Proteasomal Interference Prevents Zona Pellucida Penetration and Fertilization in Mammals

doi: 10.1095/biolreprod.104.032532The ubiquitin-proteasome pathway has been implicated in the penetration of ascidian vitelline envelope by the fertilizing spermatozoon (Sawada et al., Proc Natl Acad Sci U S A 2002; 99:1223-1228). The present study provides experimental evidence demonstrating proteasome involvement in the penetration of mammalian zona pellucida (ZP). Using porcine in vitro fertilization as a model, penetration of ZP was completely inhibited by specific proteasomal inhibitors MG-132 and lactacystin. Three commercial rabbit sera recognizing 20S proteasomal core subunits b-1i, b-2i, a-6, and b-5 completely blocked fertilization at a very low concentration (i.e., diluted 1/2000 to 1/8000 in fertilization medium). Neither proteasome inhibitors nor antibodies had any effects on sperm-ZP binding and acrosome exocytosis in zona-enclosed oocytes or on fertilization rates in zona-free oocytes, which were highly polyspermic. Consistent with a possible role of ubiquitin-proteasome pathway in ZP penetration, ubiquitin and various a and b type proteasomal subunits were detected in boar sperm acrosome by specific antibodies, immunoprecipitated and microsequenced by MALDITOF from boar sperm extracts. Antiubiquitin-immunoreactive substrates were detected on the outer face of ZP by epifluorescence microscopy. This study therefore provides strong evidence implicating the ubiquitin-proteasome pathway in mammalian fertilization and zona penetration. This finding opens a new line of acrosome/ZP research because further studies of the sperm acrosomal proteasome can provide new tools for the management of polyspermia during in vitro fertilization and identify new targets for contraceptive development

Semen levels of spermatid-specific thioredoxin-3 correlate with pregnancy rates in ART couples

Spermatid specific thioredoxin-3 (SPTRX3 or TXNDC8) is a testis/male germ line specific member of thioredoxin family that accumulates in the superfluous cytoplasm of defective human spermatozoa. We hypothesized that semen levels of SPTRX3 are reflective of treatment outcome in assisted reproductive therapy (ART) couples treated by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Relationship between SPTRX3 and treatment outcome was investigated in 239 couples undergoing ART at an infertility clinic. Sperm content of SPTRX3 was evaluated by flow cytometry and epifluorescence microscopy, and correlated with clinical semen analysis parameters, and data on embryo development and pregnancy establishment. High SPTRX3 levels (>15% SPTRX3-positive spermatozoa) were found in 51% of male infertility patients (n¿=¿72), in 20% of men from couples with unexplained, idiopathic infertility (n¿=¿61) and in 14% of men from couples previously diagnosed with female-only infertility (n¿=¿85). Couples with high SPTRX3 produced fewer two-pronuclear zygotes and had a reduced pregnancy rate (19.2% pregnant with >15% SPTRX3-positive spermatozoa vs. 41.2% pregnant with 15% of SPTRX3-positive spermatozoa, a cutoff value established by ROC analysis, had their chance of fathering children by IVF or ICSI reduced by nearly two-thirds. The percentage of SPTRX3-positive spermatozoa had predictive value for pregnancy after ART. Gradient purification and sperm swim-up failed to remove all SPTRX3-positive spermatozoa from semen prepared for ART. In summary, the elevated semen content of SPTRX3 in men from ART couples coincided with reduced incidence of pregnancy by IVF or ICSI, identifying SPTRX3 as a candidate biomarker reflective of ART outcomeThis study was funded by grant number 1R21HD066333-01A1 from the National Institutes of Health, NICHD, grant number CB000414 from the Research Board of the University of Missouri, seed funding from the Food for The 21st Century Program of the University of Missouri and Undergraduate Summer Research Internship, College of Agriculture, Food and Natural Resources (CAFNR), University of Missouri.Peer Reviewe

Ubiquitin C-terminal hydrolase-activity is involved in sperm acrosomal function and anti-polyspermy defense during porcine fertilization

The 26S proteasome, which is a multi-subunit protease with specificity for substrate proteins that are postranslationally modified by ubiquitination, has been implicated in acrosomal function and sperm-zona pellucida (ZP) penetration during mammalian fertilization. Ubiquitin C-terminal hydrolases (UCHs) are responsible for the removal of polyubiquitin chains during substrate priming for proteasomal proteolysis. The inhibition of deubiquitination increases the rate of proteasomal proteolysis. Consequently, we have hypothesized that inhibition of sperm acrosome-borne UCHs increases the rate of sperm-ZP penetration and polyspermy during porcine in vitro fertilization (IVF). Ubiquitin aldehyde (UA), which is a specific nonpermeating UCH inhibitor, significantly (P < 0.05) increased polyspermy during porcine IVF and reduced (P < 0.05) UCH enzymatic activity measured in motile boar spermatozoa using a specific fluorometric UCH substrate, ubiquitin-AMC. Antibodies against two closely related UCHs, UCHL1 and UCHL3, detected these UCHs in the oocyte cortex and on the sperm acrosome, respectively, and increased the rate of polyspermy during IVF, consistent with the UA-induced polyspermy surge. In the oocyte, UCHL3 was primarily associated with the meiotic spindle. Sperm-borne UCHL3 was localized to the acrosomal surface and coimmunoprecipitated with a peripheral acrosomal membrane protein, spermadhesin AQN1. Recombinant UCHs, UCHL3, and isopeptidase T reduced polyspermy when added to the fertilization medium. UCHL1 was detected in the oocyte cortex but not on the sperm surface, and was partially degraded 6-8 h after fertilization. Enucleated oocyte-somatic cell electrofusion caused polarized redistribution of cortical UCHL1. We conclude that sperm-acrosomal UCHs are involved in sperm-ZP interactions and antipolyspermy defense. Modulation of UCH activity could facilitate the management of polyspermy during IVF and provide insights into male infertility

Sperm Proteasomes Degrade Sperm Receptor on the Egg Zona Pellucida during Mammalian Fertilization

Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP) remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP) solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and significantly reduced the fertilization/polyspermy rates after IVF, accompanied by en-mass detachment of zona bound sperm. Thus, the sperm borne 26S proteasome is a candidate zona lysin in mammals. This new paradigm has implications for contraception and assisted reproductive technologies in humans, as well as animals

A Journal of Newar Studies - Number 4, NS 1121 / 2000-2001

Number 4, NS 1121 / 2000-200

A Journal of Newar Studies - Number 2, NS 1119 / 1998-1999

Number 2, NS 1119 / 1998-199

A Journal of Newar Studies - Number 3, NS 1120 / 1999-2000

Number 3, NS 1120 / 1999-200

Peroxiredoxin 2 and Peroxidase Enzymatic Activity of Mammalian Spermatozoa

10 páginas, 6 figuras, 2 tablas.Peroxiredoxin 2 (PRDX2) is a highly efficient redox protein that neutralizes hydrogen peroxide, resulting in protection of cells from oxidative damage and in regulation of peroxide-mediated signal transduction events. The oxidized form of PRDX2 is reverted back to the reduced form by the thioredoxin system. In the present study, we investigated the presence of PRDX2 in mouse and boar spermatozoa and in mouse spermatids using proteomic techniques and immunocytochemistry. Sperm and spermatid extracts displayed a 20-kDa PRDX2 band on Western blotting. PRDX2 occurred as a Triton-soluble form in spermatids and as a Triton-insoluble form in mature spermatozoa. Boar seminiferous tubule extracts were immunoprecipitated with PRDX2 antibody and separated by SDS-PAGE. Peptide mass fingerprinting by matrix-assisted laser desorption ionization-time of flight (TOF) and microsequencing by nanospray quadrupole-quadrupole TOF tandem mass spectrometry revealed the presence of PRDX2 ions in the immunoprecipitated band, along with sperm mitochondria-associated cysteine-rich protein, cellular nucleic acid-binding protein, and glutathione peroxidase 4. In mouse spermatocytes and spermatids, diffuse labeling of PRDX2 was observed in the cytoplasm and residual bodies. After spermiation, PRDX2 localization became confined to the mitochondrial sheath of the sperm tail midpiece. Boar spermatozoa displayed similar PRDX2 localization as in mouse spermatozoa. Boar spermatozoa with disrupted acrosomes expressed PRDX2 in the postacrosomal sheath region. Peroxidase enzyme activity of boar sperm extracts was evaluated by estimating the rate of NADPH oxidation in the presence or absence of a glutathione depletor (diethyl maleate) or a glutathione reductase inhibitor (carmustine). Diethyl maleate partially inhibited peroxidase activity, whereas carmustine showed an insignificant effect. These observations suggest that glutathione and glutathione reductase activity contribute only partially to the total peroxidase activity of the sperm extract. While the specific role of PRDX2 in the total peroxidase activity of sperm extract is still an open question, the present study for the first time (to our knowledge) shows the presence of PRDX2 in mammalian spermatozoa. Peroxidase activity in sperm extracts is not due to the glutathione system and therefore possibly involves PRDX2 and other peroxiredoxins.Supported in part by the Food for the 21st Century Program of the University of Missouri and by National Research Initiative Competitive Grant No. 2007-01319 from the USDA CSREES to P.S.Peer reviewe

Semen levels of spermatid-specific thioredoxin-3 correlate with pregnancy rates in ART couples.

Spermatid specific thioredoxin-3 (SPTRX3 or TXNDC8) is a testis/male germ line specific member of thioredoxin family that accumulates in the superfluous cytoplasm of defective human spermatozoa. We hypothesized that semen levels of SPTRX3 are reflective of treatment outcome in assisted reproductive therapy (ART) couples treated by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Relationship between SPTRX3 and treatment outcome was investigated in 239 couples undergoing ART at an infertility clinic. Sperm content of SPTRX3 was evaluated by flow cytometry and epifluorescence microscopy, and correlated with clinical semen analysis parameters, and data on embryo development and pregnancy establishment. High SPTRX3 levels (>15% SPTRX3-positive spermatozoa) were found in 51% of male infertility patients (n = 72), in 20% of men from couples with unexplained, idiopathic infertility (n = 61) and in 14% of men from couples previously diagnosed with female-only infertility (n = 85). Couples with high SPTRX3 produced fewer two-pronuclear zygotes and had a reduced pregnancy rate (19.2% pregnant with >15% SPTRX3-positive spermatozoa vs. 41.2% pregnant with <5% SPTRX3-positive sperm; one-sided p<0.05). The average pregnancy rate of all 239 couples was 25.1%. Live birth rate was 19.2% and lowest average SPTRX3 levels were found in couples that delivered twins. Men with >15% of SPTRX3-positive spermatozoa, a cutoff value established by ROC analysis, had their chance of fathering children by IVF or ICSI reduced by nearly two-thirds. The percentage of SPTRX3-positive spermatozoa had predictive value for pregnancy after ART. Gradient purification and sperm swim-up failed to remove all SPTRX3-positive spermatozoa from semen prepared for ART. In summary, the elevated semen content of SPTRX3 in men from ART couples coincided with reduced incidence of pregnancy by IVF or ICSI, identifying SPTRX3 as a candidate biomarker reflective of ART outcome