Departments of 1 Pediatrics, 2 Anatomy and Neurobiology, and 3 Pathology and Immunology, and 4 Center for Immunology, Washington University School of Medicine, St. Louis, Missouri 63110; Departments of 5 Pediatrics and 6 Physiology, University of Turku, 20520 Turku; and 7 Children's Hospital, University of Helsinki, 00290 Helsinki, Finland
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ABSTRACT |
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The calcium/calmodulin-dependent protein kinase type IV/Gr (CaMKIV/Gr) is expressed in male germ cells and spermatids and has been implicated in controlling the differentiation of germ cells into mature spermatozoa. The function of CaMKIV/Gr in spermatogenesis was investigated using CaMKIV/Gr-deficient mice generated by targeted gene disruption. CaMKIV/Gr-deficient males exhibited normal spermatogenesis, and their fertility was similar to that of wild-type littermates. Notwithstanding the function of CaMKIV/Gr as an activator of cAMP response element (CRE)-dependent transcription, mRNA levels of several testis-specific CRE modulator (CREM)-regulated genes were unaltered. These results indicate that CaMKIV/Gr is not essential for spermatogenesis or for CRE-regulated gene transcription in the testis.
calcium signaling; calmodulin-dependent protein kinase; spermiogenesis; cyclic adenosine monophosphate response element modulator
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INTRODUCTION |
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SPERMATOGENESIS, THE DIFFERENTIATION of progenitor male germ cells or spermatogonia into spermatozoa, is a complex process that occurs in seminiferous tubules and proceeds through several ordered steps (20). It is initiated upon the commitment to differentiation of spermatogonia present at the periphery of the seminiferous tubule. Committed spermatogonia undergo mitotic division to give rise to spermatocytes. These in turn undergo meiosis to give rise to postmeiotic spermatids. Spermiogenesis, the differentiation of spermatids into spermatozoa, involves complex morphological differentiation including acrosomal formation and nuclear condensation.
A number of molecular mechanisms have been implicated in
regulating the progression of male germ cell differentiation. Of particular interest has been the role of a calcium-signaling pathway involving the calcium/calmodulin-dependent protein kinase type IV/Gr
(CaMKIV/Gr) (13, 15, 16). Several attributes of CaMKIV/Gr have marked it as a candidate regulator of germ cell differentiation. In the mouse testis, CaMKIV/Gr is expressed in male germ cells in a
stage-specific manner. It locates in spermatogonia and spermatids (but
not spermatocytes) and associates with chromatin and the nuclear matrix
of elongating spermatids (25). The CaMKIV/Gr gene encodes
two different CaMKIV/Gr isoforms, a shorter - and a longer
- that extends an extra 28 amino acids at the
NH2-terminal (12, 18, 23). CaMKIV/Gr
gene expression is under distinct, tissue-specific regulation in the
three tissues in which it is enriched; only the
-isoform is
expressed in the testis, whereas both
- and
-isoforms are
expressed in neurons and T lymphocytes (8, 9, 16, 25).
Moreover, the CaMKIV/Gr gene encodes a second protein, calspermin,
which is expressed exclusively in spermatids. Calspermin is
composed of 164 amino acids that correspond to the calmodulin-binding
and the carboxyl-terminal-associative domains of CaMKIV/Gr, and it is
transcribed from a separate internal promoter in the 10th intron of the
CaMKIV/Gr gene (15, 17, 22, 23).
Although the function of CaMKIV/Gr in male germ cell
differentiation has remained undefined, it has been suspected of
serving both transcriptional and posttranscriptional regulatory
functions. CaMKIV/Gr has been demonstrated to activate members of the
cAMP response element (CRE) binding protein (CREB) family of
transcription factors, including the testis-specific CRE modulator-
(CREM
) (11, 21, 22, 24). This factor, which is
activated by both cAMP- and calcium-dependent signaling pathways, has
been revealed as a master molecular switch that is indispensable to the
initiation of spermiogenesis (19). CREM
regulates the
expression of several haploid germ cell-specific genes, including those
encoding protamine-1 and -2 and transitional protein-1 and -2 as well
as calspermin itself. Mice deficient in CREM
suffer from postmeiotic
arrest in the first step of spermiogenesis, leading to complete lack of
spermatozoa and resultant infertility (2, 14). Expression of CaMKIV/Gr and calspermin in spermatids may also serve
posttranscriptional regulatory functions at later stages in
spermatogenesis (25).
The present report makes use of CaMKIV/Gr-deficient mice generated by targeted gene disruption to analyze the function of this kinase in male germ cell development. We find that CaMKIV/Gr is dispensable for spermatogenesis.
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MATERIALS AND METHODS |
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Mice.
CaMKIV/Gr-deficient mice were derived by targeted disruption of the
79-bp-long exon III of CaMKIV/Gr gene, corresponding to bp 195-273
of murine CaMKIV/Gr cDNA
(5'-GGGTGCTACATCCATTGTGTACAGATGCAAACAGAA
GGGGACCCAGAAGCCCTATGCTCTCAAAGTGTTAAAGAAAACA-3') (10). Details of the targeting strategy have been reported
elsewhere (9). Germline transmission of the mutant
CaMKIV/Gr gene was carried out by breeding male chimeric mice with both
inbred C57BL/6 and outbred Black Swiss mice. Mice were back-crossed for
at least five generations on both backgrounds. Wild-type (WT),
heterozygous (HET), and homozygous deficient (KO) littermate mice were
derived by mating heterozygous animals. The mice used in these studies were housed on a 12:12-h light-dark cycle with ad libitum access to
rodent chow. All mouse protocols were in accordance with National Institutes of Health guidelines and were approved by the Animal Care
and Use Committee of Washington University School of Medicine.
Histopathology and TdT-mediated nick end labeling. Testes were fixed in Bouin's fixative and embedded in paraffin. Sections were cut at 5 µm and stained with periodic acid Schiff-hematoxylin. TdT-mediated nick end labeling (TUNEL) staining was performed as described (27). Briefly, after rehydration, the sections were incubated in 2× SSC at 80°C for 20 min followed by washing twice with water and once with proteinase K buffer (20 mM Tris · HCl, pH 7.4, 2 mM CaCl2) for 5 min each. The slides were then treated with proteinase K (10 µg/ml, Roche Molecular Biochemicals, Indianapolis, IN) in proteinase K buffer at 37°C for 30 min. An aliquot of 3'-end labeling reaction mixture containing 4 µl of 5× terminal deoxynucleotidyl transferase (TdT) buffer (Promega), 0.1 µl of digoxigenin-11-ddUTP (10 nmol/µl; Roche Molecular Biochemicals), 0.2 µl of ddATP (5 mM; Promega), 1 µl of TdT (Roche Molecular Biochemicals), and 14.7 µl nuclease-free water (Promega) was applied to one section. The slides were kept in a humidified box, incubated at 37°C for 1 h, and then washed three times with TBST buffer (10 mM Tris · HCl, pH 8.0, 100 mM NaCl, and 0.1% Tween-20) for 10 min each. An anti-digoxigenin-horseradish peroxidase monoclonal antibody (DAKO, 1:200 dilution in TBST containing 1% BSA) was applied, and the slides were incubated in the humidified box at room temperature for 1 h and then washed three times with TBST for 5 min each time. Finally, the labeled cells were visualized by 3,3'-diaminobenzidine tetrahydrochloride (Sigma) for 0.5-2 min.
Immunoblotting.
Homogenates of testes of adult WT, HET, and KO littermate mice were
cleared by high-speed centrifugation, and 50-µg protein samples were
resolved by SDS-PAGE and then transferred to nitrocellulose membranes
for immunoblotting. Membranes were blocked in fat-free milk and then
probed with one or more of the following antibodies as indicated: mouse
monoclonal anti-CaMKIV/Gr catalytic domain antibody, anti-CaMKII
antibody, and anti-protein kinase A (PKA) catalytic subunit (PKAc)
antibody (Transduction Laboratories); goat polyclonal anti-CaMKIV/Gr
COOH-terminal peptide antibody, rabbit polyclonal anti-ERK or anti-CREM
antibodies, and mouse monoclonal anti-Rsk-2 or anti-CREB antibodies
(Santa Cruz Biotechnology). The blots were developed using horseradish
peroxidase-conjugated secondary antibodies and enzyme-linked enhanced
chemiluminescence (ECL, Amersham Pharmacia Biotech).
Northern blot analysis. Total RNA was extracted from mouse testis using RNAzol. RNA (10 µg/lane) was loaded onto a formaldheyde gel, resolved by electrophoresis, and transferred to Hybond-XL membranes (Amersham Pharmacia Biotech). Membranes were hybridized with the indicated probes overnight at 42°C in ULTRAhyb solution (Ambion). The membranes were washed, exposed, and then stripped and reprobed for glyceraldheyde 3-phosphate dehydrogenase (GAPDH) transcripts to confirm equal loading. Probes for the following transcripts were obtained as expressed sequence tag clones from Human Genome Systems (St. Louis, MO): protamine-1 (GenBank accession no. AA064036), protamine-2 (AI528784), transitional protein-1 (AA144629). GAPDH and CaMKIV/Gr full-length cDNA were derived by PCR from mouse brain cDNA. A DNA fragment corresponding to CaMKIV/Gr cDNA bp 39-791 (5'-end cDNA) was derived by EcoRI digestion (10). An exon III-specific single-stranded oligonucleotide probe was synthesized that spans bp 195-243 of murine CaMKIV/Gr cDNA, 5'-GGGTGCTACATCCATTGTGTACAGATGCAAACAGAAGGGGACCCAGAAG-3' (10). cDNA probes were labeled with [32P]dCTP by use of a Prime It II kit (Stratagene), and the exon III-specific oligonucleotide probe was labeled with [32P]dATP by means of the Starfire kit (Integrated DNA Technologies).
RT-PCR analysis and sequencing. cDNA was derived from total testis RNA of WT, HET, and KO mice by reverse transcription using an oligo dT primer and then subjected to RT-PCR analysis with the use of the following exon III sequence flanking primers: 5'-TCCTCTGGGCGATTTCTTCG-3' (sense primer; bp 153-172 of murine CaMKIV/Gr cDNA) and 5'-CTGATTTCTGTGGGGGTTTCG-3' (antisense primer; bp 377-357 of murine CaMKIV/Gr cDNA) (10). PCR conditions were 95°C for 1 min followed by 35 cycles at the following settings: 94°C for 30 s, 62°C for 30 s, and 72°C for 30 s. Amplified products were sequenced using an ABI 377 sequencer with dye-labeled dideoxy terminators.
Sperm count. The caudae epididymides of WT and KO mice were dissected, and their sperm content was released into phosphate-buffered saline medium. Sperm number and motility (measured as percent sperm with active flagella) were determined using a hemocytometer.
Fertility assessment. In this study, 6-wk-old WT and KO littermate males were derived by mating heterozygous F5-F7 Black Swiss parents. These were mated with WT F5-F7 Black Swiss females. One female was placed with each male, and the number of days leading to the first two deliveries was counted to a maximal follow-up period of 11-wk. Litter sizes were counted at birth.
Testosterone assay. Fifty microliters of serum were collected from males by retroorbital bleeding. Serum testosterone levels were determined using an RIA kit (Diagnostic Products, Los Angeles, CA) following the manufacturer's recommendations.
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RESULTS |
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CaMKIV/Gr-deficient mice were generated by targeted
disruption of the 79-bp-long exon III of CaMKIV/Gr gene
(9). The targeting strategy aimed to disrupt expression
of CaMKIV/Gr - and
-isoforms while sparing that of
calspermin by avoiding targeting of CaMKIV/Gr or calspermin promoter
sequences or of calspermin coding sequences. Replacement of
CaMKIV/Gr exon III with a neomycin resistance gene was predicted
to result in the generation of out-of-frame, alternatively spliced
CaMKIV/Gr transcripts that lacked the deleted exon III sequence. The
mutant transcripts were predicted to encode a catalytically inactive
55-amino acid peptide before terminating at a premature stop codon.
These predictions were verified by several criteria. First, Northern
blot analysis with a full-length CaMKIV/Gr cDNA probe (bp 39-1466)
or a cDNA probe spanning the cDNA 5' region (bp 39-791) revealed
normal levels of CaMKIV/Gr and calspermin transcripts in testes of
mutant mice (Fig. 1A). Levels
of CaMKIV/Gr transcripts were similarly unaffected in brains and thymi
of mutant mice (data not shown). However, both Northern blot analysis
and RT-PCR confirmed the lack of exon III sequence from CaMKIV/Gr transcripts of KO mice. Testicular CaMKIV/Gr transcripts of KO mice
failed to hybridize to an oligonucleotide probe specific for exon III
sequence, whereas transcripts of HET mice hybridized at 50% of the
level noted for those of WT littermates. Furthermore, RT-PCR analysis
with flanking primers confirmed the absence of the targeted exon III
sequence in transcripts of KO mice, whereas HET mice manifested both WT
and exon III-deficient transcripts (Fig. 1B).
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The lack of exon III sequence in CaMKIV/Gr transcripts of KO animals was further verified by direct sequencing of exon III-flanking RT-PCR products. Figure 1C demonstrates that, although WT transcripts showed the predicted transition from exon II to exon III sequence at codon 50, mutant KO transcripts skipped directly from exon II to exon IV sequence. The loss of exon III sequence rendered the KO transcripts out of frame. This resulted in a garbled 5-codon sequence downstream of codon 50 followed by a premature stop codon. On the basis of our previous studies on NH2-terminal, catalytically inactive CaMKIV/Gr fragments, such a peptide would fail to express in targeted cells due to instability/degradation (4).
Expression of CaMKIV/Gr protein in gene-targeted animals was evaluated
by immunoblotting with the use of antibodies directed at different
domains of CaMKIV/Gr, including the catalytic and the COOH-terminal
associative domains. CaMKIV/Gr protein expression was found to be
totally absent in KO mice and was reduced by 50% in HET mice compared
with WT littermates (Fig. 1D) (9). In contrast, expression of the testis-specific calspermin protein, as
detected by an anti-CaMKIV/Gr COOH-terminal peptide antibody, was
normal (9). Similarly, there was normal expression in
CaMKIV/Gr KO mice of other protein kinases, such as CaMKII,
extracellular signal-regulated kinase-1 and -2, receptor-activated
signal kinase-2, PKA, and the CaMKIV/Gr substrates CREB and CREM
(Fig. 1D).
Transmission of WT and mutant alleles was analyzed in weaned mice derived by mating of heterozygous parents. One thousand eighteen mice were examined in this way, pooled from matings of parents on predominantly outbred (F4-F5 Black Swiss), inbred (F6-F8 C57BL/6J), or mixed backgrounds (F2-F5 129× Black Swiss or 129× B57BL/6J). Of those, 245 (24%) carried only the WT allele, 529 (52%) had one copy of the mutant allele, and 244 (24%) carried two copies of the mutant allele. The ratio of the three groups of mice is consistent with Mendelian transmission of both alleles. Analysis of mutant gene transmission in subgroups of out- and inbred populations yielded similar results. These findings indicated that the presence of the mutant CaMKIV/Gr allele did not impair fetal development or early postnatal survival.
Measurement of testis weights revealed no significant difference
between WT and KO mice (Table 1).
Testosterone levels were also found similar in the two groups (Table
1). Sperm count and motility of CaMKIV/Gr KO mice were examined and
compared with those of WT littermates; results revealed no significant
differences between WT and KO mice in the total sperm counts or in the
proportion of motile sperm. To examine the fecundity of CaMKIV/Gr KO
male mice, WT and KO males derived from matings of F5-F7 Black Swiss HET parents were mated with WT and HET females of similar background. The results, shown in Table 2, revealed
no significant difference between WT and KO groups in the proportion of
females attaining pregnancy, the number of days it took to deliver the
first and second litters, or the litter sizes (with the exception of
modestly larger 2nd litter sizes in the KO group). The progeny appeared normal.
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Examination of periodic acid Schiff-hematoxylin-stained,
paraffin-embedded sections of testes from 3-mo-old WT and KO mice on
both outbred (Black Swiss) and inbred (C57BL/6J) backgrounds showed
normal histological features in the KO group (Fig.
2A). The seminiferous tubules
of KO mice appeared normal in diameter and lumen size. They also
appeared to contain normal numbers of Sertoli cells, spermatogonia,
spermatocytes, and round and condensed spermatids. Spermatogenesis
proceeded normally until step 16 spermatids, and the lumen contained
mature sperm. To determine whether CaMKIV/Gr deficiency was associated
with enhanced apoptosis of male germ cells, in situ TUNEL was
performed to detect DNA-free ends, a cardinal feature of
apoptosis. Figure 2B demonstrates that similar numbers of TUNEL-stained cells were detected in WT and KO testes, indicating that CaMKIV/Gr deficiency was not associated with enhanced male germ cell apoptosis. The results demonstrating the normal histology, spermatogenesis, and apoptosis in the KO testes are consistent with the adequate sperm count observed in these animals.
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Because CaMKIV/Gr has been implicated as an activator of CREM, we
examined the expression in KO testis of CREM
-regulated gene
transcripts including those encoding protamine-1 and transitional protein-1 and -2. No difference in the expression of these transcripts was observed between WT and KO mice (Fig.
3). These results, together with the
normal levels of calspermin in KO mice, are consistent with the lack of
an effect of CaMKIV/Gr deficiency on CREM
-mediated transcription.
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DISCUSSION |
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Products of the CaMKIV/Gr gene, including CaMKIV/Gr and calspermin, are expressed in male germ cell in a developmentally regulated manner, raising the possibility of a role for these products in Ca2+-mediated regulatory events during spermatogenesis. In this study, we have analyzed mice in which CaMKIV/Gr expression was specifically abrogated while that of calspermin was maintained. We found that CaMKIV/Gr deficiency did not compromise fetal development or early postnatal survival. Also, CaMKIV/Gr-deficient males were fertile and had normal testis weight and serum testosterone levels and normal number and motility of sperm. Testis histology and the progression of spermatogenesis appeared normal, and apoptosis of the germ cells was not increased in KO mice relative to WT controls. It thus appeared that CaMKIV/Gr deficiency did not affect spermatogenesis or male fertility.
CaMKIV/Gr has been suspected of playing a role in spermatogenesis due
in part to its capacity to activate CREM-dependent transcription
(23, 24). This activation may involve phosphorylation by
CaMKIV/Gr of a regulatory serine residue that is conserved among CREB
family members (Ser133 of CREB, Ser117 of
CREM
) and is targeted by CaMKIV/Gr phosphorylation (6, 11,
21). It may additionally involve activation by CaMKIV/Gr of
co-activator proteins such as p300/CREB binding protein (CBP) (5). Consistent with the role of CaMKIV/Gr
as a CREB activator, neuronal Ser133 CREB phosphorylation
and CREB-dependent gene expression were markedly depressed in CaMKIV/Gr
KO mice (9). In addition, several forms of CREB-dependent
neuronal plasticity models were also impaired, including a late phase
of cerebellar long-term depression and hippocampal CA1 long-term
potentiation (1, 3, 9). However, we found no evidence of
impaired CREM
function in the testis. Transcription of
CREM
-regulated genes including calspermin itself, protamine-1 and
-2, and transitional protein-1 was normal. Sparing of testicular
CREM
activation was also inferred by the finding of normal
spermiogenesis in CaMKIV/Gr KO, whereas spermiogenesis is arrested in
CREM
KO mice.
It is possible that CREM function in CaMKIV/Gr KO mice is rescued by
activation of other signaling pathways, including cAMP-dependent PKA
and mitogen-activated protein kinases (6). Alternatively, testicular CREM
activation may proceed by
phosphorylation-independent mechanisms involving the testis-specific
CREM
co-activator ACT (activator of CREM in testis)
(7). Unlike p300/CBP, the capacity of ACT to
associate with and transactivate CREM
is not regulated by
phosphorylation but is constitutive (7). Both CREM
and ACT accumulate synchronously in the adult testis and co-localize in
spermatids. Hence, the sparing of CREM
activity in testes of
CaMKIV/Gr KO mice would follow from the regulation of testicular CREM
function at the level of CREM
and ACT expression rather than
by phosphorylation-induced activation.
Recently, Wu et al. (26) have reported the generation by
targeted gene disruption of CaMKIV/Gr-deficient mice. Similar to the
mice reported herein, those of Wu et al. exhibited no abnormality in
CREM-dependent transcription; however, they suffered from impaired
spermiogenesis and male sterility. The reason for the discrepant
phenotypes of the CaMKIV/Gr-deficient mice of Wu et al. and those
described in this report is not clear. The difference cannot be
ascribed to residual CaMKIV/Gr expression in the mice utilized in this
report, which is totally lacking not only in the testis but in all
other tissues examined, including the brain and T-lymphocytes. The
normal testicular phenotype is in contrast with the manifestation by
the same mutant mice of both neuronal and lymphoid abnormalities that
are consistent with CaMKIV/Gr deficiency. These include the
aforementioned deficits in synaptic plasticity, CREB activation, and
Ca2+-dependent gene transcription (9) as well
as impaired positive selection and Ca2+-dependent gene
transcription in developing T-cells (V. Raman and T. Chatila,
unpublished results). Potential explanations for the discrepancy
between the results of Wu et al. and those reported herein include
differences in the embryonal stem cell clones utilized in the
respective study, the gene-targeting strategies, or the mouse strains
on which studies were carried out. Of note, the gene-targeting strategy
of Wu et al. resulted in the deletion of the promoter sequences of both
- and
-CaMKIV/Gr together with exons I and II. These sequences
were spared in our mice. It remains to be determined whether these or
other factors may have contributed to the different outcomes in the two studies.
Finally, our study does not rule out a latent role for CaMKIV/Gr in spermatogenesis that may be uncovered under special conditions including deficiency of other regulatory pathways implicated in spermatogenesis. Also presently unclear is the function in spermiogenesis of calspermin, whose expression was left unaffected by our targeting strategy. Derivation of mice that suffer from combined deficiencies of CaMKIV/Gr and other regulatory pathways as well as the selective ablation of calspermin expression by gene-targeting approaches may further clarify the function of products of the CaMKIV/Gr gene in male germ cell differentiation.
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ACKNOWLEDGEMENTS |
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We thank Dr. Louis Muglia for critical review of the manuscript. This work was supported by National Institutes of Health Grant HD-35694 (to T. A. Chatila) and by a grant from the Academy of Finland (to M. Heikinheimo and J. Toppari).
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. A. Chatila, Division of Immunology/Rheumatology, Dept. of Pediatrics, Washington University School of Medicine, 1 Children's Pl., St. Louis MO 63110 (E-mail: chatila{at}kids.wustl.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 11 April 2001; accepted in final form 8 June 2001.
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