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Department of Veterinary Biosciences,3
Division of Nutritional Sciences,4 University of Illinois, Urbana, Illinois 61802
Curriculum in Genetics and Molecular Biology5 and
Department of Cell and Developmental Biology,6 University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599
Laboratory of Cellular Biology,7 Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, 31270-901 Belo Horizonte, Brazil
Department of Biochemistry and Molecular Genetics,8 University of Illinois, Chicago, Illinois 60607
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CDKN1A, CNKN1B, cyclin-dependent kinase inhibitors, testis, thyroid hormone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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FSH, androgen, and thyroid hormone are important regulators of Sertoli cell proliferation. FSH is mitogenic for neonatal rodent Sertoli cells [1, 2]. However, this mitogenic response is limited to an early developmental window as continual FSH exposure does not extend Sertoli cell proliferation [4] and FSH is not mitogenic for either juvenile or adult Sertoli cells [5, 6]. Mice lacking a functional androgen receptor (Tfm) or treated with an antiandrogen have reduced adult Sertoli cell number [7, 8]. Importantly, androgens appear to act indirectly because Sertoli cell numbers in mice that have a Sertoli cell-selective null mutation for the androgen receptor were not different from wild-type (WT) mice [9].
In contrast, a body of literature suggests that thyroid hormone is an inhibitor of Sertoli cell proliferation in rodents. For example, neonatal hypothyroidism results in unprecedented increases in final Sertoli cell number, adult testis size, and daily sperm production (DSP) [2, 10–12]. Hypothyroidism extends Sertoli cell proliferation, while hyperthyroidism shortens the period of Sertoli cell mitogenesis [2, 13, 14]. Because of the critical role of Sertoli cells in establishing sperm production, these increases in Sertoli cell number cause concomitant increases in the overall germ cell population and eventually sperm numbers once euthyroidism is reestablished.
Recently, we and others have shown that thyroid hormone may regulate Sertoli cell proliferation through increasing the expression of cyclin-dependent kinase inhibitors (CDKIs) [15, 16]. In mammals, progression through the cell cycle is regulated by the association of cyclins and their cyclin-dependent kinase partners. Activity of these cyclin/cyclin-dependent kinase complexes is inhibited by CDKIs that are able to bind the cyclin-dependent kinase complexes and inhibit progression through the G1 restriction point of the cell cycle. Western blotting and immunohistochemical analysis of Sertoli cells from neonatal hyperthyroid mice showed that CDKN1B levels, commonly referred to as p27Kip1, were significantly higher compared with euthyroid controls, whereas expression of p27 was lower in neonatal Sertoli cells following hypothyroidism [15]. A concomitant report showed that 6-day-old rat Sertoli cells cultured with triiodothyronine (T3) had decreased proliferation accompanied by increased expression of p27 as well as p21Cip1 (also known as CDKN1A) [16]. In combination, these data suggest that T3 effects on terminating Sertoli cell proliferation may be through stimulating p27 and p21 expression.
In addition to data indicating that T3 regulates p27, other evidence indicates that p27 may be involved in Sertoli cell proliferation. Testis expresses high levels of p27 protein, predominately in Sertoli cells [17], suggesting that its primary testicular site of p27 action is Sertoli cells. Furthermore, expression of p27 correlates closely with proliferative state of the Sertoli cell. Levels of p27 are minimal in rapidly proliferating neonatal Sertoli cells [17]. Conversely, p27 expression is maximal in postmitotic adult Sertoli cells and neoplasia in Sertoli cells is accompanied by both reinitiation of proliferation and a sharp decrease in p27 [17, 18]. These findings, along with extensive literature indicating that p27 is an important determinant of the cell's ability to remain in or exit the cell cycle, suggest that the low p27 levels during early neonatal life could allow rapid proliferation of Sertoli cells, while high p27 levels seen later may cause a cessation of Sertoli cell proliferation.
Knockout mice that lack Cdkn1b and Cdkn1a and therefore their respective proteins p27 (p27KO) or p21 (p21KO) have been generated, as well as double-knockout mice (DBKO) lacking both of these genes. In contrast to p21KO mice [19], in which no testicular or other phenotype has been described, p27KO mice have increased testicular size [20–22]. Testes from DBKO mice have not been described, though these mice have alterations in proliferation of granulosa cells, the female homologue of Sertoli cells [23]. If either p27 or p21 are important regulators of Sertoli cell proliferation, then loss of either or both of these should result in changes in overall testis growth, Sertoli cell number, and sperm production. In this study, we examined effects of loss of p27 and/or p21 on Sertoli cell number and testis development. Our results indicate that both of these CDKIs are important inhibitors of Sertoli cell proliferation, and loss of these CDKIs leads to large increases in adult Sertoli cell populations, as well as increases in DSP and testis weight.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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WT and p27 single-knockout animals were generated by crossing mice heterozygous for Cdkn1b (designated thenceforth as p27). Cdkn1a null mutant mice (referred to hereafter as p21) are fertile, so p21KO mice were obtained by breeding homozygous knockouts. DBKO mice were obtained by mating mice that were homozygous null mutants for p21 and heterozygous for p27 (Cdkn1b±/Cdkn1a–/–). Between 8 and 12 days of age, DNA was isolated from tail tissues using DirectPCR Lysis Reagent (Viagen Biotech Inc., Los Angeles, CA), and used to determine genotype as described [20, 23]. Mice were housed at 25°C with 12L:12D cycles and given water and a standard rodent diet ad libitum. All animal experiments were approved by the IACUC of the University of Illinois and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Body and Testis Weights, Sperm Production, and Serum Hormone Analyses
Body weights of WT, p21KO, p27KO, and DBKO males were measured every 2 wk starting at Postnatal Day 14. Between 115 and 120 days of age, mice were given a lethal injection of ketamine. One testis was excised for determination of testis weight and DSP as described previously [2].
Blood was collected from a separate pool of 90- to 120-day old WT, p21KO, and DBKO mice (n = 12–15/genotype) for determination of growth hormone (GH) and IGF1 concentrations by radioimmunoassay at the National Hormone and Peptide Program (Los Angeles, CA). The p27KO mice were not evaluated because values have already been reported previously [20]. IGF1 binding proteins were extracted before IGF1 analyses. Briefly, 50 µl of serum was diluted with 200 µl of 87.5% ethanol:12.5% 2 N HCl (v/v) solution. Samples were vortexed, incubated for 30 min at 4°C, then centrifuged for 15 min at 13 000 x g to isolate the supernatant. Supernatants were transferred into a new tube and 80 µl of 0.86 M Tris base was added to all samples and mixed. Samples were incubated for 1 h at –20°C, recentrifuged, and supernatants collected for RIA IGF1 analyses.
Determination of Sertoli Cell Number and Proliferation and Epididymal Histology
The remaining testis and epididymis were fixed by vascular perfusion using 10% neutral buffered formalin or by immersion in Bouin fixative, embedded in paraffin, sectioned at 4 µm, and stained with hematoxylin and eosin or periodic acid Schiff and hematoxylin for determination of Sertoli cell number and histological examination. Total Sertoli cell number per testis was determined as described [12], and a subset of the samples was independently reanalyzed using the point-count method [24] to verify the original results. Seminiferous tubular diameter and volume and height of the epithelium were measured as described previously [24].
To determine if the length of Sertoli cell proliferation in knockouts and WT mice was similar, testes (n = 4–8/genotype) from 16-day-old WT, p21KO, and p27KO were fixed in 10% neutral buffered formalin for 1 wk at room temperature before dehydration and paraffin embedding. Tissues were serially sectioned at 4 µm, then deparaffinized and rehydrated. To facilitate antigen detection, slides were placed in boiling 10 mM sodium citrate buffer (pH = 6.0) for 10 min, then cooled to ambient temperature. Endogenous peroxidase activity was quenched by incubation with 0.3% H2O2 for 20 min. Sertoli cells were identified by immunodetection of Wilm tumor protein (WT-1) using a rabbit polyclonal IgG to human WT-1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). On an adjacent serial section, cell proliferation was detected using mouse anti-human monoclonal IgG to human Ki-67 (BD Transduction Laboratories, Lexington, KY). Binding of primary antibody was localized by using a horseradish peroxidase-Vectastain ABC Kit (Vector Laboratories, Burlingame, CA) for each species and DAB Substrate Kit (Vector) according to the supplier's instructions. Negative control tissue sections were processed with normal goat serum instead of primary antibody to determine nonspecific staining. Following immunostaining, tissues were counterstained with Hematoxylin QS (Vector). Ki-67 staining, indicative of cell proliferation, was determined in 500 Sertoli cells identified by positive WT-1 staining per testis, and results were expressed as percentage labeling.
Statistical Analysis
All data are presented as mean ± SEM. Seminiferous tubule diameter and volume and height of seminiferous epithelium were analyzed using one-way ANOVA followed by Student-Newman-Keuls test for multiple comparisons. All other data were analyzed by least-square means using the MIXED procedure of SAS (SAS Institute, Cary, NC) and Student t-test where appropriate. Means and SEM were calculated using the MEAN procedure of SAS. Differences are considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The p21KO mice had significant increases in body weight compared with WT on days 84 and 98, after which no significant differences were observed even though p21KO animals had a trend toward an increase in body weight compared with controls (Fig. 1). Consistent with previous reports [20–22], loss of p27 significantly increased (P < 0.04) body weight compared with WT controls from Day 56 onward. Furthermore, loss of both p27 and p21 significantly increased body weight (P < 0.002) compared with WT, p21KO, or p27KO mice from as early as 42 days postnatal until the conclusion of the study. As in female mice [25], the increases in body weight in DBKO mice reflect both a 30% increase in lean body mass and a sixfold increase in adipose tissue.
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Loss of p27 and p21 Results in Elevated IGF1 Serum Levels
Serum growth hormone concentrations in p21KO (n = 13) and DBKO (n = 14) mice (2.3 ± 0.5 and 1.8 ± 0.4 ng/ml, respectively) did not differ from WT values (2.4 ± 0.7 ng/ml; n = 15), although DBKO mice showed a trend toward a decrease. As in p27KO mice [20], serum IGF1 levels in p21KO mice (60 ± 2 ng/ml; n = 12) also did not differ from WT levels (54 ± 1 ng/ml; n = 14). However, serum IGF1 levels were significantly elevated in DBKO mice (67 ± 4 ng/ml; n = 14) compared with WT mice (P < 0.0006) and had a tendency to be different from p21KO mice (P < 0.06).
Sertoli Cell Number Increases in Mice Lacking p27 and/or p21
Sertoli cell number in WT testes (3.20 ± 0.44 x 106/ testis; n = 7) was comparable with previously reported values [26]. Increases in Sertoli cell numbers were observed in p21KO, p27KO, and DBKO males (Figs. 2 and 3). The p21KO mice had a 48% increase in Sertoli cell number (4.75 ± 0.34 x 106/testis; n = 8) compared with WT controls. This difference was significant when analyzed by the Student t-test (P < 0.02; Fig. 3), but not by ANOVA (P = 0.09). Sertoli cell numbers in testes from p27KO (7.22 ± 0.69 x 106/testis; n = 9) and DBKO (7.24 ± 0.75 x 106/ testis; n = 8) mice were more than doubled compared with WT controls (Fig. 3) and 52% greater than p21KO Sertoli cell numbers. Sertoli cell numbers in p27KO and DBKO testes did not differ but were significantly greater than those in WT (P < 0.0001) and p21KO (P < 0.007) mice. Results obtained with the point-count method were consistent with the results above (data not shown).
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Length of Sertoli Cell Proliferation Is Not Extended in Knockout Mice
Sertoli cell proliferation was minimal in Postnatal Day 16 WT mice (labeling index 0.8% ± 0.2%), which was consistent with previous reports [2, 14]. Sertoli cell proliferation in 16-day-old p21KO (1.5% ± 0.4%) and p27KO (0.3% ± 0.2%) mice was low and not significantly different from WT. By Postnatal Day 25, Sertoli cell proliferation had ceased completely in all genotypes (not shown).
Seminiferous Tubule Volume Increases in Knockout Mice
Seminiferous tubule volume did not differ (P = 0.14) between p21KO (125 ± 4 µl) and WT (108 ± 7 µl) mice, though there was a trend toward increases. However, loss of p27 or both p27 and p21 significantly increased the volume of seminiferous tubules to 135 ± 4 µl (P < 0.05) and 185 ± 15 µl (P < 0.0002), respectively, compared with WT. In contrast, seminiferous tubule diameter and seminiferous epithelium height were similar in p21KO, p27KO, and DBKO mice compared with WT controls (data not shown).
Testicular Organomegaly in CDKI Knockout Testes
At 115–120 days of age, testicular size was increased with loss of p21 and/or p27 (Fig. 4). The p21KO testes had a 29% increase in wet weight (129 ± 5 mg; n = 9) compared with control testes (100 ± 3 mg; n = 14; P < 0.001). Loss of p27 resulted in even larger increases in testis weight (141 ± 3 mg; n = 10), which differed significantly from both WT and p21KO genotypes (P < 0.001 and P < 0.05, respectively). Maximal testicular weight was seen with the loss of both p21 and p27; DBKO testes were larger (185 ± 7 mg; n = 7; P < 0.001) than all other genotypes.
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Loss of p27 and/or p21 Increases Daily Sperm Production
Increased Sertoli cell number is typically associated with an increased DSP. To establish whether the loss of p21 and/ or p27 affected sperm production, we quantitated DSP in all genotypes (Fig. 5). Significant increases (P < 0.004) in DSP occurred in both p21KO (6.84 ± 0.23 x 106; n = 9) and p27KO testes (6.93 ± 0.47 x 106; n = 10) compared with WT (5.43 ± 0.20 x 106; n = 13). Sperm production in DBKO testes was higher (9.72 ± 0.51 x 106; n = 6; P < 0.001) than either single knockout or WT controls. However, the increase in DSP in knockout mice was not attributed to an increase in Sertoli cell efficiency. The number of spermatids per Sertoli cell was 12.5 ± 0.8 and 10.5 ± 0.8 in p21KO (n = 4) and p27KO (n = 6) mice, respectively, and did not differ from WT mice (11.4 ± 0.8, n = 5; P = 0.39). DBKO mice (n = 4) showed no difference in the number of spermatids per Sertoli cell (12.3 ± 1.3) compared with WT.
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All WT and p21KO testes analyzed were histologically normal, with 0.5% or less of the seminiferous tubules exhibiting any spermatogenic defect. In comparison, 5% and 1.5% of p27KO and DBKO seminiferous tubules, respectively, had spermatogenic defects compared with only 0.5% of the WT seminiferous tubules. The extent of spermatogenic defects was variable between animals (n = 4–6 mice/ genotype). Defects ranged from selective sloughing of late-stage spermatids to extensive vacuolation of the seminiferous epithelium (Fig. 6, A and B). Some testes had focal regions that were severely affected, while surrounding seminiferous tubules were normal in appearance (Fig. 6A). Most notably, some p27KO and DBKO testes displayed focal areas of Sertoli cell-only phenotype (Fig. 6B).
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Developing Germ Cells Found in the Epididymis of p27KO Mice
The processes of germ cell development, spermatogenesis, and spermiation normally occur within the seminiferous tubules of the testis, and the resulting newly formed spermatozoa are released for passage into the epididymis for further maturation. Because loss of p27 was associated with a variety of germ cell abnormalities, epididymides from p27KO mice were examined for developing germ cells that were prematurely sloughed from the seminiferous epithelium and passed into the epididymis. In WT mice, only spermatozoa are observed within the epididymis (Fig. 7A). In p27KO mice, particularly those with extensive testicular defects, pyknotic spermatogenic cells were found within the caput and cauda epididymis, and fewer spermatozoa were present (Fig. 7B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our results show that p27 is a primary regulator of Sertoli cell proliferation, as evidenced by the 125% increase in adult Sertoli cell population in p27KO mice. These increases in Sertoli cell number seen in p27KO mice may result from the inability of thyroid hormone to signal through p27 to induce a cessation of proliferation. However, it has been reported that loss of p27 results in a 50% increase in serum FSH in 8-wk-old mice [18, 27]. This increase in FSH could promote mitogenesis in Sertoli cells through the stimulation of cyclin D2 synthesis [28] and could be contributory to the overall Sertoli cell effect. Furthermore, the significance of these findings is highlighted by a recent report showing that somatic gonadal precursor cells were increased in Caenorhabditis elegans lacking CKI-1, the p27 orthlog [29], indicating that the role of p27 in somatic cells of the gonad is evolutionarily conserved.
Our preliminary results indicate that p27KO, p21KO. and DBKO mice may have reduced androgen (Mukai et al., unpublished results). Therefore, androgen could decrease Sertoli cell number in the p27KO or other knockout mice [7, 8], which could result in underestimations of the actual stimulatory effects of p27 and/or p21 deficiency on Sertoli cell populations.
The role of p21 in controlling Sertoli cell proliferation appears to be secondary to that of p27 because p27KOs had an increase in Sertoli cell number that was 150% greater than the increase seen in p21KOs. Furthermore, the lack of an additional increase in overall Sertoli cell number in the DBKO compared with the p27KO is also consistent with the idea that p21 plays a secondary role to p27 in regulating Sertoli cell mitogenesis. In combination, these results support the concept that p27 may be the dominant regulator of Sertoli cell proliferation because the doubling of Sertoli cell number in p27KO mice suggests that, even when p21 is present, these Sertoli cells have increased proliferation. In contrast, p27 regulation of Sertoli cell proliferation in p21KOs is more critical, such that loss of p21 in Sertoli cells still expressing p27 leads to a much smaller increase in overall Sertoli cell number. However, the lack of a further increase in overall Sertoli cell number in the DBKO compared with the p27KO suggests that these CDKIs possibly regulate Sertoli cell mitogenesis through a redundant pathway.
Despite the role of both p27 and p21 in Sertoli cell proliferation, Sertoli cells cease dividing at approximately the same time in the p27KO and p21KO as in the WT. These results demonstrate that the rate of Sertoli cell mitogenesis must be greater during the normal postnatal proliferative period with loss of p27 and p21 to establish a larger adult Sertoli cell population within a similar length of time. In addition, because Sertoli cells in single-knockout mice become mitotically quiescent at the normal time and we have seen no evidence of Sertoli cell proliferation in juvenile DBKOs, there must be another factor(s) that regulate(s) the cessation of Sertoli cell proliferation even in the absence of p21 and p27, such as the Ink4 family member p18 or other cell cycle proteins [30].
An enlarged Sertoli cell population typically produces similar increases in germ cell numbers and ultimate sperm production, leading to bigger testes [10–12]. In this study, even though the number of spermatids per Sertoli cell did not differ among genotypes, the 28% and 80% increases in DSP in p27KO and DBKO mice, respectively, were less than the 125% increases in Sertoli cell number in these knockouts. The smaller increase in DSP relative to Sertoli cell numbers may reflect increased spermatogenic defects, including the presence of Sertoli cell-only seminiferous tubules in both p27KO and DBKO testes. This is consistent with the presence of immature germ cells within p27KO epididymides, which also may account for the smaller increase in DSP compared with Sertoli cell number when p27 is absent. Interestingly, mean sperm counts/cauda epididymis were not different between p27KO (25.90 ± 4.56 x 106, n = 9) and WT mice (28.96 ± 2.72 x 106, n = 18), consistent with variable germ cell loss in the testes of p27KO males. However, in contrast with p21, which is expressed in Sertoli and germ cells, p27 expression is most abundant in Sertoli and Leydig cells of the adult testis [17]. Therefore, the role of p27 in normal spermatogenesis appears to be primarily through effects on Sertoli and Leydig cells. To definitively determine if p27 may also have some direct effects on germ cell development, Sertoli cell- and germ cell-specific knockouts for p27 are needed.
The p27 expression is maximal in adult Sertoli cells and corresponds with Sertoli cell maturation [17, 18]. Loss of p27 may impair Sertoli cell differentiation and maturation, leading to a diminished ability to support spermatogenesis. This hypothesis is supported by previous results showing that, after in utero exposure to a Sertoli cell toxicant, dibutyl phthalate, Sertoli cell p27 expression was prominent in those seminiferous tubules exhibiting full spermatogenesis, whereas p27 expression in adjacent Sertoli cell-only tubules was low [31]. Similar to dibutyl phthalate treatment, both p27KO and DBKO mice show focal areas of Sertoli cell-only tubules. However, normal spermatogenesis did occur in adjacent tubules, suggesting that, although loss of p27 or both p27 and p21 can produce abnormalities in spermatogenesis, they are not obligatory for Sertoli cells to sustain spermatogenesis.
Full spermatogenesis is dependent on adequate serum testosterone levels [32], and chronic testosterone deficiency in rats resulted in 
60% overall reduction in spermatogonial and spermatocyte cell populations [33]. Furthermore, a Sertoli cell-specific knockout of the androgen receptor [9] resulted in spermatogenic arrest in meiosis and emphasized the role of androgen action on Sertoli cells in maintaining spermatogenesis. Preliminary analyses suggest that adult p27KO and DBKO mice may have a testosterone deficiency (Mukai et al., unpublished results). Thus, the areas of Sertoli cell-only syndrome within some p27KO and DBKO testes may reflect decreased testosterone levels as well as the effects of loss of p27 on spermatogenesis. Mice lacking p27 have increased incidence of pituitary hyperplasia and tumors [20, 30] and disturbances in the hypothalamic-pituitary-gonadal axis [20], suggesting that pituitary and/or other endocrine changes may have an effect on testicular function. Therefore, the greater increase in Sertoli cell number relative to DSP in p27KO and DBKO mice may not only reflect depressed testosterone levels but changes to the endocrine axis as well. In addition to potential spermatogenic impairments resulting from lack of testosterone or impaired Sertoli cell maturation, the more than doubling of the Sertoli cell population in the DBKO may increase Sertoli cell density and impair concomitant increases in DSP due to physical limitations and cell crowding within the seminiferous tubules.
The increased Sertoli cell numbers and DSP resulted in testicular organomegaly in mice of all three genotypes (p21KO, p27KO, and DBKO) when compared with WT. Although an earlier report on p21KO mice describes normal testis histology [19], our data are the first to document phenotypic changes, such as increased testis weight, Sertoli cell number, and DSP in p21KO mice. The testicular enlargement observed in p27KO mice was consistent with previous accounts describing testicular organomegaly in these animals [22]. However, our results indicate that loss of both p27 and p21 have greater effects on testis weight and DSP than loss of p27 alone. Synergistic effects of p27 with other CDKIs have been reported previously by others, where loss of p27 along with p18 resulted in a doubling of testis size, while absence of either p27 or p18 alone increased testis weights 30% compared with WT controls [30].
Our data also suggest that the roles of these CDKIs in regulating overall testis size may differ. DBKO testis weight increased compared with p27KO testes even though both genotypes had an equal Sertoli cell population, so the additional loss of p21 on a p27-deficient background in the DBKO results in approximately threefold greater increases in DSP than loss of p27 alone. These results suggest that the role of p21 in testicular development may include regulation of spermatogenesis and the large increase in DSP in the DBKO compared with the smaller DSP increase in the p27KOs may result from increased spermatogenesis. The p21 is expressed in Sertoli cells as well as in mid- and late-pachytene spermatocytes [34], thus indicating a possible role for p21 during meiotic prophase.
To establish whether increased testis weight in knockout mice was a nonspecific result of increased body growth, we have previously measured lean body mass using dual energy X-ray absorbtiometry (DEXA) analysis. DEXA analysis showed that loss of both p27 and p21 resulted in only a 30% increase in lean body mass despite the 50% increase in body weight. Therefore, the large difference seen in weight gain in DBKO mice compared with WT or either single knockout reflects in large part the sixfold increase in adipose tissue after loss of these CDKIs [25]. In the DBKO testis, Sertoli cell number rose 125% over WT controls despite only a 30% increase in lean body mass. Taken together, these results prove that the increase in DBKO testis weight is not simply allometric. Similar effects are seen in p21KO and p27KO mice, where overall body weight increased less than 10% but Sertoli cell number and testis weights showed much larger increases.
The roles of p21 or p27 in regulating overall body growth do not appear to be redundant because loss of p21 or p27 results in 6% and 9% increases in body weight compared with WT, respectively, while DBKO animals are almost 50% heavier. The rapid weight gain seen in p21KO mice within the first month of age corresponds to the time when p21 is highly expressed in mice [35, 36], and our data are the first to demonstrate the long-term effects of loss of p21 on body weight in males. As expected, loss of p27 caused even larger increases in adult body weight, in agreement with earlier reports [20–22]. Finally, loss of both p21 and p27 led to a large weight gain over the course of the experiment, which mainly can be attributed to hyperplasia in adipose and other tissues where either or both of these CDKIs are critical to proper development.
Differences in overall body weight in the various groups could not be attributed to increased somatotrophic effects of GH because GH levels did not differ by genotype in the WT, p21KO, and DBKO, in agreement with earlier results indicating that GH production appeared normal in p27KO mice [20, 23]. IGF1 is induced by GH, and the growth-promoting effects of IGF1 on various tissues may involve both local autocrine/paracrine as well as endocrine effects of IGF1 [37, 38]. In contrast with GH levels, IGF1 levels were elevated 26% in DBKO mice compared with WT. Adipose tissue is one source of IGF1, and previous results have shown that obesity is associated with an increase in free IGF1 [39]. Therefore, the large increase in adipose tissue in DBKO mice might contribute to the observed increase in circulating IGF1 in these animals. However, it is not clear whether the increase in systemic IGF1 is merely a result of the increased overall growth of tissues in the DBKO, which could result in greater IGF1 production, or whether the increase in IGF1 could be contributory to the increased growth of some organs in the DBKO mice.
In summary, using knockout mice, we were able to determine the roles of p27 and p21 in Sertoli cell proliferation. Our results demonstrate that these cell cycle proteins regulate overall Sertoli cell number. Both p27 and p21 have a negative influence on Sertoli cell proliferation, and loss of either results in significant increases in adult Sertoli cell number during the normal period of postnatal growth, with subsequent increases in DSP and testis weight. Clearly, p27 serves as the primary regulator of proliferation because p27KO mice have 2.5-fold more Sertoli cells than p21KO mice. However, loss of p27 alone had a negative impact on spermatogenesis, with more abnormalities and Sertoli cell-only tubules than seen in either p21KO or DBKO. The p21 may also affect spermatogenesis in addition to its inhibitory effects on Sertoli cell proliferation. Regardless of the magnitude of their roles in establishing Sertoli cell number and consequent DSP, p27 and/or p21 clearly are critical for Sertoli cell proliferation and normal testis development, such that loss of either or both results in permanent increases in the Sertoli cell population.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Paul S. Cooke, Department of Veterinary Biosciences, 2001 S. Lincoln Ave., University of Illinois at Urbana-Champaign, Urbana, IL 61802. FAX: 217 244 1652; p-cooke{at}uiuc.edu
Received: 26 January 2005.
First decision: 9 February 2005.
Accepted: 17 February 2005.
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