Differential expression and regulation of the retinoblastoma family of proteins during testicular development and spermatogenesis: roles in the control of germ cell proliferation, differentiation and apoptosis

Abstract

Normal spermatogenesis is highly dependent on well-balanced germ cell proliferation, differentiation, and apoptosis. However, the molecular mechanisms that govern these processes are largely unknown. Retinoblastoma family proteins (pRb, p107 and p130) are potentially important regulators of cell growth, differentiation and apoptosis. pRb has been shown to be expressed in the rat testis and involved in the regulation of spermatogenesis. In the present study, the expression and localization of the other two pRb family members, p107 and p130, were analysed at both mRNA and protein levels during testicular development and spermatogenesis using Northern, Western blotting, immunohistochemistry, and in situ hybridization. Furthermore, changes of levels and phosphorylation status of pRb family proteins in response to growth suppression and/or apoptosis induction were investigated using a seminiferous tubule culture system and three animal models. Our data suggest that: (1) pRb family proteins are differentially expressed in the rat testis and they function in a cell-type-specific manner during testicular development and spermatogenesis; (2) they participate in the control of germ cell cycle and act in a cell cycle-phase-specific fashion during germ cell proliferation, and (3) they are also involved in the regulation of apoptosis of germ cells and Leydig cells.

Introduction

The retinoblastoma susceptibility gene, Rb, was the first tumor suppressor gene described (Friend et al., 1987). pRb has been well characterized as an important regulator of cell proliferation, apoptosis and differentiation (for recent review see Lundberg and Weinberg, 1999; Kaelin, 1999). It functions mainly at transcriptional level by binding transcription factors (TFs) in its hypophosphorylated form. During cell cycle it acts through regulating a group of TFs that belong to E2F family. When the hypophosphorylated pRb is phosphorylated by cyclinD/CDK4/6 complexes during G1/S transition and becomes hyperphosphorylated, it loses its ability to bind E2F family of TFs. Release of E2F TFs results in activation of S phase genes and thus permits the progression of cell cycle into S phase. pRb also interacts with several differentiation-specific TFs, such as Myo D and NF-IL6, and activates transcription of genes that are required for cell cycle exit and onset of differentiation program. The discovery of two relatives of pRb, p107 and p130, added complexity to our understanding of the regulatory roles of pRb in the control of cell cycle, apoptosis, and differentiation (Huppi et al., 1996; LeCouter et al., 1996; Mayol et al., 1993; Pertile et al., 1995). These three proteins share a highly homologous region that is composed of domains A, B, and a spacer in between. It is also called pocket region and therefore they are often called pocket proteins. The pocket region is the functional domain of the proteins, since viral oncoprotein binding, tumorigenic mutations and transcription factor interaction all occur there. Intensive studies have been carried out to analyse the physiological roles of this family of proteins over the past decade. Studies using knock-out models suggest that three pocket proteins are functionally redundant and inactivation of one pocket protein could be compensated by the other two (LeCouter et al., 1998a,b; Mulligan and Jacks, 1998). On the other hand, a number of in vitro studies show that these three proteins have distinct biochemical and biological features in terms of binding partners of the E2F family TFs and timing of phosphorylation during cell cycle progression and differentiation (Dyson, 1998; Mayol et al., 1995). However, differences in their physiological roles and potential coordination among the three members of pRb family still remains poorly understood.

Spermatogenesis is a complex process in which germ cells undergo a number of mitotic divisions and two meiotic divisions, and finally differentiate into a population of spermatozoa within the seminiferous epithelium. Normal spermatogenesis requires well balanced cell proliferation, differentiation and apoptosis. Regulation of spermatogenesis involves endocrine, paracrine, and autocrine factors, which form a complex signaling network. Germ cells receive signals that are mediated by these factors and finally converge on the regulatory machinery that governs cell cycle entry, progression, and exit, as well as programmed cell death. However, the nature of the regulatory machinery is still largely unknown. Previously, we have studied the expression and phosphorylation pattern of pRb during testicular development and spermatogenesis (Yan et al., 1997). pRb appears to be involved in the regulation of spermatogonial proliferation and Sertoli cell differentiation in the adult rat testis. In the present study, we made use of rat spermatogenesis as a model to analyse: (1) how the two retinoblastoma family proteins, p107 and p130, are expressed and regulated during germ cell proliferation, differentiation, and apoptosis and (2) how their levels and phosphorylation status change during germ cell cycle progression and in response to growth-stimulating/inhibiting and apoptosis-inducing signals, both in vitro and in vivo.

Results

Expression of pocket proteins p107 and p130 during testicular development

Both p107 mRNA and protein were detected throughout the testicular development, and the expression levels of p107 mRNA and protein were correlated with each other (Figure 1A,B). Levels of both p107 mRNA and protein were higher at days 10 and 20 of postnatal life than at other ages. The levels of hyperphosphorylated form of p107 changed synchronously with those of total protein. In the adult rat testis, p107 protein was detected in all stages of the seminiferous epithelial cycle (Figure 1C). However, the hyperphosphorylated form of p107 appeared to be distributed in a stage-specific fashion, with higher levels at stages XII–XIV and lower levels at all other stages.

Figure 1
figure1

Expression of mRNA and protein of pRb family members during testicular development and spermatogenesis. The figures show: Northern analysis of p107 (A) and p130 (D) mRNA expression during testicular development; Western blotting analysis of p107 (B), p130 (E), and pRb (G) expression and phosphorylation during testicular development; expression and phosphorylation of p107 (C) and p130 (F) at 10 stages of epithelial cycle of the seminiferous tubules. The hyperphosphorylated forms of pRb, p107, and p130 are shown as pRb-p, p107-p and p130-p. Roman Numerals represent stages of the rat seminiferous epithelium. Beta-actin was used as a loading control. NB, newborn; 1d–60d ; days 1–60 after birth

The expression profiles of p130 mRNA and protein were similar to those of p107 during testicular development, with a peak at days 10–20 of postnatal life (Figure 1D,E). Levels of p130 mRNA correlated well with those of protein. p130 protein could be detected at all stages of the epithelial cycle with a stage-specific phosphorylation pattern. More hyperphosphorylated forms were present at stages I–VI than at other stages (Figure 1F).

The expression levels of pRb mRNA during testicular development and protein levels and phosphorylation status of pRb have been reported earlier (Yan et al., 1997). Here we analysed pRb expression and phosphorylation in the immature and mature rat testes using a different pRb antibody. pRb levels were continuously reduced during the testicular development, with higher levels prior to day 20 of postnatal life and lower levels afterwards (Figure 1G). The stage-specific expression and phosphorylation pattern in the adult rat testis was similar to that previously reported (Yan et al., 1997); (data not shown).

Localization of mRNAs and proteins of pRb family members in the immature and mature rat testes

In situ hybridization signals of p107 mRNA were detected near the basal compartment of seminiferous tubule (Figure 2A-A′). Higher magnifications showed that the signals were mainly located in spermatocytes and spermatogonia (Figure 2B-B′). p130 mRNA was detected near the basal membrane of the seminiferous epithelium (Figure 3A-A′). Higher magnifications revealed that they were localized in Sertoli cells and spermatogonia (Figure 3B-B′). Sense controls gave weak and homogenous background signals (Figure 2C,C′,D,D′ and Figure 3C,C′,D,D′). We also performed in situ hybridization for pRb mRNA using a 32P-UTP-labeled riboprobe and the results were similar to that of our previous study using non-radioactive in situ hybridization method (Yan et al. (1997); data not shown).

Figure 2
figure2

p107 mRNA localization in the rat testis. The bright field is shown in the left panel and the corresponding dark field is shown on the right. The specific signals are located in the basal compartment of the seminiferous epithelium (A, A′). Higher magnification (B, B′) shows the specific signals in spermatogonia (Sg) and spermatocytes (Sp). The background signals were evaluated according to the sense probe (C, C′, D, D′). Roman Numerals represent stages of the rat seminiferous epithelium. Bars=50 μm

Figure 3
figure3

p130 mRNA localization in the rat testis. The bright field is shown in the left panel and the corresponding dark field is shown on the right. The specific signals are confined to the basal membrane of the seminiferous epithelium (A, A′). Higher magnification (B, B′) displays the specific signals in Sertoli cells (Sc). The background signals were monitored according to the sense probe (C, C′, D, D′). Roman Numerals represent stages of the rat seminiferous epithelium. Bars=50 μm

In the immature rat testis at the age of 5 days of postnatal life (P5), pRb was detected in a proportion of Sertoli cells and spermatogonia; p107 was found in all Sertoli cells and some spermatogonia; p130 was expressed in all Sertoli cells, spermatogonia, and Leydig cells (1st panel, Figure 4). Upon P20, all three proteins were expressed in Sertoli cells at a higher level and in spermatogonia and spermatocytes at a lower level (2nd panel, Figure 4). Upon p40, the localization of these three proteins started to differentiate: pRb was found in Sertoli cells and a proportion of spermatogonia; p130 was only expressed in somatic cell types including Sertoli cells, Leydig cells, and peritubular myoid cells; p107 was localized to spermatogonia and meiotically dividing germ cells including preleptotene, leptotene, pachytene spermatocytes (3rd panel, Figure 4).

Figure 4
figure4

Immunohistochemical detection of pocket proteins in the immature and mature rat testes. The ages of the testes are marked on the left. Sc, Sertoli cell; Sg, spermatogonia; Sp, spermatocyte; Lc, Leydig cell; Mc, peritubular myoid cell. Bars=50 μm

The differential localization patterns remained as such until adulthood and all three proteins displayed highly stage- and cell-specific expression pattern in the adult rat testis (4th and 5th panels, Figure 4). The localization and the mRNA and protein levels of these three pocket proteins were schematically summarized in Figure 5. In Sertoli cells at stages VII–VIII pRb expression levels were the highest, while in spermatogonia of all stages the pRb levels were relatively constant (Figures 4 and 5A). The highest levels of p107 were detected in spermatogonia of types A single (As) and A paired (Apr) at stages I–III. p107 levels were higher in spermatogonia of type A aligned (Aal), type A4 (A4), intermediate spermatogonia (In), and type B spermatogonia (B) at stages I–VI. The expression levels of p107 were relatively lower in spermatocytes than in spermatogonia. However, from preleptotene to late pachytene the expression levels of p107 increased and p107 protein expression ceased in step 1 spermatids right after meiotic cell divisions (Figures 4 and 5B). Higher expression levels of p130 were found in Sertoli cells at stages I–VI than at other stages (Figures 4 and 5C).

Figure 5
figure5

Schematic illustration of localization and expression pattern of pRb (A), p107 (B), and p130 (C) in the rat seminiferous epithelium. Cells within the frame express a pocket protein and the width of the frame represents the relative abundance of the protein. The specific cell associations in the vertical columns represent specific stages (Roman numerals) of the epithelial cycle. Sc, Sertoli cells; A1-4, type A spermatogonia; In, intermediate spermatogonia; B; type B spermatogonia; Pl, preleptotene spermatocytes; L, leptotene spermatocytes; Z, zygotene spermatocytes; P, pachytene spermatocytes; Di, diplotene spermatocytes; m, meiosis. Cells that are not marked are spermatids. Cells that undergo mitotic division are marked with asterisks

To see whether the undifferentiated stem spermatogonia expressed p130, thin (3 μm) and consecutive sections were stained with antibodies against p27kip1 (a Sertoli cell-specific protein, Beumer et al., 1999) and p130. Some flatten-shaped nuclei on the basal membrane are p27kip1 negative, but p130-positive, indicating that undifferentiated spermatogonia did express p130 (Figure 6). Furthermore these cells were c-kit negative (Figure 6).

Figure 6
figure6

Immunohistochemical staining of three consecutive sections (3 μm thick) using antibodies against p27Kip1 (A), p130 (B), and c-kit (C). Sc, Sertoli cells; Sg, spermatogonia. Two bigger arrows point to two p130-positive, but p27- and c-kit-negative stem spermatogonia. All micrographs are in the same magnification. Bar=50 μm

Expression levels and phosphorylation status of pocket proteins in the cultured seminiferous tubules in the presence or absence of SCF

When the seminiferous tubules from stage XII of the epithelial cycle were cultured in the presence of SCF (100 ng/ml), the [3H]-thymidine uptake was significantly elevated after 48 and 72 h culture in vitro in comparison to the controls (Hakovirta et al., 1999). Moreover, in the absence of SCF, the number of apoptotic germ cells was much higher than in the presence of stem cell factor during 8–72 h culture (Yan et al., 2000c). To see how the expression levels and phosphorylation status of pocket proteins change in response to SCF/c-kit-mediated growth stimulating and pro-survival signals in vitro, Western blotting analyses were performed using proteins isolated from the cultured seminiferous tubules at different time points under the same culture conditions as described previously (Yan et al., 2000c). Total levels of p107 and pRb increased and they both became more hyperphosphorylated after 48 h of culture in the presence of SCF (Figure 7). In contrast, total protein levels of p107 and pRb decreased dramatically and they became more hypophosphorylated in the absence of SCF. No significant change was found in p130 levels and phosphorylation status (data not shown).

Figure 7
figure7

Expression and phosphorylation of p107 (A) and pRb (C) in the seminiferous tubule segments at stage XII of the epithelial cycle cultured in the presence (SCF+) or absence (SCF−) of stem cell factor (100 ng/ml). Quantification of Western results of three independent experiments (B for p107 and D for pRb). Each bar represents mean±s.e.m. (n=3). ADU, arbitrary densitometric unit. *P<0.05, as compared with controls (0 h)

Expression levels and phosphorylation status of pocket proteins in response to inhibition of spermatogonial proliferation and induction of spermatogonial apoptosis by ACK-2 treatment

Administration of a monoclonal anti-c-kit antibody, ACK-2, causes reduction of BrdU-positive spermatogonia in number and induction of apoptosis of differentiating spermatogonia (Tajima et al., 1994; Yan et al., 2000a; Yoshinaga et al., 1991). As reported previously, the number of BrdU-positive spermatogonia started to decrease at 48 h after ACK-2 administration and was significantly reduced after 72 h. Concomitantly, TUNEL-positive spermatogonia significantly increased in number 48 h after ACK-2 treatment (Yan et al., 2000a). pRb level decreased significantly and it became more hypophosphorylated after 48 h of ACK-2 treatment (Figure 8A,B). Total protein levels of p107 remained unchanged, but p107 became more hypophosphorylated 72 h after the treatment (Figure 8C,D). p130 was not significantly changed (data not shown).

Figure 8
figure8

Changes of expression levels and phosphorylation of p107 (A) and pRb (C) in response to inhibition of spermatogonial proliferation and induction of spermatogonial apoptosis by ACK-2 treatment. Histograms (B for p107 and D for pRb) show quantitative analysis of the Western results of three independent experiments. Each bar represents mean±s.e.m. (n=3). ADU, arbitrary densitometric unit. *P<0.05, as compared with controls

Expression levels and phosphorylation status of pocket proteins during spermatocyte apoptosis induced by methoxyacetic acid (MAA)

Methoxyacetic acid (MAA) is able to induce specifically spermatocyte apoptosis within 24 h when applied orally at a dose of 650 mg/kg in the rat (Krishnamurthy et al., 1998; McKinnell and Sharpe, 1997; Suter et al., 1998). TUNEL staining was performed to monitor the time course of induction of spermatocyte apoptosis (Figure 9A). Spermatocytes at stages VII–XIV started to be TUNEL-positive at 12 h after administration and the number of TUNEL-positive spermatocytes peaked at 24 h after MAA treatment (Figure 9A). In the testis 12 h after MAA treatment, most of pachytene spermatocytes appeared to be weakly stained by anti-p107 antibody and some were even negative, while in the control testis pachytene spermatocytes were all strongly stained (Figure 9B). Western blotting analysis revealed that both total p107 protein levels and the hyperphosphorylated form of p107 decreased significantly at 12 and 24 h after MAA administration (Figure 9C). Levels of pRb and p130 were slightly elevated but the phosphorylation status was not changed significantly during the same time period (data not shown).

Figure 9
figure9

Changes of expression levels and phosphorylation status of p107 during spermatocyte apoptosis induced by methoxyacetic acid (MAA). (A) TUNEL staining of the rat testes at 0 h (Control), 6, 12 and 24 h after MAA treatment. (B) p107 expression in the testis 12 h after MAA administration. Arrows point to p107-negative pachytene spermatocytes at stage IX in the MAA-treated testis (right), which are strongly p107-positive in the control testes (left). (C) Western analysis of p107 levels and phosphorylation status during induction of spermatocyte apoptosis by MAA. (D) Quantitative analysis of Western blotting results from three independent experiments. Each bar represents mean±s.e.mean (n=3). ADU, arbitrary densitometric unit. *P<0.05, as compared with controls

Expression levels and phosphorylation status of pocket proteins in EDS-treated rat testis

Ethylene dimethane sulfonate (EDS) induces specifically depletion of Leydig cells through apoptosis within 4 days and precursor Leydig cells start to proliferate from day 2 after EDS treatment, and finally, Leydig cell population can be fully regenerated around day 40 after the treatment (Yan et al., 2000d). Following Leydig cell depletion, germ cells undergo massive apoptosis during days 7–15 due to androgen withdrawal (Figure 10A). BrdU incorporation study showed that spermatogonial proliferation was inhibited during days 4–20 (Figure 10B,C). After day 20, the number of BrdU-positive spermatogonia and preleptotene and leptotene spermatocytes started to recover and reached the control levels upon day 40 (Figure 10B,C).

Figure 10
figure10

Expression and phosphorylation status of pocket proteins in EDS-treated rat testis. (A) TUNEL staining of testes at different time points after EDS treatment. (B) Quantitative analysis of BrdU content in germ cells at stages VIII and I after EDS treatment. Insert A in (b) shows immunohistochemical detection of incorporated BrdU in seminiferous epithelium of stage VIII. Insert B in (b) shows a representative result of Southern-Western-based BrdU content assay for stages VIII and I. (C) Quantitative analysis of BrdU content in seminiferous tubules at different time points after EDS treatment. Insert shows a representative result of Southern-Western-based BrdU content assay. D–I Show Western analysis of protein levels, phosphorylation status, and quantification of the Western results (D, E for p130, F, G for p107, and H, I for pRb). Each bar represents mean±s.e.m. (n=3). ADU, arbitrary densitometric unit. *P<0.05, as compared with controls

Levels of p130 declined dramatically and it became more hypophosphorylated during the first 4 days after EDS administration, when Leydig cells were undergoing massive apoptosis. Total protein levels and the hyperphosphorylated form of p130 started to increase at day 20 after EDS treatment, when regenerated Leydig cell population became prominent and some of them started to produce testosterone (Figure 10D,E). Total p107 levels decreased significantly and p107 became more hypophosphorylated during days 3–10 after EDS treatment (Figure 10F,G). Levels of total pRb did not change significantly, but the hyperphosphorylated form of pRb declined significantly during days 7–20 after EDS treatment (Figure 10H,I).

Discussion

In the present study, we found that pocket proteins pRb, p107, and p130 are tightly and differentially regulated during testicular cell proliferation in the immature testis and germ cell proliferation and differentiation during spermatogenesis in the adult testis. On the basis of our findings, three major conclusions could be drawn. First, three pocket proteins act differentially during germ cell cycle progression in terms of changes in their expression levels and phosphorylation status. p107 appears to be the only pocket protein that is expressed during meiosis. Secondly, pRb family proteins function in a cell-type-specific fashion during testicular development and spermatogenesis. For example, Leydig cells only express p130 and p130 is the only pocket protein that is involved in Leydig cell proliferation and differentiation. Thirdly, different settings of changes in expression levels and phosphorylation status of pocket proteins correspond to germ cell proliferation, differentiation, and apoptosis.

Differential expression and phosphorylation of pocket proteins during germ cell cycle progression in the rat testis

Rat spermatogenesis can be divided into 14 stages according to specific cellular associations at specific locations of the seminiferous tubules. Each stage contains mitotically proliferating spermatogonia, meiotically proliferating spermatocytes and differentiating spermatids. Germ cells at different stages are in different phases of cell cycle. Therefore, accurate staging enables us to determine the cell cycle phase of a specific germ cell. For example, all pachytene spermatocytes are in G2 phase of meiosis and all type A spermatogonia are at stage I, including A single (As), A paired (Ap), A align (Aal), and A4 spermatogonia (A4), are in S phase of mitosis (de Rooij and Grootegoed, 1998). All spermatids have exited from cell cycle and all are in differentiation status (G0). Preletotene spermatocytes at stages VII–VIII are in S phase of meiosis.

p130

Our in situ hybridization and immunohistochemical staining clearly showed that p130 was only expressed in terminally differentiated somatic cells including Sertoli cells, Leydig cells, and peritubular myoid cells in the adult rat testis, suggesting that p130 may be responsible for the exit of cell cycle and maintenance of the quiescent G0-state of the cells. Previously it has been shown that cell cycle exit caused by serum deprivation leads to an accumulation of active hypophosphorylated p130 protein and p130/E2F-4 complexes in numerous cell systems (Stiegler et al., 1998). Our finding further supports that p130 is the main representative of the pRb family in G0-state cells. However, it is noteworthy that both total protein levels and phosphorylation status of p130 were stage-specific, with higher expression levels and more hyperphosphorylated states at stages I–VI than at the rest of the stages of the epithelial cycle. It implies that p130 might be involved in the regulation of function of these somatic cell types during spermatogenesis. p130 has been identified as a binding partner of HBP1, a TF that plays an essential role during differentiation of myoblasts. More and more p130 binding partners are being discovered, which are mostly TFs and function in regulating tissue-specific gene expression. It will be of interest to see whether p130 could interact with these TFs and thereby control testis-specific gene expression in testicular somatic cells.

p107

In the adult rat testis, strong expression of p107 in mitotically and meiotically cycling germ cells, and absence in quiescent and differentiated cells strongly support that p107 is the most prominent pocket protein during late G1 phase through G2/M. Interestingly, p107 levels were higher in the mitotically dividing spermatogonia than in the meiotically dividing spermatocytes. The levels of p107 increased with the progression of the meiotic cell cycle from G1 to S, G2 and M phases. These variable expression levels were more prominent in spermatogonia than in spermatocytes. This feature makes it different from pRb, which is also expressed in mitocally dividing spermatogonia and showed relatively constant levels during cell cycle progression. Expression of p107 has been shown to be strictly regulated by its E2F-dependent promoter (Nevins, 1998). Our finding appears to support a current model in which p130 can actually repress p107 expression, and accumulation of p130 upon cell cycle exit may account for the absence of p107 in quiescent or differentiated cells (Grana et al., 1998; Stiegler et al., 1998).

pRb

The retinoblastoma protein was detected not only in all mitotically dividing spermatogonia, but also in terminally differentiated Sertoli cells by using the monoclonal antibody raised against amino acids 332–344 of human pRb. This result is partially consistent with our previous data showing that pRb was mainly in the nuclei of Sertoli cells and spermatogonia and was also found in the cytoplasm of steps 14–19 spermatids using a polyclonal antibody raised against, c-terminus of pRb (Yan et al., 1997). The discrepancy appears to be caused by the different epitopes that these two antibodies were raised against, since in situ hybridization data of the present ([35S]-radioactive in situ hybridization) and previous (Dig+-based non-radioactive in situ hybridization) studies consistently showed that Rb mRNA were present in Sertoli cell, spermatogonia, and spermatocytes, as well as early spermatids. As reported in our earlier study (Yan et al., 1997), a shorter transcript (2.7 kb) of pRb mRNA could be stored in spermatocytes and early spermatids and translated into protein later in elongated spermatids. pRb expression levels did not change during cell cycle progression of spermatogonia. However, drastic elevation of pRb protein levels in Sertoli cells at stages VII–VIII may suggest a potential role in the regulation of Sertoli cell function. Numerous studies have shown that pRb could interact with a number of proteins that are responsible for terminating cell cycle and inducing tissue-specific gene expression and thereby regulating function of the tissue, such as myoD, HBP-1 (Shih et al., 1998; Tevosian et al., 1997; Yee et al., 1998). It has been recently established that in the prostate, pRb could directly bind androgen receptor (AR) and regulate AR-mediated gene transcription (Gao et al., 1999; Lu and Danielsen, 1998; Yeh et al., 1998). In the rat seminiferous epithelium, stages VII and VIII are preferentially regulated by androgens (Parvinen et al., 1986). More intriguingly, the stage-specific expression pattern of pRb in Sertoli cells (highest at stages VII–VIII) disappeared during days 2–20 after EDS treatment (data not shown), suggesting that pRb levels might be affected by androgens, since testosterone levels were undetectable during this period of time (Henriksen et al., 1995; Tena-Sempere et al., 1994). Therefore, it would be of interest to analyse whether pRb could also interact with AR in Sertoli cells and thereby regulate Sertoli cell function.

Cell type-specific expression of pocket proteins during testicular development and spermatogenesis

Rat spermatogenesis is activated soon after birth. During the first 5 days of postnatal life, mitotically arrested gonocytes within the testicular cords re-enter the cell cycle, differentiate into spermatogonia and proliferate. At approximately P10, spermatogonia start to differentiate into spermatocytes and initiate meiosis. By P20, round spermatids become evident and by P35–40, spermatozoa appear in the lumen of seminiferous tubules. Subsequent spermatogenic waves are maintained through continuous stem spermatogonial self-renewal and re-entry of the proliferating pool of spermatogonia. On the basis of differential localization in the immature and mature testes, we found that the pocket proteins function in a cell-type-specific manner. In germ cells, undifferentiated spermatogonia that are in quiescent state express p130 but not pRb. Once they are activated to enter cell cycle G1 phase, those type A spermatogonia start to express pRb and p107 instead of p130. Therefore, p130 could be a marker for undifferentiated germ cells. During mitotic cell cycle progression, both pRb and p107 were expressed and the levels of pRb remained relatively constant, but p107 levels increased continuously with the progression of cell cycle. These findings are consistent with earlier report (Garriga et al., 1998; Yan et al., 1997) and suggest that pocket proteins may function not only through phosphorylation, but through changes in expression levels as well. When type B spermatogonia differentiate into preleptotene spermatocytes and meiotic S phase starts, p107 remained to be the only pocket protein. After meiotic cell divisions, spermatocytes become haploid spermatids. Neither p130 nor p107 was detected in spermatids, but pRb could be detected as reported previously (Yan et al., 1997). p107 appears to be the only pocket protein that is expressed during meiosis and pocket proteins function in a cell type-specific manner in the rat testis.

During testicular development, all somatic cells, including Sertoli cells and Leydig cells, are actively proliferating. Sertoli cells cease proliferation and start to differentiate by P20 in the rat. In proliferating Sertoli cells, three pocket proteins were all expressed, while p107 was absent when Sertoli cells ceased proliferation and started to differentiate upon puberty. This finding supports the current thought that p107 is not expressed in terminally differentiated cells, where p130 and pRb are frequently present (Stiegler et al., 1998).

Mesenchymal stem cells, present in the earliest stage of testicular development, serve as a source of Leydig cells. Some of them differentiate into fetal type of Leydig cells, while some proliferate and give rise to adult type of Leydig cells through a series of maturation steps including progenitor Leydig cells at P14–28, immature Leydig cells at P28–56, and finally adult type of Leydig cells from p56 onward in the rat (Hardy et al., 1989). Interestingly, p130 was the only pocket protein that was expressed in Leydig cells in both immature and mature testes. During days 3–40 after EDS treatment, Leydig cell population could be regenerated by precursor Leydig cells. During precursor Leydig cell proliferation, we failed to detect either pRb or p107. P130 was steadily expressed in proliferating precursor Leydig cells. This further supports that p130 may have cell-specific function in controlling Leydig cell proliferation and differentiation. Stage-specificity of p130 expression in all somatic cells within the testis during spermatogenesis strongly implicates that p130 may regulate functions of these cells.

Stem cell factor enhances pRb expression and phosphorylation in vitro and blockade of SCF/c-kit interaction in vivo suppresses levels and phosphorylation of pRb: correlation with spermatogonial proliferation and apoptosis

It is known that in the presence of stem cell factor (SCF) [3H]-thymidine incorporation of the seminiferous tubules from stage XII was enhanced and apoptosis was suppressed significantly after 48–72 h culture in vitro in comparison to controls (Hakovirta et al., 1999). Recent studies also show that in the presence of SCF both mitotic and meiotic DNA synthesis of germ cells could be maintained in vitro for at least 72 h and germ cells could pass through meiosis and further differentiate into spermatids (Vincent et al., 1998). In the present study, pRb and p107 levels increased and they became more hyperphosphorylated during 24–72 h of culture in the presence of SCF. In contrary, their levels decreased and they became more hypophosphorylated in the absence of SCF. Since pRb is mainly expressed in proliferating spermatogonia, it is likely that the changes of pRb expression levels and phosphorylation status reflect the growth-stimulating and pro-survival effect of SCF. Similarly, changes in p107 levels and phosphorylation status might be due to the similar effect of SCF on meiotically dividing germ cells. Administration of a monoclonal anti-c-kit antibody, ACK-2, could inhibit BrdU incorporation of spermatogonia and preleptotene spermatocytes, and induces apoptosis of differentiating spermatogonia including type A1–4 spermatogonia (Yoshinaga et al., 1991). In response to ACK-2 treatment, both pRb and p107 became more hypophosphorylated, in accordance with the reduced number of BrdU-positive spermatogonia at stages I–III, IV, VI and IX–XIV and preleptotene spermatocytes at stages VII and VIII. Severely reduced levels of pRb were correlated with massive spermatogonial apoptosis and unchanged levels of p107 might be due to lack of spermatocyte apoptosis. This observation supports two recent studies showing that the stimulatory effect of SCF/c-kit on spermatogonial proliferation is actually mediated through PI3 kinase/Akt/p70 S6 kinase/cyclin D3/pRb pathway (Blume-Jensen et al., 2000; Feng et al., 2000; Kissel et al., 2000). The signaling pathway that is used for connecting SCF/c-kit interaction with germ cell apoptosis machinery remains unknown currently.

Reduced levels and phosphorylation of p107 correspond to spermatocyte apoptosis in MAA-treated rats

The apoptotic nature of the spermatocyte depletion by MAA has been shown by a number of groups (Krishnamurthy et al., 1998; McKinnell and Sharpe, 1997; Suter et al., 1998). MAA-treated rats have become a good model to study apoptosis of spermatocytes although the mechanism by which MAA induces apoptosis of spermatocytes in such a specific fashion remains unclear. Only p107 changed in both protein levels and phosphorylation status, further confirming that it is the only pocket protein that was expressed by spermatocytes. Significantly reduced levels of total protein and more hypophosphorylated status correspond to massive apoptosis of spermatocytes. Earlier we have shown that during spermatocyte apoptosis induced by MAA, pro-apoptotic members, including Bax and Bak, were upregulated and pro-survival Bcl-2 family members, e.g. Bcl-w, was down regulated (Yan et al., 2000a). Changes in p107 levels and phosphorylation status seem to support a role of p107 in protecting spermatocytes from apoptosis. A certain amount and more phosphorylated status of p107 correspond to meiotic cell cycle progression of spermatocytes. Otherwise, they may undergo apoptosis. pRb has a role in protecting cells from apoptosis (Harbour and Dean, 2000; Kasten and Giordano, 1998) although the mechanism has not been fully clarified. However, release of E2F-1 might at least partially account for the induction of apoptosis by inactivation of pRb (Kasten and Giordano, 1998). p107 and p103 preferentially bind E2F-4 and E2F-5, while pRb binds E2F-1-3. E2F-1 could initiate apoptosis, while other members of E2F family have not been shown to play a similar role. The present finding that p107 might be involved in spermatocyte apoptosis raised the possibility that either p107 could also interact with E2F-1, or E2F-4/5 has an apoptosis-inducing effect as well, or other p107 binding partner(s) may be capable of inducing apoptosis.

Reduced levels and less phosphorylated states of pocket proteins correlate with proliferation inhibition and/or apoptosis of Leydig cells and germ cells in the EDS-treated rats

Leydig cells are depleted within 4 days and testosterone declined to undetectable levels at day 2 after EDS treatment (Yan et al., 2000d). Precursor Leydig cells start to proliferate soon after mature Leydig cells undergo apoptosis, and the proliferation of Leydig cells peaks at days 2–7, day 10 and day 20 (Yan et al., 2000d). The Leydig cell population is finally regenerated around day 40 after EDS treatment. As a consequence of testosterone withdrawal, germ cells undergo massive apoptosis between days 7–15. A BrdU incorporation experiment revealed that DNA synthesis is inhibited during the same period of time. Since levels of both gonadotropins, LH and FSH, are higher than normal levels between days 2–20 after EDS treatment (Yan et al., 2000d), the germ cell abnormalities appear to result from testosterone withdrawal. It is noteworthy that during the first 2 days after EDS treatment, when massive Leydig cell apoptosis took place, only p130 declined dramatically and became more hypophosphorylated. The changes suggest that p130 might be capable of protecting Leydig cells from apoptosis in a normal situation. Since Sertoli cells and other somatic cells in the testis are morphologically and functionally normal, it is unlikely that the changes of p130 were also contributed by other somatic cells, even though it can not be excluded. More interestingly, significantly reduced levels and more hypophosphorylated states of p107 and pRb corresponded very well with inhibited DNA synthesis of germ cells and massive germ cell apoptosis during days 7–20 after EDS treatment. The changes of p107 could be ascribed to spermatogonia and spermatocytes, and changes of pRb may be due to reduced number of BrdU-positive spermatogonia and increased number of TUNEL-positive spermatogonia. It is not surprising to observe that p130 levels and phosphorylation status declined during days 7–20 after EDS treatment since precursor Leydig cells were actively proliferating and expression levels of p130 were lower than in differentiated status. However, it is indeed surprising that during precursor Leydig cells proliferation, neither p107 nor pRb was detected. Therefore, p130 appears to function alone during Leydig cell cycle progression, exit, and differentiation of precursor Leydig cells. The repopulation process of Leydig cells after EDS treatment mimics the adult-type Leydig cell development in the immature testis (Teerds, 1996). Therefore, we revisited the latter process and found that indeed neither process involved p107 or pRb. It is further confirmed that p130 is the only pocket protein that is expressed in Leydig cells at least in the rat testis. Both p107 and p130 preferentially bind E2F-4/E2F-5. However E2F-4 and -5 have not been demonstrated to be able to induce apoptosis. It is therefore intriguing to find that both p107 and p130 declined in expression levels and became more hypophosphorylated when cells undergo apoptosis, since it suggests that either E2F-4 or E2F-5 could possibly induce apoptosis, or p130 and p107 could also bind E2F-1 or other factors, which could induce apoptosis. Clearly, studies on E2F family of proteins during spermatogenesis are required to get more insight into the potential pro-survival function of p107 and p130.

Taken together, we have shown in the present study that the retinoblastoma family proteins are differentially expressed in the testis and they function in a cell type-specific way. Their levels and phosphorylation status are modulated during progression of cell cycle and apoptosis. Reduced levels and more hypophosphorylated states correspond to suppression of proliferation and/or apoptosis of germ cells and Leydig cells in the rat testis. This family of proteins may serve as markers for evaluation of cell cycle status of germ cells during spermatogenesis.

Materials and methods

Experimental animals and treatments

Sprague-Dawley male rats at the ages of 1 day, 5 days, 10 days, 20 days, 30 days, 40 days, and 2–3 months, were used as experimental animals. They were housed two per cage in a controlled environment at 21°C with a 14 h lightness and 10 h darkness cycle with free access to water and food.

All animal experiments were approved by the Turku University Committee on Ethics of Animal Experimentation.

To inhibit proliferation of spermatogonia and induce apoptosis of differentiating spermatogonia by blocking SCF/c-kit interaction (Tajima et al., 1994; Yoshinaga et al., 1991), a function-blocking anti-c-kit antibody, ACK-2 (kindly provided by Dr T Kunisada, Department of Immunology, Faculty of Medicine, Tottori University, Japan) was injected i.v. at 3.5 mg/kg BW in physiological saline. The detailed procedure was described previously (Yan et al., 2000d). Briefly, injection was given twice, once every 48 h. Rats that received ACK-2 injection were sacrificed at 24, 48, 72 and 96 h after the first injection, respectively. To monitor germ cell proliferation, 8-bromodeoxyuridine (BrdU, Boehringer Mannheim, Germany) was i.p. injected at a dose of 50 mg/kg B.W. 1 h before sacrifice. One testis was snap frozen in liquid nitrogen and then stored at −70°C for protein preparation, the other was fixed overnight at 4°C in 4% paraformaldehyde (PFA) followed by dehydration and embedding into paraffin for immunohistochemistry and TUNEL staining.

For induction of spermatocyte apoptosis, methoxyacetic acid (MAA, Aldrich Chemie, Steinheim, Germany) was diluted in physiological saline and administered orally at a dose of 650 mg/kg B.W. (Krishnamurthy et al., 1998; McKinnell and Sharpe, 1997; Suter et al., 1998). The control rats received physiological saline. The rats were sacrificed at 6, 12 and 24 h, respectively. Sample collection was performed as described above.

To study the response of pocket proteins to Leydig cell apoptosis after EDS treatment, and proliferation inhibition and apoptosis of germ cells due to testosterone withdrawal after EDS treatment, the rats were i.p. injected with a single dose of EDS (75 mg/kg BW). EDS was synthesized as previously described (Jackson and Jackson, 1984) and dissolved in dimethylsulfoxide (DMSO)-water (1 : 3, Vol./Vol.). Control animals (three rats/time point) for all time points received injection of vehicle. Rats (n=3/group) were killed by cervical dislocation under CO2 anesthesia at 1, 2, 3, 4, 7, 10, 20 and 40 days after administration of EDS. To monitor germ cell proliferation, 8-bromodeoxyuridine (BrdU, Boehringer Mannheim, Mannheim, Germany) was i.p. injected at a dose of 50 mg/kg B.W. 1 h before sacrifice. Sample collection was performed as described above.

Riboprobe preparation

The pRb cDNA was kindly provided by Dr Rene Bernards (The MGH Cancer Center, USA). A 985-bp-long fragment (SacI/PstI) was sub-cloned into pGEM 4Z vector (Promega, Madison, WI, USA) for preparation of riboprobes for Northern and in situ hybridization experiments. Plasmids containing p130 and p107 cDNA were gifts from Dr Michael A Rudnicki (McMaster University, Hamilton, Ontario, Canada) and Dr Loren J Field (Indiana University School of Medicine, Indiana, USA), respectively. A 2551-bp-long fragment from p130 cDNA and a 1845-bp-long fragment from p107 cDNA were subcloned into pGEM 4Z vector. p130/pGEM4Z (XhoI/PuvII) and p107/pGEM 4Z (DraI/PstI) were linearized for preparation of antisen/sense probes for Northern and in situ hybridization. For Northern hybridization, [32P]-UTP (Amersham, Aylesbury, UK) was used for labeling the antisense riboprobe. For in situ hybridization, [35S]-UTP (Amersham) was used for labeling both antisense and sense probes.

Northern hybridization

RNA preparation, gel fractionation, and hybridization were performed as described previously (Yan et al., 1999).

In situ hybridization

Five-μm-thick sections were cut from paraffin-embedded testis samples and mounted onto SuperForst® Plus glass slides (Menzel-Gläser, Steinheim, Germany). The slides were incubated at 37°C overnight and then stored at 4°C before use. In situ hybridization was performed as described previously (Yan et al., 2000a).

Western blotting

Western blotting was performed as described in details previously (Yan et al., 2000b). The primary antibodies used included a monoclonal mouse anti-human pRb antibody (at 1 : 500 dilution; Pharmingen, San Diego, CA, USA), a polyclonal rabbit anti-p130 antibody (at 1 : 250, C-20; Santa Cruz, CA, USA), or a polyclonal rabbit anti-p107 antibody (at 1 : 200 dilution, C-19; Santa Cruz, CA, USA), as well as a mouse anti-Actin monoclonal antibody (at 1 : 1000; ICN Biomedicals Inc., Aurora, OH, USA).

Tissue culture and stimulation with Stem cell factor

Transillumination-assisted microdisection and culture of seminiferous tubule segments from stage XII were performed as described previously (Yan et al., 2000c). SCF was purchased from Genzyme Transgenics Corp. (Cambridge, MA, USA) and the concentration used was 100 ng/ml.

Immunohistochemistry

Immunohistochemical staining was carried out as described previously (Yan et al., 2000a). The dilutions of the antibodies were 1 : 1000 for anti-pRb, 1 : 500 for anti-p130, and 1 : 250 for anti-p107. Immunohistochemical detection of incorporated BrdU was performed using a monoclonal anti-BrdU antibody (at 1 : 200; Boehringer Mannheim, Mannheim, Germany). A mouse anti-p27kip1 monoclonal antibody (F-8) and a rabbit anti-c-Kit polyclonal antibody (M-14) were purchased from Santa Cruz Biotechnology Inc. (San Diego, CA, USA). The primary antibodies preabsorbed with 100× excess amount of corresponding immunizing peptides (from the sources where the antibodies were purchased) were used for negative controls.

TUNEL staining

TUNEL staining was performed as described previously (Yan et al., 2000c).

Southern–Western-based detection of incorporated BrdU

DNA isolation, slot blotting, antibody incubation, and chemiluminescent detection were performed as described previously (Yan et al., 2000d).

Quantitative analysis of Northern hybridization and Western blotting results

Scanning and densitometric analysis were carried out as described previously (Yan et al., 2000b). After normalization to β-actin protein, the densitometric value of an extra control sample was designated as 100% (excluded from statistical analysis) and values of other controls and treated samples were expressed as the percentages of the designated one.

Statistical analysis

The values from three independent experiments were pooled for the calculation of the means and their standard errors (s.e.m.) and for one-way analysis of variance and Duncan's new multiple range test to determine the significant differences between different experimental groups by using StatView 4.51 statistic program (Abacus Concepts Inc., Berkeley, CA, USA). The P values less than 0.05 were considered statistically significant

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Acknowledgements

We would like to thank Drs Rene Bernards (The MGH Cancer Center, USA), Michael A Rudnicki (McMaster University, Hamilton, Ontario, Canada), and Loren J Field (Indiana University School of Medicine, Indiana, USA), for providing pRb, p107 and p130 cDNA plasmids respectively. Johanna Vesa is acknowledged for excellent technical assistance. This work was supported by the Academy of Finland, Turku University Central Hospital, and EU contract.

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Correspondence to Jorma Toppari.

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Yan, W., Kero, J., Suominen, J. et al. Differential expression and regulation of the retinoblastoma family of proteins during testicular development and spermatogenesis: roles in the control of germ cell proliferation, differentiation and apoptosis. Oncogene 20, 1343–1356 (2001). https://doi.org/10.1038/sj.onc.1204254

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Keywords

  • retinoblastoma protein family
  • spermatogenesis
  • cell cycle
  • apoptosis
  • differentiation

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