Bull semen was collected artifically and vitamin A was determined in the intact spermatozoa, the acrosomes and the sperm tails. The results show that over 90% of the total vitamin A in the spermatozoa was present in the acrosomes and that the sperm tails were completely devoid of vitamin A. The concentration of vitamin A varied from 224 to 364 ng/10-9 spermatozoa. (+info)
Impact of epididymal maturation on the tyrosine phosphorylation patterns exhibited by rat spermatozoa.
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As mammalian spermatozoa migrate through the epididymis, they acquire functionality characterized by the potential to express coordinated movement and the competence to undergo capacitation. The mechanisms by which spermatozoa gain the ability to capacitate during epididymal transit are poorly understood. The purpose of this study was to investigate the impact of epididymal maturation on the signal transduction pathways regulating tyrosine phosphorylation, because this process is thought to be central to the attainment of a capacitated state and expression of hyperactivated motility. Western blot and immunocytochemical analyses demonstrated that epididymal maturation in vivo is associated with a progressive loss of phosphotyrosine residues from the sperm head. As cells pass from the caput to the cauda epididymis, tyrosine phosphorylation becomes confined to a narrow band at the posterior margin of the acrosomal vesicle. Epididymal maturation of rat spermatozoa was also associated with an acquired competence to respond to high levels of intracellular cAMP by phosphorylating tyrosine residues on the sperm tail. Immature caput spermatozoa were incapable of exhibiting this response, despite the apparent availability of cAMP and protein kinase A. These findings help to clarify the biochemical changes associated with the functional maturation of spermatozoa during epididymal transit. (+info)
Requirement of phospholipase Cdelta4 for the zona pellucida-induced acrosome reaction.
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Several phospholipase C (PLC) isoforms have been found in male and female mammalian gametes, and splicing isoforms of PLCdelta4 are predominantly expressed in testis. Here we report that male mice in which the PLCdelta4 gene had been disrupted either produced few small litters or were sterile. In vitro fertilization studies showed that insemination with PLCdelta4-/- sperm resulted in significantly fewer eggs becoming activated and that the calcium transients associated with fertilization were absent or delayed. PLCdelta4-/- sperm were unable to initiate the acrosome reaction, an exocytotic event required for fertilization and induced by interaction with the egg coat, the zona pellucida. These data demonstrate that PLCdelta4 functions in the acrosome reaction that is induced by the zona pellucida during mammalian fertilization. (+info)
A highly conserved sequence essential for translational repression of the protamine 1 messenger rna in murine spermatids.
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Translational regulation of the protamine 1 mRNA is mediated by sequences in its 3' untranslated region. In this study, we demonstrate that a highly conserved sequence, the translational control element, is solely responsible for protamine 1 translational regulation. Mutation of the conserved sequence causes premature translation of a transgene containing a fusion between the human growth hormone coding sequence and the protamine 1 3' untranslated region. Temporal expression of the transgene was monitored in prepubertal animals by Northern and Western blotting and in adult animals by immunocytochemistry. Messenger RNAs lacking the translational control element sediment in the messenger ribonucleoprotein particle and ribosomal fractions of polysome gradients, suggesting that the translational control element is required for translational repression but not for incorporation of mRNAs into ribonucleoprotein particles. (+info)
Characterization of a fucose-binding protein from bull sperm and seminal plasma that may be responsible for formation of the oviductal sperm reservoir.
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Oviductal sperm reservoirs have been found in cattle, mice, hamsters, pigs, and horses. In cattle (Bos taurus), the reservoir is evidently formed when sperm bind to fucosylated ligands resembling Le(a) trisaccharide on the surface of oviductal epithelium. The aim of this study was to characterize the fucose-binding protein on bull sperm. Fresh ejaculated sperm were extracted with 0.5 M KCl in Hepes-balanced salts. Extracts were subjected to affinity chromatography using immobilized Le(a) trisaccharide (alpha-L-Fuc[1,4]-beta-D-Gal[1,3]-D-GlcNAc). Two-dimensional PAGE of the affinity chromatography eluates revealed a prominent protein of approximately 16.5 kDa and a pI of 5.8. This protein inhibited binding of sperm to oviductal explants. A similar analysis of proteins extracted from capacitated sperm (which do not bind to oviductal epithelium) showed a reduction in the amount of the 16.5-kDa protein. When examined by epifluorescence microscopy, live uncapacitated sperm labeled over the acrosome with a fucose-BSA-fluorescein isothiocyanate (FITC) conjugate, while capacitated sperm did not. When capacitated sperm were treated with 16.5-kDa protein, labeling with fucose-BSA-FITC was partially restored. The comparative ease with which the protein was removed from sperm and its apparent reassociation with sperm suggested that it could be a peripheral protein derived from epididymal or accessory gland fluids. Blots of SDS-PAGE gels of seminal plasma proteins revealed the presence of a Le(a)-binding protein with an apparent mass of 16.5 kDA: Amino acid sequencing of two tryptic fragments of the protein purified from sperm extracts identified it as PDC-109 (BSP-A1/A2), a product of the seminal vesicles. (+info)
Teratozoospermia in mice lacking the transition protein 2 (Tnp2).
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It is believed that the transition proteins (Tnp1 and Tnp2) participate in the removal of the nucleohistones and in the initial condensation of the spermatid nucleus. Later in spermatogenesis, Tnp1 and Tnp2 are replaced by the protamines 1 and 2. In an effort to elucidate the physiological role of Tnp2, we have disrupted its locus by homologous recombination. Breeding of the Tnp2(-/-) males on different genetic backgrounds revealed normal fertility on the mixed background C57BL/6Jx129/Sv, but total infertility on the inbred 129/Sv background. Light and electron microscopy showed that the germ cells were capable of undergoing chromatin condensation, although many spermatozoa exhibited head abnormalities with acrosomes not attached to the nuclear envelope. Furthermore, migration of Tnp2(-/-) spermatozoa from the uterus into the oviduct was reduced. These results suggest that male infertility of the Tnp2(-/-) mice is a result of sperm head abnormalities and reduced sperm motility. The increased level of the Tnp1 transcript in testes of the Tnp2-deficient mice raises the possibility that a deficiency created through the disruption of the Tnp2 gene can be compensated for by recruitment of the Tnp1. (+info)
Semen parameters, including WHO and strict criteria morphology, in a fertile and subfertile population: an effort towards standardization of in-vivo thresholds.
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In this study, the semen analysis results of a fertile population were compared with those from a subfertile population, in order to establish normal cut-off values for the standard semen parameters with the aid of receiver operating characteristic (ROC) curve analysis. The fertile group comprised healthy males (n = 107) without any history of fertility problems, the partners of whom had had a spontaneous pregnancy within one year of unprotected intercourse and were pregnant at the time of the male's inclusion into the study. A total of 103 males from couples attending the infertility clinic, and with an initial sperm count of <20x10(6)/ml were recruited to form the subfertile population. The best discriminating parameter between the two populations was sperm morphology evaluated according to WHO criteria at a cut-off point of 31% normal spermatozoa. The other cut-off values were at 8% for the acrosome index, 45% for motility, and 4% normal spermatozoa for strict criteria. Recalculating the ROC curve cut-off values based on an assumed 50% prevalence of subfertility in an assisted reproductive setting, the cut-off points were reduced to 21% and 3% normal spermatozoa for WHO and strict criteria respectively. For motility, the new cut-off value was at 20% motile spermatozoa, for motility quality at 3.5 (on a scale of 1-6), the acrosome index at 3% normal acrosomes, and the teratozoospermia index at 2.09. (+info)
Effect of seminal phospholipid-binding proteins and follicular fluid on bovine sperm capacitation.
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Bovine seminal plasma (BSP) contains a family of novel phospholipid-binding proteins (BSP-A1/-A2, BSP-A3, and BSP-30-kDa; collectively called BSP proteins) that potentiate sperm capacitation induced by heparin or by serum high-density lipoprotein (HDL). BSP proteins stimulate lipid efflux from sperm that may occur during the early events of capacitation. Here, we investigated the role of BSP proteins, bovine follicular fluid (FF), and bovine follicular fluid HDL (FF-HDL) in sperm capacitation. FF and FF-HDL alone stimulated epididymal sperm capacitation (19.5% +/- 0.8% and 18.2% +/- 2.8%, respectively, control, 9.0% +/- 1.9%) that was increased by preincubation with BSP-A1/-A2 proteins (30.2% +/- 0.4% and 30.9% +/- 1.5%, respectively). In contrast, lipoprotein-depleted follicular fluid (LD-FF) alone was ineffective, and a preincubation with BSP-A1/-A2 proteins was necessary before sperm capacitation was stimulated (up to 22.8% +/- 1.4%). The interaction of BSP proteins with FF components was analyzed using ultracentrifugation, Lipo-Gel electrophoresis, SDS-PAGE, and gel filtration. We established that the BSP proteins interact with factors present in FF including FF-HDL. Additionally, we obtained evidence that BSP proteins, found associated with FF-HDL, were released from the sperm membrane during capacitation. These results confirm that the BSP proteins and the FF-HDL play a role in sperm capacitation. (+info)