A burst of genomic innovation at the origin of placental mammals mediated embryo implantation.

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.

MicroRNAs emerging coordinate with placental mammals alter pathways in endometrial epithelia important for endometrial function.

We tested the hypothesis that conserved placental mammal-specific microRNAs and their targets facilitate endometrial receptivity to implantation. Expression of miR-340-5p, -542-3p, and -671-5p was regulated by exposure of endometrial epithelial cells to progesterone (10 µg/ml) for 24 h coordinate with 1,713 of their predicted targets. Proteomic analysis of cells transfected with miRNA mimic/inhibitor (48 h: n = 3) revealed 1,745 proteins altered by miR-340-5p (mimic; 1,369, inhibitor; 376) of which 171 were predicted targets and P4-regulated. MiR-542-3p altered 2,353 (mimic; 1,378, inhibitor; 975) 100 which were mirDB predicted, including 46 P4-regulated. MiR-671-5p altered 1,744 proteins (mimic; 1,252, inhibitor; 492) 95 of which were predicted targets and 46 P4-regulated. All miRNAs were detected in luteal phase endometrial biopsies, irrespective of pregnancy outcomes. miR-340-5p expression increased in biopsies from individuals suffering previous and subsequent miscarriage compared to those with subsequent live birth. Dysfunction of these miRNAs and their targets contribute to endometrial-derived recurrent pregnancy loss.

Sex bias in utero alters ovarian reserve but not uterine capacity in female offspring†.

Environmental stressors to which a fetus is exposed affect a range of physiological functions in postnatal offspring. We aimed to determine the in utero effect of steroid hormones on the 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 analyzed with a Levene's test, while data were analyzed using mixed 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 higher numbers of primordial follicles with opposite trends seen in pigs from female-biased litters. Sex bias held no effect on endometrial gland development. A lower birth weight 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 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.

Repeat Detector: versatile sizing of expanded tandem repeats and identification of interrupted alleles from targeted DNA sequencing.

Targeted DNA sequencing approaches will improve how the size of short tandem repeats is measured for diagnostic tests and preclinical studies. The expansion of these sequences causes dozens of disorders, with longer tracts generally leading to a more severe disease. Interrupted alleles are sometimes present within repeats and can alter disease manifestation. Determining repeat size mosaicism and identifying interruptions in targeted sequencing datasets remains a major challenge. This is in part because standard alignment tools are ill-suited for repetitive and unstable sequences. To address this, we have developed Repeat Detector (RD), a deterministic profile weighting algorithm for counting repeats in targeted sequencing data. We tested RD using blood-derived DNA samples from Huntington's disease and Fuchs endothelial corneal dystrophy patients sequenced using either Illumina MiSeq or Pacific Biosciences single-molecule, real-time sequencing platforms. RD was highly accurate in determining repeat sizes of 609 blood-derived samples from Huntington's disease individuals and did not require prior knowledge of the flanking sequences. Furthermore, RD can be used to identify alleles with interruptions and provide a measure of repeat instability within an individual. RD is therefore highly versatile and may find applications in the diagnosis of expanded repeat disorders and in the development of novel therapies.

Expanded CAG/CTG repeats resist gene silencing mediated by targeted epigenome editing.

Expanded CAG/CTG repeat disorders affect over 1 in 2500 individuals worldwide. Potential therapeutic avenues include gene silencing and modulation of repeat instability. However, there are major mechanistic gaps in our understanding of these processes, which prevent the rational design of an efficient treatment. To address this, we developed a novel system, ParB/ANCHOR-mediated Inducible Targeting (PInT), in which any protein can be recruited at will to a GFP reporter containing an expanded CAG/CTG repeat. Previous studies have implicated the histone deacetylase HDAC5 and the DNA methyltransferase DNMT1 as modulators of repeat instability via mechanisms that are not fully understood. Using PInT, we found no evidence that HDAC5 or DNMT1 modulate repeat instability upon targeting to the expanded repeat, suggesting that their effect is independent of local chromatin structure. Unexpectedly, we found that expanded CAG/CTG repeats reduce the effectiveness of gene silencing mediated by targeting HDAC5 and DNMT1. The repeat-length effect in gene silencing by HDAC5 was abolished by a small molecule inhibitor of HDAC3. Our results have important implications on the design of epigenome editing approaches for expanded CAG/CTG repeat disorders. PInT is a versatile synthetic system to study the effect of any sequence of interest on epigenome editing.

The role of CAPG in molecular communication between the embryo and the uterine endometrium: Is its function conserved in species with different implantation strategies?

During the preimplantation period of pregnancy in eutherian mammals, transcriptional and proteomic changes in the uterine endometrium are required to facilitate receptivity to an implanting blastocyst. These changes are mediated, in part, by proteins produced by the developing conceptus (inner cell mass and extraembryonic membranes). We hypothesized that this common process in early pregnancy in eutheria may be facilitated by highly conserved conceptus-derived proteins such as macrophage capping protein (CAPG). 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. A recombinant bovine form of CAPG (91% sequence identity between bovine and human) was produced and bovine endometrial epithelial (bEECs) and stromal (bESCs) and human endometrial epithelial cells (hEECs) were cultured for 24 hours with and without recombinant bovine CAPG (rbCAPG). RNA sequencing and quantitative real-time PCR analysis were 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 alterations in the abundance of 1052 transcripts (629 increased and 423 decreased) compared to vehicle controls. 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 < .05). In conclusion, we have demonstrated that CAPG, a highly conserved protein in eutherian mammals, elicits a transcriptional response in the endometrial epithelium in species with different implantation strategies that may contribute to pregnancy success.

Strigolactone synthesis is ancestral in land plants, but canonical strigolactone signalling is a flowering plant innovation.

BACKGROUND: Strigolactones (SLs) are an important class of carotenoid-derived signalling molecule in plants, which function both as exogenous signals in the rhizosphere and as endogenous plant hormones. In flowering plants, SLs are synthesized by a core pathway of four enzymes and are perceived by the DWARF14 (D14) receptor, leading to degradation of SMAX1-LIKE7 (SMXL7) target proteins in a manner dependent on the SCFMAX2 ubiquitin ligase. The evolutionary history of SLs is poorly understood, and it is not clear whether SL synthesis and signalling are present in all land plant lineages, nor when these traits evolved. RESULTS: We have utilized recently-generated genomic and transcriptomic sequences from across the land plant clade to resolve the origin of each known component of SL synthesis and signalling. We show that all enzymes in the core SL synthesis pathway originated at or before the base of land plants, consistent with the previously observed distribution of SLs themselves in land plant lineages. We also show that the late-acting enzyme LATERAL BRANCHING OXIDOREDUCTASE (LBO) may be considerably more ancient than previously thought. We perform a detailed phylogenetic analysis of SMXL proteins and show that specific SL target proteins only arose in flowering plants. We also assess diversity and protein structure in the SMXL family, identifying several previously unknown clades. CONCLUSIONS: Overall, our results suggest that SL synthesis is much more ancient than canonical SL signalling, consistent with the idea that SLs first evolved as rhizosphere signals and were only recruited much later as hormonal signals.