Preimplantation maternal stress impairs embryo development by inducing oviductal apoptosis with activation of the Fas system

Abstract

Study question: What are the mechanisms by which the preimplantation restraint stress (PIRS) impairs embryo development and pregnancy outcome?

Summary answer: PIRS impairs embryo development by triggering apoptosis in mouse oviducts and embryos,and this involves activation of the Fas system.

What is known already: Although it is known that the early stages of pregnancy are more vulnerable than later stages to prenatalstress, studies on the effect of preimplantation stress on embryo developmentare limited. Furthermore, the mechanisms by which psychological stress impairs embryo development are largely unknown. These issues are worth exploring using the mouse PIRS models because restraint of mice is an efficient experimental procedure developed for studies of psychogenic stress.

Study design, size and duration: Mice of Kunming strain, the generalized lymphoproliferative disorder (gld) mice with a germline mutation F273L in FasL in a C57BL/6J genomic background and the wild-type C57BL/6J mice were used. Female and male mice were used 8-10 weeks and 10-12 weeks after birth, respectively. Female mice showing vaginal plugs were paired by weight and randomly assigned to restraint treatments or as controls. For restraint treatment, an individual mouse was put in a micro-cage with food and water available. Control mice remained in their cages with food and water during the time treated females were stressed.

Participants/materials, setting, methods: Female mice were exposed to PIRS for 48 h starting from 16:00 on the day of vaginal plug detection. At the end of PIRS, levels of glucorticoids (GC), corticotropin-releasing hormone (CRH)and redox potential were measured in serum, while levels of GC, GC receptor (GR), CRH, CRH receptor (CRHR), Fas and Fas ligand (FasL) protein, mRNAs for brain derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1), oxidative stress (OS) and apoptosis were examined in oviducts. Preimplantation development and levels of GR, Fas, redox potential and apoptosis were observed in embryos recovered at different times after the initiation of PIRS. The gld mice were used to confirm a role for the Fas system in triggering apoptosis of embryos and oviducts.

Main results and the role of chance: Compared to those in control mice, while the number of blastocysts/mouse (5.0 ± 0.7 versus 11.1 ± 0.5), cell number/blastocyst (49.1 ± 1.3 versus 61.5 ± 0.9), percentages of term pregnancy (37.5% versus 90.9%) and litter size (3.7 ± 0.1versus 9.6 ± 0.6) decreased, blood CRH (560 ± 23 versus 455 ± 37 pg/ml), cortisol (27.3 ± 3.4 versus 5 ± 0.5 ng/ml) and OS index (OSI: 2.8 versus 1.7) increased significantly (all P < 0.05) following PIRS. In the oviduct, while levels of CRH (1175 ± 85 versus 881 ± 33 pg/100 mg), cortisol (28.9 ± 1.7 versus14 ± 4 ng/g), CRHR (2.3 ± 0.3 versus 1.0 ± 0.0), FasL (1.31 ± 0.06 versus 1.08 ± 0.05 ng/g), Fas (1.42 ± 0.13 versus 1.0 ± 0.0) and apoptotic cells (19.1 ± 0.5% versus 8.4 ± 0.4%) increased, levels of GR proteins (0.67 ± 0.14 versus 1.0 ± 0.0) and Igf-1 (0.6 ± 0.09 versus 1.0 ± 0.0) and Bdnf (0.73 ± 0.03 versus 1.0 ± 0.0) mRNAs decreased significantly (all P < 0.05 versus control) after PIRS. Mouse embryos expressed GR and Fas at all stages of preimplantation development and embryo OS (GSH/GSSG ratio: 0.88 ± 0.03 versus 1.19 ± 0.13) and annexin-positive cells (blastocysts: 31.4 ± 3.8% versus 10.96 ± 3.4%) increased significantly (P < 0.05) following PIRS. Furthermore, the detrimental effects of PIRS on embryo development and oviductal apoptosis were much reduced in gld mice. Thus, PIRS triggered apoptosis in oviductal cells with activation of the Fas/FasL system. The apoptotic oviductal cells promoted embryo apoptosis with reduced production of IGF-1 and BDNF and increased production of FasL.

Limitations, reasons for caution: Although important, the conclusions were drawn from limited results obtained using a single model in one species and thus they need further verification using other models and/or in other species. Furthermore, as differences in stressed samples were modest and sometimes not significant between gld and wild-type mice whereas differences between control and stressed samples were always present within gld mice, it is deduced that signaling pathways other than the Fas/FasL system might be involved as well in the PIRS-triggered apoptosis of oviducts and embryos.

Wider implications of the findings: The data are important for studies on the mechanisms by which psychological stress affects female reproduction, as FasL expression has been observed in human oviduct epithelium.

Large scale data: Not applicable.

Study funding and competing interests: This study was supported by grants from the National Basic Research Program of China (Nos. 2014CB138503 and 2012CB944403), the China National Natural Science Foundation (Nos. 31272444 and 30972096) and the Animal breeding improvement program of Shandong Province. All authors declare that their participation in the study did not involve factual or potential conflicts of interests.

Keywords: Fas/FasL; apoptosis; embryo development; oviduct; preimplantation stress.