234 MORPHOLOGICAL CHARACTERIZATION OF TESTES FROM INSULIN RECEPTOR SUBSTRATE (IRS) 2-DEFICIENT MICE

Abstract

In mammals, reproduction is controlled by the hypothalamic-pituitary-gonadal axis but it is also affected by changes in energy homeostasis and metabolism. Insulin exhibits pleiotropic effects that are tissue and development dependent. In humans, sperm from males with diabetes exhibit severe structural defects and lower motility and concentration. Insulin receptor substrate 2 (IRS2) is a key mediator in the insulin/insulin-like growth factor-1 signalling pathway. Deletion of IRS2 in mice leads to diabetes and causes female infertility and impaired male fertility. The objective of this study was to determine if the absence of IRS2 in male mice causes a testicular phenotype. Mice were categorized into 3 groups based on genotyping and blood glucose concentrations: wildtype (WT), knockout normal (KOn), or knockout diabetic (KOd); n = 6 mice per group. Testes weighed significantly less in KOn (107.5 ± 10.4 mg) and KOd (112.9 ± 11.1 mg) mice, which corresponds to a reduction in testes size of 46 and 43%, respectively, compared with WT (197.8 ± 29.9 mg; P < 0.001). To determine the difference between the size of the testes in KO and WT mice, the diameter of the seminiferous tubules and the length of the seminiferous epithelium were measured. In both KO groups, both parameters were significantly reduced (P < 0.001). The diameter of the seminiferous tubules of KOn (123.0 ± 12.6 μm) and KOd (137.1 ± 13.1 μm) was reduced by 42 and 35%, respectively, compared with WT (210.5 ± 16.5 μm). The length of the seminiferous epithelium of the KOn (44.0 ± 5.5 μm) and KOd (41.5 ± 6.9 μm) was reduced by 43 and 45%, respectively, compared with WT (76.6 ± 7.25 μm). Because the number of Sertoli cells determines the size of the testes and the number of germ cells that can be supported during spermatogenesis, Sertoli cells were counted. The number of Sertoli cells per cross section of seminiferous tubule was significantly lower in KO v. WT mice (KOn 13.7 ± 2.5, KOd 15 ± 2.8, WT 24.1 ± 2.5; P < 0.001), corresponding to a 43% reduction in KOn and a 38% reduction in KOd. To determine how the differences in morphology affect spermatogenesis, epididymal sperm were counted. As expected, there were fewer sperm in KO mice than WT mice (KOn 3.80 × 10⁶, KOd 8.58 × 10⁶, WT 1.34 × 10⁷). After in vitro capacitation, sperm from all groups were motile; however, sperm from WT mice displayed a more characteristic motility pattern. In summary, there was a reduction in all morphological criteria studied in the testes of KO mice compared with WT. Although IRS2-deficient mice are initially able to breed, when they become severely diabetic, they are infertile. It appears that reductions in testicular morphology and declining numbers of sperm are likely causes of the reduced fertility. The data suggest that insulin signalling in the testes is important for normal testicular development and function and that metabolic abnormalities such as diabetes lead to reduced fertility.