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Abstract Background Currently, there is a high demand for efficient pig embryo cryopreservation procedures in the porcine industry as well as for genetic diversity preservation and research purposes. To date, vitrification (VIT) is the most efficient method for pig embryo cryopreservation. Despite a high number of embryos survives in vitro after vitrification/warming procedures, the in vivo embryo survival rates after embryo transfer are variable among laboratories. So far, most studies have focused on cryoprotective agents and devices, while the VIT effects on porcine embryonic gene expression remained unclear. The few studies performed were based on vitrified/warmed embryos that were cultured in vitro (IVC) to allow them to re–expand. Thus, the specific alterations of VIT, IVC, and the cumulative effect of both remained unknown. To unveil the VIT-specific embryonic alterations, gene expression in VIT versus (vs.) IVC embryos was analyzed. Additionally, changes derived from both VIT and IVC vs. control embryos (CO) were analyzed to confirm the VIT embryonic alterations. Three groups of in vivo embryos at the blastocyst stage were analyzed by RNA–sequencing: (1) VIT embryos (vitrified/warmed and cultured in vitro), (2) IVC embryos and (3) CO embryos. Results RNA–sequencing revealed three clearly different mRNA profiles for VIT, IVC and CO embryos. Comparative analysis of mRNA profiles between VIT and IVC identified 321, differentially expressed genes (DEG) (FDR

Abstract Background The success of early reproductive events depends on an appropriate communication between gametes/embryos and the oviduct. Extracellular vesicles (EVs) contained in oviductal secretions have been suggested as new players in mediating this crucial cross-talk by transferring their cargo (proteins, mRNA and small ncRNA) from cell to cell. However, little is known about the oviductal EVs (oEVS) composition and their implications in the reproductive success. The aim of the study was to determine the oEVs content at protein, mRNA and small RNA level and to examine whether the oEVs content is under the hormonal influence of the estrous cycle. Results We identified the presence of oEVs, exosomes and microvesicles, in the bovine oviductal fluid at different stages of the estrous cycle (postovulatory-stage, early luteal phase, late luteal phase and pre-ovulatory stage) and demonstrated that their composition is under hormonal regulation. RNA-sequencing identified 903 differentially expressed transcripts (FDR 2). Our data revealed proteins related to early embryo development and gamete-oviduct interactions as well as numerous ribosomal proteins. Conclusions Our study provides with the first molecular signature of oEVs across the bovine estrous cycle, revealing marked differences between post- and pre-ovulatory stages. Our findings contribute to a better understanding of the potential role of oEVs as modulators of gamete/embryo-maternal interactions and their implications for the reproductive success.

The endometrium undergoes marked functional changes during estrous cycle and pregnancy. As the adjacent environment of the conceptus, it represents the maternal interface for embryo-maternal communication, which is essential to maintain pregnancy. Transcriptome studies provide the unique opportunity to assess molecular profiles changing in response to endocrine or metabolic stimuli or to embryonic pregnancy recognition signals. Here we review the current state of transcriptome profiling techniques and the results of a series of transciptome studies comparing bovine endometrium samples during the estrous cycle or endometrium samples from pregnant vs. non-pregnant animals. These studies revealed specific mRNA profiles which are characteristic for the functional status of the endometrium. Transcriptome studies of endometrial samples recovered during the pre-attachment period identified many interferon-stimulated genes, genes that are possibly involved in embryo-maternal immune modulation ( C1S, C1R, C4, SERPING1, UTMP, CD81, IFITM1, BST2), as well as genes affecting cell adhesion ( AGRN, CD81, LGALS3BP, LGALS9, GPLD1, MFGE8, and TGM2) and remodeling of the endometrium ( CLDN4, MEP1B, LGMN, MMP19, TIMP2, TGM2, MET, and EPSTI1). The results of these transcriptome studies were compared to those of similar microarray analyses in human, mouse and Rhesus monkey to identify similarities in endometrial biology between mammalian species and species-specific differences. Future studies will cover dynamic transcriptome changes between different stages of early pregnancy, the relationship between metabolic problems in dairy cows and the functionality of reproductive tissues as well as endometrium transcriptome profiles in recipients of somatic cell nuclear transfer embryos. [ABSTRACT FROM AUTHOR]

Placental abnormalities account for a high proportion of pregnancy loss after transfer of cloned (SCNT) bovine embryos. The high rate of pregnancy failure has been linked to the finding of abnormal placental formation and function. Because bovine SCNT embryos were shown to exhibit altered trophoblast differentiation (Arnold DR et al.2006 Reproduction 132, 279–290), we hypothesized that placental abnormalities in bovine clone pregnancies may originate from disturbed embryo-maternal communication in the pre-implantation period. To test this hypothesis, we evaluated the response of the endometrium to SCNT v. IVF embryos. The SCNT embryos were produced from fibroblast cultures derived from 4 different fetuses to exclude specific effects of a particular donor cell culture. After SCNT or IVF, embryos were cultured under identical conditions. Two SCNT or IVF blastocysts (grade 1) were transferred per recipient heifer (Day 8 of estrous cycle). Ten days later the recipients were slaughtered, the uteri were recovered, and pregnancy was verified by the presence of at least one normally developed embryo. Endometrium samples of 9 SCNT and 10 IVF pregnancies were used for transcriptome profiling with a custom cDNA microarray (BOE array; Bauersachs S et al.2007 J. Dairy Sci. 90, 4420–4423). In total, 58 transcripts were found to be differently abundant between endometrium samples from SCNT v. IVF pregnancies (SAM, FDR 5.24%). Interestingly, for some of them an important role in implantation and/or placentation has already been shown. NR2F2, encoding the orphan nuclear receptor superfamily member NR2F2 (COUP-TFII), was downregulated in endometrium from SCNT pregnancies (1.5-fold; P< 0.01). Uterine-specific Nr2f2mutant mice are infertile due to implantation failure (Kurihara I et al.2007 PLoS Genetics 3, e102). Another interesting candidate is GJA1, encoding connexin 43 (Cx43), with 1.8-fold (P< 0.001) downregulated transcript levels in SCNT pregnancies. A striking increase of stromal Cx43 has been observed in the ovine intercaruncular and caruncular endometrium during intensification of the feto-maternal contact (Gabriel et al.2005 Placenta 25, 287–296), suggesting that reduced GJA1mRNA expression in bovine clone pregnancies may negatively affect placentation. In view of the well-orchestrated spectrum of transcriptome changes in endometrium during the peri-attachment period (Bauersachs S et al.2006 Reproduction 132, 319–331), these findings suggest that placental failure in bovine clone pregnancies may originate from abnormal embryo-maternal communication already in the pre- or peri-implantation period.This study was supported by the Deutsche Forschungsgemeinschaft (FOR 478). [ABSTRACT FROM AUTHOR]

Uterine secretion from the endometrial glandular epithelium provides optimal conditions for early embryonic development. The uterine milk protein (UTMP), a member of the serine proteinase inhibitor superfamily, has been demonstrated to be a major progesterone-induced glycoprotein secreted by the endometrium during pregnancy. Previous transcriptomic analysis revealed that UTMP was highly abundant at estrus in the bovine endometrium (Bauersachs et al. 2005 J. Mol. Endocrinol. 34, 889–908). Here we describe a detailed characterization of UTMP mRNA expression at several time points during the bovine estrous cycle and the pre-implantation period. Simmental heifers were monitored with respect to serum progesterone (P4) and estradiol-17? (E2), and slaughtered at estrus or 3.5, 12, 15, or 18 days after estrus, or at Day 15 or 18 of pregnancy (n = 4 per group). The uterus was divided into corpus and caudal, middle and cranial parts of the ipsilateral uterine horn for sampling of intercaruncular endometrium. In addition, effects of steroid hormones were investigated by stimulating an endometrial cell culture obtained from Day 8 animals (n = 4) with physiological doses of P4 or E2. In all cases, UTMP mRNA was quantified by real-time RT-PCR. Pronounced changes of UTMP mRNA abundance were detected during the estrous cycle. Expression was highest at estrus, followed by a remarkable decrease at Day 3.5. There was no difference between pregnant and non-pregnant animals at Day 15. Cycling animals displaying a high P4 and low E2 content (P4 > 2 ng mL-1 and E2 < 1 pg mL-1) revealed a lower expression of UTMP at Day 18 compared to the pregnant heifers, whereas animals at Day 18 progressing toward estrus (P4 < 1.5 ng mL-1 and E2 > 3 pg mL-1) exceeded the mRNA expression of the pregnant group. Following stimulation with estradiol-17?, the in vitro UTMP transcripts increased significantly. These results indicate that two different interfering stimulatory events might take place. While estradiol-17? appeared to increase UTMP mRNA expression at estrus, a second factor, most probably embryo-derived or embryo-induced, is assumed to be responsible for the UTMP rise during early pregnancy. The distinct gradient from the cranial uterine horn to the corpus at estrus was less pronounced at Day 3.5 and absent at Days 12, 15, and 18, pointing toward functional implications regarding the passing gametes, particularly sperm. An antibody raised against bovine UTMP will further validate the observed mRNA regulations on the protein level. In conclusion, bovine UTMP seems to play a decisive role for precise cyclic regulation of the bovine uterine milieu and during early embryo-maternal communication.This work was supported by the DFG FOR 478. [ABSTRACT FROM AUTHOR]

Major problems in breeding of modern dairy cows include increasing rates of stillbirth and dystocia, associated with elevated costs for veterinary intervention (caesarean section) and loss of production animals (cows and calves). Rates of stillbirth as high as 12% have been reported, primarily caused by fetal overgrowth and increased birth weight. In order to discover molecular markers for selection against stillbirth, we developed a model for the inheritance of fetal growth traits. A Fleckvieh bull segregating for paternal stillbirth was used for insemination of 36 cows that were slaughtered at Day 150 of pregnancy in order to recover and phenotype the fetuses. The mode of inheritance indicated involvement of imprinting. Mapping results suggested an imprinted region on chromosome 9 as candidate for the stillbirth QTL. Due to the complexity of the trait, we opted for a holistic approach that is not restricted to the QTL candidate region but allows identification of genes and networks that influence fetal growth. Transcriptome profiles of liver and cotyledon samples from the fetuses with the highest (n = 10) and the lowest total weight n = 10) were analyzed using Affymetrix Bovine GeneChips (Affymetrix, Inc., Santa Clara, CA, USA). Analysis with the program SAM showed 41 up- and 4 down-regulated genes in liver samples of the heavy-weight group. Most of these genes are involved in immune response. Interestingly, many of these genes are reported to be regulated by vitamin D. Furthermore, vitamin D is closely connected to the IGF1 system and thus the most important fetal growth regulation circuit. Seasonal effects on vitamin D levels could mostly be excluded by the experimental design and did not correlate with growth traits. Most likely, the mRNA levels of our candidate genes were influenced by alterations in the IGF1/vitamin D circuit and did not cause the observed weight differences. The imprinted candidate genes showed no correlation with fetal weight. Gene set enrichment analysis indicated enhanced metabolic activity in the liver of heavy-weight fetuses. Genes from the QTL region showed a clear enrichment in correlation with fetal weight, confirming their involvement in fetal growth. The gene with the best correlation, GHITM (growth hormone inducible transmembrane protein), could give an explanation for the enhanced metabolic activity, as it is reported to function as a metabolic regulator. Simultaneous analysis of the data sets for liver and placenta in a linear model (R-package LIMMA; Smyth 2004 Stat. Applic. Genet. Mol. Biol. 3, art. 3) yielded essentially the same differentially expressed genes for liver and a higher number of differentially expressed genes for placenta (89 up- and 114 down-regulated), with little overlap between the two tissues.This work was supported by Grant BMBF FUGATO-Fertilink. [ABSTRACT FROM AUTHOR]

The endometrium provides the optimal conditions for the transport of sperm to the oviduct, to the site of fertilization, and later on for the reception of the embryo. This is reflected by specific morphological and functional changes during the estrous cycle, which are mainly regulated by the hormones progesterone, estradiol, and oxytocin. To study these changes on the level of messenger RNA, a microarray analysis of endometrial tissue samples was performed. Tissue samples were collected from 20 cyclic heifers (Deutsches Fleckvieh, between 17 and 31 months of age) after slaughter at Days 0, 3.5, 12, and 18 of the estrous cycle. The Day 18 group split into two subgroups, one with high and one with low progesterone levels in peripheral blood. Altogether there were 4 heifers in each experimental group. RNA was extracted from these tissue samples and analyzed with a custom-made bovine oviduct and endometrium (BOE) cDNA array (Bauersachs et al. 2007 J. Dairy Sci. 90, in press). The cDNAs present on the array were derived from several previously conducted differential gene expression studies of bovine endometrium between different stages of the estrous cycle, during early pregnancy, and from studies of bovine oviduct epithelial cells. In all of these studies, cDNAs of differentially expressed genes were identified using a combination of subtracted cDNA libraries and cDNA array hybridization. Redundant cDNA clones were removed, resulting in 1440 cDNA fragments on the array, representing 950 different genes. Twenty radioactively (33P) labeled cDNA samples (n = 4 for each cycle stage) were hybridized with the BOE array. After normalization of raw data (using BioConductor open source software) and significance analysis (SAM, FDR 1%), 272 mRNAs were identified that showed alterations of their concentration during the estrous cycle. Expression data from cDNAs with significant changes during the estrous cycle were used for cluster analyses with MultiExperiment Viewer 4.0 (Saeed et al. 2003 Biotechniques 34, 374–378). The main clusters represented genes upregulated either during the estrus or during the diestrus phase. Quantitatively enriched Gene Ontology (GO) categories were identified to find relevant functional groups and prominent biological processes. At estrus, e.g., GO categories extracellular matrix, cytoskeleton organization, and growth factor activity indicate changes in the composition of the endometrium during the estrous cycle. At diestrus, only a few overrepresented GO categories were found, mostly related to immune response and metabolism. The genes of known function were further analyzed in the context of interaction and regulatory networks. One of a number of central factors was TGF-β, which controls the expression of 12 genes upregulated at estrus and 8 at diestrus. In conclusion, the present study extended the results of our previously conducted analysis of bovine endometrium between the estrus and diestrus stages (Bauersachs et al. 2005 J. Mol. Endocrinol. 32, 449–466), revealed distinct temporal expression profiles, and identified additional genes differentially expressed during the estrous cycle.This work was supported by Grant BMBF FUGATO-Fertilink. [ABSTRACT FROM AUTHOR]

Background: In vitro embryo production is increasingly used for genetic improvement in cattle but bypasses the oviduct environment and exposes the embryos to oxidative stress with deleterious effects on further development. Here we aimed to examine the effect of oviduct epithelial spheroids (OES) on embryo development and quality in terms of morphology and gene expression during two co-culture times (4 days: up to embryonic genome activation at 8-16 cell stage vs. 7 days: up to blastocyst stage) and under two oxygen levels (5% vs. 20%)., Methods: Bovine presumptive zygotes produced by in vitro fertilization (day 0) using in-vitro matured oocytes were cultured in droplets of synthetic oviductal fluid (SOF) medium with or without (controls) OES for 4 or 7 days under 5% or 20% oxygen (4 treated and 2 control groups). Cleavage rates were evaluated on day 2 and blastocyst rates on days 7-8. Expanded blastocysts on days 7-8 were evaluated for total cell numbers and gene expression analysis by RNA-sequencing., Results: Under 20% oxygen, blastocyst rates and total cell numbers were significantly higher in the presence of OES for 4 and 7 days compared to controls (P < 0.05), with no difference according to the co-culture time. Under 5% oxygen, the presence of OES did not affect blastocyst rates but increased the number of cells per blastocyst after 7 days of co-culture (P < 0.05). Both oxygen level and OES co-culture had a significant impact on the embryonic transcriptome. The highest number of differentially expressed genes (DEGs) was identified after 7 days of co-culture under 20% oxygen. DEGs were involved in a wide range of functions, including lipid metabolism, membrane organization, response to external signals, early embryo development, and transport of small molecules among the most significantly impacted., Conclusion: OES had beneficial effects on embryo development and quality under both 5% and 20% oxygen, mitigating oxidative stress. Stronger effects on embryo quality and transcriptome were obtained after 7 than 4 days of co-culture. This study shows the impact of OES on embryo development and reveals potential molecular targets of OES-embryo dialog involved in response to stress and early embryonic development., (© 2024. The Author(s).)

Background: Increasing evidence points to an active role of oviductal extracellular vesicles (oEVs) in the early embryo-maternal dialogue. However, it remains unclear whether oEVs contribute to the recognition of the presence of embryos and their quality in the oviduct. Hence, we examined whether the molecular cargo of oEVs secreted by bovine oviduct epithelial cells (BOEC) differs depending on the presence of good (≥ 8 cells, G) or poor (< 8 cells, P) quality embryos. In addition, differences in RNA profiles between G and P embryos were analyzed in attempt to distinguish oEVs and embryonic EVs cargos., Methods: For this purpose, primary BOEC were co-cultured with in vitro produced embryos (IVP) 53 h post fertilization as follows: BOEC with G embryos (BGE); BOEC with P embryos (BPE); G embryos alone (GE); P embryos alone (PE); BOEC alone (B) and medium control (M). After 24 h of co-culture, conditioned media were collected from all groups and EVs were isolated and characterized. MicroRNA profiling of EVs and embryos was performed by small RNA-sequencing., Results: In EVs, 84 miRNAs were identified, with 8 differentially abundant (DA) miRNAs for BGE vs. B and 4 for BPE vs. B (P-value < 0.01). In embryos, 187 miRNAs were identified, with 12 DA miRNAs for BGE vs. BPE, 3 for G vs. P, 8 for BGE vs. GE, and 11 for BPE vs. PE (P-value < 0.01)., Conclusions: These results indicated that oEVs are involved in the oviductal-embryo recognition and pointed to specific miRNAs with signaling and supporting roles during early embryo development., (© 2024. The Author(s).)

Most in vitro models of oviduct epithelial cells (OEC) used thus far to gain insights into embryo-maternal communication induce cell dedifferentiation or are technically challenging. Moreover, although the presence of developing embryos has been shown to alter gene expression in OEC, the effect of embryos on OEC physiology remains largely unknown. Here, we propose a model based on bovine oviduct epithelial spheroids (OES) with specific shape and diameter (100-200 μm) criteria. The aims of this study were to i) determine the appropriate culture conditions of bovine OES cultured in suspension by evaluating their morphology, total cell number, viability, and activity of ciliated cells; ii) monitor gene expression in OES at the time of their formation (day 0) and over the 10 days of culture; and iii) test whether the vicinity of developing embryos affects OES quality criteria. On day 10, the proportions of vesicle-shaped OES (V-OES) were higher in M199/500 (500 μl of HEPES-buffered TCM-199) and synthetic oviduct fluid (SOF)/25 (25-μL droplet of SOF medium under mineral oil) than in M199/25 (25-μL droplet of M199 under mineral oil). The proportion of viable cells in V-OES was not affected by culture conditions and remained high (>80%) through day 10. The total number of cells per V-OES decreased over time except in SOF/25, while the proportions of ciliated cells increased over time in M199/500 but decreased in M199/25 and SOF/25. The movement amplitude of OES in suspension decreased over time under all culture conditions. Moreover, the gene expression of ANXA1, ESR1, HSPA8, and HSPA1A in OES remained stable during culture, while that of PGR and OVGP1 decreased from day 0 to day 10. Last, the co-culture of developing embryos with OES in SOF/25 increased the rates of blastocysts on days 7 and 8 compared to embryos cultured alone, and increased the proportion of V-OES compared to OES cultured alone. In conclusion, M199/500 and SOF/25 provided the optimal conditions for the long-time culture of OES. The supporting effect of OES on embryo development and of developing embryos on OES morphology was evidenced for the first time. Altogether, these results point OES as an easy-to-use, standardizable, and physiological model to study embryo-maternal interactions in cattle., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

Progesterone is pivotal to maintain pregnancy in the first trimester and low concentration (<4 ng/mL) has been associated with early pregnancy loss. Measurement of progesterone is widely used in practice to determine whether a mare needs progestin supplementation. Therefore, the objective of the present study was to determine progesterone concentration and the luteal tissue area in mares non-bred, and those bred becoming pregnant, and those failing to become pregnant. We hypothesized that pregnant mares have greater progesterone concentration than non-pregnant mares. Fifty-two cycles of mares (n = 14) were monitored by ultrasonography every other day until detection of a pre-ovulatory follicle. Then deslorelin acetate was administered to induce ovulation. Twenty-four hours later, mares were bred (∼2 billion progressively motile sperm extended in 50 mL; n = 37 cycles) or a sham-bred (50 mL of extender; n = 15 cycles). Ovulation was confirmed and number of corpora lutea and the luteal tissue area were recorded daily until 10-days post-ovulation. Progesterone concentration was assessed daily from the day of the ovulation up to 10-days post-ovulation. Pregnancy diagnosis was carried out at 10- and 13-days post-ovulation. Of the bred mares, 20 of them became pregnant and 17 did not. Data were analyzed with a mixed model, Tukey's test as post-hoc, and Pearson's coefficient of correlation. Progesterone concentration and luteal tissue area varied with time (P = .001) but not with group (P > .05). Multiple ovulations were associated with greater progesterone concentration and luteal tissue area (P = .0001). There was a moderate positive association between the number of ovulations and luteal tissue area (r = 0.54; P = .0001). The lack of change in the progesterone concentration and luteal tissue area between bred and non-bred mares suggests that horse seminal plasma does not affect luteal function in mares. As all mares had progesterone above 4 ng/mL after 5-days post-ovulation; it is possible that if mares with abnormal progesterone concentration were used, the results could have been different. In conclusion, pregnancy was not associated with greater progesterone concentration or luteal tissue area., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

During initial maternal recognition of pregnancy (MRP), the equine embryo displays a series of unique events characterized by rapid blastocyst expansion, secretion of a diverse array of molecules, and transuterine migration to interact with the uterine surface. Up to date, the intricate transcriptome and proteome changes of the embryo underlying these events have not been critically studied in horses. Thus, the objective of this study was to perform an integrative transcriptomic (including mRNA, miRNAs, and other small non-coding RNAs) and proteomic analysis of embryos collected from days 10 to 13 of gestation. The results revealed dynamic transcriptome profiles with a total of 1311 differentially expressed genes, including 18 microRNAs (miRNAs). Two main profiles for mRNAs and miRNAs were identified, one with higher expression in embryos ≤5 mm and the second with higher expression in embryos ≥7 mm. At the protein level, similar results were obtained, with 259 differentially abundant proteins between small and large embryos. Overall, the findings demonstrated fine-tuned transcriptomic and proteomic regulations in the developing embryo associated with embryo growth. The identification of specific regulation of mRNAs, proteins, and miRNAs on days 12 and 13 of gestation suggested these molecules as pivotal for embryo development and as involved in MRP, and in establishment of pregnancy in general. In addition, the results revealed new insights into prostaglandin synthesis by the equine embryo, miRNAs and genes potentially involved in modulation of the maternal immune response, regulation of endometrial receptivity and of late implantation in the mare., Competing Interests: The authors declare no conflict of interest., (©2022 The Authors FASEB BioAdvances published by The Federation of American Societies for Experimental Biology.)

In contrast to other domestic mammals, the embryo-derived signal(s) leading to maternal recognition of pregnancy (MRP) are still unknow in the mare. We hypothesize that these embryonic signals could be packed into uterine extracellular vesicles (uEVs), acting as multi-signal messengers between the conceptus and the maternal tract, and contributing to MRP. To unveil these signals, the RNA and protein cargos of uEVs isolated from uterine lavages collected from pregnant mares (P; day 10, 11, 12 and 13 after ovulation) and cyclic control mares (C; day 10 and 13 after ovulation) were analyzed. Our results showed a fine-tuned regulation of the uEV cargo (RNAs and proteins), by the day of pregnancy, the estrous cycle, and even the size of the embryo. A particular RNA pattern was identified with specific increase on P12 related to immune system and hormonal response. Besides, a set of proteins as well as RNAs was highly enriched in EVs on P12 and P13. Differential abundance of miRNAs was also identified in P13-derived uEVs. Their target genes were linked to down- or upregulated genes in the embryo and the endometrium, exposing their potential origin. Our study identified for first time specific molecules packed in uEVs, which were previously associated to MRP in the mare, and thus bringing added value to the current knowledge. Further integrative and functional analyses will help to confirm the role of these molecules in uEVs during MRP in the mare., (© 2022. The Author(s).)

Oviductal extracellular vesicles (oEVs) have been identified as important components of the oviductal fluid (OF) and have been pointed to as key modulators of gamete/embryo-maternal interactions. Here, we determined the functional impact of oEVs on embryo development and the embryonic transcriptome in porcine. Experiment 1 examined the effect of oEVs and OF on embryo development. In vitro-produced embryos were cultured with oEVs or OF for 2 or 7 days using an in vitro sequential system or without supplementation (control). Experiment 2 analyzed transcriptomic alterations of EV-treated embryos versus control and the oEVs RNA cargo by RNA-sequencing. Two days of EV treatment enhanced embryo development over time when compared to other treatments. Different RNA expression profiles between embryos treated with EVs for two or seven days and untreated controls were obtained, with 54 and 59 differentially expressed (DE) genes and six and seven DE miRNAs, respectively. In oEV RNA cargo, 12,998 RNAs and 163 miRNAs were identified. Integrative analyses pointed to specific oEV components that might act as modulators of the embryonic transcriptome, such as S100A11 , ANXA2 or miR-21-5p. Overall, the findings suggested that oEVs could be a potential strategy to improve porcine IVP outcomes, particularly by using two days of EV treatment.

The canine corpus luteum (CL) is able to synthetise, activate and deactivate 17b-estradiol (E2) and also expresses nuclear estrogen receptors in a time-dependent manner during diestrus. Nevertheless, we are still missing a better comprehension of E2 functions in the canine CL, especially regarding the specific roles of estrogen receptor alpha (ERa) and ERb, encoded by ESR1 and 2 , respectively. For that purpose, we analyzed transcriptomic data of canine non-pregnant CL collected on days 10, 20, 30, 40, 50 and 60 of diestrus and searched for differentially expressed genes (DEG) containing predicted transcription factor binding sites (TFBS) for ESR1 or ESR2 . Based on biological functions of DEG presenting TFBS, expression of select transcripts and corresponding proteins was assessed. Additionally, luteal cells were collected across specific time points during diestrus and specificity of E2 responses was tested using ERa and/or ERb inhibitors. Bioinformatic analyses revealed 517 DEGs containing TFBS, from which 67 for both receptors. In general, abundance of predicted ESR1 targets was greater in the beginning, while abundance of ESR2 targets was greater in the end of diestrus. ESR1 / ESR2 ratio shifted from an increasing to a decreasing pattern from day 30 to 40 post ovulation. Specific receptor inhibition suggested an ERa-mediated positive regulation of CL function at the beginning of diestrus and an ERb-mediated effect contributing to luteal regression. In conclusion, our data points toward a broad spectrum of action of E2 and its nuclear receptors, which can also act as transcription factors for other genes regulating canine CL function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bonfim Neto, Cardoso, Silva, Sousa, Giometti, Binelli, Bauersachs, Kowalewski and Papa.)

This study aimed to investigate the susceptibility to persistent breeding-induced endometritis (PBIE). Cytobrush samples were collected from 81 broodmares 1-3 days before artificial insemination (AI). Susceptibility to PBIE was evaluated by the presence of ≥ 2 cm of intrauterine fluid 24 h after AI, besides the fertility was determined by a sonographic pregnancy diagnosis 2 weeks after ovulation. RNA expressions were compared between susceptible non-pregnant (SNP) mares (n=9) and resistant pregnant (RP) mares (n=9) as well as between susceptible pregnant (SP) mares (n=9) and susceptible non-pregnant (SNP) mares. 66 differentially expressed genes (DEGs) were identified between SNP and RP mares and 60 DEGs between SP and SNP mares. In SNP compared to RP mares, transcript levels of genes regulating steroid hormone metabolism and neutrophil chemotaxis were lower, while higher for genes participating in uterine inflammation.Transcripts of genes related to extracellular matrix degradation, tissue adhesions, and fibrosis were lower in SP mares than in SNP mares, while higher for genes related to uterine cell proliferation, differentiation, and angiogenesis in SP mares than SNP mares. In conclusion, increased transcript levels of apolipoprotein E (APOE) and roundabout 2 (ROBO2), cluster domain 44 (CD44), integrin beta 3 (ITGB3), and epidermal growth factor (EGF) are possible biomarkers for susceptibility to PBIE. While higher expression of fibroblast growth factor 9 (FGF9), kinase domain receptor (KDR), and C-X-C motif chemokine ligand (CXCL) 16, collagen type V alpha 2 (COL5A2) and fibronectin (FN1) are suggested indicators of fertility in susceptible mares if they receive proper breeding management., (Copyright © 2021 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

Background: Currently, there is a high demand for efficient pig embryo cryopreservation procedures in the porcine industry as well as for genetic diversity preservation and research purposes. To date, vitrification (VIT) is the most efficient method for pig embryo cryopreservation. Despite a high number of embryos survives in vitro after vitrification/warming procedures, the in vivo embryo survival rates after embryo transfer are variable among laboratories. So far, most studies have focused on cryoprotective agents and devices, while the VIT effects on porcine embryonic gene expression remained unclear. The few studies performed were based on vitrified/warmed embryos that were cultured in vitro (IVC) to allow them to re-expand. Thus, the specific alterations of VIT, IVC, and the cumulative effect of both remained unknown. To unveil the VIT-specific embryonic alterations, gene expression in VIT versus (vs.) IVC embryos was analyzed. Additionally, changes derived from both VIT and IVC vs. control embryos (CO) were analyzed to confirm the VIT embryonic alterations. Three groups of in vivo embryos at the blastocyst stage were analyzed by RNA-sequencing: (1) VIT embryos (vitrified/warmed and cultured in vitro), (2) IVC embryos and (3) CO embryos., Results: RNA-sequencing revealed three clearly different mRNA profiles for VIT, IVC and CO embryos. Comparative analysis of mRNA profiles between VIT and IVC identified 321, differentially expressed genes (DEG) (FDR < 0.006). In VIT vs. CO and IVC vs. CO, 1901 and 1519 DEG were found, respectively, with an overlap of 1045 genes. VIT-specific functional alterations were associated to response to osmotic stress, response to hormones, and developmental growth. While alterations in response to hypoxia and mitophagy were related to the sum of VIT and IVC effects., Conclusions: Our findings revealed new insights into the VIT procedure-specific alterations of embryonic gene expression by first comparing differences in VIT vs. IVC embryos and second by an integrative transcriptome analysis including in vivo control embryos. The identified VIT alterations might reflect the transcriptional signature of the embryo cryodamage but also the embryo healing process overcoming the VIT impacts. Selected validated genes were pointed as potential biomarkers that may help to improve vitrification., (© 2022. The Author(s).)

Background: Subclinical mastitis, the inflammation of the mammary gland lacking clinical symptoms, is one of the most prevalent and costly diseases in dairy farming worldwide. Milk microRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) have been proposed as potential biomarkers of different mammary gland conditions, including subclinical mastitis. However, little is known about the robustness of EVs analysis regarding sampling time-point and natural infections. To estimate the reliability of EVs measurements in raw bovine milk, we first evaluated changes in EVs size and concentration using Tunable Resistive Pulse Sensing (TRPS) during three consecutive days of sampling. Then, we analysed daily differences in miRNA cargo using small RNA-seq. Finally, we compared milk EVs differences from naturally infected udder quarters with their healthy adjacent quarters and quarters from uninfected udders, respectively., Results: We found that the milk EV miRNA cargo was very stable over the course of three days regardless of the health status of the quarter, and that infected quarters did not induce relevant changes in milk EVs of adjacent healthy quarters. Chronic subclinical mastitis induced changes in milk EV miRNA cargo, but neither in EVs size nor concentration. We observed that the changes in immunoregulatory miRNAs in quarters with chronic subclinical mastitis were cow-individual, however, the most upregulated miRNA was bta-miR-223-3p across all individuals., Conclusions: Our results showed that the miRNA profile and particle size characteristics remained constant throughout consecutive days, suggesting that miRNAs packed in EVs are physiological state-specific. In addition, infected quarters were solely affected while adjacent healthy quarters remained unaffected. Finally, the cow-individual miRNA changes pointed towards infection-specific alterations., (© 2022. The Author(s).)

During the period of maternal recognition of pregnancy (MRP) in the mare, the embryo needs to signal its presence to the endometrium to prevent regression of the corpus luteum and prepare for establishment of pregnancy. This is achieved by mechanical stimuli and release of various signaling molecules by the equine embryo while migrating through the uterus. We hypothesized that embryo's signals induce changes in the endometrial gene expression in a highly cell type-specific manner. A spatiotemporal transcriptomics approach was applied combining laser capture microdissection and low-input-RNA sequencing of luminal and glandular epithelium (LE, GE), and stroma of biopsy samples collected from days 10-13 of pregnancy and the estrous cycle. Two comparisons were performed, samples derived from pregnancies with conceptuses ≥ 8 mm in diameter (comparison 1) and conceptuses ≤ 8 mm (comparison 2) versus samples from cyclic controls. The majority of gene expression changes was identified in LE and much lower numbers of differentially expressed genes (DEGs) in GE and stroma. While 1253 DEGs were found for LE in comparison 1, only 248 were found in comparison 2. Data mining mainly focused on DEGs in LE and revealed regulation of genes related to prostaglandin transport, metabolism, and signaling, as well as transcription factor families that could be involved in MRP. In comparison to other mammalian species, differences in regulation of genes involved in epithelial barrier formation and conceptus attachment and implantation reflected the unique features of equine reproduction at the time of MRP at the molecular level., (© 2021. The Author(s).)

Embryonic diapause in mammals leads to a reversible developmental arrest. While completely halted in many species, European roe deer ( Capreolus capreolus ) embryos display a continuous deceleration of proliferation. During a 4-mo period, the cell doubling time is 2 to 3 wk. During this period, the preimplantation blastocyst reaches a diameter of 4 mm, after which it resumes a fast developmental pace to subsequently implant. The mechanisms regulating this notable deceleration and reacceleration upon developmental resumption are unclear. We propose that amino acids of maternal origin drive the embryonic developmental pace. A pronounced change in the abundance of uterine fluid mTORC1-activating amino acids coincided with an increase in embryonic mTORC1 activity prior to the resumption of development. Concurrently, genes related to the glycolytic and phosphate pentose pathway, the TCA cycle, and one carbon metabolism were up-regulated. Furthermore, the uterine luminal epithelial transcriptome indicated increased estradiol-17β signaling, which likely regulates the endometrial secretions adapting to the embryonic needs. While mTORC1 was predicted to be inactive during diapause, the residual embryonic mTORC2 activity may indicate its involvement in maintaining the low yet continuous proliferation rate during diapause. Collectively, we emphasize the role of nutrient signaling in preimplantation embryo development. We propose selective mTORC1 inhibition via uterine catecholestrogens and let-7 as a mechanism regulating slow stem cell cycle progression., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Background: Subfertility is a major problem in modern horse breeding. Especially, mares without clinical signs of reproductive diseases, without known uterine pathogens and no evidence of inflammation but not becoming pregnant after several breeding attempts are challenging for veterinarians. To obtain new insights into the cause of these fertility problems and aiming at improving diagnosis of subfertile mares, a comparative analysis of the intrauterine transcriptome in subfertile and fertile mares was performed. Uterine cytobrush samples were collected during estrus from 57 mares without clinical signs of uterine diseases. RNA was extracted from the cytobrush samples and samples from 11 selected subfertile and 11 fertile mares were used for Illumina RNA-sequencing., Results: The cytobrush sampling was a suitable technique to isolate enough RNA of high quality for transcriptome analysis. Comparing subfertile and fertile mares, 114 differentially expressed genes (FDR = 10%) were identified. Metascape enrichment analysis revealed that genes with lower mRNA levels in subfertile mares were related to 'extracellular matrix (ECM)', 'ECM-receptor interaction', 'focal adhesion', 'immune response' and 'cytosolic calcium ion concentration', while DEGs with higher levels in subfertile mares were enriched for 'monocarboxyl acid transmembrane transport activity' and 'protein targeting'., Conclusion: Our study revealed significant differences in the uterine transcriptome between fertile and subfertile mares and provides leads for potential uterine molecular biomarkers of subfertility in the mare.

Successful pregnancy establishment in mammals depends on numerous interactions between embryos and the maternal organism. Estradiol-17β (E2) is the primary embryonic signal in the pig, and its importance has been questioned recently. However, E2 is not the only molecule of embryonic origin. In pigs, prostaglandin E2 (PGE2) is abundantly synthesized and secreted by conceptuses and endometrium. The present study aimed to determine the role of PGE2 and its simultaneous action with E2 in changes in porcine endometrial transcriptome during pregnancy establishment. The effects of PGE2 and PGE2 acting with E2 were studied using an in vivo model of intrauterine hormone infusions, and were compared to the effects of E2 alone and conceptuses' presence on day 12 of pregnancy. The endometrial transcriptome was profiled using gene expression microarrays followed by statistical analyses. Downstream analyses were performed using bioinformatics tools. Differential expression of selected genes was verified by quantitative polymerase chain reaction. Microarray analysis revealed 2413 differentially expressed genes (DEGs) in the endometrium treated simultaneously with PGE2 and E2 (P < 0.01). No significant effect of PGE2 administered alone on endometrial transcriptome was detected. Gene ontology annotations enriched for DEGs were related to multiple processes such as: focal adhesion, vascularization, cell migration and proliferation, glucose metabolism, tissue remodeling, and activation of immune response. Simultaneous administration of E2 and PGE2 induced more changes within endometrial transcriptome characteristic to pregnancy than infusion of E2 alone. The present findings suggest that synergistic action of estradiol-17β and PGE2 resembles the effects of pregnancy on endometrial transcriptome better than E2 alone., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Extracellular vesicles (EVs) have been identified in the uterine fluid in different species and have been pointed as key players in the embryo-maternal dialogue, maternal recognition of pregnancy and establishment of pregnancy. However, little is known about the uterine EVs in the mare. Therefore, the present study aimed at characterizing EVs from uterine lavage of cyclic mares by comparing five EVs isolation methods and the combination of them: (1) ultracentrifugation (UC); (2) concentration of lavage volume by Centricon ultrafiltration (CE); (3) the use of CE with different washing steps (phosphate-buffered saline with or without trehalose); (4) size-exclusion chromatography with iZON-qEV columns, and (5) a combination of the methods with best results based on EVs yield, purity, and protein cargo profiles. Transmission electron microscopy and Western blotting confirmed the isolation of EVs by all methods but with quantitative and qualitative differences. Mass spectrometry provided differences in protein profiles between methods, number of identified proteins, and protein classes. Our results indicate that the combination of CE/trehalose/iZON/UC is an optimal method to isolate equine uterine EVs with good yield and purity that can be applied in future studies to determine the role of equine uterine EVs in embryo-maternal interactions.

Background: The use of sex-sorted sperm in cattle assisted reproduction is constantly increasing. However, sperm fertility can substantially differ between unsorted (conventional) and sex-sorted semen batches of the same sire. Sperm microRNAs (miRNA) have been suggested as promising biomarkers of bull fertility the last years. In this study, we hypothesized that the miRNA profile of cryopreserved conventional sperm is related to bull fertility after artificial insemination with X-bearing sperm. For this purpose, we analyzed the miRNA profile of 18 conventional sperm samples obtained from nine high- (HF) and nine low-fertility (LF) bulls that were contemporaneously used to produce conventional and sex-sorted semen batches. The annual 56-day non-return rate for each semen type (NRR conv and NRR ss , respectively) was recorded for each bull., Results: In total, 85 miRNAs were detected. MiR-34b-3p and miR-100-5p were the two most highly expressed miRNAs with their relative abundance reaching 30% in total. MiR-10a-5p and miR-9-5p were differentially expressed in LF and HF samples (false discovery rate < 10%). The expression levels of miR-9-5p, miR-34c, miR-423-5p, miR-449a, miR-5193-5p, miR-1246, miR-2483-5p, miR-92a, miR-21-5p were significantly correlated to NRR ss but not to NRR conv . Based on robust regression analysis, miR-34c, miR-7859 and miR-342 showed the highest contribution to the prediction of NRR ss ., Conclusions: A set of miRNAs detected in conventionally produced semen batches were linked to the fertilizing potential of bovine sperm after sex-sorting. These miRNAs should be further evaluated as potential biomarkers of a sire's suitability for the production of sex-sorted sperm.

Extracellular vesicles (EVs) have emerged as novel cell-to-cell communication mediators in physiological and pathological scenarios. Their ability to transfer their molecular cargo (RNAs, proteins and lipids) from one cell to another, in the vicinity or far from the cell of origin, together with their capacity of exerting a functional impact on the target cell make them valuable diagnostic tools as well as therapeutic vectors in a variety of diseases. In the reproductive field, there is a growing interest in the role of EVs in gamete/embryo-maternal communication and their potential implications in the reproductive success. In this review, we provide current knowledge of EVs secreted by the oviduct (oEVs) and embryos (eEVs), since both have been proposed as key players in the crucial two-way dialogue between the oviduct (lining epithelium and secretions) and the embryo that ensures successful pregnancy. Both oEVs and eEVs molecular cargos and their potential role as multi-signal messengers in the gametes/embryo-oviduct cross-talk and in the embryo-to-embryo communication in different species are also addressed. Eventually, a comparative analysis between oEVs and eEVs has been performed to shed some light on common and specific cargos responsible for their functions supporting the early reproductive events and as prime candidate molecules for improving fertility and assisted reproductive technologies outcomes., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020.)

Human toll-like receptor 8 (TLR8) activation induces a potent T helper-1 (Th1) cell response critical for defense against intracellular pathogens, including protozoa. The receptor harbors two distinct binding sites, uridine and di- and/or trinucleotides, but the RNases upstream of TLR8 remain poorly characterized. We identified two endolysosomal endoribonucleases, RNase T2 and RNase 2, that act synergistically to release uridine from oligoribonucleotides. RNase T2 cleaves preferentially before, and RNase 2 after, uridines. Live bacteria, P. falciparum-infected red blood cells, purified pathogen RNA, and synthetic oligoribonucleotides all required RNase 2 and T2 processing to activate TLR8. Uridine supplementation restored RNA recognition in RNASE2 -/- or RNASET2 -/- but not RNASE2 -/- RNASET2 -/- cells. Primary immune cells from RNase T2-hypomorphic patients lacked a response to bacterial RNA but responded robustly to small-molecule TLR8 ligands. Our data identify an essential function of RNase T2 and RNase 2 upstream of TLR8 and provide insight into TLR8 activation., Competing Interests: Declaration of Interests W.B. is an employee of IFM Therapeutics. The other authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Preimplantation horse conceptuses require nutrients and signals from histotroph, the composition of which is regulated by luteal progesterone and conceptus-secreted factors. To distinguish progesterone and conceptus effects we shortened the period of endometrial progesterone-priming by asynchronous embryo transfer. Day 8 embryos were transferred to synchronous (day 8) or asynchronous (day 3) recipients, and RNA sequencing was performed on endometrium and conceptuses recovered 6 and 11 days later (embryo days 14 and 19). Asynchrony resulted in many more differentially expressed genes (DEGs) in conceptus membranes (3473) than endometrium (715). Gene ontology analysis identified upregulation in biological processes related to organogenesis and preventing apoptosis in synchronous conceptuses on day 14, and in cell adhesion and migration on day 19. Asynchrony also resulted in large numbers of DEGs related to 'extracellular exosome'. In endometrium, genes involved in immunity, the inflammatory response, and apoptosis regulation were upregulated during synchronous pregnancy and, again, many genes related to extracellular exosome were differentially expressed. Interestingly, only 14 genes were differentially expressed in endometrium recovered 6 days after synchronous versus 11 days after asynchronous transfer (day 14 recipient in both). Among these, KNG1 and IGFBP3 were consistently upregulated in synchronous endometrium. Furthermore bradykinin, an active peptide cleaved from KNG1, stimulated prostaglandin release by cultured trophectoderm cells. The horse conceptus thus responds to a negatively asynchronous uterus by extensively adjusting its transcriptome, whereas the endometrial transcriptome is modified only subtly by a more advanced conceptus.

Fibroblast growth factors (FGF) play an important role during embryo development. To date, the role of FGF and the respective receptors (FGFR) during the preimplantation phase in cattle are not fully characterized. We examined FGF1, FGF2, FGFR1, FGFR2, and FGFR3 in cyclic and early pregnant heifers at Days 12, 15, and 18 after insemination (Day 0). Endometrial FGF1 mRNA transcript abundance in heifers varied significantly with respect to the day after insemination, the pregnancy status, and their interaction. The expression was higher in nonpregnant than in pregnant heifers at Day 18. The conceptus transcripts abundance of FGFR2 and FGFR3 were significantly lower at Day 15 than 18. In the endometrium, FGF1 protein abundance significantly decreased from Day 12 onwards and FGF2 protein abundance showed a minor, but a significant increase at Day 15 in comparison to Days 12 and 18. We concluded that the decrease in FGF1 mRNA expression in pregnant heifers at Day 18 points towards a potential contribution of FGF1 in the preimplantation process. Additionally, successful embryo elongation might require a spatiotemporal FGF2 protein increase in the endometrium.

Oviductal extracellular vesicles (oEVs) are emerging as key players in the gamete/embryo-oviduct interactions that contribute to successful pregnancy. Various positive effects of oEVs on gametes and early embryos have been found in vitro. To determine whether these effects are associated with changes of embryonic gene expression, the transcriptomes of embryos supplemented with bovine fresh (FeEVs) or frozen (FoEVs) oEVs during in vitro culture compared to controls without oEVs were analyzed by low-input RNA sequencing. Analysis of RNA-seq data revealed 221 differentially expressed genes (DEGs) between FoEV treatment and control, 67 DEGs for FeEV and FoEV treatments, and minor differences between FeEV treatment and control (28 DEGs). An integrative analysis of mRNAs and miRNAs contained in oEVs obtained in a previous study with embryonic mRNA alterations pointed to direct effects of oEV cargo on embryos (1) by increasing the concentration of delivered transcripts; (2) by translating delivered mRNAs to proteins that regulate embryonic gene expression; and (3) by oEV-derived miRNAs which downregulate embryonic mRNAs or modify gene expression in other ways. Our study provided the first high-throughput analysis of the embryonic transcriptome regulated by oEVs, increasing our knowledge on the impact of oEVs on the embryo and revealing the oEV RNA components that potentially regulate embryonic development., Competing Interests: The authors declare no conflict of interest.

Estradiol-17β (E2) is a key hormone regulating reproductive functions in females. In pigs, E2, as the main conceptus signal, initiates processes resulting in prolonged corpus luteum function, embryo development, and implantation. During early pregnancy the endometrium undergoes morphological and physiological transitions that are tightly related to transcriptome changes. Recently, however, the importance of E2 as a primary conceptus signal in the pig has been questionable. Thus, the aim of the present study was to determine the effects of E2 on the porcine endometrial transcriptome in vivo and to compare these effects with transcriptome profiles on day 12 of pregnancy. Microarray analysis revealed differentially expressed genes (DEGs) in response to E2 with overrepresented functional terms related to secretive functions, extracellular vesicles, cell adhesion, proliferation and differentiation, tissue rearrangements, immune response, lipid metabolism, and many others. Numerous common DEGs and processes for the endometrium on day 12 of pregnancy and E2-treated endometrium were identified. In summary, the present study is the first evidence for the effect of E2 on transcriptome profiles in porcine endometrium in vivo in the period corresponding to the maternal recognition of pregnancy. The presented results provide a valuable resource for further targeted studies considering genes and pathways regulated by conceptus-derived estrogens and their role in pregnancy establishment.

Vascular endothelial growth factor A (VEGFA) plays a critical angiogenic role in the endometrium of placentalia during preimplantation. The role of VEGFA and its receptors is not fully characterised in bovine reproduction. We analysed the mRNA expression of VEGFA isoforms 121, 165 and 189, and VEGF receptors 1 and 2 in three experimental settings (A, B and C). We compared intercaruncular endometrium of cyclic to pregnant heifers at Days 12, 15 and 18 post insemination (Day 0), and between Day 15 and Day 18 conceptuses (A). We further compared caruncular versus intercaruncular endometrium at Day 15 (B), and endometrium of heifers carrying embryos originating from somatic cell nuclear transfer (SCNT) versus in vitro fertilisation (IVF) at Day 18 (C). Endometrial VEGFA protein was localised and quantified. Pregnant heifers displayed lower intercaruncular endometrial mRNA expression of VEGFA-121 ( p = 0.045) and VEGFA-189 ( p = 0.009) as well as lower VEGFA protein abundance ( p < 0.001) at Day 15. The VEGFA protein was localised in intercaruncular luminal, glandular epithelium and in tunica muscularis of blood vessels. At Day 15, caruncular endometrium displayed higher VEGFA mRNA expression than intercaruncular endometrium ( p < 0.05). Intercaruncular endometrial VEGFA protein at Day 18 was higher in abundance in SCNT than in IVF ( p = 0.038). Therefore, during preimplantation in cattle, there may be a need for timely physiological reduction in intercaruncular endometrial VEGFA expression in favour of the caruncular area to facilitate a gradient towards the implantation sites. A higher expression of VEGFA in SCNT may predispose for later placentation abnormalities frequently observed following SCNT.

Background: During the preimplantation phase in the pig, the conceptus trophoblast elongates into a filamentous form and secretes estrogens, interleukin 1 beta 2, interferons, and other signaling molecules before attaching to the uterine epithelium. The processes in the uterine endometrium in response to conceptus signaling are complex. Thus, the objective of this study was to characterize transcriptome changes in porcine endometrium during the time of conceptus attachment considering the specific localization in different endometrial cell types., Results: Low-input RNA-sequencing was conducted for the main endometrial compartments, luminal epithelium (LE), glandular epithelium (GE), blood vessels (BV), and stroma. Samples were isolated from endometria collected on Day 14 of pregnancy and the estrous cycle (each group n = 4) by laser capture microdissection. The expression of 12,000, 11,903, 11,094, and 11,933 genes was detectable in LE, GE, BV, and stroma, respectively. Differential expression analysis was performed between the pregnant and cyclic group for each cell type as well as for a corresponding dataset for complete endometrium tissue samples. The highest number of differentially expressed genes (DEGs) was found for LE (1410) compared to GE, BV, and stroma (800, 1216, and 384). For the complete tissue, 3262 DEGs were obtained. The DEGs were assigned to Gene Ontology (GO) terms to find overrepresented functional categories and pathways specific for the individual endometrial compartments. GO classification revealed that DEGs in LE were involved in 'biosynthetic processes', 'related to ion transport', and 'apoptotic processes', whereas 'cell migration', 'cell growth', 'signaling', and 'metabolic/biosynthetic processes' categories were enriched for GE. For blood vessels, categories such as 'focal adhesion', 'actin cytoskeleton', 'cell junction', 'cell differentiation and development' were found as overrepresented, while for stromal samples, most DEGs were assigned to 'extracellular matrix', 'gap junction', and 'ER to Golgi vesicles'., Conclusions: The localization of differential gene expression to different endometrial cell types provided a significantly improved view on the regulation of biological processes involved in conceptus implantation, such as the control of uterine fluid secretion, trophoblast attachment, growth regulation by Wnt signaling and other signaling pathways, as well as the modulation of the maternal immune system.

Background: Porcine embryos undergo rapid differentiation and expansion between Days 8 and 12 before attaching to the maternal uterine epithelial surface after Day 13. It is known that maternal recognition of pregnancy and successful implantation are driven by mutual interactions between the elongated conceptus and the maternal endometrium. While most of the genes involved in regulation of embryo development are located on autosomal chromosomes, gene expression on sex chromosomes is modulating development through sex-specific transcription. To gain more insights into the dynamic transcriptome of preimplantation embryos at the onset of elongation and into X-linked gene expression, RNA-seq analyses were performed for single female and male porcine embryos collected on Days 8, 10, and 12 of pregnancy., Results: A high number of genes were differentially expressed across the developmental stages (2174 and 3275 for Days 8 vs 10, and 10 vs 12, respectively). The majority of differentially expressed genes (DEGs) were involved in embryo elongation, development, and embryo-maternal interaction. Interestingly, a number of DEGs was found with respect to embryo sex (137, 37, and 56 on Days 8, 10 and 12, respectively). At Day 8, most of these DEGs were X-linked (96). Strikingly, the number of DEGs encoded on the X chromosome dramatically decreased from Day 10 to Day 12., Conclusions: The obtained results deepen the understanding about temporary transcriptomic changes in porcine embryos during the phase of conceptus elongation, meanwhile reveal dynamic compensation of X chromosome in the female and distinct transcriptional differences between female and male embryos.

The uterine microenvironment during pre-implantation presents a pro-survival milieu and is essential for embryo elongation in ruminants. The European roe deer (Careolus capreolus) pre-implantation embryo development is characterised by a 4-month period of reduced development, embryonic diapause, after which the embryo rapidly elongates and implants. We investigated the uterine fluid proteome by label-free liquid chromatography tandem mass spectrometry at four defined stages covering the phase of reduced developmental pace (early diapause, mid-diapause and late diapause) and embryo elongation. We hypothesised that embryo development during diapause is halted by the lack of signals that support progression past the blastocyst stage. Three clusters of differentially abundant proteins were identified by a self-organising tree algorithm: (1) gradual reduction over development; (2) stable abundance during diapause, followed by a sharp rise at elongation; and (3) gradual increase over development. Proteins in the different clusters were subjected to gene ontology analysis. 'Cellular detoxification' in cluster 1 was represented by alcohol dehydrogenase, glutathione S-transferase and peroxiredoxin-2. ATP-citrate synthase, nucleolin, lamin A/C, and purine phosphorylase as cell proliferation regulators were found in cluster 2 and 'cortical cytoskeleton', 'regulation of substrate adhesion-dependent cell spreading' and 'melanosome' were present in cluster 3. Cell cycle promoters were higher abundant at elongation than during diapause, and polyamines presence indicates their role in diapause regulation. This study provides a comprehensive overview of proteins in the roe deer uterine fluid during diapause and forms a basis for studies aiming at understanding the impact of the lack of cell cycle promoters during diapause.

Maternal exposure to estrogens can induce long-term adverse effects in the offspring. The epigenetic programming may start as early as the period of preimplantation development. We analyzed the effects of gestational estradiol-17β (E2) exposure with two distinct low doses, corresponding to the acceptable daily intake "ADI" and close to the no-observed-effect level "NOEL", and a high dose (0.05, 10, and 1000 μg E2/kg body weight daily, respectively). The E2 doses were orally applied to sows from insemination until sampling at day 10 of pregnancy and compared to carrier-treated controls leading to a significant increase in E2 in plasma, bile and selected somatic tissues including the endometrium in the high-dose group. Conjugated and unconjugated E2 metabolites were as well elevated in the NOEL group. Although RNA-sequencing revealed a dose-dependent effect of 14, 17, and 27 differentially expressed genes (DEG) in the endometrium, single embryos were much more affected with 982 DEG in female blastocysts of the high-dose group, while none were present in the corresponding male embryos. Moreover, the NOEL treatment caused 62 and 3 DEG in female and male embryos, respectively. Thus, we detected a perturbed sex-specific gene expression profile leading to a leveling of the transcriptome profiles of female and male embryos. The preimplantation period therefore demonstrates a vulnerable time window for estrogen exposure, potentially constituting the cause for lasting consequences. The molecular fingerprint of low-dose estrogen exposure on developing embryos warrants a careful revisit of effect level thresholds., (© The Author(s) 2018. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

Previous endometrial gene expression studies during the time of conceptus migration did not provide final conclusions on the mechanisms of maternal recognition of pregnancy (MRP) in the mare. This called for a cell type-specific endometrial gene expression analysis in response to embryo signals to improve the understanding of gene expression regulation in the context of MRP. Laser capture microdissection was used to collect luminal epithelium (LE), glandular epithelium and stroma from endometrial biopsies from Day 12 of pregnancy and Day 12 of the oestrous cycle. RNA sequencing (RNA-Seq) showed greater expression differences between cell types than between pregnant and cyclic states; differences between the pregnant and cyclic states were mainly found in LE. Comparison with a previous RNA-Seq dataset for whole biopsy samples revealed the specific origin of gene expression differences. Furthermore, genes specifically differentially expressed (DE) in one cell type were found that were not detectable as DE in biopsies. Overall, this study revealed spatial information about endometrial gene expression during the phase of initial MRP. The conceptus induced changes in the expression of genes involved in blood vessel development, specific spatial regulation of the immune system, growth factors, regulation of prostaglandin synthesis, transport prostaglandin receptors, specifically prostaglandin F receptor (PTGFR) in the context of prevention of luteolysis.

Next-generation sequencing technologies and the availability of an increasing number of mammalian and other genomes allow gene expression studies, particularly RNA sequencing, in many non-model organisms. However, incomplete genome annotation and assignments of genes to functional annotation databases can lead to a substantial loss of information in downstream data analysis. To overcome this, we developed Mammalian Annotation Database tool (MAdb, https://madb.ethz.ch) to conveniently provide homologous gene information for selected mammalian species. The assignment between species is performed in three steps: (i) matching official gene symbols, (ii) using ortholog information contained in Ensembl Compara and (iii) pairwise BLAST comparisons of all transcripts. In addition, we developed a new tool (AnnOverlappeR) for the reliable assignment of the National Center for Biotechnology Information (NCBI) and Ensembl gene IDs. The gene lists translated to gene IDs of well-annotated species such as a human can be used for improved functional annotation with relevant tools based on Gene Ontology and molecular pathway information. We tested the MAdb on a published RNA-seq data set for the pig and showed clearly improved overrepresentation analysis results based on the assigned human homologous gene identifiers. Using the MAdb revealed a similar list of human homologous genes and functional annotation results regardless of whether starting with gene IDs from NCBI or Ensembl. The MAdb database is accessible via a web interface and a Galaxy application., (© The Author(s) 2019. Published by Oxford University Press.)