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Endocrinology Vol. 143, No. 8 3001-3008
Copyright © 2002 by The Endocrine Society

ARTICLE

Pituitary Hypoplasia and Lactotroph Dysfunction in Mice Deficient for Cyclin-Dependent Kinase-4

David S. Moons, Siwanon Jirawatnotai, A. F. Parlow, Geula Gibori, Rhonda D. Kineman and Hiroaki Kiyokawa

Departments of Molecular Genetics (D.S.M., S.J., H.K.), Physiology and Biophysics (G.G.), and Medicine (R.D.K.), University of Illinois College of Medicine, Chicago, Illinois 60607; and 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
 
The lactotroph undergoes dynamic regulation of cell cycle progression during pregnancy, as well as throughout the development of the pituitary. We recently reported that female mice with targeted disruption of Cdk4, one of the G1-regulatory cyclin-dependent kinases, are unable to support embryo implantation because of defective progesterone secretion from the corpus luteum. In this study, we demonstrate that this phenotype is not attributable to a primary defect in the corpus luteum but is a consequence of defective prolactin (PRL) production caused by inappropriate development of the pituitary lactotroph population. Specifically, the pituitary of Cdk4-deficient mice is extremely hypoplastic. Lactotrophs and somatotrophs of prepubertal Cdk4-deficient mice were 80% decreased in number, relative to those in wild-type mice, whereas gonadotrophs were unaffected. Lactotrophs of Cdk4-deficient mice did not proliferate in response to estrogen administration, whereas estrogen could induce the expression of galanin, an estrogen-responsive factor required for lactotroph proliferation. The reduction in lactotroph numbers was reflected by markedly diminished serum PRL levels in both prepubertal and postcoital Cdk4-deficient mice. Administration of PRL, after mating, significantly increased serum progesterone levels and restored implantation in Cdk4-deficient female mice. These observations demonstrate that Cdk4 is required for normal proliferation of the lactotroph population.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROLIFERATION-STIMULATORY AND INHIBITORY signals regulate the G1 phase of the cell cycle, governing the transition between proliferation and quiescence (1). Cyclin-dependent kinases (Cdks), in complex with the regulatory cyclin subunits, form central machinery of cell cycle progression (2, 3, 4, 5, 6). In G1 progression, cyclin D- and cyclin E-dependent kinases play independent (but interacting) roles. D-type cyclins, D1, D2, and D3, activate Cdk4 or Cdk6, whereas cyclin E activates Cdk2. The temporal activation of cyclin D- and cyclin E-dependent kinases is crucial for proper transition from G1 to S. Cyclin D-dependent phosphorylation of retinoblastoma protein (Rb) converts the transcription factor E2F from a repressor to activator form, leading to transactivation of cyclin E and other S-phase specific genes (7, 8). The coordinated control of the G1 Cdks involves physical association with not only cyclins but also a number of Cdk inhibitor proteins. The Cdk inhibitor family consists of the Kip/Cip-type inhibitors, p21, p27, and p57, and the Ink4-type inhibitors, p16, p15, p18, and p19 (9, 10).

To investigate the role of G1 Cdk regulation in tissue development and function, we recently generated and characterized mice with targeted disruption of the Cdk4 gene (11, 12). Cdk4-null mice exhibit growth retardation, and all homozygous females are sterile. Cdk4-deficient females undergo successful ovulation and fertilization but are unable to support implantation (12). Implantation in Cdk4-null females is restored by injections of progesterone, at first suggesting that sterility may be the result of defective luteal function. However, reduced levels of serum prolactin (PRL) are also observed in Cdk4-null females after mating. Because PRL is required for the maintenance of luteal function during the early part of pregnancy (13), these findings suggested that infertility of the Cdk4-null mouse could be attributable to a primary failure of the pituitary to synthesize and secrete PRL. Here, we report that PRL administration stimulated progesterone production and rescued implantation failure, confirming that the sterility in Cdk4-/- mice is a consequence of defective PRL secretion. Decreased PRL secretion of the Cdk4-null mouse is directly associated with a decrease in pituitary size, with a dramatic decrease in the number of lactotrophs. Estrogen-induced proliferation of the lactotroph is almost abrogated in Cdk4-/- mice. Therefore, Cdk4 plays an essential role in regulation of cell cycle progression of the lactotroph, both during development and in the adult, in response to hormonal stimuli.


   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 (11). 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, as described (12). Because a fraction of Cdk4-/- mice develop diabetes mellitus by 8–10 wk of age (11), 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 examine the effect of exogenous PRL, mice were injected sc with 75 µg purified mouse PRL dissolved in 50% polyvinylpyrrolidone solution (Sigma, St. Louis, MO), at 1000 and 0600 h daily from d 2 p.c., as described previously (14). Progesterone administration was performed as described previously (12). To stimulate lactotroph proliferation, mice were injected sc with 0.5 mg estradiol (E2) dissolved in sesame oil, once a week for 3 wk, as previously described (15). To examine the effect of estrogen on galanin expression in the pituitary, ovariectomized mice at 6 wk of age were injected sc with E2 (100 µg/kg body weight) dissolved in sesame oil, once daily for 7 d, as described previously (16). Mice were used in compliance with the NIH/Association for Assessment and Accreditation of Laboratory Animal Care and with the approval of the animal care review committee of the University of Illinois.

Hormone measurements
Mice were anesthetized with an ip injection of avertin, and blood was collected by retroorbital sinus puncture at 1200 h. Precautions were taken to minimize the stress of animals during sampling. Sera were prepared by centrifugation, at 10,000 x g for 10 min, and stored at -70 C until analysis. Progesterone and E2 were assayed using RIA kits (Diagnostic 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.

Pituitary cell preparation and immunocytochemistry
Macroscopic pictures of pituitaries in situ were taken using a Leica Corp. GZ7 dissection microscope (Leica Microsystems, Inc., Buffalo, NY) with a JVC GCQX-5HD digital camera (Victor Co. of Japan, Ltd., Tokyo, Japan) installed. Dispersed pituitary cells were prepared from Cdk4+/+ and Cdk4-/- mice, as described previously (15, 17). Pituitaries from two mice with the same genotype were dissected out, combined, and cut into small pieces in Eagle’s minimum essential medium supplemented with 0.1% BSA. Cells were incubated at 37 C for 33 min with 0.2% trypsin, in the same medium, and then dispersed by gentle trituration with a siliconized glass pipette. Monodispersed cells were counted using a hemocytometer, and 50,000 cells were plated on a glass slide. Cells were then incubated, at 37 C for 45 min for adhesion to the slide, and fixed with a solution of 220 mM mercuric chloride, 90 mM acetic acid, and 3.7% formaldehyde for 10 min. The slides were then treated with the following solutions for 1 min each, in order: 95% ethanol, distilled water, Lugol’s iodine, distilled water, and 0.08M sodium thiosulfate. Immunocytochemistry for pituitary hormones was performed, as described previously (18), using the following primary antibodies: antirat PRL (1:5,000 dilution), antirat GH (1:100,000), antirat TSH (1:10,000), antirat ACTH (1:5,000), antirat FSH (1:5,000), and antirat LH (1:20,000) antibodies. The National Hormone and Pituitary Program of NIDDK provided these polyclonal antibodies. Signals were visualized using the Vectastain Elite ABC kit and biotinylated antirabbit Ig antibody (1:200) (Vector Laboratories, Inc., Burlingame, CA), according to the manufacturer’s instruction. At least 1000 dispersed pituitary cells were counted to determine the percentage of cells stained for each hormone. Microscopic images were captured using a JVC GCQX-5HD digital camera installed onto a Leica Corp. DM/E phase microscope.

RT-PCR
Total RNA (250 ng) 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 deoxynucleotide triphosphates, 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 29 cycles. The primers used are: for Pit-1, 5'-TTCAGTCAAACAACCATCTGTCGA-3' and 5'-GGTTGCAGAACCACACTCGTACTAC-3'; for galanin, 5'-GCGTTATCCTGCTAGGCTGG-3' and 5'-AGTGCGGACAATGTTGCTCTC-3'; and 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., Hercules, CA). The amplified products were 300 bp, 256 bp, and 195 bp for Pit-1, galanin, and L19, respectively.

Statistics
Data are expressed as mean ± SEM. The differences between groups were evaluated with ANOVA and Student’s t test, with a significance level of P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Hypoplastic pituitaries in Cdk4-null mice
Perturbed luteal function with hypoprolactinemia in Cdk4-/- female mice (12) prompted us to examine the effect of Cdk4 deficiency on the pituitary. Pituitaries of Cdk4-null mice were significantly smaller than wild-type pituitaries. The weights of pituitaries of 3-wk-old Cdk4-/- females were 61% lower than those of Cdk4+/+ females, whereas pituitary weights of 3-wk-old Cdk4-/- males were decreased by 53% in comparison with Cdk4+/+ males (Fig. 1AGo). Because Cdk4-/- mice exhibited growth retardation, as described previously (11), the pituitary weight of each mouse was normalized by its body weight (Fig. 1BGo). The normalized pituitary weights were still significantly lower, by 40% and 26%, in Cdk4-/- females and males, respectively, relative to wild-type controls. To determine whether the reduced size of the Cdk4-deficient pituitary is caused by a hypoplastic or hypotrophic change, we harvested pituitaries of Cdk4+/+ and Cdk4-/- mice and prepared monodispersed cells, using standard procedures (15, 17). The numbers of monodispersed pituitary cells prepared from Cdk4-/- mice were 50% lower than those from Cdk4+/+ mice (1.01 ± 0.14 x 105 vs. 2.00 ± 0.21 x 105, n = 3, P < 0.05). Thus, pituitaries of Cdk4-/- mice were hypoplastic at the prepubertal age.



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  		Figure 1. Smaller pituitaries in Cdk4-deficient mice. A, The wet weights of pituitaries from 3-wk-old Cdk4+/+ (open columns) and Cdk4-/- (closed columns) mice. B, The wet weights of pituitaries from 3-wk-old Cdk4+/+ (open columns) and Cdk4-/- (closed columns) mice, normalized as ratios to the body weights. The data are expressed as mean + SEM (n = 3). *, P < 0.05 vs. Cdk4+/+ mice.

 
Marked hypoplasia of the lactotroph and somatotroph of Cdk4-null mice
To determine whether particular cell types of the pituitary are affected by Cdk4 deficiency, we examined monodispersed pituitary cells from 3-wk-old Cdk4+/+ and Cdk4-/- mice, by immunocytochemistry, for pituitary hormones (Fig. 2Go, A–C). The numbers of lactotrophs (PRL+) were most dramatically reduced in Cdk4-/- mice, exhibiting 80% and 85% decreases in females and males, respectively. The numbers of somatotrophs (GH+) were similarly affected in Cdk4-/- mice, showing 76% and 80% decreases in females and males, respectively. These changes are more dramatic than the 2-fold decrease in total numbers of pituitary cells. In contrast, the numbers of LH+ cells and FSH+ cells in Cdk4-/- mice were comparable with those in wild-type mice in both sexes. Consequently, the percentages of LH+ cells and FSH+ cells in total pituitary cells were even higher in Cdk4-/- mice, relative to those in Cdk4+/+ mice. The numbers of TSH+ cells and ACTH+ cells in Cdk4-/- pituitaries were reduced by 58% and 47%, respectively, compared with Cdk4+/+ pituitaries (data not shown). These changes were proportional to the decrease in total pituitary cell numbers. These data suggest that the hypoplastic effect of Cdk4 deficiency on the lactotroph and somatotroph is relatively specific, whereas the gonadotroph is maintained virtually unaffected.



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  		Figure 2. Hypoplastic lactotrophs and somatotrophs in pituitaries of Cdk4-deficient mice. A, Monodispersed pituitary cells from 3-wk-old Cdk4+/+ and Cdk4-/- female mice were examined, by immunocytochemistry, for PRL expression. The arrows indicate PRL+ cells. B, Monodispersed pituitary cells from 3-wk-old female mice were examined, by immunocytochemistry, for the hormones indicated; and the numbers of cells expressing each hormone are shown. C, Monodispersed pituitary cells from 3-wk-old male mice were analyzed as described above. Open columns, Cdk4+/+ mice; closed columns, Cdk4-/- mice. Pituitaries from two mice with each genotype were analyzed together, and cell counts normalized per mouse are shown. Data from three independent experiments are expressed as mean + SEM. *, P < 0.05 vs. Cdk4+/+ mice.

 
Decreased Pit-1 expression in the pituitary of Cdk4-null mice
The lineage-specific development of the lactotroph, somatotroph, and thyrotroph depends on the POU-domain transcriptional factor Pit-1 (19, 20). To determine whether the expression of Pit-1 is altered in hypoplastic pituitaries of Cdk4-/- mice, we performed RT-PCR analysis for Pit-1 mRNA in pituitaries of 3-wk-old Cdk4+/+ and Cdk4-/- female mice (Fig. 3Go). The Pit-1 mRNA level normalized by the L19 mRNA level was 85% lower in Cdk4-/- mice than that observed in Cdk4+/+ mice. Because Pit-1 expression is restricted to the lactotroph, somatotroph, and thyrotroph in normal adult pituitaries, these data are consistent with the specific decreases in these pituitary cell populations, especially the lactotroph and somatotroph, in Cdk4-null mice.



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  		Figure 3. Reduced Pit-1 mRNA expression in Cdk4-deficient mice. Total RNA was prepared from the pituitaries of female mice at 3 wk of age, and the mRNA levels were analyzed using semiquantitative RT-PCR. The PCR products from two different mice for each genotype are shown in the panel. The chart indicates levels of Pit-1 mRNA normalized by levels of L19 control. Data from three independent experiments are expressed as mean + SEM. *, P < 0.05, for comparison of Cdk4-/- mice vs. Cdk4+/+ mice.

 
Impaired PRL secretion in Cdk4-deficient mice
The hypoplastic change in lactotrophs of Cdk4-null mice is consistent with our previous finding that serum levels of PRL were very low in Cdk4-/- females after mating (12). In contrast, apparently intact gonadotrophs are correlated with normal levels of serum FSH, LH, and E2 and spontaneous ovulation in Cdk4-/- females (12). Thus, in subsequent investigations, we focused on the relationship between the lactotroph function and sterility in Cdk4-/- mice. First, we examined serum PRL levels in prepubertal mice. Cdk4-/- females at 3 wk of age showed significant hypoprolactinemia, with serum PRL levels 85% lower than those in wild-type mice (Fig. 4Go). Cdk4-/- immature males also showed severe hypoprolactinemia, consistent with hypoplastic lactotrophs (Fig. 2CGo). It has been well established that PRL secretion from the lactotroph of mature female rats is increased at proestrus and dramatically enhanced by cervical stimulation during mating (21, 22, 23). However, serum PRL levels in 6-wk-old Cdk4-/- females on d 1 p.c. were 92% lower than those in wild-type mice (Fig. 4Go) (12). Thus, Cdk4-/- mice displayed marked hypoprolactinemia under both prepubertal and postcoital conditions. These observations suggest that Cdk4-deficient mice have a severe defect in PRL secretion, correlated with lactotroph hypoplasia.



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  		Figure 4. Hypoprolactinemia in Cdk4-deficient mice. Serum PRL levels were determined, by RIA, in female and male Cdk4+/+ and Cdk4-/- mice at 3 wk of age (immature), or in females at 6 wk of age after mating (d 1 p.c.); n = 6 for each group. *, P < 0.05 vs. Cdk4+/+ mice. The data are expressed as mean + SEM.

 
Impaired proliferation of Cdk4-deficient lactotrophs
Increased PRL secretion after mating is, at least in part, associated with proliferation of lactotrophs in response to cervical stimuli. To determine whether postcoital hypoprolactinemia in Cdk4-deficient mice is related to impaired proliferation of the lactotroph, we first examined pituitaries of adult Cdk4-/- females after mating (Fig. 5AGo). Pituitaries in 6-wk-old Cdk4-/- mice on d 2 p.c. were 61% smaller in weight and contained 62% fewer cells than wild-type controls (Fig 5BGo). The numbers of lactotrophs in Cdk4-/- mice were 85% decreased, relative to those in Cdk4+/+ mice, indicating severe hypoplasia of the lactotroph.



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  		Figure 5. Severe hypoplasia of the lactotroph in postcoital Cdk4-deficient female mice. A, Cdk4+/+ and Cdk4-/- females at 6 wk of age were mated with fertile wild-type males, and the pituitaries were examined at d 2 p.c. B, Monodispersed pituitary cells from these mice were analyzed, by immunocytochemistry, for PRL. Pituitaries from two mice with each genotype were analyzed together, and the numbers of total pituitary cells and PRL+ cells per mouse are shown. Data from three independent experiments are expressed as mean + SEM. *, P < 0.05 vs. Cdk4+/+ mice.

 
Estrogen is a major factor that promotes lactotroph proliferation, involved in lactotroph hyperplasia during pregnancy (24, 25). To determine whether Cdk4 deficiency perturbed estrogen-dependent proliferation, we injected mature Cdk4+/+ and Cdk4-/- females with 0.5 mg E2 once a week for 3 wk (Fig. 6Go). The pituitary weight of E2-treated Cdk4+/+ mice was 2.33 ± 0.16 mg, whereas that of E2-treated Cdk4-/- mice was 0.64 ± 0.04 mg. The E2 administration increased the numbers of pituitary cells by 59% in Cdk4+/+ mice but did not affect the number of pituitary cells in Cdk4-/- mice, compared with vehicle-treated controls. E2 increased the numbers of lactotrophs in Cdk4+/+ mice by 220%, whereas E2 increased the numbers of Cdk4-/- lactotrophs by only 15%. Thus, lactotrophs in Cdk4-deficient mice exhibited essentially no proliferation response to estrogen.



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  		Figure 6. Impaired proliferative response of Cdk4-deficient pituitary cells in response to estrogen. Randomly cycling Cdk4+/+ (open columns) and Cdk4-/- (closed columns) female mice at 8 wk of age were injected with 0.5 mg E2 dissolved in sesame oil or oil only (vehicle control), given once a week for a 3-wk period. Monodispersed pituitary cells were prepared and analyzed, by immunocytochemistry, for PRL. Pituitaries from two mice of each group were analyzed together, and the numbers of total pituitary cells and PRL+ cells per mouse are shown. Data from three independent experiments are expressed as mean + SEM. *, P < 0.05, for comparison of Cdk4-/- mice vs. Cdk4+/+ mice; **, P < 0.05, for comparison of control vs. E2-treated Cdk4+/+ mice.

 
Galanin has been shown to play a critical role in the lactotroph proliferation response to estrogen (25), and mice deficient for galanin display a defect in estrogen-induced proliferation similar to that observed in Cdk4-null mice (15). To determine whether estrogen can induce galanin expression in Cdk4-null mice, we injected mature Cdk4+/+ and Cdk4-/- female mice with E2, once daily for 7 d (Fig. 7Go), according to the procedure described previously (15). Total mRNA was obtained from the pituitary, and galanin mRNA levels were analyzed by semiquantitative RT-PCR. The basal levels of galanin expression without E2 treatment were comparable in Cdk4+/+ and Cdk4-/- mice. A significant increase in galanin mRNA was observed in Cdk4-/- mice in response to E2 treatment, although the levels were 40% lower than those observed in E2-treated Cdk4+/+ mice. These data suggest that lactotrophs in Cdk4-null do not proliferate in response to estrogen, despite their ability to increase galanin expression.



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  		Figure 7. Estrogen-dependent induction of galanin mRNA in pituitaries of Cdk4-deficient mice. Ovariectomized Cdk4+/+ (open columns) and Cdk4-/- (closed columns) mice at 6 wk of age were injected sc with E2 (100 µg/kg body weight) dissolved in sesame oil or oil only (vehicle control), once daily for 7 d. Total RNA was prepared from each pituitary, and the mRNA levels of galanin and L19 were analyzed by semiquantitative RT-PCR. The galanin RT-PCR products from two (controls) or three (E2-injected) different mice for each genotype are shown in the panel. The chart indicates levels of galanin mRNA normalized by levels of L19 control. Data from three independent experiments are expressed as mean + SEM. *, P < 0.005, for comparison of Cdk4+/+ control vs. Cdk4+/+ E2-treated mice; **, P < 0.05, for comparison of Cdk4-/- control vs. Cdk4-/- E2-treated mice; ***, P < 0.05, for comparison of Cdk4-/- mice vs. Cdk4+/+ mice.

 
Female sterility caused by lactotroph dysfunction in Cdk4-null mice
Because PRL is a major luteotropin during early pregnancy in rodents (13, 26, 27, 28), luteal failure in Cdk4-null mice could be a consequence of perturbed lactotroph function. To further address this question, we injected Cdk4-/- females with 75 µg PRL, twice a day from d 2 p.c., and measured serum progesterone levels (Fig. 8Go). On d 4 and 6 p.c., serum progesterone levels in Cdk4-/- females were lower than in Cdk4+/+ mice by 5- to 10-fold, as reported previously (12). By d 6 p.c., PRL injections significantly restored serum progesterone concentrations, up to levels similar to those in wild-type.



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  		Figure 8. Hypoprogesteronemia in Cdk4-deficient mice rescued by PRL administration. Cdk4+/+ (open columns) and Cdk4-/- (closed columns) females at 6 wk of age were mated with wild-type fertile males. Seventy-five micrograms of PRL was sc injected into Cdk4-/- females (hatched columns), twice a day from d 2 p.c. Blood was sampled on d 4 and 6 p.c. for progesterone measurement by RIA. Data are indicated as mean ± SEM (n = 6). *, P < 0.05, for comparison of Cdk4-/- vs. Cdk4+/+ mice; **, P < 0.05, for comparison of untreated and PRL-treated Cdk4-/- mice.

 
When examined on d 8–12 p.c. (Table 1Go), none of the Cdk4-/- females without progesterone or PRL injections (n = 20) demonstrated implantation. In contrast, most of the progesterone-injected Cdk4-/- females had successful embryo implantation, as shown previously (12). PRL injections were able to rescue implantation as effectively as progesterone injections, demonstrating numbers of implantation sites per mouse similar to those observed with progesterone treatment. The numbers of implantation sites observed in Cdk4-/- females after PRL or progesterone treatment were 23% and 27% lower, respectively, than those in wild-type control mice. The fewer implantation sites are likely attributable to the decrease in ovulation rate, which we previously reported for Cdk4-/- mice (12). These observations indicate that restoring serum PRL levels is sufficient to rescue Cdk4-null female mice from the implantation failure, and suggest that lactotroph dysfunction accounts for the infertility of these mutants.


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  		Table 1. Restoration of implantation in Cdk4-/- female mice by progesterone or PRL1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present results demonstrate that PRL administration can rescue both progesterone secretion and embryo implantation in the Cdk4-null female mouse, indicating the significance of lactotroph dysfunction in the sterile phenotype. PRL secretion from the lactotroph is essential for female reproductive function. PRL is a potent luteotropic factor, especially in rodents (29, 30). Daily surges of PRL secretion from the lactotroph are initiated on d 1 of pregnancy, following cervical stimuli in mating (22, 31). This lactotroph-derived PRL is critical for the maintenance of luteal function during the early part of pregnancy (13, 26), whereas PRL secreted from decidua and placental lactogens are important luteotropins during mid- and late-pregnancy (32, 33, 34). The PRL receptor (PRLR) is expressed in the corpus luteum, mammary gland, and many other cell types (29). Female mice with targeted disruption of PRL or PRLR are sterile (35, 36). PRLR-deficient mice display multiple defects in ovulation, cyclicity, fertilization, decidualization, implantation, and fetal survival (36), most of which are associated with insufficient luteal function and are rescued by progesterone treatment (27, 28). These observations, coupled with our current findings in the Cdk4-null mouse, demonstrate the essential role of PRL in maintenance of luteal function.

PRL deficiency in Cdk4-null mice is directly correlated with a dramatic decrease in lactotroph numbers. In addition, proliferation of Cdk4-deficient lactotrophs in response to estrogen administration is abrogated. Estrogen acts directly on normal lactotrophs to stimulate both PRL secretion and lactotroph proliferation (37, 38). The cell cycle-promoting action of estrogen seems to be mediated by growth factors in autocrine/paracrine manners. One such estrogen-induced lactotroph growth factor is galanin. Galanin is expressed by a subpopulation of lactotrophs, and its targeted disruption leads to the inability of lactotrophs to proliferate in response to estrogen (15). Transgenic mice with galanin expression under the PRL promoter exhibit pituitary hyperplasia and hyperprolactinemia (39). These data indicate that galanin is required for estrogen-dependent lactotroph proliferation. The basal levels of galanin mRNA in the pituitary were not statistically different between Cdk4+/+ and Cdk4-/- mice (Fig. 7Go). Estrogen administration significantly induced galanin expression in the Cdk4-deficient pituitary, to levels modestly lower than those in the wild-type pituitary. However, cells in the Cdk4-deficient pituitary displayed no proliferation in response to estrogen. One possibility is that Cdk4 function is a downstream target of galanin signaling in inducing lactotroph proliferation, which remains to be clarified.

Low circulating PRL levels and decreased lactotroph numbers are not only evident in adult Cdk4-null mice but are also evident before puberty. This observation suggests that Cdk4 is critical in the expansion of the lactotroph population during pituitary development. Because the numbers of somatotrophs are comparably reduced in Cdk4-null mice, and lactotrophs and somatotrophs have been shown to be derived from a common cell lineage requiring the expression of Pit-1 (20), it is possible that the absence of Cdk4 affects the regulation of Pit-1 expression. In fact, the phenotypes of Cdk4 deficiency, such as growth retardation and infertility, are somewhat analogous to Snell or Jackson dwarf mice. These dwarf mice carry mutations in the Pit-1 gene that are responsible for the absence of the development of the lactotroph, somatotroph, and thyrotroph (19, 40). Our RT-PCR analyses showed that the amounts of Pit-1 mRNA in pituitaries of 3-wk-old Cdk4-null mice were 85% reduced, relative to those in wild-type mice. This decrease in Pit-1 expression may be secondary to the decrease in the proportions of lactotrophs, somatotrophs, and thyrotrophs in total pituitary cells, because Pit-1 expression in normal adult pituitaries is restricted to theses cell types. Alternatively, the absence of Cdk4 may affect expression of Pit-1 during lineage determination in pituitary morphogenesis, which normally occurs around embryonic d 13.5 (20). Detailed studies on embryonic pituitary development in Cdk4-null mice and Pit-1-mutated dwarf mice are necessary to determine how Pit-1 and Cdk4 interact with each other in the control of cell cycle progression and lineage determination in pituitary morphogenesis.

Our study has revealed that Cdk4 plays a critical role in homeostasis of pituitary cells. We also reported that pancreatic ß-cells spontaneously degenerate in Cdk4-null mice after 8 wk of age (11). These observations imply that development and/or maintenance of endocrine cells may require Cdk4 in a specific manner. This tissue-specific phenotype of Cdk4-null mice is intriguing because Cdk4 is ubiquitously expressed. The selective effects of Cdk4 deficiency suggest that other factors may compensate for its absence. Intact development of most tissues in Cdk4-null mice may depend on the function of Cdk6, whose cyclin D-dependent kinase activity in vitro is indistinguishable from that of Cdk4 (41). Cdk6 is normally coexpressed with Cdk4 in most tissues (6, 11). All threeD-type cyclins are expressed in the normal rat anterior pituitary; however, different levels of expression have been reported for each pituitary cell type (42). The specific defect in proliferation of Cdk4-null pituitary cells may suggest that pituitary cells, especially lactotrophs and somatotrophs, are normally deficient in some molecule(s) that functions redundantly with Cdk4. Although Cdk6 expression is generally unaffected in several visceral tissues examined in Cdk4-null mice (11), it remains to be determined whether pituitaries of Cdk4-null mice have altered levels or activities of Cdk6 and other Cdk proteins. Mice with targeted disruption of Cdk6 will provide more insight into the molecular interaction of these G1-specific Cdks in the pituitary and other endocrine organs.

The specific requirement of Cdk4 for lactotroph proliferation implies that the development of pituitary tumors may involve aberrant activation of Cdk4. Intriguingly, various tumors, including prolactinomas, develop in mice with a targeted mutation of the Cdk4 gene that produces a mutant Cdk4 protein insensitive to the Ink4-type inhibitors (43). These observations, together with our present study, suggest that Cdk4 can be a potent target for therapeutic intervention against pituitary tumors. Pituitary tumors are identified in about 20% of the population by imaging diagnoses or autopsies. Prolactinomas are the most common pituitary tumors; and they manifest a variety of symptoms, including galactorrhea and amenorrhea. Although many patients with clinical prolactinomas respond to antidopaminergic therapies, more information about the mechanism of deregulated proliferation of the lactotroph is needed for better control of these tumors. Intriguingly, pituitary tumors arise from the intermediate lobes of mice deficient for Cdk inhibitors, such as p27Kip1 and p18Ink4c (18, 44, 45), and mice heterozygous for the Rb, a major Cdk4 substrate (46). This is of considerable interest because the intermediate and posterior lobes of adult Cdk4-null mice appear normal without major hypoplastic changes (Moons, D. S., and H. Kiyokawa, unpublished data).

In summary, Cdk4 plays a critical role in regulating function and proliferation of the lactotroph, which is essential for female fertility. Cdk4-deficient mice provide a unique tool for further investigation of the interaction of the G1 cell cycle machinery and lineage-specific development and function of the pituitary.


   Acknowledgments
 
We thank Asgi Fazleabas, Julie Kim, Roberta Franks, Jim Artwohl, Robert Streit, other members of the Kiyokawa laboratory, and the Reproductive Endocrinology Research Group at the University of Illinois for helpful discussions and suggestions. We thank NIDDK and the National Hormone and Pituitary Program for generously providing us with the antisera and purified mouse PRL used in this study.


   Footnotes
 
This work was supported, in part, by funds (to H.K.) from the NIH (HD-38085) and the American Cancer Society (RPG-00-043-01-CCG).

Abbreviations: Cdk, Cyclin-dependent kinase; E2, estradiol; p.c., post coitum; PRL, prolactin; PRLR, PRL receptor; Rb, retinoblastoma protein.

Received January 15, 2002.

Accepted for publication April 18, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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