[show abstract][hide abstract] ABSTRACT: Among its many functions, the ubiquitin-proteasome system regulates substrate-specific proteolysis during the cell cycle, apoptosis, and fertilization and in pathologies such as Alzheimer's disease, cancer, and liver cirrhosis. Proteasomes are present in human and boar spermatozoa, but little is known about the interactions of proteasomal subunits with other sperm proteins or structures. We have created a transgenic boar with green fluorescent protein (GFP) tagged 20S proteasomal core subunit α-type 1 (PSMA1-GFP), hypothesizing that the PSMA1-GFP fusion protein will be incorporated into functional sperm proteasomes. Using direct epifluorescence imaging and indirect immunofluorescence detection, we have confirmed the presence of PSMA1-GFP in the sperm acrosome. Western blotting revealed a protein band corresponding to the predicted mass of PSMA1-GFP fusion protein (57 kDa) in transgenic spermatozoa. Transgenic boar fertility was confirmed by in vitro fertilization, resulting in transgenic blastocysts, and by mating, resulting in healthy transgenic offspring. Immunoprecipitation and proteomic analysis revealed that PSMA1-GFP copurifies with several acrosomal membrane-associated proteins (e.g., lactadherin/milk fat globule E8 and spermadhesin alanine-tryptophan-asparagine). The interaction of MFGE8 with PSMA1-GFP was confirmed through cross-immunoprecipitation. The identified proteasome-interacting proteins may regulate sperm proteasomal activity during fertilization or may be the substrates of proteasomal proteolysis during fertilization. Proteomic analysis also confirmed the interaction/coimmunoprecipitation of PSMA1-GFP with 13/14 proteasomal core subunits. These results demonstrate that the PSMA1-GFP was incorporated in the assembled sperm proteasomes. This mammal carrying green fluorescent proteasomes will be useful for studies of fertilization and wherever the ubiquitin-proteasome system plays a role in cellular function or pathology.
Proceedings of the National Academy of Sciences 04/2013; · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
PLoS ONE 01/2013; 8(5):e61000. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Inorganic pyrophosphate (PPi) is generated by ATP hydrolysis in the cells and also present in extracellular matrix, cartilage and bodily fluids. Fueling an alternative pathway for energy production in cells, PPi is hydrolyzed by inorganic pyrophosphatase (PPA1) in a highly exergonic reaction that can under certain conditions substitute for ATP-derived energy. Recombinant PPA1 is used for energy-regeneration in the cell-free systems used to study the zymology of ATP-dependent ubiquitin-proteasome system, including the role of sperm-borne proteasomes in mammalian fertilization. Inspired by an observation of reduced in vitro fertilization (IVF) rates in the presence of external, recombinant PPA1, this study reveals, for the first time, the presence of PPi, PPA1 and PPi transporter, progressive ankylosis protein ANKH in mammalian spermatozoa. Addition of PPi during porcine IVF increased fertilization rates significantly and in a dose-dependent manner. Fluorometric assay detected high levels of PPi in porcine seminal plasma, oviductal fluid and spermatozoa. Immunofluorescence detected PPA1 in the postacrosomal sheath (PAS) and connecting piece of boar spermatozoa; ANKH was present in the sperm head PAS and equatorial segment. Both ANKH and PPA1 were also detected in human and mouse spermatozoa, and in porcine spermatids. Higher proteasomal-proteolytic activity, indispensable for fertilization, was measured in spermatozoa preserved with PPi. The identification of an alternative, PPi dependent pathway for ATP production in spermatozoa elevates our understanding of sperm physiology and sets the stage for the improvement of semen extenders, storage media and IVF media for animal biotechnology and human assisted reproductive therapies.
PLoS ONE 01/2012; 7(4):e34524. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Porcine oocyte-cumulus complexes (OCCs) form an expanded cumulus extracellular matrix (ECM) in response to gonadotropins during meiotic maturation. Essential components of ECM are hyaluronan (HA), tumor necrosis factor α-induced protein 6 (TNFAIP6) and heavy chains (HC) of interalpha-trypsin inhibitor. To form expanded cumulus ECM, intermediate complexes (TNFAIP6-HC) must bind to HA to allow HC transfer onto HA. Protein turnover by the ubiquitin-proteasome pathway is poorly characterized in this process. It is known that the specific proteasomal inhibitor MG132 prevents cumulus expansion and formation of ECM. To determine whether inhibition of proteasomal proteolysis with MG132 affects cumulus cell steroidogenesis and expression of the cumulus expansion-related components (hyaluronan synthase type 2, HAS2, TNFAIP6) we cultured porcine OCCs and granulosa cells (GCs) in a medium supplemented with FSH/LH. Methods performed included real-time reverse transcription PCR, immunofluorescence and RIAs. The expression of TNFAIP6 and HAS2 transcripts increased significantly after the stimulation of OCCs and GCs with FSH/LH. In contrast, treatment with MG132 reduced the expression of TNFAIP6 and HAS2. Hyaluronan was detected with biotinylated HA-binding proteins within FSH/LH-stimulated expanded OCCs but not in those treated with MG132. Progesterone production, although increased almost three times after OCCs stimulation with FSH/LH, was significantly suppressed by MG132. The FSH/LH-stimulated a 40-fold increase in progesterone secretion by GCs was inhibited in the presence of MG132. In conclusion, MG132 affects progesterone secretion and expression of cumulus expansion-related components by cumulus and GCs, suggesting the requirement of ubiquitin-proteasome pathway-regulated protein turnover for formation of ECM during cumulus expansion in the preovulatory period in the pig.
[show abstract][hide abstract] ABSTRACT: The GIRK2-containing inward-rectifying K(+) ion channels have been implicated in mammalian spermatogenesis. While the Girk2 null mice are fertile, the male weaver transgenic mice carrying a gain-of-function mutation in the Girk2 gene are infertile. To establish the exact period of spermatogenesis affected by this mutation, we performed StaPut isolation and morphological characterization of the germ cells present in the weaver testis. Germ cells representing all periods of spermatogenesis were identified. However, no spermatozoa were present, suggesting that this mutation only affected the haploid phase of spermatogenesis. Real-time PCR studies performed on StaPut purified germ cells from wild-type mice indicated that the Girk2 transcripts were exclusively expressed in spermatids. Immunofluorescence studies of mouse and boar spermatids/spermatozoa localized the GIRK2 K(+) containing channels to the acrosomal region of the sperm plasma membrane. During porcine in vitro fertilization (IVF), GIRK2-containing channels remained associated with the acrosomal shroud following zona-induced acrosome reaction. Fertilization was blocked by tertiapin-Q (TQ), a specific inhibitor of GIRK channels, and by anti-GIRK2 antibodies. Altogether, studies in two different mammalian species point to a conserved mechanism by which the GIRK2 inward-rectifying K(+) ion channels support sperm function during fertilization.
Systems biology in reproductive medicine 11/2011; 57(6):296-308. · 0.80 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
International Journal of Andrology 09/2011; 35(2):196-210. · 3.57 Impact Factor
[show abstract][hide abstract] ABSTRACT: Ubiquitin C-terminal hydrolases (UCHs) comprise a family of deubiquitinating enzymes that play a role in the removal of multi-ubiquitin chains from proteins that are posttranslationally modified by ubiquitination to be targeted for proteolysis by the 26S proteasome. The UCH-enzymes also generate free monomeric ubiquitin from precursor multi-ubiquitin chains and, in some instances, may rescue ubiquitinated proteins from degradation. This study examined the roles of two oocyte-expressed UCHs, UCHL1, and UCHL3 in murine and rhesus monkey oocyte maturation. The Uchl1 and Uchl3 mRNAs were highly expressed in GV and MII oocytes, and were associated with the oocyte cortex (UCHL1) and meiotic spindle (UCHL3). Microinjection of the UCH-family enzyme inhibitor, ubiquitin-aldehyde (UBAL) to GV oocytes prevented oocyte meiotic progression beyond metaphase I in a majority of treated oocytes and caused spindle and first polar body anomalies. Injection of antibodies against UCHL3 disrupted oocyte maturation and caused meiotic anomalies, including abnormally long meiotic spindles. A selective, cell permeant inhibitor of UCHL3, 4, 5, 6, 7-tetrachloroidan-1, 3-dione also caused meiotic defects and chromosome misalignment. Cortical granule localization in the oocyte cortex was disrupted by UBAL injected after oocyte maturation. We conclude that the activity of oocyte UCHs contributes to oocyte maturation by regulating the oocyte cortex and meiotic spindle.
Journal of Cellular Physiology 07/2011; 227(5):2022-9. · 4.22 Impact Factor
[show abstract][hide abstract] ABSTRACT: The purpose of semen quality evaluation is to predict the fertility potential of the sample in an objective, rapid and inexpensive manner. However, utilization of sperm quality biomarkers such as ubiquitin and lectin Arachis hypogaea agglutinin (PNA) for flow cytometric semen evaluation might eliminate the need for visual assessment by microscopy. Herein, we demonstrate a robust ubiquitin and PNA-based semen evaluation conducted on a simple, easy to operate, dedicated sperm flow cytometer, EasyCyte Plus (IMV Technologies, L'Aigle, France). Semen samples were collected periodically from two dairy bulls, which were subjected to temporary scrotal insults to induce variable semen quality. Samples were labeled with fluorescently-conjugated anti-ubiquitin antibodies (bind exclusively to the surface of defective sperm) and lectin PNA (binds to acrosomal surface in prematurely capacitated and acrosome-damaged sperm). Fluorescent properties of the samples were measured with a conventional flow cytometer (Becton Dickinson FACScan; Becton Dickinson Corp., Franklin Lakes, NJ, USA) and by the EasyCyte (IMV Technologies) instrument. Data from the two flow cytometers were positively correlated for the percentage of PNA-positive sperm with a damaged acrosome (r = 0.47; P < 0.001) and the percentage of ubiquitin-positive, defective sperm (r = 0.68; P < 0.001). Relative intensities of ubiquitin-induced fluorescence in cells with high ubiquitin levels were also positively correlated (r = 0.90). The proportion of sperm with abnormal morphology was positively correlated with ubiquitin-induced fluorescence measured by EasyCyte (IMV Technologies) (r = 0.63; P < 0.001). These observations provided a rationale for the adaptation of a dual ubiquitin-PNA sperm quality assay for flow cytometric semen evaluation.
[show abstract][hide abstract] ABSTRACT: Post-translational protein modification by ubiquitination, a signal for lysosomal or proteasomal proteolysis, can be regulated and reversed by deubiquitinating enzymes (DUBs). This study examined the roles of UCHL1 and UCHL3, two members of ubiquitin C-terminal hydrolase (UCH) family of DUBs, in murine fertilization and preimplantation development. Before fertilization, these proteins were associated with the oocyte cortex (UCHL1) and meiotic spindle (UCHL3). Intracytoplasmic injection of the general UCH-family inhibitor ubiquitin-aldehyde (UBAL) or antibodies against UCHL3 into mature metaphase II oocytes blocked fertilization by reducing sperm penetration of the zona pellucida and incorporation into the ooplasm, suggesting a role for cortical UCHL1 in sperm incorporation. Both UBAL and antibodies against UCHL1 injected at the onset of oocyte maturation (germinal vesicle stage) reduced the fertilizing ability of oocytes. The subfertile Uchl1(gad-/-) mutant mice showed an intriguing pattern of switched UCH localization, with UCHL3 replacing UCHL1 in the oocyte cortex. While fertilization defects were not observed, the embryos from homozygous Uchl1(gad-/-) mutant females failed to undergo morula compaction and did not form blastocysts in vivo, indicating a maternal effect related to UCHL1 deficiency. We conclude that the activity of oocyte UCHs contributes to fertilization and embryogenesis by regulating the physiology of the oocyte and blastomere cortex.
Journal of Cellular Physiology 06/2011; 227(4):1592-603. · 4.22 Impact Factor
[show abstract][hide abstract] ABSTRACT: Proteasomes are large protein complexes involved in protein degradation in eukaryotes and undergo dynamic redistribution between cellular compartments. Characterising the cellular localization of proteasomes at various stages of development and in response to stimuli is of interest. We hypothesised that porcine proteasomes could be visualised in vivo via a ubiquitously expressed transgene fusion comprising a proteasomal subunit and green florescent protein (GFP). The full-length sequence for porcine PSMA-1 was first constructed in silico from public data and was used to retrieve a GenBank expressed sequence tag (EST) sequence that appeared to be full length (accession CO946059; kind gift from R. S. Prather). Primers were designed to remove the stop codon and create homology for cloning with InFusion (Clontech, Palo Alto, CA, USA). The amplimer was inserted into pCAG-CreGFP (Addgene plasmid 13776) in place of the Cre coding region. The resulting plasmid (pKW14) was screened via restriction digest and sequenced for confirmation. This plasmid was confirmed functional in porcine fetal fibroblasts. After removal of the plasmid backbones, pKW14, a G418 resistance cassette (NEO), and the chicken egg white matrix attachment region were co-electroporated into male fetal fibroblasts (10μg of total DNA, 5:2:2 ratio, respectively). Cells were grown in DMEM with 10% fetal bovine serum (FBS) and selection was initiated 36h after transfection. Following 12 days of selection at 400mgL(-1) G418, colonies were screened by epifluorescence. Positive colonies were harvested and confirmed transgenic for all 3 input DNAs. Positive colonies were randomly pooled as sets of 3 independent integration events. Embryos were reconstructed via SCNT and transferred to 2 recipients. The fusion rates were 70 and 78%, respectively, with transfer numbers of 120 and 125 fused couplets being transferred into synchronized recipients on Day 0 of heat. Both recipients became pregnant and delivered 2 piglets each on Day 114 by Caesarean section. One live piglet was produced from each litter. Of the 2 live-born piglets, 1 survived beyond Day 3 and continues to be healthy. Transgenic status was verified by PCR. Expression was confirmed by epifluorescence of GFP-labelled proteasomes. This founder will be used to establish a model to evaluated cellular localization of proteasomes in vivo and in culture.
Reproduction Fertility and Development 01/2011; 23(1):263. · 2.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
PLoS ONE 01/2011; 6(2):e17256. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Proteolysis of ubiquitinated sperm and oocyte proteins by the 26S proteasome is necessary for the success of mammalian fertilization, including but not limited to acrosomal exocytosis and sperm-zona pellucida (ZP) penetration. The present study examined the role of PSMD4, an essential non-ATPase subunit of the proteasomal 19S regulatory complex responsible for proteasome-substrate recognition, in sperm-ZP penetration during porcine fertilization in vitro (IVF). Porcine sperm-ZP penetration, but not sperm-ZP binding, was blocked in the presence of a monoclonal anti-PSMD4 antibody during IVF. Inclusion in the fertilization medium of mutant ubiquitins (Ub+1 and Ub5+1), which are refractory to processing by the 19S regulatory complex and associated with Alzheimer's disease, also inhibited fertilization. This observation suggested that subunit PSMD4 is exposed on the sperm acrosomal surface, a notion that was further supported by the binding of non-cell permeant, biotinylated proteasomal inhibitor ZL3VS to the sperm acrosome. Immunofluorescence localized PSMD4 in the sperm acrosome. Immunoprecipitation and proteomic analysis revealed that PSMD4 co-precipitated with porcine sperm-associated acrosin inhibitor (AI). Ubiquitinated species of AI were isolated from boar sperm extracts by affinity purification of ubiquitinated proteins using the recombinant UBA domain of p62 protein. Some proteasomes appeared to be anchored to the sperm head inner acrosomal membrane, as documented by co-fractionation studies. In conclusion, the 19S regulatory complex subunit PSMD4 is involved in the sperm-ZP penetration during fertilization. The recognition of substrates on the ZP by the 19S proteasomal regulatory complex is essential for the success of porcine/mammalian fertilization in vitro.
Cell and Tissue Research 08/2010; 341(2):325-40. · 3.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human and rat endometriotic lesions synthesize and secrete tissue inhibitor of metalloproteinase 1 (TIMP1). More TIMP1 localizes in the ovarian theca in an established rat model for endometriosis (Endo) when compared to surgical controls (Sham). We hypothesized that endometriotic TIMP1 secreted into peritoneal fluid (PF) negatively affects ovarian function and embryogenesis by altering the balance of matrix metalloproteinases (MMPs) and TIMPs. Three experiments were performed modulating TIMP1 in vitro and in vivo to investigate ovarian and embryonic anomalies. The first experiment demonstrated control embryos treated in vitro with endometriotic PF concentrations of TIMP1 developed abnormally. In the second experiment where TIMP1 was modulated in vivo, TIMP1-treated Sham rats had fewer zygotes, ovarian follicles, and corpora lutea (CLs) and poorer embryo quality and development, which is analogous to the findings in Endo rats. Importantly, Endo rats treated with a TIMP1 function-blocking antibody had zygote, follicle, and CL numbers and embryo quality similar to Sham rats. In addition, more TIMP1 inhibitory activity was found in ovaries from Endo and TIMP1-treated Sham rats than in ovaries from Sham or TIMP1 antibody-treated Endo rats. In experiment three, control rats (no surgery) treated with Endo PF had fewer follicles and CLs and increased TIMP1 localization in the ovarian theca whereas treatment with Endo PF stripped of TIMP1 or with Sham PF had no effect, providing further evidence that endometriotic TIMP1 sequesters in the ovary and inhibits MMPs necessary for ovulation. Collectively, these results showed that excessive TIMP1 was deleterious to ovulation and embryo development. Thus, novel TIMP1-modulating therapies may be developed to alleviate infertility in women with endometriosis.
Biology of Reproduction 08/2010; 83(2):185-94. · 4.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mammalian spermatozoa complete their morphogenesis and acquire their fertilizing potential in the epididymis. Prominent among the hallmarks of epididymal sperm maturation is the proximal-distal migration of the cytoplasmic droplet (CD), the last remnant of the spermatogenic cell cytoplasm, down the sperm flagellum. Failure to shed the CD has been associated with male infertility. Because of the presence of the organelle degradation enzyme 15-lipoxygenase (15LOX) in sperm CD, we hypothesize that subfertile male Alox15 mice lacking the 15Lox gene display sperm CD anomalies. Caput and cauda epididymal sperm samples from seven adult Alox15 and seven wild-type (wt) males of equal age were examined by differential interference contrast microscopy (DIC) and transmission electron microscopy (TEM). Compared with wt males, Alox15 males had significantly more spermatozoa with a retained CD in both caput (P = 0.004) and cauda (P = 0.005) epididymidis. TEM and DIC analyses revealed intact mitochondria present in the CDs of epididymal Alox15 spermatozoa. The CDs of wt spermatozoa, however, had a smooth appearance and contained only hollow membrane vesicles, with no intact mitochondria embedded in their CD matrix. Epithelial lesions, phagocytosis-like figures, and missing or aberrant apical blebs were observed in the caput epididymidis of Alox15 males. Thus, the process of epididymal sperm maturation and CD migration is altered in Alox15 males. Aberrant sperm maturation might contribute to the reduced fertility and smaller litter size of Alox15 mice, a rare example of subfertile mutants displaying normal spermatogenesis but altered epididymal sperm maturation.
Cell and Tissue Research 06/2010; 340(3):569-81. · 3.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: Identification of the biomarkers of oocyte quality, and developmental and reprogramming potential is of importance to assisted reproductive technology in humans and animals.
PerkinElmer ExacTag™ Kit was used to label differentially proteins in pig oocyte extracts (oocyte proteome) and pig oocyte-conditioned in vitro maturation media (oocyte secretome) obtained with high- and low-quality oocytes.
We identified 16 major proteins in the oocyte proteome that were expressed differentially in high- versus low-quality oocytes. More abundant proteins in the high-quality oocyte proteome included kelch-like ECH-associated protein 1 (an adaptor for ubiquitin-ligase CUL3), nuclear export factor CRM1 and ataxia-telangiectasia mutated protein kinase. Dystrophin (DMD) was more abundant in low-quality oocytes. In the secretome, we identified 110 proteins, including DMD and cystic fibrosis transmembrane conductance regulator, two proteins implicated in muscular dystrophy and cystic fibrosis, respectively. Monoubiquitin was identified in the low-quality-oocyte secretome.
A direct, quantitative proteomic analysis of small oocyte protein samples can identify potential markers of oocyte quality without the need for a large amount of total protein. This approach will be applied to discovery of non-invasive biomarkers of oocyte quality in assisted human reproduction and in large animal embryo transfer programs.
[show abstract][hide abstract] ABSTRACT: Spermatid specific thioredoxin-3 protein (SPTRX-3) accumulates in the superfluous cytoplasm of defective human spermatozoa. Novel ImageStream technology combining flow cytometry with cell imaging was used for parallel quantification and visualization of SPTRX-3 protein in defective spermatozoa of five men from infertile couples. The majority of the SPTRX-3 containing cells were overwhelmingly spermatozoa with a variety of morphological defects, detectable in the ImageStream recorded images. Quantitative parameters of relative SPTRX-3 induced fluorescence measured by ImageStream correlated closely with conventional flow cytometric measurements of the same sample set and reflected the results of clinical semen evaluation. Image Stream quantification of SPTRX-3 combines and surpasses the informative value of both conventional flow cytometry and light microscopic semen evaluation. The observed patterns of the retention of SPTRX-3 in the sperm samples from infertility patients support the view that SPTRX3 is a biomarker of male infertility.
Systems biology in reproductive medicine 12/2009; 55(5-6):244-51. · 0.80 Impact Factor
[show abstract][hide abstract] ABSTRACT: The 26S proteoasome is a multi-subunit protease specific to ubiquitinated substrate proteins. It is composed of a 20S proteasomal core with substrate degradation activity, and a 19S regulatory complex that acts in substrate recognition, deubiquitination, priming and transport to the 20S core. Inhibition of proteolytic activities associated with the sperm acrosome-borne 20S core prevents fertilization in mammals, ascidians and echinoderms. Less is known about the function of the proteasomal 19S complex during fertilization. The present study examined the role of PSMD8, an essential non-ATPase subunit of the 19S complex, in sperm-ZP penetration during porcine fertilization in vitro (IVF). Immunofluorescence localized PSMD8 to the outer acrosomal membrane, acrosomal matrix and the inner acrosomal membrane. Colloidal gold transmission electron microscopy detected PSMD8 on the surface of vesicles in the acrosomal shroud, formed as a result of zona pellucida-induced acrosomal exocytosis. Contrary to the inhibition of fertilization by blocking of the 20S core activities, fertilization and polyspermy rates were increased by adding anti-PSMD8 antibody to fertilization medium. This observation is consistent with a possible role of PSMD8 in substrate deubiquitination, a process which when blocked, may actually accelerate substrate proteolysis by the 26S proteasome. Subunit PSMD8 co-immunoprecipitated with acrosomal surface-associated spermadhesin AQN1. This association indicates that the sperm acrosome-borne proteasomes become exposed onto the sperm surface following the acrosomal exocytosis. Since immunological blocking of subunit PSMD8 increases the rate of polyspermy during porcine fertilization, the activity of the 19S complex may be a rate-limiting factor contributing to anti-polyspermy defense during porcine fertilization.
Journal of Reproductive Immunology 12/2009; 84(2):154-63. · 2.34 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Biology of Reproduction 03/2009; 80(6):1168-77. · 4.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: The following experiments compared the efficiency of three fusion/activation protocols following somatic cell nuclear transfer (SCNT) with porcine somatic cells transfected with enhanced green fluorescent protein driven by the chicken beta-actin/rabbit beta-globin hybrid promoter (pCAGG-EGFP). The three protocols included electrical fusion/activation (NT1), electrical fusion/activation followed by treatment with a reversible proteasomal inhibitor MG132 (NT2) and electrical fusion in low Ca(2+) followed by chemical activation with thimerosal/dithiothreitol (NT3). Data were collected at Days 6, 12, 14, 30, and 114 of gestation. Fusion rates, blastocyst-stage mean cell numbers, recovery rates, and pregnancy rates were calculated and compared between protocols. Fusion rates were significantly higher for NT1 and NT2 compared to NT3 (P < 0.05). There was no significant difference in mean nuclear number. Pregnancy rate for NT2 was 100% (n = 19) at all stages collected and was significantly higher than NT1 (71.4%, n = 28; P < 0.05), but was not significantly higher than NT3 (82.6%, n = 23; P < 0.15). Recovery rates were calculated based on the number of embryos, conceptuses, fetuses, or piglets present at the time of collection, divided by the number of embryos transferred to the recipient gilts. Recovery rates between the three groups were not significantly different at any of the stages collected (P > 0.05). All fusion/activation treatments produced live, pCAGG-EGFP positive piglets from SCNT. Treatment with MG132 after fusion/activation of reconstructed porcine embryos was the most effective method when comparing the overall pregnancy rates. The beneficial effect of NT2 protocol may be due to the stimulation of proteasomes that infiltrate donor cell nucleus shortly after nuclear transfer.
Molecular Reproduction and Development 12/2008; 76(5):490-500. · 2.81 Impact Factor
[show abstract][hide abstract] ABSTRACT: The cause of reduced fecundity in women with endometriosis is unknown. Expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) by both ectopic and eutopic endometrium reportedly has a role in the pathogenesis of endometriosis. We hypothesize that anomalous endometriotic TIMP protein synthesis, secretion, and localization also cause reproductive pathologies resulting in reduced fecundity. An established rat model for endometriosis (Endo) compared with nonendometriotic controls (Shams) was used to investigate reduced fecundity in endometriosis. Comparing Endo and Sham rats, Endo rats had altered ovarian dynamics, including fewer ovarian follicles and corpora lutea with luteinized unruptured follicles. Furthermore, in vivo anomalies in postovulatory oocyte structure and preimplantation embryo development, including misaligned chromosomes, nuclear and cytoplasmic fragmentation, and delayed or arrested cleavage, as well as spontaneous abortions, were found only in Endo rats. A causative role for TIMP1 in these phenomena is supported by our findings that Endo rats have more TIMP1 in their peritoneal fluid as detected by ELISA and more TIMP1 immunolocalization in the theca of antral follicles as measured by computer-assisted morphometric analysis. These data suggest that in endometriosis the accumulation of TIMP1 disrupts the normal MMP/TIMP enzymatic milieu in the peritoneal cavity and negatively affects ovarian dynamics, oocyte quality, and preimplantation embryo development, thereby decreasing fecundity. Most intriguingly, daughters of Endo rats that had no experimental interventions exhibited these same reproductive abnormalities. We predict that developmental exposure to endometriosis leads to permanent epigenetic changes in subsequent generations.
Biology of Reproduction 12/2008; 80(4):649-56. · 4.03 Impact Factor