Effect of relaxin and IGF‐I on the pre‐implantation development of Mongolian gerbil (Meriones unguiculatus) embryos in vitro

Abstract

Purpose

Both relaxin and insulin‐like growth factor (IGF) are members of the insulin super family. This study aimed to investigate the effect of relaxin and IGF‐I on the pre‐implantation of Mongolian gerbil of blastocyst development in vitro.

Methods

Blastocysts and eight‐cell stage embryos were collected from female gerbils. Eight‐cell embryos and blastocysts were cultured in mM16 medium supplemented with or without relaxin or IGF‐I for 24 h. Blastocysts were counted for total, inner cell mass (ICM) and trophectoderm (TE) cell numbers, and assessed apoptosis incidence. In addition, to measure incorporation of ³H‐methionine, blastocysts were cultured for 3 h with relaxin or IGF‐I, washed with trichloroacetic acid and measured by liquid scintiration counter.

Results

Relaxin (200 ng/ml) increased total, TE and ICM cell numbers of blastocyst (P < 0.05) when it was compared with the control. IGF‐I (150 ng/ml) also has influence on total and ICM cell numbers of blastocyst when compared with control. Apoptosis incidence was relatively low, and a significant difference was not observed between each group. The effect of relaxin on incorporation of ³H‐methionine was higher than the control group (P < 0.05). Relaxin increased the developmental rate from the eight‐cell stage to blastocyst (P < 0.05).

Conclusions

In conclusion, relaxin and IGF‐I stimulated protein synthesis and increased cell numbers of blastocysts, promoting development of the gerbil embryo in vitro culture.

Introduction

Both relaxin and insulin‐like growth factors (IGFs) are members of the insulin super family [1], and they have a structure which is similar to insulin.

Relaxin is a polypeptide hormone with a molecular weight of about 6 kDa identified from the corpus luteum and seminal plasma [2]. In the porcine follicle, relaxin induces growth of granulosa and theca cells by increasing DNA synthesis and cell proliferation in vitro [3, 4]. Relaxin has important roles in oocyte maturation [5] and sperm acrosome reaction in porcine [6]. But the effect of relaxin on embryo is not yet known.

The preimplantation blastocyst is an insulin‐sensitive tissue [7, 8]. Prior studies have shown that the mouse blastocyst responds to insulin or IGF‐I by increasing glucose uptake and that this event occurs via the IGF‐I receptor. IGF‐I receptor signaling induces translocation of GLUT8 to the plasma membrane of the trophectoderm cells of the embryo [9]. Addition of IGF‐1 to culture medium increases both the proportion of embryos that develop to the blastocyst stage [10, 11]. IGF‐I prevent apoptosis incidence in embryo, increase total cell number, and improve development of embryo in mouse [12] and bovine [13].

Metabolic changes occur during pre‐implantation development, from the relative quiescence of the cleavage stages to the metabolically more active blastocyst with its high rate of glucose uptake [14]. The first embryonic cell proliferation and differentiation occurs before implantation, while the embryo is moving through the reproductive tract. The gerbil zygote cleaves and develops to a blastocyst consisting of a tight epithelial trophectoderm (TE) surrounding the inner cell mass (ICM) and blastocyst cavity on day 5. Implantation is a critical step in embryogenesis that requires preparation of a receptive uterus and activation of the blastocyst. Both of these processes proceed in a temporally and spatially coordinated manner.

Thus, we showed the effect of relaxin and IGF‐I on development of a gerbil embryo in vitro.

Materials and methods

Animals

Ten‐to‐twelve‐week‐old adult virgin female Mongolian gerbils (Meriones unguiculatus) were used at unselected times in their estrous cycles. These animals were maintained and used in accordance with the Guidelines for Regulation of Animal Experimentation (Faculty of Agriculture, Shinshu University). They were maintained under controlled light conditions (12 h light: 12 h darkness: lights on at 0600 h) and allowed free access to a pellet diet and tap water. Gerbils received 5 IU of pregnant mare serum gonadotropin (Peamex, Sankyo Ltd., Tokyo, Japan) followed 54 h later by 20 IU human chorionic gonadotropin (hCG, Puberogen, Sankyo Ltd., Tokyo, Japan). They were mated overnight with fertile males of the same colony.

Media and embryo collection

The medium used for collection of embryos was modified M16 medium (mM16) [15]. Eight‐cell stage embryos were collected 84 h after hCG injection. Blastocyst‐stage embryos were collected 124 h after hCG injection. Embryos were recovered by flushing the oviduct or uterus with mM16 medium.

Embryo culture and evaluation of cell number

Blastocyst‐stage gerbil embryos were recovered and washed three times in mM16 medium. Embryos were cultured in groups of 10 per one micro drop (20 μl) of medium which was put under mineral oil (Sigma‐Aldrich Co., St. Louis, USA) in a humidified atmosphere of 5% CO₂ in air at 37°C for 24 h. Those media contained 0, 20, 50, 100, 200, 500 ng/ml relaxin (isolated from swine ovary) or 0, 20, 50, 100, 150, 200 ng/ml IGF‐I (recombinant human IGF‐I) (Collaborative Research Inc., Clearwater, USA) added at the beginning of culturing. After culturing, embryos were stained with Hoechst 33342 (Sigma‐Aldrich Co., St. Louis, USA) and propidium iodide (PI) (Sigma‐Aldrich Co., St. Louis, USA). Embryos were stained for 30 s in PI solution (PI 0.1 mg/ml 0.2% Triton X‐100 in phosphate buffered saline (PBS), which included neither calcium nor magnesium), and overnight in Hoechst 33342 solution (25 μg/ml Hoechst 33342, 99.5% ethanol). They were then washed with glycerol, observed under fluorescence microscope (IXY‐70 Olympus Co., Tokyo, Japan), and cell numbers were counted. The ICM showed blue fluorescence and TE showed pink fluorescence under 352 nm light. Eight‐cell stage gerbil embryos were cultured with mM16 medium which included relaxin (200 ng/ml) or IGF‐I (150 ng/ml), in a humidified atmosphere of 5% CO₂ in air at 37°C for 60 h. Then embryos were observed morphology under the optical microscope IX 71 (Olympus Co., Tokyo, Japan).

Incorporation of ³H‐methinonine

Relaxin (200 ng/ml) or IGF‐I (150 ng/ml) was added to the mM16 medium. ³H‐methionine (Specific activity: 370 MBq/ml Moravek Biochemicals Inc., La Brea, USA) was also added to each media (final concentration; 9.25 kBq), and layered with mineral oil. Five blastocyst‐stage embryos were incubated at 37°C for 3 h in air of 5% CO₂. Embryonic metabolism was stopped by the addition of the same volume of 10% trichloroacetic acid (TCA) (Nacalai tesque Inc., Japan). Embryos were then recovered by Millipore filter (Millipore Co., Billerica, USA), washed with 5% TCA, and dried overnight. Afterward, dry up filter and 5 ml scintillation cocktail (0.5% (w/v) 2.5‐diphenyloxazole 0.03% (w/v) 2.2‐p‐phenylene‐bis (5‐phenyloxazole) in toluene (Nacalai Tesque Inc., Kyoto, Japan)) were added to the scintillation vial. Incorporation of ³H‐methionine was determined by the liquid scintillation counter (Beckman Coulter Inc., Fullerton, USA), and five samples were used for each determination.

Apoptosis analysis

An in situ apoptosis detection kit (Takara Bio Inc., Japan) was used to evaluate apoptosis rate by TUNEL (terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay) method. Embryos were cultured in mM16 medium which included relaxin (200 ng/ml) or IGF (150 ng/ml). After completion of culturing, embryos were collected into PBS. Embryos were washed in every progress in PBS. They were fixed with 4% p‐formaldehyde (in PBS, pH 7.4) for 20 min at room temperature, and incubated in permeabilisation buffer for 3 min on ice. They were incubated in reaction buffer and PI‐RNase solution (30 μg/ml PI and1 μg/ml ribonuclease A solution (Amresco Inc., USA)) for 1 h at 37°C. Finally, embryos were mounted on the glass, and enclosed by DABCO (Sigma‐Aldrich Co., St. Louis, USA) and glycerol. Images were obtained by Fluoview confocal laser‐scanning microscope (IXY‐70, Olympus Co., Tokyo, Japan) equipped with laser for excitation at 488 and 568 nm. We used Fluoview software for imaging (Olympus Co., Tokyo, Japan).

Statistical analysis

The data are expressed as means ± S.E. of values. Statistical analyses were conducted using one‐factor ANOVA for repeated measurements followed by Turkey–Kramer method as post hoc tests (Statcel2 software; OMS publishing Inc., Tokorozawa, Japan). For analysis of apoptosis rate, Kruskal–Wallis method was used, and for analysis of developmental rate Chi‐square for independence test. Differences were considered to be statistically significant at P < 0.05.

Results

Effect of relaxin and IGF‐I on total cell numbers of gerbil blastocyst is shown in Fig. 1. Addition of 200 ng/ml relaxin increased total cell numbers of gerbil embryo (P < 0.05), while addition of 150 or 200 ng/ml IGF‐I did the same. Therefore we used 200 ng/ml relaxin and 150 ng/ml IGF‐I for the following experiments.

Figure 1.

The effect of relaxin (a) and IGF‐I (b) on total cell number of gerbil blastocyst. Different letters mean significantly difference (P < 0.05). Values represent means ± SEM. n = 5

Blastocysts stained with Hoechst 33342 and PI are shown in Fig. 2. All nuclei were stained with Hoechst 33342, and only TE was stained with PI. The relaxin group and the IGF‐I group tended to have blastocoel larger than pre‐ incubation group and control group.

Figure 2.

Blastocysts were stained with Hoechst 33342 and PI to count cell number. a Pre‐ culture, b Control, c Relaxin, d IGF‐I*

Effect of relaxin and IGF‐I on each cell number of gerbil blastocyst is shown in Table 1. Each cultured embryo increased ICM, TE and total cell numbers through culture for 24 h with mM16. Compared to the control, ICM (P < 0.01), TE and total (P < 0.05) cell numbers significantly increased in relaxin treated blastocyst. IGF‐I also increased ICM and total cell numbers (P < 0.05).

Table 1.

Effect of relaxin and IGF‐I on each cell number of gerbil blastocyst in vitro

	Cell numbers
	Total	ICM	TE
Pre‐culture	25.0 ± 4.51a	8.0 ± 0.58A	17.0 ± 4.04α
Control	47.2 ± 3.17b	11.4 ± 0.81B	37.6 ± 2.14β
Relaxin	64.4 ± 3.30c	14.8 ± 0.58C	49.6 ± 3.17γ
IGF‐I	61.0 ± 3.18c	14.4 ± 0.51C	46.6 ± 2.79βγ

Different agreements mean significant difference (P < 0.05). Values represent mean ± SEM (n = 5)

Effect of relaxin and IGF‐I on the incorporation of ³H‐methionine by gerbil blastocyst is shown in Fig. 3. Compared to the control, incorporation of radioactive methionine was significantly increased by relaxin (P < 0.05). IGF‐I has a tendency to increase incorporation of ³H‐methionine by gerbil blastocyst in comparison with the control group (P = 0.05763).

Figure 3.

Effect of relaxin and IGF‐I on the incorporation of ³H‐methionine by blastocyst in vitro. Asterisk denotes significant difference (P < 0.05). Values represent mean ± SEM (n = 5)

Effect of relaxin and IGF‐I on apoptosis of gerbil blastocyst is shown in Table 2. Both relaxin and IGF‐I have no effect on apoptosis of blastocyst. Little cells caused apoptosis in all experimental groups.

Table 2.

Effect of IGF‐I and relaxin on the incidence of apoptosis by blastocyst in vitro

	n	Apoptosis rate (%)
Control	8	0.82
Relaxin	8	0.67
IGF‐I	8	0.76

The effect of relaxin and IGF‐I on the embryo development from eight‐cell embryo to blastocyst was shown in Table 3. Relaxin group increased blastocyst rate significantly, when they were compared with control group (P < 0.05). IGF‐I group also tended to increased blastocyst rate (P = 0.05847). No significantly deference between all groups about developmental rate to morula.

Table 3.

The effect of relaxin and IGF‐I on the embryo development from eight‐cell embryos

	n	Developed embryo (%)
		Morula	Blastocyst
Control	48	43 (89.6)	3 (6.3)a
Relaxin	49	47 (95.9)	10 (20.4)b
IGF‐I	47	45 (95.7)	9 (19.1)ab

Different letters mean significant difference (P < 0.05). Developmental rate was shown in parentheses

Discussion

In the present study, we found that relaxin was involved in the growth of the blastocyst at gerbil pre‐implantation in vitro. Both relaxin and IGF‐I stimulated blastocyst development. And pre‐implantation embryos increased blastocyst formation rate in vitro culture with relaxin or IGF‐I.

The best concentration of IGF‐I for gerbils in this study was higher than for other species like bovine [10] or mice [11]. This might be derived from the difference in species, developmental stage or culture period.

Some researchers have tried to let gerbil embryo develop into blastocyst in vitro, but were unable to produce any remarkable results. This is because the cell block prevents the gerbil embryo development to the blastocyst stage in vitro [15, 16]. There is no report on growth factor of a gerbil embryo during pre‐implantation development. Four‐cell embryos that were cultured with relaxin or IGF‐I could not develop to blastocyst (data not shown). However, relaxin and IGF‐I increased the developmental rate from eight‐cell to blastocyst and cell numbers. The present study suggested that relaxin may influence on the development of other mammalian blastocyst during pre‐implantation in vitro. Relaxin and IGF‐I are powerful supporters of the gerbil pre‐implantation embryo development.

IGF‐I receptors begin to be concurrently expressed at the compaction of the mouse embryos [17]. IGF‐I enhanced embryo development and improved embryo quality by increasing TE and ICM cell numbers. IGF‐I also had a tendency to increase incorporation of ³H‐methionine by gerbil blastocyst. IGF‐I stimulates protein synthesis of mouse blastocyst [18]. The results of this experiment corresponded to these reports. In this study, the growth and quality of blastocyst and protein synthesis were improved by relaxin and IGF treatment.

The blastocysts which were incubated with relaxin or IGF‐I tended to have larger blastocoel than embryos which were incubated with mM16 only. Both TE increasing and blastocoel expansion is very important for blastocyst hatching. It is possible that those growth factors facilitate implantation.

In this experiment, there is no remarkable difference between each group of apoptosis incidence when blastocysts were incubated in mM16 medium. It is known that relaxin increases cell number two way: prevention of apoptosis and stimulation of cell proliferation [19, 20]. Relaxin and IGF‐I may stimulate protein synthesis of the gerbil embryos and increase cell numbers of blastocyst rather than decrease apoptosis incidence.

In this report, we demonstrated the effect of relaxin and IGF‐I on gerbil embryo which was cultured in vitro. Thus, relaxin and IGF‐I may play an important role in in vitro culture pre‐implantation embryo of gerbils. This report suggests that relaxin and IGF‐I have potential for improving gerbil embryo culture in pre‐implantation in vitro.

Acknowledgments

We are thankful to Prof. T. Kohsaka, Shizuoka University, Japan for providing the relaxin which was used in this study.