Uterine Deletion of Bmal1 Impairs Placental Vascularization and Induces Intrauterine Fetal Death in Mice

Abstract

Recently, it was demonstrated that the expression of BMAL1 was decreased in the endometrium of women suffering from recurrent spontaneous abortion. To investigate the pathological roles of uterine clock genes during pregnancy, we produced conditional deletion of uterine Bmal1 (cKO) mice and found that cKO mice could receive embryo implantation but not sustain pregnancy. Gene ontology analysis of microarray suggested that uterine NK (uNK) cell function was suppressed in cKO mice. Histological examination revealed the poor formation of maternal vascular spaces in the placenta. In contrast to WT mice, uNK cells in the spongiotrophoblast layer, where maternal uNK cells are directly in contact with fetal trophoblast, hardly expressed an immunosuppressive NK marker, CD161, in cKO mice. By progesterone supplementation, pregnancy could be sustained until the end of pregnancy in some cKO mice. Although this treatment did not improve the structural abnormalities of the placenta, it recruited CD161-positive NK cells into the spongiotrophoblast layer in cKO mice. These findings indicate that the uterine clock system may be critical for pregnancy maintenance after embryo implantation.

Keywords: BMAL1; NK cell; cKO mouse; miscarriage; placental formation; vascularization.

Figure 1

Bmal1 cKO uteri showed efficient deletion of BMAL1 and no live birth. (A–C) The uterine deletion of BMAL1 was observed by (A) RT-PCR, (B) Western blotting, and (C) immunofluorescence. (D) Uterine Bmal1 cKO mice showed no live births. The bar shows SE. ACTB, ACTIN; em, endometrium; mm/i, inner circular layer of myometrium; mm/o, the outer longitudinal layer of myometrium; ND, not determined. The scale bar shows 200 µm.

Figure 2

The miscarriage rate is increased in cKO mice after implantation. (A,B) Representative of uteri and HE-stained section of implantation sites from WT and cKO on days 5 (A) and 6 (B) of pregnancy. (C) Representative of uteri of WT and cKO on day 8. (D) HE-stained section of implantation sites and area of the blood vessel (BV) on day 8. (E) Although the difference is not significant, the calculated pixel areas of the blood vessel cavities in the mesometrial region in the cKO seem decreased compared to those in WT. The scale bar shows 100 µm. Arrowheads show embryo. Circles show uterine vessels in the mesometrial region. le, luminal epithelium; ge, glandular epithelium; st, endometrial stroma; mm, myometrium.

Figure 3

cKO mice show abnormal placental formation on day 12 of pregnancy. (A) Representative of uteri from WT and cKO on day 12 of pregnancy. (B–D) HE-stained section of the placenta. Blue-dotted areas show maternal blood vessels. Red-dotted areas show fetal blood vessels which contain nucleated erythrocytes. (E) The percentage of the maternal blood vessels that contains denucleated red blood cells (RBC). The bar shows SE. Differences in the ratio of calculated areas of maternal vessels between the wild-type and cKO mice were analyzed by the unpaired t-test. p < 0.05. The scale bar shows 100 µm.

Figure 3

cKO mice show abnormal placental formation on day 12 of pregnancy. (A) Representative of uteri from WT and cKO on day 12 of pregnancy. (B–D) HE-stained section of the placenta. Blue-dotted areas show maternal blood vessels. Red-dotted areas show fetal blood vessels which contain nucleated erythrocytes. (E) The percentage of the maternal blood vessels that contains denucleated red blood cells (RBC). The bar shows SE. Differences in the ratio of calculated areas of maternal vessels between the wild-type and cKO mice were analyzed by the unpaired t-test. p < 0.05. The scale bar shows 100 µm.

Figure 4

The immune environment in cKO uteri. (A) Gene ontology analysis with microarray data. The top 11 groups are shown. (B) DBA/PAS double staining with the placenta on day 12 of pregnancy. Black arrowheads show PAS-positive and DBA-negative cells. White arrowheads show both PAS- and DBA-positive cells. (C) In cKO mice, numbers of DBA-positive and -negative uNK cells were significantly lower than those in WT. (D) CD161 staining with the placenta on day 12 of pregnancy. In the WT placenta, CD161 was expressed on uNK cells in both the decidua (upper panel, white arrowheads) and spongiotrophoblast layers (upper panel, arrows). In contrast, uNK cells in the decidua layer of cKO mice expressed CD161 (lower panel, white arrowheads) but its expression was hardly detected in uNK cells in the spongiotrophoblast layer (lower panel, arrows). Arrows show CD161 positive cells. The scale bar shows 100 µm.

Figure 5

P4 supplement rescues pregnancy failure in cKO mice. (A) Serum P4 levels in WT and cKO on days 8 and 12 of pregnancy. The mean P4 levels in the control group (WT, n = 5) and in the cKO groups with normal implantation (w/IS, n = 2) and with confirmed inhalation (w/o IS, n = 4) on day 8 of pregnancy, and those in the control group (WT, n = 4), the cKO groups with implantation (w/IS, n = 4) and without implantation (w/o IS, n = 6), and the progesterone-treated cKO group (w/P4, n = 5) on day 12 of pregnancy. The bar shows STDEV. Differences between groups were analyzed by the unpaired t-test. *, p < 0.01; **, p < 0.05. (B,C) Representative of the uterus of WT and cKO with P4 supplementation on days 8 (B) and 12 (C) of pregnancy. (D,E) Representative of HE-stained uterus of WT and cKO with P4 supplementation on days 8 (D) and 12 (E). (F) The percentage of maternal blood vessels, which contain denucleated red blood cells (RBC). The bar shows SE. Differences in the ratio of calculated areas of maternal vessels between the WT and cKO mice were analyzed by the unpaired t-test. (G) DBA/PAS double staining of the placenta from cKO with P4 supplementation on day 12 of pregnancy. Black arrowheads show PAS-positive and DBA-negative cells. White arrowheads show both PAS- and DBA-positive cells. (H) Numbers of DBA-positive and -negative uNK cells were not significantly different between cKO and cKO witn P4 supplementation. (I) CD161 staining of the placenta from cKO with P4 supplementation on day 12 of pregnancy. Arrows show CD161 positive cells in the spongiotrophoblast layers. The scale bar shows 100 µm. N.S., not significant.

Figure 5

P4 supplement rescues pregnancy failure in cKO mice. (A) Serum P4 levels in WT and cKO on days 8 and 12 of pregnancy. The mean P4 levels in the control group (WT, n = 5) and in the cKO groups with normal implantation (w/IS, n = 2) and with confirmed inhalation (w/o IS, n = 4) on day 8 of pregnancy, and those in the control group (WT, n = 4), the cKO groups with implantation (w/IS, n = 4) and without implantation (w/o IS, n = 6), and the progesterone-treated cKO group (w/P4, n = 5) on day 12 of pregnancy. The bar shows STDEV. Differences between groups were analyzed by the unpaired t-test. *, p < 0.01; **, p < 0.05. (B,C) Representative of the uterus of WT and cKO with P4 supplementation on days 8 (B) and 12 (C) of pregnancy. (D,E) Representative of HE-stained uterus of WT and cKO with P4 supplementation on days 8 (D) and 12 (E). (F) The percentage of maternal blood vessels, which contain denucleated red blood cells (RBC). The bar shows SE. Differences in the ratio of calculated areas of maternal vessels between the WT and cKO mice were analyzed by the unpaired t-test. (G) DBA/PAS double staining of the placenta from cKO with P4 supplementation on day 12 of pregnancy. Black arrowheads show PAS-positive and DBA-negative cells. White arrowheads show both PAS- and DBA-positive cells. (H) Numbers of DBA-positive and -negative uNK cells were not significantly different between cKO and cKO witn P4 supplementation. (I) CD161 staining of the placenta from cKO with P4 supplementation on day 12 of pregnancy. Arrows show CD161 positive cells in the spongiotrophoblast layers. The scale bar shows 100 µm. N.S., not significant.