Impairment of spermatogenesis has been identified as an inevitable side effect of cancer treatment. Although estrogen treatment stimulates spermatogenic recovery from the impaired spermatogenesis by suppressing the intra-testicular testosterone (ITT) level, side effects of estrogen are still major impediments to its clinical application in humans. Soybeans are rich in genistein, which is a phytoestrogen that binds to estrogen receptors and has an estrogenic effect. We investigated the effects of genistein administration on ITT levels, testis weight, and recovery of spermatogenesis in rats treated with a chemotherapeutic agent, busulfan.
Busulfan was administered intraperitoneally to rats, and then a GnRH agonist was injected subcutaneously into the back, or genistein was administered orally.
The weight of the testes was significantly reduced by the treatment with busulfan. The testis weight was partially restored after busulfan treatment by additional treatment with either the GnRH agonist or genistein. Busulfan also induced atrophy of a high percentage of the seminiferous tubules, but this percentage was decreased by additional treatment with either the GnRH agonist or genistein. Treatment with genistein was effective at suppressing and maintaining ITT levels comparable to that in the GnRH agonist group.
Genistein effectively suppressed ITT levels and stimulated the recovery of spermatogenesis in rats treated with a chemotherapeutic drug. This suggests that genistein may be a substitute for estrogens, for helping humans to recover fertility after cancer therapy without the risk of side effects.
Impairment of spermatogenesis as a result of treatment with chemotherapeutic drugs or radiation has been identified as an inevitable side effect of cancer treatment in human. Previous studies using a rat model have shown that spermatogenesis impaired by radiation or chemotherapy can be restored by treatment with GnRH agonists [
However, the side effects of estrogen treatment, such as gynecomastia and cardiovascular problems [
In the present study, we investigated the effects of genistein administration on ITT levels, testis weight, and recovery of spermatogenesis in rats treated with a chemotherapeutic agent, busulfan, to evaluate the possibility of the clinical application of genistein.
All animal housing and surgical procedures were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee of MizMedi Hospital, Seoul, Korea (Miz-ani-IRB 20090715).
A total of 150 male Sprague-Dawley rats were used for the present study, and they were maintained on a 12 hours light/dark cycle and were allowed food and water
Busulfan (Sigma, St. Louis, MO, USA), an anticancer drug, was first dissolved in a 100% dimethyl sulfoxide solution (8 mg/mL). The same volume of distilled water was added just before use to give a final concentration of busulfan (4 mg/mL). This busulfan solution was administered intraperitoneally to the rats at a dose of 25 mg/1 kg of body weight. When the body weight of the rat was 200 g, 5 mg (1.25 mL) busulfan was administered.
A GnRH agonist (leuprorelin) was obtained from Dong-gook Pharmaceutical (Seoul, Korea). GnRH injections were prepared by suspending a vial of 2.6 mg of GnRH into 1.6 mL of its solvent. 0.65 mg of the GnRH solution (0.4 mL) was injected subcutaneously into the back of the rat.
Genistein was purchased from LC Laboratories (Woburn, MA, USA), and was administered daily to the rats orally at a dose of either 50 or 100 mg/kg with a oral zonde for a period of 4 weeks (from 3 weeks to 7 weeks) (
One hundred fifty rats were divided into 5 groups, with 30 in each group; 1) control, 2) busulfan, 3) busulfan+GnRH, 4) busulfan+genistein (50 mg), and 5) busulfan+genistein (100 mg). The experimental design is shown schematically in
All of the rats (with the exception of the control group) were first weighed before being given a single injection of busulfan at experimental week 0. At week 3 and thereafter, either a single injection of the GnRH agonist was administered, or daily oral administration of genistein was initiated (depending upon the experimental group).
All of the animals were sacrificed by CO2 asphyxiation at experimental week 13. Both testes were freed of the tunica, weighed, and one testis was homogenized in 2 mL of cold water. The testicular supernatant in the homogenate was stored at -80℃ for ITT analysis. The level of ITT was assayed in samples from experiments by using T-antiserum-coated tubes (Diagnostic Systems Laboratories, Webster, TX, USA). The ITT level was estimated by chemiluminescence immunoassay and expressed as the amount per gram of testis, to reflect the actual concentration of testosterone to which the testicular cells were exposed [
Histological analysis of the testes was performed at experimental week 13. The testes were fixed in Bouin's fixative, dehydrated, and then embedded in paraffin. Thereafter, 5 µm-thick serial sections were prepared, and at least five slides from each testis were stained with hematoxylin and eosin for histological assessment. A single slice from each testis was examined under a light microscope. The condition of spermatogenesis in each seminiferous tubule was classified as follows according to the number of germ cell layers present: 'recovery' (at least five layers), 'half recovery' (three or four layers), 'poor recovery' (one or two layers), and 'atrophy' (no germ cells).
The statistically significant differences among the various treatment groups were determined using one-way ANOVA. All statistical analyses were performed using SPSS ver. 16.0 (SPSS Inc., Chicago, IL, USA). For the statistical procedure performed, a
The testes appeared to be significantly smaller in the groups treated with busulfan than in the control group. The testes were weighed bilaterally in all groups (
Seminiferous tubule cross-sections were observed and then classified as follows according to the number of germ cell layers present: 'recovery' (at least five layers), 'half recovery' (three or four layers), 'poor recovery' (one or two layers), and 'atrophy' (no germ cells). For each rat, approximately 300 seminiferous tubules were evaluated in this way (
The effect of genistein treatment on the recovery of seminiferous tubules treated with busulfan was investigated (
ITT levels per gram of testis were assessed at experimental week 13 (
In the present study, we selected busulfan as a chemotherapy drug, because it is a potent agent that preferentially kills spermatogonial stem cells [
Histological analysis of the testes revealed that the testes of rats treated with busulfan exhibited atrophy of the seminiferous tubules, which contained no apparent spermatozoa and were devoid of germ cells. This concurs with the findings of a previous study, although the percentages of 'recovery' and 'atrophy' tubules differ somewhat between the studies. The cytotoxicity of busulfan was significantly reduced by additional treatment with the GnRH agonist, which resulted in a partial recovery of spermatogenesis in the seminiferous tubules. The effect of the GnRH agonist on the recovery of spermatogenesis in testes treated with busulfan has already been reported [
While there have been no report regarding the effect of genistein on the cytotoxicity of busulfan, we observed herein that genistein treatment also significantly reduced the cytotoxicity of busulfan and induced the recovery of spermatogenesis. Genistein is one of the phytoestrogens (a group of natural selective estrogen receptor modulators, nonsteroidal, diphenolic structures found in many plants, especially soy products) that binds to estrogen receptors [
It has been noted that ITT levels are elevated after irradiation or chemical treatments, which suggests that an excess of ITT is detrimental to spermatogenesis. ITT levels are suppressed by GnRH analogs that interrupt the pituitary-gonadal axis through flare-up and down-regulation [
The mechanism underlying the favorable suppression of ITT levels thus stimulating spermatogenesis in rat cytotoxic-drug-damaged testes has yet to be established. One potential mechanism whereby high levels of ITT may be detrimental to spermatogenesis is a change in the pattern of stem cell factor (SCF) expression [
It is expected that practical hormonal treatment regimens will be developed that could help patients to recover fertility after chemotherapy. However, there may be species differences in the testicular responses to radiation and chemotherapy, GnRH analogs, or both, so that rescue protocols that are shown to be successful in rodents might not work in primates [
In conclusion, we observed that genistein effectively suppressed ITT levels and stimulated the recovery of spermatogenesis in rats treated with a chemotherapeutic drug. Genistein is a phytoestrogen that is more natural and has fewer side effects than estrogen hormone. This makes genistein a candidate substitute for GnRH analogs and estrogens, for helping to recover fertility after cancer therapy in humans. Further study is required to identify whether there are the species differences in the effects of genistein.
No potential conflict of interest relevant to this article was reported.
Schematic representation of experimental design. One hundred-fifty rats were equally divided into 5 groups. A busulfan injection was performed at week 0, and then a single injection of a GnRH agonist or oral administration of genistein for 4 weeks was performed at week 3. The rats were sacrificed by CO2 asphyxiation at experimental week 13 for evaluation of intra-testicular testosterone levels and histological analysis of the testes.
Effect of genistein treatment on the weight of testes reduced in size by busulfan treatment. All of the animals were sacrificed by CO2 asphyxiation at experimental week 13. Both testes were freed of the tunica and weighed. a,b,cNumbers in each group with different superscripts indicate significant difference (
Classification of seminiferous tubules in the chemically impaired testis according to the number of germ cell layers present. The testes were fixed in Bouin's fixative, dehydrated, and then embedded in paraffin. Thereafter, 5 µm-thick serial sections were prepared and stained with hematoxylin and eosin for histological assessment. Seminiferous tubule cross-sections were observed and then classified as follows: (A) recovery (star, at least five layers), (B) half recovery (star, three or four layers), (C) poor recovery (star, one or two layers), (D) atrophy (star, no germ cells). For each rat, approximately 300 seminiferous tubules were evaluated in this way (×200).
Effect of genistein treatment on the recovery of seminiferous tubules treated with busulfan. The effect of genistein was investigated with the percentages of the classified seminiferous tubules such as recovery (at least 5 layers), half recovery (3 or 4 layers), poor recovery (1 or 2 layers), and atrophy (no germ cells). a,b,cNumbers in each group with different superscripts indicate significant difference (
Effect of genistein treatment on intra-testicular testosterone (ITT) levels (ng/g) in rats treated with busulfan. One testis was homogenized and the level of ITT in the testicular supernatant in the homogenate was assayed at experimental week 13 by using T-antiserum-coated tubes. a,b,cNumbers in each groups with different superscripts indicate significant difference (