Ubiquitin-activating enzyme (UBA1) is required for sperm capacitation, acrosomal exocytosis and sperm-egg coat penetration during porcine fertilization.

Protein ubiquitination is a stable, covalent post-translational modification that alters protein activity and/or targets proteins for proteolysis by the 26S proteasome. The E1-type ubiquitin-activating enzyme (UBA1) is responsible for ubiquitin activation, the initial step of ubiquitin-protein ligation. Proteasomal proteolysis of ubiquitinated spermatozoa and oocyte proteins occurs during mammalian fertilization, particularly at the site of sperm acrosome contact with oocyte zona pellucida. However, it is not clear whether the substrates are solely proteins ubiquitinated during gametogenesis or if de novo ubiquitination also occurs during fertilization supported by ubiquitin-activating and -conjugating enzymes present in the sperm acrosome. Along this line of inquiry, UBA1 was detected in boar sperm-acrosomal extracts by Western blotting (WB). Immunofluorescence revealed accumulation of UBA1 in the nuclei of spermatogonia, spermatocytes and spermatids, and in the acrosomal caps of round and elongating spermatids. Thiol ester assays utilizing biotinylated ubiquitin and isolated sperm acrosomes confirmed the enzymatic activity of the resident UBA1. A specific UBA1 inhibitor, PYR-41, altered the remodelling of the outer acrosomal membrane (OAM) during sperm capacitation, monitored using flow cytometry of fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Although viable and motile, the spermatozoa capacitated in the presence of PYR-41, showed significantly reduced fertilization rates during in vitro fertilization (IVF; p < 0.05). Similarly, the fertilization rate was lowered by the addition of PYR-41 directly into fertilization medium during IVF. In WB, high Mr bands, suggestive of protein ubiquitination, were detected in non-capacitated spermatozoa by antibodies against ubiquitin; WB with anti-phosphotyrosine antibodies and antibodies against acrosomal proteins SPINK2 (acrosin inhibitor) and AQN1 (spermadhesin) revealed that the capacitation-induced modification of those proteins was altered by PYR-41. In summary, it appears that de novo protein ubiquitination involving UBA1 contributes to sperm capacitation and acrosomal function during fertilization.

Expression of mitochondrial transcription factor A (TFAM) during porcine gametogenesis and preimplantation embryo development.

Mitochondrial transcription factor A (TFAM) is responsible for stability, maintenance, and transcriptional control of mitochondrial DNA (mtDNA). We have studied the expression and distribution of TFAM in the gametes and preimplantation embryos of the domestic pig (Sus scrofa). We hypothesized that TFAM is not present in the boar sperm mitochondria to reduce the possibility of paternal mtDNA propagation in the progeny. In contrast, we anticipated that Tfam gene is expressed in a developmental stage-dependent manner in porcine oocytes and embryos. The appropriate TFAM band of 25 kDa was detected by Western blotting in ejaculated boar spermatozoa, as well as in porcine oocytes and zygotes. Boar sperm extracts also displayed several bands >25 kDa suggestive of post-translational modification by ubiquitination, confirmed by affinity purification of ubiquitinated proteins. TFAM immunoreactivity was relegated to the sperm tail principal piece and sperm head in fully differentiated spermatozoa. The content of Tfam mRNA increased considerably from the germinal vesicle to blastocyst stage and also between in vitro fertilized and cultured blastocysts compared to in vivo-derived blastocysts. TFAM protein accumulated in the oocytes during maturation and was reduced by proteolysis after fertilization. This pattern was not mirrored in parthenogenetically activated oocytes and zygotes reconstructed by SCNT, suggesting deviant processing of TFAM protein and transcript after oocyte/embryo manipulation. Thus, TFAM may exert a critical role in porcine gametogenesis and preimplantation embryo development. Altogether, our data on the role of TFAM in mitochondrial function and inheritance have broad implications for cell physiology and evolutionary biology.

High resolution light microscopic evaluation of boar semen quality sperm cytoplasmic droplet retention in relationship with boar fertility parameters.

The purpose of this study was to investigate the relationship between fertility and quantitative measures of boar semen quality, including various patterns of sperm cytoplasmic droplet (CD) retention, as determined by high power differential interference contrast (DIC) microscopy. A total of 116 ejaculates were collected from a nucleus herd of 18 Large White boars over an eight month period. Semen quality parameters were analyzed for each ejaculate by calculating the percentage of normal spermatozoa, spermatozoa possessing a CD in the proximal, distal, or distal midpiece reflex position, total spermatozoa with an attached cytoplasmic droplet, spermatozoa with non-CD related aberrations and total spermatozoa with abnormalities. Of the 116 ejaculates received, 71 ejaculates from 13 boars had corresponding fertility data from single-sire inseminations of multiparous sows. The fertility data included farrowing rate (FR) and total number born (TNB). The monthly FR encompassed one month before and one month after the date of semen collection. Detection of differences for fertility and semen quality parameters was performed by separating the boars into either an above-average or below-average group based on the mean FR (74.01 +/- 1.43%) or TNB (12.34 +/- 0.17) for the study. For FR, the boars in the below-average group had a significantly lower percentage of normal spermatozoa and significantly higher percentage of spermatozoa possessing distal CDs, total attached CDs and total abnormalities compared to the boars in the above-average group. Conversely, for TNB there were no significant differences between the above- and below-average groups for the semen quality parameters. These data suggest that the attached CD may negatively affect FR, but not TNB. The detection of relationships between the boar fertility parameters and the retention of the sperm CD after ejaculation, document the advantage of high power DIC microscopy in conventional semen evaluation.

Arachidonate 15-lipoxygenase and ubiquitin as fertility markers in boars.

Accurate semen analysis is an important issue in the swine industry. We evaluated two candidate fertility marker proteins associated with sperm cytoplasmic droplet (CD), including 15-lipoxygenase (15-LOX) and ubiquitin (UBI) in a controlled single-sire artificial insemination (AI) trial. Ejaculates (n=116) were collected from 18 fertile Large White boars monthly for 8 mo, and analyzed by semi-quantitative, densitometry-based Western blotting and flow cytometry with antibodies against 15-LOX and UBI. Data were correlated with farrowing rates (FR) and total numbers of piglets born (TNB) from 1754 AI services by 13 of 18 boars, and compared with a conventional microscopic semen analysis. In semi-quantitative Western blotting, both 15-LOX and UBI were correlated with seasonal changes in the percentage of normal (r=-0.38, P<0.01; r=-0.27, P<0.05, respectively) and CD-bearing spermatozoa (r=0.35, P<0.01; r=0.27, P<0.05, respectively). In flow cytometry, UBI and 15-LOX levels showed seasonal changes coinciding with seasonal changes of FR and TNB, representing 13 boars, 88 ejaculates and 1,232 AI services. There were correlations between flow cytometric values of UBI and FR (r=0.31; P<0.05), adjusted FR (r=0.30; P<0.05), TNB (r=-0.38; P<0.01) and adjusted TNB (r=-0.37; P<0.01). Flow cytometric measurements of 15-LOX correlated negatively with TNB (r=-0.33; P<0.05) and adjusted TNB (r=-0.34; P<0.05). These data suggested that boar fertility estimation could be achieved within a group of fertile boars by the use of objectively measurable fertility markers. Flow cytometry appeared more informative and more practical than semi-quantitative Western blotting. This technology could be further optimized for the selection of the most fertile sires in an artificial insemination program.

Centrosomal protein centrin is not detectable during early pre-implantation development but reappears during late blastocyst stage in porcine embryos.

Centrin is an evolutionarily conserved 20 kDa, Ca+2-binding, calmodulin-related protein associated with centrioles and basal bodies of phylogenetically diverse eukaryotic cells. Earlier studies have shown that residual centrosomes of non-rodent mammalian spermatozoa retain centrin and, in theory, could contribute this protein for the reconstruction of the zygotic centrosome after fertilization. The present work shows that CEN2 and CEN3 mRNA were detected in germinal vesicle-stage (GV) oocytes, MII oocytes, and pre-implantation embryos from the two-cell through the blastocyst stage, but not in spermatozoa. Boar ejaculated spermatozoa possess centrin as revealed by immunofluorescence microscopy and western blotting. Immature, GV oocytes possess speckles of centrin particles in the perinuclear area, visualized by immunofluorescence microscopy and exhibit a 19 kDa band revealed by western blotting. Mature MII stage oocytes lacked centrin that could be detected by immunofluorescence or western blotting. The sperm centrin was lost in zygotes after in vitro fertilization. It was not detectable in embryos by immunofluorescence microscopy until the late blastocyst stage. Embryonic centrin first appeared as fine speckles in the perinuclear area of some interphase blastocyst cells and as putative centrosomes of the spindle poles of dividing cells. The cells of the hatched blastocysts developed centrin spots comparable with those of the cultured cells. Some blastomeres displayed undefined curved plate-like centrin-labeled structures. Anti-centrin antibody labeled interphase centrosomes of cultured pig embryonic fibroblast cells as distinct spots in the juxtanuclear area. Enucleated pig oocytes reconstructed by electrofusion with pig fibroblasts displayed centrin of the donor cell during the early stages of nuclear decondensation but became undetectable in the late pronuclear or cleavage stages. These observations suggest that porcine zygotes and pre-blastocyst embryonic cells lack centrin and do not retain exogenously incorporated centrin. The early embryonic centrosomes function without centrin. Centrin in the blastocyst stage embryos is likely a result of de novo synthesis at the onset of differentiation of the pluripotent blastomeres.

15-Lipoxygenase is a component of the mammalian sperm cytoplasmic droplet.

Lipoxygenases (LOXs) are a family of enzymes capable of peroxidizing phospholipids. A member of the LOX family of enzymes, 15-LOX, participates in the degradation of mitochondria and other organelles within differentiating red blood cells, the reticulocytes. The present study provides biochemical and immunocytochemical evidence for the presence of 15-LOX in the sperm cytoplasmic droplet (CD). Testicular, epididymal and ejaculated spermatozoa were evaluated for the presence of 15-LOX using an affinity-purified immune serum raised against a synthetic peptide corresponding to the C-terminal sequence of rabbit reticulocyte 15-LOX. Western blotting revealed an appropriate single band of approximately 81 kDa in boar spermatozoa but not in boar seminal plasma. When ejaculated boar spermatozoa were subjected to separation on a 45/90% Percoll gradient, 15-LOX co-migrated with the immotile sperm and cellular debris/CD fractions, but not with the motile sperm fraction containing morphologically normal spermatozoa without CDs. Varied levels of 15-LOX were expressed in ejaculated sperm samples from boars with varied semen quality. By immunofluorescence, prominent 15-LOX immunoreactivity was found within the residual body in the testis and within the CDs from caput, corpus and cauda epididymal and ejaculated spermatozoa. Components of the ubiquitin-dependent proteolytic pathway, which is thought to facilitate both spermiogenesis and reticulocyte organelle degradation, were also detected in the sperm CD. These included ubiquitin, the ubiquitin-conjugating enzyme E2, the ubiquitin C-terminal hydrolase PGP 9.5, and various 20S proteasomal core subunits of the alpha- and beta-type. The 15-LOX and various components of the ubiquitin-proteasome pathway were also detected in sperm CDs of other mammalian species, including the human, mouse, stallion and wild babirusa boar. We conclude that 15-LOX is prominently present in the mammalian sperm CD and thus may contribute to spermiogenesis, CD function or CD removal.

Fate of postacrosomal perinuclear theca recognized by monoclonal antibody MN13 after sperm head microinjection and its role in oocyte activation in mice.

Monoclonal antibody (mAb) MN13 labels mouse sperm head postacrosomal perinuclear theca (PT), which is possibly involved in oocyte activation during fertilization. The antigenic site is expressed after mild sonication followed by treatment with dithiothreitol (DTT) or heat (45 degrees C), and is visible as a thick band in the postacrosomal region. The presence of protease inhibitors in the sonication medium suppresses the exposure of MN13 epitope (MN13p), suggesting the involvement of a proteolytic reaction in this process. Spermatozoa do not express MN13p after the induction of acrosome exocytosis by Ca(2+) ionophore, zona binding, or during zona penetration, a strategy that ensures safe delivery of postacrosomal PT proteins to oocytes after fusion. MN13 labeling was not detectable during fertilization by zona-free in vitro fertilization, suggesting that the antigenic site does not react with proteolytic enzymes during sperm-oocyte fusion and the antibody does not recognize the nascent epitope. Microinjection of sperm heads prepared by sonication and DTT treatment led to the activation of metaphase II oocytes. The oocyte activating function of such sperm heads was significantly diminished after labeling with MN13 prior to intracytoplasmic sperm injection (ICSI), but labeling with antiequatorin antibody MN9 activated oocytes with a frequency similar to that of unlabeled sperm heads. The sperm heads in inactive oocytes formed premature chromosome condensations (PCCs), which were invested by independent metaphase-like spindles. These observations indicate that the postacrosomal PT recognized by mAb MN13 is involved in oocyte activation. MN13p is dissociated from sperm heads during the early stages of decondensation after ICSI. In activated oocytes, MN13-labeled fine granules were redistributed in the midzone spindle region, whereas in inactive oocytes they formed a ring around the polar regions of the metaphase II and PCC spindles.

Exposure of sperm head equatorin after acrosome reaction and its fate after fertilization in mice.

Equatorin is a sperm head equatorial protein, possibly involved in sperm-oocyte fusion (Toshimori et al., Biol Reprod 1998; 59:22-29). In the present work, we have shown that equatorin contained in the posterior acrosome is detectable only after spontaneous or induced acrosome reactions following fixation and permeabilization, but not in intact spermatozoa. The presence of protease inhibitors during sonication or ionophore treatments does not inhibit the exposure of the antigenic epitope. The zona-penetrated spermatozoa lying in the perivitelline space display equatorin, similar to those of the acrosome-reacted ones. After sperm-egg fusion during in vitro fertilization (IVF), the equatorin dissociates from the sperm head equatorial region and remains at the vicinity of the decondensing male pronuclei. During pronuclear apposition stage, it is pushed away from the pronuclei, possibly by the perinuclear microtubules. After first cleavage, equatorin is inherited by one of the proembryonic cells. The residual equatorin disappears after the second cleavage. Microinjected whole spermatozoa or sperm heads into the MII stage oocytes display equatorin similar to those of the perivitelline sperm. After activation, it dissociates from the sperm nuclei in a similar manner as during IVF. The mode of equatorin degeneration during fertilization is similar to those of the sperm tail components or mitochondria, but different from those of the membrane associated proteins.

Contractile apparatus of the normal and abortive cytokinetic cells during mouse male meiosis.

Mouse male meiotic cytokinesis was studied using immunofluorescent probes against various elements of cytokinetic apparatus and electron microscopy. In normal mice, some spermatocytes fail to undergo cytokinesis after meiotic I or II nuclear divisions, forming syncytial secondary spermatocytes and spermatids. Abnormal cytokinetic cells develop sparse and dispersed midzone spindles during the early stage. However, during late stages, single and compact midzone spindles are formed as in normal cells, but localize asymmetrically and attach to the cortex. Myosin and f-actin were observed in the midzone spindle and midbody regions of normally cleaving cells as well as in those cells that failed to develop a cytokinetic furrow, implying that cytokinetic failure is unlikely to be due to defect in myosin or actin assembly. Depolymerization of microtubules by nocodazole resulted in the loss of the midbody-associated f-actin and myosin. These observations suggest that actin-myosin localization in the midbody could be a microtubule-dependent process that may not play a direct role in cytokinetic furrowing. Anti-centrin antibody labels the putative centrioles while anti-(gamma)-tubulin antibody labels the minus-ends of the midzone spindles of late-stage normal and abnormal cytokinetic cells, suggesting that the centrosome and midzone spindle nucleation in abnormal cytokinetic cells is not different from those of normally cleaving cells. Possible use of mouse male meiotic cells as a model system to study cytokinesis has been discussed.

Centrosome reduction during Rhesus spermiogenesis: gamma-tubulin, centrin, and centriole degeneration.

Centrosome reduction during spermiogenesis has been studied using anti-gamma-tubulin and anti-centrin antibodies and electron microscopy in nonhuman primates. Rhesus spermatids possess apparently normal centrosomes comprising a pair of centrioles associated with gamma-tubulin and centrin. However, they do not nucleate detectable microtubules. The spermatids discard gamma-tubulin in the residual bodies during the spermiation stage. Mature sperm do not have any detectable gamma-tubulin. About half of the centrin associated with the distal centriole degenerates during spermiogenesis and the remainder is intimately bound to the centriolar microtubules. The mature sperm possess highly degenerated distal centrioles. The centriolar microtubules degenerate in the rostral region and the ventral side of the sperm. The study indicates that the centrosome is reduced during rhesus spermiogenesis, but not completely as in mice.

Occurrence of Fusarium species and mycotoxins in nepalese maize and wheat and the effect of traditional processing methods on mycotoxin levels.

Maize (Zea mays) and wheat (Triticum aestivum) collected in the foothills of the Nepal Himalaya Mountains were analyzed for Fusarium species and mycotoxins: fumonisins, nivalenol (NIV), and deoxynivalenol (DON). Predominant species were Gibberella fujikuroi mating population A (F. moniliforme) in maize and F. graminearum in maize and wheat; G. fujikuroi mating population D (F. proliferatum), F. acuminatum, F. avenaceum, F. chlamydosporum, F. equiseti, F. oxysporum, F. semitectum, and F. torulosum were also present. Strains of G. fujikuroi mating population A produced fumonisins, and strains of F. graminearum produced NIV or DON. By immunoassay or high-performance liquid chromatography, fumonisins were >1000 ng/g in 22% of 74 maize samples. By immunoassay or fluorometry, NIV and DON were >1000 ng/g in 16% of maize samples but were not detected in wheat. Fumonisins and DON were not eliminated by traditional fermentation for producing maize beer, but Nepalese rural and urban women were able to detoxify contaminated maize by hand-sorting visibly diseased kernels.

Fusarium species from nepalese rice and production of mycotoxins and gibberellic acid by selected species.

Infection of cereal grains with Fusarium species can cause contamination with mycotoxins that affect human and animal health. To determine the potential for mycotoxin contamination, we isolated Fusarium species from samples of rice seeds that were collected in 1997 on farms in the foothills of the Nepal Himalaya. The predominant Fusarium species in surface-disinfested seeds with husks were species of the Gibberella fujikuroi complex, including G. fujikuroi mating population A (anamorph, Fusarium verticillioides), G. fujikuroi mating population C (anamorph, Fusarium fujikuroi), and G. fujikuroi mating population D (anamorph, Fusarium proliferatum). The widespread occurrence of mating population D suggests that its role in the complex symptoms of bakanae disease of rice may be significant. Other common species were Gibberella zeae (anamorph, Fusarium graminearum) and Fusarium semitectum, with Fusarium acuminatum, Fusarium anguioides, Fusarium avenaceum, Fusarium chlamydosporum, Fusarium equiseti, and Fusarium oxysporum occasionally present. Strains of mating population C produced beauvericin, moniliformin, and gibberellic acid, but little or no fumonisin, whereas strains of mating population D produced beauvericin, fumonisin, and, usually, moniliformin, but no gibberellic acid. Some strains of G. zeae produced the 8-ketotrichothecene nivalenol, whereas others produced deoxynivalenol. Despite the occurrence of fumonisin-producing strains of mating population D, and of 8-ketotrichothecene-producing strains of G. zeae, Nepalese rice showed no detectable contamination with these mycotoxins. Effective traditional practices for grain drying and storage may prevent contamination of Nepalese rice with Fusarium mycotoxins.

Highly degenerated distal centrioles in rhesus and human spermatozoa.

In humans and other mammals except rodents, the spermatozoa contribute the proximal centriole during fertilization. The inheritance of the distal centriole is not yet fully clear. In the present work, the distal centrioles of rhesus and human spermatozoa have been studied by transmission electron microscopy. The round and elongating rhesus spermatids possess both proximal and distal centrioles. The distal centriole extends posteriorly as an axoneme while the proximal centriole produces a microtubular adjunct. Ejaculated rhesus and human spermatozoa have intact proximal centrioles, but the distal centrioles have degenerated. The central pair of microtubules of the axoneme extends continuously into the distal centriolar region up to the sperm head. Serial transverse and longitudinal sections of the sperm neck region reveal few scattered microtubule duplexes or triplets in the distal centriolar region. The loss of the centriolar microtubules is more extensive on the ventral side of the neck region, the side where the proximal centriole resides. The distal centriole degenerates caudally from the rostral area. Immunogold electron microscopy with anti-beta-tubulin antibody showed that the distal centriolar regions possess 50% fewer gold particles than the proximal centrioles, indicating a significant loss of centriolar microtubules in the distal centriolar region. The A-tubules of the remaining triplets are filled with a dense material, as observed in the axoneme. Thus, rhesus and human spermatozoa introduce only proximal centrioles intact, whereas the distal centrioles are mostly disorganized in the mature spermatozoa.

Centriole and centrin degeneration during mouse spermiogenesis.

Centrosome reduction during mouse spermiogenesis has been studied by immunofluorescent microscopy using anticentrin antibody (20H5) and TEM. Centrin is detected as two spots in round spermatids, corresponding to a pair of centrioles. In elongating spermatids, centrin spots colocalize with the centrioles in the neck region, while the perinuclear ring from which manchette microtubules arise, does not label with the antibody 20H5. The proximal centriole of the elongating spermatids develops a prominent adjunct, which assembles an aster of microtubules. TEM studies after immunogold labeling revealed that centrin is associated with the distal and the proximal centrioles, but not with the adjunct. Centrin labeling in the neck region diminishes after spermiation stage, although it is not completely lost from all testicular sperm. Mature epididymal sperm do not display centrin labeling. Mouse sperm lose both distal and proximal centrioles at maturity. Loss of centrin staining appears to correlate with the degeneration of centrioles during mouse spermiogenesis.

Centrosome reduction during mouse spermiogenesis.

The sperm does not contribute the centrosome during murine fertilization. To determine the manner in which a functional centrosome is reduced, we have studied centrosome degeneration during spermiogenesis of mice. The round spermatids display normal centrosomes consisting of a pair of centrioles along with gamma-tubulin containing foci. However, they do not seem to organize microtubules. Elongating spermatids display gamma-tubulin spots in the neck region, while microtubules are organized from the perinuclear ring as the manchette. Electron microscopic studies using immunogold labeling revealed that gamma-tubulin is mainly localized in the centriolar adjunct from which an aster of microtubules emanates. Microtubules repolymerized randomly in the cytoplasm after nocodazole treatment and reversal. gamma-Tubulin dissociates from the neck region and is discarded in the residual bodies during spermiation. The distal centriole degenerates during testicular stage of spermiogenesis, while the proximal centriole is lost during epididymal stage. Loss of centrosomal protein and centrioles in mouse sperm further confirm the maternal inheritance of centrosome during murine fertilization.

Centriolar cycle of fused cells.

Ultrastructure of centrioles of fused cells containing heterophasic interphase nuclei, premature chromosome condensation, and a telophase-like nucleus were studied. The study indicated that the heterophasic cellular environments contributed by the different cell partners exert a mutually opposite effect on the structure of centrioles. The G1-cell partner suppresses replication of S-centrioles. Asynchronous replications of G1 and S-stage centrioles were observed in some G1-S, G1-G2 and S-G2 fused cells. In interphase-mitotic fused cells, centrioles of interphase stages underwent mitotic activation when their nuclei were induced to premature chromosome condensation. Daughter centrioles of G1-, S-, and G2-stages were also capable of mitotic activation independently if they were separated from their mother centriole. Inactive centrioles were observed in some cells containing G1-premature chromosome condensation. When mitotic nuclei were induced to telophase-like nucleus formation, their centrioles were also inactivated. Concomitant events of induced nuclear and centriolar changes suggest that they might have been controlled by the heterophasic cytoplasmic factors through similar pathways.

[The characteristics of the course of mitosis in polykaryons formed during cell fusion]. / Osobennosti protekaniia mitoza v polikarionakh, obrazuiushchikhsia pri sliianii kletok.

A new method of cell fusion is proposed utilizing treatment with 15% solution of DMSO in serum before and after PEG treatment. With such treatments in SPEV cell culture a higher rate of cell fusion was obtained than that with other known methods of cell fusion. In the first wave of mitoses (0.5-4 h) mainly asynchronous division of nuclei, premature chromosome condensation and formation of telophase-like nuclei were observed in polykaryons. In the period of the second wave (14-20 h), mitoses were mainly synchronous and completed with cytokinesis. Micronuclei were formed frequently as a result of such mitoses. After the first wave of mitoses the number of polykaryons with pycnotic chromosomes sharply increased, and after the second wave of mitoses the number of polykaryons with pycnotic nuclei increased. The results obtained allow to conclude that heterophasic condition of the fused cells is one of the causes of pathological mitosis of polykaryons and of their death.