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Oviduct-specific glycoprotein (OGP) displays estrus-associated regional and temporal differences in expression and localizes to the zona pellucida, perivitelline space, and plasma membrane of oviductal oocytes and embryos, suggesting that it may have a role in regulation of fertilization and/or early embryonic development. The aims of this study were to evaluate the effect of exogenous OGP on in vitro fertilization (IVF) and embryo development in the pig using a defined serum-free culture system. In vitro-matured porcine oocytes were incubated with homologous OGP (0, 1, 10, 20, and 40 microg/ml) for 3 h and then washed prior to IVF. Exposure of oocytes to 10 or 20 microg/ml porcine OGP (pOGP) significantly reduced the incidence of polyspermy compared with the control (P < 0.01) while maintaining high penetration rates. When oocytes, spermatozoa, or both were preincubated with 10 microg/ml pOGP prior to IVF, the incidence of polyspermy was similarly reduced (P < 0.01) by all three treatments without affecting penetration rates. The ability of spermatozoa to undergo calcium ionophore-induced acrosome reaction was similar with or without exposure to pOGP. However, significantly fewer spermatozoa (P < 0.01) bound to the zona pellucida when oocytes were preincubated with pOGP. To evaluate the effect of pOGP on embryo development, embryos were cultured in pOGP-supplemented medium for 48 h or 144 h. Both transient and continuous exposure to pOGP significantly enhanced cleavage and blastocyst formation rate compared with the control (P < 0.01). These data demonstrate that exposure of either in vitro-matured oocytes or spermatozoa to pOGP decreased polyspermy and spermatozoa binding while maintaining high penetration rates of pig oocytes fertilized in vitro. Furthermore, pOGP exerted an embryotrophic effect independent of effects demonstrated on spermatozoa and oocytes at fertilization.

The object of this study was to investigate the role of epidermal growth factor (EGF) and IGF-I in the regulation of preantral follicular growth, antrum formation, and granulosal cell proliferation/ apoptosis. Porcine preantral follicles were manually dissected and cultured for up to 8 d in Waymouth's (Exp. 1) or alpha-minimum Eagle's essential medium (Exp. 2 and 3) supplemented with 10 microg/mL of transferrin, 100 microg/mL of L-ascorbic acid, and 2 mU/mL of ovine FSH, in the presence (Exp. 1 and 3) or absence (Exp. 2) of 7.5% fetal calf serum. According to the experimental protocol, IGF-I (0, 1, 10, or 100 ng/mL; Exp. 1), or IGF-I (50 ng/mL), EGF (10 ng/mL) and EGF+IGF-I (Exp. 2 and 3) were added to the culture media. In Exp. 1, follicles exhibited a concentration-dependent response (P < 0.05) to IGF-I, with the highest rates of granulosal cell proliferation, follicular integrity, and recovery rate of cumulus cell-oocyte complexes and lowest incidence of apoptosis occurring at the highest IGF-I dose. In Exp. 2 serum-free medium, granulosal cell proliferation was low (1 to 5%), irrespective of whether EGF and/or IGF-I were present and cellular apoptosis was increased (P < 0.05) on d 4 and 8 in the EGF+IGF-I group compared with the addition of either factor alone. In Exp. 3, granulosal cell proliferation was high in all follicles cultured in serum-containing medium for the first 3 d, but fell sharply (P < 0.05) on d 4, except in media containing IGF-I. Collectively, EGF and IGF-I increased granulosal cell proliferation, decreased apoptosis, and promoted follicular antrum formation. These results may provide useful information for developing a preantral follicular culture system in which the oocytes are capable of fertilization and embryonic development.

The invasion has begun: Invaders are shown to recognize DNA hairpins in cell‐free assays and chromosomal DNA during non‐denaturing fluorescence in situ hybridization (nd‐FISH) experiments. As Invaders are devoid of inherent sequence limitations, many previously inaccessible DNA targets could become accessible to exogenous control with important ramifications for karyotyping, in vivo imaging, and gene regulation.

Post-acrosomal WW-domain binding protein (PAWP) is a signaling molecule located in the post-acrosomal sheath (PAS) of mammalian spermatozoa. We hypothesized that the proper integration of PAWP in the sperm PAS is reflective of bull-sperm quality and fertility. Cryopreserved semen samples from 298 sires of acceptable, but varied, fertility used in artificial insemination services were analyzed using immunofluorescence microscopy and flow cytometry for PAWP protein. In normal spermatozoa, PAWP fluorescence formed a regular band around the proximal PAS. Anomalies of PAWP labeling in defective spermatozoa were reflected in flow cytometry by varied intensities of PAWP-induced fluorescence. Distinct sperm phenotypes were also identified, including morphologically normal and some defective spermatozoa with moderate levels of PAWP; grossly defective spermatozoa with low/no PAWP; and defective spermatozoa with high PAWP. Analysis by ImageStream flow cytometry confirmed the prevalence of abnormal sperm phenotypes in the spermatozoa with abnormal PAWP content. Live/dead staining and video recording showed that some abnormal spermatozoa are viable and capable of progressive motility. Conventional flow-cytometric measurements of PAWP correlated significantly with semen quality and fertility parameters that reflect the sires' artificial insemination fertility, including secondary sperm morphology, conception rate, non-return rate, and residual value. A multiplex, flow-cytometric test detecting PAWP, aggresomes (ubiquitinated protein aggregates), and acrosomal integrity (peanut-agglutinin-lectin labeling) had a predictive value for conception rate, as demonstrated by step-wise regression analysis. We conclude that PAWP correlates with semen/fertility parameters used in the cattle artificial insemination industry, making PAWP a potential biomarker of bull fertility.

Development of probes capable of recognizing specific regions of chromosomal DNA has been a long-standing goal for chemical biologists. Current strategies such as PNA, triplex-forming oligonucleotides, and polyamides are subject to target choice limitations and/or necessitate non-physiological conditions, leaving a need for alternative approaches. Toward this end, we have recently introduced double-stranded oligonucleotide probes that are energetically activated for DNA recognition through modification with +1 interstrand zippers of intercalator-functionalized nucleotide monomers. Here, probes with different chemistries and architectures – varying in the position, number, and distance between the intercalator zippers – are studied with respect to hybridization energetics and DNA-targeting properties. Experiments with model DNA targets demonstrate that optimized probes enable efficient (C50 < 1 μM), fast (t50 < 3h), kinetically stable (> 24h), and single nucleotide specific recognition of DNA targets at physiologically relevant ionic strengths. Optimized probes were used in non-denaturing fluorescence in situ hybridization experiments for detection of gender-specific mixed-sequence chromosomal DNA target regions. These probes present themselves as a promising strategy for recognition of chromosomal DNA, which will enable development of new tools for applications in molecular biology, genomic engineering and nanotechnology.

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