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Department of Cell Biology, Utrecht University Medical School (T.L.B., H.L.R.-G., L.A.C.v.d.B., D.G.d.R.), 3584 CX Utrecht, The Netherlands; Cancer Center, University of Illinois College of Medicine (H.K.), Chigaco, Illinois 60607-7173; the Departments of Urology (T.M.T.W.L.) and Radiotherapy (I.S.G., D.H.R.), Academic Hospital Utrecht, 3584 CX Utrecht, The Netherlands; and Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center (A.K.), New York, New York 10021
Address all correspondence and requests for reprints to: Dr. Tim L. Beumer, Department of Cell Biology, Utrecht University Medical School, AZU-RM G02-525, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. E-mail: t.l.beumer{at}lab.azu.nl
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In p27kip1 knockout mice, aberrations in the spermatogenic process were observed. First, an increase in the numbers of A spermatogonia was found, and second, abnormal (pre)leptotene spermatocytes were observed, some of which seemingly tried to enter a mitotic division instead of entering the meiotic prophase. These observations indicate that p27kip1 has a role in the regulation of spermatogonial proliferation, or apoptosis, and the onset of the meiotic prophase in preleptotene spermatocytes. However, as p27kip1 is only expressed in Sertoli cells, the role of p27kip1 in both spermatogonia and preleptotene spermatocytes must be indirect. Hence, part of the supportive and/or regulatory role of Sertoli cells in the spermatogenic process depends on the expression of p27kip1 in these cells. Finally, we show that the expression of p27kip1 transiently increases by a factor of 3 after x-irradiation in whole testicular lysates. Hence, p27kip1 seems to be involved in the cellular response after DNA damage.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell cycle progression depends on the activity of a series of cyclin-dependent kinase (CDK) complexes (9). These complexes consist of a catalytic subunit, a CDK, and a controlling subunit, a cyclin. The activity of the CDKs depends on the phosphorylation state, binding of cyclins, and the presence of CDK inhibitors (CDKIs) (10, 11). At least two families of CDKIs are able to modulate CDK activity during G1/S phase transition, the Kip/Cip family and the INK4 family (12, 13). Members of the INK4 family specifically bind and inhibit cyclin D-dependent kinases (14, 15, 16), whereas members of the Kip1/Cip1 family are able to bind and regulate cyclin A-, D-, and E-dependent kinases (17, 18, 19, 20, 21, 22, 23, 24, 25).
The first CDKI found was p21Cip1/Waf1, which is under the control of the tumor suppressor p53 (17, 26, 27). Recently, both the p21Cip1/Waf1 messenger RNA and protein were found to be expressed in spermatocytes, but not in spermatogonia (28, 29). Even after irradiation when a cell cycle arrest and/or apoptosis can be expected in spermatogonia, no p21Cip1/Waf1 expression was observed. It was concluded that p21Cip1/Waf1 is not involved in radiation-induced spermatogonial apoptosis (28).
p27kip1 contains a 62-amino acid-region with homologous domains to p21Cip1/Waf1 at the N-terminal region (21) and may be directly involved in cell cycle restriction point control. First, levels of p27kip1 increase when murine keratinocytes and astrocytes differentiate (30, 31). Second, fibroblasts in which p27kip1 is down-regulated fail to become quiescent (32). Third, various antimitogens, including transforming growth factor-ß in mink epithelial cells (33), rapamycin in T lymphocytes (34), and cAMP in macrophages (35), prevent mitogen-induced p27kip1 down-regulation, allowing it to associate with and inhibit the cyclin E/CDK2 complex (36).
p27kip1 knockout mice exhibit multiple organ hyperplasia and have 2-fold larger testes than their wild-type littermates (37, 38, 39). A higher pituitary tumor incidence was reported in p27kip1 knockout mice (37, 39), indicating that loss of p27kip1 may contribute to oncogenesis and tumor progression. Nevertheless, mutations of p27kip1 are rare in human tumors (40, 41).
We now have studied the expression of p27kip1 in testicular cells before and after irradiation in young and adult mice and in human testicular biopsies, using immunohistochemistry and Western blotting techniques. The results suggest a role for p27kip1 in the regulation of gonocyte and Sertoli and Leydig cell proliferation. Using adult p27kip1 knockout mice, it was found that p27kip1 expression in adult Sertoli cells is important in the regulation of the proliferation or apoptosis of undifferentiated spermatogonia and the start of the meiotic prophase.
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Materials and Methods |
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For histology and immunohistochemistry, testes were fixed in 10% neutral buffered formalin for 4 h and postfixed in diluted Bouin’s solution [71% picric acid (0.9%), 24% formaldehyde (37%), and 5% acetic acid] for 16 h at 4 C. For histological analysis of p27kip1 knockout, heterozygous, and wild-type testes in newborn mice, 1 day postpartum (pp) mouse testes were fixed in Bouin’s solution. Tissues were dehydrated and washed in 70% ethanol before embedding them in paraffin (Stemcowax, Adamas Instruments, Ameronger, The Netherlands) or Technovit 7100 (Kulzer & Co. GmbH, Wehrheim, Germany), for histological analysis. For protein isolation, testes were frozen in liquid nitrogen and stored at -80 C.
Immunohistochemistry
Paraffin sections, 5 µm thick, of testes at different
intervals after irradiation were mounted together on a silane-coated
slide to avoid slide to slide differences. At least three series of
separate animals were used. Unmasking of the epitope was established by
boiling the sections for 10 min in 0.01 M sodium citrate
using a microwave oven (H2500, Bio-Rad Laboratories, Inc.).
Endogenous peroxidase was blocked by incubation with 0.35%
H2O2 in PBS for 10 min. The slides were washed
in PBS and then incubated with 10% normal horse serum to block
nonspecific binding sites of the antibodies. Subsequently, the slides
were incubated with the primary antibody, a monoclonal
p27kip1 antibody [Neomarkers, (Lab Vision, Fremont, CA),
clone DCS-72.F6], diluted 1:200 in PBS including 5% normal horse
serum, in a humidified chamber overnight at 4 C. After extensive
washing steps in PBS, slides were incubated for 60 min with a secondary
biotinylated antimouse IgG (Elite ABC-peroxidase staining kit,
Vector Laboratories, Inc., Burlingame, CA), diluted 1:200
in PBS including 5% normal horse serum, in a humidified chamber. The
avidin-biotin complex reaction was performed according to the
manufacturer’s protocol. To visualize bound antibodies, sections were
washed in PBS and covered with 0.3 µg/µl 3,3'-diaminobenzidene in
PBS to which 0.03% H2O2 was added. Sections
were counterstained with Mayer’s hematoxylin.
Negative control sections were treated as described above, except that primary antibody was omitted during the procedure and replaced by normal rabbit serum.
Adjacent sections were used for periodic acid-Schiff (PAS) staining to identify the stages of the cycle of the seminiferous epithelium in the tubular cross-sections.
Protein gel electrophoresis and Western blotting
Total protein lysates were prepared by mincing the
testes in a membrane disrupter (microdismembrator II, B. Braun
Biotech), whereafter the cells were lysed in RIPA buffer (PBS, 1%
Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) for 30 min on
ice. Lysates were sonicated on ice and cleared by centrifugation.
Protein levels were measured using BCA analysis (Pierce Chemical Co., Rockford, IL). SDS-PAGE was performed as described by
Laemmli (42). Fifty micrograms of protein were denatured by boiling for
5 min in Laemmli-SDS sample buffer and separated on a 13% PAGE gel.
Proteins were blotted onto a polyvinylidene difluoride membrane
(Millipore Corp., Bedford, MA). After blotting, the gel
was Coomassie stained for transfer efficiency.
Western blots were blocked using Blotto-A (5% Protifar (Nutricia, Zoetermeer, The Netherlands) in Tris-buffered saline (10 mM Tris-HCl, pH 8.0, and 150 mM NaCl) including 0.05% Tween-20. Antibodies against p27kip1 [p27kip1 Ab-1, clone DCS-72.F6, mouse monoclonal, 1:200), Neomarkers; or p27(C-19), SC-528, rabbit polyclonal, 1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were diluted in Blotto-A. Incubation with a either rabbit antimouse (RAMPO, DAKO Corp., Carpenteria, CA) or goat antirabbit secondary antibody (GARPO, Pierce Chemical Co.) conjugated to horseradish peroxidase 1:10,000 in Blotto-A was performed after at least three rinses in Tris-buffered saline including 0.05% Tween-20, 5 min each.
Chemiluminescence (ECL, Amersham, Arlington Heights, IL) was used for analyzing levels of protein according to the manufacturer’s protocol. Blots were exposed to x-ray film (RX, Fuji Photo Film Co., Ltd., Tokyo, Japan). The intensity of p27kip1 protein signals was measured using an LKB Ultrascan XL enhanced laser densitometer (LKB, Bromma, Sweden).
Histological examination and
p27kip1 labeling index
For histological examination, testes were embedded in
Technovit 7100 (Kulzee & Co. GmbH, Wehrheim, Germany).
Five-micron sections were stained by the PAS reaction and
counterstained with Mayer’s hematoxylin. The stages of the cycle of
the seminiferous epithelium were classified according to the method of
Oakberg (43). Cell numbers of both A spermatogonia and preleptotene
spermatocytes in the p27kip1 knockout and wild-type C57BL/6
mice were counted in stage VIII of the seminiferous epithelium. In each
animal cell counts were performed in an area containing 500 Sertoli
cells in total.
Cell types in the developing testis were identified according to Vergouwen et al. (1). In p27kip1 immunohistochemical-stained developing and adult testis, a total of 500 Sertoli and Leydig cells were counted in 4 different animals and scored for p27kip1 staining.
Three-micron sections of p27kip1 knockout, p27kip1 heterozygous, and wild-type testes of day 1 pp newborn mice were stained by the PAS reaction and counterstained with Mayer’s hematoxylin. The numbers of gonocytes and Sertoli cells were counted in 50 tubular cross-sections in each testis. The mitotic index was obtained by determination of the percentage of gonocytes in mitosis over the total number of gonocytes in 50 tubular cross-sections using a x100 objective. The tubular diameter was determined by measuring 50 tubular cross-sections using a ocular ruler, which was calibrated using a cell finder culture slide (Microlab Holland, Leiden, The Netherlands) at a x1000 magnification. The percentage of testicular interstitium over the seminiferous tubules was determined using a raster consisting of 11 horizontal and 11 vertical lines using a x20 objective. Intersections of these lines in a total of 15 rasters/testis were determined to be interstitial or tubular, resulting in a percentage of interstitial tissue over seminiferous tubules. The volume of the testes was determined morphometrically. The area of every 20th section was determined, from which the total volume of the testis was measured, using an IBAS interactive image analysis system (Kontron/Zeiss, Eching, Germany). Sections were scanned using a b/w CCD camera (Panasonic, Haag-Techno, Den Bosch, The Netherlands), type WC-CD50 (frame size, 640 x 512 pixels; 256 gray levels) and a x1 objective.
Statistics
The statistical significance of the difference between the
p27kip1 knockout and the wild-type testes was evaluated
using Student’s t test.
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Results |
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), which were all actively
proliferating during this period. However, from E16.5 to day 3 pp,
nuclear staining of p27kip1 was observed in gonocytes (Fig. 1B), which are quiescent during that period. From E18.5 to day 3 pp,
p27kip1 staining could be determined in 20 ± 2% of
the Leydig cells. Cytoplasmic staining in interstitial cells appeared
to be nonspecific, because the control sections, to which no primary
antibody was added, also showed similar staining patterns (Fig. 1D). A
light nuclear staining was seen in 12 ± 1% of the Sertoli cells
from day E15.5 to day 3 pp. On days 3 and 4 pp, p27kip1
staining was not observed in germ cells (Fig. 1C). Some
p27kip1 staining was present in the cytoplasm of Sertoli
cells.
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, E and
F) as well as in 28 ± 2% of Leydig cell nuclei (Fig. 1E). In
contrast, spermatogonia, including A spermatogonia in epithelial stages
in which they are quiescent, did not stain for p27kip1
(Fig. 1F). In testes of p27 knockout mice, no staining was observed
(Fig. 1G).
In the adult human testis also, strong p27kip1
staining was found in Sertoli cells and Leydig cells, whereas no
p27kip1 staining was found in germ cells (Fig. 1H). When
the primary antibody was omitted, no staining could be found (Fig. 1I).
Spermatogenesis in p27kip1 knockout
mice
p27kip1-/- males are fertile, but their testes are
almost 2-fold larger than those in wild-type mice (39). Spermatogonial
cell counts in epithelial stage VIII revealed that in
p27kip1 knockout mice the number of A spermatogonia was
significantly (P < 0.05) increased by a factor 1.5
(Table 1). In addition, in stage IX
groups of spermatocytes were occasionally seen that failed to enter the
leptotene phase and were still in the preleptotene phase (Fig. 2A). Furthermore, in about 14% of the
tubular cross-sections in stage VIII abnormal (pre)leptotene
spermatocytes were seen, that tried to perform mitotic division (Table 1 and Fig. 2, B and C). In wild-type mice no abnormal (pre)leptotene
spermatocytes were observed.
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Discussion |
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In the fetal and neonatal testis, a clear correlation was found between the proliferative activity of the gonocytes and the presence of p27kip1 in these cells. In proliferating gonocytes present on E14.5 and E15.5, no p27kip1 expression was found, whereas the next day, when they had become quiescent, nuclei of gonocytes did stain for p27kip1. On day 3 after birth, after abolishment of the G0/G1 arrest and the start of spermatogenesis, p27kip1 was no longer detectable in gonocytes and newly formed spermatogonia. While p27kip1 expression is correlated with cell cycle arrest in gonocytes, the observation that these cells were also arrested in p27kip1 knockout mice suggests that this gene product may not regulate the gonocyte’s cell cycle or may play a redundant role in this process. A correlation between the proliferative activity of the spermatogonia and p27kip1 staining was not found in the adult testis. None of the various types of germ cells, including spermatogonia in G0/G1 arrest, did stain for p27kip1. This indicates that p27kip1 does not play a direct role in spermatogenesis in the adult.
We previously suggested that undifferentiated spermatogonia are identical to gonocytes (44). This hypothesis was based on two observations. First, when the morphology of these two cell types is carefully compared, there are no significant differences. Second, both cell types undergo a period of active proliferation (gonocytes E13.5–E15.5; undifferentiated spermatogonia in epithelial stages X–II) followed by a period of quiescence (gonocytes E16.5 to day 1 pp; undifferentiated spermatogonia epithelial stages III–VII). At the end of the quiescent period, in both cases differentiating type A spermatogonia are formed. However, the present data show that although gonocytes express p27kip1 during quiescence, spermatogonia do not. Consequently, although the role of p27kip1 is not known in germ cells, the similarity in behavior is no longer an argument for the supposition that gonocytes and undifferentiated spermatogonia are similar cells. Also, in view of our finding of a differentiation marker on gonocytes not present on undifferentiated spermatogonia (45), it now seems more likely that gonocytes and undifferentiated spermatogonia are different types of cells.
In the mouse, Sertoli cells proliferate from at least E14.5 until day 16 after birth (1), whereafter they terminally differentiate. As in gonocytes, p27kip1 expression correlates with the proliferative activity of the Sertoli cells. During testicular development only occasionally lightly stained Sertoli cells were seen, possibly representing cells in the G1 phase of the cell cycle (46). In the adult testis, all Sertoli cells are terminally differentiated and quiescent and heavily stain for p27kip1 in both the mouse and the human. Although this suggests that p27kip1 plays a role in the quiescence of adult Sertoli cells, in the p27kip1 knockout mice no signs of Sertoli cell proliferation were observed. Hence, the role of p27kip1 in the proliferation arrest of Sertoli cells in the adult testis is probably redundant.
In recent years, it has become abundantly clear that the number of Sertoli cells determine the amount of seminiferous epithelium and, with that, testis size. Lengthening or shortening of the period of Sertoli cell proliferation in rats by changing postnatal thyroid hormone levels (47, 48, 49) or by stimulation of Sertoli cell proliferation by knocking out the Fmr1 gene in mice (50) greatly influences adult testis size. Therefore, the increased testis size in p27kip1 knockout mice is probably caused by increased Sertoli cell proliferation. The present data indicate that the difference in testis size between p27kip1 knockout and wild-type mice does not arise untill after birth. The nature of the difference will have to be studied in further detail.
In the testes of mice from E14.5 to day 3 pp, fetal-type Leydig cells were observed. About 20% of these Leydig cells were lightly p27kip1 positive, which is in accordance with our previous observation that during this time these cells are only slowly proliferating (1). In the adult testis, approximately 28% of the adult type Leydig cells lightly stained for p27kip1. Although in the adult, Leydig cells only rarely undergo mitosis (51) the nonstaining subset of the Leydig cells may be just quiescent, whereas the p27kip1-stained Leydig cells indicate the population of terminally differentiated Leydig cells.
To investigate the role of p27kip1 in the adult testis, we studied spermatogenesis in normal and p27kip1 knockout mice. Surprisingly, a 50% increase in the number of A spermatogonia in epithelial stage VIII was found in the p27kip1 knockout testis compared with that in their wild-type littermates. As the number of A spermatogonia in stage VIII depends on the proliferative activity of the undifferentiated A spermatogonia during the preceding epithelial cycle, this suggests that this activity is enhanced in p27kip1 knockout mice or that apoptosis induction is decreased. An alternative hypothesis would be that in the p27kip1 knockout mouse, stem cell density would be increased because of enhanced proliferation of gonocytes during the fetal period due to the lack of p27kip1. The latter could lead to a higher ultimate density of gonocytes and subsequently of their daughter A spermatogonia. However, we did not find significant differences between the p27kip1 knockout and the wild-type testis on day 1 pp with respect to gonocyte density. This result does not indicate an increase in the initial numbers of spermatogonial stem cells in testes of p27kip1 knockout mice.
Also, in p27kip1 knockout mice, significant numbers of abnormal (pre)leptotene spermatocytes were seen, indicating that these cells could not properly enter the meiotic prophase. Intriguingly, some of these abnormal spermatocytes tried to carry out mitotic division instead of entering the meiotic prophase. Taken together, these results indicate that in terminally differentiated, quiescent Sertoli cells, p27kip1 plays a role in the proper functioning of the Sertoli cells in supporting and regulating the spermatogenic process. Without p27kip1 expression in Sertoli cells, there are changes in spermatogonial numbers, and some spermatocytes have difficulty entering the meiotic prophase.
The human p27kip1 gene is localized at the short arm of chromosome 12, in the 12p12–12p13.1 area (40, 52, 53), which is often the subject of multiplication and rearrangements in testicular tumors (54). One of the current hypotheses on the origin of germ cell tumors is that they have a spermatocyte origin (55). In the p27kip1 knockout testis, preleptotene spermatocytes are seen that attempt to enter mitotic division. Hence, p27kip1, via Sertoli cells, seems to play a role in the development of spermatocytes just at the time they may be vulnerable to oncogenic transformation. Therefore, p27kip1 might be a factor in male germ cell tumorigenesis. In this respect, it is important to note that in the human, too, only Sertoli cells and Leydig cells stained for p27kip1, suggesting a role for p27kip1 in the adult human testis similar to that in the mouse testis.
From in vitro studies, it has been shown that fibroblasts down-regulate p27kip1 quickly upon severe DNA damage induced by UV rays (56). In whole testicular lysates, after 4 Gy of x-rays, two waves of p27kip1 induction could be distinguished at 1.5 and 9 h postirradiation. Therefore, in vivo in the testis, p27kip1 is up-regulated rather than down-regulated as seen in the fibroblast cell line. This could be due to differences in irradiation or cell type. These transient increases in p27kip1 suggest that in vivo, p27kip1 has a fast turnover rate, and p27kip1 plays a role in the response of cells to irradiation. As also after irradiation p27kip1 is only found in terminally differentiated cells that do not go into apoptosis, the possible role for p27kip1 in the radiation response does not involve G1/S arrest or apoptosis in these cells.
In conclusion, p27kip1 levels in gonocytes and Sertoli cells were closely related to the proliferative activity of these cells. However, the role of p27kip1 in the control of the cell cycle seems to be redundant during fetal testicular development, as no differences in histology or morphology were found between the p27kip1 knockout and wild-type developing testes on the day of birth. In the adult seminiferous epithelium, p27kip1 is only seen in Sertoli cells. Nevertheless, significant effects of a lack of p27kip1 on the behavior of spermatogonia and spermatocytes were seen in p27kip1 knockout mice. This indicates that the expression of p27kip1 in Sertoli cells, apart from a possible role in inhibiting proliferation or apoptosis regulation of these cells, is important in the supportive role Sertoli cells have in spermatogenesis.
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Acknowledgments |
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Footnotes |
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Received April 27, 1998.
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