Butyrolactone I specifically inhibits M-phase promoting factor activation and prevents the resumption of meiosis. These experiments were conducted to examine effects of butyrolactone I on pig oocytes in a serum-free maturation system. The first experiment was conducted to determine the effect of butyrolactone I (0–100 μM) on nuclear maturation. At concentrations of ≥12.5 μM, germinal vesicle breakdown was prevented in >90% of the oocytes after 24 h of culture. In the second experiment, the kinetics of in vitro maturation of butyrolactone I-treated oocytes was investigated. Oocytes were treated with 0 or 12.5 μM butyrolactone I and FSH for 20 h and then cultured with LH in the absence of butyrolactone I for another 24 h. Fewer butyrolactone I-treated oocytes reached MII stage at 36 h compared with controls (5.8% vs. 62.4%, P < 0.01). However, by 44 h, 83.4% of butyrolactone I-treated oocytes reached MII compared with 88.6% of controls. In the third experiment, butyrolactone I-treated oocytes were fertilized and cultured in vitro. No differences (P > 0.05) were found between controls and treated groups in cleavage rate, blastocyst rate, or mean number of cells per blastocyst. Effects of butyrolactone I on mitogen-activated protein kinase activation and localization of microfilaments and active mitochondria were examined by Western blot analysis and laser scanning confocal microscopy, respectively. The results suggested that although butyrolactone I reversibly inhibited germinal vesicle breakdown and mitogen-activated protein kinase activation, it did not affect mitochondrial and microfilament dynamics. Butyrolactone I is a potent inhibitor of nuclear maturation of porcine oocytes, and the inhibition is fully reversible.
Seminal plasma deposited simultaneously with extended semen in the presence of neutrophils led to higher fertility in pigs and horses. The mechanism by which fertility was improved is not known, however, data suggest that seminal plasma conveys DNase activity which inhibits negative effects of neutrophils. Seminal protein extracts reduced sperm-neutrophil binding in vitro in a dose-dependent manner by hydrolyzing DNA extruded from activated neutrophils. DNase activity exists in semen from several mammalian species, and inhibits formation of sperm-neutrophil complexes. Seminal fertility-associated antigen (FAA) is a basic 31 kDa non-glycosylated heparin binding protein homologous to the family of DNase-I-like proteins (bovine FAA shares 88% identity to human DNase1L3). It is secreted by accessory sex glands and binds to sperm at ejaculation. FAA possesses two DNase-I-like signature motifs, and recombinant bovine FAA hydrolyzed DNA in vitro. FAA is a protein marker of fertility in bulls; sperm extracts containing detectable FAA indicated higher bull fertility compared to extracts lacking FAA. The objective of this study was to clone the human FAA homolog, determine whether it displayed biological activity previously described in seminal plasma, and investigate the hypothesis that human FAA modulates fertility. To that end, the human homolog to bovine FAA was cloned and expressed as a recombinant N-terminal his-tagged fusion protein. Gene-specific primers successfully amplified a 411 bp product of the human FAA gene spanning the DNase-I-like motifs. PCR product was cloned into a TOPO expression vector and transformed into BL21(DE3) cells. Expression of recombinant was induced by IPTG (4h, 37C). Optimal protein induction and solubility was determined by extraction in non-denaturing or denaturing buffers. Bacterial cell pellet was lysed and clarified extracts were purified by metal affinity chromatography. Purity was assessed by SDS-PAGE and verified by Western blotting. Blots were probed with anti-HisG-HRP conjugated antibody and with anti-bovine FAA monoclonal antibody to verify identity of the human homolog. Blots probed with anti-bovine FAA were incubated with goat anti-mouse HRP secondary antibody and developed by incubation in HRP substrate. Purified rFAA was renatured by multi-step dialysis with insoluble aggregates removed by centrifugation. DNA activity exhibited by human seminal plasma and purified recombinant human FAA was analyzed by degradation of calf thymus DNA using agarose gel electrophoresis. Seminal plasma from 12 patients displayed variation in amount of DNase activity detected. Marked degradation occurred in one-third, intermediate degradation in another third, with little to no activity in remaining samples. The expressed recombinant shared 90% nucleotide identity with bovine recombinant FAA (390 bp). Affinity-purified recombinant displayed a single band of 19.6 kDa using an anti-HisG and anti-FAA antibody. Human recombinant FAA displayed DNase activity in a dose response manner above 50 µg/ml. These data demonstrate high homology between bovine and human FAA and indicate that FAA is a primary source of intrinsic DNase activity in human semen. The recombinant human homolog to FAA can now be exploited (1) to evaluate if it modulates sperm/neutrophil binding, (2) as an antigen to develop antibodies for cytological studies, and (3) to use those antibodies to quantify FAA in semen in relation to fertility of patients.
Ejaculates from peripubertal bulls (n = 468) in 5 different populations were analyzed individually for the presence of fertility- associated antigen (FAA) using a lateral flow cassette. Bull age, scrotal circumference, ejaculate volume, sperm cells per milliliter of ejaculate, percentage of motile sperm, percentage of normal sperm, and number of sperm in the ejaculate were recorded at each collection. These data were compared in bulls with and without FAA in the ejaculate. The percentage of bulls that were FAA-positive (FAA present in the ejaculate) ranged from 65 to 86% among the 5 populations. The only variable that differed across all bulls at first semen collection was ejaculate volume with FAA-negative (FAA not detected in the ejaculate) bulls having a greater volume (P < 0.001) than FAA-positive bulls. However, among subsets of bulls (n = 134) that were serially collected (3 times at 30-d intervals) at 3 ranches, ejaculate volume was greater in FAA-negative bulls in only 1 of 3 collections at each ranch. Additionally, the fact that a bull was capable of passing a breeding soundness exam did not affect the likelihood that FAA would be detected in the ejaculate. Among bulls that had not reached puberty, 70% were FAA-positive. Ten bulls had no detectable sperm in the ejaculate at their first collection, and 70% of those bulls were FAA-positive. These data suggest that the production of FAA in semen is not affected by any variable typically measured during a breeding soundness exam. The presence of FAA can be determined in an ejaculate either with or without sperm in peripubertal bulls.
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 α-minimum Eagle's essential medium (Exp. 2 and 3) supplemented with 10 μg/mL of transferrin, 100 μg/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.
Heparin-binding proteins (HBP) recognized by a monoclonal antibody (M1) are produced by male accessory sex glands and bind to distinct regions of ejaculated bull sperm. Immunoblots of sperm proteins probed with M1 identified HBP variants of approximately 31-, 24-, and 21.5-kDa that were associated with increased fertility of bulls. The purpose of this study was to identify the 31-kDa HBP known as fertility-associated antigen (FAA). FAA was isolated by heparin-affinity chromatography and reversed-phase high performance liquid chromatography near homogeneity. Biochemical characterization indicated that FAA was an unglycosylated, basic protein. FAA protein was detected in seminal vesicle and prostate gland homogenates, and FAA extracted from sperm membranes by treatment with hypertonic media was identical biochemically to seminal fluid-derived FAA. N-terminal sequence analysis of purified FAA yielded a 26 amino acid sequence (L K I X S F N V R S F G E S K K A G F N A M R V I V) with 73% identity to a recently identified human deoxyribonuclease (DNase) I-like protein. Two internal amino acid sequences generated from lys-C digested FAA were 85% and 92% identical to the same DNase I-like protein. In conclusion, we have identified a bovine seminal heparin-binding protein that binds to sperm and is indicative of bull fertility as being similar to the family of DNase I-like proteins. These data demonstrate the presence of a novel DNase I-like protein in bull accessory sex glands and form the groundwork for the identification of a candidate genetic marker for fertility of bulls.
Heparin-binding proteins (HBP) in bull seminal fluid bind to epididymal sperm membranes at ejaculation. Peptides recognized by a monoclonal antibody (M1) correspond to proteins identified in complexes that have the greatest affinity for heparin and are present on sperm from bulls with higher fertility. Presence of specific HBP on sperm regulates the ability of sperm to bind heparin, and heparin binding to sperm correlates with the fertility potential of a bull. In these studies, the interaction of HBP with sperm from 10 bulls of proven fertility was analyzed by immunofluorescence of M1 to determine the localization of heparin-binding proteins during capacitation, and the fluorescent binding patterns were compared to bull nonreturn rates. Immunofluorescent localization of M1 binding sites revealed the existence of specific membrane domains containing HBP in acrosomal and postacrosomal regions of ejaculated but not in epididymal sperm. Monoclonal antibody recognition of HBP localized on membranes of sperm revealed variable binding patterns of M1 to the acrosomal region in sperm from bulls of known fertility. Regression analysis indicated a negative relationship between sperm displaying exclusively acrosomal fluorescence and bull nonreturn rate. These data indicate that HBP bind to sperm in distinct patterns, one of which differed among bulls of varying fertility, and indicate no apparent relocalization of these sites during cellular changes that occur in preparation for fertilization.
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