Exogenous gonadotropin-releasing hormone counteracts the adverse effect of scrotal insulation on testicular functions in bucks

Abstract

This study determined the effects of scrotal insulation on testicular functions in bucks and evaluated the impact of exogenous gonadotropin-releasing hormone (GnRH) administration before scrotal insulation on sperm production and testicular vascular dynamics. Twelve bucks were randomly divided into three groups: scrotal-insulated animals without GnRH treatment (INS), scrotal-insulated animals treated previously with GnRH (GnRH + INS), and animals without insulation as controls (CON). Doppler ultrasonography was used to evaluate testicular vascular changes, and semen samples were collected to assess seminal parameters. Testicular samples were collected from slaughtered bucks at the end of the experiment for histological investigations and immunohistochemical analysis for caspase 3 (apoptotic marker), and a vascular endothelial growth factor (VEGF; hypoxic marker) evaluation. Sperm motility drastically decreased (33%) in the INS group on day 8 compared with those in the GnRH + INS and CON groups (58% and 85%, respectively). Testicular blood flow significantly decreased for 3 and 2 weeks in the INS and GnRH + INS groups, respectively. The pulsatility index (PI) reached pretreatment values at 5 and 4 weeks after insulation in the INS and GnRH + INS groups, respectively. The resistance index (RI) values increased in both insulated groups for the first 2 weeks and decreased to control values 4 weeks after insulation. However, the maximum velocity (VP) started to increase reaching pretreatment values by the 5th and 3rd weeks after insulation in the INS and GnRH + INS groups, respectively. Histological investigations showed a marked reduction in lipid inclusions in Sertoli cells in the GnRH + INS group compared with those in the INS group. The distributions of both caspase 3 and VEGF decreased in the GnRH + INS group compared with those in the INS group. This study showed that the administration of a single dose of GnRH delayed the negative effects of scrotal insulation on different seminal traits and revealed the pivotal role of GnRH in compensating testicular insulation in bucks.

Figure 1

Photomicrographs of semi-thin sections of tubules from adult buck testes stained with toluidine blue (X1000 magnification). Testes in all groups exhibited a typical histological picture of functionally active mature seminiferous tubules. Sertoli cells showed differences in lipid inclusions and cell junctions in different experimental groups: A: Sertoli cells (S) in testis of CON showed low amount of lipid inclusion (arrow) and no vacuoles within the cytoplasm. B: INS showed marked accumulation of lipid droplets in Sertoli cells (arrow) and vacuolization (V) in both Sertoli and primary spermatocytes. C: GnRH + INS showed the Sertoli cells with scanty lipid inclusions, large cytoplasmic processes (CP) and no vacuoles in the Sertoli cells and primary spermatocytes. S: Sertoli cell, and Sg: spermatogonia, PS: primary spermatocytes, RS: round spermatid and sperm.

Figure 2

Photomicrographs of paraffin testicular sections stained with immunohistochemistry to detect the expression of active caspases-3: A, C, E (low magnification, X100), B, D, and F (higher magnification: X200). The CON group (A and B) showing a weak expression of caspases-3 immunostaining. Note: Arrowhead points to the reaction in sperm. The INS group (C and D) showing moderate expression of caspases-3 immunostaining. While, GnRH + INS group (E and F) showing the lowest expression of caspase-3 in Sertoli, germ and Leydig cells were decreased in comparison to insulated group. Abbreviation: ST: seminiferous tubules, S: Sertoli cells, P: primary spermatocytes, L: Leydig cells. The inserted squares highlight the reaction; red squares highlight the reaction in Sertoli and germ cells, and the black squares highlight the reaction in Leydig cells.

Figure 3

Numbers of apoptotic Sertoli (A), Germ (B) and Leydig (C) cells in seminiferous tubules of buck’s testis. Different letters indicate significant differences between the groups (p < 0.05). Results are presented as mean ± SEM.

Figure 4

photomicrographs of testicular paraffin sections stained with immunohistochemistry to detect expression of vascular endothelial growth factor (VEGF): Control group (CON) (A, B, C) showed a strong expression for VEGF occurred in endothelium of blood vessels (En) and weak reaction in Sertoli (S), germ (primary spermatocyte, p) and Leydig cells (L). INS group (D, E, F, G, H, I) with a strong VEGF expression that distinguished in Sertoli cells and reacted in spermatogenic cells (Primary spermatocyte (p), Leydig cell (L), endothelium of blood vessels (En)). GnRH + INS (J, K, L) showed a weak VEGF expression which detected in Sertoli cells (S) and germ cell (primary spermatocytes, p), with a moderate reaction observed in Leydig cell (L) in comparison to INS group. Abbreviation ST: seminiferous tubules, S: Sertoli cells, En: endothelium of blood vessel, L: Leydig cells. The inserted squares showing the reaction B, H expressing the reaction in Sertoli and germ cells; C, L expressing the reaction in Leydig cells. Magnifications: (A, D, G, J, X200), (B_L, X400).

Figure 5

Reciprocal staining intensity of VEGF in (A) Sertoli, (B) germ, (C) Leydig cells and (D) vascular endothelium of buck’s testis. Different letters indicate significant differences between the groups (p < 0.05). Results are presented as mean ± SEM.