Signalling of DNA damage and cytokines across cell barriers exposed to nanoparticles depends on barrier thickness.

The use of nanoparticles in medicine is ever increasing, and it is important to understand their targeted and non-targeted effects. We have previously shown that nanoparticles can cause DNA damage to cells cultured below a cellular barrier without crossing this barrier. Here, we show that this indirect DNA damage depends on the thickness of the cellular barrier, and it is mediated by signalling through gap junction proteins following the generation of mitochondrial free radicals. Indirect damage was seen across both trophoblast and corneal barriers. Signalling, including cytokine release, occurred only across bilayer and multilayer barriers, but not across monolayer barriers. Indirect toxicity was also observed in mice and using ex vivo explants of the human placenta. If the importance of barrier thickness in signalling is a general feature for all types of barriers, our results may offer a principle with which to limit the adverse effects of nanoparticle exposure and offer new therapeutic approaches.

The liver X receptor (LXR) and its target gene ABCA1 are regulated upon low oxygen in human trophoblast cells: a reason for alterations in preeclampsia?

OBJECTIVES: The Liver X receptors (LXR) alpha and beta and their target genes such as the ATP-binding cassette (ABC) transporters have been shown to be crucially involved in the regulation of cellular cholesterol homeostasis. The aim of this study was to characterize the role of LXR alpha/beta in the human placenta under normal physiological circumstances and in preeclampsia. STUDY DESIGN: We investigated the expression pattern of the LXRs and their target genes in the human placenta during normal pregnancy and in preeclampsia. Placental explants and cell lines were studied under different oxygen levels and pharmacological LXR agonists. MAIN OUTCOME MEASURES: Gene expressions (Taqman PCR) and protein levels (Western Blot) were combined with immunohistochemistry to analyze the expression of LXR and its target genes. RESULTS: In the human placenta, LXRA and LXRB expression increased during normal pregnancy. This was paralleled by the expression of their prototypical target genes, e.g., the cholesterol transporter ABCA1. Interestingly, early-onset preeclamptic placentae revealed a significant upregulation of ABCA1. Culture of JAr trophoblast cells and human first trimester placental explants under low oxygen lead to increased expression of LXRA and ABCA1 which was further enhanced by the LXR agonist T0901317. CONCLUSIONS: LXRA together with ABCA1 are specifically expressed in the human placenta and can be regulated by hypoxia. Deregulation of this system in early preeclampsia might be the result of placental hypoxia and hence might have consequences for maternal-fetal cholesterol transport.

Connexin expression pattern in the endometrium of baboons is influenced by hormonal changes and the presence of endometriotic lesions.

Experimentally induced endometriosis in baboons serves as an elegant model to discriminate between endometrial genes which are primarily associated with normal endometrial function and those that are changed by the presence of endometriotic lesions. Since connexin genes are characteristic of the hormonally regulated differentiation of the endometrium, we have examined connexin expression in baboon endometrium to delineate if they are altered in response to the presence of endometriotic lesions. Connexin expression in the endometrium of cycling baboons is similar to that of the human endometrium with Connexin(Cx)43 being primarily seen in the stromal compartment and Cx26 and Cx32 being present predominantly in the epithelium. Although Cx32 is up-regulated during the secretory phase, Cx26 and Cx43 are down-regulated. In the baboon model of induced endometriosis a change in connexin pattern was evident in the presence of endometriotic lesions. In the secretory phase, Cx26 and Cx32 are no longer present in the epithelium but Cx26 is now observed primarily in the stromal cells. Infusion of chorionic gonadotrophin in a manner that mimics blastocyst transit in utero failed to rescue the aberrant stromal expression of Cx26 that is associated with the presence of endometriotic lesions suggesting an impairment of the implantation process. The altered connexin pattern coupled with a loss of the channel protein in the epithelium and a gain of Cx26 in the stromal compartment suggests that the presence of lesions changes the uterine environment and thereby the differentiation programme. This aberrant expression of connexins may be an additional factor that contributes to endometriosis-associated infertility.

Analogous and unique functions of connexins in mouse and human placental development.

Here, we review the expression, localization and the possible role of the different connexin isoforms in placental function and development in mice and men. Connexin gene deletion in mice has shown that Cx26 is responsible for transplacental uptake of glucose in the labyrinth, and Cx31 as well as Cx31.1 for trophoblast cell lineage development. In the human placenta, it appears that Cx43 is required for the fusion process of cytotrophoblastic cells leading to the formation of the syncytiotrophoblast. Thus Cx26 and Cx43 serve different species-specific functions in the functionally analogous placental compartments, mouse labyrinth and human villous trophoblast. However, like Cx31 in the mouse, Cx40 plays a critical role in the switch from a proliferative to an invasive phenotype of the trophoblast cells invading the endometrium. Both connexin channels seem to have similar functions in analogous compartments of the placentas. Taken together, connexins are important in regulating trophoblast cell differentiation in both species. In mouse, connexin channels are specifically involved in passive transport of molecules across the placental barriers.

Implantation of the human embryo: research lines and models. From the implantation research network 'Fruitful'.

Infertility is an increasing problem all over the world, and it has been estimated that 10-15% of couples in fertile age have fertility problems. Likewise induced unsafe abortion is a serious threat to women's health. Despite advances made in assisted reproduction techniques, little progress has been made in increasing the success rate during fertility treatment. This document describes a wide range of projects carried out to increase the understanding in the field of embryo implantation research. The 'Fruitful' research network was created to encourage collaborations within the consortium and to describe our different research potentials to granting agencies or private sponsors.

EGF modulates trophoblast migration through regulation of Connexin 40.

In this study we show that decidua conditioned media (DCM) downregulate Connexin 40 (C x 40) expression in extravillous trophoblast (EVT) outgrowths and can promote EVT differentiation to the invasive phenotype resulting in switching of integrin and EGF receptor expression. This suggests that growth factors secreted by the decidua, such as EGF, mediate trophoblast migration/invasion and may do so by modulating C x 40 expression and function. To test this hypothesis we have utilized migration assays using cell lines expressing C x 40. Migration assays were performed with Jeg-3, Jeg-3 overexpressing C x 40 (JpUHD) and JAR cells seeded on fibronectin-coated inserts with 8 microm pores and incubated in the absence or presence of serum-starved decidual cells. Cell migration was only observed in the presence of DCM. Conversely overexpression of C x 40 in Jeg-3 cells resulted in inhibition of cell migration as compared to wild-type control. Addition of DCM to cultured JAR cells resulted in the downregulation of C x 40 protein. EGF is known to stimulate trophoblast migration/invasion and was detected in DCM; therefore, we investigated the action of EGF on C x 40. EGF (10 ng/mL) resulted in the downregulation of C x 40 in the JAR cell line. However, EGF had no effect on JAR cell migration. We conclude that decidual secretion of growth factors, such as EGF, may act to prime trophoblast for migration/invasion through modulation of connexin expression and function.

Vascular development in endometriosis.

Endometriosis, defined as the presence of endometrial tissue outside the uterus, is an estrogen-dependent disease which causes pelvic pain and subfertility in women of reproductive age. The condition has a dramatic impact on the professional, social and marital life of sufferers. Direct and indirect evidence suggests that angiogenesis is required for the development and persistence of endometriosis. In this review the state-of-the-art with regard to our understanding of the role of angiogenesis in the ectopic implantation and survival of menstrual endometrial tissue will be discussed.

Gene signatures of testicular seminoma with emphasis on expression of ets variant gene 4.

Gene expression patterns of testicular seminoma were analysed applying oligonucleotide microarrays in 40 specimens of different tumour stages (pT1, pT2, pT3) and in normal testes. Transcripts of maternally expressed 3 transcripts were expressed in seminoma without correlation with delta-like 1 homologue expression indicating an impaired imprinting status in seminoma. Interestingly, the transcripts of bromodomain-containing 2 and nuclear autoantigenic sperm protein associated with spermatogenesis were significantly upregulated in progressing tumour stages. Transcription factors TEA domain family member 4 and ETS variant gene 4 (ETV4), weakly expressed in normal testis, were strongly augmented during tumourigenesis. For ETV4 expression, a significant correlation with the increased expression of matrix metalloproteinase 2 and a disintegrin and metalloproteinase domain 15 was determined. The ETV4 protein was localised to nuclei of spermatogonia and revealed an intense staining in seminoma cells. Taken together, we characterised additional transcription factors and spermatogenesis-associated genes involved in the progression of seminoma.

Gap junctions are required for trophoblast proliferation in early human placental development.

Little is known about the role of gap junctional intercellular communication (GJIC) in human trophoblast differentiation, particularly during the formation of extravillous trophoblast (EVT) cell columns and their subsequent differentiation into invasive cells. We have identified transcripts for five connexin gap junction proteins in the early human placenta (Cx32, Cx37, Cx40, Cx43 and Cx45). Of these, Cx40 and Cx45 proteins immunolocalize to EVT in anchoring cell columns. Cx40 expression is prominent in the anchoring column throughout the first trimester of pregnancy (6-14 weeks gestation). We used first trimester placental villous explant cultures to determine the functional significance of the inhibition of GJIC in EVT cell proliferation and differentiation using two known GJIC inhibitors, carbenoxolone (CBX) and heptanol. The morphology of EVT outgrowths changed dramatically upon GJIC-blockade, from compact and organized outgrowths into a scattered group of rounded individual trophoblast cells, reminiscent of an early invasive phenotype. Furthermore, the inhibition of GJIC in placental explants by CBX or heptanol induced a switch away from the proliferative and towards an invasive EVT phenotype, as evident from (a) the loss of the proliferation marker Ki67 and (b) an increase in the invasive marker alpha1 integrin. We also utilized antisense oligonucleotides to inhibit Cx40 protein expression in placental explants. Cx40 antisense treatment also resulted in the abolishment of outgrowth EVT cell proliferation (as determined by Ki67 immunostaining). Together, these results suggest that gap junctions composed particularly of Cx40 channels are required for the proliferation of EVT cells in anchoring cell columns, and that a loss of GJIC contributes to differentiation to the invasive EVT phenotype.

Different regulatory pathways of endometrial connexin expression: preimplantation hormonal-mediated pathway versus embryo implantation-initiated pathway.

Transformation of the endometrium into the receptive phase is under the control of ovarian steroid hormones and is modulated by embryonic signals during implantation. We have previously shown that this differentiation process is accompanied by a suppression of gap junction connexins (Cx) 26 and 43 before implantation followed by a local induction of both connexins in the implantation chamber. In the present study, we demonstrate that connexin gene expression in the rodent endometrium is regulated via two distinct signaling pathways during these different stages of early pregnancy. During preimplantation, transcription of connexins can be induced by estrogen via an estrogen receptor (ER)-dependent pathway. Additionally, Cx26 and Cx43 are induced by embryonic signals during implantation and delayed implantation as well as during artificially induced decidualization. In contrast to the estrogen-induced expression, this embryonic/decidual-associated induction of Cx26 and Cx43 could not be blocked by antiestrogen, thus pointing to another regulatory pathway independent of the ER. Studies in ERalpha and ERbeta knockout mice confirmed these different pathways, demonstrating that in the endometrium, estrogen-mediated Cx26 gene induction, but not induction during decidualization, is dependent on functional ERalpha. To evaluate potential embryonic signals regulating Cx26 expression, uteri of pseudopregnant animals were incubated with different mediators in an organ-culture model, showing that catechol estrogen and mediators of the inflammatory cascade such as prostaglandin F(2alpha) and interleukin-1beta are able to induce Cx26 expression through the ER-independent pathway. Thus, the present study demonstrates that endometrial expression of Cx26 and Cx43 is induced via estrogen and ERalpha during preimplantation but then utilizes an ER-independent signaling pathway during embryo implantation and decidualization.

Modulation of connexin expression in sheep endometrium in response to pregnancy.

The expression pattern of two typical gap junction channel proteins, connexin 43 and connexin 26 (Cx43 and Cx26), was identified in the endometrium of sheep, a species with epitheliochorial type of implantation, by indirect immunohistochemistry during the cyclic phases, early and late pregnancy, and immediately after birth. The extent of Cx43 immunoreaction bound to endometrial stromal cells of the early implantation stage (day 15 p.c.) was comparable to the situation observed in oestrus. The subsequent intensification of feto-maternal contact correlated with a striking increase of stromal Cx43 in the intercaruncular and caruncular regions of the uterus (days 18 and 21 p.c.) and the induction of Cx26 in the glandular epithelium of late implantation (day 21 p.c.). In contrast, both gap junction proteins, coexpressed in the stroma of placentomes and interplacentomal sections on days 131 and 145 p.c., decreased during late pregnancy, while an intense and augmenting staining for Cx26 was detected at the cell borders of the glandular and luminal epithelium. The spatial and temporal distribution of both connexins suggests that, under embryonal and hormonal influences, gap junctional communication is involved in the implantation process and the regulation of endometrial tissue functions during sheep pregnancy and indicates further, that this connexin expression path resembles more the invasive type of implantation.

Responsiveness of endometrial genes Connexin26, Connexin43, C3 and clusterin to primary estrogen, selective estrogen receptor modulators, phyto- and xenoestrogens.

Phytohormones and chemical compounds revealing estrogenic effects are of increasing interest for their possible influence on the physiology of the reproductive tract. The gap junction connexin (Cx) genes Cx26 and Cx43, the plasma glycoprotein clusterin gene and the complement C3 gene are highly regulated by estrogen in rat endometrium. To test the value of these genes as markers for estrogenic responsiveness we analyzed the effects of estradiol, diethylstilbestrol, the selective estrogen receptor modulators (SERMs) raloxifene and tamoxifen, the phytoestrogens genistein and daidzein, and the industrial compounds DDT (1,1,1-trichloro-2-(2-chlorophenyl)-2-(4-chlorophenyl) ethane) and polychlorinated biphenyl (PCB) on the transcription of these genes in rat endometrium in vivo. Enhancement of Cx26 and decrease of clusterin transcripts expression by estradiol was observed at 0.03 micro g/250 g body weight (BW), and induction of C3 expression was observed at 0.05 micro g/250 g BW. A comparable effect was obtained by a tenfold higher concentration of diethylstilbestrol. Tamoxifen had a regulatory effect on this set of genes at about a 300-fold higher concentration, while raloxifen revealed much weaker estrogenic activity. No effect on Cx43 transcripts was observed with any of the compounds at the concentrations used. An effect of genistein was observed only on Cx26 expression, while PCB decreased clusterin transcripts. These results show that Cx26, C3 and clusterin reveal a comparable sensitivity to estrogens and SERMs. With respect to the phytoestrogen genistein, however, Cx26 seems to be the most sensitive gene. The analysis of clusters of estrogen-sensitive endometrial genes could help to identify estrogenic substances, assess their potency, and elucidate their mechanism of action.

Expression of MAPkinases (Erk1/2) during decidualization in the rat: regulation by progesterone and nitric oxide.

The interaction between nitric oxide (NO), progesterone and the MAPkinase signalling pathway involved in decidualization was studied using immunohistochemistry during implantation in the rat. Early pregnant rats were treated with the inhibitor of nitric oxide synthesizing enzyme iNOS, aminoguanidine, either alone or in combination with the low dose antiprogestin, onapristone. The combined treatment was most effective on days 7 and 9 post coitum leading to a complete loss of embryos. The expression pattern of activated MAPkinases, Erk1/2 and iNOS appeared to be associated with the differentiation process of decidualization. A maximum staining of both enzymes was observed on day 9 post coitum in the mesometrial decidua. In addition, Erk1/2 and iNOS were highly coexpressed around the mesometrial sinusoids. Combined treatment with aminoguanidine and onapristone for 3 days led to a transient suppression of Erk1/2 and abolished Cox2 expression. Concomitantly, angiogenesis was reduced and dilated sinusoids were missing in the mesometrial decidua. In conclusion, our study suggests that (i) the member of the mitogen-activated protein kinase (MAPK) family, Erk1/2, is activated during implantation and may play an important role during the decidualization process, and (ii) this enzyme may be regulated by both progesterone and NO.

Functional significance of gap junctional coupling in preimplantation development.

Gap junctional intercellular coupling allows cells to share low molecular weight metabolites and second messengers, thus facilitating homeostatic and developmental processes. Gap junctions make their appearance very early in rodent development, during compaction in the eight-cell stage. Surprisingly, preimplantation mouse embryos lacking the gap junction protein connexin 43 develop normally and establish full-term pregnancies despite severely reduced gap junctional coupling. It was suggested that this might be explained by the presence of at least five additional connexins known to be expressed in blastocysts. In the present study, we set out to clarify the number of connexins present in preimplantation rodent embryos and the role of gap junctional coupling, if any, in blastocyst development. We provide evidence from reverse transcription-polymerase chain reaction analysis that the genes encoding 3 additional connexins (connexin 30 or beta6, connexin 36 or alpha9, and connexin 57 or alpha10) are also transcribed in preimplantation mouse embryos. Furthermore, we show that multiple connexins are expressed in rat preimplantation embryos, indicating that multiplicity of connexin expression may be a common feature of early mammalian embryogenesis. We could detect no up-regulation of any of 3 coexpressed connexins examined in mouse embryos lacking connexin 43. Impaired intercellular coupling caused either by the loss of connexin 43 or by treatment of cultured embryos with the gap junctional coupling blocker 18alpha-glycyrrhetinic acid (AGA) had no discernable effect on either apoptosis or glucose utilization, parameters known to be affected by gap junctional coupling in other contexts. These results, taken together with the reported inability of AGA to perturb blastocyst formation, imply that gap junctional coupling is not essential during this developmental period. We propose that connexin expression and the assembly of multiple types of gap junction channels in preimplantation embryos facilitates the diversification of communication pathways that will appear during postimplantation development. New evidence of this diversification is presented using rat blastocyst outgrowths.

Peritoneal endometriosis: validation of an in-vivo model.

BACKGROUND: The current medical treatment of endometriosis, a common gynaecological disease, is still associated with a high recurrence rate. To establish an appropriate in-vivo model to evaluate new therapeutic strategies we validated the nude mouse model for the intraperitoneal cultivation of human endometrial tissue. METHODS: Human endometrium of the proliferative phase was implanted into the peritoneal cavity of normal cycling and ovariectomized athymic mice and of cycling non-obese diabetic (NOD)-severe combined immuno-deficiency (SCID) mice. Morphology, proliferation, differentiation, and angiogenesis in the ectopic endometrium at different time points after implantation was investigated. RESULTS: Adhesion of endometrial fragments was observed from day 2 onwards. The lesions persisted for up to 28 days revealing a well preserved glandular morphology. The glandular epithelium maintained cytokeratin expression even after 14 days of culture. With progressing culture, glands exhibited vimentin staining in combination with a decrease of surrounding stromal cells. Proliferation of glandular epithelium could be demonstrated throughout the investigated period of 28 days, whereas expression of oestrogen and progesterone receptors was maintained only in endometriotic lesions grown in cycling but not in ovariectomized mice. Neoangiogenesis occurred from day 4 onwards, independent from the intraperitoneal localization of the ectopic lesions. CONCLUSIONS: This in-vivo model is a promising tool to test the effect of compounds such as different hormone agonists/antagonists or anti-angiogenic factors to develop new therapeutic concepts in endometriosis.

Mutations in the connexin26/GJB2 gene are the most common event in non-syndromic hearing loss among the German population.

Congenital sensorineural hearing loss affects approximately 1/1,000 live births. Mutations in the gene encoding connexin26 (GJB2) have been described as a major cause of genetic nonsyndromic hearing impairment. Additionally, another gap junction gene, connexin30 (GJB6), was found to be responsible for hereditary hearing loss. We have studied 134 patients with severe to profound hearing loss or deafness and 13 patients with mild to moderate nonsyndromic sensorineural hearing loss in order to evaluate the prevalence of connexin26 and connexin30 mutations in Germany. Mutations in the connexin26 gene were found in 30 patients (22%) with profound to severe hearing impairment whereas only one novel single nucleotide polymorphism (396G-->A) in the connexin30 gene was detected. Among the 13 patients with mild to moderate hearing loss neither mutations in the connexin26 nor in the connexin30 gene could be detected. These results demonstrate that mutations in the connexin26 gene are also a frequent cause of hereditary non-syndromic hearing loss in Germany. Therefore a screening of mutations in the connexin26 gene should be performed in every case of non-syndromic hearing loss of unknown origin.

Expression of the gap junction connexins Cx43, Cx45 and Cx26 in human uterine leiomyomata.

Uterine leiomyomata of 34 premenopausal women undergoing leiomyomectomy or hysterectomy, and in four cases the corresponding myometrium, were collected at laparotomy or laparoscopy to investigate the ability of these benign smooth muscle cell tumors to express different connexins. Immunohistochemical and Northern blot analyses were performed for the characterization of the expression of connexins Cx43, Cx45, Cx26 and Cx32. Immunofluorescence revealed the presence of Cx43 in most leiomyomata. Only seven leiomyomata lacked Cx43 expression. Cx45 was expressed in 13, a weak Cx26 immunostaining was found in seven cases, whereas Cx32 could not be detected. No correlations between the 17 beta-estradiol or progesterone serum levels and the expression patterns of the connexins Cx43, Cx45 and Cx26 could be observed. Gonadotropin-releasing hormone (GnRH)-agonist or progestin treatment did not influence the connexin expression pattern. Northern blot analyses confirmed these results; however, transcripts of Cx26 were not detectable. Connexin transcripts between myomata and the corresponding myometrium showed no obvious differences. Our data show that uterine leiomyomata are capable of expressing different connexins comparable to the corresponding myometrium, but do not respond to different hormonal conditions. The ability to express the appropriate connexins could explain why these tumors, though developing independently of hormonal levels, are still differentiated benign smooth muscle tumors.

Intercellular communication in preimplantation development: the role of gap junctions.

Gap junctions are sites where intercellular membrane channels are clustered that allow neighboring cells to pass small molecules directly between them. Gap junctional intercellular communication has been implicated in a variety of human diseases. Gap junction channels are assembled from a large family of proteins called connexins with each type of channel having some unique properties. Preimplantation mouse and rat embryos express multiple connexins and thus potentially contain many types of gap junction channels. Based on experiments focussing on connexin43, gap junction assembly in the mouse begins during compaction in the 8-cell stage and is post-translationally regulated. Gene targeting has been used to create mice lacking individual connexins that are expressed in preimplantation embryos, but none of these experiments has yet revealed a necessary role for any single connexin before implantation. Experiments with anti-connexin antibodies and pharmacological blockers of gap junctional coupling have provided conflicting evidence as to the importance of gap junctions for preimplantation development. However, connexin knockouts have revealed important roles for gap junctional coupling in early postimplantation development. It is proposed that expression of multiple connexins in the blastocyst could prepare the implanting conceptus for rapid diversification of cell types during gastrulation and development of the placenta.

Organization and regulation of the rat Cx31 gene. Implication for a crucial role of the intron region.

The connexin31 (Cx31) gene, a member of the connexin multigene family, is expressed in a characteristic spatiotemporal pattern during placental development in rodents. To elucidate the trophoblast-specific regulation of Cx31, we have isolated the rat Cx31 gene and performed structural and functional promoter analysis. The isolated Cx31 gene contains two exons separated by an intron of 2.6 kb. The first exon of the Cx31 gene is preceded by a TATA-less promoter region. Transcription is initiated in exon 1 from two transcription start sites producting transcripts of 105 and 139 bp. The 935 bp of the 5' flanking region of exon 1 comprises five putative binding sites for the GATA transcription factors as well as a NF-kappa B element, a CAAT-box and E-box/E-box-related sequences. For functional promoter analysis, the rat choriocarcinoma cell line Rcho-1 and the mouse keratinocyte cell line Hel37, which both express Cx31, were chosen. Only constructs including exon 1 and the complete intron showed high activity in transient transfection experiments in both cell lines. All deletion fragments of the putative promoter region, but which contain the entire intron sequence, did not reveal any obvious changes in luciferase activity. However, deletion of 1.1 kb of the intron sequence downstream of the splice donor site resulted in the loss of promoter activity. The intron exhibits no enhancer activity for the gene; however, the mRNA stability was increased in the presence of the intron sequence. These results indicate that parts of the intron sequence are critical for basic promoter function of the Cx31 gene.

Connexin31-deficiency in mice causes transient placental dysmorphogenesis but does not impair hearing and skin differentiation.

Mutations in the human GJB3 gene that codes for Connexin31 (Cx31), a protein subunit of gap junction channels, have recently been reported to cause deafness and the skin disorder erythrokeratodermia variabilis. To study the function of this gene in mice, we generated animals with targeted replacement of the Cx31 gene (Gjb3) by a lacZ reporter gene. Although homozygous Cx31-deficient adult mice (Gjb3(-/-)) were found among the offspring of heterozygous Cx31-deficient parents (Gjb3(+/-)), 60% of the animals expected according to Mendelian inheritance were lost between ED 10.5 and 13.5. Placentas of Gjb3(-/-) embryos at ED 9.5 were smaller than controls as a result of severely reduced labyrinth and spongiotrophoblast size. From ED 10.5 onward, placentas of surviving Gjb3(-/-) embryos recovered progressively and reached normal size and morphology by ED 18.5. This corresponds to a time period in which another connexin isoform, Connexin43, is upregulated in spongiotrophoblast cells of Cx31-deficient and control placentas. No morphological or functional defects of skin or inner ear were observed in surviving adult Gjb3(-/-) mice. We conclude that Cx31 is essential for early placentation but can be compensated for by other connexins in the embryo proper and adult mouse.