PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of ijmsMDPIhomeThis articleThis journalInstructions for authorsSubscribeIJMS
 
Int J Mol Sci. 2010; 11(2): 719–730.
Published online 2010 February 11. doi:  10.3390/ijms11020719
PMCID: PMC2852863

Temporal and Spatial Regulation of miR-320 in the Uterus during Embryo Implantation in the Rat

Abstract

The implantation process is complex, requiring reciprocal interactions between implantation-competent blastocysts and the receptive uterus. There were reports to show that some microRNAs (miRNAs) may play a key role during embryo implantation in mouse. However, the miR-320 expression profiles in the rat uterus during peri-implantation are unknown. In the present study, we found that the expression level of miR-320 was lower on day 5 of gestation (g.d. 5) in rats than g.d.3 and g.d.4 and restored gradually from g.d.6. MiR-320 was specifically localized in glandular and luminal epithelia and decidua. The expression of miR-320 was not significantly different in the pseudopregnant uterus and decreased in the uteri of rats subjected to activation of delayed implantation. Artificial decidualization and treatment with progesterone increased the miR-320 expression. Thus, miR-320 was differentially expressed in the rat uterus during implantation. The expression level was affected by active blastocysts and decidualization during the window of implantation. Steroid hormones, progesterone stimulated miR-320 expression.

Keywords: miR-320, embryo implantation, uterus, rat, hormone, pregnancy

1. Introduction

Implantation is a highly coordinated sequence of events that begins with the attachment of an embryo to the uterine luminal epithelium and ultimately results in formation of the placenta. Implantation of the embryo to the uterine wall is regulated by various factors, for example, hormones [1,2], cytokines [3,4], growth factor [5,6], etc. Although a growing list of molecules are known to be involved in the implantation process, specifically, mechanisms associated with the onset of uterine receptivity and embryo implantation remains to be determined.

MicroRNAs (miRNAs) are small noncoding RNAs whose function as modulators of gene expression is crucial for proper control of cell growth [7,8] and are known to participate in mouse embryo implantation [9,10]. Mouse mir-320 was predicted [11] based on homology to a cloned human miRNA (MI0000542) [12] and cloned from mouse oocytes and testes [13]. The human miR-320 sequence was originally cloned from the normal mucosa-derived population of human[12] and rat miR-320 sequence was firstly found from large-scale cloning studies [14]. MiR-320 expression level was down-regulated in primary breast cancer (BC) [15], and inhibited HL-60 cell proliferation by targets transferrin receptor 1 (CD71) [16]. This implied that miR-320 may play an important role in the development of cancer. Embryo implantation shares similar phenomena and mechanisms with tumor invasion [17]. However, up to now, there is no information in the literature about how such miRNAs act on the rat uterus during embryo implantation.

Here, we report the expression pattern of miR-320 in the uterus during embryo implantation in the rat. We studied the effect of pseudopregnancy, artificial decidualization and activation of delayed implantation on the expression of miR-320. In addition, we also tested the effect of steroid hormones on miR-320 expression.

2. Results and Discussion

2.1. Differential expression of miR-320 in the rat uterus during the peri-implantation period

To study the role of miR-320 in embryo implantation, we first examined its temporal and spatial distribution in the uterus during the peri-implantation period in rat. Northern blot analysis showed that the expression level of miR-320 was lower on g.d. 5 in rats than g.d. 3 and g.d. 4 (p < 0.05), and restored gradually from g.d. 6, and was higher on g.d. 8 and g.d. 9 than g.d. 5 (p < 0.05) (Figure 1 A). The in situ hybridization results showed that the miR-320 was mainly located in the glandular, luminal epithelia and stroma on g.d. 3 and g.d. 4 (Figure 1 B a and b). This suggested that it might participate in uterine epithelial and endometrial remodeling in preparing it to receive implanting blastocysts. On g.d. 5, the miR-320 signal mainly appeared in the glandular epithelia and weak in the luminal epithelia and stroma (Figure 1 B c). A miR-320 signal was found in the deciduas, glandular and luminal epithelia on g.d. 6 (Figure 1 B d) and was strengthened in deciduas from g.d. 7 (Figure 1 B e). In rat, blastocysts entered into uterus on g.d. 5 and began to implant from g.d. 6. In this study, the implantation sites and interimplantation sites in uterus were not examined independently and whole uterus was collected to perform the experiment of Northern blot and in situ hybridization after embryo implantation. The in situ results were from interimplantation uterine regions on g.d. 6. The above-mentioned observations suggested that the presence of blastocysts may reduce the expression of miR-320 in the uterine luminal epithelia and stroma and decidualization may induce the expression of miR-320 in pregnant rats.

Figure 1.
Changes in uterine miR-320 expression during early pregnancy.

2.2. Pseudopregnancy did not change the expression of miR-320

To see whether the miR-320 expression was dependent upon the presence of embryos, uterine tissues were subjected to Northern blot and in situ hybridization analysis (Figure 2 A). The expression level of mir-320 detected by Northern blot was not significantly different in uterus during days 3–7 of pseudopregnancy. In situ hybridization showed that the miR-320 signal was mainly found in uterine glandular and luminal epithelia during days 3–7 of pseudopregnancy (Figure 2 B). The expression level of miR-320 is higher during g.d.3–4 than g.d. 5–7 in the stroma. These results suggested that the miR-320 expression is not dependent upon the presence of embryos in pregnant rats.

Figure 2.
Changes in uterine miR-320 expression during pseudopregnancy.

2.3. Delayed implantation inhibited the expression of miR-320

To test whether the miR-320 expression was dependent upon embryo implantation status, a delayed implantation model was used for Northern blot and in situ hybridization analyses. Northern blot showed a high level of miR-320 in the uterus under delayed implantation conditions, but it decreased significantly after implantation was activated with estrogen treatment (P < 0.05; Figure 3 A). In situ hybridization showed that a strong signal appeared in the uterine glandular and luminal epithelia and weak in stroma during delayed implantation (Figure 3 B a, b). After implantation was activated by estrogen treatment and the embryos had implanted, there was weak miR-320 expression in the stroma, glandular and luminal epithelia (Figure 3 B c), suggesting that down-regulation of miR-320 expression was dependent upon the presence of viable implanting blastocysts.

Figure 3.Figure 3.
The expression of miR-320 in the uterus of delayed implantation.

2.4. Experimentally induced decidualization increases the expression of miR-320

To test whether miR-320 expression was regulated by decidualization, a model of experimentally induced decidualization was used for Northern blot and in situ hybridization analyses. The expression level of miR-320 in the decidualized uterus was evidently higher than in the nonstimulated uterus on day 7 of pseudopregnancy (Figure 4 A). In situ hybridization showed strong staining in glandular and luminal epithelia in the control uterine horn on day 7 of pseudopregnancy (Figure 4 B a). However, in the oil-infused uterus, strong signals were detected in decidua but staining was weak in the luminal epithelium (Figure 4 B b). This indicated that artificial decidualization promoted the expression of miR-320 and further emphasize the importance of decidualization in regulating the dynamics of miR-320 expression in the uterus during the window of implantation.

Figure 4.
The expression of miR-320 in the uterus of artificial decidualization.

2.5. Progesterone enhances the miR-320 expression

Ovarian progesterone and estrogen are the principal hormones that direct uterine receptivity, embryo implantation and the maintenance of pregnancy in all mammals studied, and are essential for implantation in mice and rats [19,20].

In order to test the effect of steroid hormones on the miR-320 expression under physiological condition, Northern blot was performed to examine whether the miR-320 expression was regulated by steroid hormones. A low level of miR-320 expression was detected in the ovariectomized rat uterus. However, treatment with progesterone significantly increased miR-320 expression (P < 0.01) and estradiol-17β did not visibly affect the expression level of miR-320. The miR-320 expression was slightly increased by combination of both (Figure 5). All these facts indicated that progesterone can promote miR-320 expression under physiological condition.

Figure 5.
The effect of steroid hormones on uterine miR-320 expression. The effect of steroid hormones on uterine miR-320 expression steroid hormones on uterine miR-320 expression was detected by Northern blot. Hybridization was done with a 32P-labeled probe for ...

Progesterone (P4) is essential for the development of endometrial receptivity for blastocyst implantation under pregnant condition [28,29] and play an important role on the maintenance of female endocrine homeostasis by inhibiting the secretion of gonadotropin in hypothalamus. Endometrial receptivity for embryo implantation in the rat occurred on day 5 of pregnancy. The expression of miR-320 is decreased on g.d.5, but its expression was increased by progesterone under physiological condition. These results implied that the low expression of miR-320 may be benefit to formation of endometrial receptivity under pregnant condition and the high expression of miR-320 may be in favor of the maintenance of female endocrine homeostasis. In addition, there are striking similarities between the behavior of invasive placental cells and that of invasive cancer cells. Dysregulated expression of the miR-320 in many tumor [15,16] was strongly associated with tumor development, and miR-320 expression level was down-regulated greater than two fold in primary breast cancer (BC) [15]. These results imply that the action of progesterone on miR-320 expression might inhibit the excess invasion of cells from the uterus and trophectoderm.

3. Experimental Section

3.1. Experimental animals and protocols

Sexually mature, healthy female Sprague Dawley rats (220–260 g body weight) were purchased from the Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, PR China). The rats were housed in a temperature- and humidity-controlled room with a 12/12 h light/dark cycle. All animal procedures were approved by the Institutional Animals Care and Use Committee of the National Research Institute for Family Planning. The rats were caged overnight with fertile males of the same strain. The presence of a vaginal plug or sperm was considered to be day 1 of pregnancy (g.d.1). The whole uterus was collected from g.d. 3–5 rats. When embryos implant and placentae form, placentae were carefully peeled from dissected uterine horns and whole uterus including the peritoneum, myometrium and maternal decidua was collected from g.d. 6–9 rat. Divided uteri were respectively frozen in Eppendorf tubes and stored at 80 °C until processing for RNA extraction. Whole uteri or undivided uteri and placentae were fixed in 4% paraformaldehyde (PFA) solution (Sigma-Aldrich, St. Louis, MO, USA) in 0.1M phosphate buffer (pH7.4, 4 °C) for in situ hybridization analysis.

Pseudopregnancy was induced by caging adult females with vasectomized males, and mating was confirmed by checking for a vaginal plug (day 1 of pseudopregnancy). The whole uteri were collected from days 3–7 of pseudopregnancy. On day 5 of pseudopregnancy, when the uteri were optimally sensitized to deciduogenic stimuli, 100 μL olive oil (Sigma-Aldrich) was infused into the lumen of one of the uterine horns to induce artificial decidualization. The contralateral uterine horn, which was not infused with oil, served as a control. At day 7 of pseudopregnancy, the rats were sacrificed and the uterine horns were isolated.

To induce delayed implantation, the pregnant rats on g.d. 4 were ovariectomized. Progesterone (5 mg/rat, s.c.; Sigma-Aldrich) was injected to maintain delayed implantation from g.d. 5–7. The progesterone-primed delayed-implantation rats were treated with estradiol-17β (0.5 μg/rat; Sigma-Aldrich) to terminate delayed implantation. The rats were sacrificed by stunning and cervical dislocation to collect uteri 24 h after estrogen treatment. The implantation sites were also identified by i.v. injection of Chicago blue solution (Sigma-Aldrich). Delayed implantation was confirmed by flushing the blastocysts from the uterus.

To test the effects of steroid hormones on miR-320 expression, rats were treated with hormones starting 2 weeks after they were ovariectomized. The ovariectomized rats were treated with an injection of estradiol-17β (1 μg/rat) or progesterone (10 mg/rat) at intervals of 24 h for 3 d. All steroids were dissolved in olive oil and injected subcutaneously. Controls received the vehicle only (0.1 mL/rat).

3.2. Northern blot analysis

Northern blot analysis of miRNAs was performed as described previously [22]. Briefly, total RNA was isolated from the uteri of rats with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Aliquots of 40 μg of total RNA per sample were subjected to electrophoresis on a 15% urea-PAGE gel and transferred to a nylon membrane (Hybond N+; Amersham Pharmacia Biotech, St Albans, Hertford, UK). After being UV cross-linked and baked at 50 °C for 30 min, the membrane was prehybridized at 42 °C for 4 h and then hybridized with 32P-labeled probes at 40 °C overnight. Membranes were washed and exposed to PhosphorImager screens (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA). The bands were analyzed using the Quantity One software (Bio-Rad, Hercules, CA, USA). All experiments were repeated at least three times.

3.3. In situ hybridization of miR-320 with DIG-labeled LNA probes

In situ hybridization of miRNAs with DIG-labeled LNA probes was performed as described previously [23]. Briefly, sections of uterus (5 μm) were treated with proteinase K (20 g/mL) for 15 min and refixed in 4% PFA for 15 min. After acetylation with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 10 min, sections were prehybridized with hybridization buffer (Roche, Mannheim, Germany) at 40 °C for 2h and then hybridized with digoxigenin (DIG)-labeled LNA-miR-320 probe (LNA-miR-320 sequence: 5′–DIG–ttCgcCctCtCaAcCcAgCtttt–3′) at 40 °C overnight. The cells were then incubated in buffer containing anti-DIG-antibody for 2 h at 37 °C and stained with 5-bromo-4-chloro-3-indolyl phosphate (BCIP; Promega, Madison, WI, USA) and p-nitroblue tetrazolium chloride (NBT; Promega, Madison, WI, USA). The cells and sections were hybridized with a DIG-labeled LNA-scrambled probe (LNA-scrambled sequences: 5′–caTtaAtgTcGgaCaaCtcAat–3′) as a negative control [24]. Samples were viewed with an Eclipse 80i microscope (Nikon, Tokyo, Japan).

3.4. Statistical analysis

There were at least three rats in each treatment group. The results of Northern blot and in situ hybridization were repeated three times. All values are reported as the mean ± SE. Statistical analysis was performed using one-way ANOVA. When significant effects of treatments were indicated, the Student–Newman–Keuls multiple range test was applied using SPSS version 13.0 (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered statistically significant.

4. Conclusions

In conclusion, we found that the miRNA miR-320 could be detected differentially in the rat uterus during the peri-implantation period. The results obtained from our models of pseudopregnancy, artificial decidualization and delayed implantation imply an important role for implanting blastocysts and decidualization in the temporal and spatial changes of miR-320 expression in the uterus during the window of implantation. In addition, this expression of miR-320 was regulated by progesterone. Collectively, these findings will help us gain a better understanding of the role of mir-320 during pregnancy and provide a foundation for futher experimental studies on mechanisms mechanisms associated with the onset of uterine receptivity and embryo implantation.

Acknowledgments

This work was supported by the Natural Science Foundation of China (No. 30800396) and the National Basic Research Program of China (No. 2007CB511905).

References

1. Venners SA, Liu X, Perry MJ, Korrick SA, Li Z, Yang F, Yang J, Lasley BL, Xu X, Wang X. Urinary estrogen and progesterone metabolite concentrations in menstrual cycles of fertile women with non-conception, early pregnancy loss or clinical pregnancy. Hum. Reprod. 2006;21:2272–2280. [PubMed]
2. Yoshinaga K. Review of factors essential for blastocyst implantation for their modulating effects on the maternal immune system. Semin Cell Dev Biol. 2008;19:161–169. [PubMed]
3. Sun QH, Peng JP, Xia HF, Yang Y, Liu ML. Effect on expression of RT1-A and RT1-DM molecules of treatment with interferon-gamma at the maternal--fetal interface of pregnant rats. Hum. Reprod. 2005;20:2639–2647. [PubMed]
4. Xia HF, Sun QH, Peng JP. Effect of interferon-gamma treatment on the expression of interleukin-1beta at the maternal-fetal interface of pregnant rats. Reprod. Fertil. Dev. 2007;19:510–519. [PubMed]
5. Sohlström A, Katsman A, Kind KL, Roberts CT, Owens PC, Robinson JS, Owens JA. Food restriction alters pregnancy-associated changes in IGF and IGFBP in the guinea pig. Am. J. Physiol. 1998;274:E410–E416. [PubMed]
6. Roberts CT, Kind KL, Earl RA, Grant PA, Robinson JS, Sohlstrom A, Owens PC, Owens JA. Circulating insulin-like growth factor (IGF)-I and IGF binding proteins -1 and -3 and placental development in the guinea-pig. Placenta. 2002;23:763–770. [PubMed]
7. Sampson VB, Rong NH, Han J, Yang Q, Aris V, Soteropoulos P, Petrelli NJ, Dunn SP, Krueger LJ. MicroRNA let-7a down-regulates MYC and reverts MYC-induced growth in Burkitt lymphoma cells. Cancer Res. 2007;67:9762–9770. [PubMed]
8. Abdelmohsen K, Srikantan S, Kuwano Y, Gorospe M. miR-519 reduces cell proliferation by lowering RNA-binding protein HuR levels. Proc. Natl. Acad. Sci. USA. 2008;105:20297–20302. [PubMed]
9. Chakrabarty A, Tranguch S, Daikoku T, Jensen K, Furneaux H, Dey SK. MicroRNA regulation of cyclooxygenase-2 during embryo implantation. Proc. Natl. Acad. Sci. USA. 2007;104:15144–15149. [PubMed]
10. Hu SJ, Ren G, Liu JL, Zhao ZA, Yu YS, Su RW, Ma XH, Ni H, Lei W, Yang ZM. MicroRNA expression and regulation in mouse uterus during embryo implantation. J. Biol. Chem. 2008;283:23473–23484. [PubMed]
11. Weber MJ. New human and mouse microRNA genes found by homology search. FEBS J. 2005;272:59–73. [PubMed]
12. Michael MZ, O'Connor SM, van Holst Pellekaan NG, Young GP, James RJ. Reduced accumulation of specific microRNAs in colorectal neoplasia. Mol. Cancer. Res. 2003;1:882–891. [PubMed]
13. Watanabe T, Takeda A, Tsukiyama T, Mise K, Okuno T, Sasaki H, Minami N, Imai H. Identification and characterization of two novel classes of small RNAs in the mouse germline: Retrotransposon-derived siRNAs in oocytes and germline small RNAs in testes. Genes Dev. 2006;20:1732–1743. [PubMed]
14. Landgraf P, Rusu M, Sheridan R, Sewer A, Iovino N, Aravin A, Pfeffer S, Rice A, Kamphorst AO, Landthaler M, Lin C, Socci ND, Hermida L, Fulci V, Chiaretti S, Foà R, Schliwka J, Fuchs U, Novosel A, Müller RU, Schermer B, Bissels U, Inman J, Phan Q, Chien M, Weir DB, Choksi R, De Vita G, Frezzetti D, Trompeter HI, Hornung V, Teng G, Hartmann G, Palkovits M, Di Lauro R, Wernet P, Macino G, Rogler CE, Nagle JW, Ju J, Papavasiliou FN, Benzing T, Lichter P, Tam W, Brownstein MJ, Bosio A, Borkhardt A, Russo JJ, Sander C, Zavolan M, Tuschl T. A mammalian microRNA expression atlas based on small RNA library sequencing. Cell. 2007;129:1401–1414. [PMC free article] [PubMed]
15. Yan LX, Huang XF, Shao Q, Huang MY, Deng L, Wu QL, Zeng YX, Shao JY. MicroRNA miR-21 overexpression in human breast cancer is associated with advanced clinical stage, lymph node metastasis and patient poor prognosis. RNA. 2008;14:2348–2360. [PubMed]
16. Schaar DG, Medina DJ, Moore DF, Strair RK, Ting Y. MiR-320 targets transferrin receptor 1 (CD71) and inhibits cell proliferation. Exp. Hematol. 2009;37:245–255. [PubMed]
17. Murray MJ, Lessey BA. Embryo implantation and tumor metastasis: common pathways of invasion and angiogenesis. Semin. Reprod. Endocrinol. 1999;17:275–290. [PubMed]
18. Dey SK, Lim H, Das SK, Reese J, Paria BC, Daikoku T, Wang H. Molecular cues to implantation. Endocr. Rev. 2004;25:341–373. [PubMed]
19. Wang H, Dey SK. Roadmap to embryo implantation: Clues from mouse models. Nat. Rev. Genet. 2006;7:185–199. [PubMed]
20. Quinn CE, Simmons DG, Kennedy TG. Expression of Cystatin C in the rat endometrium during the peri-implantation period. Biochem. Biophys. Res. Commun. 2006;349:236–244. [PubMed]
21. Dai B, Cao Y, Liu W, Li S, Yang Y, Chen D, Duan E. Dual roles of progesterone in embryo implantation in mouse. Endocrine. 2003;21:123–132. [PubMed]
22. Zhao JJ, Hua YJ, Sun DG, Meng XX, Xiao HS, Ma X. Genome-wide microRNA profiling in human fetal nervous tissues by oligonucleotide microarray. Child’s Nerv. Syst. 2006;22:1419–1425. [PMC free article] [PubMed]
23. Zhao JJ, Sun DG, Wang J, Liu SR, Zhang CY, Zhu MX, Ma X. Retinoic acid downregulates microRNAs to induce abnormal development of spinal cord in spina bifida rat model. Child’s Nerv. Syst. 2008;24:485–492. [PubMed]
24. Chan JA, Krichevsky AM, Kosik KS. MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. Cancer Res. 2005;65:6029–6033. [PubMed]

Articles from International Journal of Molecular Sciences are provided here courtesy of Multidisciplinary Digital Publishing Institute (MDPI)