Jeffery A. Bowen

Cyclic AMP induces rapid increases in gap junction permeability and changes in the cellular distribution of connexin43

The rapid effects of cAMP on gap junction-mediated intercellular communication were examined in several cell types which express different levels of the gap junction protein, connexin43 (Cx43), including immortalized rat hepatocyte and granulosa cells, bovine coronary venular endothelial cells, primary rat myometrial and equine uterine epithelial cells. Functional analysis of changes in junctional communication induced by 8-bromo-cAMP was monitored by a fluorescence recovery after photobleaching assay in subconfluent cultures in the presence or absence of 1.0 mm 1-octanol (an agent which uncouples cells by closing gap junction channels). Communicating cells treated with 1.0 mm 8-bromo-cAMP alone exhibited significant increases in the percent of fluorescence recovery which were detected within 1–3 min depending on cell type, and junctional communication remained significantly elevated for up to 24 hr. Addition of 1.0 mm 8-bromo-cAMP to cultured cells, which were uncoupled with 1.0 mm octanol for 1 min, exhibited partial restoration of gap junctional permeability beginning within 3–5 min. Identical treatments were performed on cultures that were subsequently processed for indirect immunofluorescence to monitor Cx43 distribution. The changes in junctional permeability of cells correlated with changes in the distribution of immunoreactive Cx43. Cells treated for 2 hr with 10 μm monensin exhibited a reduced communication rate which was accompanied by increased vesicular cytoplasmic Cx43 staining and reduced punctate surface staining of junctional plaques. Addition of 1.0 mm 8-bromo-cAMP to these cultures had no effect on the rate of communication or the distribution of Cx43 compared to cultures treated with monensin alone. These data suggest that an effect of cyclic AMP on Cx43 gap junctions is to promote increases in gap junctional permeability by increasing trafficking and/or assembly of Cx43 to plasma membrane gap junctional plaques.

The Role of Integrins in Reproduction

Fertilization, implantation, and placentation are dynamic cellular events that require not only synchrony between the maternal environment and the embryo, but also complex cell-to-cell communication. This communication involves integrins, a large family of proteins involved in the attachment, migration, invasion, and control of cellular function. Over the past decade, investigators have learned that integrins participate in multiple reproductive events including fertilization, implantation, and placentation in many species. This review will describe: (i) the expression of integrins on gametes and during the establishment and development of the placenta; (ii) regulatory pathways for controlling expression of integrins in the uterus and developing placenta; (iii) the function of integrins as determined by null-mutations; and (iv) reproductive dysfunction in women related to inappropriate integrin expression in the uterus and/or placenta.

Effect of Heparin on Capacitation/Acrosome Reaction of Equine Sperm

The onset of sperm capacitation/acrosome reaction was evaluated using heparin. Equine semen was incubated at 38 degrees C for 4.5 h in culture medium with and without 10 micrograms/mL heparin and with and without 0.1 microM of Ca2+ ionophore. Sperm acrosome reaction was detected using chlortetracycline fluorescence (CTC) and transmission electron microscopy (TEM). The CTC assay provided staining patterns that corresponded with the capacitation/acrosome reaction in other mammalian species (man, mouse, guinea pig). The percentages of incapacitated sperm (PUC), capacitated acrosome-intact sperm (PC), and acrosome-reacted sperm (PAR) were evaluated following incubation times of 0.5 and 4.5 h in heparin-free and heparinized medium, and at 4.5 h only in sperm exposed to Ca2+ ionophore. The CTC assay was highly correlated with TEM for estimation of PAR. At 4.5 h, heparinized medium reduced PUC and increased PC and PAR, in comparison with heparin-free medium. Addition of Ca2+ ionophore to the medium reduced PUC and increased PC and PAR at 4.5 h, as compared with sperm in ionophore-free medium. Incubation time also affected PUC, PC, and PAR in heparin-free and heparinized medium without ionophore. The PUC was greater at 0.5 h than at 4.5 h, and PC and PAR were less at 0.5 h than at 4.5 h. It would appear that the initiation of capacitation/acrosome reaction of equine sperm in vitro is accelerated by heparin.

ENDOMETRIAL CONNEXIN EXPRESSION IN THE MARE AND PIG: EVIDENCE FOR THE SUPPRESSION OF CELL-CELL COMMUNICATION IN UTERINE LUMINAL EPITHELIUM

This investigation examines the relationship between implantation strategy and gap junction protein expression in uterine endometrium. The pattern of gap junction and connexin protein expression was analyzed in porcine and equine endometrium from cycling and pregnant animals using electron microscopy and immunocytochemistry. Functional analysis of cell‐cell communication was also monitored by laser cytometry in primary cultures of endometrial epithelial cells. Gap junctions were detected in endometrial stroma of cycling and pregnant animals, which was correlated with immunoreactive Cx43 within stromal fibroblasts and vascular elements. No Cx26, Cx32, or Cx43 immunostaining was detected in luminal endometrial epithelium in either the mare or the pig at any stage of the estrous cycle or pregnancy. In contrast, endometrial glands of the mare exhibited a spatiotemporal pattern of Cx43 expression in the apicolateral plasma membrane which, when present, colocalized with the tight junction–associated protein, ZO‐1. Uterine glandular Cx43 expression in mares was present from day 3 postovulation through day 14 of diestrus and until day 23 of pregnancy, whereas Cx43 was absent within uterine glands during seasonal anestrus, estrus, and after day 30 of pregnancy. Primary cultures of equine endometrial epithelial cells expressed both immunoreactive Cx43 and significant gap junction–mediated intercellular communication (GJIC) which was rapidly upregulated by 1.0 mM 8‐bromo‐cAMP or blocked with 1.0 mM octanol. No GJIC or connexin protein was detected in cultured porcine epithelial cells despite incubation with a variety of agents, including 8‐bromo‐cAMP, steroid hormones, retinoic acid, and/or prolactin.

Apoptosis-inducing members of the tumor necrosis factor supergene family: Potential functions in placentae

Summary Apoptosis-inducing members of the TNF supergene family are clearly involved in local maternal-fetal immunological relationships as well as placental development and function. Although significant progress has been made in mapping out the temporal and spatial distribution of the best known members of this powerful gene family, TNFα, LTα and FasL, many newly reported ligands and receptors have yet to be examined. Furthermore, it is not yet clear how broad the range of functions of these cytokines may be or whether their death-dealing property is the most important. Nor is it certain whether each factor has an individual, specific role or whether overlapping functions will be the rule. Although developing a full understanding of the cytokines that comprise this family is a daunting task as greater and greater complexities are revealed, sorting out the expression, regulation and activities of each could yield major benefits. Infertility in one in six couples is now a widely quoted figure ( ISLAT Working Group, 1998 ) and it is likely that some portion of reproductive failure is due to inappropriate PCD and ACD mediated by members of the TNF supergene family.

REGULATION OF PROTEIN AND PROSTAGLANDIN SECRETION IN POLARIZED PRIMARY CULTURES OF CAPRINE UTERINE EPITHELIAL CELLS

SummaryCaprine uterine epithelial (UE) cells were cultured on Matrigel-coated filters. Transmission electron microscopy revealed polarized UE cells characterized by basally located nuclei, apical microvilli, convoluted lateral membranes, and junctional complexes. Domain-specific secretion of prostaglandins and radiolabeled proteins provide further evidence of functional epithelial cell polarity. Two experiments were conducted to evaluate factors controlling prostaglandin E2 (PGE) and prostaglandin F2α (PGF) secretion. In experiment one, steroid-treated (estradiol, progesterone, or estradiol + progesterone) polarized UE cells were treated with interferon tau (IFNτ) and/or oxytocin (OT). Steroid treatment did not influence PGE or PGF secretion. However, analysis of variance revealed an IFNτ by OT interaction (P<.01) for both PGE and PGF. This interaction was caused by a reduction in PGE and PGF secretion by cultures receiving only IFNτ and the inability of IFNτ to block OT-induced release of PGE or PGF. In experiment 2, polarized UE cells were cultured in progesterone, with or without IFNτ, and sequentially challenged with estradiol and OT. Oxytocin stimulated the release of both PGE and PGF by polarized cUE cells (P<.01) and resulted in an increased accumulation of PGE (OT*domain; P<.01) in the basal compartment. Interferon tau did not influence PGE (P<.1) secretion. However, further analysis revealed that IFNτ reduced PGF secretion and was unable to block OT-induced PGF secretion (IFNτ*OT; P<.05) by polarized UE cells. Therefore, caprine UE cells form polarized monolayers and retain responsiveness to IFNτ and OT in vitro.

Endocrine Control of Trophoblast-Uterine Epithelial Cell Interactions

Implantation in mammals refers to a series of highly orchestrated interactions that begin with an intimate association of the conceptus and uterine endometrium and end with the formation of a placenta as a means to support embryonic/fetal development to the end of pregnancy. Before any cell-cell contacts are established, the embryo and uterine histotrophe exert a mutual influence to support further development of the conceptus and its secretion of the signal that sustains the function of the corpus luteum during the early stages of pregnancy, a process referred to as “maternal recognition of pregnancy” (see 1–3). Implantation is then initiated with an uncommon union between apical plasma membranes of the genetically distinct endometrial epithelium and embryonic trophectoderm subsequent to hatching from the zona pellucida (see 4). In some species, the blastocyst is gradually implanted in the endometrial stroma following trophoblast penetration of uterine epithelium (invasive hemochorial and endotheliochorial implantation), but in others, stromal invasion does not occur, and the intimate association between uterine epithelium and conceptus tissues is retained (noninvasive epitheliochorial implantation). In the latter situation, which is seen, for example, in the pig, horse, and all ruminants, the trophoblast and its supporting layer of extraembryonic mesoderm is simply apposed to the uterine epithelium. There is no displacement or invasion of the maternal tissues, and the conceptus remains within the uterine lumen throughout gestation (5). However, species, such as rodents and primates, in which implantation is invasive, penetration of the uterine epithelium triggers a series of stromal responses collectively identified as decidualization. During decidualization, the endometrium is transformed into a specialized tissue by hyperplasia and hypertrophy of stromal cells, secretion of prolactin and the extracellular matrix (ECM) proteins laminin and fibronectin, the invasion by large numbers of immune system cells, and the formation of extensive cell-cell contacts (6). This specialized tissue is believed to provide an important element of control of trophoblast invasion during implantation (7) in addition to a number of other endocrine and paracrine functions.

The Role of Integrins in Reproduction (44499)

Fertilization, implantation, and placentation are dynamic cellular events that require not only synchrony between the maternal environment and the embryo, but also complex cell-to-cell communication. This communication Involves integrins, a large family of proteins involved in the attachment, migration, invasion, and control of cellular function. Over the past decade, investigators have learned that integrins participate in multiple reproductive events including fertilization, implantation, and placentation in many species. This review will describe: (i) the expression of Integrins on gametes and during the establishment and development of the placenta; (ii) regulatory pathways for controlling expression of integrins in the uterus and developing placenta; (iii) the function of integrins as determined by null-mutations; and (iv) reproductive dysfunction in women related to inappropriate integrin expression in the uterus and/or placenta.