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Testis |
a Department of Biochemistry, Cell Biology and Histology,
b Faculty of Veterinary Medicine, and Department of Endocrinology, Faculty of Biology, University of Utrecht, 3584 CH Utrecht, The Netherlands
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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isoform) of protein phosphatase 2A (PP2A-A) was clearly expressed by fetal gonocytes but weakly expressed by adult single (As), paired (Apr), and aligned (Aal) A spermatogonia. The scaffolding subunit A of PP2A (PP2A-A) is the major subunit in the formation of a functional PP2A holoenzyme. In this study, we investigated the expression of PP2A-A during testicular development in more detail using in situ hybridization, immunohistochemistry, and Western blot with testes of rats of various ages from 16 days postcoitum (pc) to adulthood. The expression of PP2A-A was detected in fetal proliferative gonocytes at 16 days pc, declining thereafter during the quiescent period of the gonocytes. From the day of birth to the start of spermatogenesis (Day 4 postpartum [pp]), the number of PP2A-A-immunopositive gonocytes increased. At Day 4 pp, the first A1 spermatogonia appeared along the basement membrane; all were PP2A-A positive. In the adult, PP2A-A was upregulated during the differentiation of the As, Apr, and Aal spermatogonia to the A1 spermatogonia and expressed thereafter by all other spermatogonia. Spermatocytes from the pachytene stage onward and all spermatids in the adult testis also showed clear expression of PP2A-A. In Sertoli cells, PP2A-A was detected during their proliferative period at 19 days pc to 15 days pp. The presence of a functional enzyme was confirmed by the additional detection of the catalytic subunit C of PP2A using Western blot analyses at various ages during testicular development. This apparent pattern of expression of PP2A-A during testicular development suggests that PP2A may play an important role in the proliferation of distinct populations of testicular cells and during meiosis and sperm maturation.
developmental biology, phosphatases, signal transduction, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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isoform) of protein phosphatase 2A (PP2A-A) was highly expressed in gonocytes at 19 days postcoitum (pc) but was not or only weakly detectable in adult single A (As), paired A (Apr), and aligned A (Aal) spermatogonia [2]. PP2A is well known for its diverse roles in cell proliferation by downregulating activated signaling pathways through the dephosphorylation of kinases (for a review, see [3]). Hence, the regulation of the mitotic activity of the gonocytes may well be different from that of the adult As, Apr, and Aal spermatogonia, despite the close relationship of these cell types within the germ cell lineage [1]. This possible difference in regulation of mitotic activity prompted us to investigate in more detail the expression of PP2A during testicular ontogeny. Protein phosphatases have generally been classified as tyrosine and serine/threonine phosphatases, although phosphatases with a dual specificity have also been described [4]. The serine/threonine phosphatases are then classified as type 1 and type 2 protein phosphatases (for a review, see [5]). Based on their biochemical properties and cation sensitivity, the type 2 phosphatases are further divided into three classes, A (PP2A), B (PP2B), and C (PP2C) [6]. Whereas PP2A is active in the absence of cations, PP2B and PP2C require Ca2+ and Mg2+, respectively, to function.
The PP2A holoenzyme consists of a catalytic subunit C (PP2A-C) that is associated with the scaffolding A subunit of PP2A (PP2A-A). This scaffolding A subunit is essential for recruiting one of the many regulatory B subunits (PP2A-B) [6–8], because in vivo the majority of the PP2A enzyme exists as an ABC trimer. Although two isoforms ( and ß) of the A subunit exist, no functional differences have been detected [9].
The presence of PP2A-C in the adult testis has been demonstrated [10, 11], as has the presence of a number of regulatory B subunits [12–15]. For example, the mRNA of the 55-kDa PP2A-Bß has been found specifically in rat elongated spermatids, whereas the isoform was found in the testis by 5 days after birth [12]. However, the expression of PP2A-A during testicular ontogeny has not yet been described.
In this study, we investigated the expression of the PP2A holoenzyme during testicular development and spermatogenesis. Detection of the scaffolding A subunit is best suited for this purpose because the A subunit is known to combine the catalytic subunit C with the regulatory subunit B and thus is the key subunit in the formation of a functional holoenzyme. Therefore, we characterized the cellular expression of PP2A-A by performing in situ hybridizations and immunohistochemistry at multiple time points during rat testicular development. In addition, we confirmed the presence of a functional enzyme by the detection of the catalytic subunit C of PP2A at important ages during testicular development. The results provide a basis for performing more functional studies of the role of PP2A during testicular development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Timed pregnant (16 and 19 days pc) rats, male rats at the day of birth and at 1, 2, 3, 4, 11, and 15 days postpartum (pp), and adult male Wistar U:WU (CpB) rats were obtained from the Central Animal Facilities of the University of Utrecht (Utrecht, The Netherlands). To obtain adult vitamin A-deficient (VAD) animals, we used the protocol described by Van Pelt and De Rooij [16]. Pregnant Wistar rats (18–20 days pc) were fed a VAD diet (Teklad Trucking, Madison, WI). Newborn male rats received the same diet until their body weights decreased (usually at 9–11 wk pp). Spermatogenesis in the VAD rat is arrested at stage VII of the seminiferous epithelium [16], and only Sertoli cells, As, Apr, and Aal spermatogonia and some preleptotene spermatocytes are present within the seminiferous tubules [17]. Animals were used and maintained with permission of and according to regulations provided by the Animal Ethical Committee of the University of Utrecht.
Tissue Processing
Tissue processing was performed according to the method of Wilkinson and Green [18]. After dissection, the testes were fixed in 4% paraformaldehyde/PBS (w/v) at 4°C overnight, dehydrated, and embedded in paraffin. Sections (5 µm) were mounted on positively charged Superfrost slides (Superfrost/plus; Menzel Gläser, Braunschweig, Germany) and dried overnight at 37°C. Each slide contained sections of testes of all different ages investigated.
Probes
Digoxygenin (DIG) labeled sense and antisense PP2A-A RNA probes were made according to Boehringer Mannheim (Roche Molecular Biochemicals, Mannheim, Germany). A cDNA of about 2000 base pairs (bp) that was isolated from a fetal rat gonocyte cDNA library [2] was used for transcription of the probes. Sequencing of about 500 bp, starting from the poly(A)+ end of the cDNA, and alignment of this sequence to sequences present in GenBank and the database of expressed sequence tags revealed that this sequence was homologous to the Rattus norvegicus cDNA clone UI-R-E0-dd-e-09-0-UI 3', similar to gi|510468|emb|Z34955|SSPP2A65A Sus scrofa mRNA for protein phosphatase 2A 65-kDa regulatory subunit, isoform (accession gb|AA900212|AA900212).
In Situ Hybridization
In situ hybridization (ISH) was performed as described by Braissant and Wahli [19] with minor modifications. All aqueous solutions needed for ISH were made using diethylpyrocarbonate (DEPC)-treated deionized water or were treated directly with DEPC. Before hybridization, sections were deparaffinized, rehydrated, and postfixed with freshly prepared 4% paraformaldehyde in PBS (w/v, 10 min). They were then incubated in PBS containing 0.1% active DEPC (v/v) twice for 15 min under continuous agitation, followed by addition of triethanolamine (0.1 M, pH 8.0) containing 0.25% acetic anhydride (v/v; 10 min under continuous agitation) and 5x saline-sodium citrate (SSC; 15 min), all at room temperature. Slides were prehybridized for 2 h at 60°C with freshly denatured hybridization mix containing 50% deionized formamide, 5x SSC, and 1 µg/µl herring sperm DNA (Promega, Madison, WI) and hybridized for 40 h at 60°C with freshly denatured hybridization mix containing either 10 ng/µl sense or antisense DIG-labeled RNA probe. During prehybridization and hybridization, slides were covered by a glass coverslip and stored in a box saturated with 50% formamide and 5x SSC. After hybridization, sections were washed in 2x SSC at room temperature for 30 min, 2x SSC at 60°C for 60 min, and 0.1x SSC at 60°C for 60 min. Detection of the DIG-labeled RNA probe was performed as described by Van Pelt et al. [20], using mouse anti-DIG monoclonal antibody (Boehringer Mannheim) and diaminobenzidine (DAB; DAKO Corp., Carpinteria, CA) as a substrate. Slides were counterstained with haematoxylin and mounted.
Immunohistochemistry
Before immunohistochemical detection of PP2A-A, slides were deparaffinized, rehydrated, and heated three times to 98°C for 10 min in fresh 10 mM sodium citrate (pH 6.0) using a M722 microwave oven (Miele & Cie, Gütersloh, Germany). Slides were then equilibrated in PBS (5 min), incubated in 0.3% H2O2/PBS (v/v) for 10 min, washed in PBS/glycine (0.15% (w/v); twice for 5 min), equilibrated in Tris-buffered saline (TBS; 0.1 M Tris/0.15 M NaCl, pH 7.6; 5 min), blocked for 1 h with TBS containing 10% rabbit serum (w/v; TBS+), and incubated overnight at 4°C with polyclonal goat IgG reactive to PP2A-A ( and ß isoforms; C-20: sc-6112; 0.5 µg/ml in TBS+; Santa Cruz Biotechnology, Santa Cruz, CA). Slides were then washed with TBS containing 0.2% Tween (v/v; TBT) for 5 min and in TBS alone (twice for 5 min), incubated with biotinylated rabbit anti-goat IgG (in TBS+; Vector Laboratories, Burlingame, CA) for 1 h at room temperature, and washed with TBT (5 min) and TBS (twice for 5 min). After incubation with peroxidase-conjugated avidin/biotin complex (in TBS; Vector) for 1 h at room temperature, washing with TBS (twice for 5 min) and TBS+ (5 min), and equilibrating with 0.05 M Tris (pH 7.6) for 10 min, the reaction was visualized using DAB as a substrate. Slides were then counterstained with haematoxylin and mounted. The specificity of the immunostaining was checked by saturating the primary antibody with a 5x concentrated blocking peptide at 4°C 18 h prior to incubation (sc-6112 p; 2.5 µg/ml in TBS+; Santa Cruz Biotechnology).
Cell Counts
At 1, 2, 3, and 4 days pp, the percentage of PP2A-A immunolabeled gonocytes was investigated. Testes of three rats per age group were used. The cell counts for PP2A-A-positive versus -negative germ cells during the first 4 days of life were performed randomly by one observer in a blinded fashion using a 100x magnifying objective. One hundred large luminal cells (gonocytes) were evaluated in three cross sections of the same testis. The small germ cells that appear along the basement membrane around the start of spermatogenesis (A1 spermatogonia) were not included in these cell counts. Cross sections were chosen that were at least 100 µm apart from each other.
SDS-PAGE and Western Blotting
Protein lysates from testes of adult rats, isolated As, Apr, and Aal spermatogonia of adult VAD rats [21], and whole testes of 4-day-old rats were prepared in RIPA buffer (PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS) including 1 mM PMSF. As, Apr, and Aal spermatogonia from VAD rats were isolated as described by van Pelt et al. [21]. Fifty micrograms of each sample was separated on a 12% SDS-polyacrylamide gel with the Mini-Protean II system (Bio-Rad, Hercules, CA). After separation, proteins were blotted onto a polyvinylidene fluoride membrane (Millipore, Bedford, MA).
Western blots were blocked using Blotto-A, containing 5% Protifar (Nutricia, Zoetermeer, The Netherlands) in TBS and 0.05% Tween-20 (TBT2). The antibodies recognizing PP2A-C (C20: sc-6110; Santa Cruz Biotechnology), PP2A-A (C20: sc-6112; Santa Cruz Biotechnology or PP19; Oxford Biomedical Research, Oxford, MI) and specifically PP2A-Aß (N20: sc-8705; Santa Cruz Biotechnology) were diluted 1:100 in Blotto-A and incubated for 1 h at room temperature. Blots were washed with TBT2. After incubation with rabbit anti-goat horseradish peroxidase (DAKO, Glostrup, Denmark) diluted 1:2000 in Blotto-A, blots were incubated with ECL (Amersham Pharmacia Biotech, Little Chalfront, U.K.) and exposed to x-ray film (RX-omat, Kodak, Paris, France).
Statistical Analyses
Statistical analyses were performed using a nonparametric ANOVA.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ISH signals for PP2A-A were detected in gonocytes at 16 (Fig. 1, A and B) and 19 days pc, whereas no signal or a very weak signal for PP2A-A was detected on the day of birth (Fig. 1, C and D) or at 4 days pp. Staining of the spermatogonia was detected at 11 and 15 days pp (Fig. 1, E and F) but not in the adult testis (Fig. 1, G and H). Spermatocytes from the pachytene stage onward and all spermatids in the adult testis showed a clear staining for PP2A-A (Fig. 1, G and H).
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At 16 days pc, the Sertoli cells were not labeled for PP2A-A. At 19 days pc and on the day of birth, all Sertoli cells were clearly stained for PP2A-A, whereas at Days 4, 11, and 15 pp, staining of Sertoli cells was much weaker. In the adult testis, no staining of the Sertoli cells was observed.
Immunohistochemistry
PP2A-A-immunolabeled gonocytes were detected at 16 (Fig. 2A) and 19 days pc, whereas on the day of birth, almost all gonocytes showed no labeling or a very weak labeling (Fig. 2B). At Day 4 pp, some gonocytes showed immunostaining for PP2A-A. However, the intensity of the gonocyte labeling differed between the individual cells (Fig. 2C). At 4 days pp, the first A1 spermatogonia appeared along the basal membrane. These small germ cells were all clearly PP2A-A positive. In the VAD testis, in which all tubules are in stage VII [16] (Fig. 2E), and in the adult testis during stages IV–VII of the seminiferous epithelium (Fig. 2F), a subset of A spermatogonia showed clear staining for PP2A-A, whereas other A spermatogonia showed no labeling or much weaker labeling. A spermatogonia without clear staining were rarely detected during stages VIII–III in the adult testis. Clear immunostaining was also detected in all intermediate (In) spermatogonia at 11 days pp onward and in all B spermatogonia at 15 days pp onward (Fig. 2D). Spermatocytes from the pachytene stage onward and all spermatids in the adult animal were clearly immunolabeled for PP2A-A (Fig. 2, F–H). No staining was observed in early spermatocytes.
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Sertoli cells at 16 days pc were not labeled for PP2A-A, whereas at 19 days pc and on the day of birth all Sertoli cells showed clear staining for PP2A-A (Fig. 2C). At 4, 11, and 15 days pp, a much weaker signal was found, whereas in the adult testis no immunolabeling was detected in the Sertoli cells.
Cell Counts
The results of PP2A-A immunolabeling suggest an increase in the percentage of PP2A-A-expressing gonocytes during the development of the neonatal testis. To confirm this increase, the percentage of PP2A-A positive gonocytes in the neonatal testis at 1, 2, 3, and 4 days pp was determined. At Day 1 pp, the percentage of PP2A-A immunolabeled gonocytes was 6.1% ± 1.2% (mean ± SD). This percentage significantly increased to 19.2% ± 1.5% at Day 2 pp, 28.3% ± 4.0% at Day 3 pp, and 40.7% ± 1.6% at Day 4 pp (Fig. 3).
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SDS-PAGE and Western Blotting
The immunohistochemistry results for PP2A-A were confirmed by Western blot analysis using extracts from adult rat testes, isolated As, Apr, and Aal spermatogonia, and whole testes of 4-day-old rats (Fig. 4A). PP2A-A was detected at the expected size of 55 kDa (according to the manufacturer) in all investigated protein extracts (Fig. 4A). These results were confirmed using an antibody from a different company that also recognized PP2A-A at 55 kDa (data not shown). We also investigated the presence of specifically the ß isoform of PP2A-A. Only a very faint signal for PP2A-Aß could be detected in adult rat whole testis extracts (data not shown). To verify that the expressed PP2A-A subunit likely functions as a part of the PP2A holoenzyme complex, the expression of PP2A-C, the catalytic subunit of the functional holoenzyme, was also investigated. A clear signal was found for PP2A-C at the expected size of 30 kDa in all investigated protein extracts (Fig. 4B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Generally, the ISH and the immunolocalization studies showed similar results, except that type A, In, and B spermatogonia that were clearly labeled using immunohistochemistry showed no signal using ISH. These results may be explained by the expression of PP2A-Aß instead of PP2A-A by the A, In, and B spermatogonia. The PP2A-A antibody used for immunohistochemistry recognizes both PP2A-A and PP2A-Aß, whereas the RNA probe used for ISH specifically recognizes PP2A-A. Therefore, we investigated the presence of PP2A-Aß protein in lysates from adult whole testis and in testes of adult VAD rats and 4-day-old rats, using an antibody that specifically recognized PP2A-Aß. However, the Western blot analyses repeatedly showed no significant signals in these lysates. Therefore, PP2A-Aß probably is not expressed in the rat testis. Also in Xenopus testis, PP2A-A is the major PP2A-A isoform, as detected by Northern blotting [22]. Most likely, our ISH data are the result of such a small amount of PP2A-A mRNA in the A, In, and B spermatogonia that it was below the detection level of this technique [2].
Most but not all A spermatogonia in the adult testis were immunolabeled for PP2A-A. A spermatogonia without PP2A-A immunolabeling were particularly detected during stages IV–VII of the seminiferous epithelium. In testes of VAD rats, in which spermatogenesis is arrested at stage VII just prior to the appearance of the A1 spermatogonia, labeled and unlabeled As, Apr, and Aal spermatogonia are present. Although As, Apr, and Aal spermatogonia are present throughout all stages of spermatogenesis, during stages IV–VII, As, Apr, and Aal spermatogonia are the only A spermatogonia present. During stages VIII–III, the more differentiated A1, A2, A3, or A4 spermatogonia, which cannot be discriminated from the As, Apr, and Aal spermatogonia, by far outnumber the As, Apr, and Aal spermatogonia [23, 24]. During these stages however, hardly any unlabeled A spermatogonia could be detected. Hence, our results strongly indicate that PP2A-A protein is only expressed by a subset of As, Apr, and Aal spermatogonia, whereas the A1–A4 spermatogonia all express PP2A-A. PP2A-A appears to be upregulated during the differentiation of the As, Apr. and Aal spermatogonia to A1 spermatogonia. A similar expression pattern in the A spermatogonia has been described for the stem cell factor (SCF) receptor, c-kit [25].
On the day of birth, only a few gonocytes express PP2A-A, whereas prior to the start of spermatogenesis at 4 days pp, the number of PP2A-A-expressing gonocytes is significantly increased. PP2A-A is clearly present in a subset of these cells, as determined by counting the percentage of PP2A-A-positive gonocytes in the neonatal testis. Van Dissel-Emiliani et al. [26] concluded that the germ cells present after birth must represent the first As, Apr, and Aal spermatogonia. The results of van Haaster and de Rooij [27] indicated that the germ cells present at the start of spermatogenesis are a mixed population of As, Apr, Aal, and A1 spermatogonia [28]. The expression pattern of PP2A-A in the neonatal testis also points to the presence of a mixed population of germ cells after birth. Thus, in the neonatal testis, as in the adult testis, PP2A-A protein expression is upregulated during the differentiation of the As, Apr, and Aal spermatogonia to the A1 spermatogonia. The small germ cells that appear along the basal membrane for the first time at 4 days pp and are generally considered to represent the first A1 spermatogonia are all PP2A-A positive. A comparable expression pattern for the c-kit protein has been found prior to the start of spermatogenesis [29, 30].
The scaffolding subunit A is an essential part of the PP2A holoenzyme. It combines the catalytic subunit C with one of the many regulatory B subunits and is therefore the key subunit in the formation of a functional enzyme [31]. Western blot analysis to detect the expression of the catalytic subunit C and the scaffolding subunit A of PP2A in the testis at 4 days pp, in the adult testis, and in isolated As, Apr, and Aal spermatogonia confirmed the presence of both proteins in these extracts. Kitagawa and coworkers [10] already demonstrated the presence of PP2A-C in the rat testis. Using Northern blot analysis, they were able to detect the mRNA for this protein from 16 days pp onward. Hatano et al. [12] found PP2A-Bß mRNA in the testis at 5 days pp. Our Western blot analyses strongly indicate that the PP2A holoenzyme is already present at the start of spermatogenesis and is also expressed by the less differentiated germ cells of spermatogenesis. Recent functional studies performed by us have confirmed that a functional PP2A holoenzyme is present in the neonatal testis and during the start of spermatogenesis and that this holoenzyme has distinct functions in the germ cells and Sertoli cells at these times (unpublished results).
PP2A is particularly involved in the regulation of cell proliferation [32–34]. In the present study, PP2A-A protein was expressed by a number of proliferating cell types in the testis. The proliferating gonocytes at 16 days pc all expressed PP2A-A. This expression then declined during the quiescent phase of the gonocytes. During spermatogenic development, PP2A-A was expressed by the A1–A4 spermatogonia and the In and B spermatogonia, which are all also highly proliferative. Hence, PP2A may play a crucial role in the regulation of cell cycle progression of these cell types. A variety of growth factors are involved in the survival and proliferation of these cells (for a review, see [1]). Some of these factors bind to tyrosine kinase receptors that mediate a signaling pathway involving the extracellular regulated kinase (ERK) pathway. For example, SCF signaling in spermatogonial proliferation involves both ERK and phosphatidylinositol 3-kinase (PI3K)-dependent Akt kinase [35]. Among others, ERK and PI3K signaling pathways may both be regulated by PP2A [34, 36, 37]. Thus, PP2A has the potential to regulate the proliferation of the gonocytes and the spermatogonia via modulation of several signal transduction pathways.
In pachytene spermatocytes, the G2/M phase transition of the first meiotic division and the condensation of chiasmate bivalents during meiotic division is thought to be inhibited by PP2A [38, 39]. Okadaic acid (an inhibitor of PP2A) is able to induce the G2/M transition of meiotic division I in pachytene spermatocytes. However, preleptotene and leptotene spermatocytes are not sensitive to okadaic acid. PP2A-A was not expressed by the spermatocytes before the pachytene stage of meiosis, which supports the hypothesis that the G2/M phase transition of the first meiotic division is mediated by PP2A.
Hatano et al. [12] proposed that the 55-kDa regulatory subunit PP2A-Bß is involved in spermatid elongation. In the present study, the scaffolding subunit PP2A-A was present in spermatids. The A and B subunits probably are part of the same functional PP2A holoenzyme in these cells and mediate spermatid elongation.
PP2A-A immunolabeling in Sertoli cells could not be detected at 16 days pc but was clear at 19 days pc and on the day of birth. Immunolabeling then declined from 4 days pp to 15 days pp and was undetectable in the Sertoli cells of the adult testis. Similarly, Sertoli cell proliferation starts during the last week before birth, whereas their proliferative activity declines a few days after birth in the rat. At about 15 days pp, the Sertoli cells become quiescent [40]. Hence, PP2A may also be involved in the regulation of the proliferation of the Sertoli cells.
The expression of PP2A-A by the various cell types present in the seminiferous tubules during testicular ontogeny points to a role for PP2A in proliferation during testis development and in proliferation, meiosis, and spermatid maturation during spermatogenesis. These multiple functions of PP2A in the testis may be explained by the fact that the specificity and the actual activity of PP2A are highly dependent on the degree of phosphorylation, the type of regulatory B subunit bound to the A-C dimer, the intracellular substrates present, and the presence or absence of any modulating factors within each cell type [8, 41, 42]. The results of this study provide a basis for investigating the role of PP2A during spermatogenesis and testicular development in more detail.
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FOOTNOTES |
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Received: 22 February 2002.
First decision: 26 March 2002.
Accepted: 30 October 2002.
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REFERENCES |
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