Development of a bovine luteal cell in vitro culture system suitable for co-culture with early embryos
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velopment of a bovine luteal cell in vitro culture system suitable for co-culture with early embryos M. Batista & A. Torres & P. Diniz & L. Mateus & L. Lopes-da-Costa
Received: 10 August 2012 / Accepted: 6 September 2012 / Published online: 10 October 2012 / Editor: T. Okamoto # The Society for In Vitro Biology 2012
Abstract The cross talk between the corpus luteum (CL) and the early embryo, potentially relevant to pregnancy establishment, is difficult to evaluate in the in vivo bovine model. In vitro co-culture of bovine luteal cells and early embryos (days 2–8 post in vitro fertilization) may allow the deciphering of this poorly understood cross talk. However, early embryos and somatic cells require different in vitro culture conditions. The objective of this study was to develop a bovine luteal cell in vitro culture system suitable for coculture with early embryos in order to evaluate their putative steroidogenic and prostanoid interactions. The corpora lutea of the different stages of the estrous cycle (early, mid, and late) were recovered postmortem and enriched luteal cell populations were obtained. In experiments 1 and 2, the effects of CL stage, culture medium (TCM, DMEM-F12, or SOF), serum concentration (5 or 10%), atmosphere oxygen tension (5 or 20%), and refreshment of the medium on the ability of luteal cells to produce progesterone (P4) were evaluated. The production of P4 was significantly increased in early CL cultures, and luteal cells adapted well to simple media (SOF), low serum concentrations (5%), and oxygen tensions (5%). In experiment 3, previous luteal cell cryopreservation did not affect the production of P4, PGF2α, and PGE2 compared to fresh cell cultures. This enables the use of pools of frozen–thawed cells to decrease the variation in cell function associated with primary cell cultures. In experiment 4, mineral oil overlaying culture wells resulted in a 50-fold decrease of the P4 quantified in the medium, but had M. Batista and A. Torres contributed equally to this work. M. Batista : A. Torres : P. Diniz : L. Mateus : L. Lopes-da-Costa (*) Reproduction and Obstetrics, CIISA, Faculty of Veterinary Medicine, UTL, Avenida da Universidade Técnica, Alto da Ajuda, 1300-477 Lisbon, Portugal e-mail: [email protected]
no effect on PGF2α and PGE2 quantification. In conclusion, a luteal cell in vitro culture system suitable for the 5-d-long co-culture with early embryos was developed. Keywords Luteal cells . Progesterone . Prostaglandins . Embryo . Bovine
Introduction In bovine species, events leading to pregnancy establishment rely on a complex cross talk between the mother and the early embryo, which is still very poorly understood (Forde et al. 2011). This cross talk mainly involves three protagonists: the embryo (blastocyst), the uterus (endometrium), and the ovary (corpus luteum, CL). It is known that progesterone (P4), secreted by luteal cells, regulates the growth and elongation of the blastocyst and the secretion of interferon-tau by trophoectoderm embryonic cells, which in turn inhibits t
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