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- Materials and Methods
The proportion of infertility as a whole that is accounted for by male infertility is 40–50% and greater than generally thought. Impaired spermatogenesis is said to be the cause of 90% of male infertility; its etiology is varied and includes gene abnormalities, cryptorchidism, spermatic varicocele, anticancer drugs, radiation and physicochemical factors such as high temperatures, inflammation and circulatory failure. However, no effective treatment has ever been established and efforts are being made to elucidate its mechanisms and develop a method of treatment. We have focused our attention on c-kit, whose gene abnormalities are known to result in infertility.
The proto-oncogene c-kit encodes a protein that is a member of the tyrosine kinase receptor family and, as the receptor for its ligand stem cell factor, it has been found to play an important role in early differentiation processes, including in the hematopoietic and reproductive systems. Moreover, in recent years attention has also been focused on research in the field of regenerative medicine, including the regeneration of myocardium by c-kit-positive cardiac stem cells and experiments on induced pluripotent stem cells in which c-kit has been used.
The c-kit gene was first identified in 1988 as a gene located at the W gene locus. It is located on chromosome 5 in mice and on chromosome 4 in humans; in both mice and humans the size of the protein is 976 amino acids and the gene contains 21 exons. The receptors for platelet-derived growth factor and macrophage colony-stimulating factor have similar structures: they all have five extracellular domains and an intracellular kinase region with a transmembrane region between them.
C-kit is said to be present in primordial germ cells in the embryo, in spermatogonia and Leydig cells in the testis, and in oocytes and interstitial cells in the ovary; its ligand stem cell factor is said to be present in the Sertoli cells of the testis and in the granulosa cells of the ovary. The c-kit receptor forms a dimer by binding its ligand stem cell factor, and, as a result, tyrosine kinase activity is induced and the signal is transmitted.
The c-kit homomutant (W/Wv) mouse is known to be infertile because of a lack of germ cells. A variety of studies have already been conducted on c-kit, and its role is gradually becoming clearer. Based on the results of experiments in which antibodies were administered, Yoshinaga et al. reported that c-kit is an essential molecule in the early stage of spermatocyte differentiation, particularly in the differentiation process associated with the support and proliferation of type A spermatogonia. In addition, in the early 1990s a method of transplantation into seminiferous tubules was developed by Brinster et al. as a way of treating impaired spermatogenesis related to male infertility. When spermatogonia containing normal c-kit were injected into the seminiferous tubules of homomutant mice (W/Wv) with a c-kit mutation, spermatogenesis was restored and natural pregnancy by mating became possible. Thus, c-kit appears to have an important role in the process of spermatogenesis; however, few papers have mentioned the expression of c-kit in impaired spermatogenesis.
Cryptorchidism,[6-8] administration of drugs, such as anticancer drugs,[9-12] and x-irradiation,[13-15] are some of the known methods of experimentally impairing spermatogenesis. Impaired spermatogenesis in humans pursues a chronic course and in view of the ease of producing models of impairment, we produced a model of chronically impaired spermatogenesis in mice by long-term, low-dose administration of the anticancer drug doxorubicin (DXR).
The purpose of this experiment was to histologically elucidate chronically impaired spermatogenesis in mice exposed to DXR and to assess its relationship to the infertility factor c-kit.
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Many studies have been conducted in relation to c-kit, but there have never been any assessments of c-kit expression in testes that have been damaged. In the present study we showed that c-kit expression was decreased in mouse testes in which impaired spermatogenesis had been induced by long-term low-dose exposure to DXR.
Doxorubicin is said to selectively damage various types of spermatocytes in the process of spermatogenesis in mice, especially type A spermatogonia and to damage stem cells more severely than other anticancer drugs, and we used it as the anticancer drug to induce impaired spermatogenesis in our experiment. DXR is said to inhibit DNA and RNA polymerase reactions by binding to DNA, to cause cell damage by inhibiting DNA and RNA synthesis and to inhibit DNA synthesis via intercalation, as well as generating toxic reactive oxygen species.[19, 20]
Sudo et al. conducted a histological and functional analysis of impaired spermatogenesis induced by DXR in mice and the results suggested the possibility of using them as a model of chronically impaired spermatogenesis.[21, 22]
There were no significant differences in body weight or the gross appearance or weight of any of the major organs except the testes during the period of the experiment. Based on these findings it seemed that long-term administration of the 0.15 mg/kg dose of DXR in the present study would specifically damage the testes alone.
We used the same method in the past and reported on telomerase activity in chronically impaired spermatogenesis and so on.[24, 25] Another study showed that the LD50 of DXR for differentiated spermatogonia in mice was 1 mg/kg when administered as a single injection.
Sperm counts, sperm motility and the fertilization rate were significantly lower and abnormalities were significantly higher in the DXR group. The processes of spermiogenesis and fertilization involve various factors that differ from those related to spermatogonial differentiation. An increase in abnormal sperm is generally considered to represent damage during the spermatogenic process, but when such damage actually occurs is not clear. In our present morphologic evaluation of sperm, the head and tail were often markedly deformed. Abnormalities have been characterized to occur mainly at the neck-forming stage. Decreased motility has been attributed to abnormalities in Sertoli cells.
Mice in the DXR group had few seminiferous tubules showing differentiation beyond the stage of spermatogonia, and numerous tubules contained only Sertoli cells, thus demonstrating the toxicity of DXR for stem cells. Although DXR has been reported to damage stem cells, including spermatogenic cells, the mechanism by which DXR damages stem cells is not clear. Nambu and Kumamoto reported that spermatogenic disorder resulted from the interaction between impaired DNA synthesis in stem cells and the dysfunction of Sertoli cells.
Many studies on reproductive function in which DXR has been used have already been reported.[27, 28] An experiment in which Kato et al. administered long-term, low-dose DXR to rats showed a dose-dependent decrease in testicular weight, abnormal sperm morphology, decreased sperm motility and a decrease in the number of spermatogonia, suggesting that it had a negative impact on male reproductive capacity. Moreover, based on testicular weight and the pathological findings in the testes, the male reproductive toxicity of DXR has been reported to be the most suitable for modeling impaired spermatogenesis.
In the control group, c-kit protein was detected in the spermatogonia and Leydig cells. Treatment with DXR suppressed spermatogenesis. In most seminiferous tubules, no c-kit protein expression was found in the spermatogonia; however, c-kit expression was observed in the Leydig cells, similar to the results in the control group. Our results for c-kit expression in the testis support those of Manova et al.
It is said that c-kit expression does not occur during all of the periods from the time the individual is generated until maturity, but is restricted to periods that depend on the stage of differentiation, that it is especially strongly expressed in immature cells such as spermatogonia in the embryo stage and after birth, and that its expression grows weaker as they mature. Since the mice in this study were in a somewhat mature stage and c-kit expression was weak, it seemed that further modifications would be necessary, particularly to evaluate protein expression.
C-kit is present in spermatogonia and Leydig cells and forms a dimer as a result of binding to its ligand, Sertoli cells. Tyrosine kinase activity is then induced and the signal is transmitted. C-kit is an essential molecule in the support of type A spermatogonia and in the differentiation process that accompanies proliferation. By contrast, it has been reported that although type B spermatogonia express c-kit, their differentiation process is not dependent on c-kit and that its cause is unknown.
The fact that tissue damage from the spermatogonia onward and decreases in c-kit protein and c-kit mRNA expression were observed in this study suggests the possibility of some sort of abnormal transmission of the c-kit signal in type A spermatogonia, in which c-kit is expressed and in Sertoli cells, which are its ligand.
In addition, there is a report that transcription of the c-kit gene also occurs in spermatocytes after meiotic cell division; however, the transcription product is shorter than normal c-kit mRNA and it is specific to spermatocytes. These transcripts lack extracellular regions, membrane-penetrating regions and ATP-binding sites. The function of such defective c-kit gene transcripts remains unknown.
The expression of c-kit mRNA in the testis was markedly reduced in mouse testes with impaired spermatogenesis resulting from DXR exposure. The total RNA was extracted from testicular tissue, including germ cells, Sertoli cells and Leydig cells. Thus, whether the reduction in c-kit mRNA expression is related to the reduction in spermatogonia cannot be determined. To elucidate this point, the extraction of spermatogonia is required.
There has never been a report on c-kit expression in a model of chronically impaired spermatogenesis induced by long-term, low-dose DXR administration and the investigation of the behavior of c-kit, which is an infertility factor and closely related to the differentiation of spermatogonia; this study was a very significant experiment from the standpoint of elucidating impaired spermatogenesis. Expression of c-kit was correlated with the decreases in the numbers of spermatogonia, but questions remain in regard to explaining how c-kit is involved in the pathogenetic mechanism of the damage to the spermatogonia and it seems that doing so will be a future research task.