Review of psychological stress on oocyte and early embryonic development in female mice

Generally, the embryo becomes implanted into the proximal endoderm and initiates early development. Around embryonic day 6.5 (E6.5), some ectodermal cells transform into the precursor cells of primordial germ cells (PGCs) [28]. With the inhibition of somatic cell expression module, the precursor cells of PGCs complete the specification process, subsequently migrate into the hindgut endoderm, and ultimately colonize the genital ridges (~E10.5). Following embryonic development, PGCs gradually enter meiosis and differentiate into oogonia at approximately E13.5, some of which end up with primordial follicles. Most of these primordial follicles are kept dormant, with only a fraction recruited to activate the growth process driven by bidirectional communication between the oocytes and the surrounding somatic cells [29], after which the primordial follicles further develop into secondary follicles and finally antral follicles [30]. The production of antral follicles largely depends on the secretion of multiple sex hormones, including gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH), luteinizing hormone (LH) and oestrogens (E2) [31]. Most of the follicles undergo atretic degeneration, and only a subset of antral follicles (known as the dominant follicles) are ready to ovulate, which is triggered by LH. In addition, the granulosa and theca cells form the corpus luteum secrete P4 to maintain pregnancy [32, 33] (Fig. 1, oogenesis).

In the mouse, fertilization is achieved when a haploid sperm merges with an oocyte to form a diploid zygote, which is the starting point of new life [34]. Following fertilization in the oviduct, the zygote grows to a morula, which consists of approximately 8–16 cells after several cycles of mitotic divisions [35, 36]. The morula enters the uterine lumen and transforms into a blastocyst, which could be divided into two distinct layers: the inner cell mass (ICM) layer, i.e., a cluster of pluripotent cells attached to one inner side of the trophectoderm; and the outer epithelial cell layer, known as the trophectoderm (TE) (the precursor of trophoblast cells) [31] (Fig. 1, early embryo formation and development). Subsequently, embryos mature and escape from the zona pellucida, acquire implantation competence, and start post-implantation development.

Currently, the chronic unpredictable mild stress (CUMS) model is widely accepted for psychological stress studies, especially in oocytes and early embryonic development. The CUMS paradigm consists of a series of mild and unpredictable stressors, including restraint, isolation or crowded housing, food or water deprivation, disruption of the dark-light cycle, and dampened bedding. Although different patterns of stress responses were triggered by various responses in animals, no invasive physical procedure or tissue trauma was allowed in psychological stress models [37‐41]. Importantly, many researchers have demonstrated that CUMS leads to long-lasting changes in behavioural, neurochemical, neuroimmune and neuroendocrinological activities, which resemble dysfunctions observed in depressed patients [42].

Oocytes are largely influenced by psychological stress. The number of atretic antral follicles increased in mice under psychological stress, which was accompanied by impaired follicular development due to a lack of corpus luteum, pyknotic granulosa cells, and an irregular oestrous cycle [43]. Tan and colleagues investigated the effects of psychological stress on female mice using two methods, repeated restraint stress for 23 days or CUMS twice a day for 4 days, and found that the degree of damage to the oocytes following psychological stress was related to the duration and severity of the stress. Moreover, the accumulating effects of stress impair the developmental potential of oocytes, and the oocytes in preantral follicles are less sensitive than those in antral follicles [44]. Maternal separation (MS) as a model of early life stress causes a certain degree of psychological stress for offspring. The developmental parameters of in vitro cultured pre-antral follicles (PF), such as survival, growth, formation of antrum cavity, ovulation, as well as oocyte maturation were affected. This study also reported that psychological stress decreased follicular development by altering the oxidative status, which might lead reduced adult reproductive potential [45].

Using different stress animal models, psychological stress was found to inhibit both secondary and antral follicular development, as well as increase follicular atresia, which may be due to the suppressed expression of growth and differentiation factor 9 (GDF9) in mice [38]. Similarly, additional studies reported that the expression of brain-derived neurotropic factor (BDNF, a stress-responsive intercellular messenger protein) in antral follicles is reduced when mice are exposed to psychosocial stress [46].

Germinal vesicle breakdown (GVBD) is the key event conferring oocyte quality [47]. Following a 4-week psychological stress treatment, oocytes with abnormal metaphase II spindles (MII spindles) and non-surrounded nucleolus type (NSN-type) were increased, and the progression into M phase were delayed, suggesting that psychological stress compromised the meiotic competence of oocytes [41]. In addition, the percentage of surrounded nucleolus (SN) oocytes were significantly decreased during in vitro culture when female mice were subjected to psychological stress for 2 or 23 days [48]. Another investigation demonstrated that oocyte aneuploidy at MII stage was higher in maternal mice exposed to psychological stress, which was caused by impaired metaphase I (MI) spindle assembly [22].

Rat studies revealed that psychological stress decreased the number of secondary and antral follicles [49]. Rats in the CUMS groups showed irregular oestrous cycles caused by marked changes in the antral follicles and atretic follicles, which was partly attributed to BDNF-mediated PI3K/Akt pathway regulation [50]. Interestingly, more cystic follicles were found in the ovaries after similar psychological stress lasted for 12 weeks, suggesting that psychological stress may lead to polycystic ovary (PCO) [51]. Wang et al. reported that CUMS may cause premature ovarian failure (POF) in female rats. Thus, the CUMS model may be suitable for further assessment of the pathogenesis of psychological stress-induced POF [52] (Fig. 1, key events, and psychological stress exposure).

Given the sensitive nature, an early embryo is more vulnerable to prenatal stress than at later stages [53]. According to previous reports, restraint stress in mice impairs the blastocyst activation and hatching, as well as uterine receptivity in a time-dependent manner, and implantation impairment caused by stress is closely connected to both the embryo and the uterus [54]. Even if more embryos could be found in the oviducts, only a few embryos could reach blastocyst stage in pregnant mice exposed for 5 h per day to restraint for 3 days to model psychological stress, and with the number of implantation sites were much less when the mice were restrained for 6 days [23]. Using immobilization restraint, another supporting study revealed that mice exposed to psychological stress had a 40% reduction in the number of implantation sites and fewer delivered litters than controls [55]. Likewise, psychological stress that occurs during pregnancy influenced the developmental capacities of embryos, delayed blastocyst hatching, and even implantation failure [54], and one of those studies further revealed that psychological stress exposure decreased the number of ICM and TE cells in mouse blastocysts unevenly, and resulted in a higher ICM/TE ratio [56, 57]. Less retrieved oocytes, a lower fertilization rate, and less 2-cell embryos and blastocysts were detected in female mice when exposed for 4 weeks to nine stressors. This study also demonstrated that heat shock proteins 70 (HSP70), a cellular stress protein, plays a protective role in mice subjected to CUMS [58]. Additionally, maternal restraint stress at the oocyte pre-maturation stage resulted in higher percentages of aneuploid 2- to 4-cells, which subsequently led to lower percentages of parthenotes developing to either 4-cell or blastocyst stages [22]. In vivo embryo development of oocytes derived from mice restrained for 23 days showed that both the number of blastocysts obtained per mouse and the cell counts per blastocyst were significantly lower, and the average number of young per recipient decreased after embryo transfer compared to unstressed mice [48]. An in vitro study by Liu and collaborators provided evidence that the blastocyst formation rates in psychologically stressed mice are significantly decreased [46].

Applying a novel psychological stress model (mice were placed in oblong micro-cages in which they could not turn around) also resulted in reduced rates of blastocyst formation and cell counts per blastocyst both in vitro and in vivo. Notably, the number of young and their birth weights from stressed donors decreased significantly compared to those of control donors after embryo transfer [21], which was consistent with the results observed in mice exposed to natural predators [20]. Furthermore, systematic studies have shown that intrauterine growth retardation, disrupted organogenesis, faulty sex differentiation and external malformations were found in pregnant mice subjected to severe restraint procedures during early pregnancy [44]. Additional evidence suggests that the activation of the Fas system is required for detrimental effects to embryo development, as well as apoptosis in oviducts and embryos triggered by preimplantation restraint stress (PIRS) in mice. Increased tumour necrosis factor alpha (TNF-α) levels in oviducts and embryos implies that proinflammatory cytokines are also involved in apoptosis [59] (Fig. 1, key event, and psychological stress exposure).