased in the Hoxa11 mutant as evidenced by real-time PCR. Epidermal growth factor receptor expression is dramatically decreased as evidenced by both real-time PCR and immunohistochemistry results. These findings suggest that Hoxa11 is required for proper cellular proliferation and apoptotic responses in the developing neonatal uterus and that the regulation of epidermal growth factor receptor is critical to these processes.