Nutritional and metabolic stressors on ovine oocyte development and granulosa cell functions in vitro

Abstract

The present study was undertaken to study the effect of ammonia, urea, non-esterified fatty acid (NEFA), and β-hydroxybutyric acid (β-OHB) on oocyte development and granulosa cell (GC) growth parameter of ovine (Ovis aries). Ovine oocytes were matured in vitro in the presence of different concentration of ammonia, urea, NEFA, and β-OHB for 24 h, in vitro inseminated and evaluated for cleavage and blastocyst yield. Same concentrations of ammonia, urea, NEFA, and β-OHB were examined on growth parameters and hormone secretion activity of granulosa cells in vitro. Real-time reverse transcription polymerase chain reaction was used to evaluate the expression of steroidogenic genes (steroidogenic cytochrome P-450 (CYP11A1, CYP19A1)), cell proliferation-related genes (GDF9, FSHr), and apoptosis-related genes (BCL-2 and BAX). The maturation, cleavage, and blastocyst production rates were significantly lowered in media containing either 200 μM ammonia or 5 mM urea or high combo NEFA or 1 μM β-OHB. Exposure of granulosa cell to 400 μM ammonia or 1 μM β-OHB or very high combo or 6 mM urea significantly decreased all the parameters examined compared to lower levels of all nutritional and metabolic stressors. Elevated concentration of metabolic stressors induced GC apoptosis through the BAX/BCL-2 pathway and reduced the steroidogenic gene messenger RNA (mRNA) expression and cell proliferation gene mRNA expression. These results suggested that the decreased function of GCs may cause ovarian dysfunction and offered an improved understanding of the molecular mechanism responsible for the low fertility in metabolic stressed condition.

The Dynamic Transcription Profiles of Proliferating Bovine Ovarian Granulosa When Exposed to Increased Levels of β-Hydroxybutyric Acid

“…Hence, low doses of BHBA have been shown to exert positive effects in the treatment of certain diseases (33,34). However, if the concentration of BHBA increases excessively, this could directly or indirectly affect normal liver metabolism (35,36) and reproductive performance (e.g., granulosa cell development and oocyte maturation) (20,37,38) in ruminants such as cows.…”

“…Existing data suggest that an abnormal increase in BHBA may lead to intracellular oxidative stress and ultimately induce apoptosis in somatic cell models, such as bovine abomasum smooth muscle cells and bovine liver cells (17)(18)(19). However, metabolic stress, involving elevated concentrations of BHBA, has been shown to cause apoptosis in ovine GCs, thus inhibiting estrogen synthesis and negatively affecting fertility (20). Studies involving direct stimulation of GCs through increased BHBA to explore the mechanisms of their effects on fertility are still scarce.…”

Epidermal growth factor alleviates the negative impact of urea on frozen-thawed bovine sperm, but the subsequent developmental competence is compromised

“…We also documented that urea-incubated oocytes had a lower rate of developmental competence and higher more amino acid depletion during in vitro maturation (IVM) 17,18 . It has been shown that urea uses EGFR transactivation and induces the expression of immediate-early genes 19 and, as a toxic molecule, can induce ROS production in a variety of cells, such as oocytes 20 . There is no evidence that urea influences sperm production of ROS, but urea has been shown to cause toxicity by several mechanisms, including oxidative stress 21 .…”

Pengaruh urea dalam media maturasi in vitro terhadap tingkat maturasi oosit sapi

“…Urea yang masuk ke cairan folikel bersifat toksik dan dapat dapat merusak membran sel oosit. Sel granulosa oosit yang terpapar ammonia dengan kadar tinggi dapar mengurangi kemampuannya untuk mendukung proses maturasi oosit secara in vitro (Nandi et al, 2018). Kadar urea yang tinggi dalam darah dapat menginduksi denaturasi protein dan asam nukleat sehingga kehilangan struktur sekunder dan struktur tersiernya (Lambert dan Draper, 2012), karena perubahan konformasi ikatan hidrogen pada kutubnya (Wang et al, 2014).…”