The expression pattern of PARP-1 and PARP-2 in the developing and adult mouse testis

Although the importance of the PARP family members in the adult testis has already been acknowledged, their expression in the developing testis has not been addressed. We performed immunohistochemistry by using PARP-1 and PARP-2 antibodies on the developing mouse testis at embryonic day (E) 15.5, E17.5, postnatal day (PN) 0, PN3, PN9, PN20 and adult. Our results showed that at embryonic and early postnatal days, the expression of PARP-1 was in the nuclei of gonocytes and spermatogonia. PARP-1 was positive in interstitial cells with nuclear localization at all studied ages. At embryonic and early postnatal days, the expression of PARP-2 was in the cytoplasm of gonocytes and spermatogonia. During the progress of spermatogenesis, PARP-2 was localized in the cytoplasm of pre-leptotene spermatocytes on PN9, in the cytoplasm of pachytene spermatocytes on PN15 and in the cytoplasm of round spermatids on PN20. In the adult, PARP-2 staining can still be observed in the cytoplasm of spermatogonia, but to a much lesser degree than in the round and elongating spermatids. For all the studied ages, PARP-2 was positive in Sertoli cells and interstitial cells with cytoplasmic localization. Our results indicate that PARP proteins are present in germ and somatic cells during testis development in mice.

Introduction

Poly(ADP-ribose) polymerases (PARPs) are enzymes that trans- fer ADP-ribose groups to target proteins and thereby affect various nuclear and cytoplasmic processes. PARP-1 and PARP-2 are unique members of the PARP family, whose catalytic activity is stimulated in vitro and in vivo by DNA strand breaks, which suggests that both are involved in the cellular response after DNA damage (Ame et al., 1999; Schreiber et al., 2002). Studies over the last decade have shown that PARP-1 plays a prominent role mainly in the DNA damage repair of germ cells and apoptosis in the testicular germ cells (Whitacre et al., 1995; Di Meglio et al., 2004). During apo- ptosis, PARP-1 is cleaved into the fragments of 89 kDa and 24 kDa proteins (Gobeil et al., 2001) and the high levels of cleaved PARPs in the sperm suggests a role for PARPs in male infertility (Agarwal et al., 2009). In the case of low or moderate DNA damage, PARP-1 functions as a survival factor and is involved in the detection of DNA damage with many other checkpoint proteins (Masson et al., 1998). Although in PARP-1 knockout mice the DNA repair mechanism is still working at a reduced level, other members of the superfamily, probably PARP-2, have been proposed to compensate PARP-1 function in these animals (Ame et al., 1999). PARP-2 has been shown to be expressed at relatively high levels in fetal gonads around the time of sex determination (Schreiber et al., 2002) which begins with the expression of Sry (Gutti et al., 2008). PARP-1 pro- tein was shown consistently to be expressed both in the adult and fetal tissues of male gonads and identified as a putative Sry binding protein using an in vitro binding assay (Lau and Zhang, 1998).

It has been shown that although both PARP-1 and PARP-2 proteins are present in the primary spermatocytes of the rat, the vast majority of Poly ADP-ribose (PAR) are produced by PARP-1 (Tramontano et al., 2007). Endogenous levels of PAR have been determined at different stages of male germ cell maturation where the levels of metabolites were high in primary spermatocytes and decreased progressively during the secondary spermatocytes and spermatid stages (Atorino et al., 2001). PARP-2 deficient mice exhibit severely impaired spermatogenesis, which is characterized by massive apoptosis at the pachytene and metaphase I stages (Dantzer et al., 2006). A novel role was proposed for PARylation, which is a short-lived posttranslational modification of proteins mediated by PARP enzymes in response to DNA strand breaks, during the essential exchange of spermatid nucleoproteins (Meyer- Ficca et al., 2011). Preserving genomic integrity of germ cells is necessary in order for them to supply intact genetic information to the next generation. Although there are studies in the literature, knowledge of how germ cells preserve their genomic integrity is still limited, particularly with regard to DNA repair capacities in fetal or neonatal periods (Xu et al., 2005; Hajkova et al., 2010). Because germ line establishment involves several waves of high testicular cell proliferation activity, some DNA repair proteins may work to restore an intact DNA. A recent study has found that Rad54, an important factor in the homologous recombination pathway of DNA double-strand break repair, has a key function in maintaining genomic integrity of the developing germ cells (Messiaen et al., 2013).

Although the findings summarized above demonstrate the importance of the PARP family in spermatogenesis, the expression patterns of PARP-1 and PARP-2 are still unclear. This study describes the dynamic expression patterns of PARP family members in the developing and adult mouse testis using immunohistochemistry and Western blot analysis.

Material and methods Animals and tissue collection

Forty-eight adult female and eighteen adult (18 week old) male mice (Mus musculus), weighing 200–250 g, were obtained from Akdeniz University Central Animal Service. Groups of two females and one male were caged together overnight. If a positive vaginal plaque was observed on the following morning it was considered to indicate successful copulation. The vaginal plaque positive day was designated as day 0.5 of pregnancy. Pregnant mice and adult males were killed by inhalation of CO2 followed by cervical dis- location. Postnatal day (PN) 0, PN3, PN5, PN9, PN15, PN20 males were beheaded with scissors. For pregnant mice, the fetuses were removed and placed in ice-cold PBS (Sigma–Aldrich, Gillingham, Dorset, UK) before being decapitated and their testes removed by micro-dissection. Testis tissues at embryonic day (E) 15.5, E17.5 and PN0, PN3, PN5, PN9, PN15, PN20 and adult testis were collected and processed for routine histological procedures and Western blot analyses. All the collected samples were immediately fixed in Bouin’s solution for immunohistochemistry and snap frozen in liq- uid nitrogen for Western blot analysis. The experimental protocols were approved by the animal care and usage committee of Akdeniz University (approval no: 05.01.09/01-03) and were in accordance with the declaration of Helsinki and The International Association for the study of pain guidelines.

Immunohistochemistry

For immunohistochemical labeling, 5 µm thick tissue sec- tions were deparaffinized in xylene and sections were rehydrated through a decreasing gradient of ethanol. Antigen retrieval was per- formed in 50 mM glycine (pH 3.5; ≥90 ◦C maintained for 10 min)
and the rabbit polyclonal anti-PARP-1 (Ab6079; Abcam, Cam- bridge, UK), rabbit polyclonal anti-PARP-2 (ALX-210-899-R100; Alexis, BD, Franklin Lakes, NJ, USA), primary antibodies applied at 1.0–2.0 µg/ml for overnight incubation in 0.1% bovine serum albumin (BSA)/tris-buffered saline (TBS). Negative control sections received no primary antibody, but 0.1% BSA/TBS. Subsequent steps were performed at room temperature, with TBS washes between incubations. Primary antibody bindings were detected using a biotinylated anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA, USA; 1:500 dilution, 1 h) for anti-PARP-1 and anti-PARP-2 antibodies and then with the Vectastain Elite ABC kit according to the manufacturer’s instructions (Vector Laboratories). Antibody bindings were detected as a brown precipitate following
development with 3,3r-diaminobenzidine tetrahydrochloride (DAB), with Harris Hematoxylin used as counterstain. Sections were mounted under glass coverslips in Entellan® solution (Merck Millipore, Darmstadt, Germany). At least three independent sam- ples were examined for each antibody. Photomicrographs were taken using an Axioplan microscope (Zeiss, Oberkochen, Germany).

Western blot analyses

Testis tissues were weighed and put into homogenation buffer (10 mM Tris–HCl, 1 mM EDTA, 2.5% SDS, 1 mM phenylmethylsul- fonylfluoride, 1 µg/ml leupeptin) supplemented with CompleteR protease inhibitor cocktail (Boehringer, Mannheim, Germany). After being homogenized, samples were centrifuged at 10,000 × g
for 10 min. Supernatants were collected and stored at −70 ◦C.

The protein concentration was determined by Lowry assay and 50 µg protein was applied per lane. Prior to electrophoresis, samples were boiled for 5 min at 95 ◦C. Samples were subjected to SDS-polyacrylamide gel electrophoresis and then were trans- ferred onto PVDF membranes (162-0177; Bio-Rad, Hercules, CA, USA) in a buffer containing 0.2 M glycine, 25 mM Tris and 20% methanol overnight. Successful transfer was confirmed by Pon- ceau S (Sigma–Aldrich, St. Louis, MO, USA) staining of the blots. The membranes were blocked for 2 h with 5% non-fat dry milk (Bio-Rad) and 0.1% Tween-20 (Sigma–Aldrich) in 0.14 M TBS pH 7.2–7.4 at 4 ◦C. Blotting membranes were incubated for 2 h at room tempera- ture with 1:500 dilution of rabbit anti-Parp-1 (Ab6079; Abcam) and goat anti-Parp-2 (ALX-210-899-R100; Alexis). After washing steps, the membranes were further incubated with anti-rabbit (PI-1000; Vector Lab) horseradish peroxidase conjugate (diluted to 1:5,000 for 1 h at room temperature).
Labeling was visualized using the chemiluminescence’s based Super Signal CL-HRP Substrate System (Pierce, Rockford, IL, USA) and the membranes were exposed to Hyperfilm (GE Healthcare, Amersham). Beta-actin antibody with 1:3000 dilution (Ab8229; Abcam), as an internal control, was also examined to confirm equal loading of the proteins which were measured by using Image J Software and also reflected to histogram.

Results

Immunohistochemical localization of PARP-1 protein during testicular development On E15.5 and E17.5, the positive staining of PARP-1 was in the nuclei of the gonocytes (yellow arrows) and in some interstitial cells (bold black arrow) of the developing mice testis (Fig. 1a-a2 and b-b2, respectively).

On PN0, PN3 and PN5, PARP-1 was localized in the nuclei of undifferentiated spermatogonia (yellow arrows), PARP-1 nuclear staining was also observed in some interstitial cells (bold black arrow) (Fig. 1c-c2, d-d2 and e-e2 respectively).

From PN9 (Fig. 1f-f2) through to adult (Fig. 1i-i3), the expression of PARP-1 was in undifferentiated and differentiating spermato- gonia (yellow arrows) with nuclear localization. Interstitial cells continue to show nuclear staining for PARP-1 on these days (black bold arrows).

In the adult (Fig. 1i-i3), PARP-1 protein was in spermatogonia (yellow arrow), in round spermatids (blue arrow) with nuclear localization and also in the nuclei of Sertoli cells (serrated arrow). Note the nuclear interstitial staining for PARP-1 in adult (black bold arrow).

Immunohistochemical localization of PARP-2 protein during testicular development
On E15.5and E17.5, positive staining for PARP-2 was in the cyto- plasm of gonocytes (yellow arrows), in the plasma membrane of Sertoli cells (black dotted arrow) and also in some interstitial cells (serrated arrows) (Fig. 2a-a2 and b-b2, respectively). On postnatal ages, 0, 3 and 5, PARP-2 was localized in the cytoplasm of undif- ferentiated spermatogonia (yellow arrows) (Fig. 2c-c2, d-2, e-e2, respectively). The Sertoli cell plasma membrane (serrated arrows) was also stained positively with PARP-2 protein at these ages. Note some interstitial staining (black bold arrow) for PARP-2 protein on PN0, 3 and 5.

Fig. 1. PARP-1 protein expression in developing and adult mouse testis. On E15.5 (a) and E17.5 (b) PARP-1 is in the nuclei of gonocytes (yellow arrows; a2, b2) and in some interstitial cells (bold black arrow; a1, b1). On PN0 (c), PN3 (d) and PN 5 (e), PARP-1 expression is in the nuclei of undifferentiated spermatogonia (yellow arrows; c2, d2, e2) and some interstitial cells (black bold arrow; c1, d1, e1). On PN9 (f), PN 15 (g) and PN 20 (h), PARP-1 expression is in the nuclei of undifferentiated spermatogonia and differentiating spermatogonia (yellow arrows; f2, g2, h2) and some interstitial cells (black bold arrow; f1, g1, h1). In the adult (i), the expression was in the nuclei of spermatogonia (yellow arrow; i2), round spermatids (blue arrow; i1), Sertoli cells (black serrated arrow) and some interstitial cells (black bold arrow; i3). Scale bars = 50 µm, with insets = 100 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 2. PARP-2 protein expression in developing and adult mouse testis. On E15.5 (a) and E17.5 (b) PARP-2 is in the cytoplasm of gonocytes (yellow arrows; a2, b2), in the plasma membrane of Sertoli cells (black serrated arrow; a, b) and in some interstitial cells (black bold arrow; a1, b1). On PN0 (c), PN3 (d) and PN 5 (e), PARP-2 expression is in the cytoplasm of undifferentiated spermatogonia (yellow arrows; c2, d2, e2), in the plasma membrane of Sertoli cells (black serrated arrow; c, d, e) and some interstitial cells (black bold arrow; c1, d1, e1). On PN9 (f), PARP-2 was in the cytoplasm of spermatogonia (yellow arrow; f2) in the nuclei of pre-leptotene spermatocytes (white arrow; f2), in the plasma membrane of Sertoli cells (serrated arrow; f) and also in the interstitial cells (black bold arrow; f1). On PN 15 (g) and PN 20 (h), PARP-2 was in the cytoplasm of spermatogonia (yellow arrow; g, h2) in the nuclei of pachytene spermatocytes (red arrow; g2, h2), in the nuclei of round spermatids (blue arrow; h), in the plasma membrane of Sertoli cells (black dotted arrow) and also in the interstitial cells (black serrated arrow; g, h). In the adult (i), the expression was in the cytoplasm of spermatogonia (yellow arrow; i), round spermatids (blue arrow; i), elongating spermatids (green arrow; i2), Sertoli cells (black serrated arrow; i) and some interstitial cells (black bold arrow; i1). Scale bars = 50 µm, with insets = 100 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 3. Steady-state full length PARP-1, cleaved PARP-1, and PARP-2 protein levels are shown by Western blot analysis on days E15.5, E17.5, PN 0, PN 3, PN 5, PN 9, PN 15, PN 20 and adult mouse testis beta-actin antibody was used as an internal control (A). Results of Western blot analysis were summarized in the histogram (B). Each bar is a mean ± SD of 3 samples.

By PN9 (Fig. 2f), PARP-2 was localized in the cytoplasm of sper- matogonia (yellow arrow) and pre-leptotene spermatocytes (white arrow). The Sertoli cell plasma membrane (black serrated arrow) was also positive for PARP-2. By PN15 (Fig. 2g-g2), PARP-2 protein was in some spermatogonia (yellow arrow), pachytene spermato- cytes (red arrow) and also in the plasma membrane of the Sertoli cells (black serrated arrow). By PN20 (Fig. 2h-h2), the immunos- taining of PARP-2 was in the cytoplasm of some spermatogonia (yellow arrow), spermatocytes (red arrow) and also in the round spermatids (blue arrow). The Sertoli cell cytoplasm (black serrated arrow) was stained positively for PARP-2. On PN 9, 15 and 20, some interstitial cells (black bold arrows) were also positive for PARP-2. In the adult (Fig. 2i-i2), PARP-2 staining was observed in the cytoplasm of spermatogonia (yellow arrow), round spermatids (blue arrow) and elongating spermatids (green arrow). The plasma membrane of Sertoli cells (black serrated arrow) was positive for PARP-2 in the adult. Note the interstitial staining (bold black arrow) in the adult for PARP-2 protein.

Western blotting findings

Expressions of both full length and cleaved PARP-1 and PARP-2 proteins were detected during mouse testis development by using Western blot analysis (Fig. 3A). We found that both PARP pro- teins were present in whole testes samples and these findings reinforced the data acquired from immunohistochemistry. Com- parative expression of protein levels in the developing testis is shown in a histogram (Fig. 3B).

Discussion

This study investigates the localization of PARP-1 and PARP-2 proteins during different stages of testicular development and in the adult. Using the mouse model we identified the presence of PARP-1 in the nuclei of early germ cells and PARP-2 in the cytoplasm of more differentiated germ cells.

Our results show that during male germ cell maturation, PARP- 1 and PARP-2 proteins were expressed in the testicular stem cells with PARP-1 in the nucleus and PARP-2 in the cytoplasm. With the stimulation of spermatogenesis, although PARP-1 remained in the testicular stem cells, PARP-2 expression shifted to more differenti- ated cells. These results may suggest a role for PARP-1 and PARP-2 proteins in the maturation process of male germ cells where adult fertility is dependent on proper establishment and maintenance of these cells.

PARP-1 showed its expression in the nucleus of undifferentiated and differentiating spermatogonial stem cells during the stages of germ cell maturation. Its expression increased after birth at day 3 as indicated by Western blot data and may be due to the increased number of spermatogonial stem cells after birth. Moreover, since there are more stem cells proliferating in the gonad after birth, more PARP-1 might be needed for these cells to maintain their genomic integrity by repairing DNA breaks that may arise during their pro- liferation. These data may suggest an important role for PARP-1 to maintain the pool of spermatogonial cells, and its localization in the interstitial cells may further imply its contribution to the function and maturation of the somatic cells.

It has been shown that PARP-2 deficient mice exhibit severely impaired spermatogenesis at pachytene and metaphase I stages. (Dantzer et al., 2006). Our results reinforced the previous concept that PARP-2 protein contributes to the maintenance and differen- tiation of pachytene spermatocytes. We have observed that during early stages of germ cell maturation PARP-2 protein was localized in the cytoplasm of spermatogonial cells, but with the stimulation of meiotic progress, the expression shifted to the spermatocytes and spermatids. This dynamic expression pattern of PARP-2 suggests it has a different function from that of PARP-1 during germ cell mat- uration and PARP-2 is most likely related to the spermiogenesis process.
It is known that the PARP-1 is cleaved into the fragments of 89 kDa and 24 kDa during apoptosis (Gobeil et al., 2001). Cleavage of PARP-1 into the fragments of 89 kDa and 24 kDa has become a use- ful hallmark of cell death (Wang et al., 1997). After we investigated the localization of PARP family members by using immunohisto- chemistry in the developing testis, we also observed the expression of PARP-1, PARP-2 and cleaved PARP-1 by Western blot analysis. Our results indicate that the cleavage of a 113 kDa PARP to a 29 kDa fragment occurs during the various stages of testicular develop- ment and also in the adult testis. The cleavage of PARP-1 may prevent its over-activation, energy depletion, and apoptosis by pre- serving cellular energy that is required for the apoptotic mode of cell death. It is known that during the first wave of spermatogene- sis many germ cells undergo massive apoptosis at the spermatocyte stage, which appears to be essential for subsequent normal adult spermatogenesis (Rodriguez et al., 1997). Expression of cleaved PARP-1 decreased after puberty at day 15 and in adult testes as indicated by Western blot results. This may be due to the lesser necessity for the apoptotic elimination of germ cells after puberty and in adult as well when compared to the first waves of sper- matogenesis. Our findings suggest that both PARP-1 and PARP-2 may have roles in apoptosis and differentiation of the gonocytes.

The presence of PARP-2 in Sertoli cells suggest that PAR(ADP-ribose)ylation may play a role during Sertoli cell development. The detection of PARP-2 expression in Sertoli cells before and after puberty suggests a key role for poly(ADP-ribosyl)ation, presumably to safeguard DNA integrity during somatic cell maturation.

Our study demonstrates for the first time, the presence and localization of PARP-1, PARP-2 during mouse testis development. The expression of PARP-1 and PARP-2 and their cellular localization in male germ cells, support a key role for the poly(ADP-ribosyl)ation in the mouse testis. We suggest that both PARP-1 and PARP-2 and/or DNA dependent poly(ADP-ribosyl)ation may be necessary during embryonic and postnatal testicular development. Further studies are necessary to define the precise roles of PARP family members and their activation mechanisms during male germ cell development by PARP/HDAC-IN-1 using PARP inhibitors.