FSH Prevents Porcine Granulosa Cells From Hypoxia-Induced Apoptosis Via Activating Mitophagy

In ovarian follicle development,the capillary network is restricted to the theca cell layers outside the basement membrane,resulting in few oxygen supplied to guranulosa cells.Granulosa cells were therefore thought to grow in a hypoxic microenvironment.HIF-1α(hypoxia inducible factor-1α,HIF-1α)is one of the critical mediators regulating the adaptive response to hypoxia stress,and is widely expressed in mammalian follicles.Evidence has emerged that HIF-1α plays a vital role in the growth and development of follicles,ovulation and luteinization,and even regulates the apoptosis of granulosa cells under hypoxic stress.As a conserved degradation mechanism in eukaryotic cells,autophagy can maintain intracellular homeostasis through the circulation of materials and energy.Mitophagy can eliminate damaged mitochondria and inhibit the transmission of apoptosis signals to promote cell survival.Follicle-stimulating hormone(FSH)is the primary survival factor in follicle development by preventing the apoptosis in granulosa cells.Researches have reported that autophagy is an alternative mechanism in FSH-mediated granulosa cells protection under stress conditions,especially mitophagy has been an adaptive response to hypoxic stress via HIF-1α.Our previous date showed a role for FSH in inducing autophagy via upregulating the expression of HIF-1α.Therefore,we speculate that FSH may activate the mitophagy via HIF-1 α pathway to prevent porcine granulosa cells from apoptotic death under hypoxic stress.To prove this,this study mainly include the following aspects:(1)the effects of FSH on hypoxic porcine granulosa cells were investigated by detecting the cell viability and the expression of apoptosis-related proteins;(2)the effects of FSH on the mitochondrial integrity of hypoxic granulosa cells via detecting mitochondrial membrane potential;(3)to elucidate the relationship between the protective effect of FSH on granulosa cells and mitophagy under hypoxia stress,the expression of autophagy-related proteins and the mitophagic vesicles are detecting by WB and TEM;(4)By inhibiting the activity of HIF-1α,the relationships between the pathway of FSH-induced mitophagy,HIF-1α and hypoxia-induced mitophagy are determined;(5)By knocking down PINK1,the protective mechanism of FSH on damaged granulosa cells under hypoxia stress,and the specific molecular mechanism of FSH-induced mitophagy are further elucidated.The results are as follows:1.FSH can inhibit hypoxia-induced apoptosis in porcine granulosa cells.Porcine GCs were incubated with/without 1% of O2 in the presence or absence of FSH(2 IU/m L)for 24 or 48 hours,then collected for apoptotic-related proteins and cell viability.We found that hypoxic stress could significantly inhibit the activity of porcine GCs and increase the proportion of apoptotic cells.The expression of cleaved-caspase3 and mitochondrial apoptotic proteins was detected by Western Blot,it found that hypoxia could induce GCs apoptotic death via mitochondrial apoptotic pathway.However,the proportion of hypoxia-induced apoptotic cells was decreased markedly,and the expression of mitochondrial apoptotic proteins was also significantly inhibited with FSH treatment,suggesting that FSH protects GCs from hypoxia-induced apoptosis by inhibiting mitochondrial apoptosis.2.FSH protects hypoxic GCs via mitigating mitochondria damage.Since the activation of the mitochondrial apoptotic pathway is closely linked to the depolarization and dysfunction of mitochondria,we investigated whether FSH is required for mitochondrial integrity in GCs exposed to hypoxia by stained with TMRM /Mito Green and JC-1.The results showed that hypoxia increased the fraction of depolarized mitochondria compared with normoxia GCs,which was significantly reversed by FSH treatment.3.FSH stimulates mitophagy and mitochondrial biogenesis under hypoxia exposure.In order to explore whether mitophagy contributes the protection of FSH on hypoxic GCs,we detected the expression of autophagy-related proteins by Western Blot,and examined the distribution of mitochondria in autophagic vacuoles using transmission electron microscopy(TEM).It is showed that the induction of autophagy was markedly enhanced in hypoxic GCs subjected to FSH administration,together with the accumulation of mitochondria-containing autophagic vesicles.However,FSH did not significantly reduce mitochondrial marker protein Tom20 levels in GCs,suggesting that FSH not only mediated the mitophagic clearance of mitochondria,but might also promote the generation of new mitochondria.4.FSH acts through HIF-1α to induce mitophagy in GCs exposed to hypoxia.HIF-1 is a major effector involved in responses to hypoxia,we therefore examined whether HIF-1α might be correlated with FSH-mediated GCs mitophagy.The GCs were pretreated with PX-478 or GFP-MAP1LC3B-Ⅱ vector before the indicated treatment,the autophagy punta accumulation of hypoxic GCS induced by FSH was significantly inhibited with HIF-1α inhibition.The results of autophagy-related proteins also indicated that FSHinduced autophagy was inhibited.We performed TEM to monitor the mitophagic flux in GCs,inhibition of HIF-1α blocked the formation of mitophagic vacuoles,reduced the mitochondrial membrane potential as well as increased mitochondrial apoptotic proteins levels despite FSH treatment.These dates indicate that HIF-1α is a key downstream factor in FSH-induced mitophagy to protect hypoxic GCs.5.FSH actives mitophagy to prevent GCs from hypoxic apoptosis through HIF-1α-PINK1-Parkin signalingPINK1-Parkin system plays a critical role in mitochondrial quality control.We use PINK1 si RNA to explore whether the PINK1-Parkin signaling is relevant to the mechanisms by which FSH protects against hypoxic injury in GCs.Blocking PINK1 expression prevented the induction of autophagic response,but did not affect total mitochondrial mass,HIF-1α accumulation,or Parkin expression in FSH-treated hypoxic GCs.Silencing of PINK1 expression markedly blunted the inhibitory effects of FSH on cell viability loss,augmented proportion of apoptotic GCs as well as the expression of apoptotic proteins during hypoxia stimulation.The results of TEM also showed a dramatic decrease in the number of mitochondria-containing autophagic vesicles.Collectively,our data demonstrate that the activation of mitophagy via the HIF-1α-PINK1-Parkin signaling is an important mechanism of FSH-mediated GC protection against hypoxic injury.Taken together,our findings demonstrate that FSH activates mitophagy via the HIF-1α-PINK1-Parkin cascade to prevent porcine GCs from apoptotic death upon hypoxic conditions.Therefore,targeting this pathway using small molecular inhibitors might provide novel therapy for anovulatory disorders such as PCOS and POF.