POU5F1 is a transcription factor and master regulator of cell pluripotency with indispensable roles in early embryo development and cell lineage specification. The role of embryonic POU5F1 in blastocyst formation and cell lineage specification differs between mammalian species but remains completely unknown in cattle. The CRISPR/Cas9 system was utilized for targeted disruption of the POU5F1 gene by direct injection into zygotes. Disruption of the bovine POU5F1 locus prevented blastocyst formation and was associated with embryonic arrest at the morula stage. POU5F1 knockout morulas developed at a similar rate as control embryos and presented a similar number of blastomeres by day 5 of development. Initiation of SOX2 expression by day 5 of development was not affected by lack of POU5F1. On the other hand, CDX2 expression was aberrant in embryos lacking POU5F1. Notably, the phenotype observed in bovine POU5F1 knockout embryos reveals conserved functions associated with loss of human embryonic POU5F1 that differ from Pou5f1- null mice. The similarity observed in transcriptional regulation of early embryo development between cattle and humans combined with highly efficient gene editing techniques make the bovine a valuable model for human embryo biology with expanded applications in agriculture and assisted reproductive technologies.

In many mammals, after semen deposition, a subpopulation of the sperm is transported to the lower oviduct, or isthmus, to form a functional sperm reservoir that provides sperm to fertilize oocytes. The precise molecular interactions that allow formation of this reservoir are unclear. It is proposed that binding of sperm receptors (lectins) to their oviductal cell ligands is accomplished by glycans. Previous results indicated that Lewis trisaccharides are present in glycosphingolipids and O- and N-linked glycans of the porcine isthmus and that Le(X)-containing molecules bind porcine sperm. Immunohistochemistry indicated that the Lewis structures identified by mass spectrometry were, in fact, Lewis X (Le(X)) trisaccharides. These motifs were localized to the luminal border of the isthmus. Assays using fluoresceinated glycans showed that 3-O-sulfated Le(X) (suLe(X)) bound to receptors localized on the head of nearly 60% of uncapacitated boar sperm but that the positional isomer 3-O-sulfo-Le(A) (suLe(A)) bound to <5% of sperm. Sperm also bound preferentially to suLe(X) made insoluble by coupling to beads. Capacitation reduced the ability of suLe(X) to bind sperm to <10%, perhaps helping to explain why sperm are released at capacitation. Pretreatment of oviduct cell aggregates with the Le(X) antibody blocked 57% of sperm binding to isthmic aggregates. Blocking putative receptors on sperm with soluble Le(X) and suLe(X) glycans specifically reduced sperm binding to oviduct cells up to 61%. These results demonstrate that the oviduct isthmus contains Le(X)-related moieties and that sperm binding to these oviduct glycans is necessary and sufficient for forming the sperm reservoir.

SUMMARYDue to reduced fertility, cryopreserved semen is seldom used for commercial porcine artificial insemination (AI). Predicting the fertility of individual frozen ejaculates for selection of higher quality semen prior to AI would increase overall success. Our objective was to test novel and traditional laboratory analyses to identify characteristics of cryopreserved spermatozoa that are related to boar fertility. Traditional post-thaw analyses of motility, viability, and acrosome integrity were performed on each ejaculate. In vitro fertilization, cleavage, and blastocyst development were also determined. Finally, spermatozoa-oviduct binding and competitive zonabinding assays were applied to assess sperm adhesion to these two matrices. Fertility of the same ejaculates subjected to laboratory assays was determined for each boar by multi-sire AI and defined as (i) the mean percentage of the litter sired and (ii) the mean number of piglets sired in each litter. Means of each laboratory evaluation were calculated for each boar and those values were applied to multiple linear regression analyses to determine which sperm traits could collectively estimate fertility in the simplest model. The regression model to predict the percent of litter sired by each boar was highly effective (p < 0.001, r 2 = 0.87) and included five traits;acrosome-compromised spermatozoa, percent live spermatozoa (0 and 60 min post-thaw), percent total motility, and the number of zona-bound spermatozoa. A second model to predict the number of piglets sired by boar was also effective (p < 0.05, r 2 = 0.57).These models indicate that the fertility of cryopreserved boar spermatozoa can be predicted effectively by including traditional and novel laboratory assays that consider functions of spermatozoa.

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This review focuses on current knowledge of paternal contributions to preimplantation embryonic development with particular emphasis on large animals. Specifically, the included content aims to summarize genomic and epigenomic contributions of paternally expressed genes, their regulation and chromatin structure that are indispensable for early embryo development. The accumulation of current knowledge will summarize conserved allelic function among species to include functional molecular and genomic studies across large domestic animals in context with reference to founding experimental models.

Polycystin‐2, also known as transient receptor potential polycystin‐2 (TRPP2), is a membrane protein that regulates calcium homeostasis in renal epithelial cells. Mutations in PKD2, the gene encoding human TRPP2, cause enlarged cystic kidneys and contribute to polycystic kidney disease (PKD). Male Drosophila melanogaster with mutations in amo, the homolog of PKD2, display a mild decrease in sperm motility but have a drastic reduction in fertility due to failed sperm migration and storage within the female tract. Although TRPP2 has critical roles for Drosophila sperm function, the protein has not been described in mammalian sperm. Herein, we report the localization of TRPP2 in porcine sperm and identify functions of TRPP2 in regulating intracellular Ca2+ and motility. Porcine sperm treated with an antibody to TRPP2 in capacitating medium had reduced average path velocity and curvilinear velocity (p < .05). Blocking TRPP2 also increased sperm tail beat‐cross frequency (p < .05). After 90 min of capacitation, sperm incubated with TRPP2 antibody had decreased intracellular Ca2+ concentration compared to controls (p < .05), consistent with TRPP2 function as a plasma membrane cation channel. This is the first report that mammalian sperm contain TRPP2, which appears to regulate intracellular Ca2+ and motility patterns in porcine sperm.

Context Male exposure to environmental toxicants can disrupt spermatogenesis and sperm function. However, consequences of environmentally relevant organotin exposure to post-ejaculatory mammalian spermatozoa on fertility are poorly understood. Aims Determine the consequences of tributyltin chloride (TBT) exposure on post-ejaculatory sperm function and subsequent embryo development. Methods Frozen-thawed bovine sperm were exposed to TBT (0.1–100 nM) for 90 min (acute) and 6 h (short-term) followed by quantification of multiple sperm kinematics via computer aided sperm analysis. JC-1 dye was used to measure mitochondrial membrane potential. Sperm were then exposed to TBT for 90 min in non-capacitating conditions, washed several times by centrifugation and applied to gamete co-incubation for in vitro embryo production to the blastocyst stage. Key results 100 nM TBT decreased total motility (88 vs 79%), progressive motility (80 vs 70%) curvilinear velocity and beat-cross frequency for 90 min with similar phenotypes at 6 h (P < 0.05). Sperm mitochondrial membrane potential was lower in 10 and 100 nM groups after 6 h (P ≤ 0.05). Embryos fertilised from TBT-exposed sperm had reduced cleavage rate (80 vs 62%) and 8–16 cell morula development (55 vs 24%) compared to development from unexposed sperm. Conclusions Exposure of post-ejaculatory mammalian sperm to TBT alters sperm function through lowered motility and mitochondrial membrane potential. Fertilisation of oocytes with TBT-exposed sperm reduces embryo development through mechanisms of paternal origin. Implications Acute and short-term environmental exposure of post-ejaculatory sperm to organotins and endocrine disrupting chemicals such as TBT contribute to idiopathic subfertility and early embryo loss.

CRISPR technologies used for mammalian embryology have wide implications from basic research to applications in agriculture and biomedicine. Confirmation of successful gene editing following CRISPR/Cas9 delivery is often limited to either protein expression or sequencing analyses of embryos but not both, due to technical challenges. Herein we report an integrative approach for evaluating both protein expression and genotype of single embryos from fixed bovine embryos previously subjected to CRISPR/Cas9 microinjection. The techniques described facilitate investigation of functional genomics in bovine embryos compatible with gene editing in livestock after zygotic CRISPR microinjection. These methods avoid traditional avenues that necessitate the use of gene-edited cell lines followed by nuclear transfer that hinder efficiency, limit physiological relevance and contribute to technical challenges.