The oviduct plays a crucial role in fertilization and early embryo development providing the microenvironment for oocyte, spermatozoa, and early embryo. Since dairy cow fertility declined steadily over the last decades, reasons for early embryonic loss have gained increasing interest. Analyzing two animal models, this study aimed to investigate the impact of genetic predisposition for fertility and of metabolic stress on the protein composition of oviduct fluid. A metabolic model comprised maiden Holstein heifers and postpartum lactating (Lact) and non-lactating (Dry) cows, while a genetic model consisted of heifers from the Montbéliarde breed and Holstein heifers with low- and high-fertility index. In a holistic proteomic analysis of oviduct fluid from all groups using nano-liquid chromatography tandem-mass spectrometry analysis and label-free quantification, we were able to identify 1976 proteins, among which 143 showed abundance alterations in the pairwise comparisons within both models. Most differentially abundant proteins were revealed between low fertility Holstein and Montbéliarde (52) in the genetic model and between lactating and maiden Holstein (19) in the metabolic model, demonstrating a substantial effect of genetic predisposition for fertility and metabolic stress on the oviduct fluid proteome. Functional classification of affected proteins revealed actin binding, translation, and immune system processes as prominent gene ontology (GO) clusters. Notably, Actin-related protein 2/3 complex subunit 1B and the three immune system-related proteins SERPIND1 protein, immunoglobulin kappa locus protein, and Alpha-1-acid glycoprotein were affected in both models, suggesting that abundance changes of immune-related proteins in oviduct fluid play an important role for early embryonic loss.

Proper oocyte maturation is a prerequisite for successful reproduction and requires the resumption of meiosis to the metaphase II stage (MII). In bovine oocytes, nuclear maturation has been shown to occur in in vitro maturing cumulus-enclosed oocytes (COCs) in the absence of transcription, but their developmental capacity is reduced compared to transcriptionally competent COCs. To assess the impact of transcription during in vitro maturation of bovine COCs on the quantitative oocyte proteome, a holistic nano-LC–MS/MS analysis of GV oocytes and MII oocytes matured with or without addition of the transcription inhibitor actinomycin D (ActD) was carried out. Analyzing eight biological replicates for each of the three groups, a total of 2018 proteins were identified. These could be clearly classified into proteins depending or not depending on transcription during oocyte maturation. Proteins whose abundance increased after maturation irrespective of transcription inhibition—and hence independent of transcription—were related to the cell cycle, reflecting the progression of meiosis, and to cellular component organization, which is crucial for cytoplasmic maturation. In contrast, transcription-dependent proteins were associated with cell–cell adhesion and translation. Since a high rate of protein synthesis in oocytes has been shown to correlate with their developmental competence, oocyte maturation in transcriptionally impaired COCs is apparently disturbed. Our experiments reveal that impaired transcription during in vitro maturation of COCs has a substantial effect on specific components of the oocyte proteome, and that transcription is required for specific classes of oocyte proteins predominantly involved in translation.

Over the last decades, fertility of dairy cows has declined due to selection strategies focusing on milk yield. To study the effect of genetic merit for fertility on the proteome of the bovine uterine luminal fluid, Holstein heifers with low- and two groups of heifers with high-fertility index (high-fertility Holstein and Montbéliarde) were investigated. To focus on the maternal effect, heifers from all groups were synchronized and received on Day 7 high-quality embryos. Uterine luminal fluid from Day 19 pregnant heifers was analyzed in a holistic proteomic approach using nano-LC-MS/MS analysis combined with a label-free quantification approach. In total, 1737 proteins were identified, of which 597 differed significantly in abundance between the three groups. The vast majority of proteome differences was found comparing both high-fertility groups to the low-fertility Holstein group, showing that the genetic predisposition for fertility is prevalent regarding the uterine luminal fluid proteome. Evaluation of this dataset using bioinformatic tools revealed an assignment of higher abundant proteins in low-fertility Holstein to several metabolic processes, such as vitamin metabolic process, which comprises folate receptor alpha (FOLR1) and retinol-binding protein, indicating an involvement of disturbed metabolic processes in decreased fertility. Moreover, immune system-related proteins — lactotransferrin and chromogranin A — were enriched in low-fertility cows together with interferon tau 3 h and interferon tau-2. Our results indicate that the genetic merit for fertility leads to substantial quantitative differences at the level of proteins in uterine fluid of pregnant animals, thus altering the microenvironment for the early conceptus.

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Intensive selection strategies focusing on increased milk yield over several decades has been associated with a decline in fertility in dairy cows. To study the effect of the genetic merit for fertility and the metabolic status of the female on the oocyte, early embryo and the maternal environment, 2 animal models were established. The genetic merit model involved Holstein heifers with a low (LFH) and high fertility (HFH) index and heifers from the Montbéliarde breed (MBD), known to have good reproductive performance. The metabolic model comprised samples from maiden heifers (MH), postpartum lactating cows (Lact), and non-lactating cows (dried off immediately after calving; Dry). A common pool of Day 7 embryos recovered from superovulated and artificially inseminated Holstein heifers were transferred into synchronised recipients (1/recipient) of the above-mentioned animal models and uterine lumen fluid (ULF) of confirmed pregnant animals was recovered on Day 19 post-oestrus. As communication between the conceptus and the uterine environment is crucial for the successful establishment of pregnancy, we analysed uterine luminal fluid of pregnant cows from both models using a holistic proteomic approach. Using nano-liquid chromatography-tandem mass spectrometry analysis combined with a label-free quantification approach, we analysed the uterine luminal fluid from the uterine horn ipsilateral to the corpus luteum (where the conceptus was located in all cases). A total of 2127 proteins were quantified in all samples of both models. Among this set of proteins, 458 were found to differ significantly (P-value < 0.05) in abundance between the groups of the genetic model, and 141 were altered in abundance in the metabolic model. The majority of proteome differences in ULF samples was found comparing HFH to the LFH group (358) and between Dry and Lact cows (70) in the metabolic model. Evaluation of this dataset using bioinformatic tools comprising DAVID GO and gene set enrichment analysis revealed that the affected proteins were predominantly assigned to the terms “translation”, “monosaccharide metabolic process”, “enzyme inhibitor activity”, “lipid binding”, and “response to oxidative stress”. Our study revealed that metabolic status and genetic merit for fertility lead to quantitative molecular differences at the level of proteins in uterine fluid of pregnant animals, thus altering the microenvironment for the early conceptus. This research was funded by European Union Seventh Framework Programme FP7/2007-2013 under grant agreement no. 312097 (‘FECUND’).