Pannexin 1 inhibition delays maturation and improves development of Bos taurus oocytes

Zachary Timothy Dye, Lauren Virginia Rutledge, Silvia Penuela, Paul William Dyce, Zachary Timothy Dye, Lauren Virginia Rutledge, Silvia Penuela, Paul William Dyce

Abstract

Background: Intercellular exchange between the oocyte and its surrounding cells within the follicular environment is critical for oocyte maturation and subsequent development. In vertebrates this exchange is facilitated through gap junctions formed by connexin membrane proteins. Another family of membrane proteins called pannexins are able to form single membrane channels that allow cellular exchanges with the extracellular environment. The most ubiquitously expressed and studied member, pannexin 1 (PANX1), has yet to be described thoroughly in female reproductive tissues or functionally studied during oocyte maturation. Here, we look into the expression of pannexin 1 in bovine cumulus-oocyte complexes (COCs), as well as, its potential role in oocyte maturation and development.

Results: We show that pannexin 1 is expressed in bovine COCs and that the expression of PANX1 was significantly lower in COCs isolated from large antral follicles (> 5 mm) compared to those isolated from small antral follicles (< 2 mm). Supporting this we also found lower expression of PANX1 in oocytes with higher developmental potential when compared to oocytes with lower developmental potential. We further found that PANX1 channel inhibition during in vitro maturation resulted in temporarily delayed meiotic maturation and improved in vitro developmental outcomes while decreasing intercellular reactive oxygen species.

Conclusions: These data suggests PANX1 is differentially expressed at a critical stage of follicular development when oocytes are acquiring developmental competence, and may play a role in the timing of oocyte maturation.

Keywords: Cumulus; Developmental biology; Early development; In vitro fertilization (IVF); In vitro maturation (IVM); Oocyte development; Oocyte maturation.

Conflict of interest statement

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Figures

Fig. 1
Fig. 1
Pannexin1 expression in Bovine Cumulus-Oocyte Complexes, During Different Follicular Stages, and Varying Stages of Oocyte Developmental Competence. a Representative immunofluorescent images of bovine COCs stained with Hoechst33342 (blue, a) and labeled with PANX1 antibody (green, b). c A representative image depicting a bovine COC stained with Hoechst33342 and stained with secondary antibody only (d). Oocyte outlined with dashed circle. Bars = 100 μm. e Representative blot images depicting the levels of PANX1 expression in granulosa cells isolated from small (< 2 mm) and large (> 5 mm) antral follicles. GAPDH was used as a loading control. f Densitometry results showing the expression level of PANX1 in granulosa cells isolated from small and large antral follicles. g Representative blot images depicting the levels of PANX1 in BCB- and BCB+ COCs. h Densitometry results showing the expression of PANX1 in BCB- and BCB+ groups of COCs. *Denotes a significant difference, p < 0.05. Error bars are ± SD from mean
Fig. 2
Fig. 2
Effect of Inhibiting the Pannexin 1 Channel on Hemi-channel Function. Representative images depicting PI dye uptake (red) by untreated granulosa cells (b) and granulosa cells treated with 10Panx (e). Cells were nuclear stained with Hoechst33342 (blue, a & d) and overlayed (c & f). Bars = 400 μm. g The relative number of PI positive cells in control or 10Panx treated granulosa cells. *Denotes a significant difference, p < 0.05. Error bars are ± SD from mean
Fig. 3
Fig. 3
ROS and Maturation Effects of Pannexin 1 Inhibition. a Representative images of cumulus expansion after 22 h of IVM in control COCs and PANX1 inhibited COCs. Magnification was 20x. b Cumulus expansion in the control and 10Panx treated groups. c Meiotic maturation proportions of oocytes at the GV, GVBD, and MII stages after 6 h and 22 h of IVM with or without PANX1 inhibition. d The average icAMP concentration of COCs treated with or without 10Panx at 0, 3, 6, and 22 h of IVM. *Denotes a significant difference, p < 0.05. Error bars are ± SD from mean. Representative images depicting the staining intensity of DCFH-DA showing levels of free radicals within the oocyte after 22 h of IVM without (f) or with (h) PANX1 inhibition. Cells were nuclear stained with Hoechst33342 (blue, e & g). Bars = 200 μm. i Quantitation of the staining intensity of DCFH-DA using CTCF. *Denotes a significant difference, p < 0.05. Error bars are ± SD from mean
Fig. 4
Fig. 4
Embryo Development Post Pannexin 1 Inhibition. a Cleavage rate at day 2 post IVF with or without 10Panx treatment during IVM. b Blastocyst rate at day 7 post IVF with or without 10Panx treatment during IVM. *Denotes a significant difference, p < 0.05. +Denotes a significant trend, p < 0.055. Error bars are ± SD from mean

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