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Because ovarian granulosa cells are essential for oocyte maturation and development, we validated human granulosa HO23 cells to evaluate the ability of the DHEA to prevent cell death after starvation. The present study was aimed to investigate whether DHEA could protect against starvation-induced apoptosis and necroptosis in human oocyte granulosa HO23 cells. The starvation was induced by treatment of serum-free (SF) medium for 4 h in vitro. Starvation-induced mitochondrial depolarization, cytochrome c release and caspase-3 activation were largely prevented by DHEA in HO23 cells. We found that treatment with DHEA can restore starvation-induced reactive oxygen species (ROS) generation and mitochondrial membrane potential imbalance. In addition, treatment of DHEA prevents cell death via upregulation of cytochrome c and downregulation of BAX in mitochondria. Most importantly, DHEA is ameliorated to mitochondrial function mediated through the decrease in mitochondrial ROS, maintained mitochondrial morphology, and enhancing the ability of cell proliferation and ROS scavenging. Our present data strongly indicate that DHEA reduces programmed cell death (apoptosis and necroptosis) in granulosa HO23 cells through multiple interactions with the mitochondrion-dependent programmed cell death pathway. Taken together, our data suggest that the presence of DHEA could be beneficial to protect human oocyte granulosa HO23 cells under in vitro culture conditions during various assisted reproductive technology (ART) programs.

Free Chinese abstract: A Chinese translation of this abstract is freely available at http://www.reproduction-online.org/content/154/2/101/suppl/DC1

Pituitary adenylate cyclase-activating polypeptide (PACAP), a pleiotropic neuropeptide, has diverse functions in mammals. However, studies of the expression and function of PACAP and its receptor in fish, particularly in the reproductive system, are still limited. In this report, semi-quantitative RT-PCR and immunohistochemical staining were performed to identify expression domains of commercially important tilapia (Oreochromis mossambicus). PACAP (tpacap ₃₈) and its type I receptor (tpac ₁-r). Transcripts were detected in the brain, gallbladder, gill, heart, intestine, kidney, muscles, pancreas, spleen, stomach, testes, and ovaries, but not in the liver. Expression of tpacap ₃₈ and tpac ₁-r mRNA in brain tissue was significantly higher in both sexes compared with other tissues. Addition of exogenous ovine PACAP₃₈ (0.25–5 nM), cAMP analog (dibutyryl-cAMP, 0.25–1.5 mM) or forskolin (adenylate cyclase activator, 1–10 μM) significantly upregulated tpacap ₃₈ in the gonads via a dose- and time-dependent fashion. This effect reached a maximal level at 2 h after induction, and then decreased with prolonged culture for up to 4 or 8 h. Additionally, the expression levels of tpac ₁-r were not significantly affected by ovine PACAP₃₈ or dibutyryl-cAMP in either sex. Forskolin had a slightly inductive effect and its function could be suppressed with the addition of protein kinase A (PKA) inhibitor, H89 (10 μM), indicating involvement of the cAMP-PKA signaling pathway in the regulation of tpacap ₃₈. Expression of tpacap ₃₈ and tpac ₁-r in the gonads of tilapia suggests that PACAP may mediate gonadotropin action via paracrine/autocrine mechanisms in this bony fish.