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Interferon Tau (IFNT), the conceptus-derived pregnancy recognition signal in cattle, significantly modifies the transcriptome of the endometrium. However, the endometrium also responds to IFNT-independent conceptus-derived products. The aim of this study was to determine what proteins are produced by the bovine conceptus that may facilitate the pregnancy recognition process in cattle. We analysed by mass spectrometry the proteins present in conceptus-conditioned media (CCM) after 6 h culture of Day 16 bovine conceptuses (n = 8) in SILAC media (arginine- and lysine-depleted media supplemented with heavy isotopes) and the protein content of extracellular vesicles (EVs) isolated from uterine luminal fluid (ULF) of Day 16 pregnant (n = 7) and cyclic (n = 6) cross-bred heifers on day 16. In total, 11,122 proteins were identified in the CCM. Of these, 5.95% (662) had peptides with heavy labelled amino acids, i.e., de novo synthesised by the conceptuses. None of these proteins were detected in the EVs isolated from ULF. Pregnancy-associated glycoprotein 11, Trophoblast Kunitz domain protein 1 and DExD-Box Helicase 39A were de novo produced and present in the CCM from all conceptuses and in previously published CCM data following 6 and 24 h. A total of 463 proteins were present in the CCM from all the conceptuses in the present study, and after 6 and 24 h culture in a previous study, while expression of their transcripts was not detected in endometrium indicating that they are likely conceptus-derived. Of the proteins present in the EVs, 67 were uniquely identified in ULF from pregnant heifers; 35 of these had been previously reported in CCM from Day 16 conceptuses. This study has narrowed a set of conceptus-derived proteins that may be involved in EV-mediated IFNT-independent embryo–maternal communication during pregnancy recognition in cattle.
The molecular interactions between the maternal environment and the developing embryo that are key for early pregnancy success and are influenced by factors such as maternal metabolic status. Our understanding of the mechanism(s) through which these individual nutritional stressors alter endometrial function and the in utero environment for early pregnancy success is, however, limited. Here we report, for the first time, the use of an endometrium-on-a-chip microfluidics approach to produce a multi-cellular endometrium in vitro. Isolated endometrial cells (epithelial and stromal) from the uteri of non-pregnant cows in the early-luteal phase (Day 4-7), were seeded in the upper chamber of the device (epithelial cells; 4-6 10 4 cells/mL) and stromal cells seeded in the lower chamber (1.5-2 10 4 cells/mL). Exposure of cells to different concentrations of glucose (0.5, 5.0 or 50 mM) or insulin (Vehicle, 1 or 10 ng/mL) were performed at a flow rate of 1µL/min for 72 hr. Quantitative differences in the cellular transcriptome and the secreted proteome of in vitro-derived uterine luminal fluid (ULF) were determined by RNA-sequencing and Tandem Mass Tagging Mass Spectrometry (TMT-MS), respectively. High glucose concentrations altered 21 and 191 protein-coding genes in epithelial and stromal cells, respectively (p<0.05), with a dose-dependent quantitative change in the protein secretome (1 and 23 proteins). Altering insulin concentrations resulted in limited transcriptional changes including transcripts for insulin-like binding proteins that were cell specific but altered the quantitative secretion of 196 proteins. These findings highlight one potential mechanism by which changes to maternal glucose and insulin alter uterine function.
During the pre-implantation period of pregnancy in eutherian mammals, changes to the uterine endometrium are required (both at the transcriptional and protein level) to facilitate the endometrium becoming receptive to an implanting embryo. We know that the developing conceptus (embryo and extraembryonic membranes) produces proteins during this developmental stage. We hypothesised that this common process in early pregnancy in eutheria may be facilitated by highly conserved conceptus-derived proteins such as macrophage capping protein CAPG. More specifically, we propose that CAPG may share functionality in modifying the transcriptome of the endometrial epithelial cells to facilitate receptivity to implantation in species with different implantation strategies, such as human and bovine. A recombinant bovine form of CAPG (91% sequence identity between bovine and human) was produced and bovine endometrial epithelial (bEECs) and stromal (bESCs) cells and human endometrial epithelial cells (hEECs) were cultured for 24 h with or without rbCAPG. RNA sequencing and quantitative real-time PCR analysis was used to assess the transcriptional response to rbCAPG (Control, vehicle, CAPG 10, 100, 1000 ng/ml: n=3 biological replicates per treatment per species). Treatment of bEECs with CAPG resulted in changes to 1052 transcripts (629 increased and 423 decreased) compared to vehicle controls, including those previously only identified as regulated by interferon-tau, the pregnancy recognition signal in cattle. Treatment of hEECs with bovine CAPG increased expression of transcripts previously known to interact with CAPG in different systems (CAPZB, CAPZA2, ADD1 and ADK) compared with vehicle controls (P<0.05). In conclusion, we have demonstrated that CAPG, a highly conserved protein in eutherian mammals elicits a transcriptional response in the endometrial epithelium in two species with different implantation strategies that may facilitate uterine receptivity.
The origin of placentation in mammals required a symphony of events from protein innovation to the evolution of regulatory networks that could support successful pregnancy from implantation to full gestation. There is variation across mammals in terms of the types of implantation, degree of invasiveness of the trophoblast into the maternal endometrium, and the type of placenta formed. However, there are some signalling networks, e.g. progesterone receptor signalling, that are shared across the clade. We wished to determine what regulates the diverse molecular interactions and morphologies that underpin successful pregnancy in eutheria. Whilst the relationship between miRNAs and pathophysiology of placenta is well established, their role in early implantation and diversity of implantation strategies is not well understood. We identify a cohort of miRNAs that arose coincident with the emergence of placental mammals (mir-127, -185, -188, -28, -324, -331, -340, -378, -423, -433, -505, -542, and -671). We identify 115 genes under positive selection on the stem eutherian lineage and 88 of these are predicted to be regulated by the 13 stem lineage miRNAs. Using two species of mammal that demonstrate the extremes of implantation strategies (invasive and superficial in humans and bovine respectively), we assessed the response of the endometrial epithelium in terms of expression of these miRNAs to early pregnancy molecules. We show that the 13 stem lineage miRNAs are regulated in a species-specific manner. We propose these species-specific regulatory networks contribute to the diversity of pregnancy morphologies observed in eutheria.
The origin of embryo implantation in mammals ~148 million years ago was a dramatic shift in reproductive strategy, yet the molecular changes that established mammal implantation are largely unknown. Although progesterone receptor signalling predates the origin of mammals and is highly conserved in, and critical for, successful mammal pregnancy, it alone cannot explain the origin and subsequent diversity of implantation strategies throughout the placental mammal radiation. MiRNAs are known to be flexible and dynamic regulators with a well-established role in the pathophysiology of mammal placenta. We propose that a dynamic core microRNA (miRNA) network originated early in placental mammal evolution, responds to conserved mammal pregnancy cues (e.g. progesterone), and facilitates species-specific responses. Here we identify 13 miRNA gene families that arose at the origin of placental mammals and were subsequently retained in all descendent lineages. The expression of these miRNAs in response to early pregnancy molecules is regulated in a species-specific manner in endometrial epithelia of species with extreme implantation strategies (i.e. bovine and human). Furthermore, this set of miRNAs preferentially target proteins under positive selective pressure on the ancestral eutherian lineage. Discovery of this core embryo implantation toolkit and specifically adapted proteins helps explain the origin and evolution of implantation in mammals.
Environmental stressors to which a foetus is exposed, affect a range of physiological functions in post-natal offspring. We aimed to determine the in-utero effect of steroid hormones on reproductive potential of female offspring using a porcine model. Reproductive tracts of pigs from female-biased (>65% female, n = 15), non-biased (45–54.9% female, n = 15), and male-biased litters (<35% females, n = 9) were collected at slaughter (95–115 kg). Ovaries and uterine horns were processed for H&E or immunohistochemistry. Variability of data within groups was analysed with a Levenes test, whilst data was analysed using linear models in R. In the ovarian reserve, there was a significant birth weight by sex ratio interaction (p = 0.015), with low-birth-weight pigs from male-biased litters having a higher number of primordial follicles and the opposite trend seen in pigs from female-biased litters. Sex bias held no effect on endometrial gland development. A lower BW decreased the proportion of glands found in the endometrium (p = 0.045) and was more variable in both male-biased and female-biased litters (p = 0.026). The variability of primordial follicles from male-biased litters was greater than non- and female-biased litters (p = 0.014). Similarly, endometrial stromal nuclei had a greater range in male- and female-biased litters than non-biased litters (p = 0.028). A crucial finding was the greater variability in both primordial follicles in the ovaries from females derived from male-biased litters and stromal cell count in the endometrium of females from male- and female- biased litters. This could be inflating the variability of reproductive success seen in females from male-biased litters.
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