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Hum. Reprod. Advance Access originally published online on October 6, 2005
Human Reproduction 2006 21(2):471-476; doi:10.1093/humrep/dei319
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© The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Isolation of male germ stem cell-like cells from testicular tissue of non-obstructive azoospermic patients and differentiation into haploid male germ cells in vitro

Dong Ryul Lee1,3,*, Kye-Seong Kim2,*, Yun Hee Yang1, Hwa Soon Oh1, Sook Hwan Lee1, Tae Gyu Chung1, Jung Hyun Cho1, Hyun Joo Kim1, Tae Ki Yoon1 and Kwang Yul Cha1

1 Fertility Center of CHA General Hospital, CHA Research Institute, Pochon CHA University, 606-5 Yeoksam-dong, Gangnam-gu, Seoul, 135-081 and 2 Department of Anatomy and Cell Biology, College of Medicine, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul, 133–791, Korea

3 To whom correspondence should be addressed at: Fertility Center of CHA General Hospital, CHA Research Institute, Pochon CHA University, 606-5 Yeoksam-dong, Gangnam-gu, Seoul, 135-081, Korea. E-mail: drleedr{at}cha.ac.kr

* D.R.Lee and K.-S.Kim contributed equally to this work.


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The purpose of this study was to establish the culture conditions required to isolate, identify and expand male germ stem cell-like cells (GSC-LC) from the testicular tissue of patients with non-obstructive azoospermia (NOA). METHODS AND RESULTS: Testicular tissues obtained from patients (two with maturation arrest (MA, n = 2) and Sertoli cell-only syndrome (SCOS, n = 11) were dissociated and plated into gelatin-coated dishes. After 2–4 weeks, cultures from both MA patients (100%) and four SCOS patients (36.3%) exhibited multicellular colonies, which proliferated successfully until passage 10. GSC-LC in the colonies displayed alkaline phosphatase activity, as well as Oct-4 and integrin b1 expression after every passage. After the fifth passage, GSC-LC were differentiated by encapsulation in calcium alginate and further cultivation. At 2 and 6 weeks, cells expressed c-Kit, Scp3, testis-specific histone protein 2B (TH2B), and transition protein (TP)-1. Fluorescence in situ hybridization additionally disclosed a few tetraploid and haploid cells at 6 weeks. Human oocytes were activated in the absence of artificial activation and cleaved after the injection of presumptive spermatids. CONCLUSIONS: Our novel culture system may be useful for diagnosing the existence of germ cells and facilitating the treatment of NOA patients.

Key words: embryo production/germ stem cell-like cells/haploid male germ cells/non-obstructive azoospermic patients/spermatogenesis in vitro


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Some patients with non-obstructive azoospermia (NOA) can conceive successfully by ICSI or round spermatid injection (ROSI) in cases where sperm are derived from testicular tissues with focal spermatogenesis (Devroey et al., 1995Go; Tesarik et al., 1995Go; Silber et al., 1996Go). In general, however, artificial insemination with donor sperm is recommended for most patients exhibiting maturation arrest (MA) at a specific stage of spermatogenesis or with Sertoli cell-only syndrome (SCOS).

Tesarik et al. (1999)Go reported the birth of a normal child resulting from fertilization with haploid cells obtained by culturing germ cells arrested at the primary spermatocyte stage. Tanaka et al. (2003)Go additionally reported a co-culture system that led to the in vitro release of human male germ cells from MA. However, conception using round spermatids that develop in vitro usually fails, possibly due to cell damage during arrest, which results in apoptosis (Tesarik et al., 1998Go). Therefore, techniques employing arrested germ cells clearly need further investigation before clinical application.

Spermatogenesis is a process by which germ stem cells (GSC) proliferate and differentiate into haploid free-swimming sperm within the seminiferous tubules of the testes. As precursors of the spermatogonial lineage, male GSC must maintain the balance between mature sperm production and self-renewal. Since GSC renew themselves and produce spermatogenic cells after transplantation into the seminiferous tubules of infertile recipient males, they constitute a model system for understanding spermatogenesis and developing a novel transgenic tool (Brinster and Zimmerman, 1994Go). However, GSC are rare in the testis, constituting ~1 in 3000–4000 cells in mouse testis (Telgelenbosch and de Rooij, 1993Go). Moreover, a specific model to study the proliferation and differentiation of GSC remains to be developed. Consequently, GSC are not widely employed for treating human infertility.

Recently, mouse GSC were generated in vitro by genomic modification or co-culture with STO cells (Feng et al., 2002Go; Kanatsu-Shinohara et al., 2003Go). In addition, male haploid germ cells at the round spermatid stage have been derived from embryonic stem cells by spontaneous differentiation in vitro, which introduces the possibility of investigating germ cell development, epigenetic reprogramming, and germline gene modification (Toyooka et al., 2003Go; Geijsen et al., 2004Go). Conversely, completion of spermatogenesis in vitro from GSC through spermatocytogenesis, meiosis, and spermiogenesis has not been reported previously for any mammalian species. In addition, inducing GSC proliferation by genomic modification or co-culture using feeder cells obtained from foreign species is not suitable for human application.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Isolation of male GSC-LC from NOA patients
Testicular tissues were obtained from NOA patients subjected to the multiple testicular sperm extraction (TESE)–ICSI programme. When sperm or round spermatids were absent in dissected samples, the remaining testicular materials were donated for further diagnosis with patient consent. This study was approved by the Institutional Review Board of the CHA General Hospital (Seoul, Korea). In total, the testicular constituents of 13 patients (two with MA and 11 with SCOS) were dissociated by modified enzymatic digestion (Lee et al., 2001Go). Briefly, tissues were placed in 10 ml of enzyme solution containing 5 mg/ml collagenase (Type I; Sigma Chemical Co., St Louis, MO, USA), 10 µg/ml DNase I, 1 µg/ml soybean trypsin inhibitor (Gibco, Grand Island, NY, USA), and 1 mg/ml hyaluronidase (Sigma) in Ca2+-, Mg2+-free phosphate-buffered saline (PBS), and incubated for 30 min at ambient temperature (~25°C) for efficient dissociation. Next, dissociated testicular cells were plated and grown on gelatin-coated dishes in Dulbecco’s modified Eagle’s medium (DMEM) containing 15% fetal bovine serum (Gibco), 10 µmol/l 2-mercaptoethanol (Gibco), 1% non-essential amino acids (Gibco), 4 ng/ml basic fibroblast growth factor (Invitrogen), 10 µmol/l forskolin (Sigma), and 1500 IU/ml human leukaemia inhibitory factor (Peprotech Inc., Rocky Hill, NJ, USA) in a humidified atmosphere of 5% CO2 in air. Over the following 2–4 weeks, the large multicellular (≥100 cells) colonies that formed on the dish were sequentially subcultured every week. After the fifth passage, colony cells were divided into two groups. One was fixed for characterization, while the other was encapsulated, followed by culturing for in vitro differentiation.

In vitro differentiation of GSC-LC
After five passages, >200 GSC-LC colonies and their underlying somatic cells were trypsinized into single cells, resuspended, and encapsulated with calcium alginate (Lee et al., 2001Go). Alginate-encapsulated cells were transferred into culture medium consisting of HEPES-buffered DMEM/F-12 (Gibco) supplemented with 10 µg/ml insulin-transferring-selenium solution (Gibco), 10–4mol/l vitamin C (Sigma), 10 µg/ml vitamin E (Sigma), 3.3x10–7 mol/l retinoic acid (Sigma), 3.3x10–7 mol/l retinol (Sigma), 1 mmol/l pyruvate (Sigma), 2.5x10–5 IU recombinant human FSH (Gonal-F; Serono), 10–7 mol/l testosterone (Sigma), 1 x antibiotic–antimycotic (ABAM, containing penicillin, streptomycin and amphotericin B; Gibco), and 10% bovine calf serum (Hyclone) (modified from Weiss et al., 1997Go). Alginate-encapsulated cell aggregates were transferred to 1 ml of medium in a 24-well dish, and cultured for up to 6 weeks at 32°C in a humidified atmosphere of 5% CO2 in air. The medium was replaced on alternate days. In vitro cultured cells were decapsulated mechanically, and incubated in trypsin–EDTA for 30 min. Dispersed cells were rinsed with PBS, and divided into three groups. The first and second groups were characterized by RT–PCR and fluorescence in situ hybridization (FISH), while the third group was used for embryo production using ROSI.

Characterization of GSC-LC and spermatogenic cells
Colonies of putative male GSC-LC were fixed in 4% paraformaldehyde in Dulbecco’s PBS (Gibco) for nucleus counting using 1 µg/ml 4',6'-diamidino 2-phenyindiol (DAPI; Sigma), and to detect alkaline phosphatase activity. For visualization, a mixture of the violet chromogen S-bromo-{Delta}-chloro-3-indolyl phosphate (BCIP; Sigma) and Nitroblue Tetrazolium (NBT; Sigma) was added to the alkaline phosphatase reaction buffer. Moreover, for immunocytochemical analysis of Oct-4, integrin beta1 and c-kit expression, other fixed colonies were incubated overnight with the anti-Oct-4 antibody (Santa Cruz), anti-integrin beta1 antibody (BD/Pharmingen) and anti-c-Kit (Santa Cruz) at 1:100–500 dilution. The primary antibody was visualized with fluoroscein isothiocyanate or Cy3-conjugated secondary antibody (Zymed).

RT–PCR was performed to assess the expression of stage-specific marker genes in colonies and cells cultured from GSC-LC, specifically, integrin beta1 and Oct-4 in GSC, c-Kit in spermatogonia and spermatocytes, Scp3 and testis-specific histone protein (TH2B) in spermatocytes, and transition protein (TP)-1 in spermatids. Total RNA was extracted from 100 colonies or cultured cells using the TRIzol method (Gibco) (~100 mg was obtained from each sample type). Reverse transcription was performed using 1 µg of total RNA, 5 mmol/l MgCl2, and 1 IU of DNase I at 37°C for 30 min, after which 1 mmol/l dNTP, 2.5 µmol/l oligo-dT, and 2.5 IU reverse transcriptase (Superscript, Invitrogen) were added, and the mixture was incubated at 42°C for 1 h (Huang et al., 1996Go).

Amplification was performed in a 20 µl reaction mixture containing 10 mmol/l Tris–HCl (pH 8.3), 2 mmol/l MgCl2, 50 mmol/l KCl, 0.25 mmol/l dNTP, 3–5 pmol of each primer, and 1.25 IU Taq polymerase (Gibco). The following genes were amplified using the primers indicated in parentheses: integrin beta1 (forward: 5'-CTGCAAGAACGGGGTGAATG-3', reverse: CACAATGTCTACCAACACGCCC-3': 301 bp, GenBank accession number BC020057 [GenBank] ); Oct-4 (forward: 5'-GGAAAGGCTTCCCCCTCAGGGAAAGG-3', reverse: 5'-AAGAACATGTGTAAGCTGCGGCCC-3': 460 bp, GenBank accession number NM 002701); c-Kit (forward: 5'-AAGGACTTGAGGTTTATTCCT-3', reverse: 5'-CTGACGTTCATAATTGAAGTC-3': 345 bp, GenBank accession number L04143 [GenBank] ); Scp3 (forward: 5'-TGGAAAACACAACAAGATCA-3', reverse: 5'-GCTATCTCTTGCTGCTGAGT-3'; 344 bp, GenBank accession number AF517774 [GenBank] ); TH2B (forward: 5'-GTGCTACCATTTCCAAGAAG-3', reverse: 5'-CTCGCTATACGCTCAAAGAT-3': 217 bp, GenBank accession number AF397301 [GenBank] ); TP-1 (forward: 5'-AAGGCCTTAAATACCCAGAC-3', reverse: 5'-AGCAATGTGTGCCTAAGTTT-3': 254 bp, Gen Bank accession number M59924 [GenBank] ); and 18S ribosomal RNA (forward: 5'-TACCTACCTGGTTGATCCTG-3', reverse: 5'-GGGTTGGTTTTGATCTGATA-3': 255 bp, GenBank accession number K03432 [GenBank] ). PCR was initiated with denaturation at 94°C for 5 min, followed by 30–35 cycles of 30 s at 94°C, 30 s at 55–60°C, and 30 s at 72°C. A final extension step for 10 min at 72°C completed the amplification reaction, after which the products were separated by 2% agarose gel electrophoresis, and verified by automated nucleotide sequencing. Negative controls included mock transcription without RNA or PCR with distilled deionized water.

FISH was performed to confirm the haploidy of presumptive round spermatids derived from cultured GSC-LC colonies. Dispersed cells were placed in hypotonic solution [6 mg/ml bovine serum albumin and 0.5% sodium citrate (Sigma)] for 10 min, and fixed in Carnoy’s solution (methanol:acetic acid = 3:1) for 10 min. Next, fixed cells were spread onto precleaned glass slides, dehydrated, and examined with directly labelled DNA probes (Vysis Inc., Framingham, MA, USA), CEP 11-Spectrum Green (D11Z1) and CEP 18-Spectrum Orange (D18Z1). Hybridization was performed according to the manufacturer’s instructions.

Embryo production by ROSI and embryo transfer
After 6 weeks of in vitro differentiation, presumptive round spermatids (7–10 µm) obtained from cultured testis parenchyma were injected into mature oocytes. The spouse of each patient enrolled in the ROSI programme agreed to attempt pregnancy after round spermatids were identified in cultured materials, and underwent controlled ovarian stimulation with a GnRH antagonist protocol using recombinant FSH (Serono Inc., Switzerland). An ultrasound-guided follicular aspiration technique was used to retrieve oocytes. Expanded cumulus cells were removed by repeated pipetting with a small-bore glass pipette, followed by incubation in 0.1 mg/ml hyaluronidase for 5 min. A single round spermatid was aspirated into an injection pipette, and introduced into a denuded matured oocyte at 90° from the first polar body. Next, injected oocytes were transferred into 2 ml of P1 medium (Irvine Scientific Co., CA, USA) containing 10% synthetic serum substitute. At 72 h after the injection, cleaved embryos derived from fertilized oocytes showing two pronuclei (PN) were transferred into the uteri of the spouses. Pregnancy was assessed by measuring the serum beta-HCG levels 14 days after the transfer.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
Biopsied materials obtained from a number of NOA patients were divided into two fractions. One part was used for histological analysis, while the other was dissected to seek sperm. In 13 patients, sperm or round spermatids were not observed in the chopped material, as confirmed from histological analyses by the Pathology Laboratory of our University Hospital (Figure 1).


Figure 1
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  		Figure 1. Histological and light microscopy observation of intact or dissected testis tissues obtained from non-obstructive azoospermic patients. No spermatogenic cells and few Sertoli cells were observed. (A–C) Histological section of testis tissues from non-obstructive azoospermic patients with Sertoli cell-only syndrome (SCOS). (D) Cell suspension obtained after enzymatic digestion of testis tissues from non-obstructive azoospermic patients with SCOS. Bar = 50 µm.

 

The above 13 patients included 11 with SCOS and two with MA. Dissected material was dissociated with an enzyme solution, and cultured in vitro. After 2–4 weeks, material from both MA (100%) and four SCOS patients (36.3%) exhibited large multicellular colonies, each comprising ~100 cells, on the surface on the dish. These colonies continued to proliferate successfully (Figure 2A, B; Table I). Well-formed colonies were attached slightly to their own feeder cells (consisting of Sertoli and peritubular cells), and displayed efficient propagation and morphology at a colony size of <300 µm. Accordingly, the colonies of presumptive GSC-LC were passaged every week after dissociation into small pieces by trypsinization. Colony-like structures formed in the cultures of three other SCOS patients, but disappeared after several passages. The cultured material of the four remaining patients displayed no colony formation, and was discarded after 4 weeks (data not shown).


Figure 2
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  		Figure 2. Morphology and characterization of germ stem cell-like cell (GSC-LC) colonies formed after long-term culture of testicular cells obtained from non-obstructive azoospermic patients with Sertoli cell-only syndrome. (A) GSC-LC colony formation after 2–4 weeks of cultivation. (B) Staining of cell nuclei with DAPI. (C–D) Alkaline phosphatase activity in colonies of GSC-LC and human embryonic stem cells. (E, G, I) Immunocytochemistry revealing Oct-4, integrin beta1, and c-Kit expression in colony cells. (F, H, J) Negative controls not treated with primary antibodies of Oct-4, integrin beta1 and c-Kit. Bars = 200 µm.

 

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  		Table I. The presence of germ stem cell-like cells in testicular tissues obtained from non-obstructive azoospermic patients, and their characterization after in vitro culture and by RT–PCR

 

Following the fifth passage, colonies were divided into two samples. One was fixed and characterized, while the other was cultured to induce differentiation in vitro. Colonies were partly positive for alkaline phosphatase activity (Figure 2C). Additionally, immunocytochemical analyses revealed that cells expressed Oct-4 and integrin beta1, an immunological marker for GSC (Figure 2E and G), but not c-Kit, a marker for PGC and differentiated germ cells (Figures 2I and 3B). RT–PCR analysis of cultivated material from all 13 patients disclosed that only material sexhibiting colonies expressed the marker genes of the GSC and the stem cell, Oct-4 and integrin beta1 (Figure 3A; Table I). In contrast, cultured material from patients lacking colony formation or colony-like structures either failed to express Oct-4 and integrin beta1 completely or expressed these factors only weakly during the first one or two passages (data not shown). Notably, despite the marked stem cell characteristics of successfully proliferating GSC, the colonies detached and disappeared after ~10 passages.


Figure 3
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  		Figure 3. Expression of stage-specific genes in in vitro cultured material from non-obstructive azoospermic patients. (A) Detection of mRNA encoding Oct-4, integrin beta1, and 18S genes in 2–4 week cultured patient material. Lanes: M, mol. wt marker (100 bp ladder); 1–3, 6: patients with germ stem cell-like cell (GSC-LC) colonies; 4, 5: patients with no existing GSC-LC colonies. N, negative control. (B) Detection of Oct-4, c-Kit, Scp3, TH2B, TP-1 and 18S genes in materials cultured for an additional 2–6 weeks to induce GSC-LC differentiation into germ cells. Lanes: M, mol. wt marker; 1: 0 week; 2: 2 weeks; 3: 6 weeks; N, negative control (no cDNA).

 

After confirming the presence of GSC-LC in the colonies, we induced differentiation into spermatogenic cells by in vitro culture, with patient permission. GSC-LC and underlying somatic cells were dissociated, encapsulated with calcium alginate, and further cultured. After 6 weeks, extruded calcium alginate strands containing aggregates of germ and somatic cells did not change during culture (Figure 4A). Intercellular association with germ and somatic cells was maintained, and cell proliferation was additionally observed at the margins of encapsulated cells in culture. However, we could not histologically distinguish intercellular bridges (data not shown). Dissociation of these structures revealed the presence of several cell types varying in size from 7 to 20 µm. Cell morphology was generally spherical with apparently intact plasma membranes, based on the distinct margins of individual cells (Figure 4B). At 2 and 6 weeks of culture, Oct-4 mRNA expression by encapsulated cells was lost. In contrast, c-Kit, Scp3, TH2B and TP-1 genes were expressed at 2 and 6 weeks, respectively, indicative of the presence of spermatogonia, spermatocytes and spermatids, respectively (Figure 3B). FISH analysis additionally revealed a few dissociated tetraploid and haploid cells obtained after culturing for 6 weeks (Figure 4C, D). However, sperm-like cells were not identified in these long-term cultures.


Figure 4
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  		Figure 4. Micrographs of testicular cells from non-obstructive azoospermic patients after in vitro differentiation. (A) Calcium alginate-encapsulated testicular cells. Arrow: presumptive germ cell–somatic cell aggregates. Bar = 100 µm. (B) Cell suspension after the dissociation of alginate-encapsulated cells after 6 weeks of culture. Presumptive spermatocytes and round spermatid-like cells were distinguished according to their morphological properties (clear nuclear margin, and sizes of nuclei and cells). Arrow: presumptive round spermatid; arrowhead: presumptive spermatocytes. Bar = 20 µm. (C–D) Fluorescence in situ hybridization of cells dissociated from alginate-encapsulated cells after 6 weeks of culture with fluorochrome-labelled 11 (green), 18 (orange) chromosome-specific DNA probes, and nuclear staining with DAPI (blue). (C) Germ cell displaying tetraploidy; (D) germ cell displaying haploidy. Bar = 10 µm.

 

To confirm the capacity of haploid germ cells to generate embryos, we injected round spermatid-like cells obtained from six patients into mature human oocytes taken from their spouses by controlled ovarian stimulation, followed by culture for 3 days. Fifty-one mature oocytes from the six spouses were subjected to ICSI. At day 1, one of the 13 oocytes injected with putative spermatids from the MA patients (7.7%) displayed 1PN, while three contained 2PN (23.1%). Of the 38 oocytes injected with putative spermatids from SCOS patients, nine (23.7%) contained 1PN, and eight (21.1%) showed 2PN. At day 2, three more oocytes with no PN in SCOS were cleaved, and one oocyte displaying 2PN was non-cleaved. Cleaved embryos derived from oocytes displaying 1PN, 3PN or no PN were discarded. At day 3, three cleaved embryos from MA and seven from SCOS were transferred into the uteri of two MA patient spouses and four SCOS patient spouses, respectively. However, none resulted in implantation.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
Male GSC maintain a balance between the production of mature sperm and self-renewal, and are thus valuable tools for mechanistic studies on spermatogenesis, treating male infertility, and genetically modifying the male germ line. Previous studies have shown that some GSC proliferate when immortalized or co-cultured with feeders, followed by differentiation into spematogenic cells upon in vitro culture or transplantation (Feng et al., 2002Go; Kanatsu-Shinohara et al., 2003Go). In this study, we were able to isolate and propagate GSC-LC from the testicular tissues of NOA patients with MA or SCOS. These cells proliferated 2–3-fold during every passage in vitro (data not shown). During culture, the GSC-LC colonies that formed in vitro expressed Oct-4 and GSC-specific integrin beta1, similar to earlier observations (Shinohara et al., 1999Go, 2000Go). Moreover, the colonies displayed a partly positive signal for alkaline phosphatase activity, compared to human embryonic stem cells. After encapsulation of proliferated GSC-LC and testicular somatic cells by using calcium alginate and culturing for an additional 6 weeks, cells expressing the gene specific for haploid male germ cells were obtained (Figure 3B). Light microscopy revealed a low number of presumptive spermatocytes and/or round spermatid-like cells defined according to their morphological properties [clear nuclear margin, as well as sizes of nuclei and cells (Tanaka et al., 2003Go; Vigier et al., 2004Go)] (Figure 4B). In addition, the cells exhibited normal chromosome status (tetraploidy for primary spermatocytes and haploidy for round spermatids) (Figure 4C, D) and activated human oocytes in the absence of artificial activation after injection into the cytoplasm. To our knowledge, this is the first report describing the isolation, maintenance and differentiation of human GSC-LC from testicular materials of non-obstructive azoospermic patients.

Consistent with a number of previous reports, we could not maintain GSC-LC over long-term culture (Nagano et al., 1998Go; Brinster, 2002Go). Specifically, the colonies disappeared before the 10th consecutive passage. This may be due to the ageing of autologous feeder cells (mainly Sertoli cells) in extended cultures and inadequate culture conditions. The development of a better feeder-free system may therefore extend the longevity of GSC-LC. Support of this theory is a study showing the long-term survival and proliferation of mouse GSC upon replacement of mouse embryonic feeder cells (Kanatsu-Shinohara et al., 2003Go). The addition of glial cell-derived neurotrophic factor (GDNF), a component of the basal medium for maintenance of mouse GSC, may additionally improve cell survival (Kubota et al., 2004Go). Another drawback of our culture system is its inability to induce differentiating cells to complete the final step of spermatogenesis, i.e. spermiogenesis, as sperm are not observed. This failure was responsible for the low fertilization rate and failed pregnancies after the IVF programme, since we used spermatids instead of sperm for fertilization. Previous studies have shown that unlike transplanted GSC, cells propagated and differentiated by in vitro cultivation usually display limited spermiogenesis (Rassoulzadegan et al., 1993Go; Hofmann et al., 1994Go; Hue et al., 1998Go; Staub et al., 2000Go; Lee et al., 2001Go; Feng et al., 2002Go). Consequently, the development of suitable means to induce spermiogenesis in vitro for use in the treatment of male infertility is essential.

Appropriate treatment for NOA requires accurate evaluation (Lee et al.,1998Go). Most diagnoses are made on the basis of traditional histology of testis biopsies. However, spermatogenic cells may still be present in some NOA cases diagnosed by this histological method, owing to focal spermatogenesis in the testis (Devroey et al., 1995Go; Silber et al., 1996Go). Indeed, in the present study we observed GSC-LC in 30% of the non-obstructive azoospermic patients diagnosed with SCOS. The identification of such cases should prove more informative for the application of assisted reproduction procedures.

Our in vitro culture system was thus successful in maintaining GSC-LC from the testicular tissues of some NOA patients. We therefore conclude that this culture system is useful for diagnosing the existence of germ cells, which may aid in treating NOA. Following the establishment of a method to induce in vitro spermiogenesis, our culture system should also be useful for application in IVF.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
We would like to thank Jung Jin Lim, M.Sc. for providing us with the molecular work. This work was supported, in part, by a grant (SC2060) from the Stem Cell Research Center of the 21C Frontier R&D Program funded by the Ministry of Science and Technology, and a grant from the specialized program of the University funded by the Ministry of Education and Human Resources Development, Republic of Korea.


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on May 25, 2005; resubmitted on August 26, 2005; accepted on September 1, 2005.


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