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Endocrinology Vol. 143, No. 2 647-654
Copyright © 2002 by The Endocrine Society

REPRODUCTION-DEVELOPMENT

Intact Follicular Maturation and Defective Luteal Function in Mice Deficient for Cyclin- Dependent Kinase-4

David S. Moons, Siwanon Jirawatnotai, Tateki Tsutsui, Roberta Franks, A. F. Parlow, Dale B. Hales, Geula Gibori, Asgerally T. Fazleabas and Hiroaki Kiyokawa

Departments of Molecular Genetics (D.S.M., S.J., T.T., H.K.), Physiology and Biophysics (D.B.H., G.G.), and Obstetrics and Gynecology (A.T.F.), and Research Resources Center (R.F.), University of Illinois College of Medicine, Chicago, Illinois 60607; Department of Obstetrics and Gynecology (T.T.), Osaka University Medical School, Osaka 565-0871, Japan; National Hormone and Peptide Program (A.F.P.), Harbor-UCLA Medical Center, Research and Education Institute, Torrance, California 90509

Address all correspondence and requests for reprints to: Hiroaki Kiyokawa, M.D., Ph.D., Department of Molecular Genetics, University of Illinois College of Medicine, 900 South Ashland Avenue, M/C 669, Chicago, Illinois 60607-7170. E-mail: kiyokawa{at}uic.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell cycle progression of granulosa cells is critical for ovarian function, especially follicular maturation. During follicular maturation, FSH induces cyclin D2, which promotes G1 progression by activating cyclin-dependent kinase-4 (Cdk4). Because cyclin D2-deficient mice exhibit a block in follicular growth, cyclin D2/Cdk4 has been hypothesized to be required for FSH-dependent proliferation of granulosa cells. Here we investigate ovarian function in Cdk4-knockout mice we recently generated. Cdk4-/- females were sterile, but the morphology of their ovaries appeared normal before sexual maturation. The number of preovulatory follicles and the ovulation efficiency were modestly reduced in gonadotropin-treated Cdk4-/- mice. However, unlike cyclin D2-deficient mice, Cdk4-/- mice showed no obvious defect in FSH-induced proliferation of granulosa cells. Cdk4-/- ovaries displayed normal preovulatory expression of aromatase, PR, and cyclooxygenase-2. Postovulatory progesterone secretion was markedly impaired in Cdk4-/- mice, although granulosa cells initiated luteinization with induction of p450 side-chain cleavage cytochrome and p27Kip1. Progesterone treatment rescued implantation and restored fertility in Cdk4-/- mice. Serum PRL levels after mating were significantly reduced in Cdk4-/- mice, suggesting the involvement of perturbed PRL regulation in luteal failure. Thus, Cdk4 is critical for luteal function, and some redundant protein(s) can compensate for the absence of Cdk4 in proliferation of granulosa cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE MAMMAL, the balance of growth-stimulatory and inhibitory signals regulates the G1 phase of the cell cycle, governing the transition between proliferation and quiescence (1). Cyclin-dependent kinases (Cdks), activated by the regulatory cyclin subunits, control cell cycle progression (2, 3, 4, 5). Cyclin D- and cyclin E-dependent kinases play central roles in G1 progression. During early G1, D-type cyclins, D1, D2, and D3, bind to and activate Cdk4 or Cdk6. Cyclin E activates Cdk2 in late G1. The coordinated temporal activation of cyclin D- and cyclin E-dependent kinases is crucial for proper transition from G1 to S. Cyclin D-dependent phosphorylation of proteins in the retinoblastoma gene family, such as p130 and p110Rb, increases the expression of the cyclin E gene by activating the transcription factor E2F. This forms a feed-forward control pathway (6, 7). These G1- regulatory Cdks are subject to negative regulation mediated by the Cdk inhibitor proteins. The Cdk inhibitors are categorized into two groups: the Kip/Cip-type inhibitors, p21, p27, and p57, and the Ink4-type inhibitors, p16, p15, p18, and p19 (8, 9). Recent studies using knockout mice have demonstrated that each of these Cdk inhibitors has a unique in vivo function, and they also share overlapping roles. For instance, p21-deficient mice develop apparently normally and are fertile, but they display a modest defect in establishing G1 arrest in response to DNA damage (10, 11). In contrast, p27-deficient mice exhibit gigantism with multitissue hyperplasia, female infertility, and benign adenomas in the intermediate lobe (melanotroph) of the pituitary (12, 13, 14). The p57-deficient mice die perinatally with various developmental defects including impaired development of the intestine and bone (15, 16). These data indicate that the regulation of cyclin D- and cyclin E-dependent kinases plays complex roles in the development and function of different tissues.

In the ovary, hormonal regulation of the cell cycle machinery in granulosa cells is critical for follicular growth, ovulation, and the formation of the corpus luteum (luteinization) (17). Granulosa cells in primordial follicles are arrested in G0. During the transition from primordial to preantral follicles, granulosa cells undergo extremely slow cell cycle progression in response to undefined stimuli (18). Thereafter, in response to FSH and estrogen, granulosa cells of preantral follicles initiate very rapid cell cycle progression. This proliferation leads to the development of large preovulatory follicles within 3 d in rodents (19). These hormones are essential for the formation of preovulatory follicles because mice deficient in gonadotropins, FSH, or ER-{alpha} exhibit blockage in folliculogenesis at the preantral stage (20, 21, 22). Recently, JoAnne Richards’ group (23, 24) elegantly demonstrated that cyclin D2 plays a key role in follicular growth and maturation. FSH or bromo-cAMP induces cyclin D2 expression in cultured rat granulosa cells. Moreover, mice deficient in cyclin D2 display a similar ovarian phenotype as these mutants, with impaired proliferation of granulosa cells and anovulation. These observations suggest that FSH-dependent induction of cyclin D2 is an essential driving force for cell cycle progression from preantral to preovulatory stage in folliculogenesis.

LH surge rapidly induces G1 arrest and luteal differentiation of granulosa cells in preovulatory follicles. Granulosa cells in rat preovulatory follicles arrest in G1 within 7 h, but ovulation occurs around 12 h after LH surge (25). The cell cycle arrest coincides with rapid down-regulation of cyclin D2 expression, followed by induction of p21 and p27 expression (12, 24, 26). Although p21-deficient mice exhibit no reproductive phenotype, p27-deficient female mice are sterile (12, 13, 14). We previously demonstrated that granulosa cells in the ovary of p27-deficient mice continue to proliferate beyond 48 h after LH surge (26). These data suggest that p27 plays a rate-limiting role in establishing quiescence of luteinizing granulosa cells.

To further investigate the role of G1-Cdk regulation in tissue development and function, we recently generated mice with targeted disruption of the Cdk4 gene (27). Intriguingly, Cdk4-deficient female mice were sterile without exception. However, ovaries of adult homozygous females showed apparently normal antral follicles. Corpora lutea were also observed in Cdk4-/- ovaries. Rane et al. (28) generated another strain of Cdk4-deficient mice independently, which also showed sterility. They also reported prolonged estrus cycle and low levels of serum progesterone in the mutant mice. Although these observations indicate that Cdk4 is essential for female fertility, the mechanism of sterility in Cdk4-deficient mice has not been determined. Cdk4 is expressed almost ubiquitously, and we need to clarify the involvement of the ovary and that of extraovarian tissues in the defect. In this report, we have investigated the mechanisms of the female reproductive defect in Cdk4-/- mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
A Cdk4-null mutation, Cdk4tm1Kiyo, was created in mouse embryonic stem cells, and mice with germline transmission of this mutation were bred, as described previously (27). Mice were kept at 25 C with a 13-h light, 11-h dark cycle; they were fed a pelleted diet ad libitum. Heterozygous mutants in the C57BL/6 x 129/sv background were intercrossed to generate +/+, +/-, and -/- mice. Pups were genotyped by PCR amplification using tail DNA and the following primers: 5'-ATATTGCTGAAGAGCTTGGCGG-3', 5'-CGGAAGGCAGAGATTCGCTTAT-3', and 5'-CCAGCCTGAAGCTAAGAGTAGCTGT-3'. The wild-type allele generated a 195-bp product, and the mutant allele formed a 315-bp product. Because a fraction of Cdk4-/- mice develop diabetes mellitus by 8 wk of age (27), we examined blood and urine glucose of every Cdk4-/- mouse and used normoglycemic mice without glucosuria for the study. The day when the vaginal plug was found was taken as d 1 post coitum (p.c.). To stimulate follicular growth and ovulation (superovulation), 28-d-old females were injected ip with 5 IU of PMSG (Calbiochem, San Diego, CA), and 48 h later with 5 IU of human CG (hCG) (Calbiochem). To label cells in the S phase, mice were injected ip with 50 µg bromodeoxyuridine (BrdU) per gram body weight at 2 h before sacrificing. To examine the effect of exogenous progesterone, mice were injected sc with 2 mg progesterone (Sigma, St. Louis, MO) dissolved in peanut oil, daily from d 2 p.c. Animals were used in compliance with the NIH/AAALAC guidelines and with the approval of the animal care review committee of the University of Illinois.

Hormone measurements
Blood samples were collected at noon of the day. Mice were anesthetized with an ip injection of avertin, and blood was collected by retroorbital sinus puncture. Precautions were taken to minimize the stress of animals during sampling. Progesterone and E2 were assayed using RIA kits (Diagnostics Systems Laboratories, Inc., Webster, TX), according to the manufacturer’s protocols. PRL was measured by RIA in the laboratory of the National Hormone and Peptide Program.

Immunohistochemistry and immunoblotting
Tissue samples were fixed at 4 C overnight in 10% buffered-formalin (Sigma). Fixed samples were then dehydrated, paraffin-embedded, and sectioned with 5-µm thickness by microtome, with standard procedures. To examine follicles in the entire ovary, samples were step sectioned with 100-µm gaps and stained with hematoxylin and eosin. Immunohistochemistry for BrdU and p27 was performed as described previously (26). A polyclonal rabbit antiserum to rat p450 side-chain cleavage cytochrome (p450scc) was previously characterized (29). Anti-p27 monoclonal antibody (K25020) was obtained from Transduction Laboratories (San Diego, CA). Signals were visualized using the Vectastain Elite ABC kit (Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer’s instruction.

RT-PCR
Total RNA [250 ng for aromatase; 500 ng for cyclooxygenase-2 (COX2), PR, and L19] was reverse transcribed with poly dT and SuperScript (Life Technologies, Inc., Rockville, MD) at 37 C for 60 min. The target cDNA was amplified by PCR using 1.5 U Taq polymerase (Life Technologies, Inc.), 20 pmol of each primer, 0.25 mM dNTPs, 1.5 mM MgCl2 in a 25-µl reaction, at 94 C for 1 min, 62 C for 30 sec, 72 C for 45 sec with 27 cycles for aromatase and PR; 22 cycles for COX2 and L19. The primers used are: for aromatase, 5'-TGCACAGGCTCGAGTACTTTC-3' and 5'-ATTTCCACAAGGTGCCTGTCC-3' (30); for PR, 5'-CCCACAGGAGTTTGTCAAACT-3' and 5'-TAACTTCAGACATCATTTCC-3' (31); for COX2, 5'-TGTACAAGCAGTGGCAAAGG-3' and 5'-GCTGTGGATCTTGCACATTG-3'; for L19, 5'-CTGAAGGTCAAAGGGAATGTG-3' and 5'-GGACAGAGTCTTGATGATCTC-3'. Semiquantitative conditions for each transcript have been worked out using increasing amounts of RNA. Products were run on a 1.8% agarose gel and analyzed by the Gel Doc image analysis system (Bio-Rad Laboratories, Inc.). The amplified products were 268 bp, 326 bp, 431 bp, and 195 bp for aromatase, PR, COX2, and L19, respectively. The identity of every product was confirmed by direct sequencing.

Statistics
The significance of differences between groups was evaluated with ANOVA and t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ovulation and implantation in Cdk4-/- mice
To examine ovulation, fertilization, and implantation in Cdk4-/- female mice, 8-wk-old Cdk4+/+ and Cdk4-/- females were mated with fertile Cdk4+/+ males. On d 1 p.c., the numbers of ova found in the ampullae of Cdk4+/+ and Cdk4-/- mice were 8.6 ± 1.0 and 6.0 ± 0.6, respectively (means ± SE, n = 5). On d 4 p.c., the numbers of blastocysts found in uteri of Cdk4+/+ and Cdk4-/- mice were 6.7 ± 0.7 and 5.0 ± 0.6, respectively (means ± SE, n = 3). On d 6–8 p.c., no implantation site was observed in uteri of Cdk4-/- mice (n = 6), whereas 6.5 ± 1.0 sites (mean ± SE, n = 5) were found in Cdk4+/+ females. Cdk4-/- mice did not maintain obvious pseudopregnant status following mating and mated again usually within 7 d. Blastocysts recovered from Cdk4-/- females developed normally when transferred into uteri of pseudopregnant normal females (data not shown). These data suggest that sterility of Cdk4-/- mice is due to maternal defect(s) leading to implantation failure. Although the efficiency of ovulation was reduced in Cdk4-/- mice, ovulation did occur and ova were fertilized properly.

Follicular development in Cdk4-/- mice
Spontaneous ovulation in Cdk4-/- mice contrasts with blocked follicular growth and anovulation in cyclin D2-deficient mice (23, 24). To determine whether prepubertal follicular development was altered in Cdk4-/- mice, we first examined ovaries in 21-d-old Cdk4-/- mice. The weight of the ovary was 2.38 ± 0.14 mg and 1.88 ± 0.22 mg (mean ± SEM, n = 6) for Cdk4+/+ and Cdk4-/- mice, respectively. Thus, Cdk4-/- ovaries were approximately 20% smaller than wild-type, and the body weights of Cdk4-/- mice were 30% smaller than those of Cdk4+/+ mice (8.73 ± 1.54 vs. 12.37 ± 0.97 g). Follicles were analyzed by step sectioning of each ovary (Fig. 1Go). The distributions of primordial/primary follicles (type 1–3), preantral follicles with multiple layers of granulosa cells (type 4, 5), and small antral follicles (type 6) were quite similar between Cdk4+/+ and Cdk4-/- ovaries. No large antral or preovulatory follicles (type 7, 8) were observed in these prepubertal animals. These results suggest that there is no major defect in the follicular development in the Cdk4-deficient ovary.



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  		Figure 1. Normal ovarian development in Cdk4-/- mice. A, The morphology of ovaries from 21-d-old Cdk4+/+ and Cdk4-/- mice. Sections were stained with hematoxylin and eosin (HE). Magnification, x100; bar, 100 µm. B, The distribution of ovarian follicles at different stages according to the classification of Pederson and Peters (53 ). Ovaries from 21-d-old Cdk4+/+ mice ({square}) and Cdk4-/- mice ({blacksquare}) were analyzed by step sectioning and HE staining. The data are indicated as mean ± SEM (n = 3).

 
FSH-induced proliferation of Cdk4-/- granulosa cells
To determine whether Cdk4 deficiency affects FSH- induced proliferation, we injected 28-d-old Cdk4+/+ and Cdk4-/- mice with 5 IU PMSG. Immunohistochemistry for BrdU incorporation was performed to label cells in the S phase (Fig. 2Go). Without PMSG treatment, both Cdk4+/+ and Cdk4-/- ovaries displayed BrdU incorporation in some granulosa cells of small follicles. This is consistent with slow FSH-independent proliferation as described previously (18). After PMSG treatment, granulosa cells in both Cdk4+/+ and Cdk4-/- ovaries strongly stained with BrdU, suggesting that these cells were rapidly proliferating in response to FSH. At 48 h after PMSG treatment, we observed quite a few large (types 7 and 8) preovulatory follicles in Cdk4-/- ovaries (17.3 ± 1.7 follicles per mouse, mean ± SE, n = 3). Cdk4+/+ ovaries displayed about 30% more preovulatory follicles (25.6 ± 4.6, n = 3), which could be associated with their larger sizes. These observations indicate that unlike cyclin D2- deficient granulosa cells, Cdk4-deficient granulosa cells are capable of undergoing rapid cell cycle progression in response to FSH.



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  		Figure 2. Granulosa cells in Cdk4-/- ovaries are able to proliferate in response to FSH. Cdk4+/+ and Cdk4-/- mice at 28 d of age were injected ip with 5 IU PMSG or with saline for untreated control. Mice were injected with BrdU at 46 h after PMSG treatment and killed at 48 h. Ovaries were examined for cells in the S phase by immunohistochemistry using anti-BrdU monoclonal antibody. Bar, 100 µm.

 
Ovulation in Cdk4-/- mice following hCG treatment
Next we investigated the response of Cdk4-/- ovaries to an ovulatory dose of hCG. At 48 h after FSH treatment, 28-d-old Cdk4+/+ and Cdk4-/- females were injected with 5 IU hCG. This well-established superovulation scheme triggers ovulation normally in 12 h. Ova in the ampullae were counted at 24 h after hCG treatment (Fig. 3AGo). The number of ovulated ova in Cdk4-/- mice was 36% lower than that in wild-type mice, which is consistent with the decrease in spontaneous ovulation. The lower ovulation efficiency could be related to fewer preovulatory follicles in Cdk4-/- mice as described above. These data suggest that the absence of Cdk4 modestly affects the formation of preovulatory follicles and the efficiency of ovulation.



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  		Figure 3. Ovulatory response and periovulatory gene expression in Cdk4-/- mice. A, Cdk4+/+ and Cdk4-/- mice at 28 d of age were injected ip with 5 IU PMSG and 48 h later with 5 IU hCG. Mice were killed at 24 h after hCG treatment, and ova in the ampullae and uterine horns were collected and counted under a phase microscope. *, P < 0.05 vs. Cdk4+/+ mice. B, Total RNA was prepared from Cdk4+/+ (wild-type) and Cdk4-/- (knockout) ovaries at the times indicated in the panel. The transcripts of aromatase, Cox2/PG endoperoxide synthase-2 (Cox2/PGS2), and L19 were reverse transcribed and amplified by PCR and analyzed by agarose gel electrophoresis and ethidium bromide staining.

 
We further examined the expression of genes that are temporally expressed during preovulatory periods. Aromatase expression is increased in granulosa cells of antral follicles and decreased after ovulatory LH surge (32), consistently with its rate-limiting role in estrogen synthesis. COX2, also known as PG endoperoxide synthase-2, is induced rapidly but transiently after LH surge (33), playing an essential role in ovulation (34, 35). RT-PCR for these genes was performed on RNA prepared from ovaries at various time points after PMSG and hCG treatment (Fig. 3BGo). In both Cdk4+/+ and Cdk4-/- ovaries, aromatase expression was increased within 4 h after PMSG treatment and declined rapidly after hCG injection. No expression of COX2 was detected before hCG administration. At 4 h after hCG treatment, marked expression was detected at similar levels in both genotypes and became undetectable by 24 h after hCG. The expression of the ribosomal L19 RNA, measured as a control, was relatively constant in both Cdk4+/+ and Cdk4-/- ovaries through the times examined. We also examined the expression of PR, an essential gene for ovulation (36). PR was also regulated similarly in Cdk4+/+ and Cdk4-/- mice, peaking at 4 h after hCG treatment (data not shown). These data suggest that the preovulatory expression of these genes is regulated normally in Cdk4-deficient mice.

Luteinization in Cdk4-/- ovaries
Although hCG treatment induced ovulation in Cdk4-/- mice at modestly reduced efficiency, the morphology of their ovaries displayed a significant change. At 48 h after hCG treatment, Cdk4-/- ovaries demonstrated not only corpora lutea but a number of apparently luteinized follicles that still enclosed oocytes (Fig. 4Go). Immunohistochemistry demonstrated that luteinized granulosa cells surrounding oocytes expressed p450scc and p27. Both of these proteins are abundant specifically in luteal cells of normal ovaries (26, 37). The p450scc is a rate-limiting enzyme for progesterone synthesis, and p27 is one of the Cdk inhibitors critical for quiescence of luteal cells. These luteinized cells surrounding entrapped oocytes were postmitotic according to BrdU incorporation studies (data not shown). Cdk4-/- mice displayed 10.3 ± 0.7 luteinized follicles with oocytes entrapped (per mouse, mean ± SE, n = 3), whereas Cdk4+/+ ovaries essentially did not show such follicles. These data indicate that luteinization occurs in Cdk4-deficient ovaries upon the LH signal. However, in some follicles undergoing luteinization, ovulation may be delayed or fail for unknown mechanisms.



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  		Figure 4. Luteinization in ovaries of Cdk4-/- mice. Cdk4+/+ and Cdk4-/- mice at 28 d of age were injected ip with 5 IU PMSG and 48 h later with 5 IU hCG. Mice were killed 48 h after hCG treatment, and ovaries were analyzed by immunohistochemistry for the expression of p450scc and p27Kip1. Arrow, Oocytes entrapped in luteinized follicles; bar, 100 µm.

 
Impaired luteal function in Cdk4-/- mice
Although we observed modest reduction in ovulation efficiency of Cdk4-/- mice, it could not explain their sterility. To further investigate the reproductive defect of Cdk4-/- mice, we measured serum levels of E2 and progesterone. The levels of E2 were not significantly different between Cdk4+/+ and Cdk4-/- mice treated with PMSG and hCG (data not shown). Progesterone levels in Cdk4+/+ mice increased remarkably within 4 h after hCG treatment and gradually decreased by 48 h (Fig. 5AGo). In contrast, Cdk4-/- mice exhibited a similar increase at 4 h, but the progesterone level fell rapidly within 12 h after hCG injection. We also examined levels of serum progesterone in mice after natural mating. Cdk4-/- mice on d 3–6 p.c. showed very low levels of progesterone, whereas Cdk4+/+ mice displayed sustained high levels (>20 ng/ml) of progesterone (Fig. 5BGo). These data suggest that the maintenance of luteal function is impaired in Cdk4-/- mice. To determine whether the decrease in circulating progesterone is responsible for the failure of implantation, we injected Cdk4-/- mice with 2 mg progesterone daily from d 2 p.c. Of 18 Cdk4-/- mice treated with progesterone, 14 mice showed successful implantation, as demonstrated in Fig. 5CGo. In contrast, none of 8 Cdk4-/- mice treated with carrier oil displayed implantation. All Cdk4+/+ mice treated with or without progesterone displayed implantation sites (n = 13). Of 12 Cdk4-/- mice with progesterone observed through d 20 p.c., 9 were able to carry embryos to full term (data not shown). The restoration of pregnancy by exogenous progesterone indicates that defective maintenance of luteal function is the major cause of sterility of Cdk4-deficient mice. To obtain a clue to the mechanism of luteal dysfunction, we measured serum PRL levels in Cdk4+/+ and Cdk4-/- mice after mating (Fig. 6Go) because PRL is a potent luteotropic factor (38). On d 1 and 3 p.c., PRL levels in Cdk4-/- mice were significantly lower than those in Cdk4+/+ mice. Therefore, impaired PRL secretion seems to play a key role in luteal dysfunction of Cdk4-deficient mice.



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  		Figure 5. Implantation failure in Cdk4-/- mice is associated with perturbed progesterone secretion and can be rescued by progesterone. A, Cdk4+/+ ({square}) and Cdk4-/- ({blacksquare}) mice at 28 d of age were injected ip with 5 IU PMSG and 48 h later with 5 IU hCG. Blood was sampled at the times indicated in the panel, and serum levels of progesterone were determined. Data are indicated as mean ± SEM (n = 3). *, P < 0.05 vs. Cdk4+/+ mice. B, Cdk4+/+ ({square}) and Cdk4-/- ({blacksquare}) females at 6 wk of age were mated with Cdk4+/+ fertile males, and blood was sampled for progesterone measurement from females on the day that a vaginal plug was found (d 1) and d 2–6. Data are indicated as mean ± SEM (n = 3). *, P < 0.05 vs. Cdk4+/+ mice. C, Cdk4-/- females at 6 wk of age were mated with Cdk4+/+ fertile males, and progesterone (2 mg) was injected sc daily from d 2 p.c. Mice were killed on d 8 p.c. to examine implantation sites of the uteri. The uterus of a Cdk4+/+ mouse on d 8 p.c. is shown as a control.

 


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  		Figure 6. Low levels of serum PRL in Cdk4-/- mice after mating. Cdk4+/+ ({square}) and Cdk4-/- ({blacksquare}) females at 6 wk of age were mated with Cdk4+/+ fertile males, and blood was sampled on d 1 and 3 p.c. for PRL measurement. Data are indicated as mean ± SEM (n = 6). *, P < 0.05 vs. Cdk4+/+ mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have demonstrated that Cdk4 is essential for the maintenance of luteal function. Cdk4-/- female mice mated normally and ovulated with modestly decreased efficiency. Luteinization occurred on LH signal, as demonstrated by the induction of p450scc and p27. However, the level of serum progesterone decreased rapidly following the initial increase. Cdk4-deficient mice showed no implantation and repeatedly mated without obvious pseudopregnancy. Daily injections of progesterone rescued implantation and restored fertility. These data indicate that sterility of Cdk4-deficient mice is because of perturbed luteal function. The mechanism of the luteal dysfunction in Cdk4-deficient mice is most probably related to low levels of serum PRL. It has been well established that PRL is a potent luteotropic factor in rodents (38). PRL secreted by the pituitary in response to mating is critical for the maintenance of luteal function during the early part of pregnancy. The PRL gene is expressed also in the decidua (39, 40, 41), and both the decidual PRL and placental lactogens have important tropic function later on in pregnancy. The PRL receptor (PRLR) is expressed in the corpus luteum, mammary gland, and many other cell types (42). Female mice deficient for PRL or PRLR are sterile (43, 44). The sterility of PRLR-deficient mice is characterized by defects in ovulation, cyclicity, fertilization, decidualization, implantation, and fetal survival (44). Most of these defects are associated with insufficient luteal function and are rescued by progesterone treatment (45, 46). Thus, the luteotropic action of PRL is essential for pregnancy. The low levels of serum PRL in Cdk4-deficient mice suggest that impaired PRL secretion after mating, presumably from the pituitary, plays a role in the insufficient luteal function in these mutants. We are currently studying the morphology and function of the lactotroph and other cell types in the pituitary of Cdk4-deficient mice. In addition to perturbed PRL signals, Cdk4-deficient luteal cells might have an autonomous defect. A recent study showed that luteinization of granulosa cells is accompanied by remarkable induction of cyclin D3 expression, which remains elevated until termination of the luteal function (47). Cyclin D3 forms complexes with Cdk4 and p27 in luteal cells. This trimeric complex, which should be absent in Cdk4-/- mice, may play a role in the function of fully differentiated postmitotic luteal cells.

Sterility of Cdk4-null mice involves no defect in oocytes. We observed that ovulated oocytes in Cdk4-/- mice were properly fertilized and developed normally when transferred into the uteri of pseudopregnant Cdk4+/+ females (Moons, D. S., H. Kiyokawa, unpublished observations). Progesterone treatment rescued implantation and embryonic development in Cdk4-/- mice. Furthermore, Rane et al. (28) reported that one of five Cdk4+/+ female mice that received transplantation of Cdk4-/- ovaries yielded Cdk4+/- pups after mating with a Cdk4+/+ male. This ovary transplantation study also suggested the involvement of extraovarian factors in sterility of Cdk4-deficient mice, consistent with hypoprolactinemia demonstrated in this study. In contrast, Cdk4 deficiency did not disrupt the regulation of gonadotropin secretion. Cdk4-null females ovulated without exogenous gonadotropins, and serum levels of FSH and LH in untreated and ovariectomized Cdk4-/- mice were comparable with those in Cdk4+/+ mice (Moons, D. S., H. Kiyokawa, unpublished observations).

In contrast to cyclin D2 deficiency, Cdk4 deficiency in mice does not impair follicular maturation to the preovulatory stage. Granulosa cells in cyclin D2-deficient mice are unable to proliferate on FSH treatment and fail to form preovulatory follicles (24). Granulosa cells in Cdk4-deficient mice proliferate properly in response to FSH, leading to the formation of large preovulatory follicles. This clear difference suggests that cyclin D2 can drive granulosa cell proliferation independently of Cdk4. Cyclin D2 in Cdk4-deficient ovaries may activate other Cdks, especially Cdk6 (48, 49). Cyclin D2 is expressed in granulosa cells but not detected in other types of ovarian cells such as theca, interstitial, and germ cells (23). FSH and estrogen strongly increase the expression of cyclin D2, which triggers rapid proliferation of granulosa cells and the transition from preantral to preovulatory follicles. Cdk4 is expressed almost ubiquitously in most tissues including the ovary (47). We have observed by immunohistochemistry that Cdk4 is expressed in granulosa and luteal cells as well as theca and interstitial cells in the ovary (Moons, D. S., H. Kiyokawa, unpublished observations). Cdk6 is also expressed in granulosa cells (47). In immunoblotting studies we detected no significant change in Cdk6 expression in Cdk4-deficient ovaries (27) (Moons, D. S., H. Kiyokawa, unpublished observations). Nevertheless, it remains to be determined whether FSH regulates Cdk6 expression and/or complex formation with cyclin D2. Mice with targeted disruption of Cdk6, which we are currently generating, will provide us with more insight into the molecular interaction of these Cdks in the ovary. Although Cdk4-deficient ovaries form large preovulatory follicles, they display a number of oocytes entrapped in luteinized follicles. Follicles in cyclin D2-deficient mice, which arrest at the stage of 3–4 layers of granulosa cells, are unable to ovulate with gonadotropin injections, but their granulosa cells differentiate into luteal cells (24). Luteinized follicles with oocytes entrapped have been observed in other mouse models, including mice deficient in the nuclear corepressor Nrip1 (50), connexin 37 (51), or phosphodiesterase PDE4D ( 52). Biochemical or genetic linkage of Cdk4 with these genes is obscure at this moment.

In summary, Cdk4 is essential for female reproduction, especially for maintenance of the luteal function during the periimplantation stage of pregnancy. The mechanism of insufficient luteal function in Cdk4-deficient mice involves perturbed PRL secretion. Unlike cyclin D2, Cdk4 is not required for proliferation of granulosa cells during development of preovulatory follicles. Tissue-specific disruption of the Cdk4 gene will be informative to further investigate the role of Cdk4 in the female reproductive system.


   Acknowledgments
 
We thank Jane Chladny and other staff of the Veterinary Diagnostic Laboratory, University of Illinois at Urbana-Champaign, for preparation of histological samples. We also thank Rhonda Kineman, Jim Artwohl, other members of the Kiyokawa laboratory, and the Reproductive Endocrinology Research Group at the University of Illinois for helpful discussions.


   Footnotes
 
This work was supported in part by funds provided to H.K. by the NIH (HD-38085), American Cancer Society (RPG-00-043-01-CCG), American Cancer Society Illinois Division (98-41 and 99-54), and University of Illinois Cancer Center.

Abbreviations: BrdU, Bromodeoxyuridine; Cdk4, cyclin-dependent kinase-4; CG; COX2, cyclooxygenase-2; hCG, human CG; p.c., post coitum; p450scc, p450 side-chain cleavage cytochrome; PRLR, PRL receptor.

Received September 9, 2001.

Accepted for publication October 4, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Pardee AB 1989 G1 events and regulation of cell proliferation. Science 246:603–608[Abstract/Free Full Text]
  2. Hunter T, Pines J 1994 Cyclins and cancer. II: cyclin D and CDK inhibitors come of age. Cell 79:573–582[CrossRef][Medline]
  3. Sherr CJ 1994 G1 phase progression: cycling on cue. Cell 79:551–555[CrossRef][Medline]
  4. Wuarin J, Nurse P 1996 Regulating S phase: CDKs, licensing and proteolysis. Cell 85:785–787[CrossRef][Medline]
  5. Reed SI 1997 Control of the G1/S transition. Cancer Surv 29:7–23[Medline]
  6. Weinberg RA 1995 The retinoblastoma protein and cell cycle control. Cell 81:323–330[CrossRef][Medline]
  7. Nevins JR 1998 Toward an understanding of the functional complexity of the E2F and retinoblastoma families. Cell Growth Differ 9:585–593[Medline]
  8. Kiyokawa H, Koff A 1998 Roles of cyclin-dependent kinase inhibitors: lessons from knockout mice. Curr Top Microbiol Immunol 227:105–120[Medline]
  9. Sherr CJ, Roberts JM 1999 CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 13:1501–1512[Free Full Text]
  10. Brugarolas J, Chandrasekaran C, Gordon JI, Beach D, Jacks T, Hannon GJ 1995 Radiation-induced cell cycle arrest compromised by p21 deficiency. Nature 377:552–557[CrossRef][Medline]
  11. Deng C, Zhang P, Harper JW, Elledge SJ, Leder P 1995 Mice lacking p21CIP1/WAF1 undergo normal development, but are defective in G1 checkpoint control. Cell 82:675–684[CrossRef][Medline]
  12. Kiyokawa H, Kineman RD, Manova-Todorova KO, Soares VC, Hoffman ES, Ono M, Khanam D, Hayday AC, Frohman LA, Koff A 1996 Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27(Kip1). Cell 85:721–732[CrossRef][Medline]
  13. Nakayama K, Ishida N, Shirane M, Inomata A, Inoue T, Shishido N, Horii I, Loh DY 1996 Mice lacking p27(Kip1) display increased body size, multiple organ hyperplasia, retinal dysplasia, and pituitary tumors. Cell 85:707–720[CrossRef][Medline]
  14. Fero ML, Rivkin M, Tasch M, Porter P, Carow CE, Firpo E, Polyak K, Tsai LH, Broudy V, Perlmutter RM, Kaushansky K, Roberts JM 1996 A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27(Kip1)-deficient mice. Cell 85:733–744[CrossRef][Medline]
  15. Yan Y, Frisen J, Lee MH, Massague J, Barbacid M 1997 Ablation of the CDK inhibitor p57Kip2 results in increased apoptosis and delayed differentiation during mouse development. Genes Dev 11:973–983[Abstract/Free Full Text]
  16. Zhang P, Liegeois NJ, Wong C, Finegold M, Hou H, Thompson JC, Silverman A, Harper JW, DePinho RA, Elledge SJ 1997 Altered cell differentiation and proliferation in mice lacking p57KIP2 indicates a role in Beckwith-Wiedemann syndrome. Nature 387:151–158[CrossRef][Medline]
  17. Richards JS, Russell DL, Robker RL, Dajee M, Alliston TN 1998 Molecular mechanisms of ovulation and luteinization. Mol Cell Endocrinol 145:47–54[CrossRef][Medline]
  18. Hirshfield AN 1991 Development of follicles in the mammalian ovary. Int Rev Cytol 124:43–101[Medline]
  19. Rao MC, Midgley Jr AR, Richards JS 1978 Hormonal regulation of ovarian cellular proliferation. Cell 14:71–78[CrossRef][Medline]
  20. Mason AJ, Hayflick JS, Zoeller RT, Young III WS, Phillips HS, Nikolics K, Seeburg PH 1986 A deletion truncating the gonadotropin-releasing hormone gene is responsible for hypogonadism in the hpg mouse. Science 234:1366–1371[Abstract/Free Full Text]
  21. Kumar TR, Wang Y, Lu N, Matzuk MM 1997 Follicle stimulating hormone is required for ovarian follicle maturation but not male fertility. Nat Genet 15:201–204[CrossRef][Medline]
  22. Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach KS, Smithies O 1993 Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene. Proc Natl Acad Sci USA 90:11162–11166[Abstract/Free Full Text]
  23. Sicinski P, Donaher JL, Geng Y, Parker SB, Gardner H, Park MY, Robker RL, Richards JS, McGinnis LK, Biggers JD, Eppig JJ, Bronson RT, Elledge SJ, Weinberg RA 1996 Cyclin D2 is an FSH-responsive gene involved in gonadal cell proliferation and oncogenesis. Nature 384:470–474[CrossRef][Medline]
  24. Robker RL, Richards JS 1998 Hormone-induced proliferation and differentiation of granulosa cells: a coordinated balance of the cell cycle regulators cyclin D2 and p27Kip1. Mol Endocrinol 12:924–940[Abstract/Free Full Text]
  25. Oonk RB, Krasnow JS, Beattie WG, Richards JS 1989 Cyclic AMP-dependent and -independent regulation of cholesterol side chain cleavage cytochrome P-450 (P-450scc) in rat ovarian granulosa cells and corpora lutea, cDNA and deduced amino acid sequence of rat P-450scc. J Biol Chem 264:21934–21942[Abstract/Free Full Text]
  26. Tong W, Kiyokawa H, Soos TJ, Park M, Soares VC, Manova-Todorova KO, Pollard JW, Koff A 1998 The absence of p27Kip1, an inhibitor of G1 cyclin-dependent kinases, uncouples differentiation and growth arrest during the granulosa-luteal transition. Cell Growth Differ 9:787–794[Abstract]
  27. Tsutsui T, Hesabi B, Moons DS, Pandolfi PP, Hansel KS, Koff A, Kiyokawa H 1999 Targeted disruption of CDK4 delays cell cycle entry with enhanced p27Kip1 activity. Mol Cell Biol 19:7011–7019[Abstract/Free Full Text]
  28. Rane SG, Dubus P, Mettus RV, Galbreath EJ, Boden G, Reddy EP, Barbacid M 1999 Loss of Cdk4 expression causes insulin-deficient diabetes and Cdk4 activation results in beta-islet cell hyperplasia. Nat Genet 22:44–52[CrossRef][Medline]
  29. Hales DB, Payne AH 1989 Glucocorticoid-mediated repression of P450scc mRNA and de novo synthesis in cultured Leydig cells. Endocrinology 124:2099–2104[Abstract/Free Full Text]
  30. Orly J, Rei Z, Greenberg NM, Richards JS 1994 Tyrosine kinase inhibitor AG18 arrests follicle-stimulating hormone-induced granulosa cell differentiation: use of reverse transcriptase-polymerase chain reaction assay for multiple messenger ribonucleic acids. Endocrinology 134:2336–2346[Abstract/Free Full Text]
  31. Park OK, Mayo KE 1991 Transient expression of progesterone receptor messenger RNA in ovarian granulosa cells after the preovulatory luteinizing hormone surge. Mol Endocrinol 5:967–978[Abstract/Free Full Text]
  32. Fitzpatrick SL, Richards JS 1991 Regulation of cytochrome P450 aromatase messenger ribonucleic acid and activity by steroids and gonadotropins in rat granulosa cells. Endocrinology 129:1452–1462[Abstract/Free Full Text]
  33. Hedin L, Gaddy-Kurten D, Kurten R, DeWitt DL, Smith WL, Richards JS 1987 Prostaglandin endoperoxide synthase in rat ovarian follicles: content, cellular distribution, and evidence for hormonal induction preceding ovulation. Endocrinology 121:722–731[Abstract/Free Full Text]
  34. Pall M, Hellberg P, Brannstrom M, Mikuni M, Peterson CM, Sundfeldt K, Norden B, Hedin L, Enerback S 1997 The transcription factor C/EBP-beta and its role in ovarian function; evidence for direct involvement in the ovulatory process. EMBO J 16:5273–5279[CrossRef][Medline]
  35. Lim H, Paria BC, Das SK, Dinchuk JE, Langenbach R, Trzaskos JM, Dey SK 1997 Multiple female reproductive failures in cyclooxygenase 2-deficient mice. Cell 91:197–208[CrossRef][Medline]
  36. Lydon JP, DeMayo FJ, Funk CR, Mani SK, Hughes AR, Montgomery CAJ, Shyamala G, Conneely OM, O’Malley BW 1995 Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities. Genes Dev 9:2266–2278[Abstract/Free Full Text]
  37. Hickey GJ, Oonk RB, Hall PF, Richards JS 1989 Aromatase cytochrome P450 and cholesterol side-chain cleavage cytochrome P450 in corpora lutea of pregnant rats: diverse regulation by peptide and steroid hormones. Endocrinology 125:1673–1682[Abstract/Free Full Text]
  38. Risk M, Gibori G 2001 Mechanisms of luteal cell regulation by prolactin. In: Horseman ND, ed. Prolactin. Dordrecht: Kluwer Academic Publisher; 265–295
  39. Golander A, Hurley T, Barrett J, Hizi A, Handwerger S 1978 Prolactin synthesis by human chorion-decidual tissue: a possible source of prolactin in the amniotic fluid. Science 202:311–313[Abstract/Free Full Text]
  40. Gellersen B, DiMattia GE, Friesen HG, Bohnet HG 1989 Prolactin (PRL) mRNA from human decidua differs from pituitary PRL mRNA but resembles the IM-9-P3 lymphoblast PRL transcript. Mol Cell Endocrinol 64:127–130[CrossRef][Medline]
  41. Prigent-Tessier A, Tessier C, Hirosawa-Takamori M, Boyer C, Ferguson-Gottschall S, Gibori G 1999 Rat decidual prolactin. Identification, molecular cloning, and characterization. J Biol Chem 274:37982–37989[Abstract/Free Full Text]
  42. Bole-Feysot C, Goffin V, Edery M, Binart N, Kelly PA 1998 Prolactin (PRL) and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice. Endocr Rev 19:225–268[Abstract/Free Full Text]
  43. Horseman ND, Zhao W, Montecino-Rodriguez E, Tanaka M, Nakashima K, Engle SJ, Smith F, Markoff E, Dorshkind K 1997 Defective mammopoiesis, but normal hematopoiesis, in mice with a targeted disruption of the prolactin gene. EMBO J 16:6926–6935[CrossRef][Medline]
  44. Ormandy CJ, Camus A, Barra J, Damotte D, Lucas B, Buteau H, Edery M, Brousse N, Babinet C, Binart N, Kelly PA 1997 Null mutation of the prolactin receptor gene produces multiple reproductive defects in the mouse. Genes Dev 11:167–178[Abstract/Free Full Text]
  45. Reese J, Binart N, Brown N, Ma WG, Paria BC, Das SK, Kelly PA, Dey SK 2000 Implantation and decidualization defects in prolactin receptor (PRLR)-deficient mice are mediated by ovarian but not uterine PRLR. Endocrinology 141:1872–1881[Abstract/Free Full Text]
  46. Binart N, Helloco C, Ormandy CJ, Barra J, Clement-Lacroix P, Baran N, Kelly PA 2000 Rescue of preimplantatory egg development and embryo implantation in prolactin receptor-deficient mice after progesterone administration. Endocrinology 141:2691–2697[Abstract/Free Full Text]
  47. Hampl A, Pachernik J, Dvorak P 2000 Levels and interactions of p27, cyclin D3, and CDK4 during the formation and maintenance of the corpus luteum in mice. Biol Reprod 62:1393–1401[Abstract/Free Full Text]
  48. Meyerson M, Harlow E 1994 Identification of G1 kinase activity for cdk6, a novel cyclin D partner. Mol Cell Biol 14:2077–2086[Abstract/Free Full Text]
  49. Bates S, Bonetta L, MacAllan D, Parry D, Holder A, Dickson C, Peters G 1994 CDK6 (PLSTIRE) and CDK4 (PSK-J3) are a distinct subset of the cyclin- dependent kinases that associate with cyclin D1. Oncogene 9:71–79[Medline]
  50. White R, Leonardsson G, Rosewell I, Ann JM, Milligan S, Parker M 2000 The nuclear receptor co-repressor nrip1 (RIP140) is essential for female fertility. Nat Med 6:1368–1374[CrossRef][Medline]
  51. Simon AM, Goodenough DA, Li E, Paul DL 1997 Female infertility in mice lacking connexin 37. Nature 385:525–529[CrossRef][Medline]
  52. Jin SC, Richard FJ, Kuo WP, D’Ercole AJ, Conti M 2003 1999 Impaired growth and fertility of cAMP-specific phosphodiesterase PDE4D-deficient mice. Proc Natl Acad Sci USA 96:11998–12003[Abstract/Free Full Text]
  53. Pedersen T, Peters H 1968 Proposal for a classification of oocytes and follicles in the mouse ovary. J Reprod Fertil 17:555–557[Abstract/Free Full Text]



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