Development of a bovine luteal cell in vitro culture system suitable for co-culture with early embryos.
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 co-culture 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 (P(4)) were evaluated. The production of P(4) 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 P(4), PGF(2α), and PGE(2) 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 P(4) quantified in the medium, but had no effect on PGF(2α) and PGE(2) quantification. In conclusion, a luteal cell in vitro culture system suitable for the 5-d-long co-culture with early embryos was developed.
Reproduction and Obstetrics, CIISA, UTL, Avenida da Universidade Técnica, Alto da Ajuda, Lisbon, Portugal.
M
M
Batista
A
A
Torres
P
P
Diniz
L
L
Mateus
L
L
Lopes-da-Costa
D000818:Animals; D002417:Cattle; D060833:Cellular Microenvironment; D018920:Coculture Techniques; D003338:Corpus Luteum / Q000166:cytology*; D015925:Cryopreservation; D003470:Culture Media; D015232:Dinoprostone / Q000378:metabolism; D046149:Embryo Culture Techniques; D030762:Estrous Cycle / Q000378:metabolism; D005260:Female; D011374:Progesterone / Q000378:metabolism; D011460:Prostaglandins F / Q000378:metabolism
D003470:Culture Media; D011460:Prostaglandins F; D011374:Progesterone; D015232:Dinoprostone