Technical NotePrecision-cut luteal slices: A promising approach for studying luteal function in pigs
Introduction
In most domestic animal species, the corpus luteum (CL) acquires the capacity to undergo luteolysis (luteolytic sensitivity) between days 6 and 9 of the estrous cycle. Porcine CL acquires luteolytic sensitivity relatively late in the cycle [1], [2], i.e., near day 13. Several hypotheses on the delayed acquisition of luteolytic sensitivity in the pig have been stated, including auto-amplification of luteal production of prostaglandin (PG) F2α and differences in the activation of the PGF2α signaling pathway. However, the mechanism responsible for this delay in the pig is not fully recognized.
Studies on the acquisition of luteolytic sensitivity usually include both in vivo and in vitro experiments [3] or use an intricate CL microdialysis in vivo system [4]. The current study aimed at examining whether precision-cut tissue slices of porcine CL can be used to investigate luteal function as an alternative to in vivo experiments in pigs and other species. To meet this aim two distinct experiments were performed. In the first experiment: (1) the optimal in vitro conditions for incubation of luteal slices, and (2) time- and dose- dependent effects of luteinizing hormone (LH) on progesterone (P4) secretion were established. In the second experiment, the effects of 17β-estradiol (E2) and PGF2α on P4 secretion by luteal slices were determined under the conditions established in the first experiment.
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Materials and methods
The experiments were conducted in accordance with the national guidelines for agricultural animal care and approved by the Local Animal Ethics Committee. Crossbred gilts (7–8 months old) during their second estrous cycle were used in the study. Corpora lutea collected during the mid-luteal phase (days 10–12) or late-luteal phase (days 14–15) were cut into slices (180-μm thick, 8 mm diameter, 4–5 mg wet weight) using a Krumdick Tissue Slicer (K&F Research, Birmingham, AL, USA) (Fig. 1A–F; [5]).
Results and discussion
Although porcine CL is considered to be independent on the pituitary LH until day 12 of the estrous cycle [8], 10 and 100 ng/mL of LH increased (p < 0.01) P4 secretion by mid-luteal slices incubated for 24 h in 6 mL of medium (Fig. 2A). It should be emphasized that we have applied approximately 20 times less luteal tissue [9] and have included LH in doses 10–1000 times lower in comparison to previous reports [8], [9]. When the 4-mL volume of incubation medium was examined, only 10 ng/mL of LH
Acknowledgements
We are grateful to Mrs. Katarzyna Gromadzka-Hliwa, Mrs. Benigna Szarejko and Mr. Jan Klos for their technical assistance in the laboratory. This research was supported by grant no. 2011/01/B/NZ4/04970 from the National Science Centre (NCN). The results of this study were presented at the 9th International Conference on Pig Reproduction (ICPR), 9–12 June 2013 in Olsztyn, Poland. E. Przygrodzka was supported by the European Union within the European Social Fund (DR INNO No. 5/2013).
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