Address all correspondence and requests for reprints to: Martin M. Matzuk, MD, PhD, Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas 77030. E-mail: ude.mcb@kuztamm.

Received 2016 Aug 29; Accepted 2017 Jan 12.

Abstract

In women, the window of implantation is limited to a brief 2- to 3-day period characterized by optimal levels of circulating ovarian hormones and a receptive endometrium. Although the window of implantation is assumed to occur 8 to 10 days after ovulation in women, molecular markers of endometrial receptivity are necessary to determine optimal timing prior to embryo transfer. Previous studies showed that members of the bone morphogenetic protein (BMP) family are expressed in the uterus necessary for female fertility; however, the role of BMP7 during implantation and in late gestation is not known. To determine the contribution of BMP7 to female fertility, we generated Bmp7^-Pgr-cre^{[BMP7 conditional knockout (cKO)] mice. We found that absence of BMP7 in the female reproductive tract resulted in subfertility due to uterine defects. At the time of implantation, BMP7 cKO females displayed a nonreceptive endometrium with elevated estrogen-dependent signaling. These implantation-related defects also affected decidualization and resulted in decreased expression of decidual cell markers such as}Wnt4, Cox2, Ereg, and Bmp2. We also observed placental abnormalities in pregnant Bmp7 cKO mice, including excessive parietal trophoblast giant cells and absence of a mature placenta at 10.5 days post coitum. To establish possible redundant roles of BMP5 and BMP7 during pregnancy, we generated double BMP5 knockout/BMP7 cKO [BMP5/7 double knockout (DKO)] mice; however, we found that the combined deletion had no additive disruptive effect on fertility. Our studies indicate that BMP7 is an important factor during the process of implantation that contributes to healthy embryonic development.

Abstract

Successful implantation requires a competent blastocyst and a receptive endometrium. As the first point of contact between the mother and developing embryo, the endometrium is essential for successful reproduction. Therefore, it is crucial to understand the factors and signaling pathways that contribute to endometrial receptivity. Hormones, such as estrogen (E2) and progesterone (P4), contribute to endometrial receptivity by inducing the proliferation and differentiation of endometrial stromal and epithelial cells. Finely balanced signaling by E2 and P4 define the “window of implantation,” the period of time during which endometrial receptivity to an implanting blastocyst is optimal (1). The E2- and P4-mediated signals are transmitted via the steroid hormone receptors estrogen receptor α and progesterone receptor (2) and by other transcription factors such as COUP-TFII (3, 4) and Hand2 (5).

The TGFβ family of proteins is a group of secreted factors that controls developmental processes such as cellular differentiation and proliferation and is highly conserved throughout evolution (6). Bone morphogenetic proteins (BMPs) are members of the TGFβ family and signal by binding to a receptor complex composed of two BMP type 1 receptors (ALK2, ALK3, or ALK6) and two type 2 receptors (BMPR2, ACVR2A, or ACVR2B) (7–9). After BMP binding, the receptors phosphorylate SMAD1 and/or SMAD5, which then associate with SMAD4, and together translocate to the nucleus, where they control the expression of target genes in a context-dependent manner (10).

Signaling through TGFβ is important for female reproduction (11, 12). Mouse models have demonstrated the contributions of various members of the TGFβ family to uterine development and during pregnancy. For example, Tgfbr1/ALK5 is necessary for development of uterine smooth muscle and oviduct integrity (13). Likewise, conditional deletion of Tgfbr1/ALK5 with progesterone receptor cre (Pgr-cre) results in severe subfertility with pronounced placental defects (14). Uterine ALK4 is also important during late gestation, and its conditional deletion results in defective placental development and abnormal expansion of the giant trophoblast cells (15).

In the uterus, the BMP signaling pathway is involved in the regulation of blastocyst implantation, endometrial stromal cell decidualization, and placental development (16–19). Uterine BMP2 (17, 20) and the BMP receptor ALK2 (16) are necessary for female fertility, and their conditional deletion prevents endometrial stromal cell decidualization. Conditional deletion of BMPR2 in the female reproductive tract results in growth-restricted embryos, abnormal uterine vascularization, and defective placental development (19). ALK6 knockout (KO) mice are infertile, have cumulus cell expansion defects, and lack endometrial glands (21). Conditional deletion of SMAD1, SMAD5, and the common SMAD4 with Amhr2-cre results in female sterility; the mice have abnormal smooth muscle development in the oviduct and myometrium and defective decidualization (22). We recently showed that BMP signaling via ALK3 in the uterus is necessary for endometrial receptivity and that conditional deletion of ALK3 results in defective remodeling of the endometrial epithelium, abnormal response to E2 and P4, and sterility (18).

Despite the crucial roles of the BMP type 1 and 2 receptors during pregnancy, the specific ligands that signal via the ALK3 and BMPR2 receptors are not yet known. Previous studies demonstrated that BMP2, BMP5, and BMP7 are expressed in the endometrium during the early stages of pregnancy (23). BMP2 females are infertile and have decidualization defects that prevent embryo development beyond implantation (17). BMP5 null mice were previously identified to be viable and fertile (24). However, the role of BMP7 in the female reproductive tract and the possible redundant roles of BMP5 and BMP7 have not been assessed. Here we demonstrate that conditional deletion of BMP7 in the female reproductive tract results in female subfertility due to defects during the early stages of pregnancy. Specifically, we demonstrate that absence of uterine BMP7 results in a nonreceptive endometrium, which causes abnormal implantation that leads to additional defects throughout gestation, such as abnormal decidual gene expression, excessive expansion of the parietal trophoblast giant cells during placentation, and embryo resorption.

Abbreviations: DBA, Dolichos biflorus lectin; SMA, smooth muscle actin.

Acknowledgments

We thank Ruihong Chen, Julio Agno, and Hannele Härkman for technical support, Drs. John P. Lydon and Francesco J. DeMayo for kindly sharing the Pgr-cre mice, and Dr. James F. Martin for the Bmp7 ^flox/flox mice.

Current Affiliations: C.C. is currently affiliated with Celmatix, New York, New York 10005. J.P. is currently affiliated with Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544.

Acknowledgments

This work was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development Grant R01-HD032067 (to M.M.M.), the Institutional Research and Academic Career Development Award K12-GM084897 (to D.M.), and a Postdoctoral Enrichment Program Award from the Burroughs Wellcome Fund (to D.M.).

Author contributions: D.M., C.C., J.P., P.T.F., R.P.-H., S.J.V., and M.M.M. designed the study. D.M., C.C., J.P., P.T.F., R.P.-H., and M.M.M. performed research. D.M., C.C., J.P., P.T.F., R.P.-H., and M.M.M. analyzed data. D.M., C.C., J.P., P.T.F., R.P.-H., and M.M.M. wrote the paper.

Disclosure Summary: The authors have nothing to disclose.

Acknowledgments

Footnotes

Abbreviations:

BMP: bone morphogenetic protein
cDNA: complementary DNA
cKO: conditional knockout
DBA: Dolichos biflorus lectin
DKO: double knockout
dpc: days post coitum
E2: estrogen
EPC: ectoplacental cone
H&E: hematoxylin and eosin
Ihh: Indian hedgehog
KO: knockout
P4: progesterone
PAS: periodic acid–Schiff
PCR: polymerase chain reaction
qRT: quantitative reverse transcription
SEM: standard error of the mean
SMA: smooth muscle actin
uNK: uterine natural killer
WT: wild-type.

Footnotes

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