| Cryptorchidism, in which impaired spermatogenesis results from exposure of testes to elevated temperatures in the abdomen, is one of the most common causes of infertility in man. In most mammals, the testes are found in the scrotum outside the main body cavity, approximately 2-8℃below the core body temperature. Heat-activated impairment of spermatogenesis is due to germ cell loss. Surgery-induced cryptorchidism, in which the testes are prevented from descending into the scrotal sac, results in testicular germ cell death, and it is commonly used as an experimental tool in the study of spermatogenesis. However, there are lots of contradictory information on the status of germ cells prevalent and their process of removal in the cryptorchid testis; the molecular events underlying activation of germ cell death also remain poorly understood.In the present study, we investigate selective cell loss from surgery-induced cryptorchid rat testis by using methods of morphology, stereology and DNA flow cytometry, and explore the mechanism of spermatocytes apoptosis by determining protein and mRNA expression of Hsfl, Hsf2, and Tdag51. We also investigate the change of Leydig cells function by determining the expression of CDC25A, SCF, AR, and ERa in cryptorchid rat testis using immunohistochemical staining methods.Our morphological results show that the testis weight is reduced as previous reports after surgical induction of cryptorchidism. Interestingly, the epididymal weight is significantly increased 7 days after surgery, and the caput epididymis tubules show filling with countless round germ cells. Elongating spermatids(steps 10-13), newborn spermatids(step 1) and dividing spermatocytes are the most susceptible cells to elevated temperature, and are the first disappeared cells from the seminiferous tubules after surgery. Spermatocytes are the second disappeared cells following spermatids. Germ cells removal follows the order of starting with elongating spermatids and newborn spermatids, following by round spermatids and elongated spermatids, later extending to spermatocytes.DNA flow cytometry analysis show that the hypo-haploid cell fraction is significantly decreased as early as 3 days after surgical induction of cryptorchidism (from 42.01%±5.74% to 15.98%±3.88%), followed by a significant decrease in the haploid cell fraction at day 7. At the latter time point, an apoptotic peak of spermatocytes appears in DNA histograms just before the tetraploid peak; the percentage of aneuploid cells between diploid and tetraploid rises as high as 14.05%±2.98% of the total cells in 7-day cryptorchid testis, suggesting that a large number of spermatocytes are undergoing apoptosis. The expression of Tdag51 mRNA is significantly elevated 3 days after induction of cryptorchidism. After 7 days of cryptorchidism, Hsf1 and Tdag51 are strongly expressed in the nucleus and cytoplasm, respectively, of primary spermatocytes. Numerous apoptotic spermatocytes are also observed at this time point. These results suggest that the Hsf1/Tdag51 pathway plays an important role in the apoptosis of primary spermatocytes in cryptorchid testis. We present evidence suggesting that Hsf2 is also involved in germ cell removal in cryptorchid testis.The absolute volume of the testicular interstitium is unaltered after surgical induction of cryptorchidism, although the volume fraction of testicular interstitium is significantly increased. There are no change in Leydig cells numbers and morphology after surgery. Leydig cells exhibit strong CDC25A and SCF immunostaining in control testis, and cryptorchidism do not affect their expression. The cytoplasm of Leydig cells also exhibit weak AR immunostaining, and the expression of AR is not altered by cryptorchidism. In control testis, the expression of ERαin the Leydig cells is weak, almost no positive nucleus. But the percentage of Leydig cell with ERα-positive nucleus was significantly increased during cryptorchidism. It reaches 93.6% in 28 days cryptorchid testis. These suggest that the cryptorchidism have no significant effect on the number and light microscope morphology of Leydig cells, but it have significant effect on their function of steroid hormone synthesis.
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