Influences Of Combined Administration Of Low-dose Gossypol And Steroid Hormone On Spermatogenesis In Rat

Up to now, no even one safe, effective, reliable and reversible male contraceptive is on sale. The development of male contraceptive pill has lagged far behind that of female contraception. Through year’s studies, our laboratory makes many substantial progresses in combining low-dose gossypol and steroid hormones together as a male contraceptive. After screening many candidates, we focus on a promising regimen as a male contraceptive: gossypol acetic acid (G) combined with desogestrel (DSG)/ethinyl estradiol (EE) and testosterone undecanoate (TU) Treated with this regimen, all the rats were infertile after8weeks, and no obvious side effect was found. Though the combined administration has such a remarkable effect, its targeting sites during spermatogenesis are unknown.Adult male rats were divided into four groups randomly. Group GH:rats were administered with G (12.5mg/kg-d) and DSG (0.125mg/kg-d)/EE (0.025mg/kg-d)/TU (100mg/kg·d); Group G and Group H:rats were fed with one single dose of gossypol or steroid hormone respectively, with the same dosage as Group GH. Group C:rats only received vehicle (1%methyl cellulose) as control. The rats were killed by butaylone injection at4,6,8,10weeks, testis and epididymis were removed. Through morphological studies, homogenization-resistant sperm counting, TUNEL, oil red O staining, immunohistochemistry, western blotting, RT-PCR and seminiferous tubule segments cultured, we investigated the influences on spermatogenesis of the combined regimen.The number of homogenization-resistant elongated spermatids in per gram testes was used to evaluate the sperm production. The spermatid quantity in group GH progressively declined, from the same level as control at4w to26.4%of control value at10w. The spermatid number of group H decreased slower than group GH, but closed to group GH at10w. Meanwhile, no obvious change appeared in group G. Investigated the cross sections of seminiferous tubules, we found the elongated spermatids were gradually reduced in group GH and group H with time. Almost half seminiferous tubules had no elongated spermatids in group GH versus10%in group H at8w, but the difference between the two groups disappeared at10w. Besides elongated sperms, primary spermatocytes and round spermatids in group GH and group H were also diminished. Meanwhile, we found many round germ cells instead of mature sperms were in the canal of caput epididymis. All these observations indicated the spermatogenesis had been disrupted in group GH and group H, so the quantity of mature sperm descended in testis subsequently. However, different from the former two groups, group G had no notable change, either in spermatid production or in the morphology of the testis.Using antibody of proliferating cell nuclear antigen (PCNA), we investigated the proliferation and differentiation of spermatogenic cells in all groups. No obvious difference was found in the numbers of PCNA positive cells in stage VII-VIII seminiferous epithelium among the four groups. It suggested the proliferation and differentiation of spermatogonia were normal in all treated rats.Detected by TUNEL, we found the apoptosis increased significantly at the seminiferous epithelium of group GH and group H. The numbers of apoptotic cells in group GH and group H were66and55.8times of that in group C, respectively. That might be one reason for the fewer spermatogenic cells in the former two groups. Among the whole apoptotic cells, pachytene spermatocyte in stage VII-VIII seminiferous tubule was the major apoptotic cell type in both group GH and group H. While some apoptotic round spermatids could also be seen at the same tubules. The results of group GH were similar as of group H in many ways, including the total apoptotic number and the major type of the apoptotic cells, except the apoptotic cells in stage Ⅸ-Ⅵ tubule in group GH were slightly more than that in group H. Meanwhile, only a few of apoptotic cells appeared either in group G or in group C. These results suggested that steroid hormone withdraw might play a crucial role in cell apoptosis in group GH, and gossypol enhanced that effect. No TUNEL positive spermatogonia, Sertoli cell or Leydig cell was detected.Sertoli cells can phagocytose the apoptotic cells to form lipid droplets. With oil red O staining, we found red stained lipid droplets located at the basal compartment of seminiferous epithelium. The areas of lipid droplets in group GH and group H, but not in group G, increased markedly. The result proved indirectly that plenty of apoptotic cells in group GH and group H were phagocytosed by Sertoli cells, and the treatment had no obvious influence on the phagocytic ability of Sertoli cells.More and more round germ cells appeared in the duct of epididymis with time. The immature germ cells depleted from the seminiferous epithelium indicated the adhesion junction between Sertoli cells and spermatogenic cells might be disrupted; therefore we detected the expression of some adherent related molecules in the testis. Both of western blotting results and immunohistochemical results showed the expression of neural cadherin (N-cadherin) protein increased in group GH and group H after treatment. Positive signals between the germ cells in the middle portion of the seminiferous epithelium became stronger as time passing by. The mRNA expression of N-cadherin gene at8w in group GH and group H were slightly more than that in group C, but no significant differences among them. By immunohistochemical staining of integrin a6, we found the positive reactions were much stronger with time, between the round spermatids at stage IV-VIII seminiferous epithelium. However, the expressions of integrin β1were different from the former two molecules. The immunohistochemical reactions of integrin β1decreased significant in the middle part of seminiferous epithelium in group GH and group H, while the changes happened in group GH were more severe. Meanwhile, the expression in group G was similar to that of group C in stage I-VIII, but lower in stage IX-XIV. These results indicated steroid hormone withdraw might induce the fallen of integrin β1expression, and gossypol strengthen the effect. The integrin β1expression descended might correlate with the germ cells detachment in group GH and group H. However, the ascended expression of both N-cadherin and integrin α6might be the compensations for the germ cells loss.The step19spermatids at the luminal edge of stage VII-VIII seminiferous epithelium lost significantly from4w to6w. But some step19spermatids were still remained at the luminal edge till stage XI. Meanwhile, many elongated nuclei of spermatids retained at the middle or basal portion of the seminiferous epithelium in group GH and group H. Those retained spermatid nuclei might be phagocytosed by Sertoli cells as degeneration, but we failed to prove it because of TUNEL-negative. Also no obvious change of the three former adhesion related molecules expression was found around step19spermatids, if there was any other change happened in the adhesion junctions between step19spermatids and Sertoli cell was unclear. No notable alteration was observed in group G Through the experiment of drug influence on cultured tubule segments, we found the low hormone level and gossypol lead more step19spermatids detached from the seminiferous epithelium.Immunohistochemical staining of three activated signal transducers:p-ERK, p-JNK and p-p38MAPK were investigated in group GH and group H. We found the p-ERK expressions in Sertoli cells were increased, while the p-p38MAPK expressions between the round spermatids were increased too with time, but no obvious positive reaction of p-JNK appeared in the seminiferous tubules without elongated spermatids. The expression ways of p-ERK and p-p38MAPK were similar to the expression of N-cadherin and integrin a6, respectively. Thus, the results indicated some relations might have between the signal transducers and the adhesion related molecules.It was interesting in testis that most results in group GH were consistent with that in group H, with significant differences compared with group C. Moreover, the differences between the group GH and group C were appeared much earlier and severe than that between group H and group C. The results illustrated the gossypol had further effects on the roles of steroid hormone withdraw. The rats treated with the combined regimen got a low spermatid quantity due to germ cells apoptosis, elongated spermatids phagocytosis and germ cells detachment from the seminiferous tubules.The sperm quantities and motility in epididymis in all three groups were decreased with treatment time. At10w, the numbers of homogenization-resistant sperm in group G, group H and group GH were only44.41%,40.16%and15.43%of the control value, respectively. In the meantime, the numbers of motile sperms fallen to14%and15.43%in group G and group H, respectively, and no motile sperm could be seen in group GH. In addition, the percentage of morphological abnormal sperm increased, especially in group GH. Up to10w, more than90%sperms were abnormal in group GH, while the other two groups also had high abnormal sperm ratio,54.67%in group G, and63.83%in group H. There were different types of morphological abnormality in sperms among the three groups. Decapitated sperm was the main type of abnormal sperms in group H. Although the decapitated sperms also existed in group G, the abnormal-tail sperms elevated thought the treatment. Meanwhile, in group GH, the abnormality was first appeared on sperms tail, and then decapitated sperms turned into the majority. These result indicated that gossypol and steroid hormone might have different targets. Neither abnormal sperm head nor histopathological alteration of epididymal epithelium was observed in the three treated groups. The result in group GH was more significant different from that in group C than the other two groups. It indicated gossypol and steroid hormone both played notable roles in epididymis sperms, but they might have different target sites. With their combined effects, the percent of abnormal sperms increased significantly, while the sperm quantity and motility decreased obviously.On the whole, both in testis and epididymis, both gossypol and steroid hormone had important functions. In testis, steroid hormone played a crucial role, and gossypol enhanced the effects. However, in epididymis, both of them were identically important. Through synergistic action of the two drugs, the combination regimens made a remarkable effect in contraception.Through systematical morphologic studies, we observed the influences on spermatogenesis of combined administration. Moreover, we detected the alterations of adhesion related molecules and three signal transducers expression during drug treatment. Meanwhile, with the study in vitro, we provide some evidence to supply the conclusion we had gotten in vivo. Through these investigations, we discussed the possible target sites in spermatogenesis of the combination regimen, and the roles of gossypol and steroid hormones during treatment. It will helpful for the further understanding on the combined administration.