Haploinsufficiency of Chicken Ovalbumin Upstream Promoter Transcription Factor II in Female Reproduction
Norio Takamoto,
Isao Kurihara,
Kevin Lee,
Francesco J. DeMayo,
Ming-Jer Tsai and
Sophia Y. Tsai
Department of Molecular and Cellular Biology and Program of Development, Baylor College of Medicine, Houston, Texas 77030
Address all correspondence and requests for reprints to: Sophia Y. Tsai, Ph.D., and Ming-Jer Tsai, Ph.D., Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: stsai{at}bcm.tmc.edu or mtsai{at}bcm.tmc.edu.
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ABSTRACT
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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 progesterone 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 show 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 periimplantation period.
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INTRODUCTION
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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, whereas 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, 5, 6). COUP-TFII null mutant mice die before embryonic d 10.5 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. Because normal female reproduction requires functional coordination throughout female hypothalamo-hypophyeal-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.
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RESULTS
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COUP-TFII +/ Female Mice Exhibit Reduced Fertility
In maintaining the COUP-TFII mutant mouse colony, it was noticed that COUP-TFII +/ exhibited reduced fertility. 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 months. The numbers of litters and pups born were counted. The data are 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
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 d after birth, as compared with 27.9 d 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 Fig. 1
. Significant growth retardation of COUP-TFII +/ was clearly shown in mutants as compared with the wild-type 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 with 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.

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Fig. 1. Growth Retardation and Delayed Onset of Puberty in COUP-TFII Heterozygotes
A, Gene dosage effect of COUP-TFII on postnatal growth rate was assessed by daily monitoring of body weight of progeny from wild-type male X heterozygote female (for wild-type or heterozygous pups nursed by heterozygous mother) or heterozygous female X wild-type female (for wild-type or heterozygous pups nursed by wild-type mother). Normal growth was assumed in wild-type pups nursed by wild-type female. Significantly retarded growth rate was seen in heterozygous pups nursed by wild-type mother, but better than wild-type pups nursed by heterozygous mother. Most severe growth defect was seen in heterozygous pups nursed by heterozygous mother. B, Estrus cyclicity was assessed by daily inspection of vaginal smear of 20-wk-old virgin females. Wild-type female shows regular cyclicity, but heterozygous female showed irregular estrus cycles. P, Proestrus; E, estrus; M, metestrus; D, diestrus.
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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 Fig. 1B
. COUP-TFII +/+ females showed regular cyclicity, whereas 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.
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. Figure 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 2-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.

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Fig. 2. Western Blot Analysis of COUP-TFII Expression Level in the Uteri of COUP-TF Heterozygous and Wild-Type Mice
A, Representative data of COUP-TFII and ß-actin expression in the uterus of wild-type and COUP-TFII+/ female mice 2 wk after ovariectomized are shown. B, COUP-TFII expression level was normalized by ß-actin. Bar height represents the ratio of COUP-TFII heterozygote to control. COUP-TFII expression was reduced to about half in COUP-TFII +/ mice.
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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 because previous analysis has shown COUP-TFII is expressed in the adult female endometrium (8). However, to determine where else COUP-TFII may be acting, the expression of COUP-TFII in the female reproductive axis was assessed. This was accomplished using 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 Fig. 3
. COUP-TFII expression, as seen by X-gal staining, was found in the ventromedial hypothalamus (VMH) (Fig. 3A
), whereas 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 with that shown using X-gal staining. Thus, X-gal staining could be used as an alternative assays to assess endogenous COUP-TFII expression patterns.

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Fig. 3. Expression of COUP-TFII in Female Reproductive Tract
Expression of COUP-TFII in female reproductive tract was assessed with LacZ knock-in mice or assessed by immuohistochemical staining using COUP-TFII-specific antibodies. Cryosections of various tissues from LacZ knock-in female mice were stained for LacZ expression as described in Materials and Methods. A, Coronal section of 3-wk-old female brain showed specific staining in VMH. B, Mid-sagittal section of embryonic d 14.5 embryo brain showing abundant expression in hypothalamus (H) and mesenchyme surrounding pituitary gland, but not in pituitary gland (Pit). C, Ovary from 8-wk-old randomly cycling female showing strong expression in theca cell surrounding follicles. Note, no specific staining in corpus luteum (CL) and granulosa cells. D, Endogenous expression pattern of COUP-TFII was examined by immunohistochemical staining using COUP-TFII-specific antibody and ovarian section of 8-wk-old female. COUP-TFII was highly expressed in theca cells surrounding follicles, weakly expressed in corpus luteum, but no staining was detected in granulosa cells. E, Sagittal section of uterus from 8-wk-old randomly cycling female. High expression in both longitudinal and circular smooth muscle cells was evident, and scattered staining was seen in endometrial stromal cells. Glandular and luminal epithelial cells also showed weak staining. AM, Antimesometrium; Mes, mesometrium; arrowheads, luminal epithelium; arrows, glandular epithelial cells; smc, smooth muscle cells. F, Antimesometrial portion of gestational d 8 deciduas shows strong nuclear staining of decidualized cells.
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In the uterus of randomly cycling females, strong staining was evident in the myometrium, weaker staining was seen in endometrial stromal cells, and 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 d 8 uteri, and the decidualized cells in antimesometrium stained strongly positive (Fig. 3F
).
Because 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
), whereas treatment with 17ß-estradiol (E2) for 48 h 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 that 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 nonpregnant uterine horn of a gestational d 10 pregnant female mouse (Fig. 4D
). We also examined COUP-TFII expression after the administration of exogenous gonadotropins (Fig. 4
, EH). Twenty-four hours after pregnant mares serum gonadotropin (PMSG) injection, COUP-TFII expression in endometrial stroma was obviously decreased (Fig. 4E
), whereas 48 h 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 and 48 h after human chorionic gonadotropin (hCG) injection (Fig. 4
, GH, respectively).

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Fig. 4. Hormonal Regulation of COUP-TFII Expression in Endometrial Stroma
Effect of steroid hormones on expression of COUP-TFII in endometrial stroma was examined using ovariectomized LacZ knock-in mice of COUP-TFII. Effect of gonadotropins (PMSG and hCG), that alter endogenous ovarian steroids, were also examined using intact LacZ knock-in mice. A, COUP-TFII was highly expressed in the endometrial stroma of ovariectomized mice. B, Expression of COUP-TFII was drastically reduced in the endometrial stroma after E2 treatment for 48 h. C, Expression of COUP-TFII was restored partially in the endometrial stroma after P4 treatment in addition to E2 for 48 h. D, COUP-TFII expression in nonpregnant horn of gestational d 10 female. E, Expression of COUP-TFII was low at 24 h after PMSG. F, Expression of COUP-TFII increased at 48 h after PMSG. G, Expression of COUP-TFII became high at 24 h after hCG. H, Expression of COUP-TFII further increased at 48 h after hCG. L, Lumen; S, endometrial stroma; g, endometrial glands. I, Expression of COUP-TFII was examined by immunohistochemical staining in wild-type uterus 2 wk after ovariectomy. COUP-TFII was highly expressed in endometrial stroma and myometrium but very low in grandular and luminal epithelial cells.
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The expression pattern of COUP-TFII in the uteri was further confirmed by immunostaining using COUP-TFII-specific antibodies. Strong staining was found in endometrial stromal cells as well as myometrium, whereas 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
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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. 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 Tables 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 d 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 P4 Synthesis
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 (10). 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 low-density lipoprotein (LDL) (10). 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 whether 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 an endothelial-specific marker, platelet endothelial cell adhesion molecule (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. 5
, B 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.

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Fig. 5. Defective Vascularization of Corpus Luteum and Reduced Expression of Cyp11a1, Hsd3b1, and StAR in COUP-TFII +/ ovaries
AC, Microscopic evaluation of vascularization in superovulated COUP-TFII heterozygous and wild-type ovaries. A, Hematoxylin-eosin staining. Dilated vessels were not seen in COUP-TFII heterozygous ovaries. B, Vascularization as assessed by immunofluorescent signal using CD31 (PECAM-1-specific antibody). Reduced vascularization was seen in COUP-TFII +/ corpus luteum. C, Nuclear staining with DAPI on the same slide as B. D, The mRNA for COUP-TFII, CYP11a1, HSD3b1 and StAR were quantified with TaqMan-based RT-PCR. All mRNA quantities were normalized against 18S RNA using ABI rRNA control reagents. 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 Students t test, and the P values are present in the figure.
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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 RT-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 with 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-
-5-steroid dehydrogenase, 3ß- and steroid
-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 decidualization may greatly affect reproductive performance. 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, whereas 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 vs. control horn was calculated. As shown in Fig. 6
, uterus from COUP-TFII +/ female mice exhibited significantly reduced decidual cell reaction when compared with 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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Fig. 6. Reduced Decidual Cell Reaction in COUP-TFII Heterozygote
Decidual cell reaction was induced by traumatizing left uterine horn (L) after hormonal treatment of ovariectomized wild-type or heterozygous females. Right horn (R) was left intact for control. A, Typical deciduoma formation in the left uterine horn of wild-type female is shown on the top panel, whereas limited decidual cell reaction was seen in the left uterine horn of heterozygous female shown in the bottom panel. B, Wet weight of left horn and right horn from each animal was weighted and ratio between left stimulated horn and right control horn (L/R ratio) were calculated. There was significant decrease (P = 0.018) in L/R ratio for COUP-TFII heterozygous females.
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DISCUSSION
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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. Because 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 central nervous system. 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 IGF-I 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 steroidogenic factor 1 (SF1) (11). SF1 null mice resulted in defective development of VMH (12). Rescue experiments using SF1 null mutants indicated SF1 functions in VMH to regulate late onset obesity (13). 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 (11). 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 (11). 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 with 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 of these genes are important for the biosynthesis of P4. Thus, the reduction in the expression of these 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, whereas 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 progesterone receptor (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 preimplantation 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 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 periimplantation (8). Importantly, both IHH and COUP-TFII had been implicated in angiogenesis (7, 21). 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 periimplantation period. Detailed molecular and cellular mechanisms could be elucidated by conditional gene targeting approaches, such as using endometrial-specific Cre recombinase.
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MATERIALS AND METHODS
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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 wk old) were continuously mated in a cage for 3 months, and numbers of newborn at birth were counted. Postnatal growth was assessed by weighing pups from the above matings from postnatal d 121 daily, then weaned on d 21 and genotyped by PCR of tail DNA (7). The estrus cycle of 20-wk-old females (three wild-type and 10 heterozygous females) was monitored by daily inspection of vaginal smears (22). Vaginal openings were recorded by daily inspection from postnatal d 14 until natural opening was observed. Circulating P4 levels were analyzed by RIA at the laboratory of Dr. David Hess (Oregon Health Science University, Portland, OR).
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 min at room temperature. After washing in PBS, embryos were cryopreserved in 30% sucrose, and then embedded in OCT compound (Sakura Finetek USA, Torrance, CA). Cryostat sections (20 µm) were incubated at room temperature for 2 h with staining solution containing 0.1 M phosphate buffer (pH 7.3), 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% Nonidet P-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-d-old female virgin mice with PMSG (5 IU, ip) (Sigma) followed 48 h later with hCG (5 IU, ip) (Sigma). The mice were euthanized and ova were harvested from the oviducts 24 h after hCG administration. Induction of the artificial decidual cell reaction was induced as described previously (16). Briefly, 8-wk-old COUP-TFII +/ and COUP-TFII +/+ females were ovariectomized on d 0, treated with E2 (0.1 µg per mouse per day) from d 1012, and treated with P4 (1 mg per mouse per day) and E2 (6.7 ng per mouse per day) from d 1623. Mechanical decidualization in the left uterine horn was performed 6 h after hormone injection on d 18. The whole uterus was dissected 6 h after hormone injection on d 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-wk-old COUP-TFII +/ and COUP-TFII +/+ female mice after bilateral ovariectomy 2 wk before the commencement of the hormone treatment. Mice were then treated with E2, 5 ng sc, P4 1 mg, sc, or 0.1 ml sesame oil per animal. Animals were anesthetized with Avertin (2,2,2-tribromoethyl alcohol; Sigma) and killed by cervical dislocation.
Immunohistochemical Staining
Tissues from COUP-TFII +/+ and COUP-TFII +/ mice were fixed in 4% paraformaldehyde for 1 h at 4 C, cryopreserved in 30% sucrose, then embedded in OCT compound (Sakura Finetek USA). Cryostat sections (7 µm) were incubated in blocking buffer [PBS, 1% BSA, 5% normal donkey serum, 10 µg of Fab fragment donkey antimouse IgG (heavy and light chains) per ml, 0.02% Triton X-100] for 1 h 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 antimouse IgG (heavy and light chains; Jackson ImmunoResearch Laboratories) for 1 h at room temperature, then with horseradish peroxidase-conjugated strepavidin (Molecular Probes, Eugene, OR) for 1 h 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 h and signals were developed using TSA kit no. 22 (Molecular Probes) in accordance with the manufacturers instruction.
Western Blot Analysis
Isolated tissues were homogenized in buffer composed of 0.25 M sucrose, 50 mM Tris-HCl (pH 7.5), 25 mM KCl, 5 mM MgCl2, and 0.5 mM phenylmethylsulfonyl fluoride. Protein concentrations were determined by the BCA protein assay system (Pierce, Rockford, IL). Ten micrograms of protein were 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 followed by incubation with horseradish peroxidase-conjugated antimouse IgG antibody. Signals were visualized with ECL plus Western Blotting Detection System (Amersham Biosciences, Amersham, Buckinghamshire, UK). Intensities of the bands were analyzed with National Institutes of Health 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, Carlsbad, CA) according to manufacturers instructions. The mRNA for genes of interest were quantitated with TaqMan-based RT-PCR using the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA). RT-PCRs were performed using One-step RT-PCR Universal Master Mix reagent and TaqMan Gene Expression Assays (Applied Biosystems) according to the manufacturers instructions. Cycling conditions were 95 C for 1 min, followed by 40 cycles at 95 C for 15 sec and 60 C 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 Students t test.
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ACKNOWLEDGMENTS
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We thank Wei Qian, Chen Liu, and Grace Chen for excellent technical help.
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FOOTNOTES
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This work was supported by National Institutes of Health Grants DK55636 (to S.Y.T.), DK45641 and HD17379 (to M.J.T.), U54 HD07495 (to F.D. and S.Y.T.), and U01DK62434 (Functional Atlas) (to S.Y.T., M.J.T., and F.D.).
First Published Online May 12, 2005
Abbreviations: COUP-TFII, Chicken ovalbumin upstream promoter transcription factor II; Cyp11a1, cytochrome P450, family 11, subfamily a, polypeptide 1; E2, 17ß-estradiol; hCG, human chorionic gonadotropin; Hsd3b1, hydroxy-
-5-steroid dehydrogenase, 3ß- and steroid
-isomerase 1; IHH, Indian hedgehog; LDL, low-density lipoprotein; P4, progesterone; PECAM, platelet endothelial cell adhesion molecule; PMSG, pregnant mares serum gonadotropin; PR, progesterone receptor; SF1, steroidogenic factor 1; StAR, steroidogenic acute regulatory protein; VMH, ventromedial hypothalamus.
Received for publication January 10, 2005.
Accepted for publication May 4, 2005.
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