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Haploinsufficiency of COUP-TFII in Female Reproduction *Corresponding authors, Sophia Y. Tsai, Ph.D. and Ming-Jer Tsai, PhD, Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, Tel: 713-798-6251(SYT), 713-798-6253 (MJT), Fax; 713-798-8227, stsai@bcm.tmc.edu, mtsai@bcm.tmc.edu  The publisher's final edited version of this article is available free at Mol Endocrinol. See other articles in PMC that cite the published article. | |||||||||||||||||||||
Abstract The chicken ovalbumin upstream promoter transcription factor II, COUP-TFII, is a member of the Orphan nuclear receptor transcription factor family. Genetic ablation of COUP-TFII results in early embryonic lethality and demonstrates that this gene is required for cardiac and vascular development. Expression of COUP-TFII persists throughout postnatal life in various tissues including the female reproductive tract. However, the physiological function of COUP-TFII in female reproduction has not been extensively analyzed. Here, we provide phenotypic evidences that haploinsufficiency of COUP-TFII in mice demonstrates an important role of COUP-TFII for normal female reproduction. COUP-TFII +/− females show significantly reduced fecundity, irregular estrus cycles, delayed puberty and retarded postnatal growth. Analysis of the reduced fertility revealed that although ovarian function was normal with respect to ovulation, the ovaries have reduced ability to synthesize progesterone in response to exogenous gonadotropins. This reduction is due to the reduction of the expression of steroidogenic enzymes important for P4 synthesis and the reduction of vascularization in COUP-TFII heterozygotes. Analysis of uterine function demonstrated a reduced response to an experimentally induced decidual cell reaction indicating that the ability of the uterus to support embryo implantation was reduced. Taken together, our data shows global impact of gene dosage effects of COUP-TFII on female postnatal life and indicates requirement of COUP-TFII in normal female reproduction, in particular for uterine endometrial functions during the peri-implantation period. | |||||||||||||||||||||
Chicken ovalbumin upstream promoter transcription factors (COUP-TFs; NRF2) are orphan members of the steroid/thyroid hormone receptor superfamily (1). Two highly conserved COUP-TF genes, COUP-TFI and COUP-TFII, have been identified in mammals (1). Biochemical studies showed that these proteins bind promiscuously to direct repeat motifs, with preference of DR1 sequences and can negatively or positively regulate a large number of genes (2). The expression patterns of COUP-TFI and COUP-TFII are distinctly different. COUP-TFI is expressed at high levels in the neural system, while COUP-TFII is expressed in the mesenchyme of developing organs (3). Consistent with their expression pattern, deletion of the COUP-TFI in mice results in perinatal lethality with mutants exhibiting neuronal deficits (4–6). COUP-TFII null mutant mice die before E10.5 with angiogenic and cardiogenic defects (7). The embryo lethal phenotype of COUP-TFII impeded the analysis of the role of this transcription factor in the adult physiology, however COUP-TFII +/− mice exhibit several phenotypes. The analysis of the haploinsufficiency phenotype of the COUP-TFII +/− may shed light on the role of this orphan nuclear receptor in the adult. Haploinsufficiency that is caused by heterozygosity of a targeted null allele occasionally reveals the overall impact of a gene dosage effect as a traceable trait. Many COUP-TFII +/− mutants die before weaning suggesting a haploinsufficiency phenotype of the COUP-TFII gene (7). Upon analyzing the mating patterns of COUP-TFII +/− mice, we found that the COUP-TFII +/− females have reduced reproductive function. COUP-TFII +/− female mice exhibited reduced fecundity characterized by reduced litter size, and genotype dependent reduction in growth in the progeny. Since normal female reproduction requires functional coordination throughout female hypothalamohypophyeal-ovarian endocrine axis, as well as, an endocrinological link between ovary and uterus, subtle defects in multiple organs of this axis, could significantly alter the normal reproduction. Therefore, we investigated the ovarian and uterine physiology of the COUP-TFII +/− mice to determine the cause of this reduction in female fecundity. Here, we report on the phenotypic analysis of the COUP-TFII +/− mouse, specifically related to female ovarian and uterine function. Analysis of ovarian function was conducted by challenging COUP-TFII +/− and control mice with a superovulatory regimen of gonadotropins and assaying the ability of the ovary to ovulate and produce ovarian steroids. The COUP-TFII +/− mouse showed normal ovulation of oocytes but demonstrated a reduced capability for synthesizing sex steroid hormones. This reduction is due to the reduction of the expression of steroidogenic enzymes important for P4 synthesis and the reduction of vascularization in COUP-TFII heterozygotes. Analysis of uterine function in the COUP-TFII +/− mice was accomplished by assaying the response of the uterus to an endocrine induction of the decidual reaction. COUP-TFII +/− showed a reduced decidual cell reaction in response to the artificial stimulation indicating a defect in endometrial function. Analysis of the expression of COUP-TFII +/− in the endometrium demonstrated steroid hormone dependent changes in expression of COUP-TFII in the uterine endometrial stroma. This analysis demonstrates that COUP-TFII is important in the regulation of ovarian and uterine function and indicates that COUP-TFII is required for normal female reproductive functions. | |||||||||||||||||||||
COUP-TFII +/− female mice exhibit reduced fertility In maintaining the COUP-TFII mutant mouse colony, it was noticed that COUP-TFII +/− exhibited reduced fertility. In order to quantify the reduction of fertility the overall reproductive performance of the COUP-TFII +/− female mice was assessed by continuous mating. COUP-TFII +/+ male and COUP-TFII +/+ or COUP-TFII +/− female mice were continuously mated for 3 month. The numbers of litters and pups born were counted. The data is summarized in Table 1. Although the first litter did not show significant difference between COUP-TFII +/+ and COUP-TFII +/− females, the second and third litter from the COUP-TFII +/− females showed a significant decrease in litter size in contrast to the slight increase in COUP-TFII +/+ control. This result indicates that the heterozygous females exhibit abnormal fertility with increase in age.
Delayed puberty and growth retardation and abnormal estrus cycles in COUP-TFII +/− female mice In order to further ascertain the underlying cause of reduced fertility of COUP-TFII +/− females, we examined the timing of vaginal openings as an indication of onset of puberty. The results of this analysis are shown in Table 2. COUP-TFII +/− females exhibited slightly delayed vaginal opening at 30.6 days after birth, as compared to 27.9 days after birth in wild type females (Table 2). The delay in puberty may be a result of delayed growth of the COUP-TFII +/− mice. The postnatal growth of COUP-TFII +/+ and COUP-TFII +/− pups nursed by COUP-TFII +/+ and COUP-TFII +/− mothers was measured. The results of this analysis are shown in Figure 1. Significant growth retardation of COUP-TFII +/− was clearly shown in mutants as compared to the wild types controls nursed by either COUP-TFII +/+ or COUP-TFII +/− mothers (Fig. 1A). However, growth of COUP-TFII +/+ and COUP-TFII +/− were both significantly lower when nursed by COUP-TFII +/− as compared to COUP-TFII +/+ moms. Therefore, the growth retardation is a consequence of both the genotype of the pup, as well as, the ability of the mother to nurture the development of the pup.
In the analysis of the fecundity of the COUP-TFII +/− mice, the ability of the females to have normal reproductive cycles were monitored by recording the estrus cycles of 3 month-old females through daily inspection of vaginal smears. The diagnostic difference was facilitated by modified Papanicolou staining. A representative estrus cycle was schematized in Figure 1B. COUP-TFII +/+ females showed regular cyclicity while COUP-TFII +/− female mice showed prolonged diestrus or metestrus, or lack of obvious estrus phase. Thus, estrus cycles of female heterozygote were found to be irregular. In order to ensure the expression of COUP-TFII expression is reduced in the heterozygote, we isolated protein extracts from ovariectomized wild type and COUP-TFII+/− uteri and performed Western blot analysis using COUP-TFII specific antibodies. Fig 2 shows that the expression of COUP-TFII is greatly reduced in comparison to the control. The bar graph indicates that the expression levels of COUP-TFII are reduced approximately two fold in COUP-TFII+/− mice. Similar level of reduction in COUP-TFII expression was also shown in the ovaries of COUP-TFII +/− in comparison to the controls (data not shown). The above results indicate that COUP-TFII expression correlates with the loss a single allele in the COUP-TFII+/− mice.
Expression of COUP-TFII in female reproductive axis. The above analysis demonstrates that COUP-TFII +/− female mice shows abnormal reproductive cycles and delayed puberty. Part of this phenotype may be due to expression of COUP-TFII in the uterus since previous analysis has shown COUP-TFII is expressed in the adult female endometrium (8). However, in order to determine where else COUP-TFII may be acting, the expression of COUP-TFII in the female reproductive axis was assessed. This was accomplished utilizing a genetically engineered mouse in which the LacZ reporter had been knocked into the COUP-TFII gene (9). This mouse allows for the rapid and sensitive assay of the expression pattern of COUP-TFII. Histological sections from hypothalamus, pituitaries, ovaries and uteri from COUP-TFII-LacZ mice were analyzed by X-gal staining. The results of this analysis are shown in Figure 3. COUP-TFII expression, as seen by X-gal staining, was found in the ventromedial hypothalamus, VMH, (Fig.3A) while no specific staining was detected in the pituitary gland (Fig. 3B). In the adult ovary, strong staining was found in the theca cells, the layer surrounding the granulosa cells of ovarian follicles, but not detected in the granulosa cells. Weaker staining was detected in mesenchymal cells surrounding the corpus luteum (Fig. 3C). To ensure that the X-gal staining faithfully recapitulated the endogenous COUP-TFII expression, we used COUP-TFII specific antibody in immunostaining to assess the expression of COUP-TFII in the ovaries. Again strong staining was found in the theca cells, weak staining in the corpus luteum and no detectable staining in the granulosa cells (Fig 3D). The patterns of COUP-TFII expression using immunostaining are virtually identical to that shown using X-gal staining. Thus, X-gal staining could be used as an alternative assays to assess endogenous COUP-TFII expression patterns.
In the uterus of randomly cycling females, strong staining was evident in the myometrium, weaker staining was seen in endometrial stromal cells, while very weak positive staining was shown in glandular and luminal epithelial cells (Fig. 3E). There was no significant difference in COUP-TFII expression between mesometrium and anti-mesometrium portions of the uterus. COUP-TFII expression was assessed by LacZ activity during decidualization using gestational day 8 uteri, the decidualized cells in anti-mesometrium stained strongly positive (Fig. 3F). Since uterine gene expression is regulated by steroid hormones, we also examined the effects of exogenous steroid hormones and exogenous gonadotropins on COUP-TFII expression in the uterine endometrium (Fig.4). In ovariectomized females, COUP-TFII expression was high in endometrial stroma (Fig. 4A) while treatment with E2 for 48 hours drastically reduced COUP-TFII expression (Fig. 4B). Treatment with P4 prevented the reduction of COUP-TFII expression by E2 in the endometrial stroma (Fig. 4C). Note COUP-TFII expression in the myometrium was not obviously altered by exogenous steroid treatment. The expression of COUP-TFII after E2 and P4 treatment was similar to that observed in the non-pregnant uterine horn of a gestational Day 10 pregnant female mouse (Fig. 4D). We also examined COUP-TFII expression following the administration of exogenous gonadotropins (Fig. 4E-H). Twenty-four hours after PMSG injection, COUP-TFII expression in endometrial stroma was obviously decreased (Fig. 4E), while 48 hours after PMSG injection, COUP-TFII expression in the endometrium recovered partially (Fig. 4F). A further increase of COUP-TFII expression in the endometrial stroma was found 24 hours and 48 hours after hCG injection (Fig 4G-H, respectively).
The expression pattern of COUP-TFII in the uteri was further confirmed by immunostaining utilizing COUP-TFII specific antibodies. Strong staining was found in endometrial stromal cells as well as myometrium, while weaker staining was seen in glandular and luminal epithelial cells of the ovariectomized COUP-TFII+/− uteri (Fig 4I). The endogenous COUP-TFII expression patterns are indistinguishable from the X-gal expression pattern shown in Fig 4A. Normal ovarian function but reduced progesterone levels are found in COUP-TFII +/− females The above analysis determined that COUP-TFII is expressed in the hypothalamus, ovary and uterus. Therefore, the reduction in fertility in the COUP-TFII +/− may be due to deficiencies in any of these three organs. In order to determine whether the cause of the reduced fertilely may be due to a reduction in ovarian function, ovarian function was assayed by determining the ability of the ovaries to respond to a superovulatory regimen of hormones and determining the number of oocytes ovulated, as well as, the production of progesterone after superovulation, as well as, during pregnancy. The results of this analysis are shown in Table 3 and 4, respectively. There was no significant difference between COUP-TFII +/− and COUP-TFII +/+ in the number of oocytes ovulated in response to gonadotropins. Although the ability of the COUP-TFII +/− mice to ovulate is normal, perturbation in the level of progesterone, P4, production may contribute to the reduced fertility. As shown in Table 4, P4 levels were significantly decreased in COUP-TFII +/− females subsequent to exogenous hCG stimulation. Measurement of P4 and Pregnenolone (P5) levels in pregnant (gestational day 15) females, were shown to be lower in heterozygous females, although statistically not significant. Therefore, the ovaries of COUP-TFII +/− females are able to undergo normal ovulation but the reduction in fertility may be due to impairment of the corpus luteum of COUP-TFII +/− mouse ovaries to produce P4 in early pregnancy.
Reduction of expression levels of steroidogenic components critical to P4synthesis We showed that the predominant site of expression of COUP-TFII in the ovary is the theca interna, but not in the granulosa cells. Theca cells are responsible for synthesizing precursor C19 steroids in the preovulatory follicle in response to LH, and granulosa cells aromatize the precursor steroids in vivo under the regulation of FSH (13). In the corpus luteum, however, large luteal cells that are believed to be derived from the granulosa cells are capable of synthesizing P4 from circulating LDL (13). Thus, vascularization of corpus luteum upon ovulation is considered to be important to supply the luteal cells with the substrates to synthesize P4.Considering the importance of COUP-TFII in angiogenesis (7), we conducted microscopic examination of COUP-TFII+/− and wild type super ovulated ovaries to determine if vascularization is perturbed. Although no obvious microscopic abnormality could be detected in the COUP-TFII+/− ovaries, we noted the lumen of vessels in COUP-TFII +/− ovaries is not as dilated as the COUP-TFII +/+ control, suggesting that COUP-TFII +/− ovaries might not be well supplied with blood (Fig.5A). We then used endothelial specific marker, PECAM, in immunostaining to examine the vessel more closely. A considerable reduction in CD31 (PECAM-1) positive stained vessels was seen in the COUP-TFII +/− corpus luteum (Fig 5B and C), suggesting that vascularization is compromised in COUP-TFII +/− ovaries. This could contribute at least partly to the reduction of observable progesterone levels in super ovulated COUP-TF +/− female mice.
The impaired steroid production in the COUP-TFII+/− female mice could also be due to alteration in the expression levels of steroidogenic components critical for P4 biosynthesis, The impact of COUP-TFII haploinsufficiency on the expression levels of genes involved in steroid hormone biosynthesis in the ovaries of COUP-TFII+/− and wild type ovaries administered a superovulatory regimen of gonadotropin was assayed by quantitative reverse transcriptase PCR assays. As a control, we first examined the mRNA expression levels of COUP-TFII and showed that COUP-TFII mRNA level was reduced to 30% in the heterozygote in compared to the wild type controls (Fig 5D). Similarly, a significant reduction (40%) of cytochrome P450, family 11, subfamily a, polypeptide 1 (Cyp11a1), mRNA levels was detected (Fig 5D), In addition, steroidogenic acute regulatory protein (StAR), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (Hsd3b1) mRNA levels were also greatly reduced in COUP-TFII +/− female mice to less than 30% of the controls (Fig 5D). The reduction of mRNA expression of all these components important for P4 biosynthesis will no doubt also contribute to the observed reduction of P4 levels in the COUP-TFII+/− female mice. Defective uterine function in COUP-TFII +/− females COUP-TFII is expressed in the endometrium of the uterus, as well as, the endometrial decidual cells during pregnancy (Figs. 3 and 4). A defect in the ability of the endometrium to undergo decasualization may greatly affect reproductive performance. In order to assay the ability of the uterus to undergo decidualization, independent of hypothalamus and ovarian function, COUP-TFII +/− and COUP-TFII +/+ mice were challenged with artificial decidual stimulus. This stimulus consisted of administering a mechanical trauma, a burred needle scratch, to the uterus of ovariectomized mice treated with exogenous E2 and P4. In each animal, the left horn was given the mechanical trauma after hormonal treatment while the right horn was not traumatized and could serve as a control. After the experiments, wet weight of each horn was determined and the ratio of scratched versus control horn was calculated. As shown in Figure 6, uterus from COUP-TFII +/− female mice exhibited significantly reduced decidual cell reaction when compared to COUP-TFII +/+ mouse uteri. This above analysis indicates that the reduced fertility in COUP-TFII +/− mice may be in part due to a reduced ability of the endometrial stroma to undergo the necessary differentiation to support embryo implantation.
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The above data demonstrate that ablation of one allele of COUP-TFII significantly impacts the reproductive performance of female mice. This altered reproductive performance is seen as a delay in puberty and an age dependent reduction in litter size. Haploinsufficient phenotypes are rarely reported or result in subtle phenotypes. This phenomenon usually results if the gene in question is subject to imprinting or holds a rate limiting step in a pivotal pathway. The above COUP-TFII haploinsufficient phenotype is independent of the parent transmitting the mutant allele (data not shown). Therefore, imprinting of the COUP-TFII allele is not the cause of this phenotype. The expression level of COUP-TFII is reduced to about half as measured by Western blot analysis, corresponding to a loss of a single allele in the COUP-TFII+/−. The reduction of COUP-TFII expression could affect the function of the reproductive axis. Since female reproduction is regulated by interactions in the endocrine system at the level of the hypothalamus, pituitary, ovary and uterus and expression of COUP-TFII was found in hypothalamus (VMH), theca cells, and the uterine endometrial stroma, it is not surprising that haploinsufficiency reproductive phenotype is seen in COUP-TFII heterozygous females. There is abundant expression of COUP-TFII in the VMH of the CNS. Although the significance of VMH expression is not clear, several of the observed haploinsufficiency phenotypes (e.g. growth retardation, and delayed puberty) are consistent with the idea that the COUP-TFII +/− mouse may suffer from mild hypopituitarism. However, there was no significant decrease in IGF1 in heterozygotes (data not shown), which is indicative of other potential growth perturbation. COUP-TFII has been implicated to interfere with the function of the orphan nuclear receptor SF1 (10) SF1 null mice resulted in defective development of VMH (11). Rescue experiments using SF1 null mutants indicated SF1 functions in VMH to regulate late onset obesity (12). Thus, it might be interesting to speculate that COUP-TFII and SF1 reciprocally regulate growth in VMH. Future specific deletion of COUP-TFII could elucidate this aspect of COUP-TFII function. Although the role of COUP-TFII in the VMH is speculative, the role of COUP-TFII in the ovary was investigated. COUP-TFII mice did not demonstrate impairment in ovulation but did show impairment in the ability of P4 production. The reduced capability to synthesize P4 in response to exogenous gonadotropin suggests potential defects in the ovarian function of COUP-TFII +/−. Theca cells in the ovaries are responsible for synthesizing precursor C19 steroids in the preovulatory follicle in response to LH, and granulosa cells aromatize the precursor steroids in vivo under the regulation of FSH (13). In the corpus luteum, however, large luteal cells that are believed to be derived from the granulosa cells are capable of synthesizing P4 from circulating LDL (13). Thus, vascularization of corpus luteum upon ovulation is considered to be important to supply the luteal cells with the substrates to synthesize P4. The high expression of COUP-TFII in the theca cells, but not in granulosa cells, suggests COUP-TFII might be involved in a steroidogenic pathway or it might have a significant role during vascularization of the corpus luteum. Here we showed that vascularization as measured by PECAM immunostaining is perturbed in COUP-TFII +/− mice as compared to the controls. This might lead to inefficient delivery of LDL to the large luteal cells resulting in the reduced ability of the corpus luteum to synthesize P4. In addition, we also show that the expression levels of mRNA of CYP11a1, HSD3b1 and StAR are significantly reduced in the COUP-TFII+/− female mice. All these genes are important for the biosynthesis of P4. Thus, the reduction in the expression of theses genes will directly contribute to the observable reduced P4 levels in the COUP-TFII+/− female mice and the decrease in circulating P4 in pregnant COUP-TFII +/− females might result in the reduced ability of the uterus to support pregnancy. The reduced levels of P4 in early pregnancy may impact on COUP-TFII expression in the uterus during early pregnancy. Analysis of the expression of COUP-TFII in the uterus shows that E2 treatment caused drastic decrease of COUP-TFII expression in the endometrial stroma, while P4 treatment with E2 attenuates E2 action, restoring COUP-TFII expression in the stroma. COUP-TFII expression was low after treatment with PMSG, which induces ovarian E2 synthesis, and COUP-TFII expression in the endometrial stroma increased expression after hCG, which induces ovarian P4, are consistent with steroid hormone regulation in hormone treated ovariectomized females. The lower levels of P4 in the COUP-TFII +/− mouse combined with the loss of one COUP-TFII allele in the uterine stromal cell may be the partial cause of the haploinsufficient decrease in uterine function in these mice. However, reduced uterine stromal cell function was still observed after exogenous administration of P4 indicating that part of the haploinsufficient phenotype is specific to the uterus. During implantation, the endometrial stromal cells undergo a series of complex processes called decidualization (14,15). The process of decidualization involves concurrent cell proliferation, differentiation, as well as, angiogenesis. Based on our findings, it is evident that uterine function is significantly affected in COUP-TFII heterozygous females. The relatively high expression of COUP-TFII in endometrial stromal cells, the steroid regulation of COUP-TFII expression in the endometrial stroma and the reduced decidual cell reaction, all support this notion. Identification of the role of COUP-TFII in the process of decidualization may be discerned from the analysis of what other genes have been shown to regulate this process. Several targeted null mutant mice, including PR (16), Hoxa-10 (17) and 11 (18) exhibited defects in decidual cell reaction. Consistent with the notion that P4 is required for decidualization in human endometrium, decidual cell reaction was absent in PRKO female mice (16). One of the potential key genes that are regulated by P4 is Indian Hedgehog, IHH (8), a well-known morphogen. Hedgehog signaling is known to regulate cell proliferation and differentiation during embryonic development (19). The fact that IHH is expressed at pre-implantation endometrial epithelial cells and its expression is rapidly induced by P4 (8), further implicates its role in mediating PR function. Importantly, it has been demonstrated that COUP-TFII is regulated by Hedgehog signaling (20). One of the intriguing possibilities is that P4 regulates Indian Hedgehog (IHH) in the luminal epithelium, priming stroma cells for decidualization. COUP-TFII, a downstream target of IHH could mediate IHH signaling in the endometrium during peri-implantation (8). Importantly, both IHH and COUP-TFII had been implicated in angiogenesis (21, 7). It is our speculation that during artificially induced decidualization or repeated remodeling of endometrium after first conception, normal vascularization is moderately, but functionally significantly affected by COUP-TFII Taken together, phenotypic analysis using the haploinsufficiency model of COUP-TFII indicated several functions of this gene in the female reproductive tract. Combination of those haploinsufficiency phenotypes throughout the female reproductive axis might additively affect female reproductive function. Although use of this model might be limited for overall phenotypic analysis, it, perhaps, could reveal the global impact of a gene dosage effect (as in human genetic conditions) on female reproduction. We propose that COUP-TFII plays an important role in the endometrial stroma during the peri-implantation period. Detailed molecular and cellular mechanisms could be elucidated by conditional gene targeting approaches, such as using endometrial specific Cre recombinase. | |||||||||||||||||||||
Animals and phenotypic analysis Targeted null mutants of COUP-TFII were generated as described previously (7). The COUP-TFII-LacZ knock-in mice were generated by homologous recombination in ES cells and described, previously (9). These mice were maintained as a mixed genetic background (129S6/C57B6) under the care of the Center for Comparative Medicine at Baylor College of Medicine according to the institutional guidelines for the care and use of laboratory animals. Mating pairs of COUP-TFII +/+ male crossed with COUP-TFII +/+ females or COUP-TFII +/− females (12 weeks old) were continuously mated in a cage for 3 month, and numbers of newborn at birth were counted. Postnatal growth was assessed by weighing pups from the above matings from postnatal day 1 to 21 daily, then weaned on day 21 and genotyped by PCR of tail DNA (7). The estrus cycle of 20 week old females (3 wild type and 10 heterozygous females) was monitored by daily inspection of vaginal smears (22). Vaginal openings were recorded by daily inspection from postnatal day 14 until natural opening was observed. Circulating P4 levels were analyzed by radio-immuno assay at the laboratory of Dr. David Hess (Oregon Health Science University).LacZ staining LacZ staining of various tissues from LacZ knock-in mice were performed as described previously (23). Tissues from heterozygous for COUP-TFII-LacZ knock-in were dissected and fixed in 2% paraformaldehyde for 30 minutes at room temperature. After washing in phosphate-buffered saline, embryos were cryopreserved in 30% sucrose, and then embedded in OCT compound (Sakura Finetek USA, Torrence, CA). Cryostat sections (20 μm) were incubated at room temperature for 2 hours with staining solution containing 0.1 M phosphate buffer (pH 7.3), 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% NP-40, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 1 mg/ml X-gal. Sections were counterstained with Eosin Y (Sigma, St. Louis, MO). Nuclear localization signal used for LacZ localization in the nucleus facilitated identification of specific staining in all the tissues except for neural tissues, and control sections from wild type mice did not show any specific staining with the condition used in this study.Superovulation, artificial decidual cell reaction and steroid hormone induction. Superovulation was achieved by treating 21 day old female virgin mice with PMSG (5 IU, i.p.) (Sigma, St. Louis MO) followed 48 hours later with hCG (5 IU, i.p.) (Sigma, St. Louis MO). The mice were euthanized and ova were harvested from the oviducts 24 hours after hCG administration. Induction of the artificial decidual cell reaction was induced as described, previously (16). Briefly, Eight-week-old COUP-TFII +/− and COUP-TFII +/+ females were ovariectomized on day 0, treated with 17![[filled square]](corehtml/pmc/pmcents/x25AA.gif) -estradiol (E2) (0.1μg per mouse per day) from day 10 to day 12, and treated with Progesterone (P4) (1 mg per mouse per day) and E2 (6.7 ng per mouse per day) from day 16 to day 23. Mechanical decidualization in the left uterine horn was done 6 hours after hormone injection on day 18. The whole uterus was dissected 6 hours after hormone injection on day 23. The ratio of the weights of the stimulated to the unstimulated (control) horns was calculated. Steroid hormone regulation of COUP-TFII in the mouse uterus was analyzed by administering steroid hormones to COUP-TFII +/− and COUP-TFII +/+ female mice as follows. Hormone treatment was conducted on 6 week old COUP-TFII +/− and COUP-TFII +/+ female mice after bilateral ovariectomy two weeks prior to the commencement of the hormone treatment. Mice were then treated with E2, 5 ng s.c., P4 1 mg, s.c., or 0.1 ml sesame oil per animal. Animals were anesthetized with Avertin (2,2,2-tribromoethyl alcohol, Sigma-Aldrich, St. Louis MO) and sacrificed by cervical dislocation.Immunohistochemical staining Tissues from COUP-TFII +/+ and COUP-TFII +/− mice were fixed in 4% paraformaldehyde for 1 hour at 4°C, cryopreserved in 30% sucrose, then embedded in OCT compound (Sakura Finetek USA, Torrence, CA). Cryostat sections (7 μm) were incubated in blocking buffer (PBS, 1% bovine serum albumin, 5% normal donkey serum, 10 μg of Fab fragment donkey anti-mouse IgG [heavy and light chains] per ml, 0.02% Triton X-100) for 1 hour at room temperature followed by incubation with the primary antibody (monoclonal anti-COUP-TFII [1:100,000) overnight at 4°C. After incubation with primary antibody, slides were incubated with biotin-SP-conjugated donkey anti-mouse IgG (heavy and light chains; Jackson ImmunoResearch Laboratories) for 1 hour at room temperature, then with HRP-conjugated strepavidin (Molecular Probes) for 1 hour at room temperature. Signals were visualized using DAB Kit (Vector Laboratories, Burlingame, CA) and counterstained with hematoxylin. For immunofluorescence, cryosections were incubated with biotin-conjugated polyclonal rat anti-CD31 (PECAM-1) antibody (BD biosciences pharmingen, San Diego, CA) [1:1000] for 2 hours and signals were developed using TSA kit no. 22 (Molecular Probes) in accordance with the manufacturer's instruction.Western blot analysis Isolated tissues were homogenized in buffer composed of 0.25M Sucrose, 50mM Tris-HCl (pH7.5), 25mM KCl, 5mM MgCl2, and 0.5mM PMSF. Protein concentrations were determined by the BCA protein assay system (Pierce, Rockford, IL). 10 μg of protein was mixed with 2X Laemmli Sample Buffer (Bio-Rad Laboratories, Hercules, CA) and separated on 7.5% polyacrylamide gels, then electroblotted onto nitrocellulose membranes. Membranes were incubated with the primary antibody (monoclonal anti-COUP-TFII [1:100,000; kindly provided by Toshiya Tanaka, Department of Molecular Biology and Medicine, the University of Tokyo]) followed by incubation with HRP-conjugated anti-mouse IgG antibody. Signals were visualized with ECL plus Western Blotting Detection System (Amersham Biosciences, UK). Intensities of the bands were analyzed with NIH Image software and normalized by ß-Actin expression level.Quantitative-RT-PCR assays Total mRNA was isolated from snap frozen superovulated ovarian samples using TRIzol reagent (Invitrogen) according to manufacturer’s instructions. The mRNA for genes of interest were quantitated with Taqman-based reverse transcriptase PCR (RT-PCR) using the ABI Prism 7700 sequence detection system (Applied Biosystems). RT-PCRs were performed using One-step RT-PCR Universal Master Mix reagent and TaqMan® Gene Expression Assays (Applied Biosystems) according to the manufacturer's instructions. Cycling conditions were 95C for 1 min, followed by 40 cylces at 95C for 15 sec and 60C for 1 min. All mRNA quantities were normalized against 18S RNA using ABI rRNA control reagents. The TaqMan® Gene Expression Assays used include: Mm00490735_m1 (Cyp11a1); Mm00441558_m1 (StAR); Mm00476184_g1 (Hsd3b1); Mm00772789_m1 (Nr2f2, COUP-TFII), and Eukaryotic 18S rRNA (4319413E). All experiments were performed in triplicate and verified with a duplicate set of mice. Means for mRNA expression between WT and COUP-TFII +/− mice were compared using a Student t-test. | |||||||||||||||||||||
We thank Wei Qian, Chen Liu and Grace Chen for excellent technical help, NIH grants DK55636 to SYT, DK45641 and HD17379 to MJT, U54 HD07495 to FD and SYT and U01DK62434 (Functional Atlas) to SYT, MJT and FD for supported this work. | |||||||||||||||||||||
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