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Molecular and Cellular Biology, May 2001, p. 3244-3255, Vol. 21, No. 9
Departments of Tumor Cell
Biology1 and
Pathology3 and Howard Hughes
Medical Institute,6 St. Jude Children's
Research Hospital, Memphis, Tennessee 38105; Department of
Developmental and Molecular Biology, Albert Einstein College of
Medicine, Bronx, New York 104612;
Department of Pathology, New York University School of
Medicine, New York, New York 100164; and
Centro Nacional de Investigaciones Oncologicas Carlos III,
Majadahonda, 28220 Madrid, Spain5
Received 14 November 2000/Returned for modification 14 December
2000/Accepted 13 February 2001
Male mice lacking both the Ink4c and Ink4d
genes, which encode two inhibitors of D-type cyclin-dependent kinases
(Cdks), are infertile, whereas female fecundity is unaffected. Both
p18Ink4c and p19Ink4d are expressed in the
seminiferous tubules of postnatal wild-type mice, being largely
confined to postmitotic spermatocytes undergoing meiosis. Their
combined loss is associated with the delayed exit of spermatogonia from
the mitotic cell cycle, leading to the retarded appearance of meiotic
cells that do not properly differentiate and instead undergo apoptosis
at an increased frequency. As a result, mice lacking both
Ink4c and Ink4d produce few mature sperm, and
the residual spermatozoa have reduced motility and decreased viability.
Whether or not Ink4d is present, animals lacking
Ink4c develop hyperplasia of interstitial testicular Leydig
cells, which produce reduced levels of testosterone. The anterior
pituitary of fertile mice lacking Ink4c or infertile mice
doubly deficient for Ink4c and Ink4d produces
normal levels of luteinizing hormone (LH). Therefore, the failure of
Leydig cells to produce testosterone is not secondary to defects in LH
production, and reduced testosterone levels do not account for
infertility in the doubly deficient strain. By contrast,
Ink4d-null or double-null mice produce elevated levels of
follicle-stimulating hormone (FSH). Because Ink4d-null mice
are fertile, increased FSH production by the anterior pituitary is also
unlikely to contribute to the sterility observed in
Ink4c/Ink4d double-null males. Our data indicate that
p18Ink4c and p19Ink4d are essential for male
fertility. These two Cdk inhibitors collaborate in regulating
spermatogenesis, helping to ensure mitotic exit and the normal meiotic
maturation of spermatocytes.
Spermatogenesis in mammals is
characterized by a well-defined sequence of mitotic and meiotic
divisions that lead to the production of mature spermatozoa
(27). In newborn mice, male germ cell precursors undergo
self-renewal in the testis between days 1 and 7 postpartum (pp) (Fig.
1). From day 7 pp onward, inception of spermatogenesis begins synchronously in a cohort of precursors, starting with at least two mitotic divisions followed by one round of
meiosis. The early cell divisions lead to the development of type A and
type B spermatogonia, the latter of which undergo premeiotic replication and enter meiosis as primary spermatocytes. Meiosis I is
characterized by a prolonged prophase that allows chromatid exchange
through crossing over. Segregation of homologous chromosomes occurs at
the end of meiosis I, and resulting secondary spermatocytes then
proceed through a second meiotic division in which haploid germ cells
are generated. These differentiate to form round spermatids and,
eventually, mature elongated spermatozoa (spermiogenesis). The first
round of spermatogenesis is followed by additional waves, enabling
continuous sperm production throughout the life of the animals.
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.9.3244-3255.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Control of Spermatogenesis in Mice by the Cyclin
D-Dependent Kinase Inhibitors p18Ink4c and
p19Ink4d
and
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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FIG. 1.
Idealized timing of the first wave of spermatogenesis.
The time line from birth onward indicates the temporal sequence of
events in the first 35 days pp (27). Intervals in which
mitotic cell division, meiosis I, meiosis II, and spermiogenesis occur
are indicated above the time line, noting different stages during
prophase of meiosis I. Spermatogonia populate the seminiferous tubules
after birth, giving rise to spermatocytes, spermatids, and spermatozoa,
as indicated below the time line.
Spermatogenesis is regulated hormonally through the pituitary-gonadal axis. The anterior lobe of the pituitary gland produces the gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH). In males, FSH stimulates Sertoli cells, whose number determines the thickness of the seminiferous epithelium and, in turn, the size of the testis (36). LH induces interstitial Leydig cells to produce testosterone, a gonadal steroid necessary for spermatogenesis (19).
Cyclin-dependent kinases (Cdks) likely govern both the mitotic and meiotic divisions that characterize spermatogenesis, but it remains unclear which classes of enzymes are required for the various processes. Using immunohistochemical methods, cyclins D2 and D3 and their catalytic partner Cdk4 were seen to be expressed at the periphery of the seminiferous tubules between days 1 and 13 pp in spermatogonia undergoing mitosis (7, 22, 28, 33, 34, 46). By contrast, little cyclin D2 and Cdk4 expression was observed later in differentiated spermatocytes and spermatids (7, 28), although cyclin D3 expression was maintained (33, 46). cyclin D2-null male mice are fertile but have reduced testicular size and low sperm counts (39), whereas Cdk4-null mice are for the most part sterile at birth or become so at an early age (32, 42). Atrophic seminiferous tubules observed within the testes of Cdk4-null mice showed reduced numbers of spermatogonia and spermatocytes, suggesting that their proliferative capacity was reduced. Cdk6, the other known catalytic partner of D-type cyclins, was expressed at lower levels in testes of Cdk4-null animals (42). These findings together suggest that cyclins D2 and D3 in combination with Cdk4, and possibly Cdk6, may regulate G1 progression in spermatogonia in the postnatal testis and that cyclin D3 may play an additional role later in germ cell development.
The cyclin D-dependent kinases are negatively regulated by small polypeptide Cdk inhibitors encoded by four distinct Ink4 genes (38). Two of the Ink4 gene products, p16Ink4a and p15Ink4b, are not detectably expressed during mouse fetal development and are first observed in tissues of young adult animals (48). Disruption of either Ink4a or Ink4b leads to no developmental defects, and the young animals are healthy and fertile (25, 35). By contrast, the other Ink4 family members, p18Ink4c and p19Ink4d, are expressed during mouse embryogenesis and into adult life, particularly in the central nervous system and testis (48-50). Ink4c-null male mice are fertile and manifest no apparent testicular abnormalities, but they exhibit organomegaly and eventually develop midlobe pituitary tumors and hyperplasia of Leydig cells (16, 17, 25). Mice expressing a Cdk4 knock-in allele encoding a mutant form of the kinase (R24C) that cannot be inhibited by Ink4 proteins are fertile and also manifest Leydig cell hyperplasia (32). Ink4d-null mice are fertile and enjoy a normal life span, but they do not manifest Leydig cell abnormalities and instead display marked testicular atrophy associated with increased apoptosis in seminiferous tubules and reduced sperm counts (50). These findings suggest that expression of unrestrained cyclin D-dependent kinases can not only increase Leydig cell proliferation but can also have additional effects on male germ cell development, whether their expression is cell autonomous or not.
To address possible redundant roles for p18Ink4c and p19Ink4d in testicular development, we bred mice lacking Ink4c (25) with Ink4d-null mice (50) to derive animals lacking both genes. We show that deletion of both Ink4c and Ink4d (referred to here as Ink4cd double-null mice) results in complete infertility in males but has no effect on female reproductive function. Our data suggest that inappropriate regulation of cyclin D-dependent kinases in male germ cell progenitor cells inhibits them from undergoing meiosis.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Generation of mouse strains and mouse embryo fibroblasts. Mouse strains (C57BL/6 × 129Svj) deficient in Ink4c (25) or Ink4d (50) were intercrossed. Ink4d-deficient females were bred with Ink4c-null males to yield compound heterozygotes. Interbreeding generated wild-type and single- and double-null animals at the expected Mendelian frequencies. Genotyping was performed by Southern blotting with mouse tail DNA from weanling pups, as previously described (25, 50). Primary mouse embryonic fibroblasts (MEFs) were isolated from 13.5- to 14.5-day-old embryos as described previously and were propagated on a 3T9 schedule (48).
Sperm counts. Two-cauda epididymides from 10- to 14-week-old male mice were harvested at the same time of day (between 12:00 and 2:00 p.m.). The sperm-containing fluid was squeezed out of the cauda, which was later cut into pieces. The sperm fluid and the pieces of cauda were suspended in 1 ml of Dulbecco's modified Eagle medium containing 25 mM HEPES buffer (pH 7.5) and 4 mg of bovine serum albumin per ml and were incubated at 37°C for 20 min. Suspensions of spermatozoa (20 µl) were fixed in 480 µl of 10% formalin. We used a hematocytometer to determine the number of spermatozoa.
Histologic and immunohistochemical methods. Testes were placed in Bouin's fixative (Electron Microscopy Sciences, Fort Washington, Pa.) for a period between 6 h and overnight, depending on the size of the testis. After fixation, the testes were stored in 70% ethanol at 4°C until processed. Tissues were dehydrated in increasing concentrations of ethanol, embedded in paraffin wax, and sectioned at a thickness of 5 µm. Sections were stained with hematoxylin and eosin or were processed for immunohistochemical analysis with antibodies to either germ cell nuclear antigen 1 (GCNA1; a kind gift of George Enders, University of Kansas Medical Center) or the steroidogenic enzyme P450 side chain cleavage (P450scc; Chemicon International Inc., Temecula, Calif.). The anti-P450scc antibody was used at a dilution of 1:100. Detection of the proliferating-cell nuclear antigen (PCNA) was performed with 12-µm frozen sections of testis fixed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.6. PCNA was detected as described previously (21).
In situ hybridization and apoptosis assay.
Male mice were
anesthetized with intraperitoneal injections of ketamine and xylazine
(ratio of 0.4 to 0.6 [vol/vol]) and perfused intracardially with 4%
paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.6) for 20 min.
Isolated testes were postfixed for 4 h at 4°C, transferred to
25% sucrose solution in 0.1 M sodium phosphate buffer (pH 7.6), and
incubated at 4°C for an additional 24 h. A microtome cryostat
(Microm, Walldorf, Germany) was used to section tissue into 12-µm
cryosections, which were mounted on Fischerbrand Superfrost Plus slides
(Fischer Scientific, Pittsburg, Pa.) and stored at 
20°C. In situ
hybridization was performed as previously described (49).
Briefly, sections were hybridized with radiolabeled antisense and sense
riboprobes generated from mouse Ink4c and Ink4d
cDNAs (49), from a PstI fragment containing the
3' end of the Cdk4 gene (nucleotides 588 to 867), and from a
KpnI-SacI fragment containing the 3' end of the
Cdk6 gene (nucleotides 549 to 1000). Apoptotic cells were
visualized by the terminal deoxynucleotidyltransferase-mediated
dUTP-biotin nick 3'-end labeling (TUNEL) assay (TdT FragEL DNA
fragmentation detection kit; Calbiochem, La Jolla, Calif.).
Protein analysis. Sequential immunoprecipitation and immunoblotting were performed as described previously (49). Analyses of mouse p18Ink4c and p19Ink4d were performed with commercially available polyclonal antisera: M-167 for immunoprecipitation and immunoblotting of p19Ink4d, M-20 for immunoprecipitation of p18Ink4c, and M-168 for immunoblotting of p18Ink4c (Santa Cruz Biotechnologies, Inc., Santa Cruz, Calif.). Mouse Cdk4 was precipitated from cell lysates with antiserum (Rz) to the C-terminal peptide of mouse Cdk4 (26) and was immunoblotted with antibody C-22 (Santa Cruz Biotechnologies, Inc.). Mouse Cdk6 was detected with antiserum to a C-terminal peptide (49).
Hormone measurements.
Blood samples were collected from 10- to 14-week-old male mice (between 12:00 and 2:00 p.m.) and were kept on
ice. Serum was separated and stored at 80°C. The amounts of serum
testosterone were measured by a standard coated-tube radioimmunoassay
kit (Diagnostic Systems Laboratories, Webster, Tex.). Interassay and
intra-assay controls were included. Serum FSH measurements were carried
out by radioimmunoassays (8) performed with antibodies and
standards generously provided by the National Hormone and Pituitary
Program of the National Institute of Diabetes and Digestive and Kidney Diseases (Washington, D.C.).
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Phenotypic features of mice lacking Ink4c and Ink4d. Mice containing disrupted Ink4c alleles were bred with Ink4d-null animals, and intercrosses between heterozygotes yielded mice lacking both genes at the expected Mendelian frequency. Ink4c-deficient mice exhibit organomegaly (16, 25), whereas Ink4d-null animals do not differ from their wild-type littermates in size (50). Female mice doubly deficient for both Ink4c and Ink4d exhibited a mean increase in body weight relative to wild-type and Ink4d-null female littermates but still remained 8% smaller than Ink4c-null animals (data not shown). In contrast, Ink4cd double-null males were similar in size to Ink4c-deficient males. Despite expression of high levels of Ink4c and Ink4d in the developing mouse brain (49), the doubly deficient mice displayed no overt neurological defects. However, we recently recognized that Ink4d-null mice are deaf (B. Chen, F. Zindy, M. F. Roussel, and N. Segil, unpublished observations). Other Cdk inhibitors may well compensate for the lack of these Ink4 proteins in the brain, as animals lacking p19Ink4d and p27Kip1, but neither alone, exhibit profound neurologic defects leading to the death of the animals in the first few weeks of life (47).
Ink4c-null animals develop progressively expanding adenomas of the intermediate lobe of the pituitary gland with nearly complete penetrance by 8 to 10 months of age, and by 14 months at least half of the animals die as a consequence (16, 25). Ink4c-null mice also develop pheochromocytomas at a low but significant frequency (ca. 10%) (17, 25). At least one independently derived strain exhibits hyperplasia of pancreatic
-islet cells (25), mirroring observations with mice
expressing an Ink4-resistant mutant Cdk4 allele in place of the
wild-type gene (32). Ink4cd double-null mice
exhibited each of these features of the Ink4c-null strain with similar types, frequencies, and rates of tumor development. The
fact that Ink4d loss did not synergize with Ink4c
deficiency in this respect is consistent with the previous finding that
p19Ink4d is not a tumor suppressor in mice
(50).
To determine whether the combined loss of Ink4c and
Ink4d affects cell proliferation, we explanted MEFs into
culture from 13.5-day-old single- and double-null embryos that normally
express both proteins (48). Primary MEFs from the various
knockout strains could not be distinguished from those derived from
wild-type littermates with respect to their proliferative rates, their
ability to arrest in medium containing limiting concentrations of
serum, or their rate of entry into S phase following serum withdrawal
and restimulation. The cells underwent replicative senescence after 20 to 30 population doublings, associated with the induction during
progressive cell culture of various stress response proteins, including
p16Ink4a, p15Ink4b, p21Cip1, and
p19Arf (reviewed in reference 37). Like
nonimmortalized MEF strains, Ink4cd double-null cells were
resistant to transformation by oncogenic Ras. In short, the combined
loss of Ink4c and Ink4d had no overt effects on
the fibroblast cell cycle or replicative senescence.
Sterility in Ink4cd double-null males.
Ink4cd double-null females exhibited no obvious fertility
defects, but all doubly deficient males were sterile, and test matings with wild-type females failed to induce pregnancy. Atrophy of the
testes in 6-month-old Ink4cd double-null males was more
pronounced than that in males lacking Ink4d alone
(P < 0.005) (Table 1). In contrast, the testes of organomegalic Ink4c-null mice
were larger than those of their wild-type littermates (P < 0.0001). The number of spermatozoa in fluid extruded from the
epididymides of Ink4d-null males was significantly lower
than that of both wild-type (P = 0.03) and
Ink4c-null (P < 0.001) males (Table 1). More strikingly, the sperm counts of double-null males were 85% lower
than those of wild-type littermates (P < 0.001) (Table
1). In addition, the morphology of residual epididymal spermatozoa in
the double-null mice was abnormal and more than 60% of spermatozoa were nonviable (data not shown).
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Altered hormone levels in males lacking Ink4c, Ink4d, or both. A complex network of hormonal signals orchestrates spermatogenesis. Gonadotrophs in the anterior lobe of the pituitary gland secrete LH and FSH, which stimulate Leydig cells to produce testosterone. To investigate the possible contribution of the pituitary-gonadal axis to male sterility, we measured the levels of testosterone, FSH, and LH in 10- to 14-week-old males of all genotypes (Table 1).
The onset of mature Leydig cell function at puberty is characterized by their ability to terminally differentiate and produce testosterone and other androgens at levels seen in adult males. As noted above, however, spermatogenesis begins earlier in life, arguing that adequate levels of testosterone to support this process are available soon after birth. One marker of Leydig cell differentiation is P450scc, the steroidogenic enzyme responsible for conversion of cholesterol to pregnenolone, the first and rate-limiting step in the steroidogenic pathway (29). As expected, in testis sections from 1-month-old wild-type males, Leydig cells failed to show P450scc immunoreactivity (Fig. 4A) above background levels (Fig. 4B). By 3 months of age, however, Leydig cells of wild-type males showed strong P450scc immunoreactivity, as demonstrated by the red precipitate within the interstitial space of the testis (Fig. 4C). Similarly, Leydig cells of testes from adult Ink4d-null males also showed up-regulated levels of P450scc (Fig. 4D) correlating with nearly normal levels of testosterone in their serum (Table 1). However, the levels of P450ssc immunoreactivity was severely reduced in Leydig cells of Ink4c-null (Fig. 4E) and Ink4cd double-null testes (Fig. 4F), suggesting a failure of Leydig cell differentiation. In turn, levels of serum testosterone were reduced by approximately 75% in Ink4c-null and Ink4cd doubly deficient males (Table 1). The levels of LH, released by the anterior lobe of the pituitary, were equivalent in males of all genotypes (data not shown), suggesting that the apparent failure of Leydig cell differentiation in mice lacking Ink4c was not secondary to pituitary dysfunction. We presume that the hyperplasia of Leydig cells observed somewhat later in life in the two mouse strains lacking Ink4c reflects a consequence of the same pathologic process; namely, their failure to differentiate and produce testosterone. However, despite the decline in serum testosterone levels, Ink4c-null mice remain fertile.
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Expression of Ink4 proteins, Cdk4, and Cdk6 in testes.
To
understand the nature of sterility in Ink4cd double-null
males, we first studied the expression of Ink4 proteins and cyclin D-dependent kinases during testicular development in wild-type mice. We
isolated testes from animals between 7 days and 1 month pp, the period
that marks the first wave of spermatogenesis (Fig. 1), and also from
mice at 5 months of age. We then used sequential immunoprecipitation
and immunoblotting to score for the presence of the various proteins in
testicular tissue and to look for complexes between
p18Ink4c or p19Ink4d with Cdk4 and Cdk6 (Fig.
5). As controls for antibody specificity, we used testes from the Ink4cd doubly deficient animals
(Fig. 5, lanes C). Testes from at least five mice were pooled to make lysates, and analysis of the same lysates was repeated twice with similar results.
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Impaired meiotic progression in Ink4cd doubly deficient
mice.
Because p18Ink4c and p19Ink4d, when
expressed at high levels, were expected to inhibit Cdk4 activity and to
block entry of cells into S phase, we anticipated that the loss of both
inhibitors might increase premeiotic spermatogonial proliferation. To
detect mitotically active spermatogonia, sections of testes from males
of all genotypes were stained with an antibody to PCNA, a protein
expressed in S phase (6). Up to day 15 pp, PCNA-positive
cells were detected in testicular tubular cells of all genotypes (Fig.
7A through D). By day 19 pp, the number
of PCNA-positive cells in tubules from wild-type and
Ink4c-null mice was diminished, suggesting that many cells
had exited the mitotic cycle (Fig. 7E and F versus A and B,
respectively). In contrast, more PCNA-positive cells remained in some
but not all tubules of Ink4d-deficient mice and were most
evident in those of the doubly null strain (Fig. 7G and H). By
adulthood, however, germ cell proliferation was restricted to a
monolayer of cells along the basement membrane of the seminiferous tubules in males of all genotypes (Fig. 7I through L). The relative persistence of mitotic activity of the spermatogonial population in
19-day-old Ink4d-null and Ink4cd double-null
males might therefore reflect a delay in the onset of the first wave of
meiosis I.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Impaired spermatogenesis in Ink4cd double null males. Females lacking both Ink4c and Ink4d exhibit no reproductive abnormalities, whereas males lacking both Cdk inhibitors are infertile. Ink4cd double-null males produce low numbers of mature sperm, the majority of which exhibited abnormal morphology and reduced motility. Male sterility correlated with an ineffective exit of spermatogonia from the mitotic cycle and with a failure of residual postmitotic spermatocytes to correctly undergo the meiotic divisions required to generate haploid gametes. The temporally delayed entry into meiosis of Ink4cd-null germ cells was also associated with increased apoptosis of spermatocytes, leading ultimately to the production of mostly nonviable sperm (genotypes are summarized in Table 1).
By day 19 pp during the first wave of spermatogenesis, mitotic cell divisions in the seminiferous tubules of wild-type mice, marked by PCNA-stained S-phase cells, become largely confined to spermatogonial cells that reside close to the basement membrane. In contrast, intralumenal spermatocytes at a further distance from the basement membrane have exited the cell cycle to undergo meiotic division and subsequent differentiation. Upon completion of meiosis, the latter cells differentiate to spermatids and mature spermatozoa that can be most easily visualized histologically in the epididymis. GCNA staining of cells in seminiferous tubules can distinguish the densely stained premeiotic cells at the tubular periphery from the more punctately stained cells in the pachytene stage of meiosis I within the lumen. These patterns of staining were distorted in the tubules of Ink4cd double-null mice (and, to a lesser extent, in Ink4d-null males), in which an expanded intralumenal population of mitotic S-phase cells was detected and fewer pachytene cells appeared, the latter only after a significant delay. Abnormalities in stages of meiotic progression were associated with an increased apoptotic index, and in turn, spermatids and spermatozoa were not readily seen in the seminiferous tubules of the Ink4cd doubly deficient mice. Both p18Ink4c and p19Ink4d are expressed at relatively high levels in testes compared to those in other organs of neonatal mice (48). Ink4d RNA transcripts were first readily detected by in situ hybridization in the seminiferous tubules of wild-type mice by day 13 pp, consistent with the increase in overall testicular p19Ink4d protein expression observed by immunoblotting. This corresponds to the time of entry into meiosis by the first wave of developing germ cells. At later stages, Ink4d RNA expression remains restricted to tubular cells, while the overall p19Ink4d protein level in the testis is maintained as additional waves of spermatogenesis ensue. In agreement with these findings, immunohistochemical studies of human p19INK4d in testes revealed maximal levels of protein expression in primary and secondary spermatocytes (5). This argues that Ink4d is normally expressed in postmitotic spermatocytes, where it presumably functions to extinguish the activity of cyclin D-dependent kinases, thereby helping to prevent further mitotic divisions. The overall pattern of p18Ink4c protein expression was more complex, with an earlier wave of protein synthesis (days 7 to 11 pp) followed by a nadir at day 13 pp and a return of expression from day 15 pp onward. By use of in situ hybridization, we were unable to clearly localize the earliest round of Ink4c expression to tubular germ cells and instead saw a more diffuse pattern of staining throughout the testis. However, Ink4c expression eventually became restricted to seminiferous tubular cells by day 17 pp onward, implying that the protein is expressed at high levels in spermatocytes. Similar results were obtained by others who applied immunohistochemical techniques to study p18INK4c in human testes (4). Loss of Ink4d leads to modest but still clearly defined inhibition of mature sperm cell production, but so far Ink4c has not been implicated in this process. Our data now suggest that p18Ink4c collaborates with p19Ink4d in helping to ensure the transition from mitotic to meiotic cell cycles during male germ cell maturation. Evidence is accumulating that progression of meiosis I is monitored to eliminate germ cells that have sustained unrepaired DNA damage. For example, defects in genes, such as Atm (2, 12, 20, 44), ligase-4 (3, 15), and MSH 4 and MSH 5 (11, 23), that monitor or effect DNA repair routinely lead to male sterility. One possibility, then, is that Ink4 genes have effector functions in cell cycle checkpoint control, arresting cells with broken chromatids until DNA damage is repaired. TUNEL assays indicated that loss of Ink4d, or more dramatically, loss of Ink4d in collaboration with disabled Ink4c, led to increased apoptosis once spermatocytes entered prophase I. The increased number of apoptotic cells coincided with the progression of spermatocytes up to and through pachynema (31). In contrast, the germ cells of Ink4c-null mice, like those of wild-type littermates, did not undergo significantly increased apoptosis. Hence, lack of Ink4d had a greater impact than loss of Ink4c, although both genes can contribute. Unlike the two Ink4 proteins, Cdk4 is expressed at maximal levels at earlier stages of spermatogenesis, where spermatogonia undergoing mitotic cell cycles predominate. Although its level declined as the animals aged, Cdk4 was still readily detected at reduced levels thereafter. Lower levels of Cdk6 relative to those of Cdk4 were also observed from day 7 to day 15 pp during the first wave of spermatogenesis but fell below the limit of detectability of our assays at later times. This indicates that Cdk4 is the major cyclin D-dependent catalytic subunit in the mouse testis but does not formally preclude the participation of Cdk6 in spermatogenesis in adult mice. Our results demonstrate that the effects of codeletion of Ink4c and Ink4d on spermatogenesis are not equivalent to those observed in the Ink4-resistant Cdk4 R24C knock-in strain, which is fertile (32). The mechanistic basis for these differences remains unclear. Under normal circumstances, p19Ink4d interacts preferentially with Cdk4 (data shown here), whereas p18Ink4d binds more selectively to Cdk6 (18). Possibly, the presence of the Cdk4 R24C mutant in place of wild-type Cdk4 might enhance the association of p19Ink4d with Cdk6, leading to a compensatory decrease in overall cyclin D-dependent kinase activity. A detailed analysis of spermatogenesis in the Cdk4 R24C knock-in strain has not been undertaken, and it is conceivable that the mice manifest subtle defects similar to those seen in males lacking Ink4d alone. Disruption of the cyclin D2 gene, which is normally expressed in spermatogonia (7, 22, 28, 33, 34, 46), leads to testicular atrophy and reduced sperm counts (39), while loss of Cdk4 ultimately results in decreased numbers of spermatogonia and male infertility (32, 42). Together, these results indicate that cyclin D-dependent kinases are required for progression through the early mitotic divisions that characterize postnatal male germ cell development. However, their unopposed expression at later stages due to Ink4cd loss also prevents proper germ cell maturation, underscoring a requirement for precise temporal regulation of these enzymes. In humans, testicular germ cell tumors are derived from primordial germ cells. Both seminomas and other germ cell tumor types appear to arise from cytogenetically identical carcinomas in situ that progress to invasive lesions (7). p19INK4d is undetectable in fetal germ cells that are thought to be the precursors of testicular tumors, and as might be expected, deletion of INK4d has not been seen in such tumors (5). Unlike p19INK4d, however, p18INK4c expression is abundant in carcinomas in situ, and loss of INK4c is frequently associated with progression from carcinomas in situ to invasive germ cell tumors in human testes (4). These findings point to the possibility for additional roles for p18INK4c in fetal germ cell development.Effects of Ink4c and Ink4d loss on the pituitary-gonadal axis. Although the noted defects in spermatogenesis in Ink4cd double-null mice are likely to be germ cell autonomous, the loss of Ink4c and Ink4d also affects the production of hormones that regulate germ cell development. The pituitary tumors observed in Ink4c-null or Ink4cd double-null mice arise gradually throughout life but with complete penetrance and primarily involve the intermediate lobe of the gland. Pituitary tumorigenesis is unlikely to be responsible for the observed early postnatal effects on germ cell production in Ink4cd double-null animals, because mice lacking Ink4c alone are fertile and produce normal numbers of sperm and their pituitary tumors arise much later in life and occur in a region of the gland that does not produce LH or FSH.
Whether Ink4d is present or not, Ink4c-null animals, like mice expressing the Ink4-resistant mutant form of Cdk4 (32), exhibit Leydig cell hyperplasia. However, these cells produced only low levels of P450scc, the rate-limiting enzyme for steroid biogenesis (29). In turn, serum testosterone levels in Ink4c-null and Ink4cd double-null mice at 10 to 14 weeks of age were equally low. Because Ink4c-null males had normal sperm counts and were fertile, decreased testosterone production in Ink4cd double-null males was not strictly associated with infertility. In fact, previous studies have demonstrated that the amount of testosterone synthesized by the rodent testis far exceeds that required for normal spermatogenesis (1, 51). Moreover, the initial failure of male gametogenesis in Ink4cd double-null mice was manifested before puberty during the very first wave of spermatogenesis, so the effects of Ink4c loss on Leydig cell function are unlikely to contribute to the reduced meiotic progression. In agreement, we were unable to rescue defective spermatogenesis in Ink4cd double-null males by enforced administration of testosterone (data not shown). Because Ink4c-null and Ink4cd double-null animals synthesized normal levels of LH, the failure to produce normal levels of testosterone does not reflect pituitary LH insufficiency. Instead, Ink4c expression appears to control the ability of pubertal Leydig cells to properly exit the cell cycle, up-regulate steroidogenic enzyme expression, and increase testosterone production. The fact that Ink4d-null mice produced near normal levels of testosterone connotes a distinct role for p18Ink4c, but not p19Ink4d, within Leydig cells. Regardless of whether or not Ink4c was present, mice lacking Ink4d produced elevated levels of FSH. It has been suggested that FSH is required not for the initiation of spermatogenesis but rather for the maintenance of sperm viability and motility (24). Male mice lacking the FSH receptor (10) or lacking the FSH- subunit (24) and men with a
mutation in the FSH receptor (40) are fertile despite
decreased testicular mass and reduced sperm motility and numbers.
However, FSH treatment of oligospermic men results in increased
spermatogenesis rather than improved spermiogenesis, arguing that FSH
might increase the entry of spermatogonia into meiosis
(14). Despite high levels of FSH, however, mice lacking
Ink4d, or both Ink4d and Ink4c, have
small testes and a reduction in sperm counts and viability, suggesting
that the testis is unable to up-regulate spermatogenesis in the absence of the Ink4 proteins. We saw good correlation between testis size and
sperm counts, a phenomenon also found in mice lacking other genes
involved in testicular development, including cyclin D2 (39), Cdk4 (32, 42),
E2F-1 (13, 45), and Egr4
(41).
In males, FSH enhances functions of Sertoli cells that, in turn,
provide support for spermatogonial maturation (10, 36). In
females, FSH plays a similar role in sustaining granulosa cell development, and disruption of cyclin D2, an FSH-responsive
gene in these cells, leads to faulty granulosa cell function and female infertility (39). Disruption of Cdk4 also
results in female infertility, but there is no apparent defect in
granulosa cell development. Instead, female infertility appears to be
due to defects in the hypothalamic-pituitary axis, where Cdk4 loss
leads to a significant reduction in FSH production (32).
Notably, this contrasts with the elevated FSH levels observed in both
Ink4d-null and Ink4cd double-null animals.
Although we have no clear explanation for the increased FSH production
by the pituitary gland in these mice, this could well reflect a role of
Cdk4 in regulating FSH levels in males. In this regard,
Ink4d appears to play a more prominent role than
Ink4c.
The rate of meiotic progression in the testis is governed by an
inhibin-mediated feedback loop that is initiated in the Sertoli cells.
Under normal conditions, the accumulation of pre- and perimeiotic germ
cells stimulates Sertoli cells to produce inhibin, which selectively
suppresses FSH production by gonadotrophs in the anterior pituitary
(9, 10, 30). In light of the reduced rate of meiotic entry
and progression, we might expect that inhibin levels would be reduced
in Ink4cd double-null mice. This feedback mechanism would
explain the selective increase in FSH seen in Ink4cd-null mice, with serum LH concentrations remaining unaffected. We attempted to analyze this possibility by staining for inhibin in the testes of
these mice, but available commercial antibodies did not yield specific
immunohistochemical signals in Sertoli cells. This formally leaves open
the possibility that a surfeit of FSH produced by the anterior
pituitary gland of Ink4cd-null mice acts through Sertoli
cells to drive premeiotic hyperproliferation of Ink4cd-null spermatogonia. However, given that FSH levels in fertile
Ink4d-null mice and infertile Ink4cd double-null
mice are 2.0- and 2.5-fold greater, respectively, than those in
wild-type animals, and that FSH is used to improve spermatogenesis in
men with low sperm counts (14, 36), we believe that
elevated FSH levels are unlikely to explain the abnormalities in
meiotic progression observed in the Ink4cd double-null strain.
In summary, our results demonstrate that p18Ink4c and
p19Ink4d are crucial for the progression of germ cells past
the pachytene stage of spermatogenesis and are most consistent with the
idea that this defect in progression is intrinsic rather than
hormonally driven. Human INK4d and INK4c may be
candidate markers for some forms of male-only infertility, including
those caused by autosomal recessive disorders in which early meiotic
arrest is a hallmark.
| |
ACKNOWLEDGMENTS |
|---|
We are indebted to Zhen Lu, Ming Wang, Esther van de Kamp, Rose Mathew, Myriam Chang, and Liyin Zhu for excellent technical assistance. We thank Jo-Anne Croxford and Patrick Sailor from the Animal Resource Center at St. Jude Children's Research Hospital for their help in handling the mice. We thank Beatriz Sosa-Pineda for her help with the analysis of the mouse pancreas, Shengjie Wu and Xiaoping Xiong for statistical analyses, Justine Cunningham and members of the Department of Biomedical Communications for assistance with photographic production, Julia-Cay Jones for scientific editing of the manuscript, and all members of the laboratory for helpful criticisms.
This work was supported in part by NIH grants CA-71907 (M.F.R.) and CA-89617 (J.W.P.), by Cancer Center grants CA-21765 (SJCRS) and P30-13330 (AECOM), and by the American Lebanese Syrian Associated Charities (ALSAC). C.J.S. is an investigator of the Howard Hughes Medical Institute.
F.Z. and W.D.B. contributed equally to this work.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Tumor Cell Biology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105. Phone: (901) 495-3481. Fax: (901) 495-2381. E-mail: martine.roussel{at}stjude.org.
Present address: Department of Molecular Genetics, Albert Einstein
College of Medicine, Bronx, NY 10461.
| |
REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Molecular and Cellular Biology, May 2001, p. 3244-3255, Vol. 21, No. 9
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.9.3244-3255.2001
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99: 1335-1340
[Abstract] [Full Text]
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