Expression of heat shock and apoptosis genes in riverine buffalo (Bubalus bubalis) cumulus–oocyte complexes during in-vitro maturation under thermal stress conditions
Authors: Khalil Beshoy S.F. , El-Assal Salah El-Din , Ghanem Nasser 
Published: August 2024

Background and objectives
Thermal stress arising from climate change is a crucial issue that threatens the livestock worldwide. It has various and wide range of effects on livestock’s reproductive performance. Buffalo is a main livestock in the Egyptian agricultural sector, and its’ susceptibility to the ambient temperature negatively affects its reproductive performance. Thus, it is important to study how the thermal stress affects the bubaline oocytes at both cytological and molecular levels. The current study aimed to investigate the effects of thermal stress for two different periods on the maturation of bubaline oocytes under in vitro conditions and screen the expression of various genes responsible for some mechanisms related to thermal stress alleviation, cumulus expansion, and apoptosis.

Materials and methods
Cumulus–oocyte complexes (COCs) were retrieved from buffalo ovaries and divided into three groups (C, T1, and T2) and underwent in-vitro maturation after being examined for quality. During the first 2/6 h of in-vitro maturation, good-quality COCs were exposed to 40.5°C and thereafter continued their maturation at 38.5°C. The COCs were denuded from the surrounding cumulus cells 22–24 h after maturation and were either preserved for RNA isolation in −80°C freezer or fixed for molecular maturation evaluation using Hoechst staining. The total RNA was isolated from three biological replicates of the three COC groups (C, T1, and T2) using Pico-pure RNA isolation kit, followed by cDNA synthesis for the genes of interest using real-time PCR (qPCR). Statistical analysis was performed for the obtained results for discussion and conclusion.

Results
The nuclear maturation declined more in the oocytes exposed to longer period of thermal stress than those exposed to short period of thermal stress. The longer the oocytes exposed to thermal stress, the higher was the expression of heat shock genes. The expression of heat shock genes was more expressed in cumulus cells in different groups than their corresponding oocytes. Moreover, expression of apoptosis-inducing gene (BAX) increased more in COCs exposed to long period of thermal stress than those in short period and control groups. This effect was also visible more in cumulus cells than in their corresponding oocytes. Although the cumulus expansion showed no significant change in pattern, the cumulus marker genes showed reverse relation with the period of the thermal stress, suggesting alteration in extracellular matrix proteins.

Conclusion
Heat stress affected negatively the nuclear maturation of buffalo oocytes by downregulation of cumulus expansion (PTX3, TNFAIP6, and HAS2) genes and upregulation of proapoptotic (BAX) gene under in vitro conditions. In response to this harmful situation, the cumulus cells surrounding oocytes undergo complex molecular mechanisms to adapt to the thermal shock by upregulation of heat shock transcripts (HSF1, HSF2, HSP90, and HSP70) and antiapoptotsis gene (BCL2) to provide protection against thermal stress and sustain oocyte viability.