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Cell Biology International (2007) 31, 2429 (Printed in Great Britain)
Nitric oxide affects preimplantation embryonic development in a rotating wall vessel bioreactor simulating microgravity
Yu‑jing Caoa1, Xun‑jun Fanab1, Zheng Shena, Bao‑hua Maac and En‑kui Duana*
aState Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 25 Beisihuan Xi Lu, Haidian, Beijing 100080, People's Republic of China
bGraduate School of the Chinese Academy of Sciences, 19 Yu-quan Road, Beijing 100039, People's Republic of China
cCollege of Animal Science and Technology, Northwest A&F University, 3 Taicheng Road, Yangling, Shaanxi 712100, People's Republic of China
Microgravity was simulated with a rotating wall vessel bioreactor (RWVB) in order to study its effect on pre-implantation embryonic development in mice. Three experimental groups were used: stationary control, rotational control and clinostat rotation. Three experiments were performed as follows. The first experiment showed that compared with the other two (control) groups, embryonic development was significantly retarded after 72
Keywords: Simulated microgravity, Embryonic development, Nitric oxide, Nitric oxide synthase, Apoptosis.
1These authors contributed equally to this work.
*Corresponding author. Tel.: +86 10 6255 8112 (Off), +86 10 6263 1831 (Lab); fax: +86 10 6263 1831.
Research over several years has shown that the absence or reduction of gravity affects human physiological functions. Bone loss occurs in weight-bearing bones under microgravity (Vico et al., 2000; Holick, 2000), and cardiovascular deconditioning has been described in astronauts (Carlsson et al., 2003). Space flight can have profound effects on the immune system (Borchers et al., 2002); a highly significant increase in geometric mean antibody titers against Epstein–Barr virus antigen has been reported in astronauts after space flight (Stowe et al., 2000). However, there are few studies on the effects of space flight on mammalian reproduction, a potentially important theme for the long-term future (Zhang and Duan, 2001).
Nitric oxide (NO), recently identified as a pivotal messenger molecule, is involved in many physiological processes (Furchgott et al., 1987). The rapid diffusion of NO between cells allows it to integrate the local responses of blood vessels to turbulence, to modulate synaptic plasticity in neurons, and to control the oscillatory behavior of neuronal networks (Barrete and Stress, 1996). NO synthesis from
Some studies show that microgravity affects NOS and NO production in mammals. When bone cells are cultured in a simulated microgravity condition, they show high NOS activity and produce more NO (Klein-Nulend et al., 2003). Microgravity up-regulates the expression of iNOS mRNA in rat cardiac myocytes (Xiong et al., 2003). NO is also a free radical and an important bioregulator of apoptosis (Chung et al., 2001), thought to stimulate apoptosis by activating Fas and the Fas ligand pathway in cultured human and rat pulmonary artery smooth muscle cells (Hayden et al., 2001). However, there are no reports on the effects of NO on mouse embryonic development or embryonic apoptosis under simulated microgravity conditions.
In this study, a rotating wall vessel bioreactor (RWVB) for simulating the microgravity of the space environment was used to investigate effects on pre-implantation embryonic development in mice. We analyzed the changes in NO and NOS in the culture media of the RWVB and control groups and detected apoptosis in the mouse embryos.
2 Materials and methods
Kunming strain white mice (Experimental Animal Center, the Genetic Institute of the Chinese Academy of Sciences) were housed in the animal facility of the State Key Laboratory of Reproductive Biology and raised at room temperature (approximately 25
2.2 Rotating wall vessel bioreactor (RWVB)
Microgravity simulation experiments were performed with a horizontal RWVB (Fig. 1A) (made by the Institute of Biophysics, the Chinese Academy of Sciences), which was used to produce a vector-free gravitational environment. The RWVB rotates horizontally so that the floor of the culture chamber turns continuously around an axis perpendicular to the gravity vector. Therefore, every part of the embryo surface experiences the gravitational vector during part of the rotation cycle, and this effectively cancels the vector by continuous averaging. This resembles the microgravity encountered in space and is a suitable model for studying early mammalian embryo development in vitro. The rotational speed was set at 100
Rotating wall vessel bioreactor. (A) Clinostat rotation group. The axis of rotation is perpendicular to the vector of gravity. Therefore, during rotation, every part of the embryo in the culture chamber is exposed to the gravity vector. This effectively cancels the effect of the vector by continuous averaging; (B) rotational control group. The axis of rotation is parallel to the vector of gravity. Therefore, the embryos are exposed to the gravity vector. The arrow shows the direction of the spinning, and “*” shows the place where the embryos were loaded.
The control consisted of two groups: (1) the rotational control group (Fig. 1B), which rotated the RWVB perpendicularly to eliminate the effect of rotation on mouse embryos under normal gravitational conditions; and (2) the stationary control group, in which the embryos were continuously exposed to normal gravity without rotation.
The horizontal rotation experimental group, the rotational control group and the stationary control group were all incubated simultaneously.
2.3 In vitro embryo culture
Mouse 8-cell embryos were flushed from the oviducts with Hank's medium on day 3 of pregnancy and cultured in RPMI-1640 (HyClone) without phenol red and supplemented with 2.0
2.4 NO and NOS assays
NO and NOS kits were purchased from the Jingmei Corporation of China. The assays were performed according to the manufacturer's instructions.
2.5 Embryo apoptosis assay
An Annexin V-FITC apoptosis kit was used to assess the mouse embryos in each group after 72
2.6 Statistical analysis
Experiments were repeated three times under the same conditions. The data were presented as the means
3.1 Mouse embryonic development under simulated microgravity conditions
Fig. 2 shows the morphology of the 8-cell embryos after 72
Morphology of mouse embryos in control and experiment groups (bar
Percentage of mouse embryos developed in control and experiment groups. Percentage of mouse 8-cell embryos that developed to the morula, blastocyst and hatched blastocyst stages after 72
3.2 Changes in NO concentration and NOS activity in culture media under simulated microgravity conditions
In order to examine the effects of NO on mouse embryonic development, we compared the NO and NOS level of the clinostat rotation group to the control groups after 72
(A) NOx accumulation in cultural media of each treatment (*P
3.3 Embryos apoptosis examined by Annexin V under simulated microgravity conditions
The normal embryos (those in the stationary and rotational control groups) stained negatively for AnnexinV-FITC, but those in the clinostat rotation experiment group showing retarded development were strongly stained with green fluorescence (Fig. 5). These results show that simulated microgravity induces apoptosis in the embryo cells, which does not occur under normal conditions.
Embryo apoptosis examined by Annexin-V staining. The normal embryos (stationary (A) and rotational (B) control groups) show negative Annexin-V staining, but embryos showing retarded development (clinostat rotation group (C)) showed strong green fluorescence. Scale bar
In this study, we examined the effects of microgravity on mammalian pre-implantation embryonic development in vitro using a RWVB. Our results indicate a statistically significant decrease in the number of 8-cell embryos reaching the blastocyst and hatched blastocyst stages after 72
Here we compared a rotation control group, a stationary control group and an experimental horizontal rotational group, and found that the NO concentration and the NOS activity were significantly higher in the last of these groups. A similar result was found in basal rat myocardium after space flight. Many authors have proposed that NO is an important messenger molecule, which functions through the cGMP pathway. The ratio of cAMP to cGMP may be important in regulating pre-implantation embryonic growth and differentiation (Kumei et al., 2003; Knowles and Moncada, 1994). In normal conditions, early mammalian embryonic development needs the gravity vector (Beckman and Koppenol, 1996), and simulated microgravity is abnormal and stimulates the cGMP messenger pathway in the embryo. The enhancement of NOS activity induces an increase in NO synthesis. Several recent publications show that high concentrations of NO can induce apoptosis in several cell types such as vascular endothelial cells (Gross and Wolin, 1995) and lymphocytes (Gouge et al., 1998). The involvement of NO was established by blocking its effects by inhibiting NOS, and overexpression of the anti-apoptotic protein Bcl-2 rescued cells from apoptosis by blocking signal propagation downstream of p53 and upstream caspase activation (Brune et al., 2000). Xie et al. (1995) demonstrated that the expression of recombinant iNOS in melanoma cells was associated with apoptosis, suppression of tumorigenicity and abrogation of metastasis.
Our study showed that the embryos developed slowly in simulated microgravity and showed significantly more cell apoptosis. We inferred that the increased NO concentration was the probable reason for both these effects.
Our findings therefore indicate that high NO concentration might be one of the factors that induce embryo apoptosis and retard embryo development under simulated microgravity conditions. However, because NO is a messenger molecule, the mechanism by which it might affect mammalian embryo development in a space environment should be further determined.
The authors thank professor Pidong Jiang for providing us with the RWVB. This work was supported by the CAS Knowledge Innovation Program (KSCX2-SW-322, KACX2-SW-02-07) and the National Basic Research Program of China (Grant No. 2006CB504006).
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Received 9 February 2006/21 July 2006; accepted 4 September 2006doi:10.1016/j.cellbi.2006.09.003