| Background and purpose:The World Health Organization defines infertility as follows: a disease of the reproductive system defined by the failure to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse.In recent years,infertility has widely been accepted as a public concern along with the acceleration of industrialization and the changes of our lifestyles such as the increased psychological pressure,unhealthy diet,deteriorating environment and other factors which lead to the increased proportion of infertility.Although male infertility accounted for 40% of the total infertility in human being,the causes and therapies of male infertility are still too far to be understood.It has been reported that FK506 administration lead to spermatozoa with a rigid midpiece through inhibiting the activities of calcineurin in mouse testis.FKBP12.6,an important member in FK506 binding protein family,can combine with FK506 to form a complex that plays as an immunosuppressant through inhibiting the activities of calcineurin in a direct way,meanwhile,it can also regulate the activity of calcineurin through regulating the release of calcium from ryanodine receptor 2.Therefore,this study aimed to determine the role of FKBP12.6 in male infertility induced by FK506 using FKBP12.6 knockout mice,in which the protein might provide a new target for treatment of male infertility.Methods and results:1.Disruption of FKBP12.6 alleviates the male infertility induced by FK506 in vivo: To determine the role of FKBP12.6 in male infertility induced by FK506.WT and FKBP12.6-/-adult male mice were injected with FK506 subcutaneously.The results showed that there are many of sperm with rigid midpiece,and the rate of fertilization was reduced sharply in WT mice after FK506 treatment.However,sperm with rigid midpiece in FKBP12.6-/-mice was much less than that of WT,and the rate of fertilization was significantly improved in FKBP12.6-/-male mice after FK506 treatment,indicating that FKBP12.6 played a significant protection in the FK506-induced male infertility.2.FKBP12.6 was highly expressed in testis microsome(most of which are endoplasmic reticulum)of mice: the formation and development of sperms is in testis.In order to definite the role of FKBP12.6 and FKBP12 in development of sperm in adult male testis,total protein and microsome in testis of male mice were isolated.Western blot analysis showed that although the expression of FKBP12 was much more than that of FKBP12.6 in total protein of testis,the relative expression of FKBP12.6 in microsome was significantly higher than that of FKBP12,suggesting that FKBP12.6 may plays an important role in regulation of calcium release in testis.3.FKBP12.6 plays a dominant role in inhibiting the activity of testisspecific calcineurin(PPP3CC/PPP3R2): In order to elucidate the role and possible mechanism of FKBP12.6 and FKBP12 in inhibiting testis-specific calcineurin,mouse GST-FKBP12.6 and GST-FKBP12 fusion proteins were expressed and purified using prokaryotic expression system in vitro,respectively.Then the PPP3CC/PPP3R2 in whole lysis of testis or the PPP3CC/PPP3R2 recombinants expressed and purified in vitro were pulled down using the fusion proteins with GSTaffinity chromatography.Our results showed that the interacting of FKBP12.6 with testis-specific calcineurin is significantly higher than FKBP12,suggested that FKBP12.6 played a dominant role in the process of inhibiting the activity of calcineurin through binding to FK506.ConclusionOur study showed that deficiency of FKBP12.6 alleviates the male infertility induced by FK506 in vitro and the mechanisms may probably be involved in the disruption of FKBP12.6 cancelled the inhibition of testis-specific calcineurin in sertoli cell of testis by FK506,thus to facilitate the nature development of sperm. |
PDF Full Text Request