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1 Department of Biochemistry and
2 Division of Disease Models, Center for Neurological Disease and Cancer, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
3 Department of Health Science, Faculty of Psychological and Physical Sciences, Aichi Gakuin University, 12 Araike, Iwasaki-cho, Nisshin, Aichi 470-0195, Japan
4 Division of Basic Molecular Science and Molecular Medicine, School of Medicine, Tokai University, Bohseidai, Isehara, Kanagawa 259-1193, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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MK displays diverse physiological activities, such as promotion of the growth, survival and migration of various cells (Kadomatsu et al. 1988; Muramatsu et al. 1993; Muramatsu 2002). MK is strongly expressed in the midgestation period of mouse embryogenesis (Mitsiadis et al. 1995b). MK expression is weak in adults but increases during tissue repair, inflammation and carcinogenesis (Horiba et al. 2000; Muramatsu 2002). Evidence has accumulated of important roles for MK in vertebrate development. For example, anti-MK antibody inhibits tooth development and glomerular development in vitro (Mitsiadis et al. 1995a; Vilar et al. 2002). The involvement of MK in epithelial mesenchymal interactions has also been demonstrated by using a blood vessel model (Sumi et al. 2002). The injection of MK mRNA into Xenopus blastomeres promotes neurogenesis and suppresses mesoderm differentiation (Yokota et al. 1998). Down-regulation of a molecular species of MK in a zebra fish, which has two different MK molecules generated by gene duplication, results in malformation of the floor plate (Schafer et al. 2005). However, mice deficient in the MK gene (Mdk) do not show developmental abnormalities, except for a delay in the postnatal development of the hippocampus (Nakamura et al. 1998).
MK has 45% sequence identity with PTN, also called heparin-binding growth associated molecule (HB-GAM) (Rauvala 1989; Li et al. 1990; Rauvala & Pen 1997). PTN has functions similar to those of MK, and the receptor and signaling systems of the two factors are closely related (Muramatsu 2002). The receptors for MK consist of oversulfated proteoglycans such as receptor-type protein tyrosine phosphatase, low density lipoprotein receptor-related protein and 
4ß1 or 6ß1 integrins, also possibly anaplastic lymphoma tyrosine kinase (ALK) (Maeda et al. 1999; Muramatsu 2002; Muramatsu et al. 2004). The receptors for PTN are likely to include the molecules mentioned above and syndecan-3 (Raulo et al. 1994; Maeda & Noda 1998; Muramatsu 2002). During embryogenesis, PTN is also expressed in many tissues, and the expression of MK and PTN often overlaps (Mitsiadis et al. 1995b). Evidence obtained in vitro also indicates that PTN is involved in developmental processes. As an example, PTN induces the differentiation of renal tubular epithelial cells (Sakurai et al. 2001). Furthermore, PTN induces the differentiation of dopaminergic neurons from progenitor cells (Jung et al. 2004). However, mice deficient in the PTN gene (Ptn) exhibit developmental abnormalities only in the nervous system; the cortical layer of the cerebrum becomes thicker in these mice (Amet et al. 2001; Hienola et al. 2004). We considered that the lack of developmental abnormalities in non-neurological tissues of Mdk-deficient or Ptn-deficient mice is due to compensation of the two factors.
To understand the role of MK and PTN in development and reproduction, we produced mice deficient in both factors (DKO mice) and analyzed their phenotypes as compared to wild-type mice (WT mice). DKO mice exhibited auditory deficits, which were dealt with elsewhere (Zou et al. 2006). This investigation consists of two parts, general deficits of DKO mice (reduced birth rate and small body weight) and their female infertility. Since the latter was the most significant phenotype of DKO mice, we analyzed the mechanism causing female infertility and found multiple deficits in the activities of female reproductive organs such as oocyte maturation and estrous cycle. Therefore, this investigation revealed important roles of MK, PTN and their signal reception systems in female reproductive activities.
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Results |
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Starting with Mdk-deficient mice (Mdk–/–) and Ptn-deficient mice (Ptn–/–), we produced DKO mice, which were doubly deficient in Mdk and Ptn. Although Mdk–/– were generated previously (Nakamura et al. 1998), Ptn–/– used for this study were newly created (Fig. 1A). Southern and Northern blot analyses confirmed the deficiency of Ptn and PTN mRNA in Ptn–/– (Fig. 1B,C). DKO mice were generated by crossing hetero/hetero mice (Mdk+/–, Ptn+/–) with each other. The hetero/hetero mice were obtained by crossing Mdk–/– Ptn+/+with Mdk+/+Ptn–/–. DKO mice were confirmed to be deficient in both Mdk and Ptn (Fig. 1B), and were also deficient in the mRNA of PTN and that of MK (Fig. 1C).
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The number of female DKO mice was seven, while that of male DKO mice was 12. Although the number of mice was small to draw a definitive conclusion, the result may indicate that female DKO embryos were more severely impaired than the male ones. There was no distortion of sex ratio in single knockout mice.
Although at the time of birth there were no significant differences in their weights between DKO mice and WT mice, the DKO mice grew to be much smaller than WT mice (Fig. 2A). Three- to 4-week-old DKO mice weighed about half of what WT mice would weigh at the same age. While DKO mice grew steadily, even the adult mice weighed 75–80% of the corresponding WT mice (Fig. 2B). The difference was observed both in males and in females. Single knockout mice showed no apparent weight difference as compared to WT mice; their weight was on average between 110 and 90% of that of WT mice at any given age (our unpublished observations). High-calorie feeding brought about a greater increase in weight among WT mice than did ordinary feeding, but no apparent effect was observed in DKO mice (Fig. 2B).
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We also found that the spontaneous locomotive activity of 1-month-old DKO mice was 50–60% of that of WT mice (Fig. 2C). Both male and female mice were affected. Although the difference in activity decreased with age, the activity of 3-month-old male DKO mice was still about 80% of that of the WT mice (Fig. 2C). The DKO mice exhibit severe auditory deficits (Zou et al. 2006). Whether the auditory deficits influence the locomotive activity remains to be clarified, although to the best of our knowledge no correlation has been reported between them.
High frequency of female sterility in DKO mice
Female DKO mice had difficulty in yielding offspring. Among 24 female DKO mice examined, 19 (79%) were determined to be sterile. Male DKO mice were fertile, and no difference in reproductive capability was found between WT and DKO mice. Female Mdk–/– mice and Ptn–/– mice were fertile as reported previously (Nakamura et al. 1998; Amet et al. 2001).
We used four additional female DKO mice to examine whether superovulation would improve female fertility in these mice. They were mated with WT mice after superovulation at 8 weeks after birth, but at the first mating no plug was formed in any mouse. Two weeks thereafter, these DKO mice were mated with WT mice after the second hormone administration, but again no plug was formed in any animal. After another two weeks, the DKO mice were mated with WT mice after the third hormone administration. This time, one of four formed a plug and became pregnant. Thus superovulation did not significantly increase the fertility of female DKO mice. Among four DKO mice, the vaginal abnormality (described below) was present in one mouse, which failed to form a plug even after the three trials.
Expression of MK and PTN in the ovary and poor maturation of ova in DKO mice
As a step toward understanding the mechanism underlying the sterility of female DKO mice, we analyzed the expression of MK and PTN mRNAs in the ovary using the semiquantitative real time PCR method (Fig. 3A) and the in situ hybridization method (Fig. 3B). In the ovary of newborn mice, MK and PTN mRNAs were strongly expressed. MK mRNA decreased 2 weeks after birth, while PTN mRNA decreased 1 week after birth. Both MK and PTN were expressed strongly in the follicular epithelial cells and the granulosa cells 2 weeks after birth; they were also expressed weakly in both the cortex and medulla. Endothelial cells of blood vessels did not strongly express MK as revealed by double immunostaining of the frozen sections with anti-CD31 and anti-MK (data not shown), while endothelial cells in the injured blood vessels are known to express MK strongly (Muramatsu 2002). Concerning localization of MK and PTN mRNAs at other ages, both were broadly expressed 1 day after birth, and 1 week after birth PTN expression was similar to that in 2-week-old embryos, while MK expression was more diffuse (data not shown). In 4-week-old and 8-week-old embryos, both MK and PTN mRNAs were localized similarly to that seen in 2-week-old embryos (data not shown).
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Because of the expression of MK and PTN in the ovary, it was conceivable that the defect in the maturation of follicles was due to a lack of the growth factors in the ovary. However, the possibility remained that an abnormality in some other part of the body caused the problem. To examine possible defects in gonadotropins, we employed a DNA microarray analysis and compared the mRNA expression of follicle stimulating hormone ß subunit, luteinizing hormone ß subunit, and glycoprotein hormone subunit, between the brain including the pituitary of 2-week-old female WT mice and that of DKO mice. The levels of mRNA expression were good for all three genes and there were no significant differences between WT and DKO mice. We also examined the level of gonadotropin releasing hormone mRNA in the brain of 3-week-old mice by RT-PCR and found no difference between WT and DKO mice (data not shown).
Expression of MK and PTN mRNA in the uterus and their change with the estrous cycle
The expression of MK and PTN mRNA in the uterus was analyzed using the semiquantitative real time PCR method (Fig. 4A) and the in situ hybridization method (data not shown). Both mRNAs were strongly expressed in the uterus of newborn mice, and the degree of expression decreased in 1-week-old mice, although the expression continued even in the uteri of adult mice. In 2-week-old mice, MK mRNA was located in the lining and glandular epithelium and the lamina propria of the endometrium (data not shown). Immunohistochemical staining using anti-MK antibody revealed that at 1 week after birth, MK was present in the lining and glandular epithelium and the lamina propria of the endometrium, and 4 and 8 weeks after birth, the expression became gradually limited to the simple columnar epithelium and the glandular epithelium (data not shown). PTN mRNA was found to be strongly expressed in the glandular epithelium of the uterus glands (data not shown).
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Altered estrous cycle in DKO mice
To elucidate the role of MK and PTN in the uterus, we compared the estrous cycles of WT and DKO mice. Fig. 4D shows typical results of smear tests in WT and DKO mice. DKO mice also had estrous periods, similar to the WT mice. However, the estrous period in the WT mice spanned 2–3 days of the 6-day cycle, whereas in DKO mice, the diestrus and proestrus periods were longer and the estrus period lasted only for 1 day in the 6-day cycle (Table 2). It is possible that MK and PTN act to prolong the estrus period in the uterus.
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During vaginal smear testing, we found that many DKO mice had vaginas furcate at the opening (Fig. 5A). Histological examination revealed that these mice had narrow vaginas (Fig. 5B), which were frequently divided into two parts (Fig. 5C). Vaginal malformations at the opening occurred in 8 of 19 DKO mice examined. WT mice, Mdk–/– or Ptn–/– exhibited no such malformations. All DKO mice with an abnormal vagina were sterile, without any sign of a plug. On the other hand, 7 of 11 DKO mice with a normal vagina were sterile.
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In the vagina of 2-month-old mice, the thickness of the epithelium was not different between WT (2.35 mm2) and DKO mice (2.33 mm2) (Fig. 5C). However, in DKO mice, the multilayered structure of the epithelium was disordered, the nuclear size of the epithelial cells was not even and condensed chromatins were frequently seen (Fig. 5C). Thus, the vaginal epithelium of DKO mice exhibited abnormalities soon after maturation.
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Discussion |
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First, the birthrate of these mice was one third of that expected from Mendelian segregation, when Mdk+/– Ptn+/– were crossed with each other (Table 1). Single knockout mice (Mdk–/– Ptn+/+ or Mdk+/+Ptn–/–) were born at nearly the expected number in these crosses (Table 1); however, WT mice (Mdk+/+Ptn+/+) were born with a slightly higher ratio than expected (Table 1). Mdk–/– Ptn+/+ and Mdk+/+Ptn–/– were found to be born at 71% and 75% expected values, respectively, as compared to the birthrate of WT mice. However, the low birthrate of DKO mice (27% compared to WT mice) cannot be explained by the additive effects of the two deficiencies (around 50%). Both MK and PTN are expressed in many embryonic tissues and the sites of their expression often overlap (Mitsiadis et al. 1995b). We suggest that both MK and PTN are involved in important steps in embryogenesis, and the lack of one factor is largely compensated for by the presence of the other factor. That DKO mice were born albeit at low frequency is probably explained by the fact that signaling by MK and PTN involves PI3 kinase and ERK (Muramatsu 2002), which are involved in signaling by other factors.
Second, DKO mice were smaller than WT mice. However, until 1 week after birth, no significant difference was observed between them. From 2 to 4 weeks after birth, DKO mice grew much more slowly than WT mice and 3–4 weeks after birth, the weight of DKO mice was only about half that of WT mice. Since all these mice were weaned 4 weeks after birth, the weight difference cannot be explained by a difference in weaning. We also noticed that a high-calorie diet increased the weight of WT mice, but not that of DKO mice. Histological examination of DKO mice revealed poor growth of the mucosal epithelium in the colon, but not in other organs of the digestive tract. The colon is involved in the absorption of water and minerals. However, it is not clear whether abnormality in the colon can fully explain the poor growth of DKO mice. In addition, a systemic cause might be present. Indeed we found reduced spontaneous activity in DKO mice. Changes in feeding behavior can contribute to the weight difference. Indeed, hypothalamic syndecan 3, which binds to pleiotrophin and midkine, has been proposed to modulate feeding behavior (Reizes et al. 2001).
The low birthrate, low body weight and high incidence of female sterility all mean that DKO mice have little capability to reproduce. In other words, either MK or PTN is required for mice to breed properly. MK and PTN are present from fish to man, though Drosophila lacks them (Muramatsu 2002). MK and PTN are believed to have been created by the duplication of a primordial gene, since the genes adjacent to either ends of both the MK and PTN genes encode diacylglycerol kinases and muscarnic acetylcholine receptors (Muramatsu 2002). It is possible that at a certain stage of evolution, the ancestral gene of MK and PTN emerged to facilitate complex processes of reproduction and development.
The degree of female infertility among DKO mice was severe. Seventy-nine percent of the females were sterile. As fully described in the case of pregnancy after enforced ovulation, even fertile DKO females usually attained pregnancy after a certain period of mating. In order to find out the reason for female infertility, we first examined the ovaries, because some studies have reported the expression of MK/PTN in the ovaries of other species. A large quantity of MK and PTN is present in bovine follicular fluid (Ohyama et al. 1994) and MK is expressed in the granulosa cells of the rat ovary (Karino et al. 1995) and also in the human ovary (Nakanishi et al. 1997). Furthermore, MK promotes in vitro maturation of bovine oocytes (Ikeda et al. 2000). We found that the number of mature antral follicles was lower in DKO mice than in WT mice, while the total number of follicles was the same in both mice. The number of ovulated ova after the administration of gonadotropin was also low in DKO mice. Therefore, we concluded that follicles poorly matured in DKO mice, constituted a major reason for female sterility. As we report here, both MK and PTN were expressed in granulosa cells. It is conceivable that MK and PTN secreted from granulosa cells promote the maturation of follicles.
Vaginal abnormality at the opening was found in 42% of DKO mice. All DKO mice with a vaginal abnormality were sterile and did not exhibit a vaginal plug, which is the sign of a successful mating. Thus, another obvious reason for sterility is the inability to mate successfully due to vaginal abnormality. Vaginal opening takes place at 5 weeks after birth in mice. It is induced by oestradiol and is associated with apoptosis (Rodriguez et al. 1997). Since mature follicles are the primary source of oestradiol, it is likely that poor maturation of follicles in DKO mice is a reason for the abnormality in vaginal opening.
We also found that the estrus period of the DKO mice was short. Interestingly, as reported here, expression of MK and PTN in the uterus changed dramatically in relation to the estrous cycle. It is possible that high levels of MK or PTN during estrus contribute to the maintenance of the period. We noted that DKO mice were difficult to mate even when vaginal abnormality was absent. This might be due to the short estrus period or hormonal imbalance due to the poor maturation of follicles. In relation to the uterus, the capability for implantation in DKO mice is an interesting subject. In order to examine this function of the uterus, blastocysts of a WT mouse should be transferred to the uterus of a DKO mouse; pseudopregnancy should be induced in the DKO mouse after it has been mated with a vasectomized male WT mouse following hormonal treatment. Since DKO mice seldom mate, such an experiment has not yet been successful.
PTN as well as MK are involved in angiogenesis (Laaroubi et al. 1994; Muramatsu 2002). However, the density and the size of capillaries, which were stained by anti-CD31, in frozen sections of the ovary were not significantly different between WT and DKO mice 6 weeks after birth (our unpublished observations). Probably the persistence of other angiogenic factors compensated for the loss of MK and PTN. Furthermore, it is possible that the angiogenic activity of these factors plays a major role under pathological conditions such as tumor progression. In any event, the functional deficits of the ovary in DKO mice are not due to abnormality of angiogenesis in the organ.
As described above, the high incidence of female sterility in DKO mice was caused by a combination of poor follicular maturation, vaginal malformation and probably other factors including a short estrus period. Among the three abnormalities, the first is the most important, as poor maturation of follicles may be one of the causes of the latter two abnormalities due to low levels of estrogen secreted by poorly matured follicles. Abnormality in the vaginal epithelium of DKO mice can be similarly explained. Ovary transplantation is a definitive experiment to demonstrate that the principal cause of female sterility was the deficit in the ovary, but remains to be performed.
An obvious question is whether systemic effects caused by the lack of MK and PTN are responsible for poor follicular maturation or the absence of the factors in the granulosa cells of the ovary is responsible. An important point is whether gonadotropin levels were altered in DKO mice. We observed that the levels of mRNAs for follicle stimulating hormone and luteinizing hormone were not significantly different between the brains from 2-week-old WT and DKO mice as examined by DNA microarray analysis. The level of mRNA of gonadotropin-releasing hormone, which regulates not only the secretion but also the transcription of gonadotropins (Gharib et al. 1990), did not differ between the brains of 3-week-old WT and DKO mice either. Furthermore, changes in gonadotropin levels should affect both male and female mice. Thus, it is likely that the female sterility was caused by the lack of MK and PTN in female reproductive organs, not by a lack of the factors in the brain.
The present result is also interesting from the view point of the role of glycosaminoglycans in the human reproductive system. Both MK and PTN recognize oversulfated structures in heparan sulfate or in chondroitin sulfate, and the recognition is essential for their signaling (Raulo et al. 1994; Rauvala & Pen 1997; Maeda et al. 1999; Vacherot et al. 1999; Muramatsu 2002; Sugahara et al. 2003; Polykratis et al. 2005). Therefore, oversulfated structures in glycosaminoglycans can be inferred to be important in functions of female reproductive organs.
In conclusion, midkine, pleiotrophin and their signal reception system, including glycosaminoglycans, in the female reproductive organs play important roles in the control of reproductive activities such as the estrous cycle and oocyte maturation.
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Experimental procedures |
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The Ptn targeting vector was constructed from a basic targeting vector (Igakura et al. 1998) with MC1neo (polyoma virus thymidine kinase gene promoter and neomycin resistance gene), PGK (phosphoglucokinase gene promoter) and DTA (diphtheria toxin fragment A gene) and fragments of Ptn genomic DNA. A 10.5 kb genomic DNA clone containing Ptn was isolated from a genomic DNA library of mouse strain 129/Sv. A 0.5 kb fragment from the SmaI site in exon 3 to the SalI site in intron 3 was replaced by MC1neo in the reverse orientation relative to Ptn transcription (Fig. 1).
Aliquots of 12 µg of linearized targeting construct DNA were electroporated into 1 x 107 D3 ES cells, which were derived from 129/Sv mice. The selection of homologously recombined cells and their injection into blastocysts were performed as previously described (Igakura et al. 1998). The male chimeric mice were mated with C57BL/6 mice (SLC, Japan). Mice with the recombined genome were backcrossed to C57BL/6 mice for more than 11 generations. The heterozygotes were mated with each other to yield null mutants (Ptn–/–).
Mice lacking Mdk
Mdk–/– were produced as previously described (Nakamura et al. 1998) and mated with C57BL/6 mice. The heterozygotes were backcrossed to C57BL/6 mice for more than 11 generations.
Production of DKO mice
DKO mice were produced by crossing Mdk+/–Ptn+/– with each other, crossing Mdk–/–Ptn+/– with each other, or crossing Mdk+/–Ptn–/– with each other. In the analysis of each phenotype, DKO mice obtained from the Mdk+/–Ptn+/– cross were used initially, and then DKO mice obtained by other crossses were used to confirm the result by increasing the number of cases analyzed. The WT mice used to compare the phenotype with DKO mice were derived from the cross of Mdk+/–Ptn+/–. In experiments to examine expression patterns of MK or PTN, they were commercially available C57BL/6 mice (SLC Japan).
Southern and Northern blot analyses
Southern and Northern blot analyses were performed as previously described (Igakura et al. 1998). In the Southern blot analysis to detect deletion of Ptn, the homologously recombined genomic DNA gave a 4.1 kb band, while the wild-type DNA gave a 8.0-kb band, after digestion with PstI and hybridization with an external probe (0.5 kb) (Fig. 1B). In the Northern blot analysis, total RNA prepared from the kidney of 3-month-old mice was electrophoresed and transferred on to a nylon membrane, and then hybridized with the [32P]-labeled probe corresponding to a fragment of MK cDNA (nucleotide number 1–420) (Tomomura et al. 1990) or PTN cDNA (nucleotide number 270–773) (Merenmies & Rauvala 1990).
Polymerase chain reaction
PCR was performed as previously described (Igakura et al. 1998). For the screening of mice with homologously recombined DNA at the Ptn locus, Primers I and II were used. Primers I, forward (in Intron 2, which is between Exon 2 and 3), 5'-CACACATGATTTAGTAGCCTTAGC-3' reverse (in Exon 3), 5'-GCTCCAAACTGCTTCTTCCAGTTG-3'. The wild-type DNA gave a 400-bp band, while the recombined DNA gave no band. Primers II, forward (in Exon 3), 5'-AGGTGAAAAAGTCTGACTGTGGAG-3' reverse (in Neo), 5'-GCGAGGATCTCGTCGTGACCCATG-3'.The wild-type DNA gave no band and the recombined DNA gave a 400-bp band. Primers to detect homologous recombination at the Mdk locus were previously described (Nakamura et al. 1998). For determination of the level of gonadotropin-releasing hormone, the primers used were 5'-ATGATCCTCAAACTGATGGC-3' and 5'-CTACATCTTCTTCTGCCTGG-3'.
Preparation of RNA and semiquantitative real time RT-PCR
Total RNA from the uterus was prepared as previously described (Igakura et al. 1998) and total RNA from the ovary was isolated using Isogen (Wako, Tokyo, Japan) according to the manufacturer's directions. First-strand cDNA was synthesized from total RNA using a SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA).
Real time quantitative PCR was performed using the TaqMan Gene Expression Assays and 7500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. The expression of each gene was normalized to that of ß-actin.
DNA microarray analysis
At day 14 after birth, total RNA from whole brain of a WT or DKO female mouse was isolated with an RNeasy column (QIAGEN Sciences, Tokyo, Japan) according to the manufacturer's directions. The gene expression profile was analyzed with a DNA microarray using a CodeLink Expression Bioarray System (Amersham Biosciences) provided by Kurabo (Osaka, Japan).
Histology and immunohistochemistry
Tissues were fixed in 4% paraformaldehyde, dehydrated and embedded in paraffin. Sections were cut at a thickness of 5 µM and serial sections were mounted on slides, dried overnight and stored in an airtight box. These sections were stained with hematoxylin and eosin (HE). Sections were also treated with 0.3% H2O2 in Dulbecco's phosphate-buffered saline (PBS) for 30 min at room temperature, blocked with 1% bovine serum albumin in PBS for 20 min, and treated with rabbit anti-mouse MK antibody (Muramatsu et al. 1993) at 4 °C overnight, followed by horseradish peroxidase-labeled goat anti-rabbit IgG (Jackson ImmunoReseach, West Grove, PA, USA). The staining was visualized with a diaminobenzidine tetrahydrochloride (DAB) kit (Nichirei Corp. Tokyo, Japan).
In situ hybridization
PTN cDNA (nucleotides 270–773) (Merenmies & Rauvala 1990) was inserted into Bluescript KSII+ (Stratagene) at the BamHI site. Sense and anti-sense probes were prepared using appropriate combinations of restriction enzyme-digested templates and RNA polymerases (sense, BamHI and T7 RNA polymerase; anti-sense, HindIII and T3 RNA polymerase). The probe for MK was prepared from MK cDNA (nucleotides 1–420) (Tomomura et al. 1990). Digoxigenin (DIG)-II-UTP-labeled single-stranded RNA was prepared using a DIG RNA labeling kit (Boehringer Mannheim, Mannheim, Germany). The RNAs were treated with 40 mM NaHCO3 and 60 mM Na2CO3, pH 10.2, for 50 min at 60 °C to generate shorter fragments with an average length of 150 bases. After ethanol precipitation, the products were dissolved in 50 µL of deionized formamide and stored at –80 °C prior to use. In situ hybridization was performed as previously described (Igakura et al. 1998).
Fertility analysis
Each female mouse was mated with a fertile male during a period of at least 3 months, the male being replaced every 2–3 weeks. In cases where pregnancy was not observed after the period, the female mouse was considered to be sterile.
Induction of superovulation
WT or DKO female mice (8–10 weeks old) were injected intraperitoneally with 5 I.U. of pregnant mare serum gonadotropin (PMG; Teikokuzoki, Tokyo, Japan), followed 48 h later by 5 I.U. of human chorionic gonadotropin (hCG; Teikokuzoki, Tokyo, Japan). Twenty hours after hCG administration, a cumulus mass containing the mature ova was shed from the ampulla of the oviduct into M16 medium (Sigma) and subsequently transferred into M16 medium containing hyaluronidase (1 mg/mL) and incubated for 5 min at room temperature. Ova free from the adhering cells were counted. When the formation of a plug was examined, mice were mated immediately after the administration of hCG.
Isolation of epididymal spermatozoa and in vitro fertilization (IVF)
Mouse spermatozoa were isolated from the cauda epididymides of 2 WT males (14 weeks old) of proven fertility. Spermatozoa were released from 4 epididymides into 200 mL of TYH medium under paraffin oil, and the spermatozoa were allowed to capacitate by incubating the dish for 1.5 h at 37 °C.
DKO and WT females (6–8 weeks old) were induced to superovulate, as described above. The IVF technique was performed using a method modified from that described by Tada et al. (1990) Spermatozoa were diluted directly into the fertilization drops of medium containing oocytes surrounded by cumulus cells. The final sperm concentration was 1–2 x 106 spermatozoa/mL. After incubation for 6 h at 37 °C, only oocytes exhibiting a normal morphology were picked up and washed with M16 medium (Whittingham 1971) to remove attached spermatozoa and cumulus cells, and incubated on M16 medium on a well of a Terasaki microtest plate (No. 5260, Nunc Inc.) covered with paraffin oil for an additional 20 h. The number of oocytes that developed to the 2-cell stage was recorded.
Vaginal smear testing
Vaginal smears were obtained with an ordinary pipette, the tip of which had been flamed to a smooth and reduced aperture. A few drops of PBS were drawn into the pipette, introduced into the vagina, and retraced into the pipette. The fluid was transferred to a slide, dried and fixed in methanol for 2 min, and stained with Giemsa solution for 30 min
Behavioral analysis
Spontaneous locomotive activities were recorded automatically in a computerized activity-tracing chamber (Actimo-100, S/N 0000015, Eikou Science,Tokyo, Japan) for 24 h. The mice were kept on a 12-h light–dark cycle at a constant temperature (23 ± 1 °C).
Statistical analysis
Data are presented as the mean ± SEM. Statistical significance was assessed using Student's t-test.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: tmurama{at}dpc.aichi-gakuin.ac.jp
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References |
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Received: 2 June 2006
Accepted: 20 September 2006
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) or WT (
) mice from 1 to 12 weeks (ordinary feed) or 1–16 weeks (high-calorie feed) after birth. For a genotype, 12 male or 15 female mice were examined for ordinary feeding. Four mice were examined for high-calorie diet feeding (diet containing 50% galactose).
(C) Comparison of spontaneous locomotive activities of DKO and WT mice. The number of mice used was four per gender of a genotype. The activity was recorded for 24 h and total activities were compared. Data are presented as mean ± SEM. **P < 0.01; *P < 0.05.
