Mitsubishi Kasei Institute of Life Sciences (T.O., T.T.)
Tokyo,1948511, Japan
Laboratory of Functional Genomics
(T.O., Y.S.), Human Genome Center The Institute of Medical
Science University of Tokyo Tokyo 108-8639, Japan
Laboratory of Veterinary Physiology (G.W.) Tokyo University
of Agriculture and Technology Tokyo 183-8509, Japan
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ABSTRACT |
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INTRODUCTION |
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In rodents, semicircadian surges of PRL secretion are induced by cervical stimuli. These surges are believed to be responsible for the conversion of the corpus luteum of pregnancy (CLP) from the corpus luteum (CL) of the cycle. The CLP secretes an amount of progesterone sufficient for maintaining pregnancy (2).
The suprachiasmatic nuclei of the hypothalamus, by controlling the endogenous circadian rhythm, are responsible for the timing of the mating-induced surges of PRL (1). It has been suggested that inhibitory and stimulatory neuroendocrine factors released from the hypothalamus regulate the surges (1). Dopamine (DA) is believed to be the principal inhibitory factor for not only tonic PRL secretion but also the semicircadian surges of PRL (1, 3). However, the mechanisms and pathways from cervical stimuli to PRL secretion are still not fully understood.
Puromycin-sensitive aminopeptidase (Psa) deficient mice (Psagoku/goku) have been generated by a mouse-gene trap strategy (4). The mutant mice exhibit growth retardation as well as abnormal behavior associated with anxiety and pain that might be derived from impaired brain functions (4). Psa has been characterized and purified as a putative extracellular enkephalinase in vivo (5). However, the functions of Psa in the metabolism of enkephalins remain unclear because Psa was found to be a cytoplasmic protein (6, 7). In addition, we found that no apparent differences in the distribution patterns or intensity of expression of enkephalins in the brain could be detected between genotypes (4). These studies imply unidentified and unanticipated intracellular roles for Psa.
In the present study, we have observed that Psagoku/goku females lack the ability to form and maintain the CLP. Based on the observations reported here, it is believed Psa plays an indispensable role in the induction of the mating-induced PRL surges required for the formation and maintenance of the CLP during gestation in rodents. Although no studies thus far have addressed the endocrine relevance of Psa, the present study provides informative insights into the roles of Psa in the hormonal regulation that underlies murine pregnancy.
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RESULTS |
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We next examined the estrous cycle of seven
Psagoku/goku and five wild-type female mice at
815 weeks of age for 29 days. Each stage of the estrous cycle
was confirmed by the cytological analysis of vaginal smears as
described by Freeman (1). This examination showed that the estrous
cycle of Psagoku/goku females was
irregular (Fig. 1). A rhythmic
periodicity with a recognizable estrus is evident every 45 days in
all Psa+/+ females, while no rhythmic
periodicity of the estrous cycle in tested
Psagoku/goku females could be detected. This
observation also suggested a prolonged estrus or prolonged diestrus in
Psagoku/goku females.
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A Lack of Implantation Due to Progesterone Insufficiency in
Psagoku/goku Females
Psagoku/goku females exhibited no pregnancy
with gonadotropin treatment to induce superovulation, as mentioned
above. Therefore, we first investigated whether the defects in the
response to gonadotropins in Psagoku/goku
females caused the failure to achieve pregnancy. The gonadotropin
treatment induced superovulation in both genotypes (number of
collected oocytes; Psa+/+ mice,
13.4 ± 1.74, n = 10;
Psagoku/goku, 8.8 ± 1.26, n = 9;
statistically not significant, Students t test) at
812 weeks of age. Moreover, we found fertilized oocytes in the
oviducts of Psagoku/goku females without
exogenous hormonal treatment on the day the vaginal plugs were
identified (data not shown). Based on these observations, the ovaries
of Psagoku/goku animals can respond to
endogenous or exogenous gonadotropins to induce ovulation. These data
further indicate that the ovulated oocytes of
Psagoku/goku females can undergo
fertilization.
Next, embryo-transfer experiments were performed to examine whether the absence of pregnancy in Psagoku/goku females is due to an impaired ability in the uterine environment to recognize pregnancy. Forty-seven blastocysts were collected from Psa+/+ females on embryonic day 4.5 (day 0.5 is defined as the day when the presence of a vaginal plug is confirmed) and then transferred into four Psa+/+ females on day 4.5 after mating with a vasectomized male. All mice that underwent the transfer were pregnant, and normal development was detected on day 10.5 in 29 embryos. However, no implantations were detected in the uteri of seven Psagoku/goku animals on day 10.5 after 89 normal blastocysts of Psa+/+ mice were transferred on day 4.5 after mating with a vasectomized male. These results indicate that the uterus of Psagoku/goku animals is not supportive of implantation.
We assessed the plasma level of progesterone in
Psagoku/goku females after mating because
progesterone is essential for the establishment and maintenance of
pregnancy. The plasma progesterone level on day 10.5 in
Psagoku/goku females was approximately 10-fold
less than Psa+/+ females
(Psa+/+, 32.52 ± 3.62 mg/ml,
n = 5; Psagoku/goku, 3.03 ± 0.89
µg/ml, n = 6; P < 0.001, Students
t test). This result is consistent with the observation of
ovarian morphology on day 10.5 (Fig. 2).
A number of well developed CLP were detectable in the ovaries of
pregnant Psa+/+ females (Fig. 2A
). In
contrast, no developed CL were observed in the ovaries of
Psagoku/goku females (Fig. 2B
). These data
indicate that a major cause of infertility is the lack of formation of
the CLP, which causes progesterone insufficiency.
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Ovary Transplantation between
Psa+/+ and
Psagoku/goku Females
Ovary transplantation experiments were performed to address
whether the lack of CLP arises from intrinsic ovarian deficits or the
impaired hypothalamic-pituitary axis regulating ovarian function (Table 1 and Fig. 4
). The origin of the pups
(i.e. from donor or recipient) can be distinguished by X-gal
staining because the lacZ gene, which was introduced into
the Psa gene by a gene-trap event (4), is expressed in pups from
ovaries of Psagoku/goku females but not
Psa+/+ animals. We exchanged bilateral
ovaries between Psagoku/goku and
Psa+/+ female mice.
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These data clearly demonstrate that the ovarian function of Psagoku/goku females is restored by intact endocrine regulation and suggest a disruption of the hypothalamic-pituitary axis required for development of CLP in Psagoku/goku females.
Disruption of Semicircadian PRL Secretion during Early Pregnancy
in Psagoku/goku Homozygous Females
We measured hypophyseal hormones that regulate ovarian functions
during early pregnancy. There were no significant differences in plasma
FSH and LH levels between Psa+/+ and
Psagoku/goku animals (FSH:
Psa+/+, 5.15 ± 2.00 ng/ml,
n = 4; Psagoku/goku, 4.47 ± 2.34
ng/ml. n = 3; P = 0.834, LH:
Psa+/+, 35.85 ± 9.50 pg/ml,
n = 4; Psagoku/goku, 60.66 ± 23.47
pg/ml, n = 3; P = 0.323, Students t
test).
PRL is released in two large daily surges, the nocturnal and
diurnal surges, in mice as well as rats during early pregnancy
(10, 11, 12). We examined plasma PRL concentrations of three
Psa+/+ and five
Psagoku/goku mice every 4 h from 1400
h on day 0.5 to 1400 h on day 1.5, and three
Psa+/+ and two
Psagoku/goku mice from 0200 h to 1400
h on day 4.5 (the day of implantation), independently (Fig. 5). PRL levels in
Psa+/+ females showed the first
diurnal surge of PRL secretion around 2200 h on day 0.5, the first
nocturnal surge at 1000 h on day 1.5, and the nocturnal surge at
0600 h on day 4.5 (Fig. 5
). A one-way ANOVA test of the PRL levels
on days 0.51.5, but not on day 4.5, revealed a significant difference
among the group (F(6, 14) = 3.516,
P < 0.025). The LSD test revealed the values for
nocturnal surge on day 1.5 (1000 h) were significantly higher than
values for any other time on days 0.51.5 (P =
0.00140.0176).
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Restored Formation of CLP in Psagoku/goku
Females by Pituitary Grafts or by PRL Injection after Cervical
Stimulation
To prove that the loss of PRL surges results in infertility
in Psagoku/goku females, we first examined
whether injections of exogenous PRL can rescue the
formation and maintenance of CLP and maintain pregnancy in
Psagoku/goku females. Two
Psagoku/goku females were treated with twice
daily injections beginning the day vaginal plug formations were
observed. No onset of implantation was detectable in the uteri of the
mutants on day 10.5. However, a number of eosinophilic CL were observed
in the ovaries of the PRL-treated Psagoku/goku
females (Fig. 6A). These CL were
comparable in morphology to the CLP of the day-matched ovaries of
Psa+/+ females (Fig. 6B
). Based on
these observations, the luteal cells of
Psagoku/goku females can respond to exogenous
PRL injection. These data further indicate that the formation of CLP in
Psagoku/goku females is rescued by a mimic
injection of exogenous PRL. The mutant mice, however, display increased
anxiety and impaired pain response (4). Therefore, the distress of two
daily injections may have caused the failed implantation in
Psagoku/goku animals in this experiment.
Moreover, the possibility that the dose of the injected PRL was
insufficient to maintain pregnancy cannot be excluded (13, 14).
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These results demonstrate that deficits in the semicircadian surges of PRL secretion result in impaired formation of CLP and infertility in Psagoku/goku females, and that Psa is required for regulation of the mating-induced PRL surges during gestation.
Secretion and Gene Expression of PRL Respond to DA Exposure in
Pituitary Cells Derived from Psagoku/goku
Females
Next, we examined the response to exogenous DA exposure in primary
cultured pituitary cells (Fig. 7). DA is
a well established inhibitory neurotransmitter of PRL secretion and
gene expression (3, 19). DA is believed to be a key molecule in the
regulation of tonic PRL secretion and also to participate in
mating-induced PRL secretion (1).
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The inhibitory effect of DA on gene expression of PRL was also examined
by RT-PCR. The levels of PRL mRNA were decreased by the addition of DA
in the pituitary cells from Psa+/+ and
Psagoku/goku females (Fig. 7B).
These data demonstrated that PRL release and expression in pituitary cells regulated by DA are not affected in Psagoku/goku females.
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DISCUSSION |
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First, we considered the possibility that the production of the circadian rhythm itself is impaired. The circadian rhythm of Psagoku/goku animals, however, appeared to be intact with respect to locomotor activity (4) and body temperature (data not shown). The suprachiasmatic nuclei (SCN) are known to play a central role in the biological clock regulating locomotor activity and body temperature. The SCN have been also implicated as a master pacemaker for PRL release (1). Thus, the lack of semicircadian surges of PRL secretion may not be due to deficits in the SCN of Psagoku/goku animals.
Next, we considered the possibility that the downstream signaling of the SCN is abnormal. Mating-induced PRL surges are known to be controlled by reciprocal communication between the stimulatory and inhibitory pathways in the hypothalamic-hypophyseal portal system (20). Previous studies have addressed the components of this biological process to include inhibitory factors such as DA and stimulatory neuropeptides in the hypothalamus (21, 22, 23, 24). The results of the present study indicate that the Psa-deficient pituitary cells respond to DA and that the secretion and gene expression of PRL are inhibited. Because DA is believed to be a key inhibitory signal regulating the mating-induced PRL surges, this fact suggests that a major inhibitory function of the pituitary for the mating-induced PRL surges is intact in Psagoku/goku females.
Molecular characterization of the stimulatory and inhibitory components of mating-induced PRL secretion is not well established. Therefore, further investigation to identify the Psa-mediated signaling should provide valuable insight for understanding the regulatory mechanisms of mating-induced PRL surges.
Contribution of Psa to Proteasome-Mediated Proteolysis
The function of Psa was considered from another viewpoint. The Psa
gene contains motifs that show significant similarities to motifs in
the 26S proteasome subunits, suggesting that Psa can participate in
proteasome-mediated proteolysis (7). The proteasome appears to be
responsible for the degradation of regulatory short half-life proteins
such as transcriptional factors (25).
Psagoku/goku males display aberrant copulation and spermatogenesis, which were found to be insensitive to testosterone, which is, in part, converted to 17ß-estradiol in the brain and testis (25A ). A completely altered estrous cycle in Psagoku/goku females suggests the regulatory mechanisms of estrogen-induced PRL surges are impaired because the PRL surge in the estrous cycle regulates the length of the luteal phase in rodents (1). Estrogens are also known to contribute, in part, to the mating-induced PRL surge (26, 27). These data suggest estrogen-responsive pathways are hampered in the hypothalamic-pituitary axis of Psagoku/goku females.
The estrogen receptor (ER), like other steroid receptors, is known to act as a transcriptional factor. The intracellular signaling of ER is mediated by the chaperone-complex assembly system (28). Recent investigations also report that the regulation of the ER gene expression and proteolysis of ER itself are mediated by proteasomes (29, 30). The mechanism of ER-mediated transduction is complex and still not fully understood (31).
On the basis of these studies, Psa may regulate either the bulk of the chaperone complex turnover or the flow of the transcriptional signals by degradation of the ER-ligand complex through proteasome-mediated proteolysis. Further analyses for the involvement of Psa in ER signalings may shed further light on the metabolism of other regulatory molecules by Psa as a novel subcellular function of Psa in vivo.
In the present paper, we conclude that Psa is essential for the appearance of the mating-induced PRL surges underlying the maternal recognition of pregnancy in mice. Further analysis of Psa-deficient mice should reveal the novel molecular context of the central regulation of mammalian pregnancy and the estrous cycle.
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MATERIALS AND METHODS |
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Experiments involving animals were performed in accordance with Standard Ethical Guidelines for the Care and Use of Laboratory Animals (US NIH, 1985) and approved by the Ethical Committee of our institute.
Analysis of Estrous Cycle
Vaginal smears from five Psa+/+
and seven Psagoku/goku 8- to 15-week-old females
were collected every day for a 29-day period. Smears were streaked on
slides and stained with Giemsas solution. Estrous cycle stages were
determined as described previously (1).
Collection of Oocytes and Blastocysts
Female mice (812 weeks old) were used. Oocytes and blastocysts
were recovered from the oviducts or uterus of the mice as previously
described (32). To examine the response to gonadotropins and induction
of sexual receptivity, mice were given ip injections of PMSG (5 IU,
Serotropin; Teikoku-Zouki, Tokyo, Japan), and ovulation was
induced 48 h later with human CG (hCG, 5 IU, Gonatropin;
Teikoku-Zouki). Immediately after hCG treatment, the female mice were
housed overnight with fertile BALB/cA males (Clea Japan, Tokyo, Japan).
Mating was confirmed by the presence of a copulation plug the following
morning.
Transfer of Blastocysts into the Uterus
Blastocysts recovered from normal pregnant females on day 4.5
were transferred into the uterus of females on day 4.5 after mating
with a vasectomized male as described previously (32). Eight- to
12-week-old females were used. The treated females were decapitated on
day 10.5 and examined for the presence of implantation sites. Ovaries
and serum collected on the day of examination were used for further
histological and hormonal analyses, respectively.
Ovary Transplants
Ovary transplantation experiments were performed essentially as
described by Sterneck et al. (33). The ovaries of
Psa+/+ and
Psagoku/goku females at 46 weeks of age were
exchanged bilaterally. Treated females were mated with
Psa+/+ males 2 weeks after
surgery.
PRL Treatment and Pituitary Graft Analyses
Daily treatment of Psagoku/goku
female mice after the formation of vaginal plugs with injections of PRL
or the transplantation of PRL-secreting ectopic pituitary grafts were
performed basically as previously described (14, 15). Mutant and normal
females at 812 weeks of age with vaginal plugs were injected sc with
100 µg ovine PRL in 0.1 ml saline (0.03 M
NaHCO2, 0.15 M NaCl, pH
10.8) twice daily, between 0630 and 0700 h and between 1930 and
2000 h from day 0.5 to day 10.5, in an attempt to mimic the
endogenous PRL surges of pregnancy. Six 8-week-old females of each
genotype received transplanted pituitary homografts from
Psa+/+ mice under the kidney capsule.
This operation has been used to produce a model of hyperprolactinemia.
Treated mice were mated 2 weeks after surgery. The presence of CLP was
checked histologically in ovaries collected from the treated females on
day 10.5 that displayed vaginal plug formation. The status of the
pregnancy was examined by observing the embryos in the uterus on the
same day.
Histology and X-Gal Staining
Paraffin and frozen sections of ovaries and pituitaries were
prepared essentially as described previously (4). Briefly, for paraffin
sections, ovaries were removed and fixed in Bouins fixative, embedded
in paraffin, and sectioned at 7 µm with a microtome. Sections were
stained with hematoxylin-eosin (HE). In the ovary transplant
experiments, whole-mount X-gal staining was carried out as described
previously (34).
RIA Analysis
Murine PRL, rat LH, and rat FSH were iodinated by the
chloramine-T method, and RIAs were performed using murine PRL, rat LH,
and rat FSH RIA kits provided by the National Hormone and Pituitary
Program, NIDDK (Torrance, CA). The plasma levels of progesterone were
measured using a DPC progesterone assay kit (Diagnostic Products, Los Angeles, CA) by Koto Biken Ltd. (Tokyo,
Japan).
All blood samples were collected from mice at 812 weeks of age.
The blood samples for progesterone were collected by decapitation on
day 10.5. Blood was collected between 0600 h and 1000 h on
day 1.5 for the analysis of circulating LH and FSH levels. To
investigate the PRL surges, blood was collected in two ways: 1) serial
sampling individually from reopened tail vein incisions (shown in Fig. 5A); and 2) a quick decapitation at each time of examination (shown in
Fig. 5B
). Using method 1, 10 µl of blood were collected rapidly from
individual tail veins every 4 h during the experimental period.
Using method 2, blood samples were collected by decapitation at 4-h
intervals. To prevent elevated PRL levels due to blood odor (12) or
exogenous stress, the operations were conducted in an isolated room,
and moderate massage to collect blood from the tail vein was performed
quickly (within 12 min). In assays for murine PRL, LH, and FSH
detection, the interassay coefficients of variation (CV) were 18.7%,
13.5%, and 9.9%, respectively, and the intraassay CV values were
12.6%, 5%, and 9.2%, respectively.
Primary Cultures of Pituitary Cells
Twenty-five 30-week-old mice of each genotype were examined.
After decapitation, the pituitary was quickly collected and dissected
in DMEM (Life Technologies, Inc., Gaithersburg, MD).
Pituitary cells were dissociated with 0.05% trypsin in DMEM followed
by 500 U/ml collagenase (Nitta-Gelatin Inc., Osaka, Japan) in DMEM at
37 C for 2 h. The pituitary cells were cultured at 2 x
105 cells per 96-well dish (Corning, Inc., Corning, NY) in DMEM with 12.5% FBS (Life Technologies, Inc.) at 37 C and 5% CO2.
On the third day after starting the culture, the cells were washed with
DMEM without FBS, 20 µl of DMEM without FBS, and with DA (final
concentration, 10-6 M) added to each
well, and the cells were incubated for 24 h. Control cells were
cultured in the same media without DA for 24 h.
Western Blot Analysis
The culture media were collected and 10 µl of each medium were
suspended in 2x sample buffer (100 mM Tris-HCl, pH 6.4,
4% SDS, 0.2% bromophenol blue, and 20% glycerol) and boiled for 5
min. Samples were resolved in 15% SDS-polyacrylamide gels and
electroblotted to a nitrocellulose membrane. The filters were probed
with an antibody against murine PRL (diluted in 1:5,000, same as that
in the RIA) at 4 C overnight and incubated with horseradish
peroxidase-conjugated antibody to rabbit-IgG at room temperature
for 1 h. The immune complexes were detected by enhanced
chemiluminescence (ECL) detection (Amersham Pharmacia Biotech, Arlington Heights, IL).
RT-PCR Amplification
Cells were dissociated from the wells by 0.05% trypsin in DMEM
and collected. Total RNA was isolated using RNeasy
(QIAGEN, Valencia CA). The cDNA was synthesized using
SuperScript (Life Technologies, Inc.). Serial dilutions
(1:20, 1:100, 1:500) of the reverse transcription reaction
were PCR-amplified for 35 cycles of 94 C for 1 min, 60 C for 2 min, and
72 C for 3 min. Mouse glyceraldehyde phosphate dehydrogenase (GAPDH)
gene was used as the control. The oligonucleotide primers used were as
follows. Mouse PRL, 5'-CTCACTACATCCATACCCTGTATAC-3' and
5'-CATTTCCTTTGGCTTCAGGATAGGC-3', mouse GAPDH,
5'-GGGTGGAGCCAAACGGGT-CATC-3' and
5'-GCCAGTGAGCTTCCCGTTCAG-C-3'.
Statistical Analysis
The experimental data were analyzed by Students t
test, one-way ANOVA, or two-way ANOVA. Appropriate pairwise comparisons
between pairs of groups were carried out using Fishers least
significant difference (LSD) test after obtaining the statistical
difference by an ANOVA test. Values of P < 0.05 were
considered statistically significant. All values in the text and figure
legends are expressed as the mean ±
SEM.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Present address: The Institute for Biogenesis Research,
Department of Anatomy and Reproductive Biology, John A. Burns School of
Medicine, University of Hawaii, 1960 East-West Road, Honolulu, Hawaii
96822.
Received for publication May 31, 2000. Revision received January 22, 2001. Accepted for publication January 29, 2001.
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REFERENCES |
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