Albaha Barqawi, Herald Trummer, Randall Meacham

Testicular maldescent is a common urological disorder. In full-term infants, the incidence is 3.4 % to 5.8 % and at the time of puberty, 0.8 % -1.8 % of boys remain cryptorchid [1, 2]. Although cryptorchidism is associated with impaired spermatogenesis, the cause of the decreased sperm production in this condition is still controversial. Moreover, the mechanism of this effect remains poorly defined. In 1995, Henriksen confirmed the presence of increased germ cell apoptosis following 24-48 hours of cryptorchidism in the rat, which involved all stages of spermatogenesis except stages VI and VIII [3]. Several investigators since have reported an increase in germ cell apoptosis after creation of experimental cryptorchidism using in situ end-labeling and Electron Microscopy [4-7]. More recently, Tomomasa et al indicated a similar association over time (2-8 weeks) in the congenital undescended testes in the TS inbred rats [8]. All these studies have reported a proportional correlation between the extent of germ cell apoptotic activities and the duration of cryptorchidism in rats. In order to define the impact of cryptorchidism on spermatogenesis and better understand the underlying pathophysiology of this lesion, it is important to assess the effect of chronic cryptorchidism on germ cell apoptosis. The objective of this study is to quantify germ cell apoptosis and testicular sperm content in adult rats after prolonged periods of experimental cryptorchidism ranging from 6 hours to 56 days.

Forty five adult Sprague-Dawley rats (300-350 g) were obtained from the University of Colorado Animal Laboratory and maintained on a 12 hour dark/12 hour light cycle. All animals were housed in a certified animal care facility and given food and water ad libitum. Rats were divided into 9 groups. One group served as normal controls without any surgical intervension and the second group was the sham operated controls. The other seven groups underwent creation of experimental cryptorchidism and were sacrificed at 2, 4, 7, 14, 21, 28 or 56 days thereafter. An additional 6 rats underwent creation of experimental cryptorchidism and were sacrificed at 6 hours and 2 and 4 days and evaluated using scanning electron microscopy to confirm the presence of apoptosis.

General anesthesia was given with sodium pentobarbital 50 mg/kg intraperitoneally and the abdomen was shaved and prepared with Betadine. A midline lower abdominal incision was then made and the testes were gently manipulated into the abdominal cavity bilaterally. To prevent the testes from returning to the scrotum, the gubernaculum was divided on each side and the internal inguinal ring was closed, using non-absorbable 5-0 suture. In the sham-operated group, the testes were handled and then replaced into the normal scrotal position. At sacrifice, the rats were anesthetized and after testicular excision they were euthanized via lethal overdose of sodium pentobarbital. Immediately following excision, the testes were paraffin-fixed and underwent histochemical examinations. To confirm the presence of apoptosis, specimens were evaluated using scanning electron microscopy.

Statistical evaluation was performed using JMP IN v.4 software (SAS Institute Inc.). Analysis included comparison of individual group means. Statistical analysis was performed using ANOVA and Student paired t-test with P<0.05 considered statistically significant.

At the time of sacrifice, both testes of all rats in the cryptorchidism groups were found to be located within the abdominal cavity and the testes of all sham operated rats were in the normal scrotal position. The number of apoptotic germ cells per seminiferous tubular cross section (Table 1) was significantly increased after 2, 4 and 7 days, as well as after 56 days of cryptorchidism (P<0.05). Figure 1 denotes the dynamic change in apoptotic germ cells.

It can be seen from Figure 2 that TSC decreased progressively with longer periods of cryptorchidism. At day 2 cryptorchidism, TSC stayed at the level seen in the control animals and then began to decline rapidly at day 7. Only a few mature sperm were found in the testes of rats at day 56 cryptorchidism. No increase in apoptosis was seen in the Sertoli cells and Leydig cells in all animals. As in the normal control and sham operated controls, no MGCs were noted in the testes of rats of day 2 cryptorchidism (Table 1). At day 4, a mean of 1.8 MGCs per tubular cross section were observed; at this time point, few (8 %) of the MGC nuclei stained positive for apoptosis on TUNEL assay. At day 7, the number of MGCs was 1.3 and 35 % of MGCs stained positive for apoptosis. At days 14, 21, 28 and 56, the nuclei of the giant cells at these time points no longer stained positive for apoptosis. Transmission electron microscopy confirmed the apoptosis based upon ultrastructural morphology in the testes of rats that had undergone 6 hours and 2 and 4 days of cryptorchidism. Confirmatory morphological indicators of apoptosis that were noted on transmission electron microscopy included nuclear condensation and margination. The mean diameter of seminiferous tubular cross sections decreased from 227 mm in sham operated rats to 140 mm at day 7 cryptorchidism (P<0.05). The mean tubular diameter further declined to 103 mm at day 56; at this point, some seminiferous tubules were noted to contain a population of primary spermatocytes, which had not been observed at days 21 and 28 cryptorchidism.

Previous investigators have documented that experimental cryptorchidism causes an increase in germ cell apoptosis [6]; they noted that the rate of apoptosis increased throughout the entire observation period of 7 days. Our results showed that germ cell apoptosis peaked at day 4 of cryptorchidism with a subsequent decline in apoptosis at day 7. This decline continued at days 14 and 21 reaching a nadir at day 28. These results suggest that there is a finite reservoir of germ cells that are available to undergo apoptosis when exposed to core body temperature. It seems that the initial burst of apoptotic activity resulted from wide-spread apoptotic cell death with a decline in apoptosis as the number of vulnerable germ cells declined. Finally, after 14 to 28 days of cryptorchidism, very few germ cells remain to enter the apoptotic process. This was confirmed by the fact that histological evaluation of the testes revealed a markedly depleted seminiferous epithelium and a decrease in seminiferous tubular diameter in rats of this stage.

An alternative explanation for the decline in germ cell apoptosis after day 7 cryptorchidism is that the germ cells eventually accommodate to cryptorchidism by the depletion of the number of germ cells vulnerable to apoptosis [9]. Testicular sperm content, however, declined dramatically after prolonged periods of cryptor-chidism, eventually falling to zero after 56 days. This finding refutes the argument that the decline in germ cell apoptosis over time reflects increased germ cell survival. The finding that testicular sperm content increased to a small extent following day 2 cryptorchidism is likely explained by the fact that testicular sperm content is derived by dividing the number of sperm in a given sample of testis tissue by the weight of the sample. Although an increase in germ cell apoptosis occurs after 2 days of cryptorchidism, the apoptotic process does not eliminate mature sperm, which are resistant to apoptosis [10]. Since a significant proportion of testicular weight is comprised of immature sperm, a decrease in the number of these immature forms would decrease the denominator in the equation used to calculate TSC, thus artifactually increasing the TSC value. Following 4 days of cryptorchidism, many mature sperm would have passed into the lumina of the seminiferous tubules and left the testis. Since few immature forms remain to replace these mature sperm, TSC begins to drop at this point. Yin et al noted that spermatogonia appear to be relatively resistant to cryptorchidism induced apoptosis [11]. They hypothesized that this resistance provides a reservoir of germ cells that allows replenishment of the germinal epithelium following thermal injury. We also observed that apoptosis rarely involved spermatogonia and we suggest that the remaining spermatogonia continue to give rise to the ongoing, low levels of apoptosis seen after 14, 21 and 28 days of cryptorchidism. We noted, however, a re-population of some seminiferous tubules with primary spermatocytes after 56 days of cryptorchidism. It is unclear whether or not more prolonged periods of cryptorchidism would eventually eradicate spermatogenesis altogether. The fact that human males who undergo orchidopexy after the age of puberty typically manifest end-stage failure of the effected testis, suggests that at some point irreversible testicular damage occurs.

MGCs have been identified in the testes of a variety of mammals following a broad spectrum of gonadal insults, including cryptorchidism and germ cell cancer. One stage of the apoptotic process is phagocytosis of the effected cells by cellular elements of the immune system. It has been proposed that Sertoli cells are capable of phagocytosing and eliminating germ cells under appropriate circumstances [12]. The intimate contact maintained between Sertoli cells and immature germ cells supports this concept. An alternative explanation for the formation of such MGCs in the cryptorchid testis is the breakdown of cell membranes among germ cells with subsequent fusion and formation of MGCs. Yin et al noted that the nuclei contained in some MGCs stained positive for apoptosis, while the nuclei contained in others did not. We confirmed that observation and further noted that whether or not the nuclei within MGCs produced a positive TUNEL signal, the event was time dependent, showing a maximum in apoptotic MGCs at day 7 cryptorchidism (35 % of MGCs showed TUNEL morphologic changes in the nuclei of MGCs by electron microscopy). At day 14, 21, 28 and 56, only small numbers of MGCs were identified, all of which failed to stain positive for apoptosis. The explanation for the heterogeneity in the TUNEL staining of testicular MGCs is not clear. It is known, however, that cells undergoing apoptosis only stain positive on TUNEL assay for a relatively brief period of time [13]. It is possible that in the early phases of cryptorchidism, Sertoli cells phagocytosed germ cells prior to or in the early stages of apoptosis. During the later phases of cryptorchidism (day 7 through 10), apoptosis induced DNA cleavage occurs and the nuclei within a portion of the MGCs no longer stain positive for apoptosis. It is also possible that, cryptorchidism induced loss of germ cells may be due in part to phagocytosis by Sertoli cells in a process that is independent of germ cell apoptosis. This would explain the absence of TUNEL signal in the nuclei of some giant cells. Such a hypothesis would not, however, explain the observation that the presence of giant cells containing nuclei that stain positive on TUNEL assay was limited to rats that had undergone cryptorchidism for 10 days or less.

Clearly, the present animal model differs from the situation seen in clinical undescended testes in the human in several respects. Beyond the obvious differences between the anatomy and physiology of the rat versus the human, clinical undescended testes is a long-standing lesion, the response to which may differ from that seen in animal models. This was confirmed by the findings of the effect of experimentally created and congenital cryptorchidism on the distribution of apoptotic levels and fertility between different strains of rats [14].

In conclusion, prolonged cryptorchidism in the rat results in ongoing germ cell apoptosis. It appears that after a prolonged cryptorchidism (56 days), there is a limited resumption of spermatogenesis presumably as a result of a decrease in the maturing germ cells undergoing programmed cell death. Further studies are needed to clarify the role of prolonged cryptorchidism on human testis.

Funding for this project was provided by the Dean's Academic Enrichment Fund at the University of Colorado School of Medicine, Denver, Colorado.