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Cell Biology International (2007) 31, 24–29 (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


Abstract

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 72h in the clinostat rotation group. The second experiment showed that more nitric oxide (NO) was produced in the culture medium in the clinostat rotation group after 72h (P<0.05), and the nitric oxide synthase (NOS) activity in this group was significantly higher than in the controls (P<0.01). In the third experiment, we studied apoptosis in the pre-implantation mouse embryos after 72h in culture and found that Annexin-V staining was negative in the normal (stationary and rotational control) embryos, but the developmentally retarded (clinostat rotation) embryos showed a strong green fluorescence. These results indicate that microgravity induced developmental retardation and cell apoptosis in the mouse embryos. We presume that these effects are related to the higher concentration of NO in the embryos under microgravity, which have cause cytotoxic consequences.


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.


1 Introduction

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 l-arginine is catalyzed by nitric oxide synthase (NOS) in mammalian tissues (Moncada et al., 1991). NO and NOS have important functions in reproduction: NO regulates the secretion of the sex hormones (Barnes et al., 2002), pre-implantation murine embryos produce NO, and normal development is inhibited in embryos cultured with NOS inhibitors (Gouge et al., 1998).

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

2.1 Animals

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°C) with constant photoperiod (light:dark cycle, 14:10h). Food and water were freely available. Females were treated intraperitoneally with 5 IU of pregnant mare serum gonadotrophin (PMSG), then 48h later with 5 IU of human chorionic gonadotrophin (hCG). Following hCG injection, each female mouse was caged with the same strain male mouse. The morning when a vaginal plug was first observed was designated day 1 of pregnancy.

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 100rpm (Kojima et al., 2000), and the average force on the embryos is about 0.1g.


Fig. 1

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 2.0g/l NaHCO3, 100U/ml each of penicillin and streptomycin (Sigma), 0.4% BSA and 10−3g/l estradiol (Sigma). One hundred and fifty 8-cell embryos were put into the horizontal rotation group, the rotational control group and the stationary control group along with 30ml culture medium. The embryos were incubated at 37°C, 5% CO2 in a humidified incubator. After 72h culture, the embryos and the culture medium were collected. Embryos in each group were observed by phase-contrast microscopy (Olympus). The number of embryos that developed to various stages was counted, and photographs were taken for morphology.

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 72h culture. Annexin V, a Ca2+-dependent phospholipid-binding protein, has a high affinity for phosphatidylserine, which turns over to the outer membrane layer or the external surface of the cell during apoptosis.

2.6 Statistical analysis

Experiments were repeated three times under the same conditions. The data were presented as the means±SD, and differences were evaluated by Student's t-test. Values of P<0.05 were accepted as significant.

3 Results

3.1 Mouse embryonic development under simulated microgravity conditions

Fig. 2 shows the morphology of the 8-cell embryos after 72h culture in the horizontal rotation experiment group, the rotational control group and the stationary control group. Most of the embryos in the stationary control group developed to blastocysts and hatched blastocysts with normal morphology (Fig. 2B). Those in the rotational control group had similar development ratios to the stationary control group, but had inferior morphologies to those in the stationary control group (Fig. 2C). However, only a few embryos developed into blastocysts and hatched blastocysts in the experimental clinostat rotation group (Fig. 2D). The percentages of mouse embryos that developed to the morula, blastocyst and hatched blastocyst stages after 72h culture are shown in Fig. 3. The number of embryos reaching the blastocyst stage was significantly lower after 72h culture in the clinostat rotation group. This result indicates that embryo development is retarded under simulated microgravity conditions.


Fig. 2

Morphology of mouse embryos in control and experiment groups (bar=100μm). (A) 8-cell embryo at the beginning of culture; (B) embryos in the stationary control group after 72h culture; (C) embryos in the rotational control experiment group after 72h culture; (D) embryos in the clinostat rotation group after 72h culture. Compared to the embryos in B and C, there were only a few embryos developed to the blastocysts and hatched blastocyst (H) stage in D, and the blastocysts have smaller blastocoel (*) in D.


Fig. 3

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 72h culture.



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 72h culture (Fig. 4A, B). The results showed that both the relative accumulation of NO and the relative activity of NOS were significantly increased in the clinostat rotation group. These results indicate that microgravity induces more NO synthesis in in vitro cultured embryos.


Fig. 4

(A) NOx accumulation in cultural media of each treatment (*P<0.05). After 72h culture, the relative NOX value was enhanced in the clinostat rotation group compared to controls. (B) NOS activity in each treatment (**P<0.01). After 72h culture, the relative NOS activity was enhanced in the clinostat rotation group compared to controls.


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.


Fig. 5

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=10μm. A. Embryo in the stationary control group after 72h culture; (B) embryo in the rotational control group after 72h culture; (C) Embryo in the clinostat rotation experiment group after 72h culture.


4 Discussion

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 72h culture in a horizontal RWVB. This result was similar to that reported by Yoshiyuki (Kojima et al., 2000), who showed that simulated microgravity significantly inhibited the development of mouse embryos cultured in vitro, but they did not investigate the cause of this inhibition.

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.

Acknowledgements

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 2006

doi:10.1016/j.cellbi.2006.09.003


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)