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Previous Article | Next Article 
Molecular and Cellular Biology, November 2006, p. 8507-8514, Vol. 26, No. 22
0270-7306/06/$08.00+0 doi:10.1128/MCB.01488-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Department of Pharmacology,1 Lineberger Comprehensive Cancer Center,4 Pathology and Laboratory Medicine,2 Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 275993
Received 10 August 2006/ Accepted 31 August 2006
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ABSTRACT |
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50% homology to calmodulin and calcineurin B. CIB1 is widely expressed and binds to a number of effectors, such as integrin 
IIb, PAK1, and polo-like kinases, in different tissues. However, the in vivo functions of CIB1 are not well understood. To elucidate the function of CIB1 in whole animals, we used homologous recombination in embryonic stem cells to generate Cib1–/– mice. Although Cib1–/– mice grow normally, the males are sterile due to disruption of the haploid phase of spermatogenesis. This is associated with reduced testis size and numbers of germ cells in seminiferous tubules, increased germ cell apoptosis, and the loss of elongated spermatids and sperm. Cib1–/– testes also show increased mRNA and protein expression of the cell cycle regulator Cdc2/Cdk1. In addition, mouse embryonic fibroblasts (MEFs) derived from Cib1–/– mice exhibit a much slower growth rate compared to Cib1+/+ MEFs, suggesting that CIB1 regulates the cell cycle, differentiation of spermatogenic germ cells, and/or differentiation of supporting Sertoli cells. |
INTRODUCTION |
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subunit (PP1c) (41), and NKcc2, which encodes Na+-K+-2Cl– cotransporter (34). Here, we describe another gene, Cib1, which is essential for male mouse fertility.
CIB1 was first identified as a calcium and integrin binding protein (27). It contains four EF hand motifs, two of which bind calcium (8). We previously found that CIB1 binds to the IIb subunit of integrin IIbß3 and inhibits agonist-induced IIbß3 activation in megakaryocytes, potentially via interference with the association of talin to the integrin (48). CIB1 is widely distributed and binds to a number of potential effectors, such as the small GTPase Rac3 (11), and several serine/threonine kinases, such as the polo-like kinases Plk2 and Plk3 (12), PAK1 (p21-activated kinase) (14), and FAK (focal adhesion kinase) (26). We previously showed that CIB1 directly activates PAK1, which results in decreased cell migration via a LIM kinase- and cofilin-dependent pathway (14). Moreover, sequence analysis indicates that CIB1 may be the founding member of a distinct subfamily of EF hand-containing proteins (8). However, the role of CIB1 in vivo has never been addressed. Therefore, we generated Cib1 null mice via homologous recombination in embryonic stem (ES) cells and found that CIB1 is essential for mouse spermatogenesis.
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MATERIALS AND METHODS |
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5 kb in length. A 550-bp fragment of Cib1 genomic DNA, including complete exon 4 and the majority of exon 5, was replaced with a reversed neo gene in the Cib1 knockout construct at the indicated restriction enzyme sites (Fig. 1A). The correct targeting was verified by both PCR and Southern blot analysis in the 129S6/SvEv ES cell line. The targeted cell lines were injected into C57BL/6 blastocysts, resulting in birth of chimeric mice. PCR and Southern blot analysis verified Cib1 targeting in the offspring of F1 and F2 mice (Fig. 1B). Western blotting with a chicken polyclonal antibody against CIB1 verified a lack of CIB1 protein expression in Cib1–/– mice (Fig. 1C). The housing and breeding of mice and all experiments performed with mice are in accordance with national guidelines and regulations and were approved by the University of North Carolina Institutional Animal Care and Use Committee. The mice were analyzed during the process of backcrossing the 129S6/SvEv strain onto a C57BL/6 background (three to seven generations). The protein and mRNA samples from mice of different genotypes were compared among littermates.
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Histological and TUNEL studies of Cib1–/– mouse testis sections. Mouse testes at various stages were collected and fixed for 24 h in 4% paraformaldehyde, 4% paraformaldehyde, 4% acetic acid or Bouin's solution. Testis sections were stained with hematoxylin and eosin (HE) and/or periodic acid-Schiff's reagent (PAS), and digital images were acquired with either a Nikon Eclipse TE300 microscope equipped with a Nikon D100 camera or an Olympus BH2 microscope equipped with a Spot RT Slider camera (Diagnostic Instruments, Sterling Heights, MD). To detect apoptotic cells in the adult mouse testis sections, the fluorometric terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay kit (Promega) was used according to the manufacturer's guidelines. The fluorometric images were acquired via the Nikon Eclipse TE300 microscope with an attached CoolSnap Photometrics HQ camera from Media Cybernetics with Image-Pro Plus 5.0 software.
RT-PCR. Total mRNA from adult, postnatal day 17, and 25-day-old Cib1+/+ and Cib1–/– mouse testes were extracted with a QIAGEN RNAeasy kit and then quantified and diluted to equivalent concentrations. One-step reverse transcription-PCR (RT-PCR) was used to amplify all the specific messages (QIAGEN). Primers for each gene were synthesized to generate DNA fragments of about 250 bp in length. To avoid possible human error, all primers were first diluted to the same concentration, and the two primers of a particular gene were then mixed. A master mix was initially made for a particular mRNA sample, including every component of the RT-PCR except the primers. An equal amount of the mix was then transferred to the PCR tubes, and equal amounts of excess primer mix were added to each reaction. The RT-PCR cycle numbers for individual genes were first tested to ensure that the PCR cycle number was in a linear amplification range. We found very consistent RT-PCR results in these experiments. The figures shown are representative of at least three independent testis samples for the same genotype and age.
Lysates preparation, Western blotting, and antibodies.
All tissue and cell samples (i.e., testis tissue, purified testis cells, sperm, or MEFs) were lysed in modified 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) buffer (20 mM HEPES, pH 7.4, 0.15 M NaCl, 10 mM CHAPS, 50 mM NaF, 1 mM NaVO3, 10 mM sodium pyrophosphate, 1 mM each of CaCl2 and MgCl2, and Protease Inhibitors Cocktail Set III [Calbiochem]) on ice for 30 min, and lysates were collected after centrifugation. To obtain whole-testis lysates, testes were homogenized with CHAPS lysis buffer. To obtain mixed-testis-cell lysates, the tunica albuginea was removed and the seminiferous tubules were released into, and rinsed in, phosphate-buffered saline. The tubules were then torn with forceps in RPMI 1640 media, and a mixture of cells released from tubules was filtered through a 100-µm mesh (Sigma) and collected and lysed in CHAPS buffer for blotting. Purified spermatogenic cells were isolated from CD1 mice by unit gravity sedimentation as described previously (30). Fractions were assessed for morphology and purity by light microscopy. Sertoli cells were isolated from 17-day-old mice and were then cultured for 1 week (31) before being lysed for Western analysis. The amount of protein was quantified by the bicinchoninic acid protein assay, and protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were then transferred to a polyvinylidene difluoride membrane and subjected to Western blotting. A CIB1 chicken polyclonal antibody was used to detect mouse CIB1. Antibodies against PP1c, PP2A, and -tubulin were all from Santa Cruz Biotechnology. The anti-Cdc2 antibodies were from Santa Cruz Biotechnology and Calbiochem. Rabbit anti-phospho-Cdc2 (Tyr15) was from Cell Signaling. The Cdc2 expression levels in isolated testis cells of Cib1–/– and Cib1+/+ testes were quantified via densitometry with Quantity One software (Fluor-S Multimager; Bio-Rad) and adjusted with -tubulin as a loading control (n = 4).
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RESULTS |
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We also found that the average weight of the testis from adult Cib1–/– male mice (3 to 4 months) is 47.5% that of Cib1+/+ and Cib1+/– males (n = 6; P < 0.009). The weight of the Cib1–/– testis peaks at 40 days and then gradually declines, while the weight of the Cib1+/+ and Cib1+/– testis continues to increase until 60 days of age (Fig. 1E). Sperm production is undetectable upon dissection and analysis of the epididymis (n = 6) from Cib1–/– males. In contrast, male Cib1+/– and female Cib1–/– mice exhibited no obvious impairment of growth, development, or reproductive performance (data not shown). Since testosterone produced by Leydig cells is essential for male fertility and for maintenance of androgen-dependent tissues, such as the seminal vesicles (35), we measured seminal vesicle weights in adult males but found no difference between Cib1+/+, Cib1+/–, and Cib1–/– mice (3 to 4 month age; n = 6; P > 0.6; Fig. 1F). This suggests that Leydig cell function is normal in Cib1–/– males and is unlikely to contribute to the observed phenotype. Our combined results indicate that CIB1 is essential for male fertility in mice and suggest a defect in spermatogenesis per se and/or in Sertoli cells that support germ cell differentiation.
Cib1–/– mouse embryo fibroblasts proliferate at a decreased rate. Precise cell cycle regulation and proliferation are integral for the progression of normal spermatogenesis. To better understand the potential role of CIB1 in cell cycle regulation and cell proliferation, we generated mouse embryo fibroblasts (MEFs) from embryos (13.5 to 14.5 days postcoitum) resulting from the interbreeding of heterozygous Cib1+/– mice (F7). Growth rates of Cib1+/+, Cib1+/–, and Cib1–/– MEFs were analyzed at various passages during an 8-day period. At passage 2 (P2), two separate Cib1–/– MEF cell lines proliferated at a slower rate than Cib1+/+ MEFs (Fig. 2A). At P4, Cib1–/– MEFs continued to proliferate more slowly, and by P6, Cib1–/– MEFs showed a significant delay in the rate of proliferation compared to Cib1+/+ MEFs. However, we found that early-passage Cib1–/– MEFs were morphologically indistinguishable from Cib1+/+ MEFs (Fig. 2B, P4). These data suggest that CIB1 may affect spermatogenesis by regulating cell cycle progression in germ cells and/or Sertoli cells.
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Cdc2 is overexpressed in Cib1–/– mice. The possible regulatory roles of CIB1 in spermatogenesis were tested first by determining which gene expression patterns were altered in the Cib1–/– testis via RT-PCR. We initially examined the expression of stage-specific marker genes that appear during meiosis, i.e., Hsp70-2 (heat shock protein, chaperone) and Sprm-1 (POU homeodomain domain [5, 15, 25, 36]) but found comparable mRNA expression levels in Cib1+/+ and Cib1–/– testis samples (Fig. 4A). Moreover, the expression patterns of all postmeiotic genes examined, such as Tp1 (transition protein 1), Tp2 (transition protein 2), Prm2 (protamine 2), Gapdhs (glyceraldehyde 3-phosphate dehydrogenase-S, testis specific), Trf2 (TBP-related factor 2), and Crem (cyclic AMP-responsive element modulator) were comparable in Cib1+/+ and Cib1–/– mice (Fig. 4A; Tp1 and Gapdhs are not shown) (1, 41, 47, 49). This is in sharp contrast to Crem knockout or mutant mice and Trf2 knockout mice, which have marked defects during the round spermatid stages and altered expression of the aforementioned genes (3, 20, 28, 41, 49). Our findings therefore indicate that the spermiogenesis defect in Cib1–/– mice is unlikely to involve changes in Crem and Trf2 transcriptional regulation.
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protein, but not mRNA expression levels, were lower in adult Cib1–/– testis versus Cib1+/+ testis (Fig. 4B). However, PP1cc mRNA and corresponding PP1c protein levels were not altered in juvenile Cib1–/– testis (Fig. 4A and data not shown). |
DISCUSSION |
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We observed slower growth and division of Cib1–/– MEFs before (P2 and P4) and at crisis (P6) (Fig. 2A) in the absence of overall differences in growth and weight between Cib1–/– and Cib1+/+ mice (data not shown). This result is intriguing, since CIB1 has not been previously implicated in regulating cell growth and division. Although it is apparent that CIB1 function is not essential for somatic cell growth and division, CIB1 may facilitate the regulation of cell growth and division. CIB1 binds to cell cycle-regulatory proteins such as PLK2, PLK3, and DNA-PK (12, 13, 17, 18), and thus loss of CIB1 may lead to a delay of cell growth through one or more of these proteins.
During mouse postnatal testis development, meiosis II in males first occurs around 18 days of age, and the first set of elongated spermatids do not appear until 25 to 30 days (25, 41, 49). Analysis of gene and protein expression of testis samples at different developmental stages helped us to determine where and how defects occur during Cib1–/– testis development. We found that Cdc2 protein is significantly increased in the testis before spermatids appear, whereas postmeiotic genes such as Crem and Trf2 are expressed comparably in Cib1–/– and Cib1+/+ mice. The increase in Cdc2 protein expression in Cib1–/– mice may reflect a compensatory increase that is directly related to the loss of CIB1 or a noncausal increase that is an indirect consequence of testicular defects in these mice. Thus, it is possible that CIB1 participates in the Cdc2 signaling pathway (or vice versa) and that loss of CIB1 affects the cell cycle in germ and/or Sertoli cells.
Interestingly, we observed increases in Cdc2 expression in testis as early as days 8 and 17 (Fig. 4C). Only spermatogonia and Sertoli cells are present in seminiferous tubules at day 8, and spermatogenesis progresses to the pachytene stage of meiotic prophase by day 17 (26). Since Sertoli cells represent a larger proportion of testicular cells during this juvenile period than in the adult, the observed increase in Cdc2 in the Cib1–/– testis may reflect altered expression in Sertoli cells. Such changes in the expression of Cdc2 or other cell cycle regulators could disrupt the normal interval of Sertoli cell proliferation, causing an imbalance between the number of Sertoli cells and developing germ cells (30), which can lead to increased germ cell apoptosis and defective spermatogenesis (40).
Another possibility is that loss of CIB1 leads to defects in Sertoli cell differentiation and function that are required for the maintenance of spermatogenesis. Sertoli cells secrete a variety of endocrine and paracrine factors that regulate spermatogenesis (10). In addition, they assemble and disassemble various junctions that compartmentalize the seminiferous epithelium and ensure the progressive movement of germ cells and the proper release of sperm to the lumen (16). Apical ectoplasmic specializations, which are adhesive junctions between Sertoli cells and condensing spermatids (42), may be particularly important in relation to the formation of the multinucleated spermatids seen in Cib1–/– testes. Thus, loss of CIB1 in Sertoli cells could alter junctional complexes or the microenvironment that regulates germ cell differentiation, resulting in increased germ cell apoptosis and male infertility (16, 23, 24).
The loss of CIB1 may also have direct effects on spermatogenic cells. CIB1 protein levels remain stable throughout testis development to adulthood in Cib1+/+ mice (Fig. 4C and data not shown), probably reflecting the accumulation of spermatids concomitant with a marked reduction in the proportion of Sertoli cells. Although our CIB1 antibody was not suitable for immunohistochemistry, we detected CIB1 by Western blotting in isolated round and condensing spermatids, suggesting that CIB1 may play a role in these cell types. Cdc2 has been implicated in apoptosis, although it is not universally required for apoptosis (9), and indeed we observed an increase in germ cell apoptosis in the seminiferous tubules of Cib1–/– mice. It will be interesting to determine if there is any causal relationship between increased Cdc2 and increased apoptosis in testis cells in our mouse model.
In our initial screen, we also found that PP1c protein is reduced in adult Cib1–/– testis (Fig. 4B). Interestingly, Ppp1c –/– mice show a phenotype similar to that of Cib1–/– mice (41). In addition to severe impairment of spermiogenesis beginning at the round-spermatid stage in Ppp1c–/– males, defects in meiosis were inferred from the presence of polyploid spermatids (42). Further studies are needed to determine if reduced levels of PP1c contribute to the testicular defects observed in Cib1–/– mice.
Since the initial identification of CIB1, new CIB1-interacting proteins are continually being reported. It is now clear that CIB1 is often associated with a variety of serine/threonine kinases (12, 14, 26) as well as with other molecules, such as integrin IIb and presenilin 2 (27, 39). Therefore, CIB1 may regulate spermatogenesis via multiple kinases (identified or yet to be identified) or another mechanism(s). In initial experiments, we did not observe coimmunoprecipitation of CIB1 and Cdc2, nor did we observe altered Cdc2 kinase activity (data not shown). This may indicate that these proteins do not interact, interact very transiently, or do not act in the same signaling pathway. It should be noted that the spermatogenesis defect we observed in Cib1–/– mice could be a combined effect of CIB1-interacting proteins. We are currently investigating the potential binding partner(s) of CIB1 in order to further determine the mechanism by which CIB1 regulates spermatogenesis. Our findings here suggest that CIB1 null mice may provide a useful research model for understanding human male idiopathic infertility and thus fertility drug targets (4, 6).
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ACKNOWLEDGMENTS |
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This study was supported by NIH training grant F32 HL10381 to W.Y., HL42630 to N.M., and NICHD/NIH cooperative agreement U54-HD35041 as part of the Specialized Cooperative Centers Program in Reproduction Research to D.A.O. and 2-P01-HL45100 and 2-P01-HL06350 to L.V.P.
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FOOTNOTES |
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Published
ahead of print on 18 September 2006.
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3 for each data point) were dissected, weighed, and graphed. (F) Adult seminal vesicle weight is comparable between Cib1–/–, Cib1+/+, and Cib1+/– mice. Adult mouse testes (3 to 4 months old) of different genotypes were dissected, and the average weight of seminal vesicles was graphed (n = 6 for all genotypes; P > 0.6 when seminal vesicle mass of Cib1–/– mice is compared to that of Cib1+/+ or Cib1+/– mice; all values represent means ± standard deviations).
