[Show abstract][Hide abstract]ABSTRACT: Objective:
Validate single versus sequential culture media for murine embryo development.
Prospective laboratory experiment.
Assisted Reproduction Laboratory.
Thawed murine zygotes cultured for 3 or 5 days (d3 or d5) in single or sequential embryo culture media developed for human in vitro fertilization.
Main outcome measures:
On d3, zygotes developing to the 8 cell (8C) stage or greater were quantified using 4',6-diamidino-2-phenylindole (DAPI), and quality was assessed by morphological analysis. On d5, the number of embryos reaching the blastocyst stage was counted. DAPI was used to quantify total nuclei and inner cell mass nuclei. Localization of ubiquitin C-terminal hydrolase L1 (UCHL1) and ubiquitin C-terminal hydrolase L3 (UCHL3) was reference points for evaluating cell quality.
Comparing outcomes in single versus to sequential media, the odds of embryos developing to the 8C stage on d3 were 2.34 time greater (P = .06). On d5, more embryos reached the blastocyst stage (P = <.0001), hatched, and had significantly more trophoblast cells (P = .005) contributing to the increased total cell number. Also at d5, localization of distinct cytoplasmic UCHL1 and nuclear UCHL3 was found in high-quality hatching blastocysts. Localization of UCHL1 and UCHL3 was diffuse and inappropriately dispersed throughout the cytoplasm in low-quality nonhatching blastocysts.
Single medium yields greater cell numbers, an increased growth rate, and more hatching of murine embryos. Cytoplasmic UCHL1 and nuclear UHCL3 localization patterns were indicative of embryo quality. Our conclusions are limited to murine embryos but one might speculate that single medium may also be more beneficial for human embryo culture. Human embryo studies are needed.
Full-text · Article · Dec 2015 · Reproductive Sciences
[Show abstract][Hide abstract]ABSTRACT: Aberrant sperm phenotypes coincide with the expression of unique sperm surface determinants that can be probed by objective, biomarker-based semen analysis and targeted as ligands for semen purification. This study evaluated a nanoparticle based magnetic purification method that removes defective spermatozoa (~30% of sample) from bull semen and improves sperm sample viability and fertilizing ability in vitro and in vivo. Two types of nanoparticles were developed: a particle coated with antibody against ubiquitin, which is present on the surface of defective spermatozoa, and a particle coated with the lectin PNA, which binds to glycans exposed by acrosomal damage. In a two year artificial insemination field trial with 798 cows, a conception rate of 64.5 ± 3.7% was achieved with a 10 million sperm dose of PNA-nanopurified spermatozoa, comparable to control non-purified full dose of 20 million spermatozoa per dose (63.3 ± 3.2%), and significantly higher than a 10 million sperm dose of non-purified control semen (53.7 ± 3.2%; P < 0.05). A total of 466 healthy calves were delivered, and no negative side effects were observed in the inseminated animals or offspring. Since the method is inexpensive and can be fully integrated in current protocols for semen cryopreservation, it is feasible for use in the artificial insemination industry to improve fertility with reduced sperm dosage inseminations. Spermatology will benefit from nanopurification methodology by gaining new tools for the identification of candidate biomarkers of sperm quality such as binder of sperm protein 5 (BSP5), described in the present study.
Full-text · Article · Sep 2014 · Biology of Reproduction
[Show abstract][Hide abstract]ABSTRACT: Ubiquitination is a covalent post-translational modification of proteins by the chaperone protein ubiquitin. Upon docking to the 26S proteasome, ubiquitin is released from the substrate protein by deubiquitinating enzymes (DUBs). We hypothesised that specific inhibitors of two closely related oocyte DUBs, namely inhibitors of the ubiquitin C-terminal hydrolases (UCH) UCHL1 (L1 inhibitor) and UCHL3 (L3 inhibitor), would alter porcine oocyte maturation and influence sperm function and embryo development. Aberrant cortical granule (CG) migration and meiotic spindle defects were observed in oocytes matured with the L1 or L3 inhibitor. Embryo development was delayed or blocked in oocytes matured with the general DUB inhibitor PR-619. Aggresomes, the cellular stress-inducible aggregates of ubiquitinated proteins, formed in oocytes matured with L1 inhibitor or PR-619, a likely consequence of impaired protein turnover. Proteomic analysis identified the major vault protein (MVP) as the most prominent protein accumulated in oocytes matured with PR-619, suggesting that the inhibition of deubiquitination altered the turnover of MVP. The mitophagy/autophagy of sperm-contributed mitochondria inside the fertilised oocytes was hindered by DUB inhibitors. It is concluded that DUB inhibitors alter porcine oocyte maturation, fertilisation and preimplantation embryo development. By regulating the turnover of oocyte proteins and mono-ubiquitin regeneration, the DUBs may promote the acquisition of developmental competence during oocyte maturation.
No preview · Article · May 2014 · Reproduction Fertility and Development
[Show abstract][Hide abstract]ABSTRACT: The ubiquitin-proteasome system (UPS) controls intracellular protein turnover in a substrate-specific manner via E3-type ubiquitin ligases. Mammalian fertilization and particularly sperm penetration through the oocyte vitelline coat, the zona pellucida (ZP), is regulated by UPS. We use an extrinsic substrate of the proteasome-dependent ubiquitin-fusion degradation pathway, the mutant ubiquitin UBB(+1), to provide evidence that an E3-type ligase activity exists in sperm-acrosomal fractions. Protein electrophoresis gels from such de novo ubiquitination experiments contained a unique protein band identified by tandem mass spectrometry as being similar to ubiquitin ligase UBR7 (alternative name: C14ORF130). Corresponding mRNA was amplified from boar testis and several variants of the UBR7 protein were detected in boar, mouse and human sperm extracts by Western blotting. Genomic analysis indicated a high degree of evolutionary conservation, remarkably constant purifying selection and conserved testis expression of the UBR7 gene. By immunofluorescence, UBR7 was localized to the spermatid acrosomal cap and sperm acrosome, in addition to hotspots of proteasomal activity in spermatids, such as the cytoplasmic lobe, caudal manchette, nucleus and centrosome. During fertilization, UBR7 remained with the ZP-bound acrosomal shroud following acrosomal exocytosis. Thus, UBR7 is present in the acrosomal cap of round spermatids and within the acrosomal matrix of mature boar spermatozoa. These data provide the first evidence of ubiquitin ligase activity in mammalian spermatozoa and indicate UBR7 involvement in spermiogenesis.
No preview · Article · Mar 2014 · Cell and Tissue Research
[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.
[Show abstract][Hide abstract]ABSTRACT: Supplemental figures and tables. Figure S1. Western blotting of SPTRX3 in semen of three ART patients and three presumed fertile donors (semen parameters are shown in Table S10). Relative levels of SPTRX3 (top panel) and beta-tubulin (loading control; middle panel) are shown for three presumably fertile sperm bank donors with acceptable clinical semen parameters (lanes 1-3) and three teratozoospermic infertile men (lanes 4-6) suffering from dysplasia of the fibrous sheath. Diagram of SPTRX3/tubulin band density ratios is shown on the bottom. Note the high density of SPTRX3 band in donor #1, who was presumed fertile. Figure S2. Smoking has a modest effect on sperm SPTRX3 levels. Heavy smokers consuming 15-30 cigarettes per day had higher average levels of sperm SPTRX3 than other groups shown. Of note, two thirds (10/15) of these heavy smokers had indication/clinical diagnosis of male infertility. The numeric difference between smokers consuming 2-5 cigarettes per day and smokers consuming 15-30 cigarettes per day, was not statistically significant (p>0.1). Figure S3. Scatter diagram illustrating the relationship between subjective, light microscopic assessment of sperm SPTRX3 content (% spermatozoa with SPTRX3-positive heads; x-axis) and flow cytometry (%M3 SPTRX-value; y-axis). This simple light microscopic analysis was conducted as a potential precursor of a test suitable for clinical andrology laboratories. Samples from 150 randomly chosen donors, processed for flow cytometry, were re-evaluated by epifluorescence microscopy for the percentage of SPTRX3 positive sperm heads, sperm tails, and total % of SPTRX3 positive spermatozoa. Correlations were found between all three categories of light microscopic evaluation and % of SPTRX3-positive spermatozoa as measured by flow cytometry (%M3). The highest correlation coefficient (r = 0.46) was between % SPTRX3-positive sperm heads by light microscopy and %M3 SPTRX3 by flow cytometry (see Table S4B). Table S1. Receiver Operator Characteristic (ROC) analysis. As anticipated, Specificity increases at the expense of Sensitivity as the cutoff values increase. The area under the ROC curve is 0.74, which shows that the %M3 performs well for predicting the male infertility. Table S2. Semen SPTRX3 levels and live births. Pregnancy outcomes were available for 57/60 pregnant couples. One of those 57 pregnancies was an ectopic pregnancy in a case of female-only infertility; it is not included in the above table. Live birth was achieved in 81% (46/57) of those analyzed pregnant couples. One of those three male factor patients had a vasectomy. Table S3. Parameters of zygotic development in 239 couples divided into subgroups based on percentages of SPTRX3-positive spermatozoa (A) or clinical indication (B). SPTRX3 values, but not the clinical indication, were predictive of good zygotic development after IVF or ICSI. Couples with lowest SPTRX3 levels (A, top row, <5% M3) produced the highest percentage of normal, two-pronuclear (2 PN) zygotes out of all fertilized oocytes (A, column 8), and also when calculated based on all oocytes harvested (A, column 9). These couples also produced the most embryos suitable for transfer or cryopreservation (column 13). Note that couples with low or medium levels of SPTRX3 produced more 2PN zygotes per couple on average than couples with >15% SPTRX3 (A, column 10). Idiopathic couples in which men recorded more than 15% SPTRX3-positive spermatozoa had the lowest yields of two-pronuclear zygotes. Numbers shown in red are highest & lowest values for each column. No significant correlations were found between parameters of early embryo development and flow cytometric SPTRX3 levels. Table S4. Relationship between the clinical treatment assignment and various sperm quality parameters. Table S5. A: Pregnancy rates in 150 infertile couples, divided by percentages of SPTRX3-positive spermatozoa. In accordance with flow cytometric SPTRX3-data, couples with lowest levels of SPTRX3 had highest pregnancy rate (column 2). Also similar to flow cytometric results, the percentage of couples treated by ICSI increased progressively with sperm SPTRX3 content (column 6). Spermatozoa were evaluated for the presence of SPTRX3 in the sperm head (B) or tail (C; combined head and tail labeling has not been observed), adding up to third category of total SPTRX3-positive spermatozoa (A). Numbers shown in red are highest & lowest values for each column. With the exception of one SPTRX3 parameter (A-% of all positive spermatozoa), pregnancy rates (column #2) in light microscopic analysis were dose-dependent on SPTRX3 values. B: Correlations between light microscopic SPTRX3-evaluation (STIX assay) and % of SPTRX3-positive spermatozoa as measured by flow cytometry. Bold font indicates highest correlation coefficients for flow cytometric and light microscopic parameters. Table S6. Sperm SPTRX3 levels and oocyte quality parameters in 238 couples divided by female partners’ age, with the threshold of 35 years of age. Female age subgroups (in rows) were further divided based on male partners’ semen content of SPTRX3-positive spermatozoa (Column 4), and based on the treatment (IVF or ICSI, columns 6 & 7). Regardless of female partner’s age, the combined IVF & ICSI pregnancy rates (column 9) as well as the ICSI pregnancy rates (column 10) were numerically higher when the male partner had <10% SPTRX3-positive spermatozoa. Table S7. Analysis of subgroups divided by indication (male or female infertility) and female age. Combined and idiopathic cases were not included in analysis. Table S8. Correlations between flow cytometric SPTRX3 values and clinical semen parameters. R-values by Person’s correlation analysis are shown. Most significant values are printed bold. In general, SPTRX3 values correlate negatively with sperm count, motility, and percentage of morphologically normal spermatozoa in samples, assessed by conventional light microscopic semen evaluation. Ratio of spermatozoa to debris (%TOTAL) correlates positively with sperm count, motility and normal morphology. Bold font indicates highest correlation coefficients. Table S9. Spearman correlations between the most informative flow cytometric SPTRX3 parameters and the conventional semen parameters (the second numbers in the cells are the p-values). Table S10. Conventional semen parameters for semen samples used for Western blotting. Table S11. Discriminant analysis using SAS PROC discrim and stepdisc procedures applied to study the relationship of pregnancy rate with the sperm quality parameters and SPTRX3 levels, with consideration of treatment assignments. Table S12. Structure matrix of the discriminant analysis conducted to study the relationship of the treatment assignment, SPTRX3 and the sperm quality parameters. Table S13. Variables and treatment groups in the discriminant analysis conducted to study the relationship of the treatment assignment, SPTRX3 and the sperm quality parameters. Table S14. Canonical structure matrix of the discriminant analysis using SAS proc discrim procedure, conducted to explore the relationships between the clinically diagnosed male infertility and sperm quality parameters. Table S15. Means of the nine variables shown separately for infertile and fertile male in the discriminant analysis (SAS proc discrim) conducted to explore the relationships between the clinically diagnosed male infertility and sperm quality parameters.
[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.
Full-text · Article · Apr 2013 · Proceedings of the National Academy of Sciences
[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.
No preview · Article · May 2012 · Journal of Cellular Physiology
[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.
[Show abstract][Hide abstract]ABSTRACT: (A) Effect of PPi on sperm-zona binding. Porcine oocytes were inseminated (sperm conc. 5×105 spermatozoa/ml) with various concentrations of PPi for 30 min, fixed and stained with DNA stain DAPI. The numbers of spermatozoa bound per zona-pellucida (ZP) were counted under epifluorescence microscope. Values are expressed as the mean number ± SEM. Different superscripts a, b & c in each histogram denote a significant difference at p<0.05, meaning that column a is significantly different from columns b and c, column b is significantly different from columns a and c, column ab is not significantly different from either a or b, and column bc is not significantly different from columns b and c. Numbers of inseminated ova are indicated in parentheses. (B) The percentage of acrosome-reacted spermatozoa of panel A (PNA-FITC stained). Values are expressed as the mean percentages ± SEM. Different superscripts a & b in each group of columns denote a significant difference at p<0.05. (C) Effect of PPi supplementation on the viability of spermatozoa stored in commercial and custom made BTS extenders. Boar spermatozoa were preserved in BTS-IMV (IMV technologies, France) or BTS-HM (homemade) with/without 10 µM PPi for 7 days at room temperature. The percentage of motile spermatozoa was estimated at 38.5°C using a light microscope at 250× magnification. Higher sperm motility was observed in BTS-IMV with PPi on day 6 than in any other group. Experiments were repeated twice. Values are expressed as the mean percentages ± SEM. Different superscripts a & b in each group of columns denote a significant difference at p<0.05. (D) Excessive concentrations of PPi were added into IVF medium. Fertilization rates decreased with high concentrations of PPi. Experiments were repeated twice. Diagram indicates % monospermic (□) and % polyspermic (▪) fertilization. Values are expressed as the mean percentages of total fertilization ± SEM. Numbers of inseminated ova are indicated in parentheses. (E) Effect of PPi on fertilization with spermatozoa stored in commercial extender, BTS-IMV. Nearly 100% fertilization was achieved using spermatozoa preserved in BTS-IMV with 10 µM PPi (day 3). Diagram indicates % monospermic (□) and % polyspermic (▪) fertilization. Experiments were repeated twice. Values are expressed as the mean percentages of total fertilization ± SEM. Numbers of inseminated ova are indicated in parentheses.
[Show abstract][Hide abstract]ABSTRACT: Addition of glucose (100 µM-1 mM) did not contribute to the PPi induced fluorescence of boar seminal plasma (A; 10 µg/ml), porcine oviductal fluid (B; 10 µg/ml), boar spermatozoa (C; 1×106 spermatozoa/ml) and control (D; without spermatozoa). Experiments were repeated three times. The emitted fluorescence (no units) was measured at multiple time points to follow the kinetics of the reaction (excitation 530 nm; emission 590 nm). Values are expressed as the mean of fluorescence intensity ± SEM.
[Show abstract][Hide abstract]ABSTRACT: Negative control for Western blotting (Fig. 4). Membranes with boar, bull and mouse sperm extracts probed with immunosaturated anti-PPA1 antibody and GAR-IgG-HRP. There are no specific bands.
[Show abstract][Hide abstract]ABSTRACT: Sperm mitochondrial membrane potential during sperm storage with/without PPi. Percentages of spermatozoa with polarized (live), depolarizing (dying) and depolarized (dead) mitochondrial membranes. Experiments were repeated three times. Values are expressed as the mean percentages ± SEM.
[Show abstract][Hide abstract]ABSTRACT: Effect of PPi supplementation on the ATP content of boar spermatozoa. Semen was washed twice with PBS at room temperature, collected by centrifugation at 800×g for 5 min and adjusted to concentration of 1×106 spermatozoa/ml with PBS. Sperm ATP content was determined using a luciferase reaction kit with or without cell lysis solution according to the manufacturer's protocol (ATPliteTM, Perkin Elmer Inc., Boston, MA). Standards were prepared from ATP standard (Perkin Elmer) using serial dilutions to obtain concentrations of 1×10−7, 1×10−8, 1×10−9, 1×10−10, and 1×10−11 M. Aliquots of the ATP stock solution were stored at −20°C until use, and standard curve dilutions were prepared for each assay. Bioluminescence was measured with a Synergy 2 multi-mode microplate reader (Biotek, Winooski, VT) after addition of 100 µl sample and 100 µl luciferin-luciferase reagent. The 96-well plate was incubated at 37.5°C for 30 min, and luminescence was measured at multiple time points to follow the kinetics of the reaction. Sperm motility was examined before and after measurement by light microscopy to confirm that the luciferase reagent did not cause sperm damage. Total ATP (A) or sperm-surface ATP (ssATP) (B) content of fresh semen was measured by with/without lysis solution. Total ATP (C) or ssATP (D) contents of spermatozoa preserved for 3–5 days was measured with/without lysis solution. Experiments were repeated three times. Values are expressed as the mean ± SEM. Different superscripts a & b in each histogram denote a significant difference at p<0.05, meaning that column a is significantly different from column b, and column ab is not significantly different from either a or b.
[Show abstract][Hide abstract]ABSTRACT: Immunofluorescence and Western blotting of boar spermatozoa and sperm extracts, respectively, using immunosaturated anti-PPA1 antibody. Anti-PPA1 antibody (1 mg/ml) was co-incubated with 1 mg/ml recombinant PPA1 protein (1∶20 ratio) at 4°C overnight. Porcine oocytes were fertilized in the presence of anti-PPA1 antibody, immunosaturated anti-PPA1 antibody or non-immune rabbit sera, respectively, after then fixed and stained with GAR-TRITC (red) and DAPI (blue). (A) PPA1 localization (red) in the acrosome of boar spermatozoa by immunofluorescence (a); PPA1 fluorescence was absent after labeling with immunosaturated anti-PPA1 antibody (b) or non-immune rabbit sera (c). (B) Western blotting of the boar sperm-acrosome extract. Lanes 1 and 2 were probed with active anti-PPA1 antibody and immunosaturated anti-PPA1 antibody, respectively. (C) Comparison of IVF in the presence of anti-PPA1 antibody, immunosaturated anti-PPA1 antibody, non-immune rabbit sera or no treatment. Diagram indicates % monospermic (□) and % polyspermic (▪) fertilization. Values are expressed as the mean percentages of total fertilization ± SEM. Different superscripts a, b & c in each histogram denote a significant difference at p<0.05, meaning that column a is significantly different from columns b and c, and column b is significantly different from columns a and c. Numbers of inseminated ova are indicated in parentheses.
[Show abstract][Hide abstract]ABSTRACT: Flow cytometric scatter diagrams reflecting the changes in sperm viability and mitochondrial membrane potential, induced by PPi supplementation during semen storage. Boar spermatozoa were stored in BTS in the presence/absence of 10 µM PPi for 3 or 10 days. Sperm viability (SYBR14, live/PI, dead) and mitopotential (JC-1, live/7-AAD, dead) were measured by flow cytometry. (A) Spermatozoa preserved in BTS for 3 days. (B) Spermatozoa preserved in BTS with PPi for 3 days. (C) Spermatozoa preserved in BTS for 10 days. (D) Spermatozoa preserved in BTS with PPi for 10 days. (E) Vehicle solution, DMSO was added instead of fluorescent dyes as a control.
[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.
No preview · Article · Apr 2012 · Journal of Cellular Physiology
[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.
Full-text · Article · Jan 2012 · Domestic animal endocrinology