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Background Transzonal projections (TZPs) constitute a structural basis for the communication between the oocyte and its surrounding cumulus cells (CCs), which play critical roles in promoting the oocyte maturation. Previously we found that heat stress (HS) causes loss of TZPs in porcine cumulus-oocyte complexes (COCs) with decreased density of filamentous actin (F-actin). However, the time-course responses of F-actin and its monomeric actins (β-actin and γ-actin) during the in vitro maturation of oocytes remain unclear. Results In this study, excised porcine ovaries were exposed to HS at 41.5 °C for 1 h before COCs were isolated and matured in vitro for 44 h. HS significantly reduced oocyte quality, characterized by impaired cumulus expansion, delayed meiotic resumption and lower survival rate and polar body extrusion rate, as well as decreased expression of mitochondrial DNA-encoded genes and elevated mitochondrial reactive oxygen species concentration. Expression of β-actin and γ-actin in CCs increased gradually with oocytes maturation, which was significantly reduced in HS group, especially at 24 h and/or 44 h of in vitro maturation. By contrast, the number of TZPs and the fluorescence intensity of F-actin in zona pellucida decreased gradually during oocytes maturation, which were significantly reduced by HS at 24 h of in vitro maturation. Moreover, colocalization analyses revealed both β-actin and γ-actin contribute to the F-actin formation in porcine TZPs, and the colocalization of F-actin with GJ protein connexin 45 was significantly reduced in heat-exposed COCs. Conclusions The results indicate that the suppression of actin expressions in CCs, which may lead to the F-actin unstabilization in TZPs, will subsequently contribute to the compromised quality of oocytes under HS.

The rapid and efficient clearance of apoptotic germ cells (GCs) by Sertoli cells (SCs) is important for spermatogenesis. High mitochondrial activity in phagocytes is critical for continued clearance of apoptotic cells. However, the underlying molecular mechanism is poorly understood. Glycogen synthase kinase-3α (GSK3α) is a protein kinase that participates in the regulation of mitochondrial activity. Immunohistochemistry evidenced the predominant presence of the Ser21 phosphorylation GSK3α (inactivation) signal in SCs. Heat shock-induced apoptosis of GCs and dephosphorylation of GSK3α in SCs is a perfect model to investigate the role of GSK3α in phagocytic action. The number of apoptotic GCs was significantly lower in GSK3α inhibitor pre-treated mice with HS compared to normal control. In vitro phagocytosis assays shown that the phagocytic activity in GSK3α activated SCs was downregulated, while GSK3α inhibitor supplementation restored this process. Moreover, GSK3α activation participates in the alteration of the mitochondrial ultrastructure and activity. In particular, GSK3α activation inhibits mitochondrial fission via phosphorylation of dynamin related protein 1 at Ser637. Changes of mitochondrial activity resulted in the accumulation of lipid droplets and the alteration of metabolism pattern in SCs. In summary, our results demonstrate that inactivation of GSK3α is required for mitochondria-mediated apoptotic GCs phagocytosis in SCs.

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The oocyte is vulnerable to various environmental stressors, including heat exposure. Cumulus-oocyte complexes (COCs) comprise functional units for oocytes in vitro maturation, and the cumulus cells provide essential supports and protect the oocyte from environmental insults. Heat exposure results in varied consequences in oocyte, presumably due to different responses of cumulus cells to heat exposure. In this study, we examined whether heat exposure of different duration affects porcine oocytes quality differently, and how such effects, if any, relate to transcriptomic profiles of cumulus cells. COCs were heat-exposed for 4 h (20-24 h, COC4) and 24 h (0-24 h, COC24), respectively, and the quality of oocytes in COC24 group showed significantly impaired with disrupted cumulus expansion and extracellular matrix (ECM) structure. The transcriptomic analysis identified 749 and 1238 differential expression genes (DEGs) in COC4 and COC24, respectively. Moreover, 852 DEGs were found when COC24 was compared with COC4, and the downregulated DEGs were mainly associated with Gene Ontology terms linked with ECM and cell proliferation. In the protein-protein interaction network, HSPE1, TNFAIP6, COL12A1, and COL18A1 were identified as hub genes playing important roles in heat-induced transcriptomic responses. These results indicate that impaired cumulus proliferation and ECM structure are responsible for heat-induced damage in oocytes quality. K E Y W O R D S cumulus cells, heat exposures, pig, transcriptomes 1 | INTRODUCTION Oocytes are surrounded by multilayered cumulus cells (CCs) to form cumulus-oocyte complex (COC), a functional unit for oocyte maturation. COCs can be cultured in vitro for oocytes maturation, and porcine oocytes usually require 42-48 h to reach metaphase II in culture. Cumulus cells are essential in the maturation process of oocytes, not only transmit bioactive substances and energy materials to oocytes, but also protect the oocytes from the interference of the external environment (Gilchrist et al., 2004). During oocytes maturation, cumulus cells proliferate, differentiate, and communicate with each other via extracellular matrix (ECM) and through gap junctions (Campen et al., 2018), adherens (Mora et al., 2012), and tight junctions (Clelland & Kelly, 2010). They also send transzonal projections (Yin et al., 2019) to interact with the enclosed oocyte. ECM not only provides a physical scaffold for cell-cell interactions, but also plays a key role in normal follicular growth, oocyte development, ovulation, and luteinization (Kimura et al., 2007). Oocytes are susceptible to various environmental stresses, including heat exposure. Heat-induced alterations in oocyte quality depend on timing, duration, and the extent of the exposure. For instance, COCs aspirated from multiparous Holstein cows in cold (December-April) seasons had a higher cleavage and blastocyst rates than in hot (May-September) seasons (Gendelman & Roth, 2012).