Ruth Grümmer

Expression of the gap-junction connexins 26 and 30 in the rat cochlea.

Abstract Gap junction channels which are responsible for direct intercellular communication are composed of connexin proteins. Different connexins are distributed in a tissue-specific manner. Up to now only connexin26 has been identified to be widely expressed in the inner ear. In order to investigate the role of additional gap junction proteins, the expression of connexin30 and 43 was investigated in the rat cochlea. Connexin26 and connexin30 were both expressed in the spiral limbus, the spiral ligament, the stria vascularis and between supporting cells of the organ of Corti. Double-labeling experiments suggest that both connexins are partly colocalized between cells. Weak staining of connexin43 could only be detected in the stria vascularis, the spiral ligament and between organ of Corti supporting cells. The corresponding transcripts for connexin26, 30 and 43 could be detected by Northern blot analysis. The expression of different gap junction channels in the cochlea suggests functional diversity. Gap junctions in the inner ear may control ion concentrations of cochlear fluids or act as conduits through which glucose and other metabolites diffuse.

Expression of the imprinted genes MEST/Mest in human and murine placenta suggests a role in angiogenesis.

In the mouse fetus, Mest is widely expressed in mesoderm derived tissues. In separate studies in mice and in humans, it has been shown to be maternally imprinted, that is, only the paternally inherited allele is active. Here, we show that starting with implantation, Mest is also expressed in maternal decidua of the mouse and in placenta of both humans and mice. Expression in murine decidua was restricted to endothelial cells. After Day 7, expression in the decidua gradually decreased. Mest‐specific RT‐PCR and restriction fragment length variant (RFLV) analysis of decidualized endometrium isolated from (M. musculus × M. spretus)F1 females showed that only the paternally derived Mest allele was activated in the decidual endothelium. In the mouse extraembryonic tissues, Mest transcripts were detected in derivatives of extraembryonic mesoderm only. Here, hemangioblast precursor cells and endothelial cells were positive. At all developmental stages of the mouse, trophoblast‐derived cells were clearly devoid of Mest transcripts. In the human placenta MEST transcripts were also detected in hemangioblast precursor cells, however, MEST was also expressed in villous and invasive cytotrophoblast. In a human choriocarcinoma/trophoblastic tumour grown in a nude mouse, human MEST was expressed in the tumour cells, whereas murine Mest was expressed in endothelia of the murine capillaries. The expression pattern exhibited by both Mest and MEST in extraembryonic tissues during development and during formation of choriocarcinoma/trophoblast tumour suggests a functional role of the MEST proteins related to oncofetal angiogenesis. Dev Dyn 2000;217:1–10. ©2000 Wiley‐Liss, Inc.

Replacement of connexin43 by connexin26 in transgenic mice leads to dysfunctional reproductive organs and slowed ventricular conduction in the heart

BackgroundIn order to further distinguish unique from general functions of connexin43, we have generated mice in which the coding region of connexin43 was replaced by that of connexin26.ResultsHeterozygous mothers showed impaired mammary gland development responsible for decreased lactation and early postnatal death of the pups which could be partially rescued by wild type foster mothers. Only about 17% of the homozygous connexin43 knock-in connexin26 mice instead of 25% expected according to Mendelian inheritance, were born and only 6% survived to day 21 post partum and longer. Neonatal and adult connexin43 knock-in connexin26 mice exhibited slowed ventricular conduction in their hearts, i.e. similar but delayed electrophysiological abnormalities as connexin43 deficient mice. Furthermore, connexin43 knock-in connexin26 male and female mice were infertile and exhibited hypotrophic gonads. In testes, tubuli seminiferi were developed and spermatogonia as well as some primary spermatocytes were present, but further differentiated stages of spermatogenesis were absent. Ovaries of female connexin43 knock-in connexin26 mice revealed only few follicles and the maturation of follicles was completely impaired.ConclusionThe impaired gametogenesis of homozygous males and females can explain their infertility.

Different Regulatory Pathways of Endometrial Connexin Expression: Preimplantation Hormonal-Mediated Pathway Versus Embryo Implantation-Initiated Pathway

Abstract 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 ERα and ERβ 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 ERα. 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 F2α and interleukin-1β 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 ERα during preimplantation but then utilizes an ER-independent signaling pathway during embryo implantation and decidualization.

Connexins and E‐cadherin are differentially expressed during trophoblast invasion and placenta differentiation in the rat

We have characterized the spatial and temporal expression pattern of six different connexin genes and E‐cadherin during trophectoderm development in the rat. During the initial phase of trophoblast invasion at 6 days postcoitum (dpc), the trophoblast expressed E‐cadherin but no connexin expression could be observed. With progressing invasion of the polar trophoblast into the maternal decidua, from 7 dpc onwards E‐cadherin expression in the ectoplacental cone cells was lost and was now restricted to the extraembryonic ectoderm. In the ectoplacental cone and extraembryonic ectoderm instead connexin31 mRNA and protein could be found. This pattern was maintained up to day 10 postcoitum. The start of labyrinthine trophoblast differentiation from day 11 postcoitum onwards was characterized by persisting expression of E‐cadherin in the extraembryonic ectoderm and its derivative, the chorionic plate. In addition to E‐cadherin, from 10 dpc onwards, connexin26 started to be expressed in the chorionic plate, and both molecules remained coexpressed in the labyrinthine trophoblast of the mature placenta. During this differentiation process connexin31 remained expressed mainly in the proliferating spongiotrophoblast. From day 14 postcoitum onwards, the expression of connexin31 in the spongiotrophoblastic cells decreased, and in parallel they started to express connexin43. The trophoblastic giant cells, first characterized by connexin31, lost all of the investigated connexins during midgestation on day 12 postcoitum but started to express connexin43 from day 18 postcoitum onwards. Our studies suggest that loss of E‐cadherin and induction of connexin31 expression is correlated with the proliferative and invasive stages of the ectoplacental cone, whereas appearance of connexin26, E‐cadherin and connexin43 reflects the switch to the differentiated phenotypes of the mature placenta.

Progestins inhibit expression of MMPs and of angiogenic factors in human ectopic endometrial lesions in a mouse model.

Progestins are successfully used in the treatment of endometriosis; however, the exact mechanisms of their action are still unsolved. We here focused on the effect of different progestins on parameters of extracellular matrix degradation and angiogenesis involved in the establishment and maintenance of ectopic endometrial lesions. Human endometrium was intraperitoneally transplanted into nude mice. After 7 and 28 days of treatment with progesterone, dydrogesterone, or its metabolite dihydrodydrogesterone, respectively, ectopic lesions were evaluated for proliferation and apoptosis. Expression of estrogen receptor alpha, progesterone receptor-AB, the angiogenetic factors, cysteine-rich angiogenic inducer (CYR61), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGFA) and the matrix metalloproteinase (MMP)-2, -3, -7 and -9 was investigated. Functional impact on angiogenesis was evaluated by density of microvessels and of vessels stabilized by pericytes within the ectopic lesions. Although dydrogesterone significantly reduced proliferation of endometrial stromal cells after 28 days, suppression of apoptosis was independent from progestins. Expression of MMP-2 was significantly reduced by all progestins and MMP-3 by dydrogesterone. In the grafted endometrial tissue, transcription of bFGF was suppressed by progesterone and dihydrodydrogesterone, and VEGFA and CYR61 by dihydrodydrogesterone and dydrogesterone. In parallel, microvessel density was slightly suppressed by progestins, whereas number of stabilized vessels increased. Thus, progestins regulate factors important for the establishment and maintenance of ectopic endometrial lesions.

Choriocarcinoma Cell Spheroids: An In Vitro Model for the Human Trophoblast

Cell differentiation and cell behavior are known to be strongly influenced by cell-cell and cell-matrix interactions (Grover et al., 1983; Landry and Freyer, 1984). Accordingly, among in vitro models proposed for studies of invasion, multicellular three-dimensional systems have been found to give more reliable data than two-dimensional (monolayer) systems (Mareel et al., 1979). Spheroids offer many tissue characteristics that cannot be obtained in monolayer or suspension culture (Sutherland and Durand, 1976; Yuhas et al., 1977). They represent tissue-like structures with various types of cell-to-cell contacts and can form an organized extracellular matrix (Nederman et al., 1984).

Repetitive exposure to a 7 Tesla static magnetic field of mice in utero does not cause alterations in basal emotional and cognitive behavior in adulthood

In the past three decades, magnetic resonance imaging (MRI) has been increasingly used in obstetrics to aid diagnostics of maternal and fetal conditions and has generally been considered a safe imaging method. However, the development of higher-performance systems employing, for example, stronger fields to improve the techniques diagnostic potential, necessitates on-going safety evaluation. Rodent studies provide an excellent opportunity to investigate not only acute but also long-term effects of magnetic field exposure in a systematic manner, and a behavioral analysis might help to uncover subtler effects which might result from magnetic field exposure of the vulnerable developing brain. We conducted a comprehensive investigation of emotional and cognitive behavior in adult mice which had been repeatedly exposed to a 7 Tesla static magnetic field in utero. Using well-validated tests, we did not observe any adverse behavioral alterations regarding emotional behavior as well as spatial and emotional learning.

Postovulatory aging affects dynamics of mRNA, expression and localization of maternal effect proteins, spindle integrity and pericentromeric proteins in mouse oocytes

Abstract STUDY QUESTION Is the postovulatory aging-dependent differential decrease of mRNAs and polyadenylation of mRNAs coded by maternal effect genes associated with altered abundance and distribution of maternal effect and RNA-binding proteins (MSY2)? SUMMARY ANSWER Postovulatory aging results in differential reduction in abundance of maternal effect proteins, loss of RNA-binding proteins from specific cytoplasmic domains and critical alterations of pericentromeric proteins without globally affecting protein abundance. WHAT IS KNOWN ALREADY Oocyte postovulatory aging is associated with differential alteration in polyadenylation and reduction in abundance of mRNAs coded by selected maternal effect genes. RNA-binding and -processing proteins are involved in storage, polyadenylation and degradation of mRNAs thus regulating stage-specific recruitment of maternal mRNAs, while chromosomal proteins that are stage-specifically expressed at pericentromeres, contribute to control of chromosome segregation and regulation of gene expression in the zygote. STUDY DESIGN, SIZE, DURATION Germinal vesicle (GV) and metaphase II (MII) oocytes from sexually mature C57B1/6J female mice were investigated. Denuded in vivo or in vitro matured MII oocytes were postovulatory aged and analyzed by semiquantitative confocal microscopy for abundance and localization of polyadenylated RNAs, proteins of maternal effect genes (transcription activator BRG1 also known as ATP-dependent helicase SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4 (SMARCA4) and NOD-like receptor family pyrin domain containing 5 (NLRP5) also known as MATER), RNA-binding proteins (MSY2 also known as germ cell-specific Y-box-binding protein, YBX2), and post-transcriptionally modified histones (trimethylated histone H3K9 and acetylated histone H4K12), as well as pericentromeric ATRX (alpha thalassemia/mental retardation syndrome X-linked, also termed ATP-dependent helicase ATRX or X-linked nuclear protein (XNP)). For proteome analysis five replicates of 30 mouse oocytes were analyzed by selected reaction monitoring (SRM). MATERIAL AND METHODS GV and MII oocytes were obtained from large antral follicles or ampullae of sexually mature mice, respectively. Denuded MII oocytes were aged for 24 h post ovulation. For analysis of distribution and abundance of polyadenylated RNAs fixed oocytes were in situ hybridized to Cy5 labeled oligo(dT)20 nucleotides. Absolute quantification of protein concentration per oocyte of selected proteins was done by SRM proteome analysis. Relative abundance of ATRX was assessed by confocal laser scanning microscopy (CLSM) of whole mount formaldehyde fixed oocytes or after removal of zona and spreading. MSY2 protein distribution and abundance was studied in MII oocytes prior to, during and after exposure to nocodazole, or after aging for 2 h in presence of H2O2 or for 24 h in presence of a glutathione donor, glutathione ethylester (GEE). MAIN RESULTS AND ROLE OF CHANCE The significant reduction in abundance of proteins (P < 0.001) translated from maternal mRNAs was independent of polyadenylation status, while their protein localization was not significantly changed by aging. Most of other proteins quantified by SRM analysis did not significantly change in abundance upon aging except MSY2 and GTSF1. MSY2 was enriched in the subcortical RNP domain (SCRD) and in the spindle chromosome complex (SCC) in a distinct pattern, right and left to the chromosomes. There was a significant loss of MSY2 from the SCRD (P < 0.001) and the spindle after postovulatory aging. Microtubule de- and repolymerization caused reversible loss of MSY2 spindle-association whereas H2O2 stress did not significantly decrease MSY2 abundance. Aging in presence of GEE decreased significantly (P < 0.05) the aging-related overall and cytoplasmic loss of MSY2. Postovulatory aging increased significantly spindle abnormalities, unaligned chromosomes, and abundance of acetylated histone H4K12, and decreased pericentromeric trimethylated histone H3K9 (all P < 0.001). Spreading revealed a highly significant increase in pericentromeric ATRX (P < 0.001) upon ageing. Thus, the significantly reduced abundance of MSY2 protein, especially at the SCRD and the spindle may disturb the spatial control and timely recruitment, deadenylation and degradation of developmentally important RNAs. An autonomous program of degradation appears to exist which transiently and specifically induces the loss and displacement of transcripts and specific maternal proteins independent of fertilization in aging oocytes and thereby can critically affect chromosome segregation and gene expression in the embryo after fertilization. LIMITATION, REASONS FOR CAUTION We used the mouse oocyte to study processes associated with postovulatory aging, which may not entirely reflect processes in aging human oocytes. However, increases in spindle abnormalities, unaligned chromosomes and H4K12 acetylated histones, as well as in mRNA abundance and polyadenylation have been observed also in aged human oocytes suggesting conserved processes in aging. WIDER IMPLICATIONS OF THE FINDINGS Postovulatory aging precociously induces alterations in expression and epigenetic modifications of chromatin by ATRX and in histone pattern in MII oocytes that normally occur after fertilization, possibly contributing to disturbances in the oocyte-to-embryo transition (OET) and the zygotic gene activation (ZGA). These observations in mouse oocytes are also relevant to explain disturbances and reduced developmental potential of aged human oocytes and caution to prevent oocyte aging in vivo and in vitro. STUDY FUNDING/COMPETING INTERESTS The study has been supported by the German Research Foundation (DFG) (EI 199/7-1 | GR 1138/12-1 | HO 949/21-1 and FOR 1041). There is no competing interest.

Pre- and Postovulatory Aging of Murine Oocytes Affect the Transcript Level and Poly(A) Tail Length of Maternal Effect Genes

Maternal effect genes code for oocyte proteins that are important for early embryogenesis. Transcription in oocytes does not take place from the onset of meiotic progression until zygotic genome activation. During this period, protein levels are regulated posttranscriptionally, for example by poly(A) tail length. Posttranscriptional regulation may be impaired in preovulatory and postovulatory aged oocytes, caused by delayed ovulation or delayed fertilization, respectively, and may lead to developmental defects. We investigated transcript levels and poly(A) tail length of ten maternal effect genes in in vivo- and in vitro- (follicle culture) grown oocytes after pre- and postovulatory aging. Quantitative RT-PCR was performed using random hexamer-primed cDNA to determine total transcript levels and oligo(dT)16-primed cDNA to analyze poly(A) tail length. Transcript levels of in vivo preovulatory-aged oocytes remained stable except for decreases in Brg1 and Tet3. Most genes investigated showed a tendency towards increased poly(A) content. Polyadenylation of in vitro preovulatory-aged oocytes was also increased, along with transcript level declines of Trim28, Nlrp2, Nlrp14 and Zar1. In contrast to preovulatory aging, postovulatory aging of in vivo- and in vitro-grown oocytes led to a shortening of poly(A) tails. Postovulatory aging of in vivo-grown oocytes resulted in deadenylation of Nlrp5 after 12 h, and deadenylation of 4 further genes (Tet3, Trim28, Dnmt1, Oct4) after 24 h. Similarly, transcripts of in vitro-grown oocytes were deadenylated after 12 h of postovulatory aging (Tet3, Trim28, Zfp57, Dnmt1, Nlrp5, Zar1). This impact of aging on poly(A) tail length may affect the timed translation of maternal effect gene transcripts and thereby contribute to developmental defects.