Stereological Study Of The Effect Of Hyperglycemia And Hyperlipemia On Histological Structures In The Rat Testis And Epididymis

Background and Objective: Hyperglycemia and hyperlipidemia are very common metabolic diseases in clinical practice. There were studies on the effect of hyperlipidemia or hyperglycemia on the male reproductive function, and the results suggested that hyperlipidemia or hyperglycemia might affect the histological structures of the testis and epididymis, damage the spermatogenesis, reduce testosterone levels, and lead to reduced male fertility and even infertility. These studies mostly focused on either hyperlipidemia or hyperglycemia affecting the male reproduction. In addition, there is lack of quantitative (stereological) studies on the numbers of various cells in the testis, thus it is difficult to interpret the route or mechanism of hyperglycemia or hyperlipidemia affecting the male reproductive function. This study was therefore undertaken to establish a severe hyperglycemia and hyperlipidemia rat model, and study the effect of severe hyperglycemia and hyperlipidemia on testicular and epididymal structures, especially the effect on spermatogenesis, using stereological (quantitative) methods.Materials and Methods: 56 normal male SD rats (aged 10-11 weeks) were randomly divided into a control group (n=12) and an experimental group (n=44). Animals in the control group were given an intraperitoneal injection of vehicle while those in the experimental group received an intraperitoneal injection of streptozotocin (STZ, 60mg/kg body weight) and were, 1 week later, fed with high fat food. The experiment continued until 40 days after STZ injection. Fasting blood glucose levels were measured 4 times during experiment; at the end of experiment fasting lipid levels were also measured. By the end of experiment, 2 control animals died, and 6 animals were randomly selected from the remaining 10 control animals for study; in the experimental group, 8 animals were not hyperglycemic, 28 hyperglycemic animals died, and the remaining 8 hyperglycemic animals were used for study.The testis and epididymis were removed and immersion-fixed with the Bouin fluid, and then tissue blocks were obtained and embedded in methacrylate resin and 25μm thick sections were cut. Testicular sections were stained with PAS plus hematoxylin while epididymal sections stained with hematoxylin only. Histological structures of the testis and epididymis were observed under light microscope. The volumes of various structures in the testis or epididymis and the diameter of seminiferous tubules in the testis were estimated using point counting and other stereological methods, and a new stereological technique– the optical disector was used to estimate the numbers of various cells in the testis and sperm in the epididymis.Result: Blood glucose levels of the 6 control animals ranged from 4.2 to 6.5 mmol/L during experiment; those of the 8 experimental animals were all >14.3 mmol/L. The total cholesterol (TC), triglyceride (TG) and high density lipoprotein (HDL) levels in the experiment group were over 14, 10 and 4 times, respectively, of those in the control group.The volumes of the testis and the epididymis in the experimental group decreased significantly (P < 0.05, t-test) by 22% and 43%, respectively, in comparison with the control group. Qualitative observation under light microscope showed detained late elongated spermatids in some seminiferous tubules in the experimental group, without other obvious morphological changes; there were no obvious morphological changes in the epididymis, either. Morphometric studies of the testis demonstrated that the mean diameter of seminiferous tubules and the total volumes of seminiferous tubules and interstitial tissue in the testis were significantly decreased by 14%, 19% and 28%, respectively, in the experimental group (compared to the control group); except the preleptotene primary spermatocytes, secondary spermatocytes, mid-stage spermatids, late elongated spermatids, Sertoli cells and leukocytes (in the lumen of blood vessels), the numbers of the following types of testicular cells were also significantly reduced in the experimental group: type A spermatogonia (decreased by 27%), type B spermatogonia (33%), leptotene and zygotene primary spermatocytes (22%), pachytene primary spermatocytes (24%), myoid cells (35%) and Leydig cells (32%). Morphometric studies of the epididymis showed that the total volumes of the epididymal duct, interstitial tissue and agglomerated sperm mass were significantly decreased by 50%, 40% and 61%, respectively, and total number of sperm significantly decreased by 60% in the experimental group.Conclusion: Short-term (6 weeks) hyperglycemia and hyperlipidemia did not induce serious damage to the spermatogenesis within the testis; spermatogenesis was still active, but the numbers of spermatogenic cells and Leydig cells were decreased by 20%–30% and spermiation was markedly impaired, thus resulting in a much reduction in the number of sperm in the epididymis. Reduction in the number of spermatogenic cells might be mainly ascribed to slowdown or impairment of germ cell proliferation as the result of serious metabolic disturbance, and impairment of spermiation might be mainly associated with reduction of testicular fluid (secreted by Sertoli cells, vehicle of sperm transport) and weaker contraction of myoid cells (power source for sperm transport), which might be induced by severe metabolic disturbance. Decreased number and even decreased secretion (of testosterone) of Leydig cells might not be a key factor of hyperglycemia and hyperlipidemia-induced spermatogenic impairment.