Fat area and lipid droplet morphology of porcine oocytes during in vitro maturation with trans-10, cis-12 conjugated linoleic acid and forskolin

Abstract

Lipid droplets (LD) in porcine oocytes form a dark mass reaching almost all cytoplasm. Herein we investigated changes in fat areas, cytoplasmic tone and LD morphology duringin vitromaturation (IVM) of porcine oocytes cultured with 100 μMtrans-10,cis-12 conjugated linoleic acid (t10,c12 CLA) or 10 μM forskolin at different time periods. Four groups were constituted: control, excipient,t10,c12 CLA and forskolin, with drugs being supplemented during 44 to 48 h and the initial 22 to 24 h in Experiments 1 and 2, respectively. In Experiment 3, forskolin was supplemented for the first 2 h. Matured oocytes were inseminated with frozen-thawed boar semen and cleavage rate recorded. Before and during IVM, samples of oocytes were evaluated for LD, total and fat areas and fat gray value or for meiotic progression. Results showed that forskolin supplementation during 44 to 48 h or 22 to 24 h inhibits oocyte maturation (exp. 1: forskolin = 5.1 ± 8.0%, control = 72.6 ± 5.0%; exp. 2: forskolin = 24.3 ± 7.4%, control = 71.6 ± 5.6%) and cleavage (exp. 1: forskolin = 0.0 ± 0.0%, control = 55.4 ± 4.1%; exp. 2: forskolin = 8.3 ± 3.3%, control = 54.5 ± 3.0%). Forskolin also reduced oocyte and fat areas. In Experiment 3, forskolin negative effect on oocyte maturation and cleavage disappeared, although minor (P⩽ 0.03) LD and oocyte fat areas were identified at 22 to 24 h of IVM. Oocytes supplemented witht10,c12 CLA during 44 to 48 h presented a lighter (P⩽ 0.04) colour tone cytoplasm than those of control and forskolin. In conclusion,t10,c12 CLA and forskolin were capable of modifying the distribution and morphology of cytoplasmic LD during porcine oocyte maturation, thus reducing its lipid content in a time-dependent manner.

A Role of Lipid Metabolism during Cumulus-Oocyte Complex Maturation: Impact of Lipid Modulators to Improve Embryo Production

“…Full meiotic competence is reached in oocytes developing in ovarian follicles with a diameter of 3 mm or more [22, 25]. In vitro , pig oocytes under 90  μ m in diameter are unable to resume meiosis, while oocytes measuring 110–115  μ m can complete the first meiotic division and acquire MII [10, 25]. However, many do not attain an optimal oocyte diameter before fertilization.…”

“…A lipid specific fluorescent dye, Nile red, was used to stain cattle, pig, and murine oocytes, and different amounts of emitted fluorescent light were measured according to their cytoplasmic lipid content [38]. In particular, the gray mean value within the oocyte fat area was suggested as an appropriate tool to evaluate the lipid content of a single oocyte [10]. Moreover, as this is a noninvasive technique, it can be useful to record oocyte morphology and quality before cryopreservation or fertilization.…”

“…Regardless of LD origin, they are constantly changing their shape, volume, and location. In particular, in the lipid-rich pig oocyte the cytoplasm is filled with LD showing a considerable variation of areas, ranging from 0.3  μ m 2 to 90  μ m 2 , during the maturation process [10] (Figure 3). …”

“…Although the role of lipids during oocyte maturation and early embryo development is currently under research, their primordial importance in female gamete quality is unequivocal. Modifications of LD morphology and lipid metabolism during pig oocyte maturation may interfere with monospermic fertilization, as referred, but also with embryo development [10, 11]. Immediately after sperm penetration, differences are identified in electron density of LD.…”

“…Nevertheless, a role for lipids in energy production during preimplantation development as well as precursors in steroidogenic and eicosanoid pathways has been proposed [7–9], suggesting that modifications in oocyte intracellular lipids should be carefully estimated. The high intracellular lipid content of pig oocytes [10, 11] renders them an excellent model among mammalian and microlecithal oocytes to understand the role of lipids and fatty acid metabolism during maturation. Furthermore the oocyte activating vias seem to be closely related to those regulating the mobilization of intracellular lipid reserves in the maturating oocyte [12, 13].…”