Effects of pre- and postnatal exposure to flutamide on connexin 43 expression in testes and ovaries of prepubertal pigs

Abstract

The aim of this study was to show whether the connexin43 (Cx43) expression in gonads is affected by an anti-androgen action. To test this, pigs were prenatally (on gestational days 20-28 and 80-88; GD20, GD80), and postnatally (on days 2-10 after birth; PD2) exposed to flutamide that was given in five doses, every second day and its effect was observed in prepubertal gilts and boars. Morphology and expression of Cx43 was investigated in testes and ovaries by means of routine histology, immunohistochemistry, Western blotting, and RT-PCR, respectively. Qualitative analysis of immunohistochemical staining for Cx43 was confirmed by quantitative image analysis in which the staining intensity was expressed as relative optical density of diaminobenzidine deposits. There were statistically significant differences in Cx43 signal intensity between interstitial tissue of control and GD20 pigs (p less than 0.01), between seminiferous tubules of control and PD2 boars (p less than 0.01), between granulosa cells of preantral follicles of control and GD20 and PD2 pigs (p less than 0.01 and p less than 0.05, respectively), and between theca cells of control and GD80 and PD2 gilts (p less than 0.01). In Western blotting Cx43 appeared as a band of 43 kDa, whereas the size of the PCR-amplified product was 232 bp in all gonad tissue samples. Since we demonstrated changes in gonad morphology and in the expression of Cx43 at the level of protein of prepubertal boars and gilts, it seems possible that flutamide through blocking androgen action, causes delayed gonadal maturation in later postnatal life and, among other factors, may be involved in the regulation of Cx43 gene expression in pig gonads.

Figure 1

Testes histology of prepubertal control pigs (A) and flutamide exposed pigs at GD20 (B), GD80 (C–D), PD2 (E–F) and immunolocalization of C×43 in testes of prepubertal control pigs (G) and flutamide exposed pigs at GD20 (H), GD80 (I) and PD2 (J). Seminiferous tubules (ST); Interstitial tissue (IT). Scale bars represent 20 µm. (A) Well-developed interstitial tissue with Leydig cell clusters and seminiferous tubules with Sertoli cells (arrows); prespermatogonia (small arrows) and spermatogonia are visible. (B) Normal structure of testicular compartments is visible. (C–F) Varying degrees of tubule abnormality are present, ranging from completely normal tubules to abnormal tubules. Note a distinct increase of the interstitial space, presence of numerous Leydig cells (C), severely dilated lumina (D), numerous prespermatogonia (small arrow) in the center of seminiferous tubules (E), regressed tubules (F) and multinucleated germ cells (arrowheads) (D; at higher magnification, insert in F). (G) Linear pattern of the staining between neighboring Leydig cells (arrows) and the focal pattern between Sertoli cells and germ cells (small arrows) are visible. (H) Very strong intensity of the staining between neighboring Leydig cells (arrows), (at higher magnification, insert in H). Weak staining intensity between Sertoli cells and germ cells (small arrows). (I) Moderate to strong intensity of the staining between Leydig cells (arrows). Note a further decrease in the staining intensity between Sertoli cells and germ cells (small arrows). (J) Strong-to-very strong intensity of the staining is observed between testicular cells of both compartments. Note a distinct linear pattern of the staining between Sertoli cells and remaining germ cells. No positive staining is visible when the primary antibody is omitted (an insert).

Figure 2

Ovaries histology of prepubertal control pigs (A–B) and flutamide exposed pigs at GD20 (C), GD80 (D–E), PD2 (F) and immunolocalization of C×43 in ovaries of prepubertal control pigs (G) and flutamide exposed pigs at GD20 (H), GD80 (I), and PD2 (J). Interstitial cells (IC); Granulosa cells (G); Theca cells (T); Antrum (A); Oocyte (O). Scale bars represent 20 µm. (A) Numerous preantral follicles (small arrows) and antral follicles (arrows) with well-developed compartments are visible. (B) Granulosa and theca cell layers and antrum are seen. (C) A follicle at the preantral stage is seen. (D–E) Normal preantral follicle (arrow) and abnormal antral follicle are observed. Note a distinct increase of the interstitial cell space (insert in D). (F) Normal folliculogenesis is observed. Note some antral follicles containing apoptotic granulosa cells (arrowheads) (at higher magnification, insert in F). (G) Positive staining for C×43 between adjacent granulosa cells (arrows) and between theca cells (small arrow) of preantral (an insert) and antral follicles. (H) A decline of the staining intensity between granulosa cells (arrows) is observed. Very weak staining in theca cells is visible (small arrow). (I) Strong intensity of the staining between adjacent granulosa cells of antral follicles (arrows). Note a distinct increase in the staining intensity within theca cells (small arrows). (J) A similar pattern of the staining and its differential intensity compared to (G). Note a moderate-to-strong intensity of the staining between granulosa cells (arrows) and between theca cells (small arrow). No positive staining is visible when the primary antibody is omitted (insert in J).

Figure 3

Quantitative analysis of the intensity of C×43 staining expressed as relative optical density (ROD) of diaminobenzidine brown reaction products in testicular (A) and ovarian (B) compartments of pigs exposed to flutamide vs respective controls. The values expressed are the mean ± SD (n=10). Statistically significant difference between means from control was analysed by ANOVA (*P<0.05, **P<0.01). ST, Seminiferous tubules; IT, Interstitial tissue; GC, Granulosa cells; TC, Theca cells.

Figure 4

Western blot analysis of C×43 in ovaries and testes of prepubertal pigs. Lane H indicates pig heart used as the positive control. Further lanes indicate ovarian and testicular samples of control pigs (C) and of those exposed to flutamide at GD20, GD80, and PD2. β-actin indicates an internal control.

Figure 5

Demonstration of C×43 gene expression and GAPDH as a loading control in ovaries and testes of prepubertal pigs. Lane M contains a molecular weight marker. Lane B indicates blank samples. Further lanes indicate ovarian and testicular samples of control pigs (C) and of those exposed to flutamide at GD20, GD80, and PD2. The sizes of the PCR products are indicated on the right.