Fine-tuned adaptation of embryo-endometrium pairs at implantation revealed by transcriptome analyses in Bos taurus.

Interactions between embryo and endometrium at implantation are critical for the progression of pregnancy. These reciprocal actions involve exchange of paracrine signals that govern implantation and placentation. However, it remains unknown how these interactions between the conceptus and the endometrium are coordinated at the level of an individual pregnancy. Under the hypothesis that gene expression in endometrium is dependent on gene expression of extraembryonic tissues and genes expressed in extraembryonic tissues are dependent of genes expressed in the endometrium, we performed an integrative analysis of transcriptome profiles of paired extraembryonic tissue and endometria obtained from cattle (Bos taurus) pregnancies initiated by artificial insemination. We quantified strong dependence (|r| > 0.95, empirical false discovery rate [eFDR] < 0.01) in transcript abundance of genes expressed in the extraembryonic tissues and genes expressed in the endometrium. The profiles of connectivity revealed distinct coexpression patterns of extraembryonic tissues with caruncular and intercaruncular areas of the endometrium. Notably, a subset of highly coexpressed genes between extraembryonic tissue (n = 229) and caruncular areas of the endometrium (n = 218, r > 0.9999, eFDR < 0.001) revealed a blueprint of gene expression specific to each pregnancy. Gene ontology analyses of genes coexpressed between extraembryonic tissue and endometrium revealed significantly enriched modules with critical contribution for implantation and placentation, including "in utero embryonic development," "placenta development," and "regulation of transcription." Coexpressing modules were remarkably specific to caruncular or intercaruncular areas of the endometrium. The quantitative association between genes expressed in extraembryonic tissue and endometrium emphasize a coordinated communication between these two entities in mammals. We provide evidence that implantation in mammalian pregnancy relies on the ability of the extraembryonic tissue and the endometrium to develop a fine-tuned adaptive response characteristic of each pregnancy.

Evaluation of age, weaning weight, body condition score, and reproductive tract score in pre-selected beef heifers relative to reproductive potential.

BACKGROUND: Artificial insemination is a preferred breeding method for beef heifers as it advances the genetic background, produces a predictive and profitable calving season, and extends the heifer's reproductive life span. As reproductive efficiency in heifers is key for the success of beef cattle production systems, following artificial insemination, heifers are exposed to a bull for the remainder of the breeding season. Altogether, up to 95% of heifers might become pregnant in their first breeding season. Heifers that do not become pregnant at the end of the breeding season represent an irreparable economical loss. Additionally, heifers conceiving late in the breeding season to natural service, although acceptable, poses serious losses to producers. To minimize losses due to reproductive failure, different phenotypic parameters can be assessed and utilized as selection tools. Here, we tested the hypothesis that in a group of pre-selected heifers, records of weaning weight, age at weaning, age at artificial insemination, and age of dam differ among heifers of varied reproductive outcomes during the first breeding season. RESULTS: None of the parameters tested presented predictive ability to discriminate the heifers based on the response variable ('pregnant to artificial insemination', 'pregnant to natural service', 'not pregnant'). Heifers categorized with body condition score = 6 and reproductive tract score ≥ 4 had the greatest proportion of pregnancy to artificial insemination (49% and 44%, respectively). Furthermore, it was notable that heifers presenting body condition score = 6 and reproductive tract score = 5 presented the greatest pregnancy rate at end of the breeding season (89%). Heifers younger than 368 d at the start of the breeding season did not become pregnant to artificial insemination. Those young heifers had 12.5% chance to become pregnant in their first breeding season, compared to 87.5% if the heifers were older than 368 days. CONCLUSION: Our results suggest that beef heifers with body condition score = 6 and reproductive tract score ≥ 4 are more likely to become pregnant to artificial insemination. Careful assessment should be undertaken when developing replacement heifers that will not reach 12 months of age by the beginning of the breeding season.

Transcriptome data of peripheral white blood cells from beef heifers collected at the time of artificial insemination.

The reproductive performance of heifers within their first breeding season influences the success of beef cattle operations. Therefore, a means to identify infertile and late breeding heifers before the start of the breeding season holds great promise for the future of the beef industry. Pubertal beef heifers were subjected to estrous synchronization and fixed time artificial insemination (FTAI). We collected peripheral blood from the heifers at the time of artificial insemination (AI) and generated RNA sequencing data to characterize the transcriptome of peripheral white blood cells (PWBC). Following insemination, heifers were exposed to natural service for a defined breeding season, and pregnancy was evaluated to classify heifers into one of three groups: AI-pregnant, natural-bred (NB) pregnant, and non-pregnant. The raw transcriptome data of PWBC is available on the NCBI GEO repository (GSE103628) where the reader can also find raw read counts and normalized gene expression data. The normalized data on transcript coverage can be visualized as a genome browser at HeiferFertilityRNAseq.org.

Extraction of total RNA from single-oocytes and single-cell mRNA sequencing of swine oocytes.

OBJECTIVE: Analyses of single oocytes are essential for a fine dissection of molecular features governing developmental competence. We adapted the phenol-chloroform procedure for the purification of total RNA from single oocytes. RESULTS: Key modifications include the use of Phasemaker™ tubes, a second chloroform wash of the aqueous phase, and the precipitation of the RNA with glyclogen in a 200 µl micro-centrifuge tube. Assessment of the RNA profile from single oocytes showed distinct peaks for 18S and 28S ribosomal subunits. This approach permitted the extraction of small RNAs from single oocytes, which was evident by the presence of 5S and 5.8S rRNAs and tRNAs around 122-123 nucleotides long. The amplification of polyadenylated RNA resulted in detectable DNA products ranging from ~ 500 to ~ 5000 nucleotides. We used the amplified DNA as template for single-cell mRNA-sequencing of five swine oocytes and quantified the expression levels of 9587 genes with complete coverage of transcripts over 10,000 nucleotides in length. The coverage was similar in all oocytes sequenced, demonstrating consistent high RNA quality across samples. We isolated total RNA from single oocytes and demonstrated that the quality was appropriate for single-cell mRNA-sequencing.

Transcriptome profiles in peripheral white blood cells at the time of artificial insemination discriminate beef heifers with different fertility potential.

BACKGROUND: Infertility is a longstanding limitation in livestock production with important economic impact for the cattle industry. Female reproductive traits are polygenic and lowly heritable in nature, thus selection for fertility is challenging. Beef cattle operations leverage estrous synchronization in combination with artificial insemination (AI) to breed heifers and benefit from an early and uniform calving season. A couple of weeks following AI, heifers are exposed to bulls for an opportunity to become pregnant by natural breeding (NB), but they may also not become pregnant during this time period. Focusing on beef heifers, in their first breeding season, we hypothesized that: a- at the time of AI, the transcriptome of peripheral white blood cells (PWBC) differs between heifers that become pregnant to AI and heifers that become pregnant late in the breeding season by NB or do not become pregnant during the breeding season; and b- the ratio of transcript abundance between genes in PWBC classifies heifers according to pregnancy by AI, NB, or failure to become pregnant. RESULTS: We generated RNA-sequencing data from 23 heifers from two locations (A: six AI-pregnant and five NB-pregnant; and B: six AI-pregnant and six non-pregnant). After filtering out lowly expressed genes, we quantified transcript abundance for 12,538 genes. The comparison of gene expression levels between AI-pregnant and NB-pregnant heifers yielded 18 differentially expressed genes (DEGs) (ADAM20, ALDH5A1, ANG, BOLA-DQB, DMBT1, FCER1A, GSTM3, KIR3DL1, LOC107131247, LOC618633, LYZ, MNS1, P2RY12, PPP1R1B, SIGLEC14, TPPP, TTLL1, UGT8, eFDR≤0.02). The comparison of gene expression levels between AI-pregnant and non-pregnant heifers yielded six DEGs (ALAS2, CNKSR3, LOC522763, SAXO2, TAC3, TFF2, eFDR≤0.05). We calculated the ratio of expression levels between all gene pairs and assessed their potential to classify samples according to experimental groups. Considering all samples, relative expression from two gene pairs correctly classified 10 out of 12 AI-pregnant heifers (P = 0.0028) separately from the other 11 heifers (NB-pregnant, or non-pregnant). CONCLUSION: The transcriptome profile in PWBC, at the time of AI, is associated with the fertility potential of beef heifers. Transcript levels of specific genes may be further explored as potential classifiers, and thus selection tools, of heifer fertility.