Galectin-1 induces gene and protein expression related to maternal-conceptus immune tolerance in bovine endometrium†.

Conceptus secretory factors include galectins, a family of carbohydrate binding proteins that elicit cell adhesion and immune suppression by interacting with intracellular and extracellular glycans. In rodents, galectin-1 (LGALS1) promotes maternal-fetal immune tolerance in the decidua through expansion of tolerogenic cluster of differentiation 11c (CD11c) positive dendritic cells, increased anti-inflammatory interleukin (IL)-10, and activation of forkhead box P3 (FOXP3) positive regulatory T cells (Treg). This study characterized galectin expression in early ruminant conceptuses and endometrium. We also tested the effect of recombinant bovine LGALS1 (rbLGALS1) and progesterone (P4) on endometrial expression of genes and protein related to maternal-conceptus immune tolerance in cattle. Elongating bovine and ovine conceptuses expressed several galectins, particularly, LGALS1, LGALS3, and LGALS8. Within bovine endometrium, expression of LGALS3, LGALS7, and LGALS9 was greater on Day 16 of pregnancy compared to the estrous cycle. Within ovine endometrium, LGALS7 was greater during pregnancy compared to the estrous cycle and endometrium of pregnant sheep tended to have greater LGALS9 and LGALS15. Expression of endometrial LGALS4 was less during pregnancy in sheep. Treating bovine endometrium with rbLGALS1 increased endometrial expression of CD11c, IL-10, and FOXP3, within 24 h. Specifically, within caruncular endometrium, both rbLGALS1 and P4 increased FOXP3, suggesting that both ligands may promote Treg expansion. Using IHC, FOXP3+ cells with a leukocyte phenotype were localized to the bovine uterine stratum compactum near the uterine surface and increased in response to rbLGALS1. We hypothesize that galectins have important functions during establishment of pregnancy in ruminants and bovine conceptus LGALS1 and luteal P4 confer mechanisms of maternal-conceptus immune tolerance in cattle.

Interferon tau-dependent and independent effects of the bovine conceptus on the endometrial transcriptome†.

This study investigated bovine conceptus-induced modifications to the endometrial transcriptome related to effects of interferon tau (IFNT), conceptus origin (in vivo vs. in vitro), and conceptus sex. In vitro (IVF) or in vivo (superovulation and artificial insemination, AI) produced blastocysts were transferred into recipient heifers on day 7 of the estrous cycle. On day 15, IVF- or AI-derived conceptuses were obtained by uterine flushing and individually placed on endometrial explants in media for 6 h. Explants were also cultured with media alone as a control or media containing 100 ng/mL IFNT. Total explant RNA was analyzed by RNA-Seq. Incubation of endometrium with IFNT or IVF- or AI-derived conceptuses changed (P ≤ 0.001) expression of 491, 498, and 576 transcripts, respectively, compared to the control. Further, 369 differentially expressed genes (DEGs) were common between explants exposed to IFNT or a conceptus. A total of 240 DEGs were uniquely altered by conceptuses (IVF- and AI-derived) but not IFNT. Of these transcripts, 46 were shared between the IVF and AI groups, while 61 and 133 were specific to IVF and AI conceptuses, respectively. Five genes [melanophilin (MLPH), prominin-2 (PROM2), myeloid associated differentiation marker (MYADM), vomeronasal 1 receptor 4 like (VN1R4L) and 5-hydroxytryptamine receptor 1A (HTR1A)] were more abundant in endometrium exposed to female compared to male conceptuses (P < 0.001). A single gene [ADP-ribosylation factor like GTPase 4C (ARL4C)] was more abundant in response to male conceptuses (P < 0.001) than female conceptuses. These data support the hypothesis that conceptus regulation of gene expression in the endometrium is complex and involves factors other than IFNT that may have a biological role in pregnancy establishment.

Bovine endometrium responds differentially to age-matched short and long conceptuses†.

This study combined in vitro production of bovine blastocysts, multiple embryo transfer techniques, and a conceptus-endometrial explant co-culture system to test the hypothesis that bovine endometrium exposed to long vs. short day 15 conceptuses would exhibit a different transcriptome profile reflective of potential for successful pregnancy establishment. Bovine endometrial explants collected at the late luteal stage of the estrous cycle were cultured in RPMI medium for 6 h with nothing (control), 100 ng/mL recombinant ovine interferon tau (IFNT), a long day 15 conceptus, or a short day 15 conceptus. Transcriptional profiling of the endometrial explants found that exposure of endometrium to IFNT, long conceptuses, or short conceptuses altered (P < 0.05) expression of 491, 498, and 230 transcripts, respectively, compared to the control. Further analysis revealed three categories of differentially expressed genes (DEG): (i) commonly responsive to exposure to IFNT and conceptuses, irrespective of size (n = 223); (ii) commonly responsive to IFNT and long conceptuses only (n = 168); and genes induced by the presence of a conceptus but independent of IFNT (n = 108). Of those 108 genes, 101 were exclusively induced by long conceptuses and functional analysis revealed that regulation of molecular function, magnesium-ion transmembrane transport, and clathrin coat assembly were the principal gene ontologies associated with these DEG. In conclusion, bovine endometrium responds differently to age-matched conceptuses of varying size in both an IFNT-dependent and -independent manner, which may be reflective of the likelihood of successful pregnancy establishment.

Do differences in the endometrial transcriptome between uterine horns ipsilateral and contralateral to the corpus luteum influence conceptus growth to day 14 in cattle?

Embryo transfer to the uterine horn contralateral to the ovary containing the corpus luteum (CL) negatively impacts pregnancy establishment in cattle. Our aim was to compare the transcriptome and ability of the ipsilateral and contralateral uterine horns to support preimplantation conceptus survival and growth to day 14. In experiment 1, endometrial samples from both horns were collected from synchronized heifers slaughtered on day 5, 7, 13, or 16 post-estrus (n = 5 per time) and subjected to RNA sequencing. In experiment 2, 10 day 7 in vitro produced blastocysts were transferred into the uterine horn ipsilateral (n = 9) or contralateral to the CL (n = 8) or into both horns (i.e., bilateral, n = 9) of synchronized recipient heifers. Reproductive tracts were recovered at slaughter on day 14, and the number and dimensions of recovered conceptuses were recorded for each horn. A total of 217, 54, 14, and 18 differentially expressed genes (>2-fold change, FDR P < 0.05) were detected between ipsilateral and contralateral horns on days 5, 7, 13, and 16, respectively, with signaling pathways regulating pluripotency of stem cells, ErbB signaling pathway, and mTOR signaling pathway amongst the top canonical pathways. Site of embryo transfer did not affect recovery rate (48.0%, 168/350) or length of conceptuses (mean ± SE 2.85 ± 0.27 mm). Although differences in gene expression exist between the endometrium of uterine horns ipsilateral and contralateral to the CL in cattle, they do not impact conceptus survival or length between day 7 and 14.

Embryo development in cattle and interactions with the reproductive tract.

Embryo mortality is a major contributor to poor reproductive efficiency and profitability in cattle production systems. Coordinated interaction between the developing embryo or conceptus and the maternal reproductive tract is essential for pregnancy establishment in mammals. Up to the blastocyst stage, the embryo can grow in the absence of contact with the oviduct or uterus; however, conceptus elongation after hatching and before implantation, a characteristic of ruminant early development, is entirely maternally driven and is essential to ensure that sufficient quantities of interferon-τ (IFNT) are secreted by the developing conceptus to abrogate the mechanisms that bring about luteolysis. Surprisingly, many questions, such as the threshold level of IFNT required for pregnancy maintenance, remain unanswered. Failure of the conceptus to elongate undoubtedly results in embryonic loss and is thus believed to contribute greatly to reproductive failure in cattle.

Embryonic maternal interaction in cattle and its relationship with fertility.

Embryo mortality is a major contributor to poor reproductive efficiency and profitability in cattle production systems. While conception is achieved (i.e., the oocyte is fertilized) in the vast majority of cases if insemination is carried out correctly, a significant proportion of the resulting embryos fail to develop to term. Appropriate communication between the developing conceptus and the maternal endometrium is essential for the establishment and maintenance of pregnancy in all mammals. Up to the blastocyst stage, around Days 7-9, contact worth the female reproductive system is not required. However, the process of conceptus elongation after hatching and prior to implantation is entirely maternally driven and is essential to ensure that sufficient quantities of interferon-tau (IFNT) are secreted by the developing conceptus to abrogate the mechanisms that bring about luteolysis. While the importance of conceptus-derived IFNT in maternal recognition of pregnancy and prevention of luteolysis in cattle is unequivocal, many questions, such as the threshold level of IFNT required for pregnancy maintenance, remain unanswered. Furthermore, the precise role of IFNT-independent mechanisms in pregnancy establishment remains to be elucidated. Irrespective of this, failure of the conceptus to elongate undoubtedly results in embryonic loss and is thus believed to contribute greatly to reproductive failure in cattle. This review will address some of these answered questions and try to shed some light on those gaps in knowledge that could potentially contribute to improved embryo survival and reproductive efficiency.

Laser assisted blastomere extrusion biopsy of in vitro produced cattle embryos-A potential high throughput, minimally invasive approach for sampling pre-morula and morula stage embryos.

Whilst adoption of in vitro production (IVP) of cattle embryos and subsequent biopsy for genetic evaluation is increasing, biopsy techniques primarily used were developed to sample in vivo-produced blastocysts. This study was conducted to develop a laser-assisted blastomere extrusion approach for rapid and minimal-invasive biopsy of IVP cattle embryos at pre-morula to morula stages of development (Day 5 or 6 post-fertilisation). Embryo development into blastocysts was not compromised when ≤3 cells were collected by blastomere extrusion on Day 5 (44.4 ± 4.4 % and 34.3 ± 4.6 %) or Day 6 (58.0 ± 4.3 % and 57.5 ± 5.3 %) post-fertilisation compared with non-biopsied control embryos. Similarly, capacity to withstand cryopreservation was not different between embryos biopsied at Day 5 and 6 post-fertilisation and control-embryos (58.8 ± 6.0 %, 63.5 ± 5.6 %, and 56.0 ± 4.8 %, respectively). When more cells were collected from embryos at Day 6 post-fertilisation (≥8 compared to ≤3 cells), subsequent embryo development was not different (63.6 ± 6.1 % and 73.1 ± 6.2 %, respectively) nor was the capacity to withstand cryopreservation (67.9 ± 9.0 % and 62.5 ± 8.7 %, respectively). For biopsies on Day 6 post-fertilization, 95 % of samples produced a PCR product; however, when compared to the whole embryo PCR results, approximately 11 % of biopsy-samples classified as being from a male embryo were from female embryos (false positive), indicating DNA contamination between samples. In conclusion, results of this study indicate laser-assisted blastomere extrusion is a time efficient and minimally invasive approach to biopsy IVP morula and pre-morula cattle embryos to facilitate genetic analysis.

Variation of DNA Fragmentation Levels During Density Gradient Sperm Selection for Assisted Reproduction Techniques: A Possible New Male Predictive Parameter of Pregnancy?

Predicting the outcome of in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) is one main goal of the present research on assisted reproduction. To understand whether density gradient centrifugation (DGC), used to select sperm, can affect sperm DNA integrity and impact pregnancy rate (PR), we prospectively evaluated sperm DNA fragmentation (sDF) by TUNEL/PI, before and after DGC. sDF was studied in a cohort of 90 infertile couples the same day of IVF/ICSI treatment. After DGC, sDF increased in 41 samples (Group A, median sDF value: 29.25% [interquartile range, IQR: 16.01-41.63] in pre- and 60.40% [IQR: 32.92-93.53] in post-DGC) and decreased in 49 (Group B, median sDF value: 18.84% [IQR: 13.70-35.47] in pre- and 8.98% [IQR: 6.24-15.58] in post-DGC). PR was 17.1% and 34.4% in Group A and B, respectively (odds ratio [OR]: 2.58, 95% confidence interval [CI]: 0.95-7.04, P = 0.056). After adjustment for female factor, female and male age and female BMI, the estimated OR increased to 3.12 (95% CI: 1.05-9.27, P = 0.041). According to the subgroup analysis for presence/absence of female factor, heterogeneity in the association between the Group A and B and PR emerged (OR: 4.22, 95% CI: 1.16-15.30 and OR: 1.53, 95% CI: 0.23-10.40, respectively, for couples without, n = 59, and with, n = 31, female factor).This study provides the first evidence that the DGC procedure produces an increase in sDF in about half of the subjects undergoing IVF/ICSI, who then show a much lower probability of pregnancy, raising concerns about the safety of this selection procedure. Evaluation of sDF before and after DGC configures as a possible new prognostic parameter of pregnancy outcome in IVF/ICSI. Alternative sperm selection strategies are recommended for those subjects who undergo the damage after DGC.