Cyclic guanosine monophosphate (cGMP) improves freezing tolerance of sheep embryos by reducing lipid content.

Abstract

Embryos produced in vitro exhibit heightened cryosensitivity due to excessive lipid accumulation. Previous studies demonstrated that cyclic guanosine monophosphate (cGMP) modulates intracellular lipid metabolism through cGMP-dependent protein kinase (PKG) signaling in various cell types. This study investigated the effects of cGMP on (i) cryosurvival in sheep embryos, (ii) embryonic quality, and (iii)lipolysis-related parameters. Specifically, we quantified lipid droplet content, free fatty acid levels, and hormone-sensitive lipase (HSL) phosphorylation status as key indicators of lipolytic activity. The results showed that cGMP pretreatment (0.5 mM) for 10 min prior to slow freezing significantly improved post-thaw embryo recovery rates and upregulated the mRNA expression of key developmental genes (POU5F1, SOX2, CDX2, and NANOG). cGMP pretreatment significantly upregulated the expression of multiple lipid catabolism genes (ACSL4, HMGCR, HMGCS1, LIPE, LPL, LIPF, and PLIN2), with LIPE (encoding HSL) exhibiting the most pronounced induction (27.10-fold increase vs. control). Following cGMP pretreatment, PKG activation triggered significant increases in the intracellular Ca²⁺ level and Calcium/Calmodulin-dependent Protein Kinase II (CaMKII) phosphorylation. Given HSL's established sensitivity to calcium fluctuations, this cascade led to markedly enhanced HSL phosphorylation. Concurrently, blastocysts exhibited a significant reduction in lipid droplet content, while transient fatty acid accumulation was observed due to enhanced HSL activity. In summary, pretreating sheep blastocysts with cGMP prior to slow freezing notably elevates their cryotolerance. This enhancement is achieved by triggering the lipolytic activity of HSL via the PKG/CaMKII/Ca²⁺ signaling cascade. This pathway specificity underscores cGMP's potential as a targeted cryoprotectant adjuvant for assisted reproductive technologies, particularly for improving the cryosurvival of lipid-rich embryos.