Effect And Mechanism Of Hypoxia On Apoptosis Of Porcine Follicular Granulosa Cells

In mammalian follicles,both granulosa cells and oocytes are metabolically active,and because of lacking tight binding to the capillary network,resulting in a hypoxic state within the follicle.Studies have shown that the concentration of oxygen in follicular fluid of large follicles is lower than that of small follicles,suggesting that hypoxia occurs with the development of follicles.In addition,HIF-1αis a transcription factor in hypoxia condition and has been shown to be expressed in granulosa cell layer of non-atretic follicles.Angiogenic factors secreted by ovarian granulosa cells play an important role in follicular development and survival by inducing the angiogenesis of follicular membrane layer.Among them,vascular endothelial cell growth factor（VEGFA）contributes to follicular growth and luteal formation in mammals.Studies have shown that FSH induces follicular angiogenesis to promote follicular growth and survival.Hypoxic mediates cell damage and even death through oxidative stress,inflammation and apoptosis.Granulosa cell apoptosis is the main cause of follicular atresia,but how hypoxia regulates granulosa cell apoptosis remains to be further explored.In this study,the effect of hypoxia（1%O₂）on the apoptosis of porcine follicular GCs was studied in vitro,and it found that the JNK/FOXO1 pathway was involved in the hypoxia-induced apoptosis of porcine ovarian granulosa cells.Then the hypoxia model of mouse follicles induced by FSH was used to confirm the promoting effect of hypoxia on the apoptosis of GCs in vivo,and we found that FSH can promote the survival of GCs through the HIF-1α/VEGFA pathway.The main results are as follows:1 Involvement of JNK/FOXO1 pathway in apoptosis induced by hypoxia（1%O₂）in porcine granulosa cells（1）Hypoxia（1%O₂）has an apoptosis-promoting effect on porcine GCs in vitroIn hypoxia-treated（1%O₂）GCs,TUNEL assay and western blot of apoptosis-related protein cleaved caspase-3、BAX and BCL-2 demonstrated that hypoxia（1%O₂）induced apoptosis in porcine GCs.（2）Hypoxia activates JNK activity in porcine GCsTo test whether hypoxia-induced GCs apoptosis is correlated with JNK activation,the results of western blot showed that protein levels of phosphorylated-JNK increased over time during hypoxia exposure,suggesting that hypoxia might activate the JNK pathway in porcine GCs.Besides,JNK activity inhibitor SP600125 alleviated the pro-apoptotic effect of hypoxia on GCs,as the results showed in TUNEL assay and western blot.（3）JNK-mediated apoptosis of GCs requires the nuclear localization of FOXO1 under hypoxiaAccording to our previous researches,we thus assessed whether hypoxia affect the subcellular distribution of FOXO1 in porcine GCs.Immunofluorescence assay showed the nuclear distribution of FOXO1 was significantly increased in porcine GCs after incubation with hypoxia.Immunofluorescence staining of GCs treated with the JNK inhibitor SP600125 indicated that the nuclear localization of FOXO1 upon hypoxia stimulation was blocked after SP600125 treatment.What’s more,AS1842856,an inhibitor of FOXO1transcriptional activity,could rescue GCS apoptosis under hypoxia.2 The effect of hypoxia on the apoptosis of mouse follicular granulosa cells was verified in vivo.（1）FSH induces angiogenesis in mouse ovarian follicles and escalates the expression of VEGFA and HIF-1αin follicular granulosa cellsFSH was used to induce the development of ovarian follicles and granulosa cells in mice.The protein expression of HIF-1α,angiogenesis related genes CD31,CD34 and VEGFA were strongly increased by immunofluorescence assay and western blot in ovarian sections.HE staining of ovarian sections showed mice injected with FSH displayed more blood vessels in ovarian follicles compared with those injected with saline.Taken together,these results suggested that the angiogenic effect of FSH might be relevant to the induction of HIF-1αand VEGFA in follicular GCs.Besides,the increased expression of HIF-1αand VEGFA in follicles suggested that FSH leaded to increased hypoxia in mice follicles.（2）Inhibition of HIF-1αactivity blocks FSH-induced VEGFA expression and angiogenesis in ovarian folliclesTo investigate the involvement of HIF-1αin the regulation of blood vessel formation by FSH in the ovary,FSH-primed immature mice were intraperitoneally（i.p.）injected with PX-478,a HIF-1αantagonist that acts by inhibiting HIF-1αexpression.Both western blot analysis and q RT-PCR data showed that,PX-478 strongly abolished the increase of VEGFA expression by FSH in ovarian GCs.Immunohistochemical staining of HIF-1αand VEGFA in mouse ovarian sections further confirmed that inhibition of HIF-1αactivity repressed FSH-activated VEGFA expression within ovarian follicles.Lower angiogenic activity was also observed in FSH and PX-478 treated mice compared to FSH treated mice by evaluating the number of vessels and immunohistochemistry of CD31 and CD34.Based on these data,we proposed that the activation of the HIF-1α-VEGFA axis is required for FSH-mediated follicular angiogenesis.（3）Blocking angiogenesis leads to apoptosis of mouse follicular granulosa cellsAfter blocking angiogenesis with PX-478,Tunel staining,immunohistochemistry of caspase-3,and western blot results showed that GCs apoptosis was strongly increased in mice injected with FSH and PX-478 compared with mice injected with FSH alone.Taken together,these results suggested that FSH-induced follicular angiogenesis contributed to the normal development of follicles.It also indicated that the hypoxic environment of follicles promoted the apoptosis of granulosa cells without FSH-mediated vascular protection.In conclusion,the effect and mechanism of hypoxia on the apoptosis of follicular GCs were studied in vitro and in vivo.We found JNK/FOXO1 pathway involved in GCs apoptosis induced by hypoxia（1%O₂）in vitro and GCs apoptosis could be saved by vascular development of follicles in vivo.Therefore,this paper provides a new insight into the mechanism of follicular atresia.