Elsevier

Theriogenology

Volume 75, Issue 3, February 2011, Pages 584-588
Theriogenology

Technical note
In vitro development of bovine embryos cultured with activin A

https://doi.org/10.1016/j.theriogenology.2010.09.010Get rights and content

Abstract

The aim of this study was to investigate the effects of activin A on development, differential cell counts and apoptosis/necrosis rates of bovine embryos produced in vitro. Presumptive zygotes were cultured up to Day 8 in synthetic oviduct fluid containing aminoacids, citrate, myo-inositol and BSA. In Experiment 1, activin (10 ng mL−1) was added: 1/from Day 1 to Day 3; 2/from Day 1 to Day 8; 3/from Day 3 to Day 8; or 4/absent (control). In Experiment 2, 10 ng mL−1 activin were added either before (Day 3 to Day 5) or after (Day 5 to Day 8) the early morula stage. In Experiment 1, activin during the first 72 h of culture reduced Day 3 cleavage, 5–8 cell rates and blastocyst development, while hatching rates increased. No changes were observed within differential cell counts. In experiment 2, activin improved blastocyst development after, and had no effect before, the Day 5 morula stage. However, trophectoderm (TE) cell numbers decreased with activin both before and after the Day 5 morula stage, suggesting that activin inhibits TE differentiation. The presence of activin during the whole culture had no effect on TUNEL positive cells, but when added at shorter periods activin increased apoptotic rates. Effects of activin during in vitro bovine embryo development, depends on timing of its addition to the culture medium.

Introduction

During mammalian development, a number of cytokines play a functional role in cellular proliferation, differentiation, and morphogenesis in a spatial and temporal manner [1]. Cytokines, produced by both the female genital tract and the early preimplantation embryo itself, act on embryonic cells as paracrine/autocrine factors [2].

The early discovery of FSH and LH as distinct pituitary hormones led to the definition of activin/inhibin as substances of gonadal origin that specifically increase or decrease FSH release without altering LH release [3]. Activins are homo- or heterodimers of the βA or βB subunits of inhibin linked to one another by a single disulfide bond [4]. Dimerization of these β-subunits gives rise to three forms of activin referred to as activin A (βAβA), activin AB (βAβB), activin B(βBβB) [5], [6].

The presence of βA and βB subunits of activin has been confirmed in mouse [7] and bovine [8] embryos from zygote to the morula stage, and in bovine oviductal epithelium, ovary and granulosa cells [9], suggesting that the protein might play a role in the embryogenesis. Bovine embryos also express mRNA from activin A receptors [8]. However, the effect of activin A on in vitro culture is still controversial. Positive effects of activin A on embryo development were shown when the protein was added at the earliest stages of bovine [10] and murine [11] embryo culture, while no effects on bovine blastocysts rates were reported by Park and co-workers (2008).

Experiments with in vitro produced (IVP) embryos may include analysis for development and quality [12].

In the present work we analyze the effect of activin A (activin) on the development and quality, measured as differential cell counts and apoptosis rates, of bovine embryos cultured in vitro.

Section snippets

In vitro embryo production

Bovine IVP embryos were obtained from slaughterhouse ovaries as previously reported [13]. Briefly, IVM media consisted of TCM199, NaHCO3 (2.2 g L−1), fetal calf serum (FCS, F-4135) (10% v/v), FSHp (1 μg mL−1), LH (5 μg mL−1) and 17β-estradiol (1μg mL−1), while sperm separation was carried out using a swim-up procedure similar to that reported by Parrish and co-workers [14]. Presumptive zygotes were cultured in synthetic oviduct fluid (SOF) with 3 g L−1 BSA at 38.7 °C, 5% CO2, 5% O2 and 90% N2.

Experiment 1

A total of 1199 immature bovine CCOs were processed (Table 1). The presence of activin from Day 1 to Day 3 had a negative effect on embryo development throughout the culture. However, hatching rates increased with the earlier presence of activin. When activin was supplemented only in the last phases of culture (Day 3 to Day 8), embryo development improved in all stages but not in hatching. Activin during the whole culture generally showed an intermediate effect as compared to groups where

Discussion

In the present work, the presence of activin up to Day 3 did not affect morula and blastocyst development, although hatching rates were increased. Our results partially agree with those found by Park and co-workers whereby activin effects were analyzed early in culture [18]. However, blastocyst development improved when activin was present late in culture. These effects were observed both from Day 3 and, markedly, from Day 5 onwards. Embryonic stages comprised between Day 3 and Day 5 could

Acknowledgments

Grant Support: B. Trigal (Cajastur); M. Muñoz and D. Martín (Spanish Ministry of Science and Innovation – MICINN-; RYC08-03454 and PTA2007-0268-I, respectively). Projects: AGL2009-10059; COST ACTION FA0702 (GEMINI).

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