Expression of Promyelocytic Leukemia (PML) Protein in the Mouse Developing Spermatogonia

Background: Promyelocytic leukemia (PML) as the main protein of PML nuclear bodies regulates various physiological processes such as transcription, DNA repair, apoptosis, senescence, and several signaling pathways in different cell types. It is well known that the PML protein is involved in the regulation of stem cell properties by maintaining an open chromatin conformation for the regulatory regions of the Oct4 gene. However, there is no experimental evidence for the presence and function of PML protein in the testis tissue. Results: In this study, we show the presence of PML protein in the developing mouse testis and its co-expression with the OCT4 protein. Immunohistochemical analysis of testis mirror sections shows that PML is co-expressed with the OCT4 protein in the outermost cellular layer of seminiferous tubules, where the spermatogonial stem cells are located. Conclusions: Our ndings suggest that the PML protein might be involved in the stemness of spermatogonial stem cells at different stages of its development, even before earning the ability to produce mature sperm.

. These bodies can be the right candidates to function as molecular crowding hubs for the regulation of transcription of active genes and differential gene expression during cell differentiation (Block et al., 2006). Experimental evidence shows that PML is involved in the regulation of stem cell properties by maintaining an open chromatin conformation for the promoter region of the Oct4 gene (Chuang et al., 2011). It is well known that the OCT4, SOX2, NANOG, alkaline phosphatase, protein kinase C, and other proteins, lncRNAs, and chromatin organization are important for stemness in different  (Dann et al., 2008). Thus, the presence of PML NBs in spermatogonial stem cells might be critical for the maintenance of stemness in these cells.
In the previous study, we have shown that mature sperm and oocyte cells do not contain PML nuclear bodies (Ebrahimian et al., 2010). However, these bodies appear in the developmental stages of the newly formed mouse embryo (Ebrahimian et al., 2010). This study aimed to clarify whether the PML protein is expressed and PML NBs are present in the earlier stages of sperm formation in the mouse testis. Our results indicate that the postnatal developing spermatogonial stem cells express PML proteins before going through mitosis and generation of mature sperm.

PML expression in the outermost layer of seminiferous tubules
Our immunohistochemical ndings showed that PML protein is expressed in the outermost cells of the seminiferous tubule, where developing spermatogonial stem cells are located (Fig. 1). This pattern of expression was observed at different time points after birth and during the maturation of the testis tissue.
Immunocytochemistry on McCoy B broblasts con rmed the functionality of the antibodies, while immunohistochemical analysis of liver tissue was used as a positive control experiment for the detection of PML expression using uorescence imaging.
Oct4 and PML expression in the germinal epithelium of seminiferous tubules Since PML protein seemed to be expressed in the postnatal developing spermatogonial stem cells, we analyzed the expression of OCT4 to evaluate the co-expression of these two proteins. Immunohistochemical analysis of two mirror sections and H&E staining of the third serial section revealed that OCT4 and PML proteins are co-expressed in developing spermatogonial stem cells during the postnatal development of the testis (Fig. 2). The functionality of OCT4 and PML antibodies were tested in P19 and STO cell lines, respectively.

Discussion
In the present study, we investigated the expression pattern of PML protein in the seminiferous tubules of the mouse testis during different stages of development after birth. In our previous study, the PML NBs were invisible in the mature sperm cells. However, the PML NBs were detectable after fertilization in the 2cell stage embryo (Ebrahimian et al., 2010). Here we asked whether the PML protein is expressed and PML NBs are present in the earlier stages of sperm formation in the mouse testis. We found that the expression of PML protein is limited to the outermost cell layer of the seminiferous tubules from neonatal into mature life (Fig. 1). In the mirror sections of the testis, we noticed that PML protein is co-expressed with OCT4 (Fig. 2). Considering the anatomical location of these cells and since it is known that the OCT4 protein is expressed in and essential for the self-renewal of spermatogonial stem cells (Dann et al., 2008), we can conclude that the expression of PML protein is limited to the population of developing spermatogonial stem cells. . Our ndings show that the PML protein is present in developing spermatogonial stem cells of developing mouse testis, even before earning the ability to produce mature sperms (Fig. 3). Thus, this could an evidence for its role in the stemness and gene regulatory network during postnatal development of the spermatogonial stem cells.

Conclusions
In conclusion, we have shown that PML protein is co-expressed with OCT4 in the outermost cellular layer of seminiferous tubules, where the spermatogonial stem cells are located during the development of mouse testis. The spermatogonial stem cells originate from the primordial germ cells (PGCs) and gonocytes which are tightly controlled by OCT4. Therefore, the next question would be whether PML is present in PGCs. Further studies will be needed to reveal the function of PML in the stemness of these cells.

Tissue samples
Adult male CD1 mice (weighing 200 g-250 g; 6 weeks of age) were purchased from the animal facility of Mashhad University of Medical Sciences and housed in stainless steel cages. The experiment was performed according to the guidelines for the Animal Care Committee of Ferdowsi University of Mashhad, Mashhad, Iran. Mice were kept in a controlled environment at 25 ± 3 °C, the humidity of 40-65%, 12 h light/dark cycle, and free access to food and drinking water. The testes from one-day, two-, four-, and sixweeks old and adult (> 6 weeks of age) male mice were surgically removed and immediately placed in the xator solution.
All three cell lines were maintained at 37 °C in an atmosphere of 5% CO 2 in the air.

Immunocytochemistry
All three cell lines were cultured on sterile coverslips in the culture medium and subjected to the immunocytochemical analysis. All stages of xation, permeabilization, and immunolabelling were

Histological preparations and immunohistochemistry
Testes from neonatal (postnatal day 1), juvenile (day 14 and 4 weeks old) and adult (> 6 weeks of age) mice were dissected and xed in 4% PFA in PBS for 2 hours (for neonatal testes), or overnight (for juvenile and adult testes) at RT. Fixed tissues were processed for para n embedding. For each testis tissue, three 4 µm serial sections were cut from para n-embedded blocks. Sections were depara nized in xylene (two times, 15 min each), rehydrated in graded ethanol series (2 × 100% for 10 min, 1 × 95% for 5 min, 1 × 80% for 5 min, 1 × 70% for 5 min, 1 × 50% for 5 min) and rinsed in PBS (2 × 10 min). Then slides were incubated in the sodium citrate antigen retrieval buffer (10 mM Sodium Citrate, pH, 6.00) for 20 min at 97.5 °C and allowed to cool at room temperature. After rinsing twice in water and 1x PBS, non-speci c binding sites were blocked by incubation with the blocking buffer (1 × PBS containing 1% bovine serum albumin) for 1 h at RT. Primary antibodies including mouse anti-PML and mouse anti-OCT4 (1:300) were diluted in the blocking buffer and added to tissue sections for an overnight incubation at 4 °C in a humid chamber. After two times washing in 1x PBS for 5 min, the sections were treated with 0.3% H2O2 for 15 min at RT to block the endogenous peroxidase activity. Both desired proteins, PML and OCT4, from each mouse were incubated with the secondary antibodies (goat IgG or donkey IgG) conjugated with HRP (1:500) for 30 min at room temperature. Sections were washed two times with PBS for 5 min and treated with DAB. Also, one section from each mouse was stained by hematoxylin and eosin (H&E) which was used to evaluate testicular morphology. The slides were sealed in the mounting medium and studied under a BX51 light microscope (Olympus, Japan) equipped with UPlanSApo Plan Apochromat 20X and Plan C 40X/0.65na objectives, and a digital camera (Olympus, Japan).
For Immuno uorescence staining, the dissected testes were embedded immediately in the OCT medium (Miles Scienti c, Elkhart, USA) in the cryostat chamber (-30 °C). This system provided cut sections with 5 µm thickness which mounted on the poly-L-lysine (Sigma-Aldrich, St Louis, USA) coated slides. Before staining, the slides were warmed at RT for 30 minutes and xed in 4% PFA for 20 minutes. Then, they were air-dried for 30 minutes. After washing with 1x PBS (2 × 10 min), permeabilization was performed in 0.1% Triton X-100 in PBS at RT for 10 min, which was followed by two rinses of 1x PBS. Nonspeci c binding sites in tissue sections were blocked by incubation with the blocking buffer for 1 h at RT. The mouse anti-PML (1:300) primary antibody was diluted in the blocking buffer and added to tissue sections for overnight incubation at 4 °C in a humid chamber. After incubation, the slides were washed two times (10 min each) in 1x PBS. The secondary antibody labeling was performed using the donkey anti-mouse IgG-labelled cy3 (1:500, Jackson ImmunoResearch Laboratories, Inc. West Grove, PA, USA) for 30 min at RT. Then, slides were washed two times (10 min each) in PBS. Drops of anti-fade plus DAPI was added to the immunolabelled slides. The edges of the coverslips were sealed with the nail polish and allowed to air-dry. Slides were kept in the cold (4 °C) and dark conditions before uorescence imaging. Imaging was performed using an AH3-RFC uresence microscope (Olympus, Japan) equipped with D Plan Apochromat 20X UV and D Plan Apochromat 40X UV objectives and a DP71 digital camera (Olympus, Japan). Photomicrographs were analyzed using DP controller software.

Consent for publication
No identifying patient information is included in this report.

Availability of data and material
The data used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no con ict of interest.

Funding
This study was nancially supported by Ferdowsi University of Mashhad, Iran.   An infographic depiction of PML expression and formation of PML NBs during mouse spermatogenesis, and early embryonic development. This image summarizes our ndings in a previous study (Ebrahimian et al., 2010) and this study showing that the developing spermatogonial stem cells in seminiferous tubules contain PML NBs, while mature sperm and oocyte do not show PML NBs, even after fertilization in the 1-cell-stage embryo. The rst appearance of PML NBs is in the 2-cell-stage embryo.