Sections Of Uterus Research Articles

Expression of Gap Junctional Proteins Connexin (Cx) in Ovine Utero-Placental Tissues During Early Pregnancy: Effects of Assisted Reproductive Technology.

Compromised pregnancies may be caused by genetic, epigenetic, environmental and/or other factors. Assisted reproductive technology (ART) may have profound effects on placental and fetal development, leading eventually to compromised pregnancy. We have previously demonstrated that application of ART leads to decreased cell proliferation in maternal and fetal placenta, delayed development of blood vessels in fetal placenta, and decreased fetal size during early pregnancy. Gap junctions have been implicated in the regulation of cellular metabolism and the coordination of cellular functions during growth and differentiation of organs and tissues, and gap junctions play a major role in direct cell-cell communication. Gap junctional channels and Cx proteins have been detected in uterine and placental tissues in several species. Both, expression of gap junctions/Cx proteins and cell-cell communication mediated through gap junctional channels are likely involved in the regulation of utero-placental and fetal function and growth. Thus, we hypothesized that ART application will affect the expression of four Cx in placenta during early pregnancy in sheep. To determine the expression of Cx26, Cx32, Cx37 and Cx43 in fetal and maternal placenta after transfer of embryos generated through natural breeding (NAT-ET), in vitro fertilization (IVF) or in vitro activation (IVA; parthenotes) compared to control natural breeding (NAT), uterine tissues (n=7 per group) were collected on day 22 of pregnancy. Cross sections of uterus were fixed for immunohistochemistry. Additionally, caruncular (CAR; maternal placenta) tissues and fetal membranes (FM; fetal placenta) were snap-frozen separately for RNA extractions followed by quantitative real time RT-PCR. Protein and mRNA for all four Cx were detected in uterine tissues of NAT. In NAT, Cx26 protein was localized to surface epithelium, endometrial glands and stroma and to FM; Cx32 protein was present in surface epithelium, endometrial glands, stroma and blood vessels and FM; Cx37 protein was expressed in surface epithelium, endometrial glands and FM; and Cx43 protein was expressed in endometrial stroma and FM. In FM, expression of mRNA for Cx26 was enhanced (P<0.05) 2.7-fold in IVA compared to NAT group, and expression of Cx26 mRNA was similar in NAT, NAT-ET and IVF groups. Expression of mRNA for Cx32, Cx37, and Cx43 in FM was similar in all pregnancy types. In CAR, mRNA expression for Cx32 was decreased (P<0.004) 2-4-fold in NAT-ET, IVF and IVA groups compared to NAT group. Expression of mRNA for Cx26, Cx37, and Cx43 in CAR was similar in all pregnancy types. These data demonstrate that Cx26, Cx32, Cx37 and Cx43 are expressed in utero-placental tissues, localization of these Cx in uterine compartments differs likely reflecting differing functions, and that application of ART affects mRNA expression for Cx26 and Cx32 but not Cx37 and Cx43. Thus, ART may have a negative effect on growth and function of utero-placental and fetal tissues in sheep possibly mediated through regulation of Cx expression and likely other factors. These data will help us to 1) better understand placental regulatory mechanisms in compromised pregnancies, and 2) in the future identify strategies for rescuing such pregnancies. Supported by Hatch Project ND01712; USDA grant 2007-01215 to LPR and ATGB, NIH grant HL64141 to LPR and DAR, and NSF-MRI-ARRA grant to ATGB. (poster)

Clinical Observation on the Application of Mifepristone and Ethacridine combined with little water-pocket induced Midtrimester Termination of Pregnancy for Women labor with Cicatricail Uterus Section

Objective To evaluate the safety and efficacy of the mifepristone and ethacridine combined with little water-pocket induced Midtrimester Termination of prednancy for women labor with cicaricail uterus setion. Methods 160 women with as carried uterus who voluntarily under went pregnancy termination of 10-16weeks of gestation were randomly divided into 2 groups after the evaluation of the lower uterinesegmentthickness by ultrasonography. The method of the mifepristone and ethacridine combined with little water-pocket in palace cavity outside aminotic cavity on inducing labor were adopted in group A(80cases) , and mifepristone combined intra-amniotic injection of ethacridinelactate were used in group B servedascontrol. The efficacy and safety of induced labor of two groups were investigated. Results Induction-delivery inte rvaland to talstage of labour of group A were shorter than groupB ( P ＜ 0.05 ). The rate of labor within 24 hrs was higher in group A than in group B ( P ＜ 0.05 ). The volume of hemorrhage within two hours of postpartum showed nosignificant difference in two groups (P ＞ 0.05 ). No complications occur in both group. Conclusion The application of mifepristone and ethacridine combined with little water-pocket induced midtrimester termination of pregnancy for women labor with cicatricail uterus successful. Key words: Mifepristone; ethacridine; Little water-pocket; Cicatricial uterus; The intermidiate stage induces labor

Anti-nuclear antibodies of primary biliary cirrhosis recognize 78–92-kD and 96–100-kD proteins of nuclear bodies

The specificities of anti-nuclear antibodies (ANA) reacting with multiple nuclear dots (MND-ANA) present in about 15% primary biliary cirrhosis sera were studied by Western blot analysis with nuclear fractions from a human cell line. Reactivity with two broad bands of 78-92 kD and 96-100 kD of the insoluble fraction was present exclusively in MND-ANA-positive sera. Antibodies eluted from these proteins specifically retained the immunofluorescence reactivity of MND-ANA. Immunomorphological analysis by a pre-embedding technique revealed that the antibody specifically binds to nuclear regions resembling in size and number nuclear bodies. Since these structures are absent in immature rate endometrial cell and can be induced by diethylstilbestrol, we tested MND-ANA by immunofluorescence on cryostat sections of uteri from hormone-treated and untreated immature rats. A strong reaction of nuclear dots was observed predominantly in endometrial cells of hormone treated rats. We thus conclude that MND-ANA present in primary biliary cirrhosis sera are directed against 78-92-kD and 96-100-kD nuclear proteins located in nuclear bodies.

Reproductive Effects of Lactational Coumestrol Exposure on Adult Female Offspring Rats.

The purpose of this study is to investigate whether lactational exposure to phytoestrogen coumestrol (CM) alters reproductive development in female offspring rats. Dams were treated from postnatal days (PND) 1 to 21 with coumestrol by gavage. Female offspring rats were evaluated for estrous cycle and reproductive organ weights on PND 81-83. The paraffin sections of uteri and ovaries were examined for histology (H&E), immunhistochemistry and TUNEL assays on PND 135-140. In addition, the expression of apoptosis proteins (caspases-3 and -7) was examined by western blot. The estrous status exhibited no changes. Ovarian weights in adult of CM-exposed pups were reduced at 0.1 mg/kg/day. In western analysis, CM showed a marked dose-dependent increases in active forms of caspases-3 and -7 in ovarian cell. Caspase-3 staining was decreased in ovarian granulosa -, theaca -, and luteal cells of 10 mg/kg CM compared to the control. Apoptosis dose-dependently increased in some granulosa -, theaca -, and luteal cells. Our results indicate cell death was visibly increased in adult ovary of pups exposed to CM during the lactation. (This work was supported by the grant from NITR/Korea FDA (07141 Dockwanry 556) for Endocrine Disruptor Research)

Uterine Global Gene Expression During Resumption of Embryo Development Following Delayed Implantation in the Mink.

Delayed implantation (DI), or embryonic diapause, is a reversible arrest of the mammalian embryo development prior to implantation that occurs due to suppression of cell proliferation at the blastocyst stage of development. The mink (Mustela vison) is a species in which delayed implantation consistently occurs in each breeding season. In this species, the principal environmental signal that reactivates embryos is the increased photoperiod at the vernal equinox that induces secretion of prolactin, which then activates the ovary, reactivates the embryo and initiates embryo implantation. The aim of our study was to identify specific genes expressed in the uterus associated with the resumption of embryo development after termination of DI. We used suppressive subtractive hybrization (SSH) to generate a library of cDNA fragments differentially expressed between the DI and reactivated states. Termination of DI was synchronized by daily i.m injection of prolactin beginning at the vernal equinox. Uterine horn samples were collected from females in diapause (n=11) and from females at 3 (n=3), 5 (n=5) and 7 days (n=3) after the resumption of embryo development. In the latter group, SSH was performed on cDNA of a pool of the samples collected at 3, 5 and 7 days after embryo reactivation versus cDNA of pooled samples collected in diapause. One thousand subtracted uterine cDNA fragments were randomly cloned and differentially screened to discriminate false positives. Positive cDNA fragments were sequenced and compared against the Genbank database using the BLAST program. Among the differentially expressed cDNAs fragments between DI and reactivated states (337), 69 % of the sequences are homologous (% similitude > 89 %; E-value < 1.0 e-11; length of similarity > 100 nucleotides) to sequences of known genes. Regulation of gene expression, metabolism, cell cycle, signal transduction and transport functions in the uterus is associated with the resumption of embryo development after DI. PCR analysis revealed that five candidates were significantly upregulated at the resumption of the embryo development in a specific temporal manner: HMGN1, SPARC, CBD139, GDF3 and tTG. Except HMGN1, all encode products known to be secreted and are expected be implicated in the reactivation of the embryo after DI. Immunohistochemistry and in situ hybridization on transversal sections of uterus collected during diapause and at 3, 5 and 7 days after the resumption of embryo development is underway to characterize the spatiotemporal distribution of proteins and transcripts. Supported by the Discovery Grant from the Natural Sciences and Engineering Research Council of Canada to B.D. Murphy.

Stage-specific Integration of Maternal and Embryonic Peroxisome Proliferator-activated Receptor δ Signaling Is Critical to Pregnancy Success

Successful pregnancy depends on well coordinated developmental events involving both maternal and embryonic components. Although a host of signaling pathways participate in implantation, decidualization, and placentation, whether there is a common molecular link that coordinates these processes remains unknown. By exploiting genetic, molecular, pharmacological, and physiological approaches, we show here that the nuclear transcription factor peroxisome proliferator-activated receptor (PPAR) delta plays a central role at various stages of pregnancy, whereas maternal PPARdelta is critical to implantation and decidualization, and embryonic PPARdelta is vital for placentation. Using trophoblast stem cells, we further elucidate that a reciprocal relationship between PPARdelta-AKT and leukemia inhibitory factor-STAT3 signaling pathways serves as a cell lineage sensor to direct trophoblast cell fates during placentation. This novel finding of stage-specific integration of maternal and embryonic PPARdelta signaling provides evidence that PPARdelta is a molecular link that coordinates implantation, decidualization, and placentation crucial to pregnancy success. This study is clinically relevant because deferral of on time implantation leads to spontaneous pregnancy loss, and defective trophoblast invasion is one cause of preeclampsia in humans.

Kinetics of murine decidual dendritic cells

Dendritic cells (DCs) are professional antigen presenting cells (APC) capable of induction of primary immune responses as well as immunologic tolerance. Myeloid and lymphoid subsets of murine DCs are able to shift cytokine responses of T cells toward Th2 and Th1 profiles respectively. Thus, DCs would be suitable candidates to mediate the balance of maternal immune responses to conception. We analyzed pregnancy-related variations in uterus and splenic DCs in a murine model. C57BL/6-mated Balb/c female mice with vaginal plugs were scarified at early, middle, and late pregnancy. Frozen sections of uterus and spleen at each stage of pregnancy were immunostained with CD11c- and MHC-II-specific antibodies. Two-color immunohistochemistry was also carried out using anti-CD11c and one of the antibodies against CD11b, CD8alpha, CD86, and DEC-205. Using morphometric analysis, the average density of DCs and relative percentage of myeloid (CD11c+, CD11b+) and lymphoid DCs (CD11c+, CD8a+) were determined at each stage. Our results showed that DCs are present throughout the pregnancy in decidua. The average density of decidual DCs at early pregnancy was significantly higher relative to middle and late gestation or to those of endometrial DCs of non-pregnant mice. Interestingly, the average density of decidual and splenic DCs, followed the same variations at different stages of pregnancy. The relative percentage of decidual lymphoid DCs (LDC) was significantly higher at mid-gestation when compared with other stages of pregnancy or non-pregnant mice. Inversely, the frequency of myeloid DCs (MDC) and the MDC/LDC ratio were statistically lower at the middle stage of pregnancy. A majority of decidual DCs expressed MHC-II and CD86. At early pregnancy, DCs were more concentrated subadjacent to the luminal epithelial layers, whereas at mid-or late gestation, DCs were randomly distributed in the stroma and around the epithelium. Mid-pregnancy period was a critical point with regard to splenic DCs kinetics, as both the average density of DCs and the frequency of MDCs decreased significantly when compared with early or late pregnancy, although the relative percentage of splenic LDCs did not change. Our data suggest that the balance of MDC and LDC is finely tuned throughout pregnancy, pointing an eminent immunoregulatory role of DCs in the maintenance of pregnancy.

Analysis of endometrial myeloid and lymphoid dendritic cells during mouse estrous cycle

This study was performed to evaluate the frequency and localization of endometrial myeloid (CD11c + CD11b +) and lymphoid (CD11c + CD8α +) dendritic cells (DCs) at different stages of murine estrous cycle. To address the systemic effect of ovarian hormones fluctuations during estrous cycle, the same variables were studied in splenic DCs as well. Stages of the estrous cycle of Balb/c mice were determined by examination of vaginal smears. Frozen sections of uterus and spleen at each stage of estrous cycle were stained for CD11c and MHC-II. Two-color immunohistochemistry was also carried out using anti-CD11c with one of the antibodies against CD11b, CD8α, CD86, and DEC-205. The average density of DCs and relative percentage of myeloid and lymphoid DCs (MDCs and LDCs) were determined at each stage of estrous cycle by morphometric analysis. Our results showed that DCs were present throughout the estrous cycle in mice endometrium, but their frequency was highest at estrus and lowest at proestrus ( P < 0.005). The lymphoid subset of DCs was more prominent at estrus relative to those at other stages ( P < 0.005). Conversely, the relative percentage of myeloid DCs at estrus was significantly lower compared to other stages ( P < 0.005). Nearly all endometrial and splenic DCs expressed CD86 and MHC-II. At proestrus, and particularly at estrus, DCs were more concentrated subadjacent to the luminal and glandular epithelial layers with some scattered throughout the stroma whereas, at metestrus and diestrus, DCs were randomly distributed in stroma and around the glandular and luminal epithelial layers. The number and immunophenotype of splenic DCs were not statistically different between stages of estrous cycle. Our results suggest that endometrial but not splenic myeloid and lymphoid DCs are influenced by steroid hormones during estrous cycle.

Updates on the cytogenetics and molecular genetics of bone and soft tissue tumors: leiomyoma

Leiomyomas (also known as uterine fibroids or fibromas) are the most common neoplasms of the female genital tract, accounting for approximately 30% of all hysterectomies performed in the United States annually. From a study of serial sections of uteri, it has been estimated that up to 77% of women of reproductive age have leiomyomas [1]. The clinical sequelae of leiomyomas, which may depend on their location within the uterus [2], may be associated with a spectrum of symptoms, including excessive menorrhagia, severe abdominal pain, urinary incontinence, and constipation. Leiomyomas may lead to infertility, spontaneous abortions, premature labor, or dystocia [3]. Progression of leiomyoma to malignant leiomyosarcoma (LMS) is very rare (frequency of 0.1%). Leiomyomas rarely involve deep soft tissue, i.e., somatic soft tissues (primarily extremities) and retroperitoneal-abdominal areas [4]. These can be identified using stringent histologic criteria, as proposed by Billings et al [4]. Leiomyomas may occur at a number of anatomic sites [5,6] and are most common in the uterus; thus, the major part of this Update will be devoted to uterine leiomyomas and the term “leiomyoma” will refer to the uterine tumors unless indicated otherwise.

211 TEMPORAL AND SPATIAL EXPRESSION PROFILE OF THE UTERINE MILK PROTEIN — A MEMBER OF THE SERINE PROTEASE INHIBITOR SUPERFAMILY — IN THE BOVINE ENDOMETRIUM

A combination of subtracted cDNA libraries and cDNA array hybridization was applied to identify differentially expressed genes in the bovine intercaruncular endometrium at estrus and diestrus. Six cyclic Simmental heifers were cycle-synchronized and slaughtered the morning after standing heat occurred (n = 3) or 12 days after estrus (n = 3), respectively. The uterus was divided into seven sections: corpus, plus the caudal, middle, and cranial parts of both uterine horns. Samples were collected from the epithelial layer of the lamina propria of the intercaruncular endometrium. Two subtracted libraries were prepared and analyzed by array hybridization with probes produced from the 42 tissue samples. More than 130 differentially expressed genes were identified. Among these genes, the mRNA for the uterine milk protein (UTMP) showed a much higher expression level at estrus versus diestrus. Furthermore, the expression level differed markedly between the uterus sections. Statistical significance was tested using ANOVA and Student Newman Keuls post-hoc test (GeneSpring® version 6.1, Silicon Genetics, Redwood City, CA, USA). There was a pronounced expression gradient from both cranial uterine horns (highest) to the corpus (lowest) at estrus. UTMP mRNA level was highest in the ipsilateral cranial horn. A more accurate quantification was done by quantitative real-time RT-PCR using the same RNA samples as for array hybridization and, in addition, samples from further time points of the estrous cycle and early pregnancy. The results obtained by array hybridization were clearly confirmed. UTMPs, also known as uterine serpins (US), were previously shown to be the major products secreted by the uteri of cattle, sheep, and pigs during pregnancy. The US are glycoproteins belonging to the serpin (serine proteinase inhibitor) superfamily of proteins. However, no inhibitory activity to any serine proteinase has been found. The ovine uterine serpin (OvUS) has been shown to inhibit a wide variety of immune responses, including mixed lymphocyte reaction, T cell-dependent antibody production, and NK (natural killer)-like activity, indicating a major role in protecting the conceptus from maternal immune reactions. The four members of the US family form a clade distinct from other serpins, which have probably adopted important biological functions in the reproductive biology of Artiodactyla. Therefore, the identified expression gradient of the UTMP mRNA at estrus may be of particular importance, e.g. for sperm selection, and deserves further investigation at the protein level and in functional studies. This work was supported by the Deutsche Forschungsgemeinschaft (Research Unit “Mechanisms of Embryo-Maternal Communication”; FOR 478/1).

SSR126768A (4-chloro-3-[(3R)-(+)-5-chloro-1-(2,4-dimethoxybenzyl)-3-methyl-2-oxo-2,3-dihydro-1H-indol-3-yl]-N-ethyl-N-(3-pyridylmethyl)-benzamide, hydrochloride): a new selective and orally active oxytocin receptor antagonist for the prevention of preterm labor.

4-chloro-3-[(3R)-(+)-5-chloro-1-(2,4-dimethoxybenzyl)-3-methyl-2-oxo-2,3-dihydro-1H-indol-3-yl]-N-ethyl-N-(3-pyridylmethyl)benzamide, hydrochloride (SSR126768A), a new potent and selective, orally active oxytocin (OT) receptor antagonist was characterized in several biochemical and pharmacological models. In binding studies, SSR126768A showed nanomolar affinity for rat and human recombinant and native OT receptors (K(i) = 0.44 nM) and exhibited much lower affinity for V(1a), V(1b), and V(2) receptors. In addition, it did not interact with a large number of other receptors, enzymes, and ion channels (1 microM). In autoradiographic experiments performed on at-term human pregnant uterus sections, SSR126768A dose dependently displaced [I(125)]d(CH(2))(5)[Tyr(Me)(2), Thr(4), Orn(8) (125)I-Tyr-NH(2)(9)]VT in situ labeling to OT receptors highly expressed in these tissues. In functional studies, SSR126768A behaved as a full antagonist and potently antagonized OT-induced intracellular Ca(2+) increase (K(i) = 0.50 nM) and prostaglandin release (K(i) = 0.45 nM) in human uterine smooth muscle cells. In rat isolated myometrium, OT-induced uterine contractions were competitively antagonized by SSR126768A (pA(2) = 8.47). Similarly, in human pregnant myometrial strips, SSR126768A inhibited the contractile uterine response to OT. In conscious telemetrated rats, oral administration of SSR126768A (1-10 mg/kg) produced a competitive inhibition of the dose response to OT on uterine contractions up to 24 h at 3 mg/kg p.o.; no tachyphylaxis was observed after 4-day repeated treatment. Finally, SSR126768A (30 mg/kg p.o.) significantly delayed parturition in pregnant rats in labor similar to ritodrine (10 mg/kg p.o.). Thus, SSR126768A is a potent, highly selective, orally active OT receptor antagonist with a long duration of action. This molecule could find therapeutic application as a tocolytic agent for acute and chronic oral management of preterm labor.

Expression of Interferon Receptor Subunits, IFNAR1 and IFNAR2, in the Ovine Uterus1

Interferon-tau (IFN-tau) is the antiluteolytic factor released by concepti of ruminant ungulate species prior to implantation. All type I interferons, including IFN-tau, exert their action through a common receptor, which consists of two subunits, IFNAR1 and IFNAR2c, but the distribution of the two polypeptides in uterine endometrium has not been examined. In situ hybridization and immunohistochemistry on sections from pregnant and nonpregnant ovine uteri at Days 14 and 15 after estrus and mating showed that both IFNAR1 and IFNAR2 mRNA and protein were strongly expressed in endometrial luminal epithelium (LE), superficial glandular epithelium (GE), and stromal cells, within but not outside caruncles. Similar staining patterns were noted in pregnant and nonpregnant uteri for both subunits. Western blot analysis of membrane fractions from cell lines derived from endometrial LE, GE, and stromal cells, and affinity cross-linking experiments with radioactively labeled IFN-tau performed on crude endometrial membranes indicated the presence of both high ( approximately 110 kDa) and low (75-80 kDa) molecular mass forms of the two receptor subunits. To localize where IFN-tau binds when it is introduced into the uterine lumen, immunohistochemistry with an antiserum against IFN-tau was performed on sections of uteri from Day 14 nonpregnant ewes whose uteri had previously been infused with IFN-tau. Staining was concentrated on the LE and superficial GE cells, and was absent from the deeper regions of the glands and from the stromal tissues. These studies demonstrate the heavy concentration of IFNAR1 and IFNAR2 in cells of the LE and superficial GE, which appear to be the main targets for IFN-tau.

Requirement for C3G-dependent Rap1 activation for cell adhesion and embryogenesis.

C3G is a guanine nucleotide exchange factor (GEF) for Rap1, and is activated via Crk adaptor protein. To understand the physiological role of C3G, we generated C3G knockout mice. C3G(-/-) homozygous mice died before embryonic day 7.5. The lethality was rescued by the expression of the human C3G transgene, which could be excised upon the expression of Cre recombinase. From the embryo of this mouse, we prepared fibroblast cell lines, MEF-hC3G. Expression of Cre abolished the expression of C3G in MEF-hC3G and inhibited cell adhesion-induced activation of Rap1. The Cre-expressing MEF-hC3G showed impaired cell adhesion, delayed cell spreading and accelerated cell migration. The accelerated cell migration was suppressed by the expression of active Rap1, Rap2 and R-Ras. Expression of Epac and CalDAG-GEFI, GEFs for Rap1, also suppressed the accelerated migration of the C3G-deficient cells. This observation indicated that Rap1 activation was sufficient to complement the C3G deficiency. In conclusion, C3G-dependent activation of Rap1 is required for adhesion and spreading of embryonic fibroblasts and for the early embryogenesis of the mouse.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) targets myeloid leukocytes in the uterus during the post-mating inflammatory response in mice

Factors in seminal plasma elicit a surge of GM-CSF expression in uterine epithelial cells after mating in mice. This study investigates the nature of the endometrial cell populations targeted by epithelial GM-CSF. In quantitative RT-PCR studies, expression of the α-subunit of the GM-CSF receptor (GM-CSF-R) parallelled GM-CSF expression, being maximal during the 48 h period after mating and declining thereafter. Expression of mRNA encoding β-common chain (AIC2B) also increased after mating and remained high until the time of embryo implantation on day 4 of pregnancy. Cells expressing GM-CSF receptors were identified in sections of uterus on the day after mating using 125I-GM-CSF, and were located predominantly in the endometrial stroma subjacent to the luminal epithelium, co-localising with abundant populations of myeloid leukocytes. Cells expressing GM-CSF receptor were identified as macrophages, granulocytes and putative dendritic cells by flow cytometric analysis using lineage and receptor subunit specific antibodies. Recombinant GM-CSF injected into the uterine lumen of ovariectomised mice was found to elicit a dose-dependant accumulation of macrophages and granulocytes in the endometrium, in a pattern of distribution comparable to that seen in uteri after natural mating. Together, these data indicate a role for epithelial cell-derived GM-CSF in mediating the recruitment and potentially in modifying the behaviour of uterine leukocytes during the post-mating inflammatory response in mice.

Detection of viral antigen in placenta and fetus of cattle acutely infected with bovine viral diarrhea virus.

The reproductive organs and fetuses of seven Norwegian Red heifers were investigated for the presence of bovine viral diarrhea virus (BVDV) antigen during the time of initial transplacental transmission of the virus. The heifers were inoculated with a noncytopathogenic BVDV at day 85/86 of gestation and were slaughtered at day 7, 10, 14, 18, or 22 postinoculation (pi). Cryostat sections of uterus, ovaries, placentomes, intercotyledonary fetal membranes, and fetal organs were examined using immunohistochemical techniques. A double immunofluorescence technique was used to identify cells that showed staining with antibodies against the leukocyte common antigen CD45 or the intermediate filament vimentin and BVDV antigens. The earliest stage of infection at which BVDV antigen could be detected in the fetuses was 14 days pi. At this stage, BVDV antigen was detected in cells of mesenchymal origin in the lungs and in large cells that morphologically resembled immature megakaryocytes in the liver. In the intercotyledonary fetal membranes and in the placentomes, BVDV antigen was not detected until 18 and 22 days pi, respectively. BVDV antigen was not detected in maternal tissue from any of the heifers. The present results indicate that fetal infection with BVDV can take place without preceding or simultaneous high concentrations of BVDV in uterus or placenta of acutely infected heifers.

Absence of mRNA Encoding Estrogen Receptor in the Rat Cochlea

Based on changes in hearing thresholds and tinnitus that are correlated with the menstrual cycle, it has been suggested that the cochlea may respond directly to estrogen. For this to occur, the cochlea should express estrogen receptors. In situ mRNA hybridization was performed on normal female rat cochleas, using radiolabeled RNA probes complementary to mRNA encoding estrogen receptor, to determine whether estrogen receptors are present in the cochlea. Strong hybridization of the riboprobes to sections of uterus and hypothalamus indicated that the technique detected estrogen receptor mRNA. No hybridization to any cochlear tissues was observed. The results indicate that estrogen receptors are not expressed on cochlear cells, at least in rats. This in turn suggests that variation in cochlear responses during the estrus cycle are not the result of the direct effect of estrogen on the cochlea. Such variation may, however, be caused by systemic changes in fluid regulation induced by estrogen receptors at a distant site, or by other hormone receptors.

Uterine cytokine production during the menstrual cycle and preimplantation stages in mice.

Female reproduction is the only system subjected to well defined periodic changes. The final stage of the menstrual cycle in mammals is the maturation of the ovum and the preparation of the female organism to support fetal development fertilization. Once pregnancy occurs, both maternal and fetal sites emit regulatory signals to ensure embryo development and maternal protection against a graft versus host (GvH) reaction initiated by the semi-allogeneic fetus. We and others have previously shown that each day of fetal development in mice is characterized by different cytokine production, detected not only at the proximity of the feto-placental unit (decidua, uterus), but also in maternal lymphoid organs (spleen), as well as in the serum. In the present study, we concentrated on the menstrual cycle and the preimplantation stages of pregnancy and defined the levels of GM-CSF, IL-10, IL-6, and IL-3 in the murine uterus during anoestrus, proestrus, oestrus, and second and third day of gestation. We show by immunofluorescence and ELISA techniques that GM-CSF is maintained at high levels during anoestrus, proestus, oestrus, and the second day of pregnancy while dropping on the third day. IL-3 levels are found elevated during proestrus, second and third day of gestation, IL-6 increases essentially during proestrus, whereas the production of IL-10 was detected during oestrus and the early stages of pregnancy. Immunoperoxidase staining on frozen sections of uteri during the early gestational period localize GM-CSF and IL-3 production in the endometrium, IL-10 in the endometrium on the second day of pregnancy, and endometrium/myometrium on the third day. Low levels of IL-6 could be detected in the endometrium/epithelium on the second day and endometrium/myometrium on the third day of gestation. The role of IL-3, IL-10, and, to a lesser degree, IL-6 is fortified by the embryo itself, since these cytokines were found to be produced by blastocysts as well. These results demonstrate the existence of a specific distribution of lymphokines within the uterine tissue, the role of which is being discussed.

Immunohistochemical and ultrastructural localization of prostaglandin H synthase in the preimplantation mouse embryo.

The immunohistochemical and ultrastructural localization of prostaglandin H synthase (PGH synthase) was studied in the Albino Swiss CF-1 mouse at different stages of embryonic development (two-cell stage, four-eight cell stage, morula and blastocyst). Flushed embryos and sections of uteri and oviducts containing embryos were treated with a mouse IgG monoclonal anti-PGH synthase antibody. The second antibody (rabbit anti-mouse) was conjugated with peroxidase or fluorescein isothiocyanate for light microscopy, fluorescence microscopy and confocal scanning. For reflection contrast microscopy and transmission electron microscopy a second antibody, goat anti-mouse, was conjugated with ultrasmall gold particles. Controls without anti-PGH synthase were used concurrently. All embryos demonstrated PGH synthase reactivity. Immunostaining appeared to be more intense at the two-cell stage, four-eight cell stage embryos and morulae than in blastocysts. Further examination indicated an intracytoplasmic location for PGH synthase, which was confirmed by stereoscopic photographs made during confocal scanning microscopy and by the immunostaining patterns observed with reflection contrast microscopy and transmission electron microscopy. Transmission electron microscopy immunostaining patterns support the localization of PGH synthase in the endoplasmic reticulum. This is the first demonstration of the ultrastructural localization of PGH synthase in the mouse embryo. Its presence before the apposition with the endometrial epithelium supports the hypothesis that arachidonic acid metabolism via the PGH synthase pathway may be crucial for implantation.

Cellular localization and hormonal regulation of inducible nitric oxide synthase in cycling mouse uterus

Nitric oxide (NO), a potent and versatile free radical, is synthesized in macrophages and mast cells as well as in other types of cells by the inducible form of nitric oxide synthase (iNOS). In this study, cells containing iNOS were identified in the uteri of cycling mice by using a rabbit antibody generated to an iNOS-specific peptide. Macrophages were identified in semiserial sections of the same tissues with the monoclonal antibody, F4/80, and mast cells were identified by toluidine blue staining. In tissue sections of uteri obtained from mice in the four stages of the estrous cycle (8 to 11 mice per stage), iNOS immunoreactivity was strongest in diestrus-I uteri and weakest in diestrus-II uteri. Myometrial mast cells and endometrial epithelial cells were prominent locations of iNOS, and specific protein was also present in myometrial smooth muscle and macrophage-like cells in the endometrial stroma. Because cyclic variations suggested regulation of iNOS expression by ovarian steroid hormones, studies were done using ovariectomized mice. Seven days after ovariectomy, immunoreactive iNOS was low but detectable in mast cells and luminal epithelial cells. In the uteri of ovariectomized, estradiol-17 beta (E2)-treated mice, mast cells were iNOS+ after 24 h whereas epithelial cells were negative; the reverse was observed in progesterone (P4)-treated mice. Both mast cells and epithelial cells were iNOS+ in the uteri of mice that had received a combination of E2 + P4. These results indicate that several types of uterine cells produce iNOS and that expression of this enzyme in specific cell lineages is governed by ovarian steroid hormones. The data are consistent with the postulate that NO derived from uterine leukocytes and other types of cells plays a role in uterine cyclicity and preparation for pregnancy.

Compartmentalized reactivity of M3/38 (anti-Mac-2) and M3/84 (anti-Mac-3) in the uterus of pregnant mice

Changes in the expression of antigens on mouse uterine or embryonic tissues were examined by enzyme immunocytochemistry. Cryostat sections of uteri from days 1, 8 and 15 of pregnancy were probed with the monoclonal antibodies M3/38 and M3/84, originally defined by their reactivity with macrophage surface antigens (Mac-2 and Mac-3, respectively). In the uterus of pregnant mice, reaction of these antibodies was not limited to leucocytes. M3/38 did not react at detectable levels with cells in the uterus on day 1 but did react with decidual cells immediately surrounding the embryo on day 8. By day 15, the placenta, decidua basalis and metrial gland were intensely positive but the embryo was negative. M3/84 reacted with the luminal side of the endometrium on day 1, the entire decidual mass on day 8, and with all maternal and fetal tissues on day 15. M3/38 was detected in saline-soluble preparations of uterine tissue and had a molecular mass of approximately 32-35 kDa as determined by SDS-PAGE and western blot analysis. The pattern of expression of these molecules suggests a functional relationship to developmental changes that occur at the maternal-fetal interface.