Puromycin-Sensitive Aminopeptidase Is Essential for the Maternal Recognition of Pregnancy in Mice

Tomoharu Osada1, Gen Watanabe, Yoshiyuki Sakaki and Takashi Takeuchi

Mitsubishi Kasei Institute of Life Sciences (T.O., T.T.) Tokyo,194–8511, 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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Maternal recognition of pregnancy in rodents requires semicircadian surges of hypophyseal PRL secretion during early gestation, which are required for the formation of the corpus luteum of pregnancy (CLP). Here we show that puromycin-sensitive aminopeptidase (Psa)-deficient mice display female infertility that results from impaired formation of CLP. Transplantation of mutant ovaries into normal females restored fertility but not vice versa. Psa-deficient females revealed no semicircadian surges of PRL induced after mating stimuli. Pregnancy in the mutant females was restored by grafting intact pituitaries to elevate circulating levels of PRL. Psa is thus required for the appearance of the semicircadian surges of PRL secretion that are crucial for maintaining pregnancy in rodents.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Female reproduction in mammals requires hormonal regulation by the hypothalamo-pituitary-gonadal axis. The axis functions both in ovulation during the estrous cycle and in recognition and maintenance of pregnancy after mating. While the hormones that participate in these processes have been well identified and characterized, the molecules that take part in the cross-talk of the axis have remained equivocal (1).

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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Infertility and Irregular Estrous Cycle in Psagoku/goku Female Mice
Heterozygous mice (Psa+/goku) are fertile and homozygous mutant mice (Psagoku/goku) are produced by heterozygous crossings (4). Psagoku/goku mice are infertile. No litters were recorded when Psagoku/goku females were housed for more than 1 month with normal BALB/cA males. To examine whether a lack of mating was the primary cause of infertility, Psagoku/goku females were housed with vasectomized BALB/cA males for 24 days and monitored for the presence of vaginal plugs. All mutant mice showed vaginal plug formation at least once within this period, while the frequency was irregular. These results suggest that while the sexual receptivity of the mutant females is not affected, the estrous cycle is affected.

We next examined the estrous cycle of seven Psagoku/goku and five wild-type female mice at 8–15 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. 1Go). A rhythmic periodicity with a recognizable estrus is evident every 4–5 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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Figure 1. Analysis of the Estrous Cycle in Psa+/+ and Psagoku/goku Mice

Representative samples of the estrous cyclicity of Psa+/+ (A and B) and Psagoku/goku (C and D) animals. Each stage of the estrous cycle was determined by the predominant presence of either lymphocytes, nucleated epithelial cells, or keratinocytes. E, Representative patterns of vaginal smears for 6 days from Psa+/+ (left) and Psagoku/goku (right) mice. Smears were stained with Giemsa’s solution. The normal estrous cycle has a characteristic periodicity, with an estrus every 4–5 days. Diestrus is characterized by the presence of lymphocytes (small nucleated cells) in the vaginal fluid (days 2 and 3 for the Psa+/+ female). During the proestrous stage, nucleated epithelial cells are observed in the vaginal smear (days 4 and 5 for the Psa+/+ female). The vaginal smear contains only keratinocytes at estrus (days 1 and 6 for the Psa+/+ female). Conversely, vaginal smears collected from Psagoku/goku mice reveal the prolonged presence of lymphocytes and random appearance of keratinocytes, indicating a complete alteration of the estrous cycle. The bar represents 100 µm.

 
Next, we induced estrus and ovulation by injecting exogenous gonadotropins into Psagoku/goku females at 8–15 weeks of age. We confirmed that Psagoku/goku females had mated with normal BALB/cA male mice by the presence of vaginal plugs the following day. However, female mice that mated did not appear to be pregnant and no pups were delivered.

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, Student’s t test) at 8–12 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, Student’s t test). This result is consistent with the observation of ovarian morphology on day 10.5 (Fig. 2Go). A number of well developed CLP were detectable in the ovaries of pregnant Psa+/+ females (Fig. 2AGo). In contrast, no developed CL were observed in the ovaries of Psagoku/goku females (Fig. 2BGo). 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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Figure 2. Impaired Formation of CLP on Day 10.5 in Psagoku/goku Females

Morphology of ovaries of each genotype on day 10.5. Sections were stained with HE. The ovary collected from Psa+/+ (A) has several eosinophilic CLPs (asterisks). The ovary of Psagoku/goku (B), however, contains no CLP. The detected hematoxyphilic CL (arrowheads) appears to be degenerated.

 
Impaired Formation of CLP in Psagoku/goku Females
The CLP, which secretes progesterone sufficient for the recognition of pregnancy, is induced by mating (8). We investigated the status of CL, which is converted to CLP by the mating stimuli, in unmated and mated Psagoku/goku females (Fig. 3Go). Histological observation of the ovaries of unmated females revealed that the number of CL is lower in Psagoku/goku females (Fig. 3Go, A and B). In some cases, no CL were observed in the ovaries of mutant females. In contrast, the ovaries of Psa+/+ females contained three or more generations of CL (Fig. 3AGo) because their morphological life span is 3–4 times longer than each estrous cycle (1). Together with the altered estrous cycle of Psagoku/goku females, the decreased number of CL is not likely to reflect a malformation of CL, but rather the irregular estrous cyclicity of Psagoku/goku females.



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Figure 3. Impaired Conversion of CL to CLP in Psagoku/goku Females

Sections of ovaries from Psa+/+ (A, C, E, and G) and Psagoku/goku females (B, D, F, and H) were stained with HE. A and B, Ovaries collected from untreated females. Ovary from a Psa+/+ mouse (A) contained CL at various stages. Conversely, few CL were observed in the ovary of a Psagoku/goku female (B). Mice were treated with gonadotropins and mating was confirmed by the presence of vaginal plugs. Sections of ovaries from each genotype were prepared on days 0.5. (C and D). 2.5 (E and F), and 4.5 (G and H). From day 0.5 to 2.5, nonfunctional CL (asterisks) were detectable in the ovaries from both genotypes (C–F). On Day 4.5, morphological analyses revealed CLP in the Psa+/+ female (double-asterisks). In contrast, CL began to degrade in the ovary of the Psagoku/goku female (crosses). No CLPs were observed in Psagoku/goku females. Bar represents 150 µm.

 
Next, we examined the development of CLP in Psagoku/goku female mice from day 0.5 to day 10.5 after mating. From day 0.5 to day 2.5, the CL were observed in the ovaries of both Psa+/+ and Psagoku/goku females, and there were no apparent differences in morphology between these two genotypes, although the number of CL in Psagoku/goku females was lower (Fig. 3Go, C–F). These data support the results of the intact ovarian response to gonadotropins. On days 4.5 and 10.5, morphological differences were apparent. The ovaries of Psa+/+ females revealed a number of eosinophilic CL (Fig. 3GGo). The observed CL was morphologically coincident with CLP, which secretes sufficient progesterone to support pregnancy (9). On the other hand, sections from the ovaries of Psagoku/goku females displayed apparently regressed CL (Fig. 3HGo). The degenerative status of the CL reflects the decreased circulating level of progesterone in Psagoku/goku females. This observation indicates that CL do not develop into CLP in the ovaries of Psagoku/goku females.

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 1Go and Fig. 4Go). 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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Table 1. Ovary Transplantation between Psa+/+ and Psagoku/gokuFemales

 


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Figure 4. Morphology of Transplanted Ovaries of Each Genotype on Day 10.5 after Mating

Sections were stained with HE. A, Ovary from a Psagoku/goku mouse transplanted into a Psa+/+ female. B, Ovary from a Psa+/+ mouse transplanted into a Psagoku/goku female. Apparent CLP were detectable in A, but no developed CL were observed in B. Bar represents 100 µm.

 
First, four of six Psa+/+ females carrying ovaries from a Psagoku/goku female became pregnant and carried the embryos to term at least once (Table 1Go). All pups were derived from the ovaries of the Psagoku/goku females because they expressed the lacZ gene (data not shown). Moreover, CLP were detected in the ovaries of Psagoku/goku females transplanted into Psa+/+ recipients (Fig. 4AGo). In the reciprocal experiments (donors of ovaries: Psa+/+; recipients: Psagoku/goku), females were treated with gonadotropins to stimulate ovulation, and vaginal plugs were detected in three of eight females after being housed with Psa+/+ males. None of these females, however, became pregnant (Table 1Go). The implanted ovaries derived from Psa+/+ animals were found to exist in the capsule of all eight Psagoku/goku females, but CLP-like eosinophilic morphologies were not detectable (Fig. 4BGo).

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, Student’s 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. 5Go). 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. 5Go). A one-way ANOVA test of the PRL levels on days 0.5–1.5, 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 0.5–1.5 (P = 0.0014–0.0176).



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Figure 5. Loss of Semicircadian Secretion of PRL in Psagoku/goku Females

A and B, Pattern of plasma PRL concentration of Psa+/+ (solid squares) and Psagoku/goku (open diamonds) on days 0.5–1.5 and 4.5, respectively. Each point on the graph represents the mean circulating PRL concentration for Psa+/+ mice (days 0.5–1.5, n = 3; day 4.5, n = 3) and Psagoku/goku females (days 0.5–1.5, n = 5, day 4.5, n = 2). Vertical lines for all data indicate SE. The black bars on the abscissa denote the dark period of the light/dark schedule, 2000–0800 h). *, P < 0.05; **, P < 0.01; Student’s t test (Psa+/+ vs. Psagoku/goku).

 
In contrast, the PRL surges were not apparent in Psagoku/goku homozygous females on days 0.5–1.5 and day 4.5 (Fig. 5Go). A one-way ANOVA test of the PRL levels on days 0.5–1.5 and day 4.5 did not reveal a significant difference among the group (F(6, 28) = 0.473, P = 0.8225; F(3, 4) = 1.463, P = 0.3509, respectively). A two-way ANOVA test of the levels between genotypes on days 0.5–1.5 and day 4.5 revealed a significant difference (F(1, 42) = 24.779, P < 0.001; F(1, 12) = 6.192, P < 0.05, respectively). Student’s t test indicated that the differences in values at 0200 h on day 1.5, 1000 h on day 1.5 and 0600 h on day 4.5 were significant between genotypes (P < 0.05, P < 0.01, and P < 0.05, respectively). Basal levels (intersurge) of PRL were, however, comparable in both genotypes (P > 0.05, Student’s t test). These data demonstrate that the semicircadian surges of PRL secretion are not in- duced during early gestation in Psagoku/goku females, even though the basal level of PRL secretion remains intact.

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. 6AGo). These CL were comparable in morphology to the CLP of the day-matched ovaries of Psa+/+ females (Fig. 6BGo). 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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Figure 6. Conversion of CL to CLP and Induction of Pregnancy in Psagoku/goku Females by PRL Injections or Pituitary Grafts

A and B, Sections of ovaries from PRL-treated Psagoku/goku female (A) and Psa+/+ female (B) mice on day 10.5 after cervical stimulation. Several eosinophilic CL, which are characteristic of activated CL, are detectable in panels A and B. C and D, Sections of ovaries on day 10.5 from a bilateral pituitary-grafted Psagoku/goku female and untreated Psagoku/goku female, respectively. A number of well developed CLP were detectable in panel C, but not in panel D. E, Gross morphology of the uterus on day 10.5 from the mouse shown in panel C. Developed decidua were detectable. F, The presence of embryos was confirmed by dissection of the uterus of panel E. Bars represent 100 µm (A–D), 1 cm (E), and 200 µm (F).

 
Accordingly, we next grafted ectopic pituitaries from Psa+/+ mice into Psagoku/goku females. Pituitary-grafted mice have been used as a model of hyperprolactinemia because the isografts of the pituitary under the kidney capsule secrete tonic PRL without inhibitory influence of hypothalamic DA (15, 16). By grafting ectopic pituitaries in rats or mice, serum PRL levels in the recipient reached levels comparable to that during pregnancy within 3 weeks (17, 18). Moreover, the elevated PRL level is capable of supporting luteal function in mice with hereditary PRL deficiency (13). We obtained a pregnant Psagoku/goku female displaying CLP in the ovaries after bilateral pituitary grafting (Fig. 6CGo). Moreover, this mouse exhibited implantation, and the intact embryos were detected in the uterus on day 10.5 (Fig. 6Go, E and F). In contrast, an untreated Psagoku/goku female showed neither the formation of CLP (Fig. 6DGo) nor the onset of uterine implantation (data not shown).

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. 7Go). 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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Figure 7. Response of PRL Secretion and PRL Gene Expression to Dopamine in Pituitary Cells from Psagoku/goku Females

A, Western blot analysis for PRL in the cultured medium of pituitary cells. B, RT-PCR analysis for PRL gene expression in cultured pituitary cells. An aliquot of the reverse transcription reaction was used for serial dilutions of 1:5 and 1:25. DA (-) and DA (+) represent samples from cultures without and with DA, respectively. +/+ and goku/goku represent samples from Psa+/+ and Psagoku/goku females, respectively. In both experiments, the intensity of the signals was decreased by DA exposure for both genotypes.

 
PRL secretion was examined by analyzing the immunoreactivity against an anti-PRL antibody in the medium using Western blot analysis. PRL immunoreactivity decreased in both genotypes when exogenous DA was added to the culture media (Fig. 7AGo).

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. 7BGo).

These data demonstrated that PRL release and expression in pituitary cells regulated by DA are not affected in Psagoku/goku females.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Regulatory Pathways in which Psa Functions for Mating-Induced PRL Secretion
The results of the present study indicate that a lack of the semicircadian surges of PRL secretion in response to mating results in infertility in Psagoku/goku females. We hypothesize that the regulatory pathways for the semicircadian surges are impaired in Psagoku/goku females.

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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mice
Psagoku/goku females were generated by intercrosses of heterozygous mice described previously (4). The 20th generations of the heterozygous mice were used as foster parents to produce homozygous mice. Mice were maintained in cages with sawdust at constant temperature (23 ± 1 C) with chow and water ad libitum.

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 Giemsa’s solution. Estrous cycle stages were determined as described previously (1).

Collection of Oocytes and Blastocysts
Female mice (8–12 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 4–6 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 8–12 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 Bouin’s 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 8–12 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. 5AGo); and 2) a quick decapitation at each time of examination (shown in Fig. 5BGo). 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 1–2 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 Student’s t test, one-way ANOVA, or two-way ANOVA. Appropriate pairwise comparisons between pairs of groups were carried out using Fisher’s 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.


    ACKNOWLEDGMENTS
 
The authors would like to thank the National Hormone and Pituitary Program (Torrance, CA) for providing the RIA materials for mouse PRL, rat FSH, and rat LH, and Dr. H. Kawano (Tokyo Metropolitan Institute for Neuroscience, Fuchu, Japan) and Dr. T. Mori (University of Tokyo, Tokyo, Japan) for their informative advice during the course of this work. The critical review of the manuscript by Dr. S. Hayashi of Yokohama City University (Yokohama, Japan) is also greatly appreciated.


    FOOTNOTES
 
Address requests for reprints to: Takashi Takeuchi, Ph. D., Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194-8511, Japan. E-mail: take{at}libra.ls.m-kagaku.co.jp

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. Back

Received for publication May 31, 2000. Revision received January 22, 2001. Accepted for publication January 29, 2001.


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