| BackgroundIt is key objective of male contraceptive research to develop the safe,high active, recoviable and receptiable contraceptives for men, at present.Hormonal contraception has been shown to be the best outlook as male method.Combined administration with progesterone or transient testicularhyperthermia enhances the suppression of spermatogenesis induced byandrogen, by the enhanced germ cell apoptosis. Hypothesized hormonaltreatment as "hit 1" and heat exposure as "hit 2" induced germ cell apoptosisvia different death inducing and/or execution pathways, and induced diffentstage- and cell-specific apoptosis, the combination of the two "hits" willresult in more complete and rapid but reversible suppression ofspermatogenesis. However, molecular mechanism of male hormonalcontraception, by inducing apoptosis, is still unknown. To characterizequalitatively and quantitively some proteins which principally involved inregulation of the apoptotic pathways, inhibition of proliferation or stimulation of sperm retention by germ cell apoptosis, it may provide usnovel targets for contraceptive development in the longer term. These targetscan be used for molecular modeling of new drug development resulting inspecific agonists or antagonists. Such new drugs, proteins, used alone orcombination, may synergistically turn accelerated apoptosis/proliferation/spermiation "on" or "off" to achieve simple, safe, complete and reversiblemale contraception and for the treatment of male infertility. In addition,understanding the mechanisms of transient testicular hyperthermia and/orhormones on spermatogenesis may have implications on environmentalimpact (e.g., heat, toxins, endocrine disruptors) on male reproductive health.ObjectivesIn present study, we observed and compared whether the enhancedeffect of androgen alone or combined with transient testicular hyperthermiaor progesterone on the suppression of spermatogenesis, so as to achivesimple, safe, complete and reversible male contraception. Second objective isto study the mechanism of male contraception of hormone(s) with protemicsand molecular methods, which is for understanding the related cellularprocess and the network of proteins showing changes after hormonaltreatment. Key proteins among this network will be selected and analyzed byWestern blotting and Immunohistochemical analysis. These protein involvedin regulation of germ cell apoptosis pathways may provid us new targets fordeveloping male contraception and treating male infertility. Subjectives, materials and methodsThe study was designed including observation period for 4 weeks,treatment period for 18 weeks, and recovery period for 12 weeks. Totally 72healthy male volunteers were divided randomly into 4 groups (n=18 eachgroup), as follows. 1) Testosterone undecanoate (TU) group: TU 1000 mgIM as the first dose followed by 500 mg IM every 6 weeks, means at 6 weeksand 12 weeks during treatment, without any more treatment after that; 2)Heat group: Submersion of scrota (testes) at 43℃water for 30 minutes for 6consecutive days, one time every day, during the second week of treatmentperiod, without any more treatment after that; 3)TU+Heat group: TU andscrota hyperthermia treatment as above; 4) TU+LNG group: TU injection asabove plus oral levonorgestrel (LNG) 250μg per day for 18 weeks. Atdifferent time, pretreatment, every 3 weeks during treatment, every 4 weeksduring recovery, semen parameters and blood biochemistry and hormonallevels (LH, FSH, T, E2, SHBG) were assayed, and physic examination andthe questionnaires of sexual function and mood were also performed.Testicular biopsy was performed in 4 men at pretreatment, and 4 men atposttreatment 2 weeks and 4 men at 9 weeks in each group for routinemorphological observation. We investigated the differential proteinexpression in human testicular biopsies 2-wk after TU treatment, comparedwith pretreatment, with two-dimensional gel electrophoresis (2-DE) andmatrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF), bioinformation assay, Western blotting andimmunohistochemistry. Other tissues were stored in liquid nitrogen for thefuture study.Results1. Clinical Study on the contraception of androgenSperm concentration decreased significantly in all groups at 3 weeksafter treatment except the TU alone group. TU alone suppressed spermoutput very slowly and mean sperm concentration was 62.4 (decreased byonly 14.9, 19.3%) million/ml at 6 weeks, and sperm output reachedmaximum suppression at week 18, which was 20.9 (decreased by only 59.1,74.9%) million/ml. Sperm concentration in heat group decreasedmaximally to 20.9(decreased by 59.1, 74.9%) million/ml at 6 weeks, andthen gradually returned to baseline at 12 weeks. No men in this group hadsperm concentration below 3 million/ml at any time. The combination oftesticular heating and TU injections suppressed mean sperm concentrationmarkedly and quickly to 10.9 (decreased by 64.8, 85.6%) million/ml at 6weeks and then remained suppressed at very low levels until the end oftreatment. At both 3 and 6 weeks, Heat only and TU + Heat groups hadsuppressed significantly mean sperm output more than TU alone group(p＜0.0001), but these two groups receiving heat did not differ significantly.At 9 weeks, Heat group's sperm count recoved a little, while TU + Heat andTU alone groups had further suppression (69.3%, 35.3%). Thus separately TU and Heat provided similar (p=0.12) suppression at 9 weeks that wassignificantly (p＜0.0005) less for each treatment alone than TU and Heatcombination. Compared with TU group, TU+LNG suppressed sperm outputmuch more strongly. At weeks 6, TU+LNG suppressed sperm concentrationsignificantly greater than TU alone (p＜0.0001), which was 37.3 (decreasedby 39.4, 51.4%) million/ml, but less than TU plus heat (p＜0.0001). TU plusLNG kept on suppressing spermout to very low level until the end oftreatment (0.06 million/ml, decreased by 76.6, 99.9%). During the recoveryperiod, mean sperm concentration in all groups returned to levels above 20million/ml by week 30, 12 weeks after stopping treatment.All subjects with sperm concentration＜1 million/ml reached toazoospermia following time, except one subject in the TU+Heat group atweek 18. Azoospermia was achieved at end of treatment (week 18) by 7/18,6/18, and 17/18 men in the TU, TU+Heat, and TU+LNG groups, respectively.Additionally the TU+Heat group had significantly more men achievingoligozoospermia compared to each of the other groups at week 9.Mean serum total T and free T did not change in heat group, butincreased three weeks after each injection in the TU alone, TU+Heat andTU+LNG groups, which did not differ from each other (p=0.20). SeraSHBG was unchanged in the Heat only group, but showed a small butsignificant decrease in the TU alone and TU plus heat groups and asignificantly greater reduction (to 45% of baseline levels) in the TU plus LNG group compared to the other TU groups (p＜0.002). Following theincreased total and free T after TU injection, mean serum LH and FSH levelsshowed significant decreases 3 weeks after each TU injection in the TUalone, TU+Heat and TU+LNG groups (p＜0.05～0.0001). while FSH and LHlevels were maintained below 0.5 IU/l from week 3 to 18 in TU+LNG group,being significantly (p＜0.003) lower than the other groups.Sera self-antibodies of sperm (ASAbs) in those men after biopsy raisedtransiently in small range, without any clinical significance. Changes insexual function, blood chemistry and biochemistry were not found duringwhole treatment and recovery.There were no discernible changes in testicular morphology after 2weeks of treatment from TU alone or TU+LNG treatment when comparedwith control. Germ cell exfoliation was found, at 2 weeks, in Heat group andTU+Heat group. However, obvious changes in testicular morphology werenoted after 9 weeks of treatment with TU, TU+Heat and TU+LNG. In theTU group, changes included reduction in tubular diameter, and a markeddecrease in the number of spermatocytes and spermatids at 9 weeks. Theoverall testicular morphology of the TU+LNG group was similar to that ofthe TU group except for an additional decrease in germ cell numbers. Themorphological appearance of the testis after 9 weeks in Heat alone group wassimilar to that of control, suggesting the tissue reparation.2. Proteomic study on the contraception of androgen We chosed those tissues from control and TU treated group at 2 weeksfor the protemics study, while other tissues were stored for the future work.By examining 2-DE gels with ImageMasterTM 2D Platinum Software, wefound 17 spots in TU treated group that were significantly(P＜0.05) differentfrom the control group and fourteen spots were identified. These 14 spotscorresponded to 13 known proteins. Eight proteins were observed toparticipate in the complex functional network. And there both were fiveproteins, which occupied 62.5% of proteins in the network, participating inthe events of assemble and cell survival respectively.Four proteins, including calretinin (CALB2), SOD2, hnRNP K andPSMF1 were considered as key proteins among the functional networkassociated with TU treatment because they all participated in most ofcellular/molecular events induced by exogenous testosterone treatment,including assemble, cell survival, proliferation, and death. Western blotsshown that levels of CALB2 and Rbp1 decreased and that SOD2 and DDX4increased at 2 weeks after hormonal treatment, which were consistent withthe results determined by ImageMasterTM 2D Platinum Software.Immunohistochemistry shown a moderate cytosolic expression of CALB2 inthe Leydig cells and the Sertoli cells with little or no immunostaining in thegerm cells in control. No discernible changes in CALB2 immunoreactivitywere noted in both germ cells as well as in the Leydig cells 2-wk after TUtreatment. The intense cytosolic SOD2 immunoreactivity was noted in the Leydig cells and a moderate staining in the Sertoli cells in control. Amonggerm cells, SOD2 immunostaining was most striking in spermatogonia andearly spermatocytes, with moderate staining was noted in mid and latespermatocytes and in spermatids. There was an apparent increase in theexpression of SOD2 in mid and late spermatocytes and in spermatids afterTU treatment. DDX4 was expressed in germ cells and mainly localized inspermatocytes and round spermatids. No discernible increase of DDXD4immunoreactivity was observed in TU-treated testes. A cytosolic expressionof Rbpl protein was detected both in the Leydig and in the Sertoli cells incontrol, and a weaker expression was noted in germ cells in control. Wefound decreased expression of Rbp1 in Leydig cells after TU treatment.ConclusionExogenous androgen suppressed spermatogenesis, while transientscrotal hyperthermia suppressed spermout more quickly and recovable.Combined administration of TU (hit 1) with testicular hyperthermia (hit 2)enhanced and ccelerated the suppression of spermatogenesis induced by TUalone. Progesterone also enhanced the action of TU, which is slower thanheat. The hypothesis of "two hits", inducing suppression of spermatogenesis,was comfirmed by the clinical practice.Suppression of spermatogenesis induced by exogenous testosterone mayact on events of cell assembly and cell survival and apoptosis, etc. The initialchanges in testes induced by exogenous testosterone were the altered expression of series proteins, like 14 protein regulated in this study, amongwhich four proteins (CALB2, SOD2, hnRNP K and PSMF1) wereinvestigated on expression and function. These proteins may be candidateproteins and potential molecular targets responsible for suppression ofspermatogenesis. Further study on their function in the testis will providemore information about the exact mechanisms of male hormonecontraception. At the same time, it may provide new avenues formanagement of human infertility, and provide novel targets for contraceptivedevelopment.
|
PDF Full Text Request