Roles Of ERK1/2 And PI3K/AKT Signaling Pathways On Reproductive Damage In Hypothyroid Male Rats

Objectives:The hypothyroid rat-model induced by propylthiouracil（PTU）was established in this study to further explore the roles of estrogen receptor（ER）α,βand G protein-coupled receptor 30（GPR30）,extracellular signal regulated kinase（ERK1/2）and phosphoinositide 3-kinase（PI3K）/protein kinase B（AKT）signaling pathways,and mitochondria-mediated apoptosis in testes to better understand the potential molecular mechanism of testicular dysfunction in hypothyroidism rats.Methods:1.According to the body weight（240-270 g）,male Wistar rats were randomLy divided into control（C）,low-（L）and high-hypothyroidism（H）groups[1mL/100g BW/day 0,0.1 mg/kg and 10 mg/kg propylthiouracil（PTU）respectively]by intragastrical gavage for 60 days.All rats were killed to collect the testis tissues after hemospasia from hearts.2.Thyroid hormone levels in serum including the levels of triiodothyronine（T3）,thyroxine（T4）and thyroid stimulating hormone（TSH）were measured by radioimmunoassay.3.Testicular homogenate was made for further study.The mRNA expressions of ERα,ERβand GPR30 were quantified by real-time polymerase chain reaction（real-time PCR）.The pathway related protein expressions of ERK1/2,PI3K/AKT,cell cycle and apoptosis including ERK,p-ERK1/2,PI3K p85,p-PI3K p85,AKT,p-AKT Thr308,p-AKT Ser473,Ras,Raf-1,p-Raf-1 Ser259,p-Raf-1 Ser338,cyclin D1,Bad,Bax,Bcl-2,cytochrome c,caspase 9 and caspase 3were detected by Western blot.Results:1.The results showed that T₃ and T₄ levels were decreased,and TSH level was increased significantly in H group after PTU exposure compared with C and L groups.2.Compared with C and L groups,there weren’t significant changes of mRNA expressions of ERα,ERβand GPR30（p>0.05）,whereas the protein expressions of ERα,ERβand GPR30 were decreased significantly in H groups（p<0.05）.3.When compared with C group,the significantly decreased protein expressions of p-ERK1/2,p-PI3K p85 and p-AKT Ser473 in L and H groups,as well PI3K p85 and p-AKT Thr308 in H group were observed（p<0.05）,while the protein expression of ERK1/2 was not changed（p>0.05）.4.Compared with C group,the protein expressions of Ras,p-Raf-1 Ser259,p-Raf-1 Ser338 and cyclin D1 in L and H groups were significantly decreased（p<0.05）.5.Meanwhile,there were significant increase of Bad protein expression in L and H groups,significant increases of Bax/Bcl-2,caspase 9 and cleaved caspase 3 and significant decrease of total caspase 3protein expression in H group when compared with C group（p<0.05）.Conclusions:The potential mechnism of reproductive damage in hypothyroid male rats might be that the hypothyroidism suppresses the protein expressions of ERα,ERβand GPR30,then leads to the inhibition of signal related proteins of ERK1/2 and PI3K/AKT signaling pathways,further down-regulates the protein expression of cyclin D1,and ultimately activates the mitochondria-mediated apoptosis in testes.