| This study was undertaken to study effect of orally administrating the attenuated Salmonella choleraesuis-mediated inhibin recombinant DNA vaccine (PXAIS) on rat spermatogenesis, testicular morphology and histology, concentrations of serum hormones, expression of genes and proteins related to spermatogenesis by adopting the technique of enzyme-linked immunoassay(ELISA), histological section technology, real-time quantitative PCR and western-blotting, with the purpose of clarifing the mechanism of PXAIS on spermatogensis in rats.1 Effect of orally administered inhibin gene vaccine PXAIS on immune response in ratsDifferent concentrations of the inhibin gene vaccine PXAIS (1010CFU/mL 109CFU/mL,108CFU/mL) were used to immunize male rats for three times(one primary immunization followed by two boosters), ELISA was used to detect level of serum antibody absorbance for the different given periods and the results showed that:(1) The serum antibody levels in all the immunized groups were increased, which were higher in the middle dose group (1.64±0.21)than the other groups, and there was a remarkable difference between the medium group and the control group in the first week after the second immunity (P<0.01); (2) There was an obvious difference for the serum antibody levels in the high-dose group (1.49±0.30)in comparison with the control group in the first week after the second immunization (P<0.05); (3) There, was an extreme difference for the P/N value in the low-dose group (1.56±0.35) when compared with the control group(1.56±0.35) after the first booster immunization (P<0.05)2 Effect of orally administered inhibin gene vaccine PXAIS on spermatogensis in ratsChanges of organ coefficient, organ volume, number of the sperms, rate of sperm deformation were studied by decapitating the rat, and the results showed that:(1) testicular volume in the tested groups were increased, and there was a significant difference in the low dose group and middle-dose group when compared with control group (P<0.05), but there was no obvious effect on the volume of epididymis (P>0.05); (2) The coefficients of the testis, epididymis and prostatewere increased, and there was a significant difference for the testicular coefficient in the high-dose group(0.93±0.06) in contrast to the controlgroup(.P<0.01), and a difference in the coefficient of epididymis(P<0.05), but no obvious effect on the seminal vescile (P>0.05);(3) There was a remarkable increase in the number of sperms (9.58±2.19) and decrease in the rate of sperm deformation(18.50±2.30), and difference was obvious in the medium dose groupcompared with the control and the empty plasmid group (P< 0.01).3 Effect of orally administered inhibin gene vaccine PXAIS on histological structure of the testis in ratsTesticular morphological changes in rats were observed after HE staining. The results showed that:(1) Structures of seminiferous tubules in each group were clear, with intact basical membranes and normal development of the spermsl; (2) There were different number of spermatogenic cells in the different groups, and the layer number of spermatogenic cells, the thickness of the basical membranes on the outside of the tubules, size of the lumens in middle-dose group and lacuna of residual cytoplasm were completely different compared to the control group.4 Effect of orally administered inhibin gene vaccine PXAIS on endocrine hormones in ratsThis experiment was to detect changes of concentrations of InhibinB(INHB), follicle stimulating hormone(FSH), luteinizing hormone(LH) and testosterone (T) using ELISA. The results showed that:(1) Contents of the serum inhibin B in the three immunized group were decreased, there was difference in the low-dose group (15.66±0.51)compared with the control group on the 2th week after the primary immunization (P< 0.05), a minimum value was produced in the middle dose group(14.41±1.10) on the first week after the second immunization in comparison with the control group and the empty plasmid group (P<0.01); (2) Contents of FSH were significantly increased in the three experimental groups compared with the control group after immunization, which in the middle dose group (6.02±0.12)and high dose group (5.92±0.35)were remarkably higher compared with the control group and the empty plasmid group on the second week after the primary immunization (P<0.01), and which in the low dose group(5.53±0.16) was higher than the control group (P<0.05) on the third week after the primary immunization. (3)Contents of LH were decreased in the three experimental groups, which in the middle dose group (14.48±0.67)were lower compared with the empty plasmid group on the first week after the primary immunization (P<0.05). Contents of the LH continued to decrease on the secondweek after the primary immunization, which in the low dose group, there was an obvious difference in both the medium(14.01±0.63) and low dose groups(13.84 ± 0.40) in contrast to the empty plasmid group (P<0.05) on the second week after the primary immunization. In addition, there was an obvious difference between the low dose group and the control one(P<0.05). (4) Changes of the T contents in the control group and the empty plasmid group were not clear from the first to the seventh week, which in the low-dose group (74.82±4.50)was lower in the second week after the second immunization when compared with the control group (P<0.05). The contents of T were apparently increased in the middle dose group (76.41±3.75)on the first week after the primary immunization, which were much higher than those in the control group (P<0.05). Besides, there was sharp difference for the contents of T in the middle group (86.57±6.29)compared to either the control or the empty plasmid group(P< 0.01).5 Effect of orally administered inhibin gene vaccine PXAIS on expression of genes and proteins related to spermatogenesis in ratsChanges of expression of genes and proteins of INHβB、Vimentin、SMAD4 related to spermatogenesis in rats were detected by RT-PCR and Western blot,respectively. The results showed that:(1) Expression of INHβB gene was decreased after pXAIS immunization, which in the middle dose group was lower than the control (P< 0.05); Compared to the control group, there was a significant difference in the expression of proteins of INHβB in both middle and high dose group(P< 0.01) and low dose group(,P< 0.05). (2) Expression of Vimentin gene was increased after pXAIS immunization in all the immunized groups, there was an obvious difference in the low dose group(20.14±2.74, P< 0.05) in contrast to the control group(17.65±1.49), and difference was remarkable in the middle group(22.01±1.60, P< 0.01) and high dose group(21.09±2.11, P< 0.05) when compared to the control and the empty plasmid group; Expression of Vimentin was increased and the difference was highly significant in comparison with the control group (P< 0.01). (3) Expression of Smad4 gene was increased after pXAIS immunization, there was extreme difference in the middle dose group(25.32±3.11, P< 0.01) and high dose group(22.36±1.98, P< 0.05) in contrast to the control group(19.93±1.15) or the empty plasmid group(19.92±0.89), respectively; However, there was no difference between the low dose group and the control one (P>0.05). For the protein expression, expression of Smad4 was increased in all the immunized groups, and difference was markedly obvious in the middle and high dose groups (P< 0.01) and low dosegroup(P<0.05).In conclusion, inhibin gene vaccine can stimulate the immune response, also lead to an increase of the number of the sperms and a decrease fo the rate of the sperm deformation, consequently promoting spermatogenesis. There are decreases in levels of inhibin B or LH and increases in FSH or T after inhibin gene vaccine immunization. In addition, immunization against inhibin gene vaccine can stimulate expression of Smad4 and Vimentin and reduce expression of INHβB gene. |
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