Acetilation of α-tubulin and expression of zinc transporters ZIP6, ZIP10 and ZIP14 as markers of structural and functional heterogenity of human spermatozoa

Muška neplodnost je primarni ili doprinoseći uzrok u 40% slučajeva neplodnosti para. Pored trenutne dijagnostike utvrđivanja muške neplodnosti svakodnevno se nameće potreba za novim načinima procene fertilizaciono sposobnih spermatozoida. Pokretljivost spermatozoida, odnosno njihova sposobnost za oplodnju, strukturno zavisi od tubulinskog koda − ekspresije specifičnih tipova tubulina koji grade centriole i flagelum, i acetilacije tubulina kao najzastupljenije post-translacione modifikacije (PTM). Funkcijski, pokretljivost spermatozoida zavisi i od cink koda koji je regulisan ekspresijom i lokalizacijom specifičnih transportera cinka, importera – ZIP i eksportera – ZnT. Cilj rada bilo je utvrđivanje ćelijske i molekulske osnove heterogenosti spermatozoida na nivou tubulinskog i cink koda. Korišćeni su uzorci sperme pacijenata u postupku biomedicinski potpomognute oplodnje i to intrauterine inseminacije koji su klasifikovani prema uputstvu Svetske zdravstvene organizacije kao normo- i astenozoospermični. U analizi su korišćeni nativni i uzorci obrađeni po modifikovanom metodu centrifugiranja u gradijentu. Imunocitohemija je korišćena za prikaz ekspresije i lokalizacije α-tubulina i acetilovanog α-tubulina putem svetlosne mikroskopije. Imunofluorescencija je korišćena za karakterizaciju ekspresije i lokalizacije α-tubulina, acetilovanog α-tubulina i tri različita tipa ZIP transportera u ejakulisanim spermatozoidima normo- i astenozoospermičnih uzoraka sperme. Detekcija imunofluorescencom je pokazala heterogenu lokalizaciju i zastupljenost imunopozitivnosti α-tubulina i acetilovanog α-tubulina. Acetilovani α-tubulin je bio prisutan u repu i jedru normozoospermičnih spermatozoida. Za acetilovani α-tubulin je više imunopozitivnosti bilo u repu, a manje u jedru astenozoospermičnih u poređenju sa normozoospermičnim uzorcima. ZIP6, ZIP10 i ZIP14 su pokazali heterogenu ekspresiju spermatozoida i različitu distribuciju u subkompartmentima spermatozoida. U oba tipa uzoraka, ZIP6 i ZIP14 su bili pretežno lokalizovani u glavi, dok je ZIP10 lokalizovan isključivo duž repa spermatozoida. Astenozoospermija nije uticala na lokalizaciju ni jednog od ZIP transportera u kompartmentima spermatozoida, međutim kod lokalizacije subkompartmenta u regionima glave spermatozoida, za ZIP6, astenozoospermija je snizila imunoekspresiju žir/polmesecečaste forme. ZIP14 imunolokalizacija je izmenjena u korist forme nalik polumesecu. Imunolokalizacija acetilovanog α-tubulina može doprineti heterogenosti jedara između spermatozida u odnosu na imunolokalizaciju i različitu ekspresiju i distribuciju ZIP transportera u subkompartmentima spermatozoida, a naši rezultati pokazuju da postoji heterogenost između spermatozoida u normozoospermičnim i astenozoospermičnim uzorcima. Buduća detaljna analiza tubulinskih PTM u humanim spermatozoidima mogla bi pružiti dodatne informacije o funkcijama zasnovanim na mikrotubulama u ćelijama. Mnoga pitanja ostaju vezana za mehanizam transporta Zn2+, posebno zato što je njegova zastupljenost u vezi sa različitim uslovima životne sredine, kako fiziološkim tako i patološkim i možda može poslužiti u utvrđivanju i/ili tretmanu muške neplodnosti. Usled sve zastupljenijeg individualizovanog pristupa pacijentu u medicini, dodatna istraživanja su potrebna kako bi dala doprinos identifikaciji novih biomarkera heterogenosti koji bi se mogli iskoristiti u dijagnostici u cilju poboljšanja procedura biomedicinski potpomognute oplodnje.Male infertility is the primary or contributing cause in 40% of couples' infertility cases. In addition to the current diagnosis of male infertility, the question of new ways of assessing sperm fertilizing capacity arises. Sperm motility, that is, its` role in fertilization structurally depends on the tubulin code – the expression of specific types of tubulin that build centrioles and the flagellum, and tubulin acetylation, as the most abundant post-translational modification (PTM). Functionally, sperm motility depends on zinc code as well and is regulated by expression and localization of specific zinc (Zn2+) transporters – ZIP and – ZnT. The aim of this study was to determine the cellular and molecular basis of heterogeneity of the spermatozoa at the level of tubulin and zinc code. The sperm samples that were used were from the patients that were undergoing the process intrauterine insemination and were classified according to the WHO instructions as normo- and asthenozoospermic. In the analysis, native and processed samples were used according to the modified gradient centrifugation method. Immunocytochemistry was used to show expression and localization of α-tubulin and acetylated α-tubulin by light microscopy. Immunofluorescence was used to characterize the expression and localization of α-tubulin, acetylated α-tubulin, and three different types of ZIP transporters in ejaculated sperm of normo- and asthenozoospermic sperm samples. Immunofluorescence detection showed heterogeneous localization and representation of α-tubulin and acetylated α-tubulin immunopositivity. Acetylated α-tubulin was present in the tail and nucleus of normozoospermic sperm samples. For acetylated α-tubulin, more immunopositivity was found in the tail and less in the nucleus of asthenozoospermic compared to normozoospermic samples. ZIP6, ZIP10, and ZIP14 showed heterogeneous sperm expression and different distribution in sperm subcompartments. In both types of samples, ZIP6 and ZIP14 were predominantly localized in the head, while ZIP10 was found along the tail of the sperm. Asthenozoospermia did not affect the localization of ZIPs. With the localization of the subcompartment in the regions of the sperm head, by ZIP6, asthenozoospermia decreased the immunoexpression of acorn / crescent formation. ZIP14 immunostaining was altered in favor of crescent-like formations. Immunolocalization of acetylated α-tubulin may contribute to heterogeneity of nuclei between spermatozoa with respect to immunolocalization and different expression and distribution of ZIPs in sperm subcompartments, and our results show that there is heterogeneity between spermatozoa in normozoospermic and asthenozoospermic samples. Future detailed analysis of tubulin PTMs in human sperm could provide additional information on microtubule-based functions in cells. Many issues remain related to the mechanisms of Zn2+ transport, especially because they are induced by different environmental conditions, both physiological and pathological, and may be useful in the identification and/or treatment of male infertility. Due to individualised approach to patients growing more common, additional research is needed for contribution in identifying new biomarkers of heterogeneity that could be used in diagnostics with the aim of improving biomedically assisted fertilization procedures