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Ida Björkgren and Petra Sipilä

The epididymis is necessary for post-testicular sperm maturation as it provides the milieu required for spermatozoa to gain the ability for progressive movement and fertilization. In the epididymis the sperm protein, lipid and small RNA content are heavily modified due to interaction with luminal proteins secreted by the epididymal epithelium and extracellular vesicles, epididymosomes. This review focuses on epididymal proteins demonstrated to have an effect on sperm functions, such as motility, capacitation, acrosome reaction, sperm-zona pellucida binding and sperm-egg binding, as well as on embryonic development.

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Srinivas V Seekallu, Behzad M Toosi, Anna T Grazul-Bilska, and Norman C Rawlings

Treatment of non-prolific western white-faced ewes with prostaglandin F (PGF) and medroxyprogesterone acetate (MAP) increases the ovulation rate as a result of ovulations from the penultimate wave in addition to the final wave of the cycle. The objective of the current study was to evaluate the expression of markers of vascularization/angiogenesis, a marker of intercellular communication, and cellular proliferation and apoptosis in follicles from the penultimate and final waves. On day 8 of the estrous cycle, 15 ewes were administered a single injection of PGF and an intravaginal MAP sponge, which remained in place for 6 days. Two days after sponge removal, ovaries which contained follicles from the penultimate and final waves were collected and processed for immunohistochemistry followed by image analysis, and for quantitative real-time RT-PCR. Expression of factor VIII (marker of vascularization), proliferating cell nuclear antigen, and GJA1 (Cx43; marker of gap junctional communication) was greater (P<0.05) in follicles from the final wave compared with follicles from the penultimate wave. For theca cells, mRNA expression for vascular endothelial growth factor (VEGF) was greater (P<0.05) and tended to be greater (P≤0.1 and ≥0.05) for GJA1 and endothelial nitric oxide synthase in follicles from the final wave compared with follicles from the penultimate wave. For granulosa cells, the mRNA expression for GJA1 was greater (P<0.05) and tended to be greater (P≤0.1 and ≥0.05) for VEGF in follicles from the final wave compared with follicles from the penultimate wave. In conclusion, extension of the lifespan of follicles in the penultimate wave reduces follicular viability in the ewe.

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Felix R Graubner, Alois Boos, Selim Aslan, Ibrahim Kücükaslan, and Mariusz P Kowalewski

For many years, modifications of the uterine extracellular matrix (ECM) during gestation have not been considered as critical for successful canine (Canis lupus familiaris) pregnancy. However, previous reports indicated an effect of free-floating blastocysts on the composition of the uterine ECM. Here, the expression of selected genes involved in structural functions, cell-to-cell communication and inhibition of matrix metalloproteinases were targeted utilizing qPCR and immunohistochemistry. We found that canine free-floating embryos affect gene expression of FN1, ECM1 and TIMP4. This seems to be associated with modulation of trophoblast invasion, and proliferative and adhesive functions of the uterus. Although not modulated at the beginning of pregnancy, the decrease of structural ECM components (i.e. COL1, -3, -4 and LAMA 2) from pre-implantation toward post-implantation at placentation sites appears to be associated with softening of the tissue in preparation for trophoblast invasion. The further decrease of these components at placentation sites at the time of prepartum luteolysis seems to be associated with preparation for the release of fetal membranes. Reflecting a high degree of communication, intercellular cell adhesion molecules are induced following placentation (Cx26) or increase gradually toward prepartum luteolysis (Cx43). The spatio-temporal expression of TIMPs suggests their active involvement in modulating fetal invasiveness, and together with ECM1, they appear to protect deeper endometrial structures from trophoblast invasion. With this, the dog appears to be an interesting model for investigating placental functions in other species, e.g. in humans in which Placenta accreta appears to share several similarities with canine subinvolution of placental sites (SIPS). In summary, the canine uterine ECM is only moderately modified in early pregnancy, but undergoes vigorous reorganization processes in the uterus and placenta following implantation.

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M Fofana, C Travert, S Carreau, and D Le Goff

Sertoli cells and germ cells are separated from the interstitial blood capillaries by an extracellular matrix and the peritubular cells, which constitute a barrier to the movement of plasma lipoproteins. The present study was undertaken to evaluate in vivo and in vitro the high density lipoprotein (HDL) cholesteryl ester transfer from plasma to seminiferous tubule cells in the testis of 30-day-old rats. Firstly, the transfer of HDL cholesteryl oleate from plasma to testicular compartments was evaluated and, secondly, the role of apolipoproteins A-I and E in the uptake of cholesteryl ester by Sertoli cells was investigated. At 2 h after the administration of HDL reconstituted with [3H]cholesteryl ester, dimyristoyl phosphatidylcholine and apolipoproteins, the tissue space in the interstitial cells (740 +/- 60 microliters g-1 cell protein) was fourfold higher than that in the seminiferous tubule cells (170 +/- 10 microliters g-1). Sertoli cells were isolated and incubated with [3H]cholesteryl ester HDL reconstituted with apolipoprotein A-I or E to evaluate the mechanisms of cholesteryl ester influx. At the same apolipoprotein concentration (50 micrograms apolipoprotein ml-1 medium), the uptake of [3H]cholesteryl oleate from phospholipid-apolipoprotein E vesicles was twofold higher than that with phospholipid-apolipoprotein A-I vesicles. The presence of heparin reduced the uptake of cholesteryl ester from apolipoprotein E vesicles but not with apolipoprotein A-I vesicles, indicating that uptake of apolipoprotein A-I vesicles via a secretion of apolipoprotein E by the cells themselves was not involved. These results demonstrate that plasma lipoprotein cholesterol is able to cross the testis lamina propria and that Sertoli cells take up cholesteryl ester for seminiferous tubule cell metabolism mainly via an apolipoprotein E pathway.

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Néstor Saiz and Berenika Plusa

During mammalian preimplantation development, the fertilised egg gives rise to a group of pluripotent embryonic cells, the epiblast, and to the extraembryonic lineages that support the development of the foetus during subsequent phases of development. This preimplantation period not only accommodates the first cell fate decisions in a mammal's life but also the transition from a totipotent cell, the zygote, capable of producing any cell type in the animal, to cells with a restricted developmental potential. The cellular and molecular mechanisms governing the balance between developmental potential and lineage specification have intrigued developmental biologists for decades. The preimplantation mouse embryo offers an invaluable system to study cell differentiation as well as the emergence and maintenance of pluripotency in the embryo. Here we review the most recent findings on the mechanisms controlling these early cell fate decisions. The model that emerges from the current evidence indicates that cell differentiation in the preimplantation embryo depends on cellular interaction and intercellular communication. This strategy underlies the plasticity of the early mouse embryo and ensures the correct specification of the first mammalian cell lineages.

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H. Funahashi and B. N. Day

The effect of hormone supplements on cytoplasmic maturation in vitro was examined by incubating oocyte–cumulus complexes in a medium with PMSG (10 iu ml−1), hCG (10 iu ml−1) and oestradiol (1 μg ml−1) for various periods and then transferring them to medium without added hormones for the remainder of the maturation period. Exposure of oocyte–cumulus complexes to hormone supplements for 2 h improved only germinal vesicle breakdown and maturation rates compared with complexes not exposed to added hormones. The removal of hormone supplements at 20 h after the start of culture enhanced the ability of oocytes to form male pronuclei 10 to 12 h after insemination. Further, the effects of transfer of intact and oocytectomized oocyte–cumulus complexes to hormone-free medium at 20 h on cumulus expansion were examined. The diameter and morphology of the intact oocyte–cumulus complexes were improved after the removal of oocyte–cumulus cell complexes from hormonal exposure. The responses of oocytectomized oocyte–cumulus complexes to hormone were similar to those of intact oocyte–cumulus complexes with the exception of corona radiata expansion. The results suggest that the removal of hormone supplements from maturation media at 20 h after culture enhanced cytoplasmic maturation and cumulus expansion. Further, cumulus expansion does not appear to depend on intercellular communication between cumulus cells and oocytes. Oocytectomy did influence expansion of the corona radiata during culture.

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Daniel Herr, Inga Bekes, and Christine Wulff

In a developing human corpus luteum, a closely regulated cellular communication system exists between the luteal steroidogenic cells and endothelial cells. This system guaranties the vascularization process during luteal formation. The process is combined with rapid release of large amounts of progesterone into the bloodstream. The regulation of endothelial proliferation and permeability by LH and human chorionic gonadotropin (hCG) is integral to this process. On the cellular level, endothelial permeability is regulated by intercellular junctions, such as adherens junctions (AJ) and tight junctions (TJ), which act as zipper-like structures between interacting endothelial cells. Several cell junctional proteins are localized to the corpus luteum, including Occludin, Nectin 2, Claudin 1, and Claudin 5, as well as, vascular endothelial (VE)-Cadherin. It has been assumed that regulation of AJ- and TJ-proteins is of particular importance for permeability, and accordingly, for the functionality of the corpus luteum in early pregnancy, because treatment with hCG induces downregulation of juntional proteins in the luteal vessels. The effect of hCG on the adhesive molecules is mediated by VE growth factor (VEGF). On a functional level, the hCG-dependent and VEGF-mediated decrease in junctional proteins causes a decrease in the density of cell–cell closure and, accordingly, an increase in endothelial permeability. In doing so, the different junctional proteins are not only directly influenced by VEGF but also interact among themselves and influence each other reciprocally. Disturbances in this strictly, regulated interactions may explain the development of pathologies with increased vascular permeability, such as the ovarian hyperstimulation syndrome.

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GM Kidder and AA Mhawi

Gap junctions are collections of intercellular membrane channels that allow adjacent cells to share small molecules (< 1 kDa). Gap junction channels are composed of connexins, a homologous family of more than 20 proteins. In developing follicles, gap junctions couple the growing oocyte and its surrounding follicle cells into a functional syncytium. This review summarizes evidence on the expression of various connexins in developing follicles and the likely roles that some of the connexins play, on the basis of findings from gene targeting experiments in mice. Gap junctions between cumulus cells contain predominantly connexin43, and this connexin has also been detected using immunoelectron microscopy in a small minority of gap junctions at the oocyte surface. The importance of connexin43 for granulosa cell function is demonstrated by the fact that follicles lacking this connexin arrest in early preantral stages and produce incompetent oocytes. Connexin37 appears to be the only connexin contributed by oocytes to the gap junctions coupling them with granulosa cells, and loss of this connexin interferes with the development of antral follicles. The expression of multiple connexins in developing follicles is thus likely to reflect the multiple functions served by gap junctional communication in folliculogenesis.

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C Vozzi, A Formenton, A Chanson, A Senn, R Sahli, P Shaw, P Nicod, M Germond, and JA Haefliger

In ovarian follicles, cumulus cells provide the oocyte with small molecules that permit growth and control maturation. These nutrients reach the germinal cell through gap junction channels, which are present between the cumulus cells and the oocyte, and between the cumulus cells. In this study the involvement of intercellular communication mediated by gap junction channels on oocyte maturation of in vitro cultured bovine cumulus-oocyte complexes (COCs) was investigated. The stages of oocyte maturation were determined by Hoechst 33342 staining, which showed that 90% of COCs placed in the maturation medium for 24 h progress to the metaphase II stage. Bovine COC gap junction communication was disrupted initially using n-alkanols, which inhibit any passage through gap junctions. In the presence of 1-heptanol (3 mmol l(-1)) or octanol (3.0 mmol l(-1) and 0.3 mmol l(-1)), only 29% of the COCs reached metaphase II. Removal of the uncoupling agent was associated with restoration of oocyte maturation, indicating that treatment with n-alkanols was neither cytotoxic nor irreversible. Concentrations of connexin 43 (Cx43), the major gap junction protein expressed in the COCs, were decreased specifically using a recombinant adenovirus expressing the antisense Cx43 cDNA (Ad-asCx43). The efficacy of adenoviral infection was > 95% in cumulus cells evaluated after infection with recombinant adenoviruses expressing the green fluorescence protein. RT-PCR performed on total RNA isolated from Ad-asCx43-infected COCs showed that the rat Cx43 cDNA was transcribed. Western blot analysis revealed a three-fold decrease in Cx43 expression in COCs expressing the antisense RNA for Cx43. Injection of cumulus cells with Lucifer yellow demonstrated further that the resulting lower amount of Cx43 in infected COCs is associated with a two-fold decrease in the extent of coupling between cumulus cells. In addition, oocyte maturation was decreased by 50% in the infected COC cultures. These results indicate that Cx43-mediated communication between cumulus cells plays a crucial role in maturation of bovine oocytes.

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Z P Reliszko, Z Gajewski, and M M Kaczmarek

Circulating miRNAs were proposed to be indicators of normal or complicated pregnancies. Based on this knowledge and our recent transcriptomic approach showing expression of miRNAs in the porcine endometrium, conceptuses and uterine extracellular vesicles during pregnancy, we have hypothesized that signs of ongoing local embryo-maternal crosstalk involving miRNAs can be detected in the circulation of pregnant gilts as early as a few days after maternal recognition of pregnancy. By applying several molecular biology techniques that differ in dynamic range and precision in maternal serum of Day 16 pregnant pigs, we were able to show for the first time increased levels of several miRNAs, previously reported to be expressed in either conceptuses and extracellular vesicles (miR-26a and miR-125b) or pregnant endometrium (miR-23b). Our results clearly showed that real-time RT-PCR and digital PCR are the most reliable methods, being able to detect small-fold changes of low-abundant circulating miRNAs. Further validation in a separate group of gilts confirmed an increase in miR-23b and miR-125b levels. In silico analyses identified pregnancy-related biological processes and pathways affected by these miRNAs. Target prediction analysis revealed hundreds of porcine transcripts with conserved sites for these miRNAs, which were classified into signaling pathways relevant to pregnancy. We conclude that a unique set of miRNAs can already be observed in the circulation of pigs during the first weeks of pregnancy, as a result of the initiation of embryo-maternal communication.