There is a well-established link between abnormal sperm chromatin states and poor motility, however, how these two processes are interdependent is unknown. Here, we identified a possible mechanistic insight by showing that Protamine 2, a nuclear DNA packaging protein in sperm, directly interacts with cytoskeletal protein Septin 12, which is associated with sperm motility. Septin 12 has several isoforms, and we show, that in the Prm2-/- sperm, the short one (Mw 36 kDa) is mis-localized, while two long isoforms (Mw 40 and 41 kDa) are unexpectedly lost in Prm2-/- sperm chromatin-bound protein fractions. Septin 12 co-immunoprecipitated with Protamine 2 in the testicular cell lysate of WT mice and with Lamin B1/2/3 in co-transfected HEK cells despite we did not observe changes in Lamin B2/B3 proteins or SUN4 expression in Prm2-/- testes. Furthermore, the Prm2-/- sperm have on average a smaller sperm nucleus and aberrant acrosome biogenesis. In humans, patients with low sperm motility (asthenozoospermia) have imbalanced histone-protamine 1/2 ratio, modified levels of cytoskeletal proteins and we detected retained Septin 12 isoforms (Mw 40 and 41 kDa) in the sperm membrane, chromatin-bound and tubulin/mitochondria protein fractions. In conclusion, our findings present potential interaction between Septin 12 and Protamine 2 or Lamin B2/3 and describe a new connection between their expression and localization, contributing likely to low sperm motility and morphological abnormalities.
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- abstrakt z konference MeSH
Juno and CD9 protein, expressed in oolemma, are known to be essential for sperm-oocyte binding and fusion. Although evidence exists that these two proteins cooperate, their interaction has not yet been demonstrated. Here in, we present Juno and CD9 mutual localization over the surface of mouse metaphase II oocytes captured using the 3D STED super-resolution technique. The precise localization of examined proteins was identified in different compartments of oolemma such as the microvillar membrane, planar membrane between individual microvilli, and the membrane of microvilli-free region. Observed variance in localization of Juno and CD9 was confirmed by analysis of transmission and scanning electron microscopy images, which showed a significant difference in the presence of proteins between selected membrane compartments. Colocalization analysis of super-resolution images based on Pearson's correlation coefficient supported evidence of Juno and CD9 mutual position in the oolemma, which was identified by proximity ligation assay. Importantly, the interaction between Juno and CD9 was detected by co-immunoprecipitation and mass spectrometry in HEK293T/17 transfected cell line. For better understanding of experimental data, mouse Juno and CD9 3D structure were prepared by comparative homology modelling and several protein-protein flexible sidechain dockings were performed using the ClusPro server. The dynamic state of the proteins was studied in real-time at atomic level by molecular dynamics (MD) simulation. Docking and MD simulation predicted Juno-CD9 interactions and stability also suggesting an interactive mechanism. Using the multiscale approach, we detected close proximity of Juno and CD9 within microvillar oolemma however, not in the planar membrane or microvilli-free region. Our findings show yet unidentified Juno and CD9 interaction within the mouse oolemma protein network prior to sperm attachment. These results suggest that a Juno and CD9 interactive network could assist in primary Juno binding to sperm Izumo1 as a prerequisite to subsequent gamete membrane fusion.
- Publikační typ
- časopisecké články MeSH
Male germ cells experience a drastic chromatin remodeling through the nucleo-histone to nucleo-protamine (NH-NP) transition necessary for proper sperm functionality. Post-translational modifications (PTMs) of H4 Lys5, such as acetylation (H4K5ac), play a crucial role in epigenetic control of nucleosome disassembly facilitating protamine incorporation into paternal DNA. It has been shown that butyrylation on the same residue (H4K5bu) participates in temporal regulation of NH-NP transition in mice, delaying the bromodomain testis specific protein (BRDT)-dependent nucleosome disassembly and potentially marking retained nucleosomes. However, no information was available so far on this modification in human sperm. Here, we report a dual behavior of H4K5bu and H4K5ac in human normal spermatogenesis, suggesting a specific role of H4K5bu during spermatid elongation, coexisting with H4K5ac although with different starting points. This pattern is stable under different testicular pathologies, suggesting a highly conserved function of these modifications. Despite a drastic decrease of both PTMs in condensed spermatids, they are retained in ejaculated sperm, with 30% of non-colocalizing nucleosome clusters, which could reflect differential paternal genome retention. Whereas no apparent effect of these PTMs was observed associated with sperm quality, their presence in mature sperm could entail a potential role in the zygote.
- MeSH
- acetylace MeSH
- chromatin * metabolismus MeSH
- histony metabolismus MeSH
- lidé MeSH
- myši MeSH
- nukleozomy * metabolismus MeSH
- posttranslační úpravy proteinů MeSH
- protaminy metabolismus MeSH
- restrukturace chromatinu MeSH
- sperma metabolismus MeSH
- spermatidy metabolismus MeSH
- spermatogeneze fyziologie MeSH
- spermie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Gamete fusion is a critical event of mammalian fertilization. A random one-bead one-compound combinatorial peptide library represented synthetic human egg mimics and identified a previously unidentified ligand as Fc receptor-like 3, named MAIA after the mythological goddess intertwined with JUNO. This immunoglobulin super family receptor was expressed on human oolemma and played a major role during sperm-egg adhesion and fusion. MAIA forms a highly stable interaction with the known IZUMO1/JUNO sperm-egg complex, permitting specific gamete fusion. The complexity of the MAIA isotype may offer a cryptic sexual selection mechanism to avoid genetic incompatibility and achieve favorable fitness outcomes.
- Publikační typ
- časopisecké články MeSH
Out of millions of ejaculated sperm, a few reach the fertilization site in mammals. Flagellar Ca2+ signaling nanodomains, organized by multi-subunit CatSper calcium channel complexes, are pivotal for sperm migration in the female tract, implicating CatSper-dependent mechanisms in sperm selection. Here using biochemical and pharmacological studies, we demonstrate that CatSper1 is an O-linked glycosylated protein, undergoing capacitation-induced processing dependent on Ca2+ and phosphorylation cascades. CatSper1 processing correlates with protein tyrosine phosphorylation (pY) development in sperm cells capacitated in vitro and in vivo. Using 3D in situ molecular imaging and ANN-based automatic detection of sperm distributed along the cleared female tract, we demonstrate that spermatozoa past the utero-tubal junction possess the intact CatSper1 signals. Together, we reveal that fertilizing mouse spermatozoa in situ are characterized by intact CatSper channel, lack of pY, and reacted acrosomes. These findings provide molecular insight into sperm selection for successful fertilization in the female reproductive tract.
- MeSH
- akrozomální reakce MeSH
- glykosylace MeSH
- molekulární zobrazování metody MeSH
- myši MeSH
- regulace genové exprese MeSH
- spermie fyziologie MeSH
- vápníková signalizace MeSH
- vápníkové kanály genetika metabolismus MeSH
- ženské pohlavní orgány diagnostické zobrazování MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Di-(2-ethylhexyl)-phthalate (DEHP) is a compound widely used as a plasticizer, which can leach from plastics into the environment and thus influence human health. The aim of this study was to analyze whether exposure to an environmentally relevant dose of DEHP during mice fetal development or puberty can cause long-lasting changes detectable month/s after the last exposure. We used a DEHP concentration relevant to a daily human intake of 2.4-3 μg/kg of body weight/day. CD1 outbred mice were treated either in utero or postnatally during puberty and analyzed in adulthood. Analyzing fertility parameters using morphometric, histologic, genomic and proteomic methods we showed that DEHP exposure leads to decreased sperm concentration and quality, in both experimental groups. Moreover, the changes in anogenital distance, seminal vesicle weight, and testicular gene expression suggest a disturbance of androgen signaling in exposed animals. In conclusion, we hereby present, that the prenatal and pubertal exposure to a low dose of DEHP negatively influenced reproductive endpoints in male mice, and some of the effects were persistent until adulthood.
- MeSH
- anální kanál anatomie a histologie účinky léků MeSH
- diethylhexylftalát toxicita MeSH
- endokrinní disruptory toxicita MeSH
- maternofetální výměna látek MeSH
- mužské pohlavní orgány anatomie a histologie účinky léků MeSH
- myši inbrední ICR MeSH
- pohlavní dospělost účinky léků MeSH
- spermie účinky léků MeSH
- těhotenství MeSH
- testis anatomie a histologie účinky léků MeSH
- vývojová regulace genové exprese účinky léků MeSH
- zpožděný efekt prenatální expozice chemicky indukované genetika MeSH
- zvířata MeSH
- zvláčňovadla toxicita MeSH
- Check Tag
- mužské pohlaví MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Capacitation involves physiological changes that spermatozoa must undergo in the female reproductive tract or in vitro to obtain the ability to bind, penetrate and fertilize the egg. Up to date, several methods have been developed to characterize this complex biological process. The goal of the presented study is to mutually compare several fluorescent techniques, check their ability to detect changes in molecular processes during the capacitation progress and determine their ability to predict the percentage of acrosome reacted (AR) sperm after the exposure to solubilized zona pellucida (ZP). The capacitation process was analyzed using four fluorescent techniques: 1. chlortetracycline (CTC) staining, 2. anti-acrosin antibody (ACR.2) assay, 3. anti-phosphotyrosine (pY) antibody assay, 4. fluorescein isothiocyanate-conjugated phalloidin (FITC-phall) assay. All these methods were tested using fluorescent microscopy and flow cytometry. RESULTS: All selected methods are capable to detect the capacitation progress of boar sperm in vitro, but there are significant differences in their outcome when using fluorescent microscopy or flow cytometry experimental arrangements and subsequent statistical analysis (KW-ANOVA). Also, the ability to predict the absolute numbers of sperm which will undergo ZP-induced AR differ significantly (CTC and ACR.2 gave the best predictions). CONCLUSIONS: Our study compared four largely used methods used to characterize capacitation process, highlighted their differences and showed that all are able to detect capacitation progress, CTC and ACR.2 are furthermore able to accurately predict the percentage of AR sperm after ZP-induced AR.
- MeSH
- akrozomální reakce fyziologie MeSH
- faloidin MeSH
- fluorescein-5-isothiokyanát MeSH
- fluorescenční barviva * MeSH
- fluorescenční mikroskopie * metody MeSH
- fluorescenční protilátková technika MeSH
- kapacitace spermií fyziologie MeSH
- průtoková cytometrie * metody MeSH
- spermie fyziologie MeSH
- Sus scrofa fyziologie MeSH
- vápník analýza MeSH
- zona pellucida fyziologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Recent studies have shown that infertility affects estimated 15% of all couples. Male infertility is the primary or contributory cause in 60% of these cases. Consequently, the application of assisted reproduction is increasing. These methods could benefit from an extended evaluation of sperm quality. For this reason, we analyzed sperm proteins from 30 men with normal spermiograms and 30 men with asthenozoospermia. Ejaculates of both groups were tested by flow cytometry (FCM) and fluorescence with a set of well-characterized anti-human sperm Hs-monoclonal antibodies (MoAbs), which were generated in our laboratory. No statistically significant differences were found between normospermics and asthenospermics in the expression of the sperm surface protein clusterin, evaluated with Hs-3 MoAb, and semenogelin, evaluated with Hs-9 MoAb. However, FCM revealed quantitative differences in the acrosomal proteins between normozoospermic and asthenozoospermic men, namely, in glyceraldehyde-3-phosphate dehydrogenase, evaluated with Hs-8 MoAb, valosin-containing protein, evaluated with Hs-14 MoAb, and ATP synthase (cAMP-dependent protein kinase II, PRKAR2A), evaluated with MoAb Hs-36. Asthenozoospermic men displayed a highly reduced expression of intra-acrosomal proteins, with a likely decrease in sperm quality, and thus a negative impact on successful reproduction. Asthenozoospermia seems to be a complex disorder involving intra-acrosomal proteins.
- MeSH
- astenozoospermie imunologie metabolismus MeSH
- fertilizace in vitro MeSH
- intracytoplazmatické injekce spermie MeSH
- lidé MeSH
- monoklonální protilátky imunologie MeSH
- proteiny metabolismus MeSH
- průtoková cytometrie MeSH
- spermie imunologie metabolismus MeSH
- těhotenství MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Publikační typ
- abstrakt z konference MeSH
