Oncogene (2001) 20, 1343 ± 1356
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Di€erential expression and regulation of the retinoblastoma family of
proteins during testicular development and spermatogenesis: roles in the
control of germ cell proliferation, di€erentiation and apoptosis
Wei Yan1,2, Jukka Kero1,2, Janne Suominen1 and Jorma Toppari*,1,2
1
Department of Physiology, University of Turku, Kiinamyllynkatu 10, 20520, Turku, Finland; 2Department of Pediatrics,
University of Turku, Kiinamyllynkatu 10, 20520, Turku, Finland
Normal spermatogenesis is highly dependent on wellbalanced germ cell proliferation, di€erentiation, 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, di€erentiation 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 di€erentially expressed in the rat testis and they function in a
cell-type-speci®c manner during testicular development
and spermatogenesis; (2) they participate in the control
of germ cell cycle and act in a cell cycle-phase-speci®c
fashion during germ cell proliferation, and (3) they are
also involved in the regulation of apoptosis of germ cells
and Leydig cells. Oncogene (2001) 20, 1343 ± 1356.
Keywords: retinoblastoma protein family; spermatogenesis; cell cycle; apoptosis; di€erentiation
Introduction
The retinoblastoma susceptibility gene, Rb, was the ®rst
tumor suppressor gene described (Friend et al., 1987).
pRb has been well characterized as an important
regulator of cell proliferation, apoptosis and di€erentiation (for recent review see Lundberg and Weinberg,
1999; Kaelin, 1999). It functions mainly at transcriptional level by binding transcription factors (TFs) in its
*Correspondence: J Toppari, Department of Physiology, University
of Turku, Kiinamyllynkatu 10, FIN-20520, Turku, Finland
Received 13 November 2000; revised 5 January 2001; accepted 9
January 2001
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 di€erentiation-speci®c TFs, such as
Myo D and NF-IL6, and activates transcription of genes
that are required for cell cycle exit and onset of
di€erentiation 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 di€erentiation
(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 di€erentiation (Dyson, 1998; Mayol et al., 1995).
However, di€erences 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 ®nally di€erentiate into a
population of spermatozoa within the seminiferous
epithelium. Normal spermatogenesis requires well
balanced cell proliferation, di€erentiation and apoptosis. Regulation of spermatogenesis involves endocrine,
paracrine, and autocrine factors, which form a complex
Pocket proteins in the testis
W Yan et al
1344
signaling network. Germ cells receive signals that are
mediated by these factors and ®nally 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 di€erentiation 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, di€erentiation,
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-speci®c fashion, with higher levels at stages
XII ± XIV and lower levels at all other stages.
The expression pro®les 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-speci®c 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 di€erent 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 stagespeci®c expression and phosphorylation pattern in the
adult rat testis was similar to that previously reported
(Yan et al., 1997); (data not shown).
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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 magni®cations 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 magni®cations
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
32
P-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).
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 di€erentiate: 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).
The di€erential localization patterns remained as
such until adulthood and all three proteins displayed
highly stage- and cell-speci®c 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).
To see whether the undi€erentiated stem spermatogonia expressed p130, thin (3 mm) and consecutive
sections were stained with antibodies against p27kip1
Pocket proteins in the testis
W Yan et al
1345
Figure 1 Expression of mRNA and protein of pRb family members during testicular development and spermatogenesis. The
®gures 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
(a Sertoli cell-speci®c protein, Beumer et al., 1999) and
p130. Some ¯atten-shaped nuclei on the basal
membrane are p27kip1 negative, but p130-positive,
indicating that undi€erentiated spermatogonia did
express p130 (Figure 6). Furthermore these cells were
c-kit negative (Figure 6).
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Pocket proteins in the testis
W Yan et al
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Figure 2 p107 mRNA localization in the rat testis. The bright
®eld is shown in the left panel and the corresponding dark ®eld is
shown on the right. The speci®c signals are located in the basal
compartment of the seminiferous epithelium (A, A'). Higher
magni®cation (B, B') shows the speci®c 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 mm
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 signi®cantly 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 di€erent time
points under the same culture conditions as described
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Figure 3 p130 mRNA localization in the rat testis. The bright
®eld is shown in the left panel and the corresponding dark ®eld is
shown on the right. The speci®c signals are con®ned to the basal
membrane of the seminiferous epithelium (A, A'). Higher
magni®cation (B, B') displays the speci®c 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 mm
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
signi®cant change was found in p130 levels and
phosphorylation status (data not shown).
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
di€erentiating spermatogonia (Tajima et al., 1994;
Yan et al., 2000a; Yoshinaga et al., 1991). As reported
Pocket proteins in the testis
W Yan et al
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Figure 4 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 mm
previously, the number of BrdU-positive spermatogonia started to decrease at 48 h after ACK-2 administration and was signi®cantly reduced after 72 h.
Concomitantly, TUNEL-positive spermatogonia signi®cantly increased in number 48 h after ACK-2
treatment (Yan et al., 2000a). pRb level decreased
signi®cantly 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 signi®cantly
changed (data not shown).
Expression levels and phosphorylation status of pocket
proteins during spermatocyte apoptosis induced by
methoxyacetic acid (MAA)
Methoxyacetic acid (MAA) is able to induce
speci®cally 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 signi®cantly 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 signi®cantly during the same time period
(data not shown).
Expression levels and phosphorylation status of pocket
proteins in EDS-treated rat testis
Ethylene dimethane sulfonate (EDS) induces speci®cally depletion of Leydig cells through apoptosis within
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Figure 6 Immunohistochemical staining of three consecutive
sections (3 mm 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-kitnegative stem spermatogonia. All micrographs are in the same
magni®cation. Bar=50 mm
Figure 5 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 speci®c cell associations in the vertical columns
represent speci®c 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
4 days and precursor Leydig cells start to proliferate
from day 2 after EDS treatment, and ®nally, Leydig
cell population can be fully regenerated around day 40
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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).
Levels of p130 declined dramatically and it became
more hypophosphorylated during the ®rst 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
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W Yan et al
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Figure 7 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 (SCF7) of stem cell factor (100 ng/ml). Quanti®cation 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. *P50.05, as compared with controls (0 h)
Figure 8 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. *P50.05, as compared with controls
and some of them started to produce testosterone
(Figure 10D,E). Total p107 levels decreased signi®cantly and p107 became more hypophosphorylated
during days 3 ± 10 after EDS treatment (Figure
10F,G). Levels of total pRb did not change
signi®cantly, but the hyperphosphorylated form of
pRb declined signi®cantly during days 7 ± 20 after
EDS treatment (Figure 10H,I).
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Figure 9 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. *P50.05, as
compared with controls
Discussion
In the present study, we found that pocket proteins
pRb, p107, and p130 are tightly and di€erentially
regulated during testicular cell proliferation in the
immature testis and germ cell proliferation and
di€erentiation during spermatogenesis in the adult
testis. On the basis of our ®ndings, three major
conclusions could be drawn. First, three pocket
proteins act di€erentially 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-speci®c 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 di€erentiation. Thirdly, di€erent settings of changes in expression
levels and phosphorylation status of pocket proteins
correspond to germ cell proliferation, di€erentiation,
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 speci®c cellular associations at speci®c
locations of the seminiferous tubules. Each stage
contains mitotically proliferating spermatogonia, meioOncogene
tically proliferating spermatocytes and di€erentiating
spermatids. Germ cells at di€erent stages are in
di€erent phases of cell cycle. Therefore, accurate
staging enables us to determine the cell cycle phase
of a speci®c 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 di€erentiation
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 di€erentiated 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 ®nding further supports that
p130 is the main representative of the pRb family in G0state cells. However, it is noteworthy that both total
protein levels and phosphorylation status of p130 were
stage-speci®c, with higher expression levels and more
hyperphosphorylated states at stages I ± VI than at the
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W Yan et al
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Figure 10 Expression and phosphorylation status of pocket proteins in EDS-treated rat testis. (A) TUNEL staining of testes at
di€erent 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 di€erent 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
quanti®cation 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. *P50.05, as compared with controls
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W Yan et al
1352
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 identi®ed as a binding partner of HBP1, a TF
that plays an essential role during di€erentiation of
myoblasts. More and more p130 binding partners are
being discovered, which are mostly TFs and function in
regulating tissue-speci®c gene expression. It will be of
interest to see whether p130 could interact with these TFs
and thereby control testis-speci®c 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 di€erentiated 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 di€erent 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 ®nding 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 di€erentiated 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 di€erentiated 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 di€erent 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 nonradioactive 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
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have shown that pRb could interact with a number of
proteins that are responsible for terminating cell cycle
and inducing tissue-speci®c 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 stagespeci®c 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 a€ected 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 ®rst 5 days of postnatal life, mitotically
arrested gonocytes within the testicular cords re-enter
the cell cycle, di€erentiate into spermatogonia and
proliferate. At approximately P10, spermatogonia start
to di€erentiate 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 di€erential
localization in the immature and mature testes, we
found that the pocket proteins function in a cell-typespeci®c manner. In germ cells, undi€erentiated
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 undi€erentiated 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
®ndings 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 di€erentiate 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
Pocket proteins in the testis
W Yan et al
and pocket proteins function in a cell type-speci®c
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 di€erentiate 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 di€erentiate upon puberty.
This ®nding supports the current thought that p107 is
not expressed in terminally di€erentiated 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 di€erentiate 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 ®nally
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-speci®c function in
controlling Leydig cell proliferation and di€erentiation.
Stage-speci®city 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 signi®cantly 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 di€erentiate 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 re¯ect the growth-stimulating and pro-survival
e€ect of SCF. Similarly, changes in p107 levels and
phosphorylation status might be due to the similar
e€ect 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 di€erentiating 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 e€ect of SCF/c-kit on spermatogonial
proliferation is actually mediated through PI3 kinase/
Akt/p70 S6 kinase/cyclin D3/pRb pathway (BlumeJensen 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.
1353
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 speci®c
fashion remains unclear. Only p107 changed in both
protein levels and phosphorylation status, further
con®rming that it is the only pocket protein that was
expressed by spermatocytes. Signi®cantly 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 prosurvival 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
clari®ed. 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
®nding that p107 might be involved in spermatocyte
Oncogene
Pocket proteins in the testis
W Yan et al
1354
apoptosis raised the possibility that either p107 could
also interact with E2F-1, or E2F-4/5 has an apoptosisinducing e€ect 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 ®nally 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 ®rst 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, signi®cantly 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 di€erentiated 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 di€erentiation 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 con®rmed
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
Oncogene
have not been demonstrated to be able to induce
apoptosis. It is therefore intriguing to ®nd 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 di€erentially expressed in the testis and they function in a cell
type-speci®c 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 218C 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 di€erentiating 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).
Brie¯y, injection was given twice, once every 48 h. Rats that
received ACK-2 injection were sacri®ced at 24, 48, 72 and
96 h after the ®rst 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 sacri®ce. One testis was snap frozen in liquid
nitrogen and then stored at 7708C for protein preparation,
the other was ®xed overnight at 48C in 4% paraformaldehyde
(PFA) followed by dehydration and embedding into paran
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 sacri®ced 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
Pocket proteins in the testis
W Yan et al
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 sacri®ce.
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-mm-thick sections were cut from paran-embedded
testis samples and mounted onto SuperForst1 Plus glass
slides (Menzel-GlaÈser, Steinheim, Germany). The slides were
incubated at 378C overnight and then stored at 48C 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.
1355
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 (F8) 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 1006
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 b-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 signi®cant
di€erences between di€erent 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 signi®cant.
Acknowledgments
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.
Oncogene
Pocket proteins in the testis
W Yan et al
1356
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