| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
c Department of Urology, Osaka University Medical School, Suita 565-0871, Japan
d Genome Information Research Center, Osaka University, Suita 565-0871, Japan
e Department of Food Science and Nutrition, Mukogawa Women's University, Nishinomiya 663-8558, Japan
f Department of Science for Laboratory Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, Suita 565-0871, Japan
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
male reproductive tract, reproductive technology, spermatogenesis, testis, testosterone
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Male mice homozygous for the jsd gene (jsd/jsd) are sterile and undergo no spermatogenesis except for the first wave of spermatogenesis [5, 6]. However, they retain undifferentiated type A spermatogonia in the seminiferous tubules after 8–10 wk of age, showing a phenotype of testicular atrophy [5, 6]. The failure of differentiation is ascribed to the spermatogonia themselves [7], which undergo active proliferation accompanied by apoptosis, as seen in the cryptorchidism or Sl17H mutant [7, 8]. The mechanism of the defect can be explained by hypersensitivity of the mutant germ cells to high levels of testosterone [9]. Upon reduction of the testosterone level using a GnRH antagonist, the mutant resumes spermatogenesis and produces spermatocytes [10], as happens with many other infertility models such as those of radiation- and chemotherapy-induced testicular damage [11]. However, no further progress beyond the spermatocyte stage has been reported using this treatment. Upon transplantation of wild-type spermatocyte nuclei into oocytes, the differentiation can proceed further to meiosis, and microfertilization eventually produces a newborn mouse [12]. However, at present the efficiency of this process is low and there are technical difficulties in applying this approach to human therapy. Therefore, the arrest of spermatocyte differentiation caused by a reduction of the testosterone level is a serious obstacle to rescuing azoospermic males. If we could find a way to promote further differentiation of such spermatocytes to spermatids, the ICSI technique could be used for some azoospermic males.
Administration of GnRH antagonists was reported to induce apoptosis of preleptotene and pachytene spermatocytes and spermatids in wild-type rats [13]. Furthermore, in gonadotropin-deficient mice, no germ cell differentiation beyond the spermatocyte stage is observed [14]. Thus, gonadotropin, testosterone, or both seem to be necessary for the progress of meiosis and spermiogenesis [13, 14]. To develop methods for rescuing the fertility of azoospermic males and for the treatment of patients, we have carried out induction of spermatogenesis in jsd mutant mice first to spermatocytes and then to elongated spermatids by suppression and subsequent reelevation of testosterone levels, respectively, and have successfully rescued male sterility by ICSI.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
C57BL/6(B6)-jsd/jsd mice were derived from original stocks obtained from the Jackson Laboratory (Bar Harbor, ME). B6-jsd/jsd males were identified by scrotal palpation at 5–7 wk of age of their smaller testes compared with those of wild-type mice.
Careful selection of the recipient in the experiments was important because the pups are the end result of the entire experiment. We used ICR mice because they are calm and excellent mothers. Mice that had been plugged by vasectomized males were used as pseudopregnant mothers [15]. B6D2F1 (C57BL/6 x DBA/2) mice were used as oocyte donors because they ovulate more oocytes and their oocytes are more robust than those of the inbred strains. ICR and B6D2F1 mice were purchased from Japan SLC (Hamamatsu, Japan).
The mice were maintained in the laboratory animal facilities of the Research Institute for Microbial Diseases (Osaka University) under controlled conditions (lights-on 0600–1800 h, 24°C), with free access to mouse chow and water. The animals were killed by cervical dislocation, and blood was collected by cardiac puncture. The whole body and bilateral testes were weighed. The right testes were used for histologic examination, and the left testes were used for measurement of intratesticular testosterone (ITT) levels. The experiments were approved by the Animal Care Committee of the Research Institute for Microbial Diseases.
Hormone Treatment
The GnRH antagonist Nal-Glu ([AC-D2-Nal1, D4Cl-Phe2, D3-Pal3, Arg5, D-Glu6 (AA), D-Ala10] GnRH) was supplied by Dr. H.K. Kim of the Contraception and Reproductive Health Branch of the NICHD (Rockville, MD). Nal-Glu was dissolved in distilled water and given to mice through miniosmotic pumps (Alzet Model 2004; ALZA Corp., Palo Alto, CA) at 2500 µg kg-1 day-1. The pumps were implanted under the back skin of the mice.
Testosterone was given by s.c. implantation of a pellet releasing a total of 15 mg of testosterone per 60 days (Innovative Research of America, Sarasota, FL).
Chronological Changes of Spermatogonial Cell Differentiation Induced by Nal-Glu Administration and Withdrawal
Eight-week-old male jsd/jsd mice were administered Nal-Glu by miniosmotic pumps, and the changes in spermatogonial cell differentiation were observed after 2, 3, 4, 6, and 8 wk of treatment. Each group consisted of 4 mice (Fig. 1A).
|
For observation of the effect of withdrawal of Nal-Glu, the miniosmotic pumps were removed after 4 wk of Nal-Glu treatment, and the mice were killed at 2, 4, or 8 wk after the withdrawal (6 mice/group, Fig. 1B). Five untreated jsd/jsd mice were used as a control group.
Induction of Meiosis and Spermiogenesis by Testosterone Supplementation
To determine whether the meiosis and spermiogenesis induced by the withdrawal of GnRH antagonist were caused by reelevation of the testosterone level, we administered testosterone to the Nal-Glu-treated jsd/jsd mice. Nal-Glu at a dose of 2500 µg kg-1 day-1 was administered to 5 male jsd/jsd mice at 8 wk of age. After 4 wk of administration, a testosterone pellet was implanted s.c., and the mice were treated for another 2 wk with Nal-Glu (Fig. 1C). Mice treated with Nal-Glu alone for 6 wk were used as controls (n = 4).
Histologic Evaluation
The right testis was fixed in Bouin solution overnight and embedded in paraffin. Five-micrometer-thick histologic sections were stained with Mayer hematoxylin and eosin. All cross-sections of the seminiferous tubules were examined, and tubules containing more than 2 spermatocytes, round spermatids, or elongated spermatids were counted as differentiated tubules. The average percentage of differentiated tubules was obtained from 2 cross-sections containing a total of approximately 200 seminiferous tubules/testis.
Hormone Assays
The left testis was homogenized in 0.5 ml of water on ice using a Teflon homogenizer that was fitted into a microfuge tube. Each sample was centrifuged at 10 000 rpm for 10 min. The supernatant was removed, frozen in liquid nitrogen, and stored at -80°C until assayed. Blood was collected soon after each mouse was killed, and serum was then frozen in liquid nitrogen and stored at -80°C until assayed. Serum and ITT levels were measured by RIA using a total testosterone kit (Diagnostic Products Co., Los Angeles, CA), and serum LH and FSH levels were determined using Biotrak rat LH [125I] assay system RPA552 and Biotrak rat FSH [125I] assay system RPA550 (Amersham Life Science, Buckinghamshire, England), respectively, according to the manufacturer's protocols. The ITT level was expressed per testicular weight (ng/g testis). The limit of detection of the assay for testosterone was 0.04 ng/ml. Therefore, the limit of detection for ITT was 0.02 ng/whole testis and 2 ng/g testis when the testicular weight was 10 mg. The limits of detection of the assays for LH and FSH were 0.8 ng/ml and 0.9 ng/ml, respectively. The inter- and intra-assay coefficients of variation were 5.9–11.4% and 3.9–11.2% for testosterone, 7.7–10.9% and 6.5% for LH, and 6.1–11.1% and 4.2% for FSH, respectively.
Intracytoplasmic Injection of Spermatids Induced by Hormonal Treatment
Preparation of oocytes B6D2F1 females (8–12 wk old) were injected with 5 IU of eCG (Teikoku Hormone Mfg. Co., Tokyo, Japan) followed by 5 IU of hCG (Teikoku) 48 h later. Oocytes collected from oviducts between 14 and 15 h after hCG injection were freed from cumulus cells by treatment with hyaluronidase (700 units/ml) in modified Hepes-CZB medium [16]. Denuded oocytes were rinsed and kept in modified Kosei medium [17] at 37°C under 5% CO2 in air and were used for ICSI experiments within 2–3 h.
Preparation and injection of spermatids Nal-Glu was administered at a dose of 2500 µg kg-1 day-1 to 2 male 8-wk-old jsd/jsd mice and then withdrawn after 4 wk of treatment. The mice were killed by cervical dislocation approximately 4 wk (26 or 30 days) after withdrawal of Nal-Glu. Testes of these mice were placed in cold PBS. The tunica albuginea was removed, and seminiferous tubules were carefully separated from each other with watchmaker's forceps. The separated tubules were examined under a dissecting microscope to identify a region where a sufficient number of spermatids could be recovered for ICSI. One-millimeter-long segments of tubules were cut from selected regions that were slightly darkened in the innermost part of the tubule, and the contents of the tubules were squeezed out with forceps. Spermatogenic cells, including elongated spermatids, were transferred to modified Hepes-CZB medium containing 12% polyvinylpyrrolidone and subjected to ICSI. A sperm head was detached from the tail by a Piezo pulse and was injected into an oocyte using a Piezo injector (Prime Tech, Tokyo, Japan) as described by Kimura and Yanagimachi [18]. The injected eggs were cultured for 24 h in KSOM medium [17], and embryos that developed to the 2-cell stage were transferred into oviducts of Day 0.5 pseudopregnant female ICR mice.
Statistical Analysis
Data obtained were expressed as arithmetic means ± SEM. A Welch t-test was used to examine the significance of the differences between the group treated with Nal-Glu alone and the group treated with Nal-Glu in combination with testosterone. An ANOVA with a Fisher PLSD test was performed to examine the significance of differences among the untreated group, the Nal-Glu withdrawal groups (2, 4 wk), and the groups treated with Nal-Glu alone (6, 8 wk) at each time point. A Welch t-test was performed to examine the significance of differences between the Nal-Glu withdrawal group (8 wk) and the untreated group.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Administration of the GnRH antagonist Nal-Glu induced spermatogonial differentiation (Fig. 2, A and B), as reported previously [10]. Although the spermatogonial differentiation was already induced within 2 wk, the maximum effect was observed at 4–6 wk after starting the antagonist treatment, and thereafter the number of differentiated tubules decreased and no haploid spermatids were observed (Fig. 3A).
|
|
To induce further progress of germ cell differentiation, administration of Nal-Glu was stopped by surgically removing the miniosmotic pumps after 4 wk of treatment. Because of marked individual differences in percentage of differentiated tubules among the animals treated with Nal-Glu for 6 wk, the pumps were removed after 4 wk of treatment. Two weeks after this operation, spermatocytes and haploid germ cells were observed in cross-sections of seminiferous tubules. Thereafter, the number of spermatocytes decreased and the number of late elongated spermatids increased, although round spermatids were predominant among the haploid germ cells (Fig. 2C). Four weeks after Nal-Glu withdrawal, elongated spermatids became predominant in all of the 6 mice examined (Fig. 2, D and E). The changes of the percentage of seminiferous tubules containing round, elongated, and total spermatids were significant at both 2 and 4 wk (P < 0.01 for all cell types at 2 wk, P = 0.01 for round spermatids and P<0.01 for elongated and total spermatids at 4 wk compared with untreated jsd mice or mice treated with Nal-Glu, respectively). Thereafter, the differentiated germ cells disappeared and only a few differentiating cells were seen in seminiferous tubules 8 wk after Nal-Glu withdrawal (Fig. 2F) (P = 0.25, 0.39, 0.39, 0.17 for spermatocytes and round, elongated, and total spermatids compared with untreated jsd mice, respectively). Chronologic changes in the percentage of seminiferous tubules containing differentiating germ cells are shown in Figure 3B. Two weeks after withdrawal of Nal-Glu, hormonal levels increased to those of the untreated control jsd/jsd mice, and no significant changes were observed thereafter (Table 1).
|
Effect of Testosterone Supplementation after Nal-Glu Treatment on Spermatogenesis
Supplementation with testosterone for the last 2 wk of Nal-Glu treatment induced meiotic division and production of haploid spermatids in 13.8% ± 6.5% of the seminiferous tubules, effects similar to those in the group withdrawn from Nal-Glu for 2 wk (Fig. 4). In contrast, germ cell development proceeded only as far as production of spermatocytes, and no haploid spermatids were observed after administration of Nal-Glu alone for 6 wk (P < 0.01). Serum LH and FSH levels were not affected by testosterone supplementation (P = 0.09 and P = 0.08, respectively). Although the serum testosterone level was elevated, the difference was not significant (P = 0.06), whereas the ITT level was significantly elevated compared with the levels in mice treated with Nal-Glu alone (P < 0.01) (Table 1).
|
Intracytoplasmic Injection of Spermatids Induced by Biphasic Treatment (Administration and Withdrawal) with GnRH Antagonist
Biphasic treatment with Nal-Glu (4-wk administration followed by 4-wk withdrawal) induced germ cell differentiation into elongated spermatids in some parts of the seminiferous tubules, as indicated by a darkened central area seen by transillumination microscopy. Such tubules contained elongated spermatids that could be squeezed out using forceps. Cells with a crescent-shaped head were carefully chosen from the recovered spermatids (Fig. 5) and injected into oocytes of B6D2F1 female mice using a Piezo injector (Prime Tech). A total of 94 elongated spermatids induced by this treatment were injected into oocytes, resulting in the birth of 8 healthy offspring (6 males and 2 females) (Table 2).
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Although Nal-Glu administration induced spermatogonial differentiation in jsd/jsd mice, the differentiated germ cells were arrested at the spermatocyte stage, and no haploid cells were observed in the seminiferous tubules [10]. In the case of gonadotropin-deficient mice, germ cell differentiation beyond the spermatocyte stage was also limited and therefore meiosis did not occur but could be induced by testosterone supplementation [14]. The administration of GnRH antagonist induced apoptosis in the preleptotene and pachytene spermatocytes and spermatids in rats [13]. Thus, the recovery of meiosis induced in our study by reelevation of the testosterone level may be due to a decrease in the rate of apoptosis of differentiating cells such as preleptotene and pachytene spermatocytes and spermatids. Furthermore, the kinetics of the germ cell population after the administration and subsequent withdrawal of Nal-Glu suggests that only 1 new wave of spermatogenesis was induced by this experimental procedure (Fig. 3B). No further differentiation of spermatogonial stem cells occurred because of reelevation of the ITT level. One possible explanation for the absence of spermatozoa in these mice is that the lack of new spermatogenesis of spermatogonia negatively influences the spermiation of elongated spermatids. Another possible explanation is that the lack of ability to complete spermiation is due to defects in the mutant germ cells themselves.
Two weeks of administration of testosterone and Nal-Glu induced meiosis to the same extent as withdrawal of Nal-Glu for 2 wk. In testosterone-supplemented mice, the serum FSH level was much lower than that in mice from which Nal-Glu was withdrawn. Although a positive effect of FSH on meiosis cannot be completely ruled out because the levels of testosterone and ITT were different in these groups, it is clear that meiosis can occur in a low-FSH environment.
Recently, novel methods have been reported as alternative therapies for male infertility [19–21]. When donor germ cells were injected into the testis of a sterile mouse, the germ cells populated the testis, produced spermatozoa [19], and restored fertility in the infertile mouse [20]. Tesarik et al. [21] reported that in vitro culturing of immature germ cells subjected to maturation arrest at the primary spermatocyte stage, followed by ICSI, resulted in the successful birth of 2 babies.
Elevated ITT levels are commonly observed in some other infertile animal models [8, 9], and suppression of the testosterone level in such animals reduces the spermatogenic damage induced by radiation or toxic reagents [11]. Thus, a common mechanism appears to exist among these animal models and may also exist in humans with male infertility. As we have demonstrated here, testosterone suppression therapy in conjunction with an assisted-reproduction technique such as ICSI might be a new alternative therapy not only for animal models but also for some human patients suffering from severe disturbance of spermatogenesis.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Correspondence: Kiyomi Matsumiya, Department of Urology, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. FAX: 81-6 6879 3539; kmatsu{at}uro.med.osaka-u.ac.jp 
2 These two authors contributed equally to this study.
Accepted: August 16, 2001.
Received: April 11, 2001.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  K. Tokuhiro, M. Hirose, Y. Miyagawa, A. Tsujimura, S. Irie, A. Isotani, M. Okabe, Y. Toyama, C. Ito, K. Toshimori, et al. Meichroacidin Containing the Membrane Occupation and Recognition Nexus Motif Is Essential for Spermatozoa Morphogenesis J. Biol. Chem., July 4, 2008; 283(27): 19039 - 19048. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Rohozinski and C. E. Bishop The mouse juvenile spermatogonial depletion (jsd) phenotype is due to a mutation in the X-derived retrogene, mUtp14b PNAS, August 10, 2004; 101(32): 11695 - 11700. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Ohta, A. Tohda, and Y. Nishimune Proliferation and Differentiation of Spermatogonial Stem Cells in the W/Wv Mutant Mouse Testis Biol Reprod, December 1, 2003; 69(6): 1815 - 1821. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. L. Meistrich and G. Shetty Inhibition of Spermatogonial Differentiation by Testosterone J Androl, March 1, 2003; 24(2): 135 - 148. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
