Mol. Cells, Vol. 22, No. 2, pp. 189-197
Molecules
and
Cells
©KSMCB 2006
Changes in Reproductive Function and White Blood Cell
Proliferation Induced in Mice by Injection of a
Prolactin-expressing Plasmid into Muscle
Jung-Sun Lee, Bo-Young Yun, Sang-Soo Kim2, Chunghee Cho1, Yong-Dal Yoon2, and Byung-Nam Cho*
Department of Life Science, The Catholic University of Korea, Bucheon 420-743, Korea;
1
Department of Life Science Gwangju Institute of Science and Technology, Gwangju 500-712, Korea;
2
Department of Life Science, Hanyang University, Seoul 133-791, Korea.
(Received May 18, 2006; Accepted July 24, 2006)
Prolactin (PRL) is a pituitary hormone involved in various physiological processes, including lactation, mammary development, and immune function. To further
investigate the in vivo and comparative endocrine roles of
PRL, mouse PRL cDNA fused to the cytomegalovirus
promoter, was introduced into muscle by direct injection.
Previously we studied the function of rat PRL using the
same protocol. PRL mRNA was detected in the muscle
following injection by RT-PCR and subsequent Southern
blot analysis. PRL was also detected and Western blot
analysis revealed a relatively high level of serum PRL. In
the pCMV-mPRL-injected female mice, the estrous cycle
was extended, especially in diestrus stage and the uterus
thickening that was shown in normal estrous stage was
not observed. In the pCMV-mPRL-injected male mice,
new blood vessels were first found at 5 weeks of age and
fully developed blood vessels were found after 8 weeks in
the testis. The number of Leydig cells increased within
the testis and the testosterone level in serum was observed high. Finally, the number of white blood cells
(WBCs) increased in the pCMV-mPRL-injected mice.
The augmentation of WBCs persisted for at least 20 days
after injection. When injection was combined with
adrenalectomy, there was an even greater increase in
number of WBCs, especially lymphocytes. This increase
was returned normal by treatment with dexamethansone.
Taken together, our data reveal that intramuscularly
expressed mouse PRL influences reproductive functions
in female, induces formation of new blood vessels in the
testis, and augments WBC numbers. Of notice is that the
Leydig cell proliferation with increased testosterone was
conspicuously observed in the pCMV-mPRL-injected
* To whom correspondence should be addressed.
Tel: 82-2-2164-4358; Fax: 82-2-2164-4765
E-mail: [email protected]
mice. These results also suggest subtle difference in function of PRL between mouse and rat species.
Keywords: Angiogenesis; Cell Proliferation; Prolactin;
Reproductive Function.
Introduction
Prolactin (PRL) is a peptide hormone synthesized and secreted by the lactotrophic cells of the anterior pituitary under the control of dopamine (Maurer, 1980), and steroid
and peptide hormones (Maurer, 1982; Raymond et al.,
1978). Synthesis of PRL is not, however, limited to the
pituitary, as numerous extra-pituitary sites of PRL expression have been identified, including the placenta (Lee and
Markoff, 1986), lymphocytes (Montgomery et al., 1990;
Pellegrini et al., 1992), and breast cancer cells of epithelial
origin (Clevenger et al., 1995; Ginsberg and Vonderhaar
1995). PRL is known to participate in regulation of reproduction (Leong et al., 1983), osmoregulation (Neill, 1988),
and immununomodulation (Bole-Feysot et al., 1998).
In females, PRL is known for its action on the ovarian
function. In rodent, luteotropic and luteolytic actions of
PRL have been recognized for a number of years. In general, the luteotropic action of PRL involves stimulation of
progesterone production by luteal cells (Matsuyama et al.,
1990). In mammals, depending on the stage of the cycle,
luteolytic effects of PRL have also been reported (Loudon
et al., 1990). In males, the physiological role of PRL is
unclear. The absence of PRL signaling in PRL-receptordeficient mice is not detrimental to male testicular function and to fertility (Binart et al., 2003) although PRL
increases LH receptor numbers (Dombrowicz et al., 1992)
Abbreviations: PRL, prolactin; WBC, white blood cells.
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Reproductive Function and WBC Proliferation by Prolactin
and steroidogenesis (Gunasekar et al., 1988) in Leydig
cells, and stimulates angiogenesis in the testis (Ko et al.,
2003).
PRL and PRL-related proteins play a role in angiogenesis (Clapp et al., 1993; Elias and Weiner, 1984; Jackson et
al., 1994), a vital aspect of many physiological processes
including wound healing and organ regeneration (Hanahan
and Folkman, 1996) as well as of pathological conditions
such as tumor growth and metastasis (Folkman, 1995). It
has been controversially reported that direct arterial vascularization of PRL-secreting pituitary tumors (Elias and
Weiner, 1984) and angiogenesis in the testis (Ko et al.,
2003) were observed whereas intact 23 kDa rat PRL had
no effect on angiogenesis (Ferara et al., 1991). However,
it seemed that 16-kDa N-terminal fragments of PRL and
related PRL family proteins including human GH, GH
variants, and placental lactogen, were anti-angiogenic
(Clapp et al., 1993; Struman et al., 1999). On the other
hand, proliferin, a PRL-related proteins, stimulated angiogenesis, whereas proliferin-related protein inhibited it
(Jackson et al., 1994).
PRL is known as an in vitro co-mitogen for T and B cells
of human or murine origin (Bernton et al., 1988; Clevenger
et al., 1990; Russell et al., 1984) although controversial
results contradicting these findings have also been reported
(Gala and Shevach, 1997). PRL regulates lymphocyte proliferation by modulating the expression of gene products
necessary for cell cycle regulation (Clevenger et al., 1992)
via the T and B lymphocyte PRL receptor (Pellegrini et al.,
1992).
Recently, transgenic mice have been generated that
overexpress PRL (Wennbo et al., 1997), as well as others
with targeted disruptions of PRL (Horseman et al., 1997;
Steger et al., 1998) or the PRL receptor (Bouchard et al.,
1999; Ormandy et al., 1997). However, relatively little
information is presently available about these mice. Consequently, many long-standing controversies regarding
the role of PRL in hematopoietic or angiogenic processes
remain unclear. In our recent study, we revealed that the
ectopically expressed rat PRL induced the angiogenesis
and WBC proliferation in vivo (Ko et al., 2003). Although
there is 85% homology in the primary structure of the
PRL between rat and mouse (Sinha, 1995), functional
difference of PRL between two species have been expected. Thus, we have investigated the general and distinct functions of mouse PRL and comparative endocrine
aspect of PRL using a direct gene transfer method that
permits rapid expression of foreign genes (Danko et al.,
1997).
Materials and Methods
Animals and experimental design ICR mice at 2 months of
age were purchased from the Daehan Animal Center and main-
tained with 14 h light, 10 h dark illumination at 23°C, and food
and water ad libitum. For adrenalectomy, a small incision was
made in the skin and muscle layer of the back. The left and right
adrenal glands were removed sequentially and suturing carried
out. No attempt at haemostasis was made, as bleeding was slight.
Plasmid DNA for injection was purified using a slightly modified alkaline lysis method (Ko et al., 2003). To measure PRL
mRNA and protein (Figs. 1 and 2), muscle tissue was harvested
4 days after a single injection of DNA. For the reproductive
studies (Figs. 3 and 4), the first injection of 200 μg pCMVmPRL in 50 μl of 10% sucrose in saline was performed at 10:00
A.M. on diestrus II and the second 7 days later in female. In
male, two injection was made, 7 days apart into the quadriceps
of mice (Figs. 5 and 6). Tissues were harvested and at 5, 8, and
11 weeks for angiogenesis assays after injection. For the angiogenesis studies (Fig. 7), tissues were harvested at 5, 10, 15, and
20 days for WBC proliferation assays after injection. For the
replacement study, dexamethasone (Fluka, Switzerland) of 0.3,
3, 30, 300 μg/g body weight in 200 μl of PBS was injected intraperitonically into mice 2 days after adrenalectomy, which
was performed 1 day before second injection of plasmid. Control mice were injected with pcDNA3 vector or vehicle. All
experiments were performed at least four times if not otherwise
noted, and representative results are shown.
Construction of the pCMV-mPRL plasmid To generate pCMVmPRL (7.0 kb), a 1.6-kb mouse PRL cDNA (a kind gift from Dr.
D.H. Linzer, Northwestern University) was digested with HindIII and BamHI cloned into the corresponding sites of vector
pcDNA3 (Invitrogen, USA) which has a CMV early promoter
and a bovine growth hormone polyadenylation site (Fig. 1A).
Reverse transcription-polymerase chain reaction (RT-PCR)
and Southern blot hybridization RNA was purified as previously described (Ko et al., 2003; Lee et al., 2003). Briefly, muscles were homogenized with denaturing solution [4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7), 0.5% N-lauryl
sarcosine, 0.1 M 2-mercaptoethanol]. The homogenate was phenol/chloroform extracted, and RNA precipitated. RNA was
quantified with a UV spectrophotometer (U.V.2000, Pharmacia).
A260/A280 ranged from 1.8 to 2.0. Ten micrograms of total RNA
were used after quantification in duplicate. RT-PCR and Southern blot hybridization were performed as described (Cho et al.,
2001). RNA was then treated with DNase I (5 U, Promega,
USA) at 37°C for 10 min in order to remove genomic and transfected plasmid DNA, and reverse transcribed at 42°C using
random hexamer primers and AMV reverse transcriptase
(Promega, USA) in a 20 μl reaction. A mixture of oligonucleotide primers (500 ng each), dNTP, and Taq DNA polymerase
(2.5 U) was added to each reaction, the total volume was
brought to 100 μl with 1× PCR buffer [10 mM Tris (pH 8.3), 50
mM KCl, 1.5 mM MgCl2 and 0.01% gelatin] and the sample
was overlaid with light mineral oil. Amplification was performed for 30 cycles using an annealing temperature of 65°C on
an Omn-E thermal cycler (Hybaid Limited, UK). For the PRL
Jung-Sun Lee et al.
gene, the primers were designed to generate a 561 bp PCR
product. The 5′ primer sequence is 5′-GGTCAGCCCAGAAAGCAGG-3′ and the 3′ primer sequence 5′-GGGCAATTT GGCACCTCAGGA-3′. After amplification, samples were chloroform
extracted, dried, resuspended in 10 μl TE buffer (10 mM Tris,
pH 8.0, 1 mM EDTA), and electrophoresed on a 1.2% agarose
gel. The gel was photographed after ethidium bromide staining.
The PCR products were then denatured with sodium hydroxide
and transferred to Nytran filters (0.45 μm, Schleicher & Schuell,
Germany) under vacuum. They were hybridized with dixogenein-labeled mouse PRL cDNA, blotted with anti-dixogeneinAP (1:1000) (Roche, Germany), washed, and exposed to X-ray
file after blotting with CSPD (Roche, Germany).
Protein blot analysis Tissues were removed, homogenized in
400 μl of protein extraction buffer [0.1 M NaCl, 0.01 M Tris-Cl
(pH 7.6), 1 mM EDTA (pH 8.0), 0.1% TritonX-100, 1 μg/ml
aprotinin, 100 ng/ml phenylmethylsulfonyl fluoride], and centrifuged four times. The homogenates were then mixed with an
equal volume of 2× SDS-loading buffer [100 mM Tris-Cl (pH
6.8), 200 mM DTT, 4% SDS, 0.2% BPB, 20% glycerol], placed
in boiling water for 10 min, and centrifuged. The supernatants
were transferred to fresh tubes. Samples of each extract containing 10 μg protein (10 μg) were heated at 70°C for 10 min, electrophoresed on a 12% acrylamide gel and transferred onto Nytran filteras in 1× transfer buffer (39 mM glycine, 48 mM Tris
base, 0.037% SDS, 20% methanol). The blots were incubated
overnight in blocking solution (5% nonfat dried milk, 0.02%
sodium azide, 0.02% Tween) with shaking at 4°C, followed by
exposure to primary PRL antibodies (1:400) (Biogenesis, UK)
overnight. They were washed in milk-TBS-Tween for 30 min
and incubated with secondary anti-rabbit Ig horseradish peroxidase-linked whole donkey antibody (1:100) (Amersham Pharmacia Biotech) in azide-free blocking solution [5% nonfat dried
milk, 150 mM NaCl, 50 mM Tris-Cl (pH 7.5)] for 2 h. The secondary antibody was detected with an ECL kit (Amersham
Pharmacia Biotech, USA).
Blood cell counts, histology, and hormone measurement For
blood cells counts, fresh tail blood containing anticoagulant was
collected; 0.5 μl was smeared onto a slide, stained with Wright’s
staining solution, and then counted under a microscope. WBCs
were recognized by their nuclear morphology and size. Average
values for WBC numbers were obtained from at least five mice
in each group, each of which was counted in quadruplicate. For
the histological studies, excised tissues from injected and control mice were examined for gross appearance. They were immediately fixed in fresh 4% paraformaldehyde in PBS, pH 7.4.
Following overnight fixation, they were dehydrated in ethanol
and embedded in paraffin. Seven-micrometer sections were
prepared using a microtome (Nippon Optical Works, Japan). The
sections were de-parafinized with xylene, dehydrated in absolute ethanol, and rehydrated in water. Sections were stained with
hematoxylin, counterstained with eosin, and observed under a
microscope (Olympus IX-70, Japan). For the hormone meas-
191
A
B
C
Fig. 1. pCMV-mPRL structure and PRL expression. A. Diagram
of the pCMV-mPRL construct. Functional elements include the
cytomegalovirus (CMV) promoter, the mouse PRL cDNA, and
human growth hormone (hGH) poly(A). B-C. RT-PCR (B) and
Southern blot analysis (C) were performed as described in Materials and Methods. RNAs from the control, and from pCMVmPRL-injected mice without reverse transcription, were used as
normal and internal controls, respectively. The pCMV-mPRL
plasmid was used as a positive control.
urement, testosterone level was determined by RIA. The sensitivity of testosterone assay was about 3.9 pg/tube. The intra- and
inter-assay coefficients of variation were approximately 5.1 and
7.8%, respectively.
Results
Intramuscular expression of the PRL gene In initial
studies we asked whether intramuscular injection was an
effective means of expressing PRL in mice. Using RTPCR and Southern blot analysis, PRL mRNA was detected in mouse muscle after injection of the pCMVmPRL construct (Fig. 1A); a PCR product of 561 bp was
obtained. Subsequent hybridization with labeled PRL
cDNA confirmed that the PCR product was PRL mRNA
(Figs. 1B and 1C). Western blot analysis showed that ma-
192
Reproductive Function and WBC Proliferation by Prolactin
A
B
Fig. 2. PRL detection. Protein blot analysis was performed as
described in Materials and Methods. Proteins from control
muscle and pituitary were used as negative control, and positive
control, respectively. The Western blot is a representative of
four independent experiments. RT, reverse transcription; pCMVmPRL, pCMV-mPRL-injected mice.
ture PRL protein (23 kDa) was formed in the muscle of
the injected mice (Fig. 2), along with a larger MW species
that probably corresponds to the large PRL species reported previously (Walker et al., 1995). The level of serum PRL was substantially elevated in the pCMV-mPRL
mice when we measured serum PRL with semi-quantitative
Western blot method (Fig. 2). Thus, PRL mRNA and protein were successfully expressed in muscle and secreted
into the serum by this approach.
Fig. 3. Change in the estrous cycle in PRL-expressing mice. A.
Each stage of the estrous cycle was identified by daily examination of vaginal cytology at 9:30 A.M. at a 100× magnification (n
= 6). The first injection was carried out after confirming at least
two normal estrous cycles, and the second injection followed 4
days later. Note that diestrus was extended after the second injection. Asterisks denote values that are significantly different
from the mean control value (Student’s t-test, P < 0.01). Values
shown are means ± standard deviations. B. A photograph of one
example. Note that total absence of epithelial cells at times corresponding to the proestrus or estrus stages. DI, diestrus I; DII
(1)-DII(7), day 1−day 7 of diestrus II; P, proestrus; E, estrus.
Effect of PRL on reproductive function in females PRL
has been known for its confusing role in the ovarian function. To investigate whether the ectopically expressed
PRL influences the estrous cycle, we examined daily the
vaginal cytology at 9:30 A.M after injecting pCMVmPRL into female mice 2 months of age. After two injections the diestrus II stage in the subsequent cycle 3 was
approximately triple the average length (Fig. 3A). In one
unusual case it was extended to 7 days (Fig. 3B). Because
diestrus II was extended, presumably due to lack of estrogen, we asked whether this was associated with any
change in the ovary and uterus. Whereas there was no
conspicuous change in the ovary (data not shown), the
uterus was markedly shrunk in proestrous and estrous
stages and the endometrium was not well developed, resulting in small uterine lumen (Fig. 4).
angiogenic function of mouse PRL. The new blood vessels were first observed 5 weeks after injection and maintained until 11 weeks (Fig. 5). Histological examination
of cross-sections of the testis revealed no conspicuous
new blood vessels within testis (Fig. 6). Instead the abundant Leydig cells were observed among seminiferous tubules (Fig. 6). When we measure the testosterone level in
serum, it was increased 3.8 fold in the pCMV-mPRL injected mice (Control: 187.09 ± 104.68 pg/ml, pCMVmPRL: 710.52 ± 119.09a pg/ml, n = 6) (a p < 0.01 compared to control). In addition, the size and morphology of
the seminiferous tubules, sperm, and spermatocytes were
no different from those of control mice.
Angiogenic action of PRL in the testis Although there is
a controversy over the action of PRL in angiogenesis in
the region of the hypothalamus-pituitary, our previous
study using the rat PRL revealed that new blood vessels
containing abundant red blood cells was observed on the
surface of the testis (Ko et al., 2003). We reconfirmed the
WBC proliferation by PRL Since PRL was known as a
co-mitogen for T and B cells and increased proliferation
of WBCs in number was observed in rat PRL-overexpressing mice (Ko et al., 2003), the effect of injection on
cells of the immune system was investigated using mouse
PRL. As expected, proliferation of WBCs was observed
Jung-Sun Lee et al.
Fig. 4. Histology of the uterus. Uteri were isolated from vehicle
(control) and pCMV-mPRL injected mice (pCMV-mPRL) at
each stage after the second injection, fixed in 4% paraformaldehyde, embedded in paraffin, transversely sectioned at 7 μm, and
stained with hematoxylin and eosin (20× magnification). Experiments were performed five times and representative results
are shown. * Uterine lumen.
193
Fig. 6. Histology of the testis. Testes were isolated from vehicle
(control) and pCMV-mPRL injected mice (pCMV-mPRL) at 5,
8, and 11 weeks after injection, fixed in 4% paraformaldehyde,
embedded in paraffin, transversely sectioned at 7 μm, and
stained with hematoxylin and eosin (200× magnification).
Since adrenalectomy may exert its effect by removing endogenous glucocorticoid, which is known to inhibit PRLinduced cell proliferation (Olazabal et al., 2000), we tested
whether this increase could return normal by treatment with
synthetic glucocorticoid, dexamethasone. As a result, the
increased WBC in number was returned normal (Fig. 7C),
indicating the suppressive function of glucocorticoid.
When the mice were injected with various doses of dexamethasone, the number of WBC decreased as a function of
dose, accompanying by paralleled decrease of lymphocytes.
However, the neutrophils revealed peak at an injection
concentration of 3 µg/g body weight (Fig. 7C).
Discussion
Fig. 5. Angiogenesis induced by PRL. The gross morphology of
the testis is shown for vehicle (control) and pCMV-mPRL injected mice (pCMV-mPRL) at 5, 8, and 11 weeks after injection.
after 5 days and elevated WBC numbers persisted until at
least 20 days after plasmid injection (Fig. 7A). A combination of pCMV-rPRL injection and adrenalectomy induced even more pronounced proliferation of WBCs (Fig.
7A). Among WBCs, the lymphocytes were mainly influenced by overexpressed PRL and adrenalectomy (Fig. 7B).
We have shown that direct injection of pCMV-mPRL
plamsid DNA into mouse muscle leads to the appearance
of PRL mRNA and protein, and has a variety of consequences, including change in estrous cycle and uterine
histology, proliferation of WBCs, Leydig cell proliferation, and formation of new blood vessel in the surface of
testis. These finding imply that functional mouse PRL is
released from the muscle, and is implicated in reproduction, hematopoiesis, angiogenesis, and cell proliferation.
In the non-pregnant females, the importance of PRL in
reproductive function has been poorly understood while
PRL has been demonstrated to be required during lacto-
194
Reproductive Function and WBC Proliferation by Prolactin
A
B
C
Fig. 7. Stimulation of WBC proliferation. A. Fresh blood samples from the tail were collected 5, 10, 15, and 20 days after
pCMV-mPRL injection in four groups: control, pCMV-mPRL
injected mice, adrenalectomized mice, and adrenalectomized
plus pCMV-mPRL injected mice. The cells were stained with
Wright’s solution and observed under a microscope (400× magnification). Values are means ± the standard deviation. B. Fresh
blood samples from the tail were collected 10 days after pCMVmPRL injection in four groups as explained above. Then, lymphocyte and neutrophil cells were countered. C. Various doses
of dexamethasone in 200 μl of PBS or vehicle were injected
intraperitonically into the adrenalectomized plus pCMV-mPRL
injected mice as described in Materials and Methods. Fresh
blood samples from the tail were collected 10 days after second
pCMV-mPRL injection. Asterisks denote values significantly
different from mean control values (ANOVA, * p < 0.05; ** p <
0.01 compared to control). B.W., body weight.
genesis in the pregnant females. It was known that there is
no functional luteal phase in the estrous cycle of mice, as
is present in humans and many other mammals although
PRL is required for maintenance of the corpus luteum in
mice. Recently, it was reported that the female PRLdeficient mice had irregular estrous cycles and did not
become pregnant when mated to stud males (Horseman et
al., 1997). Our results revealed that the estrous cycle was
extended, especially in diestrus stage (Fig. 3), suggesting
that PRL could act on the estrous cycles whether it in-
duced irregular or extended pattern. Regarding the direct
effect of PRL on the gonad, no obvious defects were observed in the histology of the ovaries (data not shown).
However, histological changes in uterus were observed
including uterine shrinkage with poorly developed endometrium and small lumen. At this juncture, the change in
estrous cycle by PRL with accompanying noticeable uterine change is not well correlated with non-change in ovarian histology. Further studies are needed to find the cause
to explain the relatively irregular and poor fertility.
In male, PRL levels in the blood increase in humans with
ages (Berry et al., 1984; Hammond et al., 1977) and PRL
could induce the new blood (Ko et al., 2003), suggesting
some uncovered, but presumably interesting role. In this
study, PRL also evidently induced angiogenesis in testis
after 5 to 11 weeks with branching on the surface of the
testis. Angiogenesis is an aspect of many physiological
processes (Hanahan and Folkman, 1996) as well as of
pathological conditions such as tumor growth and metastasis (Folkman, 1995). As explained in introduction, it is
still controversial that intact 23 kDa prolcatin has a stimulatory role in angiogenesis. Our result indicated that PRL
had a stimulatory function on angiogenesis at the testis in
case of a chronic treatment of PRL. Moreover, PRL induced proliferation of the Leydig cells which is well correlated with increased testosterone. This change, however,
gradually appeared, culminating around 11 weeks after
plasmid injection, suggesting that PRL also has a chronic
effect on the Leydig cell. This seemed one of the chronic
effects of PRL which have been expected from the gradually increased level of PRL in old aged humans. Further
studies including histological changes are undergoing in
our research. In addition, with respect to the site of action
of PRL within the testis, PRL receptor mRNA has been
observed in the testis as well as the liver, ovary, prostate
gland (Boutin et al., 1988). With regard to PRL binding,
we obtained evidence for binding to Leydig cells within
the testis using PRL-EGFP (Ko et al., 2003). At present,
the relationship between the angiogenic and reproductive
functions of PRL remains unexplored. In addition, as to
the extended period over which new blood vessels formed
in the testis, it is possible that a high blood level of PRL
may have been sustained in our experiments for a time
comparable to the 22 weeks reported in related experiments (Danko et al., 1997).
The in vitro mitogenic effects of PRL on T and B cells
of human or murine origin (Bernton et al., 1988;
Clevenger et al., 1990; Russell et al., 1984) as well as NK
(natural killer) cells and macrophages (Bernton et al.,
1988) are well known. During interleukin 2-driven T
lymphocyte proliferation, PRL is required for mitogenesis,
and after binding the PRL receptor, PRL is internalized
and translocated to the nucleus (Clevenger et al., 1990;
1991). Besides its role as a required co-mitogen, PRL also
acts as a survival factor. During periods of stress, in-
Jung-Sun Lee et al.
creased levels of PRL are released from the pituitary, approximately in parallel with the secretion of corticotrophin releasing factor (Kant et al., 1992), which stimulates
the release of glucocorticoids. Although glucocorticoids
are necessary for the maintenance of overall metabolism
and survival during periods of stress, one of their potentially undesirable effects is inhibition of immune responses. However, it is known that either pretreatment or
concomitant treatment of Nb2 cells or normal murine
thymocytes with PRL significantly inhibits glucocorticoid-induced apoptosis (Fletcher-Chiappini et al., 1993;
Witorsch et al., 1993). It was shown recently that glucocorticoids inhibit PRL-induced cell proliferation by a
pathway involving JNK (c-Jun amino terminal kinase)
and AP-1 (Olazabal et al., 2000). However, the role of
PRL is not clear. On the one hand, PRL receptor-deficient
mice showed no alteration their content of thymic or
splenic cells, or in the composition of lymphocyte subsets
in primary (bone marrow and thymus) or secondary
(spleen and lymph nodes) lymphoid organs (Bouchard et
al., 1999). Moreover examination of PRL-deficient mice
has led to the conclusion that PRL does not play an indispensable role in primary lymphocyte development and
homeostasis, or in myeloid differentiation (Horseman et
al., 1997). On the other hand, our results showing that
induction of PRL stimulates WBC proliferation support a
mitogenic role for PRL. Moreover, removal of endogenous glucocorticoid by adrenalectomy together with induction of PRL markedly increases WBC proliferation
whereas this increased WBCs in number by PRL plus
adrenalectomy decreased as a function of the dexametasone replaced (Figs. 7A−7C). Thus, our result evidently
supports the idea of an inverse relationship between PRL
and glucocorticoid in in vivo mitogenesis.
In contrast to transgenic mice overexpressing the PRL
gene that developed dramatic enlargements of the prostate
gland (Wennbo et al., 1997), we found no change in prostate volume in this study using mouse PRL gene (data not
shown). This difference may be due to the fact that the
PRL was produced locally in the prostate in the transgenic
experiments (Nevalainen et al., 1997; Wennbo et al.,
1997). The contradictory findings of increased PRL levels
in patients with prostate hyperplasia in one report (Saroff
et al., 1980), and unchanged levels in another (Harper et
al., 1976), as well as our own results with no change in
prostate, suggest that the physiological condition of the
experimental mice may be important. It should be also
noted that in our study we observed neither the abnormalities of mammary gland development (Horseman et al.,
1997) nor the pituitary weight changes reported in PRL
gene-disrupted mice (Steger et al., 1998).
In addition to their implications for the nature of PRL
function, our studies suggest significant technical advances and clinical applications as described in previous
study (Ko et al., 2003). Briefly, our approach can be ap-
195
plied to different species regardless of genetic strains. It is
also easy method to express foreign gene at any time of
development. Finally, this approach could be used for in
vivo screening of genetically engineered proteins without
producing time-consuming transgenic mice.
Acknowledgments The authors thank Sun-Kyung Koo for her
help in preparation of the manuscript. This work was supported
by a grant (No. R05-2003-000-12020-0) from the Basic Research
Program of the Korea Science and Engineering Foundation.
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