First evidence of prothymosin alpha localization in the acrosome of mammalian male gametes

Abstract

Prothymosin α (PTMA) is a highly acidic intrinsically unstructured protein. Its expression in male gonads is evolutionary conserved; in rat testis it is specifically localized in the cytoplasm of post-meiotic germ cells, in association with the developing acrosome system. In the present paper we investigated on PTMA localization inside the head of mammalian spermatozoa (SPZ). We chose a confocal approach to ascertain whether PTMA is expressed in the acrosome or in the perinuclear theca, two regions that are tightly linked and partially overlapped in the mature haploid cells. The obtained results showed that PTMA is specifically localized in the acrosome of rat epididymal SPZ; the same experimental approach evidenced, for the first time, PTMA presence in human ejaculated SPZ. A Western blot analysis on protein extracts from human sperm head fractions confirmed the confocal data and demonstrated that the peptide is specifically associated with the inner acrosomal membrane fraction. Finally, when the acrosome reaction was induced in vitro by progesterone treatment on both rat and human sperm, PTMA signal was retained in the apical region of reacted SPZ. In conclusion, this study confirms the conservation of PTMA distribution in vertebrate male gametes and strongly supports a role for this polypeptide in their physiology.

Prothymosin alpha expression and localization during the spermatogenesis ofDanio rerio

“…The expression of prothymosin α proteins in the testis was assessed with a polyclonal antibody against zebrafish Ptma (Ferrara et al , 2013), which, as verified trough bioinformatic epitope analysis, can recognize both proteins (Ptma from now on). Western blot analysis was performed on total protein extracts from zebrafish testis and rat testis, as a control: bands of the respective expected sizes of 12 kDa for the zebrafish Ptma and 13 kDa for the rat protein were obtained (Fig.…”

“…This pattern is conserved in the testis of the Mammal Rattus norvegicus , where PTMA is also clearly associated with the acrosome system throughout the stages of SPT differentiation (Ferrara et al , 2010). Finally, recent analyses on rat and human SPZ have shown that PTMA is distributed in the region of the inner acrosomal membrane, suggesting a possible role for the protein in gamete development and function (Ferrara et al , 2013).…”

“…Furthermore, their localization in the testis of Danio rerio is comparable, and it approximately matches the pattern that we and others had previously highlighted in other species (Aniello et al , 2002, Ferrara et al , 2009, 2010; Prisco et al , 2009), with a clear predilection for meiotic and post-meiotic germ cells. As for the proteins, given the unavailability of commercial antibodies specific to either Ptmaa or Ptmab, we used an antiserum (Ferrara et al , 2013) that, as verified by bioinformatic analysis, can recognize both proteins which share the same weight and are referred to as Ptma during the text. The immunohistochemical data clearly show that what we observed with the mRNAs holds true for the protein profile: in particular, Ptma absence in SPG suggests that it does not participate in the proliferation of staminal/mitotic phases, while its presence in primary SPC and in SPT supports its possible role during meiosis and/or during the subsequent stages of SPT differentiation into mature SPZ.…”

“…Antigen retrieval was performed by pressure-cooking slides for 5 min in 0.01M citrate buffer (pH 6.0). Non-specific binding sites were blocked with an appropriate normal serum diluted 1:5 in PBS containing 5% (w/v) BSA before the addition of the primary antibody (zebrafish Ptmaa polyclonal antibody; Ferrara et al , 2013) diluted 1:50 and overnight incubation at 4°C. After washing in PBS, slides were incubated for 30 min with the appropriate secondary antibody (Alexa Fluor 488 Goat anti-rabbit; Invitrogen; Paisley, UK) diluted 1:500 in the blocking mixture.…”

Digested disorder

“…Prothymosin α (PTMA) is a conserved, highly acidic IDP expressed in male gonads, which adopts a random coil conformation. 42 , 43 Using a confocal approach, Ferrara et al 44 verified expression of this protein in mammalian spermatozoa. The authors suggest that the unique composition of this protein may enable viability in a number of different cellular environments and the ability to engage in a number of different functional behaviors.…”