First Evidence of the Expression and Localization of Prothymosin α in Human Testis and Its Involvement in Testicular Cancers

Prothymosin α (PTMA) is a phylogenetically conserved polypeptide in male gonads of Vertebrates. In Mammals, it is a ubiquitous protein, and, possessing a random-coil structure, it interacts with many other partners, in both cytoplasmic and nuclear compartments. PTMA has been widely studied during cell progression in different types of cancer because of its anti-apoptotic and proliferative properties. Here, we provided the first evidence of PTMA expression and localization in human testis and in two testicular cancers (TC): classic seminoma (CS) and Leydig cell tumor (LCT). Data showed that its protein level, together with that of proliferating cell nuclear antigen (PCNA), a cell cycle progression marker, increased in both CS and LCT samples, as compared to non-pathological (NP) tissue. Moreover, in the two-cancer tissue, a decreased apoptotic rate and an increased autophagic flux was also evidenced. Results confirmed the anti-apoptotic action of PTMA, also suggesting that it can act as a switcher from apoptosis to autophagy, to favor the survival of testicular cancer cells when they develop in adverse environments. Finally, the combined data, even if they need to be further validated, add new insight into the role of PTMA in human normal and pathological testicular tissue.


Introduction
Prothymosin α (PTMA) is a small polypeptide with extremely peculiar features. Indeed, the lack of cysteines, methionine, and aromatic amino acids, as well as the presence, in its primary structure, of almost 50% of aspartic and glutamic residues [1], makes PTMA one of the most acidic proteins present in nature (isoelectric point 3.5). Moreover, the absence of secondary or tertiary structures [2] not only allows PTMA to interact with many cationic molecules but also to acquire transitional, biologically active conformations, depending on the molecule with which it interacts.
This defines the functional promiscuity of PTMA, also reflected by its ubiquitous localization in the nucleus (because of the presence of a bipartite nuclear localization signal at the C-terminal), in the cytoplasm, as well as in the extracellular compartment [3,4].
If, on one hand, the cytoplasmic role of PTMA is far from being completely understood, many reports demonstrated its crucial role for survival and proliferation of normal and cancer cells [5,6], such as squamous cell carcinoma of the breast [7], head and neck [8], hepatocellular carcinoma [9,10], lung cancer [10,11], gallbladder cancer [12], colorectal cancer [13], etc.

Tissue Samples
Testicular tissues biopsies were performed during inguinal exploration in patients with a suspect testis tumor based on clinical examination and ultrasonography. Biopsy was carried out from the suspect testicular lump, and the presence of cancer was confirmed with extemporaneous evaluation by an expert pathologist [40]. The number of collected samples was 5 NP, 10 CS, and 4 LCT. A written informed consent was obtained from all subjects involved in the study. Each tissue sample was then cut into two halves: one half was quickly immersed in liquid nitrogen and stored at −80 • C for Western blot (WB) analysis, and another one was fixed in 10% formalin for histochemical investigations. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of "Università degli Studi della Campania Luigi Vanvitelli" (protocol code 206 approved on 15 April 2019).

Immunofluorescence (IF) Analysis and TUNEL Assay
The fixed tissues were dehydrated in increasing alcohol concentrations before paraffin embedding. The 5 µm thick serial sections were stained with hematoxylin/eosin. Slides were viewed under an optical microscope (Leica DM 2500, Leica Microsystems, Wetzlar, Germany), and photographs were taken using the Leica DFC320 R2 digital Camera.
Apoptosis was examined by the Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-assay using the DeadEnd™ Fluorometric TUNEL System (#G3250; Promega Corp., Madison, WI, USA) following the manufacturer's protocol. The cell nuclei were marked with DAPI (#MBD0015; Sigma-Aldrich, Milan, Italy). IF and TUNEL-assay experiments have been performed in triplicate (the used images are just representative), and for the fluorescent signal analysis and the count of the percentage of TUNEL positive cells, 20 fields/samples, for a total or 300 NP, 600 CS and 240 LCT have been considered and analyzed.

Statistical Analysis
Data were reported as mean ± standard error (SEM). Differences between the groups were considered statistically significant at p < 0.05. Analyses were performed using one-way ANOVA; Tukey's post hoc t-test was applied when appropriate with Prism 5.0, GraphPad Software (version Prism 8.4.2; San Diego, CA, USA).

PTMA and PCNA Expression and Localization in NP, CS and LCT Testicular Tissue
WB analyses showed that PTMA was expressed in human NP testicular tissue ( Figure 1A,B). Interestingly, its protein level increased in the cancer condition of 72% in CS (p < 0.01; Figure 1 A,B) and of 23% in LCT (p < 0.05; Figure 1A,B) as compared to NP. captured with the optical microscope (LeicaDM5000 B + CTR 5000) with a UV lamp and saved with IM 1000 software (version 4.7.0).
Apoptosis was examined by the Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-assay using the DeadEnd™ Fluorometric TUNEL System (#G3250; Promega Corp., Madison, WI, USA) following the manufacturer's protocol. The cell nuclei were marked with DAPI (#MBD0015; Sigma-Aldrich, Milan, Italy). IF and TUNEL-assay experiments have been performed in triplicate (the used images are just representative), and for the fluorescent signal analysis and the count of the percentage of TUNEL positive cells, 20 fields/samples, for a total or 300 NP, 600 CS and 240 LCT have been considered and analyzed.

Statistical Analysis
Data were reported as mean ± standard error (SEM). Differences between the groups were considered statistically significant at p < 0.05. Analyses were performed using oneway ANOVA; Tukey's post hoc t-test was applied when appropriate with Prism 5.0, GraphPad Software (version Prism 8.4.2; San Diego, CA, USA).

PTMA and PCNA Expression and Localization in NP, CS and LCT Testicular Tissue
WB analyses showed that PTMA was expressed in human NP testicular tissue ( Figure 1A,B). Interestingly, its protein level increased in the cancer condition of 72% in CS (p < 0.01; Figure 1 A,B) and of 23% in LCT (p < 0.05; Figure 1A,B) as compared to NP.  Similarly, and not surprisingly, the protein level of PCNA, a common marker of DNA synthesis and, thus, of cell proliferation, increased 68% in CS (p < 0.01; Figure 1A,C) and 37% in LCT (p < 0.05; Figure 1A,C) as compared to NP. In fact, in Figure 2D, a similar tendency between PTMA levels and those of PCNA was evidenced, emphasizing that both are likely involved in the enhancement of cancer cell proliferation. To support these data, a TUNEL assay on all the tissues was performed ( Figure 2D). Data showed the presence of several apoptotic cells in NP, especially SPG and SPC ( Figure  2D and insets). As expected, in both CS (p < 0.001; Figure 2D and inset, 2E) and LCT (p < 0.01; Figure 2D and inset, 2E) samples, as compared to NP, apoptotic cells were drastically reduced, as just a few dispersed TUNEL-positive cells were still visible.

Autophagy in NP, CS and LCT Testicular Tissue
A crosstalk between apoptosis and autophagy in normal, cancer, and stressed cells/tissue has been demonstrated [45][46][47][48][49]. Verification of whether an autophagic pathway is activated in TC, LC3B and p62, autophagy markers was also analyzed (Figure 3).  This trend was confirmed by IF analysis. First, a haematoxylin-eosin staining on sections of NP, CS and LCT testicular tissue samples was performed ( Figure S1).
In NP, PTMA localized in the cytoplasm of mitotic spermatogonia (SPG; arrow; Figure 1E and inset) and in meiotic spermatocytes (SPC; arrowhead; Figure 1E) cells, with a more intense signal in the latter, as well as in differentiating spermatids (SPT; dotted arrow; Figure 1E). Moreover, a positive PTMA signal was detected also in the interstitial LC (triangle, Figure 1E). Finally, an evident PCNA staining in SPG and SPC nuclei was also observed Interestingly, in CS, a peculiar PTMA and PCNA signals was seen: indeed, in the cytoplasm and the nucleus of some seminoma cells clusters, their intensity was evident (asterisks; Figure 1E and inset), while in others they were completely absent (dots; Figure 1E). Moreover, the intermediate yellow-orange tint reflected the co-localization of the two proteins in many nuclei.
PTMA localization in the seminiferous epithelium of LCT did not differ from that observed in NP; however, it is worth noting its presence, in co-localization with PCNA; in addition, in the nucleus of the scattered SPC (arrowhead; Figure 1E and inset), it was still present among the abundant tumoral Leydig cells.
Finally, both PTMA and PCNA immunofluorescent signals showed a significant increase in CS and LCT as compared to those of NP (p < 0.01; Figure 1F,G).

Apoptotic Rate in NP, CS and LCT Testicular Tissue
To confirm the enhanced proliferation rate in CS and LCT, the apoptotic pathway was also investigated. WB analysis revealed a decreased Bax/Bcl-2 ratio in CS (p < 0.01; Figure 2A,B) and LCT (p < 0.05; Figure 2A,B) as compared to NP. An opposite trend concerning Caspase-3 protein level was observed, as it decreased in CS (p < 0.001; Figure 2A,C) and in LCT (p < 0.01; Figure 2A,C) as compared to NP.
To support these data, a TUNEL assay on all the tissues was performed ( Figure 2D). Data showed the presence of several apoptotic cells in NP, especially SPG and SPC ( Figure 2D and insets). As expected, in both CS (p < 0.001; Figure 2D and inset, Figure 2E) and LCT (p < 0.01; Figure 2D and inset, Figure 2E) samples, as compared to NP, apoptotic cells were drastically reduced, as just a few dispersed TUNEL-positive cells were still visible.

Autophagy in NP, CS and LCT Testicular Tissue
A crosstalk between apoptosis and autophagy in normal, cancer, and stressed cells/tissue has been demonstrated [45][46][47][48][49]. Verification of whether an autophagic pathway is activated in TC, LC3B and p62, autophagy markers was also analyzed (Figure 3). To support these data, a TUNEL assay on all the tissues was performed ( Figure 2D). Data showed the presence of several apoptotic cells in NP, especially SPG and SPC ( Figure  2D and insets). As expected, in both CS (p < 0.001; Figure 2D and inset, 2E) and LCT (p < 0.01; Figure 2D and inset, 2E) samples, as compared to NP, apoptotic cells were drastically reduced, as just a few dispersed TUNEL-positive cells were still visible.

Autophagy in NP, CS and LCT Testicular Tissue
A crosstalk between apoptosis and autophagy in normal, cancer, and stressed cells/tissue has been demonstrated [45][46][47][48][49]. Verification of whether an autophagic pathway is activated in TC, LC3B and p62, autophagy markers was also analyzed (Figure 3).  WB analysis showed a significant increase of LC3B-II protein level in both CS (p < 0.01; Figure 3A,B) and LCT (p < 0.05; Figure 3A,B) as compared to NP.
On the contrary, the p62 protein level showed an opposite behavior, since either in CS (p < 0.001; Figure 3A,C) and in LCT (p < 0.01; Figure 3A,C) its level decreased as compared to NP, confirming the increased rate of autophagy.
The activation of autophagy was further confirmed by LC3B IF staining, shown in Figure 3D. In NP, the signal specifically localized in some germ cell cytoplasm, principally in SPG (arrow; Figure 3D) and SPC (arrowhead; Figure 3D and inset), indicating a basal rate of autophagy.
In CS, an evident increase of LC3B signal in the cytoplasm of seminoma cells was evidenced (asterisk; Figure 3D and inset) as compared to NP (p < 0.001; Figure 3E). Finally, in LCT, besides the presence of the signal in SPG (arrow; Figure 3D) and SPC (arrowhead; Figure 3D and inset), as found in NP, it also appeared in SPT (dotted arrow; Figure 3D); LC3B fluorescence signal was increased (p < 0.01; Figure 3E) as compared to NP.

Discussion
In this paper, with the aim to expand the knowledge on the biology of CS and LCT, we analyzed the expression and localization of PTMA, not only for the first time in human testis, but also hypothesizing its putative involvement in TCs.
Indeed, if on one hand, the role of PTMA, as a consequence of its proliferative properties, in the insurgence/progression of several cancers has been extensively demonstrated [9][10][11][12][13][14], on the other, just a few papers described its function in the differentiative processes leading to spermatozoa production [25]. In our previous works, we proposed a role for PTMA in the meiotic and post-meiotic phases of spermatogenesis, due to its involvement in the acrosome biogenesis and acrosome-nuclear docking, as a result of its interaction with perinuclear theca proteins [28]. Moreover, we suggested a fascinating function for PTMA, not yet confirmed, in the fertilization events, because of its localization in the inner acrosome membrane in both rat and human sperm [29]. Indeed, PTMA shows high efficacy in decondensing human sperm chromatin; thus, following its entry into ooplasm, it may participate in pronuclear chromatin decondensation [16].
This paper provided two novelties concerning PTMA: (1) the first evidence of its expression and localization in human testis and (2) its increased protein levels in two of the most common TC: CS and LCT.
The specific localization of PTMA in SPC and SPT also in human testis not only supports the hypothesis of its involvement in spermatogenesis, but also the phylogenic importance of this polypeptide, whose role has been established ranging from non-mammalian vertebrate to humans.
Regarding PTMA and PCNA presence in the cytoplasm and the nucleus of cells in TC samples, we confirm other studies, reporting the increased PTMA expression, together with c-myc, another cell proliferation marker, in several human cancers and cell lines [19]. Thus, the proliferative and antiapoptotic properties of PTMA may contribute to the cell cycle progression also in TC. PTMA, because of its intrinsically disordered structure, can interact with many partners, both at the nuclear (as histone H1) [15][16][17][18] and cytoplasmic level. In this case, it has been reported that PTMA interacts also with apoptotic factors, as ANP32A, inhibiting the apoptosome formation [50,51]. However, we should also consider that, when apoptosis occurs, caspase-3 cleaves PTMA, generating a truncated, inactive form (aa 1-100) and the C-terminal fragment. Thus, we can hypothesize that, as in CS and LCT, apoptosis is downregulated, and there is no, or little, PTMA cleavage by caspase-3.
In addition, the altered hormonal environment may have another consequence on PTMA: indeed, in LCT samples, we also detected a specific localization signal in SPC nucleus that, on the contrary, was not visible in NP. In our previous paper, we found a comparable situation concerning the formin DAAM1 [65], a protein that was believed to be exclusively cytoplasmic [66,67], but that can be shuttled in the nucleus on the condition of stimulated germ cells proliferation [40,65]. Thus, we can speculate that, also in this case, the enhanced testosterone production and secretion may promote PTMA shuttling to the SPC nucleus to favor their proliferation. Further studies are needed to verify this point.
It is worth noting the crosstalk between apoptosis and autophagy during cancer progression. Noticeably, apoptosis downregulation is one of the most typical causes/manifestations of cancer; on the contrary, autophagy plays a double-edged sword in cancer biology: in the initial stages of cancer progression, it removes damaged cytosolic organelles and contents (as mutated DNA), acting as tumor suppressor [68]; conversely, autophagy can promote cancer cells growth and progression, functioning as an adaptive metabolic response, through recycling macromolecules, in hostile conditions, as poor vascularization, nutrient deprivation, and hypoxia in solid tumors [69].
In this context, considering that PTMA inhibits caspase activation and, consequently, may promote the switch from apoptosis to autophagy, the increased autophagic rate in CS and LCT samples, as showed in this paper, led us to hypothesize a putative role for PTMA as an anti-apoptotic and pro-autophagic factor.

Conclusions
In conclusion, the first evidence of PTMA expression and localization also in the human testis is reported here, and its increased protein level in two TC, CS and LCT. In addition, our results corroborate previous reports demonstrating the anti-apoptotic action of PTMA and, also, suggest it may act as a switcher from apoptosis to autophagy, to preserve and maintain the cell survival in the adverse environment in which cancers develop.
Although this study is preliminary, mainly due to the reduced number of the used samples, it highlighted interesting new insights into the CS and LCT biology, also supporting the significant role of PTMA in regulating cell cycle progression occurring before, during, and after normal and/or pathological cell differentiation. However, more studies are needed to validate all the results above.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biom12091210/s1, Figure S1: Hematoxylin-eosin staining of normal and pathological testicular tissues.  Informed Consent Statement: A written informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The authors confirm that the data supporting the findings of this study are available within the article.

Conflicts of Interest:
The authors declare no conflict of interest.