The purpose of this study was to assess the relationship between chronically impaired spermatogenesis induced by exposing mice to doxorubicin (DXR) and expression of the infertility factor c-kit.
The purpose of this study was to assess the relationship between chronically impaired spermatogenesis induced by exposing mice to doxorubicin (DXR) and expression of the infertility factor c-kit.
Eight-week-old male Institute for Cancer Research (ICR) mice were intraperitoneally treated with DXR (0.15 mg/kg, DXR group) or saline (0.15 mg/kg, control group) twice weekly for five weeks and were killed 14 weeks after initial exposure. The animals were sacrificed and bilateral testes were removed and weighed. The testes were stored for the mRNA assay and were fixed for immunohistochemistry. Some testicular samples were fixed in 10% formalin for histopathological examination.
Testicular weight (67.6 ± 9.7 mg, P < 0.05), sperm motility (18 ± 6.0%, P < 0.05) and the fertilization rate (2-to-16-cell embryos, 5%; P < 0.05) were significantly lower in the DXR group than in the control group. In the DXR group there was severe tissue damage from the spermatogonia onward, and the Sertoli cell ratio was lower in the DXR group than in the control group (38% vs. 9%, P < 0.05). In addition, there was a decrease in c-kit protein expression, and the amount of c-kit messenger ribonucleic acid (mRNA) expression according to a semiquantitative method was also decreased.
Expression of c-kit in the mice with chronically impaired spermatogenesis induced by long-term, low-dose administration of DXR correlated with the decrease in the number of spermatogonia.
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.
Seven-week-old male Institute for Cancer Research (ICR) mice were purchased from Japan SLC (Shizuoka, Japan) and housed in hanging wire mesh cages (five animals per cage) under controlled lightning conditions (12 h light, commencing at 6:00 am, and 12 h darkness) at a temperature of 20–24°C with free access to chow and water throughout the experimental period. The range of body weights at the start of dosing was 33–34 g. The animals were handled daily for a week before the experiments were performed.
The animals were allocated to a control or a DXR-treated group (n = 10, respectively) and both groups were fed a standard mouse diet. In the DXR-treated group, DXR was intraperitoneally administered at a dose of 0.15 mg/kg twice weekly for five weeks (total: 1.5 mg/kg). The control group was treated intraperitoneally with saline twice weekly for five weeks. At 14 weeks after the initiation of the experiment, the animals were killed and bilateral testes were removed and weighed. The testes were frozen immediately in liquid nitrogen and stored at −80°C for the mRNA assay or were fixed in paraformaldehyde for immunohistochemistry. Some testicular samples were fixed in 10% formalin for histopathological examination.
The experimental protocol was approved by the Animal Research and Care committee of the School of Medicine, Keio University.
A purified monoclonal rat anti-mouse CD117 antibody of the IgG2b class (Pharmingen, San Jose, CA, USA) was used to detect c-kit. As a class-matched control monoclonal antibody, we used purified anti-Mac-1 antibody, which does not bind to germ cells (YLEM, Rome, Italy).
Spermatozoa were extracted from 40 cauda epididymides obtained from 20 male mice (10 mice per group). Sperm from these cauda epididymides were minced in 200 μL of cryopreservation solution containing 18% raffinose and 3% skim milk (mouse sperm cryopreservation kit). Spermatozoa were dispersed at 37°C by shaking the dish gently for about 2 min. The sperm suspension was then divided into 20 portions and each sample (10 μL) was transferred to a sampling straw. Samples were then placed in the gas layer of a small container of liquid nitrogen for 10 min before being plunged directly into the liquid nitrogen, where they were stored for 3–6 months before thawing. At that time, the samples were removed from the liquid nitrogen and thawed at room temperature (26°C) for 15 min.
The thawed sperm suspension was gently transferred to the bottom of a tissue culture dish containing 100 μL of Human Tubal Fluid (HTF) medium at 37°C. The dish was then placed in a CO2 incubator (95% air, 5% CO2) at 37°C for 30 min without shaking and the sperm suspension was diluted slowly with medium. Spermatozoa were then allowed to swim in the HTF medium. After incubation for 1.5 h, a sample of the sperm suspension (10 μL) was placed on a glass slide under a coverslip and examined under a microscope (×100) to evaluate motility and morphology. Motility was scored as the percentage of spermatozoa actively moving forward in each sample.
Ovulation was stimulated in female ICR mice (eight weeks old; CLEA Japan, Tokyo, Japan) by the injection of 5 IU of pregnant mare serum gonadotrophin (Teikoku-zoki, Tokyo, Japan), followed 48 h later by the injection of 2.5 IU of human chorionic gonadotrophin (hCG; Teikoku Hormone Manufacturing, Tokyo, Japan). Within 1 h after thawing, morphologically normal oocytes were introduced into the HTF medium (100 μL) containing the thawed spermatozoa (1–2 μL of suspension) at a density of 0.5–1.0 × 105/mL. After incubation for 12 h, the fertilized oocytes were washed three times with HTF medium (50 μL) and examined under an inverted microscope to assess fertilization. The criteria for a fertilized oocyte were the presence of two pronuclei and a second polar body. Oocytes were then further cultured for 96 h and examined every 24 h to determine the stage of development. Inseminated ova reaching the pronuclear, 2-to-16-cell, morula and blastocyst stages were evaluated at 24, 48, 72 and 96 h after insemination, respectively.
The testes were fixed in 2% paraformaldehyde and washed in phosphate buffered saline (PBS). They were then covered with optimum cutting temperature compound, rapidly frozen and cut into 6-to-8-μm sections using a cryostat. Then the specimens were incubated with CD117 antibody at a concentration of 10 μg/mL in 0.1 mM PBS with 2% skim milk/0.1% Triton X-100 (PBSMT) overnight at 4°C. Antibody binding to the sections was detected with goat anti-rat IgG (H + L)-HRP (Zymed Labs, San Francisco, CA, USA), according to the manufacturer's recommendations. The specificity of antibody binding was assessed by comparing the samples with controls treated with non-immune rabbit serum and anti-Mac-1 antibody instead of CD117. The control sections did not show any reactivity, except for occasional connective tissue cells that most likely were anti-Mac-1-positive macrophages (not shown). Some testicular samples were fixed in 10% formalin and stained with hematoxylin and eosin for histological examination. All the slides were examined under a light microscope. For a quantitative analysis, 30 seminiferous tubules per group were randomly examined to calculate the Sertoli cell ratio (SCR). Based on the fact that Sertoli cells do not disappear, even in cases with severe testicular failure, the SCR is the ratio of the number of germ cells to the number of Sertoli cells.
Total RNA (10 μL) extracted from tissues in 10 μL of master mixture containing 8 U of RNase inhibitor (Promega, Madison, WI, USA), 0.5 U of Moloney murine leukemia virus reverse transcriptase (Promega), 2 μL of 25 mM MgCl2, 2 μL of 10 mM deoxyribonucleotide triphosphate and 10 pM of each antisense primer were incubated at 42°C for 30 min and 95°C for 5 min, and then cooled at 4 C. Six microliters of 25 mM MgCl2 were then added to the reverse-transcription reaction mixtures (10 μL) and the tubes were placed on a heat plate at 95°C for 9 min. After the addition of 34 μL of master mixture containing 0.5 μL of Taq Gold polymerase (Applied Biosystems, Foster City, CA, USA), 4 μL of 10 × polymerase chain reaction (PCR) buffer and 2.5 pM of each primer to the samples, the samples were mixed by pipetting. PCR was performed for 30 cycles (95°C for 15 sec; 60°C for 30 sec; 72°C for 60 sec) and then at 72°C for 5 min and cooled to 4°C. The sequences of the primers for c-kit were 5′-GGT GGT TCA GAG TTC CAT AG-3′ (corresponding to nucleotides 1453 to 1472 of mouse c-kit) and 5′-GTC GGA ACT GAA GGT CCT GA-3′ (corresponding to nucleotides 1939 to 1958 of mouse c-kit). For the positive control, the β-actin primer sequences were 5′-CTA AGG CCA ACC GTG AAA AGA T-3′ (corresponding to nucleotides 415 to 436 of mouse control β-actin) and 5′-CCT CTC TTT GAT GTC ACG CAC G-3′ (corresponding to nucleotides 704 to 725 of mouse control β-actin cDNA). The PCR products were separated on a 2% agarose gel, stained with ethidium bromide and photographed for densitometry-based semiquantitative analysis. By measuring the changes in mRNA levels, a baseline was established and a relative evaluation was performed.
The percentages of sperm motility, abnormality and developmental stage of ova fertilized in vitro and the percentage of seminiferous tubules are expressed as the mean ± SD. The statistical analysis was performed using an analysis of variance and a paired t-test. Differences were considered statistically significant if P < 0.05.
The total body and testes were weighed in 10 mice from each group. No significant difference was found in the body weights or the weights of the other organs (heart, liver, spleen or kidney) except for the testes. The testicular weight in the DXR-treated mice (67.6 ± 9.7 mg) was significantly less than that in the control group (127.5 ± 6.0 mg, P < 0.05), which showed a 40% decrease in the DXR-treated group compared with the controls (Fig. 1).
The mice in the control group had a normal sperm concentration (Fig. 2). The mean sperm concentration was 103 ± 22.3 × 106/mL in the control group and 70 ± 11.4 × 106/mL in the DXR-treated group. The mean sperm concentration was significantly decreased by DXR treatment (P < 0.05).
Sperm motility and abnormality, as assessed after thawing, is shown in Figure 3. In frozen-and-thawed spermatozoa, motility at the time of insemination was 65 ± 7.1% for the control mice and 18 ± 6.0% for the DXR-treated mice. The motility of the frozen-and-thawed sperm in the DXR-treated group was significantly lower than that in the control group (P < 0.05). Sperm abnormality was 10 ± 5.0% for the control mice and 90 ± 5.0% for the DXR-treated mice. The abnormality of the sperm in the DXR-treated group was significantly higher than that in the control group (P < 0.05).
Embryonic development after in vitro fertilization was observed as follows: pronucleus stage at 24 h, 2-to-16-cell stage at 48 h, morula stage at 72 h and blastocyst stage at 96 h. After hCG administration, a total of 50.0 ± 3.3 ova were recovered from the ovaries of the animals in each group. The percentage of pronuclear embryos was significantly higher in the control group (51%) than in the DXR-treated group (38%, P < 0.05). The percentage of 2-to-16-cell embryos also was significantly greater in the control group (31%) than in the DXR-treated group (5%, P < 0.05). Fertilized ova in the DXR-treated group did not develop beyond the morula stage. The fertilization rate in the DXR-treated group was significantly lower than that in the control group (P < 0.05) (Fig. 4).
Testicular size was markedly diminished in the DXR-treated group, compared with that in the control group. Seminiferous tubules showing various stages of differentiated germ cells and normal seminiferous epithelium was observed in the control group (Fig. 5). Mature cells, ranging from spermatogonia to spermatids, were observed just above the basement membrane. The number of spermatids was decreased in the DXR-treated group, while few seminiferous tubules were observed in the normal epithelium. In most of the seminiferous tubules, spermatogonia alone were observed as the germ cells that differentiated with the highest frequency, or Sertoli cells alone were observed. Leydig cells with neither atrophy nor hyperplasia were observed in the DXR-treated group.
Figure 6 shows the Sertoli cell ratio, which was significantly lower in the DXR group of mice (P < 0.05).
Immunohistochemistry detected Kit protein in all the specimens examined in the control group (Fig. 7), where staining was seen in the spermatogonia and Leydig cells. Interstitial Leydig cells expressed c-kit protein most intensely. The expression of c-kit also was seen in some spermatogenic cells in the basal layer of the seminiferous tubules; the number of Kit-positive cells varied with the tubules. The expression of c-kit was not detected on spermatocytes with meiotic divisions and spermatids (Fig. 7).
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 (Fig. 7).
RT-PCR was performed in testes from both the control and the DXR-treated groups (Fig. 8). The expression of c-kit mRNA was observed in each mouse and was analyzed semiquantitatively using densitometry. The semiquantification of c-kit gene expression in the DXR-treated group showed a significantly lower level, compared with that in the control group (Fig. 8).
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.
The authors would like to thank all the members of the Department of Biology-Oriented Science and Technology at Kinki University School for their support and technical assistance. The authors also wish to thank Dr Hisahiro Yoshida (Department of RIKEN Yokohama Research Promotion Division, Yokohama city, Kanagawa, Japan) and the members of his laboratory.
None of the authors have a relationship with any company that may have a financial interest in this manuscript.