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. 2012 Sep 19;87(5):115. doi: 10.1095/biolreprod.112.102251

The Reproductive Phenotype of Mice Null for Transcription Factor Krüppel-Like Factor 13 Suggests Compensatory Function of Family Member Krüppel-Like Factor 9 in the Peri-Implantation Uterus1

Melissa E Heard 3, John Mark P Pabona 3,4, Carol Clayberger 6, Alan M Krensky 6, Frank A Simmen 3,5, Rosalia CM Simmen 3,4,5,2
PMCID: PMC4435227  PMID: 22993382

ABSTRACT

The ovarian hormones estrogen and progesterone promote uterine receptivity and successful pregnancy through their cognate receptors functioning in concert with context-dependent nuclear coregulators. Previously, we showed that the transcription factor Krüppel-like factor (KLF) 9 is a progesterone receptor (PGR) coactivator in the uterus and that mice null for Klf9 exhibit subfertility and reduced progesterone sensitivity. The highly related family member KLF13 displays increased expression in uteri of pregnant and nonpregnant Klf9 null mice and similarly regulates PGR-mediated transactivation in endometrial stromal cells. However, a uterine phenotype with loss of Klf13 has not been reported. In the present study, we demonstrate that Klf13 deficiency in mice did not compromise female fertility and pregnancy outcome. Klf13 null females had litter sizes, numbers of implanting embryos, uterine morphology, and ovarian steroid hormone production comparable to those of wild-type (WT) counterparts. Further, pregnant WT and Klf13 null females at Day Postcoitum (DPC) 3.5 had similar uterine Pgr, estrogen receptor, and Wnt-signaling component transcript levels. Nuclear levels of KLF9 were higher in Klf13 null than in WT uteri at DPC 3.5, albeit whole-tissue KLF9 protein and transcript levels did not differ between genotypes. The lack of a similar induction of nuclear KLF9 levels in uteri of virgin Klf13(−/−) mice relative to WT uteri was associated with lower stromal PGR expression. In differentiating human endometrial stromal cells, coincident KLF9/KLF13 knockdown by small interfering RNA targeting reduced decidualization-associated PRL expression, whereas KLF9 and KLF13 knockdowns alone reduced transcript levels of WNT4 and BMP2, respectively. Results suggest that KLF9 and KLF13 functionally compensate in peri-implantation uterus for pregnancy success.

Keywords: female reproductive tract, implantation, Krüppel-like factors, pregnancy, progesterone/progesterone receptor, steroid hormone receptors

Krüppel-like factor (KLF) 9 may compensate for absence of KLF13 in maintaining uterine receptivity for embryo implantation.

INTRODUCTION

Early pregnancy failure remains a significant health problem, with one of three human pregnancies terminating before 20 wk due to implantation defects [1]. Successful embryo implantation and development within the female reproductive tract that leads to the birth of a live young requires the endometrium and the preimplantation embryo to develop in synchrony [2, 3]. A major cause of an out-of-phase uterus resulting in reproductive failure is the dysfunctional response of endometrial stromal cells to progesterone (P4) [4]. Working through the cognate progesterone receptor (PGR) isoforms PGR-A and PGR-B [5] in stromal cells, P4 mediates the production of paracrine factors that allow the timely growth and differentiation of the luminal epithelium (LE) for implantation to occur. With absent or limited PGR activity (a condition known as P4 resistance), the unopposed actions of estradiol (E2) can disrupt the normal patterns of proliferation and compromise cell differentiation in stromal cells, leading to LE that lacks the hallmarks of an embryo-receptive state [2, 3]. Reduced, complete loss of, or altered ratios of PGR isoform expression are accepted to underlie aberrant ligand-activated PGR signaling [6–10]. Nevertheless, recognition is increasing that PGR coregulators constitute critical determinants of PGR response and that their loss of function causes misregulation of P4-dependent signaling networks [11, 12]. To date, more than 200 proteins have been designated as PGR coregulators [13]. Thus, a key to understanding the etiology of early pregnancy failure consequent to reduced P4 responsiveness requires the systematic elucidation of the biology and actions of these important proteins.

Our laboratory has identified Krüppel-like factor (KLF) 9 (previously known as basic transcription element-binding protein 1 [BTEB1]) as a coregulator of PGR signaling in the uterus [14, 15]. KLF9 is one of 25 currently known members of the Sp/KLF family of GC box-binding transcription factors [16, 17]. Whereas members of this family were previously thought to function simply as silencers of Sp1 transcriptional activity, KLFs have gained increased appreciation as being clinically relevant to many human pathologies by virtue of their mediation of cross-talk among signaling pathways involved in the control of cell proliferation, apoptosis, migration, and differentiation, independent of Sp1 [16, 18, 19]. In the mouse uterus, KLF9 is expressed predominantly in endometrial stroma, with lower and no expression in glandular epithelium (GE) and LE, respectively [20]. KLF9 expression has been similarly reported in human endometrial stromaland GE cells during the menstrual cycle [21]. The extent to which KLF9 plays a role in uterine function was previously demonstrated from our studies of female Klf9 null mice. We showed that Klf9 null mutation resulted in smaller litters due to reduced numbers of implanted embryos, a shift in the normal window of embryo implantation, partial uterine P4 resistance, and enhanced uterine estrogen sensitivity [20, 22, 23]. Klf9 null mutation also dramatically influenced LE proliferative, apoptotic, and PGR expression status [22, 24], consistent with LE as a target of stromal KLF9 through paracrine signaling. Thus, early pregnancy loss in mice with Klf9 null mutation likely involves disruptions of multiple early events in LE coordinated by stromal KLF9.

The KLF family members share a DNA-binding domain comprised of three zinc fingers located in the carboxyl-terminus [16, 19]. Among the KLFs, KLF9 and KLF13 exhibit the highest sequence similarities, which extend well beyond the zinc fingers [16], suggesting similar, albeit not necessarily identical, functions. In previous studies [14, 15, 25], we showed that both KLFs can transactivate the PGR-B isoform and induce PGR expression in endometrial epithelial and stromal cells in vitro. Moreover, loss of Klf9 is accompanied by increased Klf13 expression in nonpregnant and peri-implantation mouse uterine stromal cells [20, 25]. Because the uterine phenotype of Klf9 null mice is subfertility rather than infertility, this finding raises the interesting possibility of regulatory and/or compensatory functions of the two KLFs in uterine endometrial cells in vivo. In the present study, we utilized mice null for Klf13 to evaluate the contribution of this KLF to pregnancy events and to determine whether KLF9 functionally complements KLF13 in maintaining peri-implantation uterine receptivity for successful pregnancy.

MATERIALS AND METHODS

Experimental Animals

All animal experiments were conducted according to protocols approved by the University of Arkansas for Medical Sciences Institutional Animal Care and Use Committee. Animals were allowed ad libitum access to water and standard chow diet (Harlan Laboratories) and were housed in a pathogen-free barrier facility with a 12L:12D photoperiod. Klf13(−/−) mutant mice were generated by replacing exon 1 plus 0.7-kb upstream and 0.6-kb downstream regions of the Klf13 gene with the Neo gene [26]. Klf13 null mice were rederived by Charles River Laboratories before their use in our facility. Mice were genotyped by PCR of genomic DNA prepared from mouse tails as recommended by the supplier. In timed pregnancy studies, wild-type (WT) and Klf13(−/−) females of comparable ages (6–8 wk) were monitored for stage of the estrous cycle by vaginal smears and were mated at proestrus of the second estrous cycle. The presence of a vaginal plug was designated as Day Postcoitum (DPC) 0.5, and uterine tissues were collected in the morning of DPC 3.5. In studies using 8-wk-old (Postnatal Day [PND] 56) virgin mice, reproductive tissues were harvested in the morning of diestrus after the second estrous cycle.

RNA Isolation and Relative Quantitative RT-PCR

Total RNA was isolated from whole-uterine tissues (n = 4–6 mice/genotype) with TRIzol reagent (Invitrogen) following the manufacturer's protocol. RNA concentrations were determined using an ND-1000 spectrophotometer (NanoDrop Corp.). RNA (1 μg) was reverse transcribed to cDNA using iScript cDNA synthesis kit (Bio-Rad). The cDNA was diluted 1:5 (vol/vol), and 5 μl were used in a total reaction volume of 20 μl containing SYBR Green mixture (Bio-Rad) and 0.3 μM of each primer. All primers were designed to span introns using PrimerExpress software (Applied Biosystems) and synthesized by Integrated DNA Technologies, Inc. The sequences of mouse primers for real-time quantitative RT-PCR (QPCR) amplification and the amplicon sizes for each primer set are listed in Table 1. Amplification was performed under previously described thermal conditions [25] using an ABI Prism 7000 Sequence Detection System (Applied Biosystems). For each primer set, a standard curve was generated by serial dilution of pooled cDNA stocks. Quantification of individual RNAs was performed using the standard curve (hence, relative QPCR), and values were normalized to the reference gene (TATA box-binding protein [Tbp]).

TABLE 1.

Mouse (m) and human (h) primer sequences.

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Western Blot Analysis

Whole-cell and nuclear protein fractions from whole uteri of WT and Klf13(−/−) mice were prepared following the manufacturer's instructions (whole cell: RIPA Lysis Buffer [Santa Cruz Biotechnology]; nuclear: NE-PER [Pierce Biotechnology]), and protein concentrations were quantified by bicinchoninic acid assay (Thermo Scientific) using bovine serum albumin as standard. Whole-cell extract (100 μg) and nuclear (30–40 μg) proteins were isolated from uteri of three to four mice per genotype, separated by SDS-10% PAGE, and transferred to nitrocellulose membranes (Millipore Corp). The membranes were incubated with rabbit anti-rat KLF9 (generated in-house) at 1:1000 dilution [14] and rabbit anti-human PGR (sc-7208; Santa Cruz Biotechnology) at 1:450 dilution at 4°C overnight and further incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) for 1 h at room temperature. Immunoreactive proteins were visualized using an enhanced chemiluminescence detection system (Amersham ECL Plus; GE Healthcare Life Sciences). Membranes were stripped (Restore Western Blot Stripping Buffer; Fisher Scientific) and probed for lamin A (for nuclear proteins; Sigma) or β-actin (for whole-cell lysates; Sigma) as loading controls. Densitometric values of immunoreactive bands were quantified using the GE Image Scanner III detection system and Quantity One software (Bio-Rad).

Uterine Morphometry

Whole uteri from early pregnant (DPC 3.5) and virgin (PND 56) WT and Klf13(−/−) mice were fixed in 10% neutral buffered formalin as previously described [20]. Sections (thickness, 5 mm) were mounted on poly-l-lysine-coated slides (Fisher Scientific) and processed for hematoxylin/eosin staining following standard protocols [23, 24]. LE height was quantified using the Axiovert 200M microscope equipped with a Axiocam HRc camera and Axiovision software (Carl Zeiss, Inc.). Measurements were taken from five different fields (100× magnification) per section, and data from two to three serial sections per mouse (n = 3 mice/genotype) were analyzed.

PGR Immunohistochemistry

Formalin-fixed, paraffin-embedded uterine tissue sections from virgin (PND 56) WT and Klf13(−/−) mice (n = 3–4 mice/genotype) were prepared and incubated with rabbit anti-human PGR antibody that recognizes both PGR isoforms (sc-7208; Santa Cruz Biotechnology) at 1:300 dilution overnight at 4°C, followed by incubation with secondary antibody (Vectastain ABC kit; Vector Laboratories, Inc.) for 1 h, as previously described [22, 24]. Control sections were processed similarly, but with the omission of the primary antibody. Approximately 1000 stromal and 100 LE cells were counted, on average, from at least five to six randomly selected fields (400× magnification) per slide. For each cell type and genotype, nuclear immunopositive cells were expressed as the percentage of total cells counted ([number of nuclear-staining cells/number of total cells counted] × 100). The relative staining intensity (optical density [OD]) of PGR immunopositive cells was determined by computerized quantification using MCID Elite 7.0 software (Cambridge, Inc.). An intensity threshold was determined by scanning and averaging the intensity range of five randomly selected uterine sections from WT mice, and these values were utilized to determine the relative amount of staining for concurrent slides. Intensity values (OD) were obtained for individually stained cells and scored as 1, 2, or 3 based on the set intensity range of WT samples (3 = high staining, OD ≤ 0.299; 2 = moderate staining, 0.300 ≤ OD ≤ 0.399; 1 = light staining, OD ≥ 0.400). For each genotype, the percentage of stromal and LE cells that were assigned to each intensity category was calculated ([number of cells per scoring level/total number of scored cells] × 100). All slides were scored in a blinded fashion

Small Interfering RNA Transfections

The human endometrial stromal cell (HESC) line was propagated as previously described [25]. Transfections with small interfering (si) RNAs targeting human KLF9 and human KLF13 (siGeNOME SMART pool) or nontargeting (siCONTROL [Scr]) siRNAs (Dharmacon) were performed with Lipofectamine 2000 reagent (Invitrogen) in OPTI-MEM reduced serum-containing medium (Invitrogen) when cells were approximately 60% confluent. KLF9 and KLF13 siRNAs were used at 50 nM concentration, with final concentrations of siRNAs made up to 100 nM with the addition of Scr siRNA. Six hours after transfection, cells were washed, and medium was replaced with phenol red-free Dulbecco modified Eagle medium (Sigma-Aldrich)/2% charcoal-stripped bovine calf serum (Gemini Bio-Products). Cells were incubated for 24 h and then transferred to fresh medium containing 8-bromoadenosine-cAMP (0.5 mM), progestin (medroxyprogesterone acetate [MPA], 1 μM), and estradiol-17β (E2, 10 nM) (all from Sigma-Aldrich) as previously described [25]. Cells were collected after 48 h of incubation and subjected to RNA expression analyses by QPCR using human primers shown in Table 1 or described previously [25]. Alkaline phosphatase (ALP) activity in HESCs transfected with control (Scr) or KLF13 siRNAs was determined as previously described [25].

Radioimmunoassay

Approximately 500 μl of whole blood were collected by closed cardiac puncture from pregnant (DPC 3.5) and young adult (PND 56) WT and Klf13(−/−) mice. Serum was prepared by centrifugation at 4600 × g for 1 h and was stored at −20°C before analysis. Serum E2 and P4 levels were measured using the Ultrasensitive Estradiol RIA kit (Beckman Coulter) and the Progesterone EIA Kit (Cayman Chemical), respectively.

Statistical Analysis

Statistical analysis was performed using SigmaStat software (Version 3.5; Systat Software). For comparisons of relative QPCR, Western blot density, ALP activity, uterine morphometry, and serum steroid hormone levels, data (mean ± SEM) were evaluated for statistical significance between genotypes or treatment groups by Student t-test. Nuclear PGR immunostaining scores (mean ± SEM) were analyzed by two-way ANOVA followed by Tukey test. Incidence and frequency of neonatal lethality were analyzed by chi-square test. Differences were considered to be significant at P < 0.05.

RESULTS

Lack of a Fertility Phenotype of Klf13 Null Females

To determine if loss of Klf13 expression affects pregnancy outcome, 6- to 10-mo-old WT, Klf13(+/−), and Klf13(−/−) females were bred with males of the same genotypes, and their litter sizes were determined. As shown in Table 2, Klf13(−/−) and Klf13(+/−) dams had numbers of pups (counted at birth) comparable to those of WT females (P = 0.915). Klf13(−/−) females showed no evidence of delayed labor or delivery complications previously noted in Klf9 null mice [27]. To evaluate if the number of pups born equaled the number of embryos implanted, WT and Klf13(−/−) pregnant females (n = 8 per genotype) were euthanized postimplantation (range, DPCs 6.5–12.5), and the total numbers of embryos in both uterine horns were counted. WT females had 7.50 ± 0.75 embryos, which was not significantly different from Klf13(−/−) females (7.87 ± 0.64 embryos). Importantly, the numbers of implanting embryos and the litter size (Table 2) did not differ for each genotype (WT: P = 0.203; knock-out: P = 0.205) and between genotypes (P > 0.05). Females from both genotypes showed similar normal distribution of embryos within each uterine horn (Fig. 1A). The lack of gross morphological abnormalities in peri-implantation Klf13 null uteri was further demonstrated by the histological appearance of the endometrial compartments (Fig. 1B), which showed no differences in LE height, numbers of glands, and gland areas from those of WT counterparts (Fig. 1, C–E). Serum E2 and P4 levels in WT and Klf13 null mice also did not differ on this day of pregnancy (Fig. 1, F and G). These results indicate that in contrast to Klf9 null mutants [20], loss of Klf13 in the uterus did not affect peri-implantation uterine growth, uterine receptivity to implantation, and pregnancy outcome in mice.

TABLE 2.

Litter size at birth and neonatal incidence and frequency of mortality at PNDs 1–5.

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a 

Number of litters analyzed from each mating.

b 

Litters with at least one neonatal death/total number of litters analyzed.

c 

Number of pups dead/total number of pups born from analyzed litters.

# 

P = 0.009 relative to WT.

## 

P < 0.001 relative to WT, P = 0.01 relative to Klf13(+/−).

* 

P < 0.001 relative to WT.

*

* P < 0.001 relative to WT and Klf13(+/−).

FIG. 1.

FIG. 1

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Uterine phenotype and serum steroid hormone levels of Klf13 null mice at peri-implantation. A) Representative images of implanted embryos in uteri of WT and Klf13(−/−) mice at DPC 8.5. B) Representative images of hematoxylin/eosin-stained uterine sections of WT and Klf13(−/−) mice at DPC 3.5. Each panel represents an individual mouse for each genotype. ST, stroma. Bar = 10 μm. C–E) Uterine sections from WT and Klf13(−/−) mice (n = 3 per genotype) were stained with hematoxylin/eosin and quantified for LE height (C), number of glands (D), and gland area (E). No differences for any uterine parameter were noted between genotypes. Data are expressed as the mean ± SEM (n = 3–4 mice/genotype). F and G) Serum E2 (F) and P4 (G) levels in early pregnant (DPC 3.5) WT and Klf13(−/−) females. No differences were found between genotypes for either hormone level. Data are expressed as the mean ± SEM (n = 5–7 mice/genotype).

Increased Neonatal Mortality with Loss of Klf13 Expression

The incidence and frequency of lethality within PNDs 1–5 for pups born from Klf13(−/−) × Klf13(−/−) mating pairs were higher than those from both Klf13(+/−) × Klf13(+/−) and WT × WT mating pairs (Table 2). Heterozygous Klf13(+/−) matings also had higher incidence and frequency of neonatal mortality than homozygous WT matings (Table 2). These results indicate that analogous to loss of Klf9 expression, KLF13 haplo- and total insufficiency predispose offspring to increased neonatal mortality.

Uterine Gene Expression in Early Pregnant Klf13 Null Mice

Our previous studies demonstrated that uterine Klf13 mRNA and protein levels were increased in Klf9 null females during early pregnancy [25], suggesting KLF13 compensation with KLF9 deficiency. To determine if Klf13 null mutation similarly results in a compensatory increase in KLF9 expression in the peri-implantation (DPC 3.5) uterus that might underlie the lack of fertility loss with Klf13 null mutation, we evaluated transcript levels of Klf9 and its downstream target genes in uteri of WT and Klf13(−/−) mice. Relative QPCR analysis of WT and Klf13 null uteri at DPC 3.5 showed no differences in Klf9 and family member Klf4 (Fig. 2A) expression. Relative gene expression levels for total Pgr (Pgr-a+ -b), Pgr-b, and Esr1 also did not differ between the genotypes (Fig. 2A). To determine if the lack of differential expression between genotypes was likewise observed at the protein level, whole-cell lysates and nuclear extracts prepared from DPC 3.5 uteri of the two genotypes were analyzed by Western blot. KLF9 protein levels did not differ in whole-cell extracts for WT and Klf13 null mice when normalized to the loading control β-actin (Fig. 2B). By contrast, nuclear KLF9 protein levels, normalized to lamin A as loading control, were 3-fold higher (P = 0.029) in peri-implantation Klf13 null uteri than in those of corresponding WT mice (Fig. 2C). No differences in nuclear levels of PGR-A and PGR-B proteins (Fig. 2C), concordant with their respective transcripts (Fig. 2A), were observed between genotypes.

FIG. 2.

FIG. 2

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Gene and protein expression in Klf13 null uteri at peri-implantation. A) Messenger RNA levels of KLF family members Klf9 and Klf4 and of steroid hormone receptors in WT and Klf13(−/−) uteri at DPC 3.5 were quantified by relative QPCR (RQ) and normalized to those of TATA box-binding protein (Tbp). Data (mean ± SEM) are expressed as fold-change relative to corresponding WT uteri (n = 4–6 uteri/genotype). B) Western blot analyses of whole-cell extracts (100 μg of protein) from WT and Klf13(−/−) uteri at DPC 3.5 using rabbit anti-rat KLF9 and β-actin (loading control) antibodies. A representative blot (top) and the quantified data from densitometric scans of normalized immunoreactive bands (bottom) from uteri of four individual WT and Klf13(−/−) mice are shown. C) Western blot analyses of nuclear extracts (30 μg of protein) from WT and Klf13(−/−) uteri at DPC 3.5 using rabbit anti-rat KLF9, rabbit anti-human PGR, and anti-lamin A (loading control) antibodies. A representative blot (top) and the quantified data from densitometric scans of normalized immunoreactive bands (bottom) from uteri of three individual WT and Klf13(−/−) mice are shown. *P < 0.05 (Student t-test). D) Messenger RNA levels of WNT signaling-associated genes in WT and Klf13(−/−) uteri at DPC 3.5 were quantified by RQ and normalized to those of TATA box-binding protein (Tbp). Data (mean ± SEM) are expressed as fold-change relative to corresponding WT uteri (n = 4–6 uteri/genotype) No differences in transcript levels were found as a function of genotype.

We also investigated potential changes in the expression of Wnt signaling components as a function of genotype, given their importance in embryo implantation [28] and our previous observation that a subset of these genes were subject to KLF9 regulation in human stromal cells [25, 29]. No significant differences in expression levels of Bmp2, Wnt2, Wnt4, Lrp6, Kremen1, Wisp1, Dkk1, or cMyc genes were noted between genotypes (Fig. 2D). These collective results indicate that increased nuclear KLF9 levels accompanying loss of Klf13 expression in the peri-implantation (DPC 3.5) uteri may serve to maintain normal PGR and Wnt signaling and, thus, uterine receptivity to embryo implantation.

Uterine Gene Expression in Nonpregnant Klf13 Null Mice

Uteri from WT and Klf13 null virgin (age, 8 wk; PND 56) females at diestrus were similarly evaluated for uterine morphology, histological appearance, and gene expression. Nonpregnant uteri from WT and Klf13 null mice had comparable uterine morphology, as demonstrated by similar LE height, gland numbers, and gland sizes (Fig. 3, A–D). Uterine wet weights (relative to body weight, g/g; WT: 2.42 ± 0.12 × 10−3; Klf13 null: 2.40 ± 0.36 × 10−3), ovarian weights (relative to body weight, g/g; WT: 4.83 ± 0.39 × 10−4; Klf13 null, 5.75 ± 0.21 × 10−4; n = 5 mice/genotype), and serum E2 and P4 levels (Fig. 3, E and F) also did not differ (P > 0.05) between WT and Klf13 null mice. Further, uteri from Klf13(−/−) mice demonstrated expression levels of Klf9, Klf4, and Esr1 comparable to those from WT counterparts (Fig. 3G). The lack of differences in relative transcript levels as a function of genotype was confirmed for KLF9 protein in uterine whole-cell lysates (Fig. 3H) as well as in isolated nuclear extracts (Fig. 4A).

FIG. 3.

FIG. 3

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Uterine phenotype, uterine gene expression, and serum steroid hormone levels of adult virgin (PND 56) Klf13 null mice. A) Representative images of hematoxylin/eosin-stained uterine sections of WT and Klf13(−/−) mice at PND 56. Each panel represents an individual mouse for each genotype. ST, stroma. Bar = 10 μm. B–D) Uterine sections from (PND 56) WT and Klf13(−/−) mice (n = 3 per genotype) were stained with hematoxylin/eosin and quantified for LE height (B), number of glands (C), and gland area (D). No differences for any uterine parameter were noted between genotypes. E and F) Serum E2 (E) and P4 (F) levels in (PND 56) WT and Klf13(−/−) females. No differences were found between genotypes for either hormone levels. G) Messenger RNA levels of Klf13, Klf9, and Klf4 and steroid hormone receptors in uteri of (PND 56) WT and Klf13(−/−) mice were quantified by relative QPCR (RQ) and normalized to those of TATA box-binding protein (Tbp). Data (mean ± SEM) are expressed as fold-change relative to corresponding WT uteri (n = 5–6 uteri/genotype). *P < 0.05 (Student t-test). H) Western blot analyses of whole-cell extracts (100 μg of protein) from (PND 56) WT and Klf13(−/−) uteri using rabbit anti-rat KLF9 and β-actin (loading control) antibodies. A representative blot (top) and the quantified data from densitometric scans of normalized immunoreactive bands (bottom) from uteri of WT and Klf13(-/-) mice (n = 4 uteri/genotype) are shown.

FIG. 4.

FIG. 4

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KLF9 and PGR expression in uteri of adult virgin (PND 56) Klf13 null mice. A) Western blot analyses of nuclear extracts (40 μg of protein) from (PND 56) WT and Klf13(−/−) uteri using rabbit anti-rat KLF9, rabbit anti-human PGR, and anti-lamin A (loading control) antibodies. A representative blot (top) and the quantified data from densitometric scans of normalized immunoreactive bands (bottom) from WT and Klf13(−/−) uteri of four individual mice are shown. *P < 0.05 (Student t-test). B) Representative PGR immunostaining of GE, LE, and stromal (ST) compartments in uteri of (PND 56) WT and Klf13(−/−) mice. Each panel for WT and Klf13 knock-out mice represents a uterine section from an individual mouse. Control shows lack of immunostaining (WT uteri) in the absence of primary antibody. Original magnification, ×400; bar = 10 μm. C) Uterine ST and LE of (PND 56) WT and Klf13(−/−) mice were quantified for nuclear PGR immunoreactivity by determining the percentage of nuclear PGR-staining cells (mean ± SEM); three to four mice per genotype were analyzed. D and E) The percentage of uterine ST (D) and LE (E) cells in (PND 56) WT and Klf13(−/−) mice showing high (3), medium (2), and low (1) PGR immunostaining intensities were quantified, following procedures described in Materials and Methods. *P < 0.05 by two-way ANOVA followed by Tukey test.

Interestingly, total Pgr and Pgr-b transcript levels were lower in Klf13 null than in WT uteri (Fig. 3G). The nearly 50% decrease in Pgr-b gene expression with Klf13 null mutation was confirmed for the corresponding PGR-B protein in nuclear extracts by Western blot analysis (Fig. 4A). No corresponding change was observed in PGR-A protein levels from those of WT in Klf13 null mutants (Fig. 4A).

Analyses of uterine sections from WT and Klf13 null mice using an anti-PGR antibody that recognizes both PGR isoforms indicated that the percentage of nuclear PGR immunopositive stromal and LE cells did not differ between WT and Klf13 null uteri (Fig. 4, B and C). However, the percentage of nuclear cells in the stromal compartment that displayed intense anti-PGR immunostaining was lower for Klf13 mutant than for WT uteri (Fig. 4D); conversely, a higher percentage of stromal cells was weakly immunostained for anti-PGR antibody in Klf13 null compared to WT uteri (Fig. 4D). Interestingly, the uterine luminal compartment did not differ in the distribution of cells that strongly or weakly immunostained for anti-PGR (Fig. 4E). These results suggest that in the absence of a compensatory increase in nuclear-localized KLF9 in nonpregnant mouse uterus, a reduction in stromal PGR levels occurs with Klf13 null mutation.

Coincident Loss of KLF9 and KLF13 Predicts Impaired Stromal Differentiation

To further evaluate a potential compensatory role of KLF9 on KLF13 function, we used siRNA targeting to knock down KLF9 and KLF13 mRNAs, both individually and in combination (designated siK13K9), in HESCs; control (Scr) siRNAs were used in parallel. Transfected cells were then incubated in medium containing 8-bromoadenosine-cAMP, MPA, and E2 for 2 days as previously described [25] to induce decidualization and were subsequently analyzed for relative expression of well-characterized implantation/decidualization-associated genes PRL, WNT4, and BMP2 by QPCR [25, 28, 29]. In previous studies, we showed the dramatic decrease (>90%) in KLF9 and KLF13 proteins in these cells with addition of their respective siRNAs and, importantly, that the loss of one KLF resulted in the increased expression of the other [25]. The latter finding was confirmed in the present study (Fig. 5A). Coaddition of siKLF9 and siKLF13 resulted in significant reductions in both PRL and WNT4 transcript levels, and numerically lower transcript levels for BMP2, relative to cells expressing both KLFs (i.e., transfected with Scr siRNAs) (Fig. 5B). However, knockdown of KLF9 alone significantly reduced WNT4 but not PRL and BMP2 transcript levels, whereas KLF13 knockdown alone caused a significant reduction in BMP2 but not PRL and WNT4 transcript levels (Fig. 5B).

FIG. 5.

FIG. 5

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Transcript levels of decidualization-associated genes with coincident KLF9 and KLF13 silencing. HESCs were transfected with Scr (control) siRNAs or siRNAs for KLF13 and KLF9 (50 nM each), alone and together, and treated with 8-bromoadenosine-cAMP, MPA, and E2 for 2 days. Isolated RNAs were evaluated for transcript levels of the indicated genes by relative QPCR (RQ). A) KLF9 and KLF13 transcript levels in HESCs transfected with KLF13 and KLF9 siRNAs, alone or together, or with control (Scr) siRNAs. B) PRL, WNT4, and BMP2 transcript levels in HESCs transfected with KLF13 and KLF9 siRNAs, alone or together, or with control (Scr) siRNAs. The indicated genes were quantified by RQ and normalized to those of TATA box-binding protein (Tbp). Data (mean ± SEM) are expressed as fold-change relative to corresponding Scr (control) samples. Means with different lowercase letters differed at P < 0.05 by one-way ANOVA followed by Tukey test. C) Relative KLF13 transcript levels in control or KLF13 siRNA transfected cells (mean ± SEM), which were analyzed for ALP activity. Values were normalized to those of TATA box-binding protein (Tbp) and renormalized to corresponding Scr (control) samples. D) ALP activity of cell lysates prepared from decidualizing HESCs transfected with control (Scr) or KLF13 siRNAs. Data are from two independent experiments, performed in triplicates per experiment. OD, absorbance at 410 nm. *P < 0.05 (Student t-test).

Alkaline phosphatase activity is a functional measure of stromal decidualization in vitro [30]. We previously showed that KLF9 knockdown reduced ALP activity in HESCs [25]. HESCs treated with siKLF13 under the same experimental conditions, however, did not affect ALP activity relative to control (Scr-transfected) cells (Fig. 5D).

DISCUSSION

In previous studies, we showed that reproduction in mice is influenced by the Sp-family member KLF9, in part, by virtue of its coregulation of PGR transcriptional activity in stromal cells [20, 22, 24, 25]. Loss of KLF9 expression in the uterus resulted in poor pregnancy outcome, uterine growth defects, partial uterine P4 resistance, and delayed onset of parturition [20, 22, 27]. The experiments in the present study show, to our knowledge for the first time, that loss of expression of KLF9's most closely related family member, KLF13, is not critical for the maintenance of fertility in mice, most likely by virtue of KLF9's functional compensation during this period of uterine receptivity. These findings highlight the functional hierarchy of KLF9 over KLF13 in the uterus and predict, based on the in vitro results presented here using HESCs with coknockdowns of KLF9 and KLF13 relative to individual knockdowns, that the coincident loss of both KLF family members will significantly compromise early pregnancy events. These findings further imply that whereas KLF9 may require numerous compensatory mechanisms for its loss, one of which involves a partial KLF13 takeover [25], the compensatory mechanism for loss of uterine KLF13 may be totally dependent on KLF9. To compensate for loss of KLF13 expression and, thus, maintain PGR-B expression and transactivation of target genes, KLF9 may induce the expression of and/or increase the interaction with PGR-B as well as promote the expression and/or activity of other KLF family members, such as KLFs 3, 4, 7, and 12, all of which have been recently reported as candidate mediators of PGR function in P4-responsive target tissues [31, 32].

The predominant loss of PGR expression in adult, cycling Klf13 null stromal cells (which was not observed in corresponding LE cells) in the absence of an accompanying increase in nuclear KLF9 levels (as noted in early pregnant Klf13 null uteri) is consistent with the stromal compartment being the major site of KLF9 and KLF13 expression [20, 25] and provides further support for the reliance of KLF13 on KLF9 to compensate for its function. It is therefore intriguing that we did not observe any major uterine phenotype (i.e., changes in uterine morphometry) in nonpregnant, adult Klf13 null mice. Although speculative, a possible explanation for this finding resides with PGR-A, which is highly expressed relative to PGR-B in the cycling adult uteri. In this regard, whereas PGR-A and PGR-B are considered to have distinct gene targets and PGR-A is considered to antagonize PGR-B signaling [33], PGR-A is critical to mouse uterine function [7, 34]. Nevertheless, it is premature to discount the contributory role of KLF13 to uterine physiology and pathology under other contexts.

Interestingly, whereas loss of KLF9 function was accompanied by increased KLF13 synthesis [25], the converse was not observed for uteri of Klf13 null mice. The higher nuclear levels of KLF9 in Klf13 null preimplantation (DPC 3.5) uteri, in the absence of coincident increases in transcript and total cellular protein levels, suggest enhancement of KLF9 nuclear localization and/or retention as a mechanism by which KLF9 function may be increased to ensure uterine PGR sensitivity. Like other transcription factors, the activities of numerous KLFs are regulated by their nuclear localization [35–37]. KLF family members possess one or more monopartite nuclear localization signals adjacent to or within their C-terminal DNA-binding domains and that are conserved in all KLFs [35–39]. The signal for nuclear transport or nuclear retention of KLF9 protein has not been definitively defined, but these activities may be promoted by coupling to Sin3a/b [40], similar to that recently demonstrated for KLF6 [37] or to poly(ADP-ribose) polymerase, as reported for KLF8 [41]. Further studies to evaluate the regulation of KLF9 (and KLF13) nuclear import and retention may contribute to novel mechanisms for regulating their stability and functions in pregnancy and in diseases caused by P4 resistance.

To convincingly address the compensatory functions of KLF9 and KLF13 in pregnancy will require the generation of conditional Klf9/Klf13 double-knockouts in the uterus, given the predisposition to neonatal mortality of Klf9 [27] and Klf13 (present study) null mice. Our results show that in decidualizing HESCs in vitro, siRNA targeting of KLF9 alone resulted in reduced WNT4 expression, whereas that of KLF13 alone resulted in reduced BMP2 expression. However, the significant attenuated expression of PRL with KLF9 and KLF13 siRNAs together, which was not observed with individual KLF knockdowns, raises the likelihood of a highly compromised uterus in vivo with coloss of these KLF members. Specifically, given the sequential expression of KLF9 and KLF13 in the pregnant uteri [25] and the role for KLF9 in the timing of uterine receptivity beginning at DPC 2.5 [22], we predict that the concurrent loss of KLF9 and KLF13 will drastically alter the timely progression of stromal and epithelial proliferation and differentiation that are requisite for embryo implantation, resulting in infertility. Nevertheless, the reduced ALP activity with loss of KLF9 expression [25], but not with loss of KLF13 expression (Fig. 5D), suggests the more prominent role of KLF9, relative to KLF13, in the peri-implantation uterus, consistent with the subfertility phenotype of the Klf9 null mice. Whether other KLFs might also compensate for the functions of KLF9 and KLF13 is currently unknown, albeit a possibility, given recent studies suggesting a subset of KLF members serve as integrators of E2 and P4 signaling [31, 32, 42, 43].

A common feature of global Klf9 and Klf13 knockout in female mice is the lack of an ovarian phenotype. Specifically, both Klf9 [22] and Klf13 (present study) null females exhibit normal steroid hormone production, as evidenced by their ability to normally cycle and achieve pregnancy. Although our results are not supported by the recent report that KLF9 and KLF13 are transcriptional activators of the steroidogenic genes LDLR, StAR, and CYP11A in ovarian granulosa cells [44], suggesting that loss of their respective expression should lead to reduced steroidogenesis, it is possible that the KLF9-regulated gene KLF4 [23] may take over the roles of KLF9 and KLF13 in a compensatory manner, as demonstrated for members of this family [25, 45] (present study). Alternatively, KLF9 and KLF13 may compensate for each other's function in the context of the ovary.

In conclusion, using mice null for Klf13, we report the existence of a compensatory function of uterine KLF9 for KLF13 during the critical window of uterine receptivity. Our studies provide a novel mechanism (i.e., increased nuclear localization/retention) by which KLF9 may take over the function of KLF13 to maintain the appropriate expression of PGR and Wnt signaling components. Given the lack of a uterine phenotype with loss of Klf13 expression and that KLF4 is a target of KLF9 [23] but not KLF13 regulation, we propose that KLF9 occupies a higher position relative to KLF13 in the hierarchy of PGR transactivators to mediate uterine function for optimal embryo implantation.

Footnotes

1

Supported by grants from the National Institutes of Child Health and Human Development HD21961 (R.C.M.S.), Arkansas Biosciences Institute/Arkansas Children's Hospital Research Institute (R.C.M.S.), Arkansas Children's Hospital Research Institute Student and Clinical Staff Intramural Program (M.E.H.), and University of Arkansas for Medical Sciences Translational Research Institute Grant UL1TR000039.

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