Apocynin Ameliorates Monosodium Glutamate Induced Testis Damage By Impaired Blood-Testis Barrier And Oxidative Stress ParametersⅡ

Results

3.1. Serum Testosterone, FSH, and LH Levels 

Testosterone levels in the MSG group were lower compared to the control group. Although the MSG + APO group showed an increase in testosterone levels compared to the MSG group, this increase was not statistically significant (Figure 1A). The FSH level was higher in the MSG group compared to the control group. The FSH level in the MSG + APO group was decreased compared to the MSG group (Figure 1B). Decreased levels of LH in the MSG group and increased levels of LH in the MSG + APO group were not statistically significant compared to the experimental groups (Fig 1C ).

3.2. Testicular MDA, GSH, and SOD Levels 

The tissue MDA level was increased in the MSG group compared to the control group. On the other hand, when comparing the MSG + APO group with the MSG group, a significant decrease in MDA was observed (Figure 1D). GSH and SOD levels decreased significantly in the MSG group when compared with the control and APO groups, respectively. A significant increase in GSH and SOD levels was revealed in the MSG + APO group compared with the MSG group (Figure 1E, F).

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3.3. Sperm Count, Motility, and Morphology 

Normal morphology was observed in sperm samples in the control group and APO groups (Figure 2A, B). A large number of spermatozoa with morphological abnormalities were observed in the MSG group (Figure 2C). In the MSG + APO group, few abnormal sperm were detected, as a majority of sperms were of normal morphology (Figure 2D).

The sperm count and motility were lower in the MSG group compared to the control and APO groups. Those parameters were increased significantly in the MSG + APO group compared to the MSG group (Figure 2E, F).

3.4. Testicular Weight/Body Weight Ratio 

Although the testicular weight/body weight ratio of the MSG group was observed to be lower compared to the other experimental groups, this decrease was not statistically significant. There was no statistical difference in the testicular weight/body weight ratio between the experimental groups (Figure 3E).

3.5. Histopathological Results 

Light microscopical examination of testicular tissue by H&E staining revealed normal morphology of the seminiferous tubules in the control group. (Figure 3(A1)). Intact testicular morphology was observed in the APO group (Figure 3(B1)). In the MSG group, the germinal epithelia of the seminiferous tubules were disorganized. Vacuoles indicating prominent tissue damage were detected in the basal compartment of the seminiferous tubules (Figure 3(C1)). Those morphological disturbances were reflected in a significantly high histopathological score for this group. In the MSG + APO group, the majority of seminiferous tubules reflected the normal morphology, with a limited number of degenerated seminiferous tubules (Figure 3(D1)). 

Compared with the MSG group, the histopathological score was significantly low in the control and APO groups. The histopathological score significantly increased in the MSG group and was significantly decreased in the MSG + APO group, compared to the MSG group (Figure 3F). In addition, the thickness of the seminiferous tubules in the MSG group was low compared to the other experimental groups. In the MSG + APO group, there was a significant increase in tubule thickness compared to the MSG group (Figure 3G). The control and APO groups presented a strong PAS-positivity (Figure 3(A2, B2)) for the basement membranes of seminiferous tubules with regular morphology. Deteriorated basement membranes of seminiferous tubules of MSG resulted in a decreased PAS-positivity of sections. (Figure 3(C2)). PAS-positivity of the MSG + APO group was nearly equivalent to the control group, with a small number of disturbed seminiferous tubules presenting detached basement membranes (Figure 3(D2)). The morphologies of testicular tubules and tunica albuginea were normal in the control group (Figure 3(A3)), APO group (Figure 3(B3)), and MSG + APO group (Figure 3(D3)). However, in the MSG group (Figure 3(C3)), fat tissue was observed in the tunica albuginea, and the morphology of the tubular stroma was also normal.

3.6. Results for PCNA 

A large number of PCNA-positive cells in the seminiferous epithelium, as dark brown, were observed in the control and APO groups (Figure 4(A1, B1)). In the MSG group, a decrease in the number of PCNA-positive cells of the seminiferous epithelium was noticed (Figure 4(C1)). Also, PCNA-positive spermatogenic cells in the lumen of seminiferous tubules are seen in the MSG group. The proliferation index, lowest in the MSG group, was increased in the MSG + APO group compared to the MSG group (Figure 4E). The MSG + APO group also showed an increase in PCNA-positive cells in the seminiferous epithelium (Figure 4(D1)).

3.7. Results for TUNEL Immunocytochemistry 

In the control and APO groups, TUNEL-positive cells were low (Figure 4(A2, B2)). The number of TUNEL-positive cells was higher in the MSG group than in the other experimental groups (Figure 4(C2)). TUNEL-positive cells were fewer in the MSG + APO group than in the MSG group (Figure 4(D2)). The apoptotic index was higher in the MSG group than in the other experimental groups. There was a comparative decrease in this index in the MSG + APO group (Figure 4F).

3.8. Results for NOX-2 Immunofluorescence

Weak NOX-2 immunopositivity was observed in the control (Figure 5A–C) and APO groups (Figure 5D–F). The highest immunoreactivity was observed in the MSG group (Figure 5G–I), whereas a decrease was detected in the MSG + APO group (Figure 5J–L). Negative controls of the NOX-2 immunofluorescence analysis were shown in Figure S1.

3.9. Results for ZO-1 and Occludin Immunohistochemistry 

ZO-1 and occludin positivities were detected as a dark red color in the basolateral cytoplasm of Sertoli cells located in the seminiferous tubule of the germinal epithelium. While the intensities of ZO-1 and occludin immunoreactivity were highest in the control group (Figure 6A–C, Figure 7A–C, Figure 8(A1, A2) and Figure 9A, B) and APO group (Figure 6D–F, Figure 7D–F and Figure 8(B1, B2)) and a decrease in the distribution of immunoreactivities was observed in the MSG group (Figure 6G–I, Figure 7G–I and Figure 8(C1, C2)). Increases in ZO-1 and occludin positivities were observed in the MSG+APO group, compared to the MSG group (Figure 6J–L, Figure 7J–L, and Figure 8(D1, D2)).

3.10. Transmission Electron Microscopical Results 

In the control and APO groups, normal ultrastructure of seminiferous tubular germinal epithelium presenting numerous spermatozoa, and Sertoli cells interconnected with tight junctions were observed (Figure 10A). The APO group had a similar ultrastructure as the control group (Figure 10B). In the MSG group, vacuolization and lipid droplets in the germinal epithelial cells of the seminiferous tubule, separations between the Sertoli– Sertoli cell junctions and deteriorated basal lamina of the seminiferous tubules were observed (Figure 10C). The ultrastructure of the MSG + APO group reflected the normal organization of seminiferous tubules with few lipid droplets in the cytoplasm (Figure 10D)

How does Cistanche boost testosterone?

Cistanche is an herb traditionally used in Chinese medicine to boost energy, libido, and overall vitality. It is believed that Cistanche may increase testosterone levels by inhibiting the activity of an enzyme called aromatase, which converts testosterone into estrogen. Cistanche has also been found to increase the production of luteinizing hormone (LH) in the body. LH plays a crucial role in regulating testosterone production in the testes. By increasing LH levels, Cistanche may indirectly boost testosterone production. Furthermore, Cistanche also contains several phytochemicals such as echinacoside and acteoside, which possess antioxidant properties. These compounds may help to protect the testes and other reproductive organs from oxidative damage, thereby improving their function and increasing testosterone production. Overall, the exact mechanisms by which Cistanche increases testosterone levels are not fully understood, but it is likely an interplay of inhibiting aromatase, increasing LH production, and providing antioxidant support to the reproductive system.

To be continued...

Merve Acikel-Elmas 1,*, Salva Asma Algilani 1 , Begum Sahin 1 , Ozlem Bingol Ozakpinar 2 , Mert Gecim 2 , Kutay Koroglu 3 and Serap Arbak 1 

1 Department of Histology and Embryology, School of Medicine, Acibadem Mehmet Ali Aydinlar University, Icerenkoy Mah., Kayisdagi Cad. No. 32, Atasehir, Istanbul 34752, Turkey 

2 Department of Biochemistry, Faculty of Pharmacy, Marmara University, Basibuyuk Yolu, 4/A, Basibuyuk, Istanbul 34854, Turkey 

3 Department of Histology and Embryology, School of Medicine, Marmara University, Basibuyuk Yolu No. 9 D:2, Maltepe, Istanbul 34854, Turkey