Estradiol Reduces Connexin43 Gap Junctions in the Uterus during Adenomyosis in Cows

Adenomyosis is defined as the presence of glandular foci external to the endometrium of the uterus, either in the myometrium or/and perimetrium, depending on the progress of this dysfunction. To date, we showed that steroids secretion and prolactin expression and proliferative processes are disturbed during uterine adenomyosis in cows. During endometriosis in eutopic endometrium in women, gap junctions are down regulated. The transmembrane gap junction protein, connexin (Cx43) is necessary for endometrial morphological, biochemical and angiogenic functions. The aim of this study is recognition of adenomyosis etiology by determination of the role of Cx43 in this process. Immunolocalization and comparison of Cx43 mRNA and protein expression in healthy (N=9) and adenomyotic uterine tissue (N=9), and Cx43 mRNA expression (real time PCR) in uterine stromal – myometrium co-culture under 24-hour stimulation with 17-beta estradiol (10 -7M) isolated from healthy (N=5) and adenomyotic (N=5) cows were determined. Cx43 was localized in healthy and adenomyotic uteri. mRNA and protein expression was down-regulated in uterine tissue in adenomyotic compared with healthy cows (p <0.05). Estradiol stimulated Cx43 mRNA expression in myometrial cell culture and co-culture of stromal and myomet- rial cells in adenomyotic compared with healthy cows (p<0.05). In summary, down-regulation of Cx43 expression in the junction zone might play an important role in pathogenesis of adenomyosis. Es- tradiol modulates gap junctions during adenomyosis. Key words: uterus, gap junction, connexin, adenomyosis, cow

Adenomyosis is a uterine dysfunction character- ized by the presence of endometrial glands with stromal elements in the myometrium (Azziz 1989, Campo et al. 2012) but the etiology of this disorder is still unclear (Ferenczy 1998, Korzekwa et al. 2014). Our previous studies showed that in cows the fre- Correspondence to: A. Korzekwa, e-mail: [email protected], tel.: 48 89 539 31 30 quency and progression of adenomyosis are positively correlated with age, what is consistent with studies on women. The cows older than 5 years had advanced stages of adenomyosis (approx. 40% of animals; Ko- rzekwa et al. 2013). Moreover, the presence of aden- omyosis did not differed based on the sampling loca- tion in the uterus. The glandular nests in adenomyotic cows were present in myometrial layer of both uterine Polish Journal of Veterinary Sciences Vol. 19, No. 3 (2016), 609–617 horns, ipsilateral and contralateral to ovary with cor- pus luteum (CL), and near the cervix as well (Ko- rzekwa et al. 2013, Łupicka et al. 2015). Gap junctions are intercellular channels that per- mit communication between cells by cytoplasmic ex- change of small molecules including metabolites and second messengers, as well as ions that transmit electri- cal impulses between neighboring cells (Evans and Martin 2002). A gap junction channel is composed of two hemi-channels (connexons), each of which is com- posed of six protein subunits (connexins -Cxs) arranged around a pore. Connexin43 (Cx43) and Cx32 are the two most abundant Cxs and are expressed in numerous tissues including the ovary, placenta and decidua in several species, including cattle (Wiesen and Midgley 1993, 1994, Mayerhofer and Garfield 1995, Wrenzycki et al. 1995, van Engelen et al. 2009, Xu et al. 2015). In addition, a channel-independent role of Cxs in intracellular signaling by interacting with other proteins has been suggested (Saez et al. 2003). Expression of t h ed i f f e r e n tC x sm u s tb ep r o p e r l yc o n t r o l l e da st h e i r misregulation may contribute to diseases. As a conse- quence, mutations in Cx proteins resulting in modula- tion of channel properties are associated with a large variety of specific pathologies (Araya et al. 2005). During adenomyosis in cows, protein expression of the 17-beta estradiol (E2) receptor α (ERα) is in- creased and blood and endometrial E2 concentrations are elevated, indicating hormonal abnormalities dur- ing this condition (Campo et al. 2012). Invasion by adenomyotic stromal cells was higher after E2 treat- ment in an in vitro model of stromal and myometrial co-culture of human cells (Taylor et al. 2014). Es- tradiol increases Cx43 mRNA expression in rat en- dometrium (Gru¨mmer et al. 1994) but also in the my- ocardium (Chen et al. 2010). Moreover, E2 stimulated Cx43 mRNA expression in human myometrial uterine cells (Di et al. 2001). The existence of estrogen re- sponse elements within the Cx43 gene and evidence implicating E2 in the induction of Cx43 transcription has been reported (Petrocelli and Lye 1993, Lye et al. 1993) providing a molecular basis for the reported estrogenic stimulation of Cx43 synthesis (Chung et al. 2004). Therefore, we designed an in vitro model using co-culture of stromal and myometrial uterine cells iso- lated from healthy and adenomyotic cows for com- paring E2 effect on Cx43 mRNA expression. The aims of the study were: (1) Cx43 immu- nolocalization and comparison of Cx43 mRNA and protein expression in healthy and adenomyotic uter- ine tissues, (2) determination of Cx43 mRNA express- ion in co-cultured uterine stromal and myometrial cells isolated from healthy and adenomyotic cows un- der stimulation with E2.

Material collection All procedures were approved by the Local Ani- mal Care and Use Committee, Olsztyn, Poland (agreement no. 83/2012/N). From a total of 30 examined cows, 18 Hol- stein/Polish Black and White cows (75%/25%, re- spectively), 5-7 years old, were used in the experi- ments for post mortem collection of uteri (days 8-10 of the oestrous cycle). These days of the estrous cycle were selected for experiments because this study is continuation of our concerning adenomyosis (Ko- rzekwa et al. 2013, 2014, Łupicka et al. 2015) and adenomyosis has been observed during the luteal stage of the estrous cycle in cattle (Moreira et al. 2007). Uterine tissues were obtained at the Meat Pro- cessing Plant „Warmia” (Biskupiec, Poland) and transported on ice to the laboratory within 40 min. Day of the oestrous cycle was evaluated by macro- scopic observation of the ovaries and uterus (Miyamoto et al. 2000) and confirmed by determina- tion of progesterone (P4) levels in peripheral blood plasma collected from the jugular vein using radioim- munoassay (RIA). The level of P4 ranged from 15 to 20 ng/ml. Just before slaughter, each animal was examined by a veterinarian via per rectum ultra- sound-guided examination to exclude abnormal ovary structure. The reasons for culling animals from the herd were of economic nature and herd renewal, none of used for experiments cows was earlier treated for endometritis. Tissue fragments (cross-sections of the uterine wall, i.e., endometrium and myometrium) were ob- tained from the middle segment of the uterine horn ipsilateral to the corpus luteum and were divided into three pieces: the first one was fixed in 4% paraformal- dehyde (PFA) in 0.1 M PBS (pH 7.4) for histo- and immunohistofluorescence staining, the second was frozen and stored at -86 oC for further mRNA and protein expression determination in uterine tissue (endometrium with myometrium), and the third piece was used for immediate isolation and culture of uter- ine cells. The tissues for experiments were divided into nor- mal (uteri without endometrial glands within the my- ometrial layer, n=9) and adenomyotic (endometrial foci present in the myometrium, n=9), based on microscopic observation of hematoxylin and eosin- -stained uterine cross-section slices. A.J. Korzekwa et al.610 Uterine cell isolation and in vitro culture Endometrial stromal cells were isolated by enzy- matic dissociation as previously described (Łupicka et al. 2015). After endometrial cell isolation, the my- ometrial layer of the uterus was dissected with scis- sors. About 4 cm-long fragments of muscle tissue were chopped up with scissors into a homogeneous material. Approximately 5 g of the chopped tissue was digested in 50 ml of M199 medium (Sigma, M2520, St. Louis, MO, USA) containing 0.1% of bovine serum albumin (BSA; Sigma, A2058), 20 μg/ml of gentamicin (Sigma, G1271), 2 mg/ml of col- lagenase I (Sigma, C0130), 1 mg/ml of deoxyribonuc- lease (Sigma, D5025) and 2 mg/ml of dispase (Life Technologies, 17105-041, Paisley, UK). The enzyme solution with myometrial tissue was held at 37.5 oC with stirring for 30 min. After digestion, the cell sus- pension was filtered through a mesh to remove un- digested tissue fragments, then the cells were washed by centrifugation (10 min at 100 x g, at 4 oC). Cells were resuspended in culture medium (DMEM; Sigma, D5796) supplemented with 10% of fetal calf serum (FCS; Sigma, 12133C) and antibiotics (gen- tamicin/amphotericin B; Life Technologies, 1153727). Uterine cells were plated in a co-culture system, in which myometrial cells were cultured at the bot- tom of wells in 6-well culture plates (basal compart- ment) and stromal cells were cultured on col- lagen-coated inserts (Biocoat ® Cell Culture Inserts Collagen Type I, BD Biosciences, Bedford, MA; api- cal compartment) at 37.5 oCi nah u m i d i f i e da t m o s - phere of 5% CO 2, 95% air. Both cell types were cul- tured in a phenol red-free DMEM medium contain- ing 10% of fetal calf serum (FCS; Sigma, 12133C) and antibiotics. The medium (DMEM with 10% FCS and antibiotics) was changed every 2 days until 70% confluence was reached (approx. on the 4 th day of culture). For the last 24 h of culture, inserts with stromal cells were replaced with myometrial cells. After changing the medium for DMEM containing 0.1% BSA and antibiotics, the co-cultures were stimulated with E2 (10 -7M). Total mRNA and cell lysates were collected from the cultures. Cell culture homogeneity was confirmed using real-time PCR for determination of mRNA expression of vimentin and desmin for stromal and myometrial cells, respective- ly, in separate cultures, before establishing co-cul- tures (Zeiler et al. 2007). Experimental procedures Experiment 1. Localization, mRNA and protein expression of connexin43 in uterine tissues of healthy and adenomyotic cows Cx43 immunolocalization (immunofluorescence) and comparison of Cx43 mRNA (real time PCR) and p r o t e i n( W e s t e r nb l o t t i n g )e x p r e s s i o ni nh e a l t h y (N=9) and adenomyotic uterine tissue (N=9) was proceeded. Experiment 2. The effect of 17-beta estradiol on Cx43 mRNA expression in uterine stroma – myometrium co-culture of healthy and adenomyotic cows Cx43 mRNA expression (real time PCR) was evaluated in uterine stromal, myometrial cells and in myometrial cells co-cultured with stromal cells after 24 h stimulation with E2 in healthy (N=5) and aden- omyotic (N=5) cows. The stroma – myometrium co-cultures, after re- ceiving the 70% of confluence, were stimulated for 24 h with E2 (10 -7M). Cells were isolated from healthy (N=5) and adenomyotic (N=5) cows. The effective dose of E2 was selected based on the pre- liminary study. Histochemical staining Uterine tissue was fixed in 4% PFA and pro- cessed for a standard haematoxylin and eosin stain- ing protocol. Stained cross-sections of the tissue were observed under a light microscope (Nikon FXA, Tokyo, Japan). Animals were classified as de- scribed previously (Korzekwa et al. 2013, Łupicka et al. 2015); briefly, if uterine glands were present only in the endometrium, and if the endometrial-myomet- rial border was clearly visible, cows were classified as normal/control. Whereas, if the glands penetrated the myometrial layer of the uterus, animals were classified as adenomyotic (according to the classifica- tion of Katkiewicz et al. 2005, data not shown). Immunofluorescence staining Immunohistofluorescence was used to localise Cx43 in uterine tissues. Cross-sections of uterine horn samples were fixed in 4% PFA in 0.1 M PBS (pH 7.4), and cryoprotected in 18% sucrose. Im- munostaining was carried out on consecutive 7 μm Estradiol reduces connexin43 gap junctions... 611 Table 1. Oligonucleotide sequences used for real-time PCR. Gene Oligonucleotide sequences Product size (bp) GeneBank vimentin FWD 5’-GACCTGGAGCGTAAAGTGG-3’ REV 5’-GACATGCTGTTCTTGAATCTGG-3’ 108 BC118269 desmin FWD 5’-GACCCAGGCAGCCAACAAG-3’ REV 5’-GTCGATCTCGCAGGTGTAGG-3’ 100 BC133410 cx43 FWD 5’-TGAGTGCCGTTTACACTTGC-3’ REV 5’-GGCAAGAGACACCAATGACA-3’ 125 J05535 GAPDH FWD 5’-CACCCTCAAGATTGTCAGCA-3’ REV 5’-GGTCATAAGTCCCTCCACGA-3’ 103 BC102589 cryostat sections. To block endogenous peroxidase, the sections were treated with hydrogen peroxide in methanol and washed in 0.1 M PBS. The sections were blocked with 10% normal donkey serum (Sigma, G9023) for 1 h at room temperature (ap- prox. 23 oC; RT), and incubated overnight at RT with a 1:500 dilution of anti-Cx43 (Sigma, C8093), Next, the cells were washed 3x with PBS and incubated 1 h at RT with secondary antibodies conjugated with cyanine 3 (CY 3; Jackson ImmunoResearch, West Grove, PA, 715-165-150). Connexin43 was visualized with confocal imaging using a Nikon C1 confocal microscope. Total RNA isolation Total RNA was extracted from uterine tissues (ap- prox. 30 mg) and from cultured cells using TRI-Re- agent (Sigma, T9424) according to the manufacturer’s instructions. The content and purity of RNA was as- sessed on a NanoDrop 1000 (Thermo Fisher Scien- tific, ND-1000, Wilmington, DE, USA). One micro- gram of each sample of total RNA was reverse-tran- scribed to cDNA with the QuantiTect Reverse Tran- scription kit (Life Technologies, 205313), as described in the supplier’s protocol. The cDNA obtained was stored at -20 oC until real-time PCR was applied. Real-time PCR quantification mRNA expression for Cx43 in tissues and cells was determined by quantitative real-time PCR. The experiments were performed using the Applied Bio- systems 7900 (Applied Biosystems, Foster City, CA, USA) with SensiFAST SYBR Hi-ROX Kit (Bioline Reagents, BIO-92002, London, UK) according to the manufacturer’s instructions. The real-time PCR re- action mix (20 μl) contained 19 μlo fS e n s i F A S T SYBR Hi-ROX Master Mix, 0.5 μMo fs e n s ea n d antisense primers, and 1 μl of reverse-transcribed cDNA (50 ng). Primer sequences used for determi- nation of Cx43 and glyceraldehyde 3-phosphate de- hydrogenase ( GAPDH) mRNA expression are de- tailed in Table 1. Standard curves consisting of serial dilutions of the appropriate cDNA were plotted for efficiency evaluation. Amplification was initiated by an initial enzyme activation step (2 min, 95 oC). The PCR steps were as follows: 40 cycles of denaturation (5 sec, 95 oC), then annealing and extending (20 sec, 60oC). After amplification, melting curves were ac- quired by stepwise increases of temperature from 50 to 95oC to ensure that a single product was amplified and no primer-dimer structures were formed. Con- trol reactions in the absence of the template or primers were performed to confirm that products were free from genomic DNA contamination. Disso- ciation curves analysis was carried out after each re- altime experiment to confirm the presence of only one amplification product. Data were normalized us- ing the ΔΔ C t method. Samples were amplified in du- plicates. Data are shown as the average fold increase, with S.E.M., and are expressed relative to the house- keeping gene GAPDH. Western blotting Protein expression for Cx43 in the tissues and cells was determined by Western blotting. Proteins from homogenized tissues and in vitro cultured cells were released with lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1% TritonX-100, 0.5% sodium deoxycholate and protease inhibitors (Sigma, P8340). The lysates were stored at -86 oC until further analy- sis. Protein concentrations were measured by the Bradford’s method. Western blot analysis was performed as previous- ly described (Korzekwa et al. 2011). Equal amounts of protein were dissolved in SDS gel-loading buffer, heated to 95 oC for 4 min and separated in 10% SDS-PAGE gel. Separated proteins were electrob- A.J. Korzekwa et al.612 A) B) C) D) 20 µm Fig. 1. Immunfluorescence detection of Cx43 in uterine tissues from control cows and from cows with adenomyosis. A – nega- tive control, B – Cx43 in adenomyotic tissue, C – in healthy tissue. Arrows indicate the most intense histochemical reactions. Scale bars: 20 μm. lotted onto 0.2 μm nitrocellulose membranes in transfer buffer. After blocking in 5% non-fat dry milk in TBS-T buffer for 1.5 h at RT, the membranes were incubated overnight with a 1:500 dilution of anti-Cx43 (Sigma, C8093) antibodies; GAPDH (Sigma, G8795; monoclonal anti-glyceraldehyde-3- -phosphate dehydrogenase antibody produced in m o u s e )e x p r e s s i o nw a su s e da sar e f e r e n c e .P r o t e i n s were detected by incubating the membranes with a 1:20,000 dilution of secondary polyclonal anti-mouse alkaline phosphatase-conjugated anti- body (Sigma, A 3562) for 1.5 h at RT. Western blots were quantitated using the Kodak 1 D program (Eastman Kodak, Rochester, NY, USA). Hormone determination Measurements of P4 in blood plasma were per- formed using a direct radioimmunoassay (RIA; DI- ASource ImmunoAssays S.A., Nivelles, Belgium). The standard curve ranged from 0.12 to 36 ng/ml and the effective dose for 50% inhibition (ED 50) of the assay was 0.05 ng/ml. The intra- and inter-assay coeffi- cients of variation (CV) were 6.5% and 8.6%, respectively. Statistical analysis In Experiment 1, statistically significant differen- ces in mRNA and protein expression between healthy and adenomyotic uterine tissue were detected using Student’s t-test (GraphPad PRISM Version 5.00, San Diego, CA, USA). The data are shown as the mean ± SEM of values obtained in nine separate experi- ments. The results were considered significantly dif- ferent when p<0.05. In Experiment 2, statistically significant differen- ces in mRNA expression between healthy and aden- omyotic uterine cells and between control and E2 stimulated cells were performed using two-way ANOVA followed by the Bonferroni multiple com- parison test (GraphPad). All data were expressed as means ± SEM. Differences were considered signifi- cant at p<0.05.

Preliminary division of the material Based on microscopic examination of hematoxylin and eosin stained tissue slices, bovine uteri were divided into two groups: control and adenomyotic. Estradiol reduces connexin43 gap junctions... 613 0.0 0.5 1.0 1.5 2.0 2.5 * 0.0 0.2 0.4 0.6 0.8 healthy adenomyotic cows * GAPDH (37 kDa) Cx43 (43 kDa) a) b) Cx43/GAPDH protein expression (arbitary units) Cx43/GAPDH mRNA expression (arbitary units) Fig. 2. mRNA (a) and protein (b) expression of Cx43 in uterine tissues obtained from control cows and from cows with adenomyosis. Data were normalized against glyceral- dehyde-3-phosphate dehydrogenase ( GAPDH). Bars repre- sent the mean ± SEM. Asterisks indicate statistical differ- ence between uterine normal and adenomyotic tissues (p<0.05), as determined by Student’s t-test. Representative blots for Cx43 and GAPDH are shown below the graphs. Experiment 1. Localization, mRNA and protein expression of connexin43 in uterine tissues of healthy and adenomyotic cows Immunofluorescence revealed the localization of Cx43 in uteri of healthy and adenomyotic cows. The protein was localized in the endometrium as well in the myometrium layer of uteri. Figure 1A represents control tissue with Cx43 antibody omitted. In the case of adenomyotic tissues, immunoreactivity was ob- served near the glands located in endometrium and myometrium, characteristic for adenomyosis 0.0 0.2 0.4 0.6 0.8 1.0 2 4 6 estradiol (10 -7 M) control 0 1 2 3 3 4 5 6 * a b 0 1 2 3 4 5 6 * a b a b control E2 control E2 healthy adenomyosis a) STROMAL CELLS b) MYOMETRIAL CELLS c) COCULTURE CELLS Cx43/GAPDH mRNA expression (arbitrary units) Fig. 3. mRNA expression of Cx43 in stromal (a), myometrial (b) and co-cultured, stromal and myometrial (c) uterine cells isolated from control cows and from cows with adenomyosis. Data were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Statistical differences were ana- lysed by two-way ANOVA followed by the Bonferroni multiple comparison test (GraphPad). All data were ex- pressed as means ± SEM. Differences were considered sig- nificant at p <0.05. Asterisks indicate statistical differences between uterine normal and adenomyotic groups and differ- ent letters (a, b) between control and E2 treatment (* p<0.05). (Fig. 1B). In healthy cows Cx43 immunoreactivity was observed in endometrium, and near blood vessels in myometrium (Fig. 1C,D). mRNA and protein expression of Cx43 was de- creased in adenomyotic uterine tissue compared with normal uteri (p<0.05, Fig. 2A,B). A.J. Korzekwa et al.614 Experiment 2. The effect of 17-beta estradiol on Cx43 mRNA expression in uterine stroma – myometrium co-culture of healthy and adenomyotic cows The expression of Cx43 was not different between healthy and adenomyotic cows and E2 did not influ- ence Cx43 expression in stromal cells (p >0.05; Fig. 3A). In myometrial cells, expression of Cx43 increased after E2 stimulation in the adenomyotic group and the effect of E2 was higher in adenomyotic myometrial cells compared with healthy cells (p<0.05; Fig. 3B). In co-culture of stromal and myometrial cells, mRNA expression of Cx43 was elevated after E2 stimulation (p <0.05). Moreover the increase in ex- pression after E2 treatment was higher in aden- omyotic compared with healthy cell co-cultures (p<0.05; Fig. 3C).

The present study demonstrated, for the first time, Cx43 mRNA and protein expression and localization in uterine tissue and cells during the estrous cycle in cows. mRNA and protein expression of Cx43 was de- creased in adenomyotic uterine tissue compared with normal uteri. Moreover differences in Cx43 express- ion during adenomyosis including the effect of E2 were examined. In uterine stroma – myometrium co-culture of adenomyotic cows, E2 caused increase of Cx43 mRNA expression with healthy cell co-cul- tures. So far, Cx43 function have been described in con- nection with pregnancy and labor in human (Laws et al. 2008), porcine (Romek and Karasiński 2011) and mouse (Doring et al. 2006) uteri. All these studies reported an increase in Cx43 in the myometrium dur- ing term and preterm delivery as essential for the de- velopment of uterine contractions. Xu et al. (2015) have showed lately that in human uterine myometrial cells prostaglandin F2alpha directly stimulated Cx43 protein expression. Thus, prostaglandins as main fac- tors influencing on uterine function regulate Cx43 and this regulation may also differ during adenomyosis but it demands further study. During pathological conditions, gap junctions in the uterus are supposed to be dysregulated. Connex- in43 immunohistostaining and protein expression were inhibited in uterus with diagnosed endometriosis comparing with uteri of healthy women (Yu et al. 2014). In our study, we demonstrated decreased Cx43 mRNA and protein expression in adenomyotic uteri, which is in accordance with the results concerning Cx43 expression during endometriosis in women. Nevertheless, in our in vitro experiment, Cx43 mRNA expression was similar in stromal, myometrial and co-cultured cells of healthy and adenomyotic cows. The discrepancies between results obtained from uterine tissue and cells may arise because Cx express- ion is regulated by other factors that are not present during culture of pure cells. The pathogenesis and aetiology of both endomet- riosis and adenomyosis are still unclear but during both disorders E2 overproduction has been observed (Takahashi et al. 1989, Chen et al. 2010, Korzekwa et al. 2013). In our study, E2 stimulated Cx43 mRNA expression in myometrial and co-cultured both my- ometrial and stromal cells collected from adenomyotic cows as well as in co-cultures of cells derived from healthy animals. Estradiol is necessary to provoke Cx43 expression if the putative estrogen response elements are located in the Cx43 gene region (Lefebvre et al. 1995). The connection between Cx43 and endometriosis of uterus were described by Regidor et al. (1997). These authors observed Cx43 by immunohistostaining, which was correlated with a high E2 serum level in women. Our results indicate that during adenomyosis, especially in myometrial cells, E2 which is a potential inducer of this disease increases gap junction forma- tion in the bovine uterus, which may support the mi- gration of glands from the stroma to the myometrium. In conclusion, our results revealed that during ad- enomyosis in the bovine uterus, gap junctions are ex- pressed at a lower level than in healthy cows in the mid-luteal stage. Moreover, Cx43 expression is stimu- lated by E2 in adenomyotic myometrial and co-cul- tured stromal and myometrial uterine cells. These data suggest the involvement of gap junctions in de- velopment of adenomyosis and a correlation between E2 influence and gap junction increase. However, elu- cidating the connections between steroids and gap junctions and the molecular mechanism of E2 action during adenomyosis of the uterus in cattle will require further study.

The authors wish to thank W. Krzywiec for techni- cal support and tissue collection and M. Domin – the owner of the slaughterhouse (Meat Processing Plant „Warmia”, Biskupiec, Poland) for permitting collec- tion of the material. We are grateful to dr. G. Bodek (Laboratory in vitro, Institute of Animal Reproduc- tion and Food Research of PAS, Olsztyn, Poland) for making pictures of uterine Cx43 histostaining ob- served under fluorescence confocal microscope. Estradiol reduces connexin43 gap junctions... 615

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