Influences of Supplementing Selective Members of the Interleukin-6 Cytokine Family on Bovine Oocyte Competency

Abstract

This work explored whether supplementing selective members of the interleukin-6 (IL6) cytokine family during in vitro bovine oocyte maturation affects maturation success, cumulus-oocyte complex (COC) gene expression, fertilization success, and embryo development potential. Human recombinant proteins for IL6, IL11, and leukemia inhibitory factor (LIF) were supplemented to COCs during the maturation period, then fertilization and embryo culture commenced without further cytokine supplementation. The first study determined that none of these cytokines influenced the rate that oocytes achieved arrest at meiosis II. The second study identified that LIF and IL11 supplementation increases AREG transcript abundance. Supplementation with IL6 supplementation did not affect AREG abundance but reduced HAS2 transcript abundance. Several other transcriptional markers of oocyte competency were not affected by any of the cytokines. The third study determined that supplementing these cytokines during maturation did not influence fertilization success, but either LIF or IL11 supplementation increased blastocyst development. No effect of IL6 supplementation on subsequent blastocyst development was detected. The fourth experiment explored whether each cytokine treatment affects the post-thaw survivability of cryopreserved IVP blastocysts. None of the cytokines supplemented during oocyte maturation produced any positive effects on post-thaw blastocyst re-expansion and hatching. In conclusion, these outcomes implicate IL11 and LIF as potentially useful supplements for improving bovine oocyte competency.

Keywords: cow; cytokine; in vitro embryo production; interleukin; oocyte.

Figure 1

The progression of oocytes to metaphase II is not affected by supplementation with LIF, IL6, or IL11. Cumulus–oocyte complexes were matured in vitro in the presence of 25 ng/mL rhLIF, rhIL6, or rhIL11. Oocytes were harvested, denuded of cumulus cells, and processed for detection of first polar body extrusion and chromatin alignment after 16 h (left side graph) or 22 h (right side graph) (94–119 oocytes/treatment over six replicates). Different superscripts within each panel represent differences (p < 0.05).

Figure 2

Effect of LIF, IL6, or IL11 supplementation on selective transcript abundances in COCs. Cumulus oocyte complexes were exposed to either no treatment (CONT) or to 25 ng/mL of either rhLIF, rhIL6, or rhIL11 during in vitro maturation, then COCs were harvested either 4 h (Panels (A–D); n = 4 replicate studies) or 22 h (Panels (E–H); n = 3replicate studies) later and processed to extract RNA and complete qRT-PCR. Shown are transcript abundances for AREG (Panels (A,E)), CX37 (Panels (B,F)), CX43 (Panels (C,G)), and HAS2 (Panels (D,H)) relative to an internal control transcript (HPRT1) that are expressed as a fold-difference relative to the CTR value for each transcript. Different superscripts within each graph represent differences (p < 0.05).

Figure 3

Effects of LIF, IL6, and IL11 supplementation during oocyte maturation on subsequent cleavage rate and blastocyst development at days 3, 7 and 8 post-fertilization. Cumulus–oocyte complexes were exposed to either no treatment (CONT) or to 25 ng/mL of either rhLIF, rhIL6, or rhIL11 during in vitro maturation, then treatments ended, and oocytes were fertilized and cultured to day 8 (n = 50–75 COCs/treatment/replicate; eight replicate studies). Shown here are the percentage of putative zygotes that cleaved by day 3 post-fertilization (Panel (A)) and that achieved the blastocyst stage by day 7 (Panel (B)) or day 8 (Panel (C)). Also shown are the distributions of regular, expanded, and hatched blastocysts on day 7 (Panel (D)) and day 8 (Panel (E)). Different superscripts within each graph represent differences (p < 0.05). The asterisks (*) indicates a tendency between the treatment group and the control group (p = 0.07).

Figure 4

Post-thaw survival of cryopreserved bovine blastocysts after supplementation of LIF, IL6, or IL11 during oocyte maturation. Cumulus–oocyte complexes were exposed to either no treatment (CONT) or to 25 ng/mL of either rhLIF, rhIL6, or rhIL11 during in vitro maturation 8 (n = 5–15 blastocysts/treatment/replicated; eight replicate studies). Blastocysts were cryopreserved then thawed and incubated for 48 h to assess blastocyst re-expansion at 24 h (Panel (A)) and 48 h (Panel (B)) and hatching from the zona pellucida at 48 h (Panel (C)). Different superscripts within each graph represent differences (p < 0.05).