Contemporaneous effects of diabetes mellitus and hypothyroidism on spermatogenesis and immunolocalization of Claudin-11 inside the seminiferous tubules of mice

Abstract

Background: Diabetes and hypothyroidism produce adverse effects on body weight and sexual maturity by inhibiting body growth and metabolism. The occurrence of diabetes is always accompanied with thyroid dysfunction. Thus, it is important to take hypo- or hyper-thyroidism into consideration when exploring the adverse effects caused by diabetes. Previous reports have found hypothyroidism inhibits testicular growth by delaying Sertoli cell differentiation and proliferation. Hence, by establishing a mouse model of diabetes combined with hypothyroidism, we provided evidence that poly glandular autoimmune syndrome affected testicular development and spermatogenesis.

Results: we mimicked polyglandular deficiency syndrome in both immature and prepubertal mice by induction of diabetes and hypothyroidism, which caused decreases in serum concentrations of testosterone and insulin like growth factor 1 (IGF-1). Such reduction of growth factor resulted in inhibition of testicular and epididymal development. Moreover, expressions of Claudin-11 were observed between Sertoli cells and disrupted in the testes of syndrome group mice. We also found reduced sperm count and motility in prepubertal mice.

Conclusions: This mimicry of the diabetes and thyroid dysfunction, will be helpful to better understand the reasons for male infertility in diabetic-cum-hypothyroid patients.

Keywords: Claudin-11; Diabetes; Epididymis; Hypothyroidism; Spermatogenesis; Testis.

Fig. 1

The blood glucose levels and concentrations of different measured hormones under the influence of diabetes mellitus and hypothyroidism. (A) Blood Glucose level, (B) IGF-1, (C) testosterone, (D) fT4 and (E) fT3. Data are represented as mean ± SEM (n = 6), and different labels indicated significant differences among groups at P < 0.05. Abbreviations: Control (C), Diabetic (D), diabetic + hypothyroidism (Dh) and Hypothyroidism (h)

Fig. 2

Effects of STZ-diabetes and hypothyroidism on body, testes and epididymides weights in immature- to prepubertal mice. (A) Photographs of mice along with their testes at the age of 24 days old (top panel) and 56 days old (bottom panel). (B) Body weight, (C) testes weight and (D) epididymides weight under the influence of diabetes and hypothyroidism conditions. Data are represented mean ± SEM (n = 6), and different labels indicated significant differences among groups at P < 0.05. Abbreviations: Control (C), Diabetic (D), diabetic + hypothyroidism (Dh) and Hypothyroidism (h)

Fig. 3

Histo-architectural changes in testicular cells during experimental diabetes mellitus and hypothyroidism in immature and prepubertal mice. Sloughed spermatids, marked with red arrow head shown inside the lumen of tubule of Dh animals (panel: C2). Abbreviations: Control (C), Diabetic (D), diabetic + hypothyroidism (Dh) and Hypothyroidism (h), Seminiferous tubule (ST), Blood cells (BC), Leydig cells (LC), Spermatogonia (SG), Germinal epithelium (GE),. Representative images were captured at 400× magnifications. The bars are 50 μm in size

Fig. 4

Hematoxylin and eosin stained digital images of the epididymis at different age levels of mice, showing proximal caput and distal cauda under the influence of diabetes and hypothyroidism compared with control. (A1) Epididymal sections of immature mice at the age of 24 days old. Most of the epididymal tubules of treated animals possess round bodies with exfoliated germ cells, marked with red arrow heads (panels B1, D1, C2 and D2). Some of the epididymal tubules of treated mice, depicts inflammatory infiltrations marked with green arrow heads (panels C1, B2 and D2). (A2) Epididymal sections of pre pubertal mice at the age of 56 days old presenting hyperplastic changes in principal cells of caput tubules are shown with green arrow heads (panels: B3, C3 and D3). Cribriform changes are marked with red arrows (pannels: C4 and D4). Abbreviations: Control (C), Diabetic (D), diabetic + hypothyroidism (Dh) and Hypothyroidism (h), (S) Spermatozoa and (I) Interstitium. The pictures were captured at magnification of 400× and the bars are 50 μm in size

Fig. 5

Immunolocalization of the Claudin-11 in the testes of adult mice, exposed to experimental diabetes mellitus and hypothyroidism. Immunostaining was seen positive with DAB brown along with blue counter stained with hematoxylin. Significantly either week (low positive) or disrupted expressions of Claudin-11 were noticed in syndrome group mice (Dh), followed by h and D compared with control. These pictures were captured at magnification of 400× and 1000× with pasted bars of 50 and 30 μm in size at the top and bottom rows, respectively

Fig. 6

Representative digital images of histogram profile shows DAB brown staining, color pixel intensity for Claudin-11 in the testes of adult mice, exposed to experimental diabetes mellitus and hypothyroidism. (A) From top to bottom rows; panels shows the digital image masks stained with hematoxylin, DAB and threshold respectively. (B) Claudin-11 is expressed in a limited quantity at tight junction in between Sertoli cells of all treated groups that is why analyzed through score calculation. Data are representing mean ± SEM (n = 6) and different labels indicated significant differences among groups at P < 0.05

Fig. 7

Sperm concentration and motility of diabetic and hypothyroid mice during the age of 08 weeks. (A) Decreased sperm count was critically noticed in Dh and h group of animals compared to D and C. (B) Decreased rapid progressive motion of spermatozoa was recorded in all treated mice compared with control. However, sperm motility parameters were critically reduced in Dh subjects. Data represented mean ± SEM (n = 6) and different labels indicated significant differences among groups at P < 0.05