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.

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.

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.

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.

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.

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.

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).

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.

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).

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.