Supplementing IL6, IL11, and LIF to Improve Cultured Bovine Oocyte Competency

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Date
2023-07-24
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Virginia Tech
Abstract

Bovine embryos produced in vitro consistently display decreased quality in terms of their potential to reach the blastocyst stage as well as post-transfer survival. Media formulations, oocyte quality, and inferior expression of needed transcripts are all causes of this reduced developmental potential commonly present in in vitro-produced (IVP) bovine embryos. Recently our lab has confirmed interleukin-6 as an embryokine whose capabilities include increasing inner cell mass (ICM) numbers and promoting bovine blastocyst development. LIF is another family member of the IL6 cytokine family and has been shown to produce several positive effects when supplemented during oocyte in vitro maturation. IL6 has predominantly been studied as being supplemented post-fertilization. However, published transcriptomic work described receptors for IL6, IL11, and LIF as present in cumulus cells at the time COCs are removed from their follicles. Consequently, we wanted to investigate if supplementing 25 ng/ml of IL6, IL11, or LIF would improve IVM bovine oocyte competency. Several experiments were completed (4replicates/experiment; 30-60 cumulus-oocyte complexes (COCs)/replicate). In Experiment 1, COCs were in vitro matured for 16 or 22 hours, then meiotic stage was assessed after denuding, fixation, and DNA staining. No cytokine treatment influenced the percentage of oocytes that achieved metaphase II at either time point. In Experiment 2, COCs were in vitro matured for 4 hours before snap freezing. and processing to examine changes in five cumulus-expressing transcripts associated with oocyte competency (CX43, CX37, AREG, TNFAIP6, HAS2). Our chosen housekeeping gene, HPRT1, served as the internal control. An increased abundance of AREG occurred following exposure to LIF but not with the other treatments. Supplementation with IL6 and IL11 but not LIF tended to increase TNFAIP6 abundance (P<0.10). No other transcript differences were detected. In Experiment 3, we examined whether supplementing these cytokines during IVM affects subsequent fertilization and blastocyst rates. No effects were detected on cleavage rates but at day 8 increases in blastocyst yield were detected for LIF and IL11, but not IL6. LIF showed a tendency to increase hatching rates. In Experiment 4, we aimed to assess how the cytokine treatments affected cryosurvivability. Blastocysts (5-10/replicate, 9 replicate studies) were frozen at a rate of -0.6 degrees C/min until reaching -32 degrees C, then were stored in liquid nitrogen for 4-8 weeks before being thawed and incubated in conventional embryo culture medium (SOF-BE1) for 3 days. No treatment effects were noted for re-expansion, hatching, and overall survivability. In summary, these results implicate IL11 and LIF as potential mediators of oocyte competency. However, the evidence presented here suggest that IL6 and IL11 may function differently than LIF when provided during COC maturation.

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Keywords
Embryo, oocyte, blastocyst, cytokine, cryo-survivability
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