Biomarker-based human and animal sperm phenotyping: the good, the bad and the ugly.

Conventional, brightfield-microscopic semen analysis provides important baseline information about sperm quality of an individual; however, it falls short of identifying subtle subcellular and molecular defects in cohorts of "bad", defective human and animal spermatozoa with seemingly normal phenotypes. To bridge this gap, it is desirable to increase the precision of andrological evaluation in humans and livestock animals by pursuing advanced biomarker-based imaging methods. This review, spiced up with occasional classic movie references but seriously scholastic at the same time, focuses mainly on the biomarkers of altered male germ cell proteostasis resulting in post-testicular carryovers of proteins associated with ubiquitin-proteasome system. Also addressed are sperm redox homeostasis, epididymal sperm maturation, sperm-seminal plasma interactions and sperm surface glycosylation. Zinc ion homeostasis-associated biomarkers and sperm-borne components, including the elements of neurodegenerative pathways such as Huntington's and Alzheimer's disease, are discussed. Such spectrum of biomarkers, imaged by highly specific vital fluorescent molecular probes, lectins, and antibodies, reveals both obvious and subtle defects of sperm chromatin, DNA and accessory structures of the sperm head and tail. Introduction of next generation image-based flow cytometry into research and clinical andrology will soon enable the incorporation of machine and deep learning algorithms with the end point of developing simple, label-free methods for clinical diagnostics and high throughput phenotyping of spermatozoa in humans and economically important livestock animals.

A Non-Synonymous Point Mutation in a WD-40 Domain Repeat of EML5 Leads to Decreased Bovine Sperm Quality and Fertility.

This study is part of a concerted effort to identify and phenotype rare, deleterious mutations that adversely affect sperm quality, or convey high developmental and fertility potential to embryos and ensuing progeny. A rare, homozygous mutation in EML5 (EML5 R1654W ), which encodes a microtubule-associated protein with high expression in testis and brain was identified in an Angus bull used extensively in artificial insemination (AI) for its outstanding progeny production traits. The bull's fertility was low in cross-breeding timed AI (TAI) (Pregnancy/TAI = 25.2%; n = 222) and, in general, AI breeding to Nellore cows (41%; n = 822). A search of the 1,000 Bull Genomes Run9 database revealed an additional 74 heterozygous animals and 8 homozygous animals harboring this exact mutation across several different breeds (0.7% frequency within the 6,191 sequenced animals). Phenotypically, spermatozoa from the homozygous Angus bull displayed prominent piriform and tapered heads, and outwardly protruding knobbed acrosomes. Additionally, an increased retention of EML5 was also observed in the sperm head of both homozygous and heterozygous Angus bulls compared to wild-type animals. This non-synonymous point mutation is located within a WD40 signaling domain repeat of EML5 and is predicted to be detrimental to overall protein function by genomic single nucleotide polymorphism (SNP) analysis and protein modeling. Future work will examine how this rare mutation affects field AI fertility and will characterize the role of EML5 in spermatogenesis.

Mammalian Cell-Free System Recapitulates the Early Events of Post-Fertilization Sperm Mitophagy.

Propagation of paternal sperm-contributed mitochondrial genes, resulting in heteroplasmy, is seldom observed in mammals due to post-fertilization degradation of sperm mitochondria, referred to as sperm mitophagy. Whole organelle sperm mitochondrion degradation is thought to be mediated by the interplay between the ubiquitin-proteasome system (UPS) and the autophagic pathway (Song et al., Proc. Natl. Acad. Sci. USA, 2016). Both porcine and primate post-fertilization sperm mitophagy rely on the ubiquitin-binding autophagy receptor, sequestosome 1 (SQSTM1), and the proteasome-interacting ubiquitinated protein dislocase, valosin-containing protein (VCP). Consequently, we anticipated that sperm mitophagy could be reconstituted in a cell-free system consisting of permeabilized mammalian spermatozoa co-incubated with porcine oocyte extracts. We found that SQSTM1 was detected in the midpiece/mitochondrial sheath of the sperm tail after, but not before, co-incubation with oocyte extracts. VCP was prominent in the sperm mitochondrial sheath both before and after the extract co-incubation and was also detected in the acrosome and postacrosomal sheath and the subacrosomal layer of the spermatozoa co-incubated with extraction buffer as control. Such patterns are consistent with our previous observation of SQSTM1 and VCP associating with sperm mitochondria inside the porcine zygote. In addition, it was observed that sperm head expansion mimicked the early stages of paternal pronucleus development in a zygote during prolonged sperm-oocyte extract co-incubation. Treatment with anti-SQSTM1 antibody during extract co-incubation prevented ooplasmic SQSTM1 binding to sperm mitochondria. Even in an interspecific cellular environment encompassing bull spermatozoa and porcine oocyte extract, ooplasmic SQSTM1 was recruited to heterospecific sperm mitochondria. Complementary with the binding of SQSTM1 and VCP to sperm mitochondria, two sperm-borne pro-mitophagy proteins, parkin co-regulated gene product (PACRG) and spermatogenesis associated 18 (SPATA18), underwent localization changes after extract coincubation, which were consistent with their degradation observed inside fertilized porcine oocytes. These results demonstrate that the early developmental events of post-fertilization sperm mitophagy observed in porcine zygote can be reconstituted in a cell-free system, which could become a useful tool for identifying additional molecules that regulate mitochondrial inheritance in mammals.

NEDD4-like ubiquitin ligase 2 protein (NEDL2) in porcine spermatozoa, oocytes, and preimplantation embryos and its role in oocyte fertilization†.

The ubiquitin-proteasome system plays diverse regulatory and homeostatic roles in mammalian reproduction. Ubiquitin ligases are the substrate-specific mediators of ubiquitin-binding to its substrate proteins. The NEDD4-like ubiquitin ligase 2 (aliases NEDL2, HECW2) is a HECT-type ubiquitin ligase that contains one N-terminal HECW ubiquitin ligase domain, one C-terminal HECT ubiquitin ligase domain, one C2 domain, and two WW protein-protein interaction modules. Beyond its predicted ubiquitin-ligase activity, its cellular functions are largely unknown. Current studies were designed to investigate the content and distribution of NEDL2 in porcine spermatozoa, oocytes, zygotes, and early preimplantation embryos, and in cumulus cells before and after in vitro maturation with oocytes, and fibroblast cells as positive control by western blot and immunocytochemistry, and to examine its roles during oocyte fertilization. Multiple isoforms of NEDL2 were identified by WB. One at approximately 52 kDa was detected only in the germinal vesicle (GV) stage and metaphase II oocytes, and in early preimplantation embryos. Other isoforms were high mass bands at 91, 136, and 155 kDa, which were only detected in somatic cells. Interestingly, ejaculated spermatozoa prominently displayed the same 52 kDa band as oocytes; they also had two minor bands of 74 and 129 kDa, which were not detected in somatic cells or oocytes. By immunofluorescence, NEDL2 showed a diffused cytoplasmic localization in all cell types and accumulated in distinct foci in the germinal vesicles (GVs) of immature oocytes, in maternal and paternal pronuclei of zygotes and nuclei of embryo blastomeres and somatic cells. In blastocysts, the labeling intensity of NEDL2 was stronger in the inner cell mass than in trophoblast, indicating higher NEDL2 content in the ICM cells than in trophectoderm. NEDL2 abundance was 10 times higher in post-maturation oocyte-surrounding cumulus cells than that of cumulus cells before in vitro maturation with hormones, indicating that NEDL2 may have a unique role in cumulus cells after ovulation. Microinjection of anti-NEDL2 antibody into oocyte before IVF did not affect the percentage of oocytes fertilized, percentage of oocytes cleaved, or blastocyst formation. However, the anti-NEDL2 antibody decreased the number of pronuclei, accelerated the formation of nuclear precursor bodies at 6 h postfertilization, inhibited sperm DNA decondensation, and resulted in more fertilized oocytes without male pronuclear formation. In summary, NEDL2 may play a key role during fertilization, especially during sperm DNA decondensation.

Sperm content of TXNDC8 reflects sperm chromatin structure, pregnancy establishment, and incidence of multiple births after ART.

Male germline-specific thioredoxin domain containing 8 (TXNDC8; alias SPTRX3) accumulates indefective human spermatozoa. We assessed the efficiency of two-step semen purification inremoving spermatozoa carrying TXNDC8, and examined the relationship of TXNDC8 with theoutcomes of assisted reproductive therapy (ART), conventional semen parameters, and sperm DNA integrity in sperm chromatin structure assay (SCSA). Semen samples (n = 255) from 91 ART couples were screened in two independent trials, both including a two-step, gradient-and-swim-up separation procedure yielding A-samples (raw semen), B-samples (gradient separated), and C-samples (gradient-and-swim-up). The C-samples were used for intracytoplasmic sperm injection (ICSI) with morphologically selected spermatozoa (IMSSI). Percentage of TXNDC8-positive spermatozoaincreased progressively from A to B/C-samples in both trials. In the first trial (35 couples), the TXNDC8 correlated positively with sperm DNA fragmentation index (%DFI; r = 0.66) measured before separation, and negatively with sperm concentration (r = -0.57) and motility (r = -0.67), also taken before separation. The high DNA stainability index (%HDS) correlated with the percentage of spermatozoa lacking TXNDC8 (r = 0.68). Both SCSA and TXNDC8 parameters showed moderate correlations (r = 0.33-0.66) with blood serum levels of hCG on day 11 (Beta 1) and day13 (Beta 2) after oocyte retrieval. In the second trial (56 couples), fathers of multiplets had a significantly lower percentage of TXNDC8-positive spermatozoa in B-sample (gradient separationonly) compared to men who conceived a singleton pregnancy (p = 0.01) and those who produced no pregnancy (p = 0.02). Those multiplets' fathers also had a significantly higher sperm concentration while their SCSA parameters did not differ from others. It is concluded that theTXNDC8 levels correlate with SCSA and conventional raw semen parameters, and are predictive of pregnancy outcome and multiple births after ART. Two-step purification does not efficiently remove TXNDC8 carrying spermatozoa. ABBREVIATIONS: ART- assisted reproductive therapy; DFI- DNA fragmentation index; FC- flow cytometry (FC); hCG: human chorionic gonadotropin; HDS: high DNA stainability index; HEPES- (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); HTF- human tubal fluid; ICSI- intracytoplasmic sperm injection; IgG- immunoglobulin G; IMSSI- ICSI with morphologically selected spermatozoa; IVF- in vitro fertilization; IU-: intrauterine insemination; NGS- normal goat serum; PBS- phosphate buffered saline; PVP- polyvinylpyrrolidone; SAB- spontaneous abortion; SCSA- sperm chromatin structure assay; SPTRX3- spermatid specific thioredoxin 3; SSS- synthetic serum substitute; TRITC- tetramethyl rhodamine isothiocyanate; TX-100- Triton X-100; TXNDC- thioredoxin domain-containing proteins; TXNDC8- thioredoxin domain containing 8; TUNEL- Terminal deoxynucleotidyl transferase dUTP nick end labeling.

Sperm Cohort-Specific Zinc Signature Acquisition and Capacitation-Induced Zinc Flux Regulate Sperm-Oviduct and Sperm-Zona Pellucida Interactions.

Building on our recent discovery of the zinc signature phenomenon present in boar, bull, and human spermatozoa, we have further characterized the role of zinc ions in the spermatozoa's pathway to fertilization. In boar, the zinc signature differed between the three major boar ejaculate fractions, the initial pre-rich, the sperm-rich, and the post-sperm-rich fraction. These differences set in the sperm ejaculatory sequence establish two major sperm cohorts with marked differences in their sperm capacitation progress. On the subcellular level, we show that the capacitation-induced Zn-ion efflux allows for sperm release from oviductal glycans as analyzed with the oviductal epithelium mimicking glycan binding assay. Sperm zinc efflux also activates zinc-containing enzymes and proteases involved in sperm penetration of the zona pellucida, such as the inner acrosomal membrane matrix metalloproteinase 2 (MMP2). Both MMP2 and the 26S proteasome showed severely reduced activity in the presence of zinc ions, through studies using by gel zymography and the fluorogenic substrates, respectively. In the context of the fertilization-induced oocyte zinc spark and the ensuing oocyte-issued polyspermy-blocking zinc shield, the inhibitory effect of zinc on sperm-borne enzymes may contribute to the fast block of polyspermy. Altogether, our findings establish a new paradigm on the role of zinc ions in sperm function and pave the way for the optimization of animal semen analysis, artificial insemination (AI), and human male-factor infertility diagnostics.

Reciprocal surface expression of arylsulfatase A and ubiquitin in normal and defective mammalian spermatozoa.

Defective mammalian spermatozoa are marked on their surface by proteolytic chaperone ubiquitin. To identify potential ubiquitinated substrates in the defective spermatozoa, we resolved bull sperm protein extracts on a two-dimensional gel and isolated a 64-65-kDa spot (p64) corresponding to one of the major ubiquitin-immunoreactive bands observed in the one-dimensional Western blots. Immune serum raised against this protein recognized a prominent, possibly glycosylated band/spot in the range of 55-68 kDa, consistent with the original spot used for immunization. Internal sequences obtained by Edman degradation of this spot matched the sequence of arylsulfatase A (ARSA), the sperm acrosomal enzyme thought to be important for fertility. By immunofluorescence, a prominent signal was detected on the acrosomal surface (boar and bull) and on the sperm tail principal piece (bull). A second immune serum raised against a synthetic peptide corresponding to an immunogenic internal sequence (GTGKSPRRTL) of the porcine ARSA also labeled sperm acrosome and principal piece. Both sera showed diminished immunoreactivity in the defective bull spermatozoa co-labeled with an anti-ubiquitin antibody. Western blotting and image-based flow cytometry (IBFC) confirmed a reduced ARSA immunoreactivity in the immotile sperm fraction rich in ubiquitinated spermatozoa. Larger than expected ARSA-immunoreactive bands were found in sperm protein extracts immunoprecipitated with anti-ubiquitin antibodies and affinity purified with matrix-bound, recombinant ubiquitin-binding UBA domain. These bands did not show the typical pattern of ARSA glycosylation but overlapped with bands preferentially binding the Lens culinaris agglutinin (LCA) lectin. By both epifluorescence microscopy and IBFC, the LCA binding was increased in the ubiquitinated spermatozoa with diminished ARSA immunoreactivity. ARSA was also found in the epididymal fluid suggesting that in addition to intrinsic ARSA expression in the testis, epididymal spermatozoa take up ARSA on their surface during the epididymal passage. We conclude that sperm surface ARSA is one of the ubiquitinated sperm surface glycoproteins in defective bull spermatozoa. Defective sperm surface thus differs from normal sperm surface by increased ubiquitination, reduced ARSA binding, and altered glycosylation.

COP9 signalosome complex subunit 5, an IFT20 binding partner, is essential to maintain male germ cell survival and acrosome biogenesis†.

Intraflagellar transport protein 20 (IFT20) is essential for spermatogenesis in mice. We discovered that COPS5 was a major binding partner of IFT20. COPS5 is the fifth component of the constitutive photomorphogenic-9 signalosome (COP9), which is involved in protein ubiquitination and degradation. COPS5 is highly abundant in mouse testis. Mice deficiency in COPS5 specifically in male germ cells showed dramatically reduced sperm numbers and were infertile. Testis weight was about one third compared to control adult mice, and germ cells underwent significant apoptosis at a premeiotic stage. Testicular poly (ADP-ribose) polymerase-1, a protein that helps cells to maintain viability, was dramatically decreased, and Caspase-3, a critical executioner of apoptosis, was increased in the mutant mice. Expression level of FANK1, a known COPS5 binding partner, and a key germ cell apoptosis regulator was also reduced. An acrosome marker, lectin PNA, was nearly absent in the few surviving spermatids, and expression level of sperm acrosome associated 1, another acrosomal component was significantly reduced. IFT20 expression level was significantly reduced in the Cops5 knockout mice, and it was no longer present in the acrosome, but remained in the Golgi apparatus of spermatocytes. In the conditional Ift20 mutant mice, COPS5 localization and testicular expression levels were not changed. COP9 has been shown to be involved in multiple signal pathways, particularly functioning as a co-factor for protein ubiquitination. COPS5 is believed to maintain normal spermatogenesis through multiple mechanisms, including maintaining male germ cell survival and acrosome biogenesis, possibly by modulating protein ubiquitination.

Ubiquitin A-52 residue ribosomal protein fusion product 1 (Uba52) is essential for preimplantation embryo development.

Ubiquitin A-52 residue ribosomal protein fusion product 1 (Uba52), a ubiquitin-ribosomal fusion gene, is a major source of ubiquitin protein for covalent modification of proteinaceous substrates recycled by ubiquitin-proteasome system (UPS). Its role in early embryo development has not been studied. Using the CRISPR/Cas9 gene editing tool, the objective of this study was to determine if UBA52 protein is required for mammalian embryogenesis. Matured metaphase II porcine oocytes were injected with CRISPR Cas9+guide RNAs (Uba52 gRNA) or Cas9 without gRNAs as control, followed by in vitro fertilization (IVF) and embryo culture to day 7. Injection of Cas9+gRNAs affected embryo development. On day 4 of embryo culture, the proportion of 2-, 4- and 8-cell stage embryos was significantly different between the Uba52 gRNA and control group (P<0.05), with more 8-cell stage embryos in the control and more 4- and 2-cell stage embryos in the Uba52g RNA group. This delay in the development of Uba52 gRNA embryos occurred at the transition from the 4- to 8-cell stages, around the time of major zygotic genomic activation. The percentage of blastocyst formation on day 7 and the cell number per blastocyst were significantly lower in the Uba52 gRNA group than in the control (P<0.05). Genotyping by PCR and DNA gel electrophoresis analysis showed that 91.8% of embryos that failed to develop to blastocyst had either a monoallelic or a biallelic modification of the Uba52 gene. In comparison, only 24.4% of embryos that reached blastocyst had a monoallelic modification and biallelic editing was not found in any of the blastocysts. Based on immuno-labeling intensity, both UBA52 and proteasome protein levels on days 4 and 7 of culture were significantly lower in the Uba52 gRNA group than in the control (P<0.05), in agreement with UBA52 western blotting-densitometry of day 4 embryos. Morphological examination of blastomere nuclei revealed abnormal nuclear structure in the Uba52 gRNA group, such as reduced size, irregular shapes, nucleus fragmentation and uneven DNA distribution at all stages of embryo development. Nuclear morphology studies of embryos injected with Cas9+gRNAs and co-injected with plasmid DNA encoding nuclear localized GFP further supported these observations. In conclusion, our data indicate that the Uba52 gene is essential in early embryogenesis.

In Utero and Postnatal Exposure to High Fat, High Sucrose Diet Suppressed Testis Apoptosis and Reduced Sperm Count.

Obesity affects male fertility and maternal diabetes affects the offspring sperm epigenome. However, the effects of in utero exposure to maternal glucose intolerance in combination with postnatal high fat, high sucrose (HFHS) diet consumption on offspring spermatogenesis is not clear. The present study was designed to test these effects. One week before and during pregnancy, dams were fed either control or HFHS diet to induce gestational glucose intolerance, and returned to standard diet during lactation. Male offspring from each maternal group were split into control and HFHS-fed groups for eight weeks prior to sacrifice at 11, 19 or 31 weeks of age, and reproductive tissues were harvested for analysis of testicular germ cell apoptosis and sperm output. Postnatal HFHS diet suppressed spermatogonia apoptosis in all age groups and maternal HFHS diet reduced testosterone levels at 11 weeks. At 31 weeks of age, the postnatal HFHS diet increased body weight, and reduced epididymis weight and sperm count. The combination of in utero and postnatal exposure impacted sperm counts most significantly. In summary, HFHS diet during pregnancy puts male offspring at greater risk of infertility, particularly when combined with postnatal high fat diet feeding.

Zinc ion flux during mammalian sperm capacitation.

Sperm capacitation, the ultimate maturation event preparing mammalian spermatozoa for fertilization, was first described in 1951, yet its regulatory mechanisms remain poorly understood. The capacitation process encompasses an influx of bicarbonate and calcium ions, removal of decapacitating factors, changes of pH and sperm proteasomal activities, and the increased protein tyrosine phosphorylation. Here, we document a novel biological phenomenon of a unique zinc (Zn2+) ion redistribution associated with mammalian sperm in vitro capacitation (IVC). Using image-based flow cytometry (IBFC), we identified four distinct types of sperm zinc ion distribution patterns (further zinc signature) and their changes during IVC. The zinc signature was altered after sperm capacitation, reduced by proteasomal inhibitors, removed by zinc chelators, and maintained with addition of external ZnCl2. These findings represent a fundamental shift in the understanding of mammalian fertilization, paving the way for improved semen analysis, in vitro fertilization (IVF), and artificial insemination (AI).

Modifications of the 26S proteasome during boar sperm capacitation.

Protein ubiquitination is a stable, reversible post-translational modification, targeting proteins for degradation/recycling by the 26S proteasome in a well-characterized enzymatic cascade. Studies have revealed the role of UPS in the regulation of fertilization, including sperm-zona pellucida interactions and the early event of sperm capacitation. The present study investigates the changes in proteasome compartmentalization, subunit composition and post-translational modifications during in vitro capacitation of fresh boar spermatozoa. We observed capacitation-dependent shedding of both 20S core and 19S regulatory particles from the acrosome that was associated with decreased plasma membrane integrity, independent of proteasomal inhibition. Subunits PSMA1-7 of the 20S core did not appear to undergo post-translational modifications during capacitation, based on invariant molecular masses before and after capacitation; however, we observed multiple PSMD4 forms of 19S regulatory particles (50, 53, 70, 115-140, 160 and >176 kDa) sequentially released from spermatozoa. PSMD4 subunit was found to be post-translationally modified during the course of capacitation, resulting in changes of apparent molecular mass, some of which were dependent on proteasomal inhibition. These results show that the sperm proteasomes are being modified during sperm capacitation. Additional studies of individual 26S proteasome subunits will be required to elucidate these modifications and to understand how UPS modulates sperm capacitation.

SIRT1-dependent modulation of methylation and acetylation of histone H3 on lysine 9 (H3K9) in the zygotic pronuclei improves porcine embryo development.

BACKGROUND: The histone code is an established epigenetic regulator of early embryonic development in mammals. The lysine residue K9 of histone H3 (H3K9) is a prime target of SIRT1, a member of NAD+-dependent histone deacetylase family of enzymes targeting both histone and non-histone substrates. At present, little is known about SIRT1-modulation of H3K9 in zygotic pronuclei and its association with the success of preimplantation embryo development. Therefore, we evaluated the effect of SIRT1 activity on H3K9 methylation and acetylation in porcine zygotes and the significance of H3K9 modifications for early embryonic development. RESULTS: Our results show that SIRT1 activators resveratrol and BML-278 increased H3K9 methylation and suppressed H3K9 acetylation in both the paternal and maternal pronucleus. Inversely, SIRT1 inhibitors nicotinamide and sirtinol suppressed methylation and increased acetylation of pronuclear H3K9. Evaluation of early embryonic development confirmed positive effect of selective SIRT1 activation on blastocyst formation rate (5.2 ± 2.9% versus 32.9 ± 8.1% in vehicle control and BML-278 group, respectively; P ≤ 0.05). Stimulation of SIRT1 activity coincided with fluorometric signal intensity of ooplasmic ubiquitin ligase MDM2, a known substrate of SIRT1 and known limiting factor of epigenome remodeling. CONCLUSIONS: We conclude that SIRT1 modulates zygotic histone code, obviously through direct deacetylation and via non-histone targets resulting in increased H3K9me3. These changes in zygotes lead to more successful pre-implantation embryonic development and, indeed, the specific SIRT1 activation due to BML-278 is beneficial for in vitro embryo production and blastocyst achievement.

The ART and science of sperm mitophagy.

This article discusses the historical perspective and the new findings of autophagy and ubiquitin-proteasome system cooperation during the post-fertilization sperm mitophagy, a process that eliminates potentially damaged paternal mitochondrial DNA from an early embryo. New insight into the mechanism that promotes clonal, maternal inheritance of mitochondrial genes may be helpful for managing mitochondrial disease and infertility in humans, as well as reproductive performance and production traits in agriculturally important domestic animals.

Autophagy and ubiquitin-proteasome system contribute to sperm mitophagy after mammalian fertilization.

Maternal inheritance of mitochondria and mtDNA is a universal principle in human and animal development, guided by selective ubiquitin-dependent degradation of the sperm-borne mitochondria after fertilization. However, it is not clear how the 26S proteasome, the ubiquitin-dependent protease that is only capable of degrading one protein molecule at a time, can dispose of a whole sperm mitochondrial sheath. We hypothesized that the canonical ubiquitin-like autophagy receptors [sequestosome 1 (SQSTM1), microtubule-associated protein 1 light chain 3 (LC3), gamma-aminobutyric acid receptor-associated protein (GABARAP)] and the nontraditional mitophagy pathways involving ubiquitin-proteasome system and the ubiquitin-binding protein dislocase, valosin-containing protein (VCP), may act in concert during mammalian sperm mitophagy. We found that the SQSTM1, but not GABARAP or LC3, associated with sperm mitochondria after fertilization in pig and rhesus monkey zygotes. Three sperm mitochondrial proteins copurified with the recombinant, ubiquitin-associated domain of SQSTM1. The accumulation of GABARAP-containing protein aggregates was observed in the vicinity of sperm mitochondrial sheaths in the zygotes and increased in the embryos treated with proteasomal inhibitor MG132, in which intact sperm mitochondrial sheaths were observed. Pharmacological inhibition of VCP significantly delayed the process of sperm mitophagy and completely prevented it when combined with microinjection of autophagy-targeting antibodies specific to SQSTM1 and/or GABARAP. Sperm mitophagy in higher mammals thus relies on a combined action of SQSTM1-dependent autophagy and VCP-mediated dislocation and presentation of ubiquitinated sperm mitochondrial proteins to the 26S proteasome, explaining how the whole sperm mitochondria are degraded inside the fertilized mammalian oocytes by a protein recycling system involved in degradation of single protein molecules.

Improved Murine Blastocyst Quality and Development in a Single Culture Medium Compared to Sequential Culture Media.

OBJECTIVE: Validate single versus sequential culture media for murine embryo development. DESIGN: Prospective laboratory experiment. SETTING: Assisted Reproduction Laboratory. ANIMALS: Murine embryos. INTERVENTIONS: 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. RESULTS: 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. CONCLUSIONS: 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.

Protein deubiquitination during oocyte maturation influences sperm function during fertilisation, antipolyspermy defense and embryo development.

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.

Increased conception rates in beef cattle inseminated with nanopurified bull semen.

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 peanut agglutinin, which binds to glycans exposed by acrosomal damage. In a 2 yr artificial insemination field trial with 798 cows, a conception rate of 64.5% ± 3.7% was achieved with a 10 × 10(6) sperm dose of peanut agglutinin-nanopurified spermatozoa, comparable to a control nonpurified full dose of 20 × 10(6) spermatozoa per dose (63.3% ± 3.2%) and significantly higher than a 10 × 10(6) sperm dose of nonpurified 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. Because 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.

Identification and characterization of RING-finger ubiquitin ligase UBR7 in mammalian spermatozoa.

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.

Protein expression pattern of PAWP in bull spermatozoa is associated with sperm quality and fertility following artificial insemination.

Post-acrosomal WW-domain binding protein (PAWP) is a signaling molecule located in the post-acrosomal sheath (PAS) of mammalian spermatozoa. We hypothesized that the proper integration of PAWP in the sperm PAS is reflective of bull-sperm quality and fertility. Cryopreserved semen samples from 298 sires of acceptable, but varied, fertility used in artificial insemination services were analyzed using immunofluorescence microscopy and flow cytometry for PAWP protein. In normal spermatozoa, PAWP fluorescence formed a regular band around the proximal PAS. Anomalies of PAWP labeling in defective spermatozoa were reflected in flow cytometry by varied intensities of PAWP-induced fluorescence. Distinct sperm phenotypes were also identified, including morphologically normal and some defective spermatozoa with moderate levels of PAWP; grossly defective spermatozoa with low/no PAWP; and defective spermatozoa with high PAWP. Analysis by ImageStream flow cytometry confirmed the prevalence of abnormal sperm phenotypes in the spermatozoa with abnormal PAWP content. Live/dead staining and video recording showed that some abnormal spermatozoa are viable and capable of progressive motility. Conventional flow-cytometric measurements of PAWP correlated significantly with semen quality and fertility parameters that reflect the sires' artificial insemination fertility, including secondary sperm morphology, conception rate, non-return rate, and residual value. A multiplex, flow-cytometric test detecting PAWP, aggresomes (ubiquitinated protein aggregates), and acrosomal integrity (peanut-agglutinin-lectin labeling) had a predictive value for conception rate, as demonstrated by step-wise regression analysis. We conclude that PAWP correlates with semen/fertility parameters used in the cattle artificial insemination industry, making PAWP a potential biomarker of bull fertility.