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Endocrinology Vol. 141, No. 12 4777-4783
Copyright © 2000 by The Endocrine Society

ARTICLES

Female Fertility Is Reduced in Mice Lacking Ca2+/Calmodulin-Dependent Protein Kinase IV1

Joy Y. Wu, Ignacio J. Gonzalez-Robayna, JoAnne S. Richards and Anthony R. Means

Department of Pharmacology and Cancer Biology (J.Y.W., A.R.M.), Duke University Medical Center, Durham, North Carolina 27710; Department of Cell Biology (I.J.G.-R., J.S.R.), Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: Anthony R. Means, Ph.D., Duke University Medical Center, Department of Pharmacology, Box 3813, C238 LSRC Lasalle Street, Durham, North Carolina 27710. E-mail: meansool{at}mc.duk.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) is a serine/threonine protein kinase with limited tissue distribution. CaMKIV is highly expressed in the testis, where it is found in transcriptionally inactive elongating spermatids. We have recently generated mice deficient in CaMKIV. In the absence of CaMKIV, the exchange of sperm nuclear basic proteins in male spermatids is impaired, resulting in male infertility secondary to defective spermiogenesis. The involvement of CaMKIV in female fertility has not been addressed. Here we report that female fertility is markedly reduced in CaMKIV-deficient mice due to impaired follicular development and ovulation. CaMKIV is expressed in the ovary, where it is localized in granulosa cells. We further find that in cultured granulosa cells, CaMKIV expression and subcellular localization are hormonally regulated. As granulosa cells differentiate, CaMKIV levels decrease and the kinase translocates from the nucleus into the cytoplasm. Our results demonstrate a critical role for CaMKIV in female reproduction and point to a potential function in granulosa cell differentiation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CA2+/CALMODULIN-DEPENDENT protein kinase IV (CaMKIV) is a multifunctional serine/threonine protein kinase expressed in a limited number of tissues including brain, thymus, bone marrow, and testis. It is encoded by the Camk4 gene, which also expresses calspermin, an abundant testis protein that consists of the C-terminal 164 amino acids of CaMKIV, including the calmodulin binding domain. Studies have localized CaMKIV to the nucleus, where it is associated with protein phosphatase 2A (1). Although the function of CaMKIV and its substrates in vivo have not yet been elucidated, CaMKIV has been implicated in mediating CREB-mediated transcription in several cell types. In hippocampal neurons, CREB is phosphorylated in response to an electrical stimulus in a CaMKIV-dependent manner (2). Transgenic mice expressing catalytically inactive CaMKIV in thymic T lymphocytes demonstrate an inability to phosphorylate CREB and reduced immediate early gene expression (3). In the testis, CaMKIV had been proposed to play a similar role in phosphorylating and activating the testis-specific transcription factor CREM{tau}, which would in turn stimulate transcription of male germ cell-specific genes including calspermin (4). However, our recent findings suggest that CaMKIV may also function in nuclear processes independent of transcription, because in the testis CaMKIV is highly expressed in postmeiotic transcriptionally inactive spermatids, where it is tightly associated with both chromatin and the nuclear matrix (5).

Our laboratory has generated mice deficient in CaMKIV. These mice demonstrate ataxia, impaired growth and survival, as well as defective Purkinje cell maturation and lymphocyte differentiation. Inspection of male mice reveals that CaMKIV is required for spermatogenesis, as males are infertile secondary to a halt in spermiogenesis, the differentiation of postmeiotic germ cells into immature spermatozoa (6). Consistent with the localization of CaMKIV in elongating spermatids, we found that CaMKIV is not required for CRE-dependent gene expression in male germ cells. Rather, Camk4-/- males exhibit a defect in the sequential replacement of histones by transition proteins and protamines during nuclear condensation (6). Protamine 2 is lost from late elongating spermatids, with prolonged retention of transition protein 2. Protamine 2 can be phosphorylated by CaMKIV in vitro, suggesting that CaMKIV may be involved in the exchange of nuclear basic proteins in differentiating male germ cells.

Despite its involvement in male fertility, the expression of CaMKIV in the ovary and a potential function in female reproduction have not been investigated. Here we report that female mice lacking CaMKIV have significantly reduced fertility. We further find that CaMKIV is expressed in the ovary, where it is localized to granulosa cells and may play a role in follicular development or ovulation. These results may offer new insights into the mechanisms governing female fertility.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Camk4-/- mice
Mice deficient in CaMKIV were generated as previously described (6). Camk4-/- mice were maintained on a 50% C57BL/6J:50% 129/Sv background. Mice were housed at the Duke University Vivarium under a 12-h light, 12-h dark cycle. Food and water were provided ad libitum and care was given in compliance with NIH guidelines on the use of laboratory and experimental animals.

Fertility assays
Camk4+/+, Camk4+/-, and Camk4-/- females were caged with fertile wild-type C57BL/6J males for up to 6 months. The number of litters produced and pups per litter were counted for each female.

Histology
Ovaries were fixed in formalin-buffered saline and paraffin embedded. Seven-micrometer sections were cut and stained with hematoxylin and eosin.

Granulosa cell cultures
Two culture systems were used in this study. In the first, immature rats were primed with estradiol (1.5 mg/0.2 ml propylene glycol once a day for 3 days) as described (7, 8). Granulosa cells were harvested and cultured at 1 x 106 cells/3 ml serum-free medium in serum coated dishes. Cells were treated with FSH and testosterone plus forskolin as indicated in the figure legend.

In the second system, immature female rats were injected with 0.15 IU hCG sc twice daily for 2 days to stimulate the growth of preovulatory (PO) follicles. The PO follicles were dissected before or 6 h after injection of 10 IU hCG. Granulosa cells were harvested from follicles by needle puncture and cultured in DMEM-F12 with 1% FBS as described (9, 10).

Cell extracts and Western blot analyses
Total cell extracts were prepared as described by Ginty et al. (11). Hot (100 C) Tris buffer containing 10% SDS and ß-mercaptoethanol were added to each well. The cells were rapidly scraped into an Eppendorf tube at 100 C for 5 min. Extracts were analyzed by SDS-PAGE, electrophoretically transferred to nylon filter, washed briefly in PBS, and blotted with either 3% BSA or 5% milk at room temperature for 1 h. Anti-CaMKIV antibody was added at 1:5000 dilution. Immunoreactive proteins were visualized with enhanced chemiluminescence (ECL) (Pierce Chemical Co., Rockford, IL). The CaMKIV antibody used does not react with other CaM kinases and behaves very similarly to the antibody used by Wu and Means (5).

Immunocytochemistry
Granulosa cells in each model system were also cultured on glass coverslips for various times in the presence or absence of FSH and T or forskolin. Cells were fixed in fresh 4% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA) in PBS for 30 min at room temperature, blocked with PBS containing 10 mM glycine, and then washed three times with PBS. The fixed cells were permeabilized with 0.5% NP-40 in PBS for 10 min and then blocked with 4% BSA in PBS for 1 h at room temperature. The cells were incubated at 4 C for 18 h with anti-CaMKIV antibody at 1:100. After several PBS washes, cells were incubated with fluorescein-labeled goat antirabbit IgG (1:20, Pierce Chemical Co.) in 4% BSA in PBS for 1 h at room temperature, then visualized on an Axiophot microscope (Carl Zeiss, Thornwood, NY).

Superovulation
Twenty-five-day-old female mice were injected ip with 5 IU PMSG. After 48 h they were injected with 5 IU hCG. The mice were killed following another 48 h and their ovaries harvested for histology.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Camk4+/+, Camk4+/-, and Camk4-/- female mice were bred for up to 26 weeks to wild-type males. Camk4-/- mice demonstrate markedly reduced survival, with less than half surviving into puberty. Overall, Camk4-/- females have significantly decreased fertility. Two of the 6 mice produced no litters at all. Figure 1AGo shows that of the mice still alive after 12 weeks of breeding, the Camk4-/- females produced fewer litters of smaller size than their control littermates. The offspring of Camk4-/- females consisted of similar numbers of male and female pups. For all mice, regardless of length of survival, we compared the average number of pups per litter and found the average litter size for Camk4-/- females to be reduced to 45% of wild-type controls (Fig. 1BGo). Camk4+/- mice are indistinguishable from wild-type in numbers and sizes of litters produced.



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  		Figure 1. Fertility is reduced in Camk4-/- females. A, Of the mice which survived at least 12 weeks of breeding, the average number of litters (hatched bars), pups (black bars) and pups/litter (white bars) produced during these 12 weeks are shown for Camk4+/+ (+/+), Camk4+/- (+/-), and Camk4-/- (-/-) mice. B, The average litter size is indicated for all mice, regardless of length of survival.

 
Histological analysis of ovaries from Camk4-/- mice revealed several abnormalities. The ovaries of females that had produced no offspring contained numerous preantral and preovulatory follicles but no corpora lutea, indicating a failure of ovulation (Fig. 2Go, A and B). Ovaries from subfertile females did contain corpora lutea; however, in these mice several corpora lutea were found with retained oocytes or antral fluid (Fig. 2CGo). A number of Camk4-/- ovaries also contained multiple oocytes within a follicle enclosed by a single layer of granulosa cells (Fig. 2Go, D and E). Such aberrant oocytes or follicles were found in ovaries from 4 of 5 Camk4-/- females taken for histology, and in none of their wild-type littermate controls. These findings suggest that CaMKIV may play a role in follicular development or ovulation.



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  		Figure 2. Histological abnormalities in Camk4-/- ovaries. A, Ovary from a 6-week-old Camk4+/+ female, with several corpora lutea (arrows). B, Ovary from a 6-week-old Camk4-/- female. Note the absence of corpora lutea. C, Retained oocyte (arrow) in a corpus luteum from a Camk4-/- ovary. D, Section demonstrating the presence of two oocytes within a single follicle. E, Three oocytes within a single primordial follicle.

 
To determine whether these defects reflect the presence of CaMKIV within the ovary, immunohistochemical analysis was performed with an antibody raised against CaMKIV. Positive signal was detected in the granulosa cells (Fig. 3AGo). Staining of granulosa cells was blocked by preincubating the antibody with antigen (Fig. 3BGo) and was absent in Camk4-/- mice (data not shown). Western blot analysis of cultured granulosa cells revealed only a single band corresponding in size to CaMKIV confirming that granulosa cells express only CaMKIV (Fig. 4Go).



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  		Figure 3. Immunohistochemical analysis of CaMKIV expression in the ovary. A, Immunohistochemistry was performed on a section from a wild-type ovary, demonstrating that CaMKIV is expressed in the granulosa cells of ovarian follicles (arrows). B, Staining of granulosa cells is absent on a section incubated with CaMKIV antibody that has been preabsorbed with CaMKIV protein.

 


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  		Figure 4. CaMKIV levels decrease with differentiation in granulosa cells. A, Granulosa cells from estrogen-primed immature rats were cultured in serum-free medium. FSH (50 ng/ml) and testosterone (10 ng/ml) were added for varying lengths of time as indicated in the presence and absence of 10 µM forskolin. Cell extracts were harvested and immunoblotted with anti-CaMKIV antibody. B, Granulosa cells were harvested from preovulatory follicles before (PO) or 6 h after hCG treatment (PO-hCG) and cultured in 1% FBS for 6 days. Cells were then treated with forskolin for up to 48 h. Cell extracts were immunoblotted for CaMKIV.

 
To gain insight into a potential function for CaMKIV in granulosa cells, we examined changes in protein level and subcellular localization in cultured granulosa cells induced to differentiate by hormonal stimulation. Two culture systems were used to compare the behavior of CaMKIV in proliferating granulosa cells to that of terminally differentiated luteinized cells. Granulosa cells cultured from preantral follicles of immature rats mimic the acute physiological response to hormonal treatment or increased cyclic AMP levels; longer term hormonal stimulation of these cells results in their differentiation into preovulatory granulosa cells (7, 8). CaMKIV levels did not change acutely in response to treatment with FSH and testosterone or forskolin (Fig. 4AGo). However, treatment with FSH/testosterone for greater than 6 h leads to a decrease in CaMKIV as a function of differentiation which results in very low levels by 48 h (Fig. 4AGo).

In a second culture system, preovulatory granulosa cells are isolated before (PO) or 6 h after an injection of hCG (PO-hCG) to induce luteinization (9, 10). Luteal cells are terminally differentiated and no longer responsive to changes in cAMP. In PO cells cultured for 48 h, CaMKIV levels are decreased, and a smaller band is detected by the anti-CaMKIV antibody at approximately 45 kDa. A similar band is seen in granulosa cells from preantral follicles cultured in serum-free media for 72 h (data not shown), and may reflect a degradation product of CaMKIV. Following luteinization, CaMKIV is nearly undetectable in PO-hCG cells, again suggesting that CaMKIV levels are regulated during differentiation (Fig. 4BGo). CaMKIV subcellular localization also changes in response to differentiation. In both immature and preovulatory granulosa cells, CaMKIV is clearly localized to the nucleus (Fig. 5Go, A and B). However, in luteinized cells CaMKIV is now found exclusively in the cytoplasm (Fig. 5CGo). This is the first demonstration of a change in CaMKIV subcellular localization in response to a cellular stimulus.



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  		Figure 5. CaMKIV subcellular localizations changes after luteinization. Granulosa cells from estrogen-primed immature rats cultured in serum-free medium (A), and PO cells harvested before (B) or 6 h after hCG treatment (C) were subjected to immunocytochemistry with anti-CaMKIV antibody. Note that CaMKIV localization changes from nuclear in A and B to cytoplasmic in C.

 
Our findings suggest that CaMKIV may play a role in the differentiation of granulosa cells, which may explain the defective follicular development and ovulation seen in CaMKIV-deficient ovaries. To better test whether ovulation might be impaired, we challenged Camk4-/- and Camk4+/+ mice with FSH and LH to induce superovulation (Fig. 6Go, A and B). Again, in a subset of Camk4-/- females, there was a complete failure of ovulation as demonstrated by the absence of corpora lutea. In the remaining mice, corpora lutea were present, but we found occasional abnormal corpora lutea with retained oocytes or antral fluid. The histologic appearance of Camk4-/- ovaries subjected to superovulating stimuli is identical with that of ovaries not exposed to exogenous hormones, suggesting that while CaMKIV-deficient follicles are able to develop to the preovulatory state, they are unable to ovulate even in response to supraphysiologic doses of hormones. These results confirm that ovulation is unable to proceed normally in the absence of CaMKIV.



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  		Figure 6. Superovulation of Camk4-/- ovaries. 25 day-old Camk4+/+ (A) and Camk4-/- (B) females were injected with 5 IU PMSG. Forty-eight hours later, the mice were injected with 5 IU hCG, then killed after another 48 h. Note the abundance of corpora lutea in (A, arrows) and absence of corpora lutea in (B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our studies have produced a number of novel findings: 1) CaMKIV is expressed in the ovary in granulosa cells and plays a role in female fertility; 2) Camk4-/- ovaries display abnormalities in follicular development and/or ovulation; 3) CaMKIV levels decrease in granulosa cells during differentiation; and 4) CaMKIV subcellular localization changes from nuclear to cytoplasmic in cells that have undergone luteinization. This is the first identification of a role for CaMKIV in female reproduction.

The histological abnormalities in Camk4-/- mice include defects in follicular maturation and ovulation. In a significant number of females, ovarian follicles seem to arrest just before ovulation. In others, ovulation can occur followed by luteinization of the follicle, but occasional corpora lutea with retained oocytes or antral fluid are noted. Unlike the testis where CaMKIV is found only in germ cells (5), within the ovary CaMKIV is localized to the granulosa cells, which are of somatic origin and play a supportive role in oocyte maturation. Granulosa cells are involved in both ovulation and luteinization, although targeted deletions of other proteins expressed in granulosa cells suggest that these two processes can be dissociated. Mice lacking cyclin D2 fail to ovulate, most likely due to the inability of granulosa cells to proliferate, although the granulosa cells are able to luteinize in response to hormonal stimulation (12). On the other hand, mice deficient for the cell cycle inhibitor p27kip cannot undergo luteinization (13, 14, 15).

Camk4-/- follicles appear to be defective in ovulation, although unlike cyclin D2-null mice the granulosa cells are capable of proliferating. The Camk4-/- phenotype shares some similarities with both the IGF-I-/- (16) and C/EBPß-/- (17) mice. IGF-1-null mice develop large antral follicles but are unable to ovulate (16). IGF-1 is not required for proliferation and may instead be involved more specifically in granulosa cell differentiation before ovulation. Ovaries from C/EBPß-/- females lack corpora lutea unless stimulated to superovulate, and retained oocytes can be found within the resulting luteinizing follicles. C/EBPß appears to function downstream of the LH receptor to regulate gene expression (17). Together, these observations suggest that the pathways that regulate the differentiation of granulosa cells or their responses to ovulating stimuli may be impaired in the absence of CaMKIV. Multiple oocytes in a single follicle have also been reported in mice lacking Ahch (18), which encodes the transcription factor Dax-1 involved in sex determination (19, 20). CaMKIV may also play a role in earlier follicular development.

In support of a role for CaMKIV in granulosa cell differentiation, we find that CaMKIV expression and localization in the ovary are regulated during hormone-induced differentiation. CaMKIV levels decrease as cultured granulosa cells assume a preovulatory phenotype, and CaMKIV is absent in granulosa cells following luteinization. We further find that, in cultured cells, CaMKIV moves from the nucleus into the cytoplasm upon hCG-induced luteinization. In the nucleus, CaMKIV may be involved in events leading to differentiation of granulosa cells. Once the cells have undergone luteinization, CaMKIV is transported out of the nucleus. This is the first demonstration of an inducible change in the subcellular localization of CaMKIV, and the mechanisms governing CaMKIV trafficking are not understood. CaMKIV contains a putative nuclear localization signal and two putative nuclear export sequences. One potential mechanism may be similar to that by which calcineurin regulates shuttling of NFAT between the cytoplasm and nucleus. Initially, it exposes the nuclear localization signal by dephosphorylation and sterically interferes with the nuclear export sequence. Inactivation of calcineurin results in rephosphorylation of NFAT, which exposes the nuclear export sequences and obscures the nuclear import signal (21, 22, 23, 24). Perhaps the phosphorylation of CaMKIV promotes nuclear entry, whereas the dephosphorylation and inactivation by PP2A (1) allows shuttling out of the nucleus.

Because CaMKIV levels decrease concurrently with its appearance in the cytoplasm, it is likely that CaMKIV is degraded once shuttled out of the nucleus, although how this occurs is unknown. One clue may be the appearance of a smaller 45-kDa band in granulosa cells from both preantral and preovulatory follicles cultured for greater than 48 h. This band may be the result of cleavage of the full-length form of CaMKIV. The only reported cleavage of CaMKIV occurs in human neuroblastoma cells induced to undergo apoptosis (25). In these cells CaMKIV is cleaved by caspase 3 to generate several smaller fragments, the largest of which is approximately 40 kDa. Caspase 3 is also expressed in the ovary, where it has been identified in granulosa cells of atretic follicles and luteal cells during luteal regression (26, 27). Granulosa cells cultured from preantral follicles eventually assume a preovulatory phenotype (28). Perhaps granulosa cells maintained at the preovulatory stage in culture for extended periods of time will initiate an apoptotic program, and the 45-kDa band results from caspase 3 activation in a subset of cells. Clarification of the mechanism underlying the appearance of this smaller band and its relevance in vivo awaits further investigation.

The means by which CaMKIV might regulate granulosa cell differentiation has not been elucidated. Based on its role as a transcriptional regulator in other tissues, one attractive hypothesis is that CaMKIV is involved in CREB-mediated transcription. CREB is expressed in the ovary, where it is phosphorylated in response to acute hormonal treatment (29). Interestingly, CREB subcellular localization also changes from nuclear in immature granulosa cells to cytoplasmic as a function of granulosa cell differentiation in a manner similar to that of CaMKIV (29). Protein kinase A is often considered the relevant CREB kinase. However, CaMKIV can also phosphorylate CREB in vitro and in cells (30), and CaMKIV can stimulate CREB-mediated transcription in a variety of cell types (2, 3, 31, 32). Furthermore, we now have evidence that cerebellar phospho-CREB is dramatically reduced in Camk4-/- mice (33), suggesting that CaMKIV may be a physiologically relevant CREB kinase in some tissues.

Our findings in the ovary highlight a general trend for a role for CaMKIV in differentiation processes. In the cerebellum, the maturation of Purkinje cells that normally occurs during the second week of postnatal life depends on CaMKIV (33) and may involve the ability of CaMKIV to stimulate transcription by the ROR{alpha} transcription factor (34). In T lymphocytes, CaMKIV has been found to play a critical role in the differentiation of naïve CD4+ T cells into Th2 cells due to its essential requirement for transcriptional activation of the IL-4 gene (Anderson, K. A., and A. R. Means, unpublished data). Monocyte and neutrophil production from myeloid progenitor cells in the bone marrow is also impaired in Camk4-/- mice (Kitsos, C. M., B. L. Harvat, and A. R. Means, unpublished data). In the testis the final steps in the differentiation of postmeiotic germ cells into sperm are defective in the absence of CaMKIV due to a unique posttranscriptional effect of CaMKIV (6). Clearly, a number of different mechanisms must be responsible for this plethora of processes that require CaMKIV but appear to involve cellular differentiation as a common theme.


   Acknowledgments
 
The authors would like to thank Claire Lo and Tom Ribar for their assistance.


   Footnotes
 
1 This work was supported by an NIH Medical Scientist Training Program award (to J.Y.W.) and NIH Grants HD-07503 (to A.R.M.) and HD-1272 (to J.S.R.) Back

Received June 28, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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