Changes in sperm quality and testicular structure in a rat model of type 1 diabetes

We studied 15 male Sprague Dawley rats from the age of 8 weeks, weighing 250–320 g, in compliance with The Animals for Scientific Purposes Act, BE 2558 (AD 2015) (Thai Government Gazette, Vol. 132, Part 18 a, 13th March 2015) under license after approval by the Center for Animal Research Naresuan University, Phitsanulok, Thailand (approval No. 58 01 002) and in conformance with the revised Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council “Guide for the Care and Use of Laboratory Animals” Washington, D.C.: National Academy Press; 1996. All rats were maintained at a constant temperature of 22 ± 1°C under dark light cycle of 12:12 h and allowed access to standard laboratory food and water ad libitum. Rats were divided into 2 groups in an unselected manner to include a vehicle control group treated with 0.1 M citrate buffer, pH 5 (control; n = 8) and a group with STZ-induced type 1 diabetes (DM; n = 7). After fasting for 8 h, rats intended for the DM group were injected intraperitoneally with a single dose of STZ (Sigma) 60 mg/kg in 0.1 M citrate buffer, pH 4.5, and were subsequently found to have a fasting blood glucose level of ≥250 mg/dL and used for the study as a model of DM. At 7–12 weeks after inducing DM, rats were humanely killed by carbon dioxide gas inhalation.

The sperm motility, normal morphology, and concentration in rats in the DM group were significantly decreased when compared with that of rats in the vehicle control group (Figure 1).

Figure 1

A significant reduction in the body weight and testicular weight was found in rats in the DM group, while the testicular weight percentage of body weight was significantly increased in rats in the DM group (Table 1).

The diameter of seminiferous tubules was significantly decreased in rats in the DM group (Figure 2).

Figure 2

We observed morphological changes in seminiferous tubules from the appearance of the tubules in vehicle-treated control rats (Figure 3A) including separation of the germinal epithelium, vacuolization, luminal sloughing of germ cells, irregular tube, and atrophy in rats in the DM group (Figure 3B–F).

Figure 3

Significant increases in the separation of germinal epithelium, vacuolization, luminal sloughing of germ cells, and atrophy of seminiferous tubules were found in rats in the DM group when the percentages of these morphological abnormalities were compared quantitatively with those in vehicle treated rats in the control group (Figure 4).

Figure 4

The mean proportion of early stage (stage I–V) seminiferous tubules was significantly increased in rats in the DM group when compared with the proportion in rats in the vehicle control group. By contrast, significant decreases in the mean proportion of late stage (stage IX–XIV) tubules were seen in rats in the DM group (Figure 5).

Figure 5

After induction of DM with STZ, the resulting rats with type 1 DM showed abnormal sperm quality including decreases in sperm motility, concentration, and normal morphology. These findings are consistent with those in previous studies of sperm quality affected by DM [11, 13, 23]. The decrease in sperm quality may be associated with hyperglycemia resulting in hyperactive oxidative stress. The plasma membrane of sperm containing high levels of polyunsaturated fatty acids and lacking anti-ROS enzymes may result in elimination of nuclear and mitochondrial DNA in sperm as a consequence of lipid peroxidation, which is implicated in sperm defects and apoptosis [24, 25].

The morphological changes of seminiferous tubules in rats with DM observed in the present study including separation of the germinal epithelium, vacuolization, luminal sloughing of germ cells, and atrophy of seminiferous tubules and may also result from hyperglycemia leading to high production of ROS. Oxidative stress has been reported to induce Leydig cell dysfunction and result in a testosterone deficiency, which plays an important role in spermatogenesis. Our present findings are consistent with those of previous studies, which found that DM induced the atrophy of seminiferous tubules by decreasing the number of spermatogenic cells [7, 8, 26].

In the present study, a decrease in testicular diameter and testicular weight was observed in association with an increase in the atrophy of tubules in the rats in the DM group, which is consistent with findings reported previously [27, 28]. These decreases may be due to a decrease in testosterone levels leading to degeneration of the Sertoli–Sertoli cell junction, which is implicated in structural changes of seminiferous tubules [29, 30].

The significant decrease in the proportion of late stage (stage IX–XIV) seminiferous tubules in rats in the DM group may reflect an arrested period of development causing impairment of spermatogenesis. This decrease may be due to decreases in testosterone levels, dysfunction of Leydig cells, changes in structure and function of Sertoli cells, or atrophy of the seminiferous tubules [17, 29]. The significant increase in the proportion of early stage tubules in rats in the DM group may also reflect nonprogressive development of the tubules due to an impairment of testosterone production from Leydig cells or changes in the structure and function of Sertoli cells.

The results of the present study are limited to type I DM, which involves impairment of insulin production. Altered fat metabolism with impaired insulin production occurs in type II DM. We cannot extrapolate the results to type II DM because we did not modify any parameters related to fat metabolism. The pathology of type II DM needs to be further elucidated. The numbers of rats in each group were not equal due to inclusion criteria for the rat model of diabetes. Nevertheless, the numbers were suitable for statistical analysis.

We reduced the number of rats used in biomedical research by using testis and epididymis from rats used for another study “Development of topical cream containing long peper extract loaded solid lipid microparticles on nerve pain relief in of rat model of diabetic neuropathy”. Thus, by this dual-purpose subproject arrangement tissues and organs were collected for further studies such as of the histopathology reported here.