Laurent Galio

Endometrium as an early sensor of in vitro embryo manipulation technologies

Implantation is crucial for placental development that will subsequently impact fetal growth and pregnancy success with consequences on postnatal health. We postulated that the pattern of genes expressed by the endometrium when the embryo becomes attached to the mother uterus could account for the final outcome of a pregnancy. As a model, we used the bovine species where the embryo becomes progressively and permanently attached to the endometrium from day 20 of gestation onwards. At that stage, we compared the endometrial genes profiles in the presence of an in vivo fertilized embryo (AI) with the endometrial patterns obtained in the presence of nuclear transfer (SCNT) or in vitro fertilized embryos (IVF), both displaying lower and different potentials for term development. Our data provide evidence that the endometrium can be considered as a biological sensor able to fine-tune its physiology in response to the presence of embryos whose development will become altered much later after the implantation process. Compared with AI, numerous biological functions and several canonical pathways with a major impact on metabolism and immune function were found to be significantly altered in the endometrium of SCNT pregnancies at implantation, whereas the differences were less pronounced with IVF embryos. Determining the limits of the endometrial plasticity at the onset of implantation should bring new insights on the contribution of the maternal environment to the development of an embryo and the success of pregnancy.

Gene expression profiles of bovine caruncular and intercaruncular endometrium at implantation

At implantation the endometrium undergoes modifications necessary for its physical interactions with the trophoblast as well as the development of the conceptus. We aim to identify endometrial factors and pathways essential for a successful implantation in the caruncular (C) and the intercaruncular (IC) areas in cattle. Using a 13,257-element bovine oligonucleotide array, we established expression profiles at day 20 of the estrous cycle or pregnancy (implantation), revealing 446 and 1,295 differentially expressed genes (DEG) in C and IC areas, respectively (false discovery rate = 0.08). The impact of the conceptus was higher on the immune response function in C but more prominent on the regulation of metabolism function in IC. The C vs. IC direct comparison revealed 1,177 and 453 DEG in cyclic and pregnant animals respectively (false discovery rate = 0.05), with a major impact of the conceptus on metabolism and cell adhesion. We selected 15 genes including C11ORF34, CXCL12, CXCR4, PLAC8, SCARA5, and NPY and confirmed their differential expression by quantitative RT-PCR. The cellular localization was analyzed by in situ hybridization and, upon pregnancy, showed gene-specific patterns of cell distribution, including a high level of expression in the luminal epithelium for C11ORF34 and MX1. Using primary cultures of bovine endometrial cells, we identified PTN, PLAC8, and CXCL12 as interferon-tau (IFNT) target genes and MSX1 and CXCR7 as IFNT-regulated genes, whereas C11ORF34 was not an IFNT-regulated gene. Our transcriptomic data provide novel molecular insights accounting for the biological functions related to the C or IC endometrial areas and may contribute to the identification of potential biomarkers for normal and perturbed early pregnancy.

MicroRNA in the ovine mammary gland during early pregnancy: spatial and temporal expression of miR-21, miR-205, and miR-200.

The mammary gland undergoes extensive remodeling between the beginning of pregnancy and lactation; this involves cellular processes including cell proliferation, differentiation, and apoptosis, all of which are under the control of numerous regulators. To unravel the role played by miRNA, we describe here 47 new ovine miRNA cloned from mammary gland in early pregnancy displaying strong similarities with those already identified in the cow, human, or mouse. A microarray study of miRNA variations in the adult ovine mammary gland during pregnancy and lactation showed that 100 miRNA are regulated according to three principal patterns of expression: a decrease in early pregnancy, a peak at midpregnancy, or an increase throughout late pregnancy and lactation. One miRNA displaying each pattern (miR-21, miR-205, and miR-200b) was analyzed by qRT-PCR. Variations in expression were confirmed for all three miRNA. Using in situ hybridization, we detected both miR-21 and miR-200 in luminal mammary epithelial cells when expressed, whereas miR-205 was expressed in basal cells during the first half of pregnancy and then in luminal cells during the second half. We therefore conclude that miR-21 is strongly expressed in the luminal cells of the normal mammary gland during early pregnancy when extensive cell proliferation occurs. In addition, we show that miR-205 and miR-200 are coexpressed in luminal cells, but only during the second half of pregnancy. These two miRNA may cooperate to maintain epithelial status by repressing an EMT-like program, to achieve and preserve the secretory phenotype of mammary epithelial cells.

Unilateral once daily milking locally induces differential gene expression in both mammary tissue and milk epithelial cells revealing mammary remodeling

Once daily milking reduces milk yield, but the underlying mechanisms are not yet fully understood. Local regulation due to milk stasis in the tissue may contribute to this effect, but such mechanisms have not yet been fully described. To challenge this hypothesis, one udder half of six Holstein dairy cows was milked once a day (ODM), and the other twice a day (TDM). On the 8th day of unilateral ODM, mammary epithelial cells (MEC) were purified from the milk using immunomagnetic separation. Mammary biopsies were harvested from both udder halves. The differences in transcript profiles between biopsies from ODM and TDM udder halves were analyzed by a 22k bovine oligonucleotide array, revealing 490 transcripts that were differentially expressed. The principal category of upregulated transcripts concerned mechanisms involved in cell proliferation and death. We further confirmed remodeling of the mammary tissue by immunohistochemistry, which showed less cell proliferation and more apoptosis in ODM udder halves. Gene expression analyzed by RT-qPCR in MEC purified from milk and mammary biopsies showed a common downregulation of six transcripts (ABCG2, FABP3, NUCB2, RNASE1 and 5, and SLC34A2) but also some discrepancies. First, none of the upregulated transcripts in biopsies varied in milk-purified MEC. Second, only milk-purified MEC showed significant LALBA downregulation, which suggests therefore that they correspond to a mammary epithelial cell subpopulation. Our results, obtained after unilateral milking, suggest that cell remodeling during ODM is due to a local effect, which may be triggered by milk accumulation.

Oleate and linoleate stimulate degradation of β-casein in prolactin-treated HC11 mouse mammary epithelial cells.

Although virtually all cells store neutral lipids as cytoplasmic lipid droplets, mammary epithelial cells have developed a specialized function to secrete them as milk fat globules. We have used the mammary epithelial cell line HC11 to evaluate the potential connections between the lipid and protein synthetic pathways. We show that unsaturated fatty acids induce a pronounced proliferation of cytoplasmic lipid droplets and stimulate the synthesis of adipose differentiation-related protein. Unexpectedly, the cellular level of β-casein, accumulated under lactogenic hormone treatment, decreases following treatment of the cells with unsaturated fatty acids. In contrast, saturated fatty acids have no significant effect on either cytoplasmic lipid droplet proliferation or cellular β-casein levels. We demonstrate that the action of unsaturated fatty acids on the level of β-casein is post-translational and requires protein synthesis. We have also observed that proteasome inhibitors potentiate β-casein degradation, indicating that proteasomal activity can destroy some cytosolic protein(s) involved in the process that negatively controls β-casein levels. Finally, lysosome inhibitors block the effect of unsaturated fatty acids on the cellular level of β-casein. Our data thus suggest that the degradation of β-casein occurs via the microautophagic pathway.

Oviduct extracellular vesicles protein content and their role during oviduct–embryo cross-talk

Successful pregnancy requires an appropriate communication between the mother and the embryo. Recently, exosomes and microvesicles, both membrane-bound extracellular vesicles (EVs) present in the oviduct fluid have been proposed as key modulators of this unique cross-talk. However, little is known about their content and their role during oviduct-embryo dialog. Given the known differences in secretions by in vivo and in vitro oviduct epithelial cells (OEC), we aimed at deciphering the oviduct EVs protein content from both sources. Moreover, we analyzed their functional effect on embryo development. Our study demonstrated for the first time the substantial differences between in vivo and in vitro oviduct EVs secretion/content. Mass spectrometry analysis identified 319 proteins in EVs, from which 186 were differentially expressed when in vivo and in vitro EVs were compared (P < 0.01). Interestingly, 97 were exclusively expressed in in vivo EVs, 47 were present only in in vitro and 175 were common. Functional analysis revealed key proteins involved in sperm-oocyte binding, fertilization and embryo development, some of them lacking in in vitro EVs. Moreover, we showed that in vitro-produced embryos were able to internalize in vivo EVs during culture with a functional effect in the embryo development. In vivo EVs increased blastocyst rate, extended embryo survival over time and improved embryo quality. Our study provides the first characterization of oviduct EVs, increasing our understanding of the role of oviduct EVs as modulators of gamete/embryo-oviduct interactions. Moreover, our results point them as promising tools to improve embryo development and survival under in vitro conditions.

Preattachment Embryos of Domestic Animals: Insights into Development and Paracrine Secretions

In mammalian species, endometrial receptivity is driven by maternal factors independently of embryo signals. When pregnancy initiates, paracrine secretions of the preattachment embryo are essential both for maternal recognition and endometrium preparation for implantation and for coordinating development of embryonic and extraembryonic tissues of the conceptus. This review mainly focuses on domestic large animal species. We first illustrate the major steps of preattachment embryo development, including elongation in bovine, ovine, porcine, and equine species. We next highlight conceptus secretions that are involved in the communication between extraembryonic and embryonic tissues, as well as between the conceptus and the endometrium. Finally, we introduce experimental data demonstrating the intimate connection between conceptus secretions and endometrial activity and how adverse events perturbing this interplay may affect the progression of implantation that will subsequently impact pregnancy outcome, postnatal health, and expression of production traits in livestock offspring.

Specific positioning of the casein gene cluster in active nuclear domains in luminal mammary epithelial cells

The nuclear organization of mammary epithelial cells has been shown to be sensitive to the three-dimensional microenvironment in several models of cultured cells. However, the relationships between the expression and position of genes have not often been explored in animal tissues. We therefore studied the localization of milk protein genes in the nuclei of luminal mammary epithelial cells during lactation as well as in two non-expressing cells, i.e., hepatocytes and the less differentiated embryonic fibroblasts. We compared the position of a cluster of co-regulated genes, encoding caseins (CSN), with that of the whey acidic protein (WAP) gene which is surrounded by genes displaying different expression profiles. We show that the position of the CSN cluster relative to various nuclear compartments is correlated with its activity. In luminal cells, the CSN cluster loops out from its chromosome territory and is positioned in the most euchromatic regions, and frequently associated with elongating RNA polymerase II-rich zones. In hepatocytes and embryonic fibroblasts, the cluster is found preferentially closer to the nuclear periphery. Interestingly, we had previously observed a very peripheral position of the CSN locus in the nuclei of HC11 mammary epithelial cells weakly expressing milk protein genes. We thus show that cultured cell lines are not fully representative of the nuclear organization of genes in a complex and highly organized tissue such as the mammary gland and propose that the spatial positioning of the locus is important to ensuring the optimum control of CSN gene activity observed in the mammary tissue.

Leptin gene in rabbit: cloning and expression in mammary epithelial cells during pregnancy and lactation

Leptin is known as a cytokine mostly produced by fat cells and implicated in regulation of energy metabolism and food intake but has also been shown to be involved in many physiological mechanisms such as tissue metabolism and cell differentiation and proliferation. In particular, leptin influences the development of mammary gland. Although leptin expression in mammary gland has been studied in several species, no data are available in the rabbit. Leptin transcripts in this species have been described as being encoded by only two exons rather than three as in other species. Our focus was to clone and sequence the rabbit leptin cDNA and to prepare the recombinant biologically active protein for validation of the proper sequence and then to describe leptin expression in rabbit mammary gland during different stages of pregnancy and lactation. The leptin sequence obtained was compared with those of other species, and genome alignment demonstrated that the rabbit leptin gene is also encoded by three exons. Additionally, we analyzed the expression of leptin during pregnancy and lactation. Leptin mRNA was weakly expressed throughout pregnancy, whereas mRNA levels were higher during lactation, with a significant increase between days 3 and 16. Leptin transcripts and protein were localized in luminal epithelial cells, thus indicating that leptin synthesis occurs in this compartment. Therefore, mammary synthesized leptin may constitute a major regulator of mammary gland development by acting locally as an autocrine and/or paracrine factor. Furthermore, our results support the possible physiological role of leptin in newborns through consumption of milk.

Milk from dams fed an obesogenic diet combined with a high-fat/high-sugar diet induces long-term abnormal mammary gland development in the rabbit

Alterations to the metabolic endocrine environment during early life are crucial to mammary gland development. Among these environmental parameters, the initial nutritional event after birth is the consumption of milk, which represents the first maternal support provided to mammalian newborns. Milk is a complex fluid that exerts effects far beyond its immediate nutritional value. The present study, therefore, aimed to determine the effect of the nutritional changes during the neonatal and prepubertal periods on the adult mammary phenotype. Newborn rabbits were suckled by dams fed a high-fat/high-sugar obesogenic (OD) or a control (CON) diet and then subsequently fed either the OD or CON diets from the onset of puberty and throughout early pregnancy. Mammary glands were collected during early pregnancy (Day 8 of pregnancy). Rabbits fed with OD milk and then subjected to an OD diet displayed an abnormal development of the mammary gland: the mammary ducts were markedly enlarged (P < 0.05) and filled with abundant secretory products. Moreover, the alveolar secretory structures were disorganized, with an abnormal aspect characterized by large lumina. Mammary epithelial cells contained numerous large lipid droplets and exhibited fingering of the apical membrane and abnormally enlarged intercellular spaces filled with casein micelles. Leptin has been shown to be involved in modulating several developmental processes. We therefore analyzed its expression in the mammary gland. Mammary leptin mRNA was strongly expressed in rabbits fed with OD milk and subjected to an OD diet by comparison with the CON rabbits. Leptin transcripts and protein were localized in the epithelial cells, indicating that the increase in leptin synthesis occurs in this compartment. Taken together, these findings suggest that early-life nutritional history, in particular through the milking period, can determine subsequent mammary gland development. Moreover, they highlight the potentially important regulatory role that leptin may play during critical early-life nutritional windows with respect to long-term growth and mammary function.