Elsevier

Theriogenology

Volume 86, Issue 9, December 2016, Pages 2254-2262
Theriogenology

Research article
Zygote arrest 1, nucleoplasmin 2, and developmentally associated protein 3 mRNA profiles throughout porcine embryo development in vitro

https://doi.org/10.1016/j.theriogenology.2016.07.013Get rights and content

Abstract

Maternal effect genes (MEGs) are expressed in oocytes and embryos and play an important role in activation of the embryonic genome. An abnormality in the expression of these genes may lead to arrest of embryonic cleavage or to altered transcription of factors responsible for further embryonic development. In vitro–produced porcine embryos have a lower developmental potential than embryos produced in vivo. We hypothesized that in vitro embryo culture conditions have an effect on the expression of MEGs at various developmental stages, which may affect their developmental potential. Here, using real-time polymerase chain reaction, we examined mRNA profiles of the MEGs, zygote arrest 1 (ZAR-1), nucleoplasmin 2 (NPM2), and developmentally associated pluripotency protein 3 (DPPA3), in porcine oocytes and embryos produced in vitro and in vivo. Further, we evaluated the effect of the combined addition of EGF, interleukin 1β, and leukemia inhibitory factor to the porcine in vitro embryo production system on mRNA profiles of selected MEGs. Finally, we studied localization of the MEG protein products in in vitro–obtained oocytes and embryos using confocal microscopy. We found that the ZAR-1 mRNA profile differed throughout in vitro and in vivo embryo development. In the embryos produced in vitro, the decrease in ZAR-1 mRNA levels was observed at the 2-cell stage, whereas in in vivo embryos, ZAR-1 mRNA levels declined significantly starting at the 4-cell stage (P < 0.05). In vitro culture conditions affected transiently also DPPA3 mRNA levels at the 4-cell stage (P < 0.05). There was no difference in the NPM2 mRNA profile during in vitro and in vivo embryo development. The ZAR-1 and DPPA3 proteins were localized in the cytoplasm of the oocytes and embryos, whereas the NPM2 protein was found both in the cytoplasm and in the nucleus. All proteins were expressed until blastocyst stage. The addition of EGF and cytokines to the culture medium decreased DPPA3 mRNA levels in 8-cell embryos (P < 0.05). This study indicated that IVC conditions affect ZAR-1 mRNA levels before the 4-cell stage, which may disturb the activation of the embryonic genome in pigs. The expression of the proteins after the 4-cell to 8-cell transition indicates that these factors play a role beyond activation of the embryonic genome. Supplementation of the culture media with EGF and cytokines affects DPPA3 mRNA levels after maternal to embryonic transition.

Introduction

Porcine embryos produced in vitro have poor developmental potential, resulting in lower blastocyst yield and total cell number when compared with their in vivo counterparts [1], [2], [3]. Culture conditions are the main factor that affects blastocyst quality, whereas the intrinsic quality of the oocyte used for IVF has a great impact on the blastocyst yield [4]. At the time of cytoplasmic maturation, the oocyte gathers a large number of transcripts and proteins (products of maternal effect genes [MEGs]), which regulate events that are crucial for oocyte nuclear maturation, the organization of pronuclei after fertilization, the first embryonic cleavages, and activation of the embryonic genome [5].

Our recent studies indicated that hormonal estrus stimulation affects maternal transcript levels, such as maternal antigen that embryo requires, bone morphogenetic protein 15 (BMP15), and zygote arrest 1 (ZAR-1), during oocyte maturation in vivo [6]. While the maternal antigen that embryo requires and BMP15 transcripts are used primarily during oocyte nuclear maturation, ZAR-1 appears to play an essential role in the oocyte to embryonic transition. In mice, the embryos from Zar-1 knockout females stopped developing before the activation of embryonic transcription [7]. Likewise, nucleoplasmin 2 (NPM2) and developmentally associated protein 3 (DPPA3) play significant roles in early embryonic development [8], [9]. Embryos from Npm2 knockout mice failed preimplantation development because of disturbances in nuclear and nucleolus organization [8], [10], whereas DPPA3 is a maternal factor that controls the proper maintenance of germline differently methylated regions after fertilization [9], [11].

The growth factors and cytokines produced by the porcine reproductive organs affect oocyte maturation and embryo development in vivo [12], [13]. According to our previous results ([14]; Wasielak et al., 2015, unpublished), the inclusion of exogenous growth factors or cytokines (insulin like growth factor [IGF], EGF, interleukin 1β [IL-1β], and leukemia inhibitory factor [LIF]) in the porcine IVP system affects the mRNA levels of developmentally important genes, including genes related to glucose transport, such as IGF-binding protein 3 and glucose transporter 8, and development-related genes, such as ZAR-1, NPM2, and DPPA3. However, recent studies highlighted an approach that uses combination of growth factors and cytokines to better reflect the local physiological environment in the embryo culture medium and that appears to be more beneficial for optimal embryonic development [15], [16], [17].

We hypothesized that IVC conditions have an effect on the expression of MEGs at various embryonic developmental stages, which may affect their developmental potential. Therefore, in this study, we compared the ZAR-1, NPM2, and DPPA3 mRNA profiles throughout porcine in vitro versus in vivo embryo development and examined whether the combined addition of EGF, IL-1β, and LIF to the culture media affected the mRNA profiles of these genes throughout embryo development in vitro. In addition, we determined ZAR-1, NPM2, and DPPA3 protein localization in porcine oocytes and embryos.

Section snippets

Collection of in vivo–produced oocytes and embryos

Pigs with similar genetic backgrounds from a single commercial herd were used in our experiments. The animals, in their second estrus, were assigned to one of the following experimental groups: pregnant or nonpregnant. The animals assigned to the pregnant group were subjected to artificial insemination at 24 hours and 36 hours after the detection of estrus symptoms, and the day of the second insemination was considered Day 0 of pregnancy. The animals were slaughtered on the following days of

Profiles of ZAR-1, NPM2, and DPPA3 mRNA throughout porcine embryo development in vitro versus in vivo

In porcine, in vitro–produced embryos, ZAR-1 mRNA levels were lower at the 2-cell stage than those in MII oocytes (P < 0.05; Fig. 1). In in vivo–produced embryos, ZAR-1 mRNA levels were significantly lower starting from the 4-cell stage than they were in MII oocytes (P < 0.05; Fig. 1). There were no differences in the ZAR-1 mRNA levels in MII oocytes and 2-cell embryos or in IM and MII oocytes produced in vivo (P > 0.05; Fig. 1). Moreover, we found that ZAR-1 mRNA levels had a tendency to be

Discussion

In vitro culture may affect the expression of developmentally important genes and, consequently embryonic development. In humans, 2390 genes that may be involved in transcription, the cell cycle, transport, and cellular protein metabolism were differentially expressed in in vitro– and in vivo–produced oocytes [23].

Our study revealed differences in ZAR-1 mRNA profiles between in vivo– and in vitro–produced embryos. We observed a decline of ZAR-1 mRNA levels starting from 4-cell embryos in vivo,

Acknowledgments

The authors would like to thank to Mr. Michal Blitek for his help in collecting ovaries and uteri from slaughterhouse and Dr. Gabriel Bodek for his assistance in the acquisition of confocal images. Confocal microscopy analysis was carried out in the In vitro and Biotechnology Laboratory of the Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences. This study was financed by National Science Center grant no. 2011/01/D/NZ4/03546.

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