Study On The Phosphorylation Of Fibrous Sheath Protein FSCB During Capacitation And Hyperactivation In Mouse Spermatozoa

Spermatozoa freshly ejaculated into the female reproductive tract undergo an array ofbiochemical changes to have fertilizing capacity, a process called spermatozoa capacitation.Spermatozoa capacitation involves increased plasma membrane fluidity, cholesterol efflux,ion flow, and tyrosine phosphorylation, hyperactive motility, and induction of the acrosomereaction. PKA-catalyzed tyrosine phosphorylation is an important post-translational event anda hallmark of spermatozoa capacitation.There are more than20proteins associated with the fibrous sheath, including AKAP3,AKAP4, TAKAP-80, GAPDS, HK1-S, GSK3β, ALDOA, LDHA, SFEC, triose phosphateisomerase, GAPDH, pyruvate kinase, LDH-C, sorbitol dehydrogenase, GSTM5, FS39,Ropporin, Rhophilin, SP17, PDE4A, FSIP1and FSIP2, ASP, and CABYR. Nevertheless, thesubstrates and mechanism(s) of action of PKA in spermatozoa capacitation are still unknown.Herein we describe findings that indicate FSCB is a novel protein expressed specificallyon the surface of the fibrous sheath of the mouse spermatozoa principle piece. We alsoobserved that FSCB can bind to spermatozoa-specific CABYR protein, a calcium-bindingprotein on spermatozoa flagella that undergoes tyrosine and serine/threonine phosphorylationduring spermatozoa capacitation. FSCB is also a calcium-binding protein, which can bephosphorylated by PKA in vitro. It is comprised of a conserved PXXP motif, proline-richregions and extensin-like regions, and is localized to the medium electron density cortex ofthe fibrous sheath of spermatozoa flagella. As FSCB is found in proximity to otherspermatozoa fibrous sheath proteins such as AKAP3, AKAP4and CABYR and is capable ofbeing phosphorylated and binding to calcium, it may be an important protein contributing tospermatozoa flagellar movement, spermatozoa capacitation and hyperactivation.The present study was aimed at exploring the basic biological properties of FSCB,whether it is phosphorylated by PKA during spermatozoa capacitation, and the effect of PKAagonist DB-cAMP and PKA antagonist H-89on FSCB phosphorylation and spermatozoa motility characteristics, in order to elucidate the molecular basis and regulatory mechanism(s)of spermatozoa capacitation. Three parts were included in this study.Part One: Serine/threonine and tyrosine phosphorylation of FSCB duringspermatozoa capacitationWe reported previously that FSCB is phosphorylated in vitro by the PKA catalyticsubunit, and that it possesses multiple phosphorylation sites. To further investigate whetherFSCB is phosphorylated during spermatozoa capacitation, male Kunming mice (12-16weeksold) were sacrificed and the spermatozoa was cultured in pre-equilibrated HTF media and M2media for two hours. Same amount of the spermatozoa were lysed and followed by proteinquantitation. Western blot analyze was conducted with anti-phospho-(Ser/Thr) PKA substrateas well as the pre-immune serum as control.The results indicated a visible270kDa positiveband at2h after capacitation, suggesting that a270kDa protein can be phosphorylated byPKA. This positive band did not appear before capacitation, or in the negative control. Toinvestigate whether this270kDa protein is just the FSCB protein, immunoprecipitation andWestern blot assays were conducted. Mouse spermatozoa before and at2h after capacitationwere lysed and subjected to immunoprecipitation with the use of anti-phospho-(Ser/Thr) PKAsubstrate, anti-phosphotyosine and anti-FSCB antibodies, respectively, followed by Westernblot using anti-FSCB antibody. The results showed that there was a270kDa band at2h aftercapacitation in all three immunoprecipitation products. However, this band was only observedin the immunoprecipitaton product of anti-FSCB antibody before capacitation. Thus, thesefindings further demonstrated that the proteins precipitated by anti-phospho-(Ser/Thr) PKAsubstrate and anti-phosphotyrosine antibodies after spermatozoa capacitation was just theFSCB protein.To further study FSCB phosphorylation over time, aliquots of spermatozoa were lysed at1,2,5,10,30,60, min, and120min during capacitation. The lysates were subjected toprotein quantification and analyzed by Western blot with anti-phospho-(Ser/Thr) PKAsubstrate and anti-FSCB antibodies, respectively.The results indicated that FSCBphosphorylation occurred as early as1min after spermatozoa capacitation, which increasedover time and remained stable after60min. The results demonstrates that the tyrosine andSer/Thr phosphorylation of FSCB occurred during spermatozoa capacitation and the extent of tyrosine phosphorylation in certain proteins was closely associated with the phase ofcapacitation.Part Two: Effect of PKA activity on FSCB phosphorylationThe above results indicated that FSCB could be phosphorylated by PKA duringspermatozoa capacitation. To further elucidate the molecular basis and regulatory mechanismof FSCB phosphorylation during spermatozoa capacitation, we investigated how PKA activityaffected FSCB phosphorylation by using the agonist and the antagonist of PKA. Spermatozoawas treated with H-89and DB-cAMP, respectively, and then the status of FSCBphosphorylation was observed. Mouse spermatozoa were cultured with HTF medium, HTFmedium containing0.5μM H-89, M2medium, and M2medium containing2.5mM DB-cAMP,respectively. Spermatozoa were cultured for1,2,5,10,30,60min and120min, whereuponan aliquot of the same volume of spermatozoa was centrifuged for PKA activity assay.Same volume of spermatozoa at30min time point after cultured in the above mediumwas lysed and the lysates were subjected to protein quantification and Western blot assay withanti-phosphorylation PKA substrate antibody. Rabbit preimmune serum was used as anegative control.The results showed that0.5μM H-89significantly decreased spermatozoa PKA activity,compared with HTF medium control. However,2.5mM DB-cAMP could markedly increasethe spermatozoa PKA activity. Western blot assays for spermatozoa lystates showed anobvious phosphorylated protein band at270kDa in the HTF medium group and the M2medium containing2.5mM DB-cAMP group, in contrast to a faint phosphorylation proteinband at270kDa in the M2medium group and the HTF medium containing0.5μM H-89group.All these results suggested that H-89suppressed PKA activity and subsequent FSCBphosphorylation, and DB-cAMP enhanced PKA activity and subsequent FSCBphosphorylation.Part Three: FSCB phosphorylation was closely associated with spermatozoamotilityTyrosine phosphorylation in specific proteins is a hallmark of spermatozoa capacitation,and it is associated with spermatozoa motility characteristics, e.g., speed and wobblingfrequency. To understand the relationship between FSCB phosphorylation and spermatozoamotility characteristics, we analyzed spermatozoa motility characteristics at various time points. Mouse spermatozoa were cultured in HTF medium, HTF medium containing0.5μMH-89, M2medium, and M2medium containing2.5mM DB-cAMP. A5μl aliquot ofspermatozoa was analyzed at37°C at1,2,5,10,20,30,60min and120min aftercapacitation using Spermatozoa Class Analyzer4.0(Spain).The results indicated that (A+B)%(%moving forward spermatozoa) was significantlyhigher in the HTF group than the HTF medium containing0.5μM H-89group at1,5,10,20,30min and60min after capacitation. In contrast,(A+B)%was higher in the M2mediumcontaining2.5mM DB-cAMP group than the M2group spermatozoa at1,5,10min and20min after capacitation. Similarly, VCL was significantly lower in the HTF medium containing0.5μM H-89group than in the HTF group at1,5,10,20,30,60min and120min aftercapacitation, while it was higher in the M2medium containing2.5mM DB-cAMP group thanin the M2group at1,5,10,20min and30min after capacitation.The characteristics of thecurves depicting these parameters were basically consistent with PKA activity changes. Allthese results demonstrated that H-89suppressed spermatozoa motility through suppressingPKA activity and phosphorylation of FSCB, and that Db-cAMP promoted such activities.