Super-resolution (SR) microscopy is a cutting-edge method that can provide detailed structural information with high resolution. However, the thickness of the specimen has been a major limitation for SR methods, and large biological structures have posed a challenge. To overcome this, the key step is to optimise sample preparation to ensure optical homogeneity and clarity, which can enhance the capabilities of SR methods for the acquisition of thicker structures. Oocytes are the largest cells in the mammalian body and are crucial objects in reproductive biology. They are especially useful for studying membrane proteins. However, oocytes are extremely fragile and sensitive to mechanical manipulation and osmotic shocks, making sample preparation a critical and challenging step. We present an innovative, simple and sensitive approach to oocyte sample preparation for 3D STED acquisition. This involves alcohol dehydration and mounting into a high refractive index medium. This extended preparation procedure allowed us to successfully obtain a unique two-channel 3D STED SR image of an entire mouse oocyte. By optimising sample preparation, it is possible to overcome current limitations of SR methods and obtain high-resolution images of large biological structures, such as oocytes, in order to study fundamental biological processes. Lay Abstract: Super-resolution (SR) microscopy is a cutting-edge tool that allows scientists to view incredibly fine details in biological samples. However, it struggles with larger, thicker specimens, as they need to be optically clear and uniform for the best imaging results. In this study, we refined the sample preparation process to make it more suitable for SR microscopy. Our method includes carefully dehydrating biological samples with alcohol and then transferring them into a mounting medium that enhances optical clarity. This improved protocol enables high-resolution imaging of thick biological structures, which was previously challenging. By optimizing this preparation method, we hope to expand the use of SR microscopy for studying large biological samples, helping scientists better understand complex biological structures.

• Klíčová slova
• 3D STED, alcohol dehydration, high refractive index medium, large biological objects, oocyte, sample preparation, super‐resolution,
• MeSH
• ethanol chemie   MeSH
• fluorescenční mikroskopie metody   MeSH
• myši MeSH
• odběr biologického vzorku * metody   MeSH
• oocyty * cytologie   ultrastruktura   MeSH
• refraktometrie metody   MeSH
• zobrazování trojrozměrné * metody   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ethanol 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.

• Klíčová slova
• CD9, Juno, MD simulation, STED, docking, oocyte, oolemma compartments, protein interaction,
• 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

Diabetes is a chronic metabolic disorder characterized by hyperglycemia and associated with many health complications due to the long-term damage and dysfunction of various organs. A consequential complication of diabetes in men is reproductive dysfunction, reduced fertility, and poor reproductive outcomes. However, the molecular mechanisms responsible for diabetic environment-induced sperm damage and overall decreased reproductive outcomes are not fully established. We evaluated the effects of type 2 diabetes exposure on the reproductive system and the reproductive outcomes of males and their male offspring, using a mouse model. We demonstrate that paternal exposure to type 2 diabetes mediates intergenerational and transgenerational effects on the reproductive health of the offspring, especially on sperm quality, and on metabolic characteristics. Given the transgenerational impairment of reproductive and metabolic parameters through two generations, these changes likely take the form of inherited epigenetic marks through the germline. Our results emphasize the importance of improving metabolic health not only in women of reproductive age, but also in potential fathers, in order to reduce the negative impacts of diabetes on subsequent generations.

• Klíčová slova
• GAPDS, TERA, diabetes, fertility, molecular biomarkers, offspring, sperm, testes,
• MeSH
• diabetes mellitus 2. typu krev   chemicky indukované   genetika   MeSH
• dieta s vysokým obsahem tuků škodlivé účinky   MeSH
• experimentální diabetes mellitus MeSH
• fenotyp * MeSH
• infertilita krev   chemicky indukované   genetika   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• paternální dědičnost účinky léků   genetika   MeSH
• spermie účinky léků   fyziologie   MeSH
• streptozocin toxicita   MeSH
• těhotenství MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• streptozocin MeSH

Integrins are transmembrane receptors that facilitate cell adhesion and cell-extracellular matrix communication. They are involved in the sperm maturation including capacitation and gamete interaction, resulting in successful fertilization. αV integrin belongs to the integrin glycoprotein superfamily, and it is indispensable for physiological spermiogenesis and testosterone production. We targeted the gene and protein expression of the αV integrin subunit and described its membrane localization in sperm. Firstly, in mouse, we traced αV integrin gene expression during spermatogenesis in testicular fraction separated by elutriation, and we detected gene activity in spermatogonia, spermatocytes, and round spermatids. Secondly, we specified αV integrin membrane localization in acrosome-intact and acrosome-reacted sperm and compared its pattern between mouse, pig, and human. Using immunodetection and structured illumination microscopy (SIM), the αV integrin localization was confined to the plasma membrane covering the acrosomal cap area and also to the inner acrosomal membrane of acrosome-intact sperm of all selected species. During the acrosome reaction, which was induced on capacitated sperm, the αV integrin relocated and was detected over the whole sperm head. Knowledge of the integrin pattern in mature sperm prepares the ground for further investigation into the pathologies and related fertility issues in human medicine and veterinary science.

• Klíčová slova
• human, male germ cells, mouse, pig, sperm, αV integrin,
• MeSH
• akrozomální reakce MeSH
• integrin alfaV metabolismus   MeSH
• lidé MeSH
• myši MeSH
• prasata MeSH
• spermie metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• integrin alfaV 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.

• Klíčová slova
• DEHP, Fertility, Gene expression, Spermatogenesis, Steroidogenesis,
• 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
• změkčovadla toxicita   MeSH
• zpožděný efekt prenatální expozice chemicky indukované   genetika   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• diethylhexylftalát MeSH
• endokrinní disruptory MeSH
• změkčovadla MeSH

Fertilization is a multiple step process leading to the fusion of female and male gametes and the formation of a zygote. Besides direct gamete membrane interaction via binding receptors localized on both oocyte and sperm surface, fertilization also involves gamete communication via chemical molecules triggering various signaling pathways. This work focuses on a mouse taste receptor, mTAS1R3, encoded by the Tas1r3 gene, as a potential receptor mediating chemical communication between gametes using the C57BL/6J lab mouse strain. In order to specify the role of mTAS1R3, we aimed to characterize its precise localization in testis and sperm using super resolution microscopy. The testis cryo-section, acrosome-intact sperm released from cauda epididymis and sperm which underwent the acrosome reaction (AR) were evaluated. The mTAS1R3 receptor was detected in late spermatids where the acrosome was being formed and in the acrosomal cap of acrosome intact sperm. AR is triggered in mice during sperm maturation in the female reproductive tract and by passing through the egg surroundings such as cumulus oophorus cells. This AR onset is independent of the extracellular matrix of the oocyte called zona pellucida. After AR, the relocation of mTAS1R3 to the equatorial segment was observed and the receptor remained exposed to the outer surroundings of the female reproductive tract, where its physiological ligand, the amino acid L-glutamate, naturally occurs. Therefore, we targeted the possible interaction in vitro between the mTAS1R3 and L-glutamate as a part of chemical communication between sperm and egg and used an anti-mTAS1R3-specific antibody to block it. We detected that the acrosome reacted spermatozoa showed a chemotactic response in the presence of L-glutamate during and after the AR, and it is likely that mTAS1R3 acted as its mediator.

• Klíčová slova
• L-glutamate, TAS1R family, acrosome reaction, chemoattractant, chemotaxis, gamete, mTAS1R3 receptor, mouse, sperm,
• MeSH
• buněčná diferenciace MeSH
• chemotaxe MeSH
• exprese genu MeSH
• glutamáty metabolismus   MeSH
• interakce spermie a vajíčka * MeSH
• messenger RNA genetika   MeSH
• mezibuněčná komunikace * MeSH
• myši MeSH
• receptory spřažené s G-proteiny genetika   metabolismus   MeSH
• spermie cytologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• glutamáty MeSH
• messenger RNA MeSH
• receptory spřažené s G-proteiny MeSH
• taste receptors, type 1 MeSH Prohlížeč

Integrins are transmembrane cell receptors involved in two crucial mechanisms for successful fertilization, namely, mammalian intracellular signaling and cell adhesion. Integrins α6β4, α3β1 and α6β1 are three major laminin receptors expressed on the surface of mammalian cells including gametes, and the presence of individual integrin subunits α3, α6, β1 and β4 has been previously detected in mammalian sperm. However, to date, proof of the existence of individual heterodimer pairs in sperm and their detailed localization is missing. The major conclusion of this study is evidence that the β4 integrin subunit is expressed in mouse sperm and that it pairs with subunit α6; additionally, there is a detailed identification of integrin heterodimer pairs across individual membranes in an intact mouse sperm head. We also demonstrate the existence of β4 integrin mRNAs in round spermatids and spermatogonia by q-RT-PCR, which was further supported by sequencing the PCR products. Using super-resolution microscopy accompanied by colocalization analysis, we located integrin subunits as follows: α6/β4-inner apical acrosomal membrane and equatorial segment; α3, α6/β1, β4-plasma membrane overlaying the apical acrosome; and α3/β1-outer acrosomal membrane. The existence of α6β4, α3β1 and α6β1 heterodimers was further confirmed by proximity ligation assay (PLA). In conclusion, we delivered detailed characterization of α3, α6, β1 and β4 integrin subunits, showing their presence in distinct compartments of the intact mouse sperm head. Moreover, we identified sperm-specific localization for heterodimers α6β4, α3β1 and α6β1, and their membrane compartmentalization and the presented data show a complexity of membranes overlaying specialized microdomain structures in the sperm head. Their different protein compositions of these individual membrane rafts may play a specialized role, based on their involvement in sperm-epithelium and sperm-egg interaction.

• Klíčová slova
• integrin heterodimers, integrins, sperm head, α3β1, α6β1, α6β4,
• MeSH
• biologické modely MeSH
• integriny chemie   metabolismus   MeSH
• kompartmentace buňky * MeSH
• multimerizace proteinu * MeSH
• myši inbrední C57BL MeSH
• podjednotky proteinů metabolismus   MeSH
• proteinové domény MeSH
• spermie metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• integriny MeSH
• podjednotky proteinů MeSH

Male infertility is a worldwide problem associated with genetic background, environmental factors, and diseases. One of the suspected contributing factors to male infertility is diabetes mellitus. We investigated the molecular and morphological changes in sperms and testicular tissue of diabetic males. The study was performed in streptozotocin-induced type 1 diabetes mouse model. Diabetes decreased sperm concentration and viability and increased sperm apoptosis. Changes in protamine 1/protamine 2 ratio indicated reduced sperm quality. The testicular tissue of diabetic males showed significant tissue damage, disruption of meiotic progression, and changes in the expression of genes encoding proteins important for spermiogenesis. Paternal diabetes altered sperm quality and expression pattern in the testes in offspring of two subsequent generations. Our study revealed that paternal diabetes increased susceptibility to infertility in offspring through gametic alternations. Our data also provide a mechanistic basis for transgenerational inheritance of diabetes-associated pathologies since protamines may be involved in epigenetic regulations.

• MeSH
• biologické markery MeSH
• diabetes mellitus 1. typu komplikace   metabolismus   MeSH
• fenotyp MeSH
• genetická predispozice k nemoci * MeSH
• meióza MeSH
• mužská infertilita etiologie   MeSH
• myši MeSH
• protaminy metabolismus   MeSH
• spermatogeneze MeSH
• spermie metabolismus   MeSH
• testis metabolismus   MeSH
• typy dědičnosti * MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biologické markery MeSH
• protaminy MeSH