Expression of TUBB4B in mouse primary spermatocyte GC-2 cells and its regulatory effect on NF-κB and MAPK signaling pathway / 南方医科大学学报

OBJECTIVE@#To explore the interaction between Tubulin beta 4B class IVb (TUBB4B) and Agtpbp1/cytosolic carboxypeptidase- like1 (CCP1) in mouse primary spermatocytes (GC-2 cells) and the role of TUBB4B in regulating the development of GC-2 cells.@*METHODS@#Lentiviral vectors were used to infect GC-2 cells to construct TUBB4B knockdown and negative control (NC-KD) cells. The stable cell lines with TUBB4B overexpression (Tubb4b-OE) and the negative control (NC-OE) cells were screened using purinomycin. RT-qPCR and Western blotting were used to verify successful cell modeling and explore the relationship between TUBB4B and CCP1 expressions in GC-2 cells. The effects of TUBB4B silencing and overexpression on the proliferation and cell cycle of GC-2 cells were evaluated using CCK8 assay and flow cytometry. The signaling pathway proteins showing significant changes in response to TUBB4B silencing or overexpression were identified using Western blotting and immunofluorescence assay and then labeled for verification at the cellular level.@*RESULTS@#Both TUBB4B silencing and overexpression in GC-2 cells caused consistent changes in the mRNA and protein expressions of CCP1 (P < 0.05). Similarly, TUBB4B expression also showed consistent changes at the mRNA and protein after CCP1 knockdown and restoration (P < 0.05). TUBB4B knockdown and overexpression had no significant effect on proliferation rate or cell cycle of GC-2 cells, but caused significant changes in the key proteins of the nuclear factor kappa-B (NF-κB) signaling pathway (p65 and p-p65) and the mitogen-activated protein kinase (MAPK) signaling pathway (ErK1/2 and p-Erk1/2) (P < 0.05); CCP1 knockdown induced significant changes in PolyE expression in GC-2 cells (P < 0.05).@*CONCLUSIONS@#TUBB4B and CCP1 interact via a mutual positive regulation mechanism in GC-2 cells. CCP-1 can deglutamize TUBB4B, and the latter is involved in the regulation of NF-κB and MAPK signaling pathways in primary spermatocytes.

The extract of "shoe flower" (Hibiscus rosea sinensis L) leaves inhibit the spermatogenesis of ddy strain mice.

This study was conducted in order to develop male contraception from plants, namely the "shoe flower" (Hibiscus rosea sinensis L) leaves. The objective of this study was to find out whether the extract of "shoe flower" leaves could inhibit the process of spermatogenesis on ddy strain mice. This research was performed in 3 groups and each group consisted of 8 mice. The control group was given 1% carboxy methyl cellulose (CMC) in 0.5 ml aquabides. The treatment group I was given the extract of "shoe flower" leaves 700 mg/kg BW and 1% CMC in 0.5 ml aquabides, and the second treatment group was given the extract of "shoe flower" leaves, 800 mg/kgBW and 1% CMC in 0.5 ml aquabides. The treatment were given for 40 days in accordance with the spermatogenesis cycle. Then, the production of histological slides of the mice testis and the observation of the slides using light microscope with magnification of 100x and 400x were done. Further, counting of the spermatogenic cells was done. At last the pictures of seminiferous tubulus cross-section of the three groups which consisted of spermatogenic cells were taken through light microscope with magnification of 100x and 400x using Fuji camera and Fuji film, 200 ASA. The results showed significant differences between the control, treatment I, and treament II group. There were decreased numbers of spermatogonia, pachyten primary spermatocytes and spermatids in treatment groups (P<0.01). The result of this study showed that the extract of "shoe flower" (Hibiscus rosea sinensis L) leaves, inhibited the process of spermatogenesis of ddy strain mice. It is hoped that the result of this study can be developed into a male contraception.