1 Department of Urology and 4 Department of Pathology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita Osaka 565-0871, 2 Department of Urology, Osaka Central Hospital, Osaka, 530-0001 and 3 Department of Urology, Osaka Police Hospital, Osaka, 543-8502, Japan
5 To whom correspondence should be addressed. Email: akitsuji{at}uro.med.osaka-u.ac.jp
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: GDNF/inhibin/microdissection TESE/SCF/Sertoli cell-only syndrome
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SCO is diagnosed when testicular biopsy reveals that seminiferous tubules are lined by Sertoli cells, with complete absence of germ cells but with normal interstitium (Rothman et al., 1982). However, 22.5% of cases of SCO are characterized by focal and minute spermatogenesis, and microdissection TESE has provided new hope for successful sperm retrieval in such cases (Tsujimura et al., 2002
). Reports prompts us to speculate that the status of the testicular tubules may depend on the function of Sertoli cells in SCO.
Sertoli cells, which interact directly with germ cells, play important roles in supporting and regulating spermatogenesis. Sertoli cells secrete soluble factors, such as inhibin B, glial cell line-derived neurotrophic factor (GDNF) and stem cell factor (SCF). Inhibin B is a dimer of a common subunit and a
B subunit, and the
subunit is immunolocalized in both Sertoli cells and Leydig cells in the normal adult testis (Marchetti et al., 2003
). Serum inhibin B levels are related to spermatogenesis (Pierik et al., 1998
; von Eckardstein et al., 1999
; Andersson et al., 2004
). GDNF promotes the survival and differentiation of undifferentiated spermatogonia including stem cells (Meng et al., 2000
), and SCF promotes the proliferation and differentiation of type A spermatogonia (Rossi et al., 1991
; Tajima et al., 1991
; Vincent et al., 1998
). Thus, expression of these factors may be related to the status of spermatogonia.
To investigate these possibilities, we conducted a study examining expression of the inhibin subunit, GDNF, and SCF in relation to the presence of testicular sperm in patients with SCO.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Inhibin immunohistochemistry
The sections were deparaffinized and rehydrated. For antigen retrieval, slides were heated for 15 min at 121°C in 0.01 mol/l citrate buffer (pH 6). After endogenous peroxidase activity was blocked with 3% H2O2, non-specific binding was blocked by a 20 min incubation with normal swine serum (DakoCytomation, USA). The primary antibody was a mouse monoclonal antibody generated against the inhibin subunit (R1; DakoCytomation, Denmark). The sections were incubated for 1 h at 37°C with primary antibody at a dilution of 1:100. Antibody was detected by the streptavidinbiotinperoxidase method with diaminobenzidine by means of a Dako LSAB+ kit (DakoCytomation, Denmark), according to the manufacturer's recommendations. Negative controls were prepared with the use of normal mouse IgG (Vector Laboratories, USA) as the primary antibody. The sections were counterstained with 10% haematoxylin, dehydrated and mounted.
GDNF immunohistochemistry
For antigen retrieval, slides were incubated in Tris-buffered saline (40 min at 98°C). After the endogenous peroxidase activity was blocked with 3% H2O2, non-specific binding was blocked by a 20 min incubation with 10% normal swine serum. The primary antibody was a goat polyclonal antibody generated against recombinant human GDNF (R&D Systems, USA). Sections were incubated overnight at 4°C with primary antibody at a dilution of 1:100. Antibody was detected by the streptavidinbiotinperoxidase method with diaminobenzidine by means of the Dako LSAB+ kit. The sections were counterstained with 10% haematoxylin, dehydrated and mounted. Negative controls were prepared with the use of normal goat IgG (Vector Laboratories) as the primary antibody. A positive control was prepared with human ileum including myenteric plexus. The myenteric plexuses of these control specimens were positive for GDNF immunostaining (Bar et al., 1997; Steinkamp et al., 2003
).
SCF immunohistochemistry
For antigen retrieval, the slides were heated by microwave for 5 min in 0.01 mol/l citrate buffer (pH 6). After endogenous peroxidase activity was blocked with 3% H2O2, non-specific binding was blocked by a 20 min incubation with normal swine serum. The primary antibody was a rabbit polyclonal antibody generated against SCF (SCF-89; IBL, Japan). Sections were incubated overnight at 4°C with the primary antibody at a dilution of 1:100. Antibody was detected by the streptavidinbiotinperoxidase method with diaminobenzidine by means of the Dako LSAB+ kit. The sections were counterstained with 10% haematoxylin, dehydrated and mounted. Negative controls were prepared with the use of normal rabbit IgG (DakoCytomation, Denmark) as the primary antibody. A positive control was prepared with human ovary including surface epithelium. The ovarian surface epithelium was positive for SCF immunostaining (Parrott et al., 2000).
Histomorphometric analysis
Quantitative microscopic analysis was performed with an Olympus BX50 microscope at x200 magnification by three independent investigators (K.F., A.T. and T.T.). Staining was considered positive if the cytoplasm of the cells appeared brown. Immunostaining was scored as intense (++), moderate (+) or negative () by visual inspection, and 100 seminiferous tubules were analysed from each section. Staining intensities for inhibin
subunit, GDNF and SCF were classified in terms of the percentage of stained seminiferous tubules of each section: group A (negative), <10% positively stained; group B > 10% positively stained and <10% intensely stained; group C, >10% intensely stained. The associations between these classifications and sperm retrieval rates was analysed statistically.
Statistical analysis
The 2-test for independence and Fisher's exact probability test were used for statistical comparisons. P<0.05 was considered statistically significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SCO is of unknown though probably various aetiologies (Campbell and Walsh, 2002) including hypogonadism, chemotherapy, and complete azoospermic factor (AZF) deletion (Kamp et al., 2001
). Sertoli cells secrete soluble factors and play important roles in spermatogenesis. Impairment of Sertoli cells is a possible cause of spermatogenic impairments including SCO.
Inhibin B consists of two subunits, and
B, linked by disulphide bridges. In the fetal human testis,
and
B subunits are localized to Sertoli and Leydig cells, whereas in the adult human testis, the inhibin
subunit is localized to Sertoli cells, and the
B subunit is localized to germ cells (Andersson et al., 1998
). Although serum levels of inhibin B are closely related to spermatogenesis (Pierik et al., 1998
) and are lower in patients with SCO (mean ± SEM, 41 ± 6 pg/ml) than that in normal men (mean ± SEM, 238 ± 32 pg/ml) (von Eckardstein et al., 1999
), Sertoli cells associated with SCO show more intense immunostaining for the inhibin
subunit than those from normal testis (Bergh and Cajander, 1990
; Marchetti et al., 2003
). Consistent with these reported findings, all of our 52 SCO-affected specimens, in which mean serum levels of inhibin B were 17.0 ± 23.4 pg/ml, showed uniformly intense staining of Sertoli cells regardless of sperm retrieval rates, and immunostaining for the inhibin
subunit was not related to sperm retrieval. It may be that in the SCO-affected testis, high FSH levels stimulate the production of the
subunit in Sertoli cells (Namiki et al., 1993
), but few germ cells produce the
B subunit. This may induce the accumulation of
subunits in Sertoli cells, with low or undetectable serum inhibin B levels.
GDNF, a distant member of the transforming growth factor-family, promotes the proliferation and differentiation of several types of neurons in the nervous system, regulates uteric branching in the embryonic kidney, enteric innervation, and promotes the proliferation of undifferentiated spermatogonia (Pichel et al., 1996a
,b
; Meng et al., 2000
). In mice with decreased GDNF expression, the testis shows disturbed spermatogenesis and subsequent change to tubules with Sertoli cells only and lack of spermatogonia (Meng et al., 2001
). Whereas GDNF is prominently expressed in embryonic mouse gonads, GDNF is not detectable in adult mouse testis (Golden et al., 1999
; Widenfalk et al., 2000
). Expression of GDNF in the human testis has not been reported. Our experiments showed no staining for GDNF in normal human adult testes. We did identify immunostaining for GDNF in SCO Sertoli cells; 55.8% of SCO specimens were immunohistochemically positive for GDNF, whereas 44.2% were negative (showed < 10% positive tubules). It is not clear whether GDNF expression is the cause or result of SCO, but it is possible that impaired regulation of GDNF expression induces SCO.
In SCF-deficient mice, spermatogenesis is arrested at the stage of undifferentiated type A spermatogonia (de Rooij et al., 1999). In the adult mouse testis, SCF produced by Sertoli cells promotes the proliferation of c-Kit-expressing differentiating spermatogonia (Koshimizu et al., 1991
). In addition, SCF is essential for the survival of type B spermatogonia before differentiation to meiotic stages and is important for the survival and development of meiotic spermatocytes (Vincent et al., 1998
). In the adult human testis, SCF is expressed in Sertoli cells, and the intensity of immunostaining for SCF increases in the SCO-affected testis (Sandlow et al., 1996
). In the current study, all specimens but one showed moderate or intense immunostaining for SCF.
GDNF promotes the proliferation of undifferentiated spermatogonia and SCF promotes the proliferation, and differentiation of spermatogonia to spermatocytes. GDNF and SCF are thought to play important roles in the recovery of these cells when they are impaired. The sperm retrieval rate was higher in SCF group C (intense immunostaining) than in SCF group A/B (no to moderate immunostaining) among GDNF group A samples (no staining). The absence of staining for GDNF suggests the existence of undifferentiated spermatogonia, and increased SCF may stimulate the proliferation of differentiated spermatogonia and the differentiation of spermatogonia to spermatocytes, resulting in successful sperm retrieval.
We have shown these results by means of immunohistochemistry, but there might be a concern about the validity of the antibodies. The myenteric plexus of intestine was positive for GDNF (Steinkamp et al., 2003) and the ovarian surface epithelium was positive for SCF (Parrott et al., 2000
). We have shown that the antibodies against GDNF or SCF were positive for these tissues. The validity of the antibodies used in our study have been also confirmed by western blotting (Hachiya et al., 2001; Del Fiacco et al., 2002; Pina Serra et al., 2002). It seems reasonable to assume that the validity of the antibodies against GDNF and SCF were confirmed.
Whereas TESE combined with ICSI is becoming the first-line treatment for NOA, the sperm retrieval rate in patients with SCO is only 6.313.0% with the use of conventional TESE (Okada et al., 2002; Tsujimura et al., 2002
). Many couples with SCO are subjected to unnecessary ovarian stimulation and risk of testicular damage (Tash and Schlegel, 2001
), and therefore predictive factors for successful sperm retrieval have been explored. It was recently reported that serum FSH and inhibin B levels are predictive factors for TESE success (Bohring et al., 2002
; Bailly et al., 2003
). However, neither high serum FSH levels nor very low serum inhibin B levels exclude the possibility of sperm retrieval (Jezek et al., 1998
; Foresta et al., 1999
). In our study, the sperm retrieval rate was high in cases with no staining for GDNF and intense staining for SCF. When simple TESE fails, azoospermic patients may opt for microdissection TESE, depending on results of immunohistochemical staining for GDNF and SCF.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Andersson AM, Petersen JH, Jorgensen N, Jensen TK and Skakkebaek NE (2004) Serum inhibin B and follicle-stimulating hormone levels as tools in the evaluation of infertile men: significance of adequate reference values from proven fertile men. J Clin Endocrinol Metab 89, 28732879.
Bailly M, Guthauser B, Bergere M, Wainer R, Lombroso R, Ville Y and Selva J (2003) Effects of low concentrations of inhibin B on the outcomes of testicular sperm extraction and intracytoplasmic sperm injection. Fertil Steril 79, 905908.[CrossRef][ISI][Medline]
Bar KJ, Facer P, Williams NS, Tam PK and Anand P (1997) Glial-derived neurotrophic factor in human adult and fetal intestine and in Hirschsprung's disease. Gastroenterology 112, 13811385.[ISI][Medline]
Bergh A and Cajander S (1990) Immunohistochemical localization of inhibin-alpha in the testes of normal men and in men with testicular disorders. Int J Androl 13, 463469.[ISI][Medline]
Bohring C, Schroeder-Printzen I, Weidner W and Krause W (2002) Serum levels of inhibin B and follicle-stimulating hormone may predict successful sperm retrieval in men with azoospermia who are undergoing testicular sperm extraction. Fertil Steril 78, 11951198.[CrossRef][ISI][Medline]
Campbell MF and Walsh PC (2002) Campbell's Urology, 8th edn. Saunders, Philadelphia, USA.
Del Fiacco M, Quartu M, Serra MP, Follesa P, Lai ML and Bachis A (2002) Topographical localization of glial cell line-derived neurotrophic factor in the human brain stem: an immunohistochemical study of prenatal, neonatal and adult brains. J Chem Neuroanat 23, 2948.[CrossRef][ISI][Medline]
de Rooij DG, Okabe M and Nishimune Y (1999) Arrest of spermatogonial differentiation in jsd/jsd, Sl17H/Sl17H, and cryptorchid mice. Biol Reprod 61, 842847.
Devroey P, Liu J, Nagy Z, Goossens A, Tournaye H, Camus M, Van Steirteghem A and Silber S (1995) Pregnancies after testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermia. Hum Reprod 10, 14571460.[Abstract]
Foresta C, Bettella A, Rossato M, La Sala G, De Paoli M and Plebani M (1999) Inhibin B plasma concentrations in oligozoospermic subjects before and after therapy with follicle stimulating hormone. Hum Reprod 14, 906912.
Golden JP, DeMaro JA, Osborne PA, Milbrandt J and Johnson EM, Jr (1999) Expression of neurturin, GDNF, and GDNF family-receptor mRNA in the developing and mature mouse. Exp Neurol 158, 504528.[CrossRef][ISI][Medline]
Hachiya A, Kobayashi A, Ohuchi A, Takema Y and Imokawa G (2001) The paracrine role of stem cell factor/c-kit signaling in the activation of human melanocytes in ultraviolet-B-induced pigmentation. J Invest Dermatol 116, 578586.[CrossRef][ISI][Medline]
Jezek D, Knuth UA and Schulze W (1998) Successful testicular sperm extraction (TESE) in spite of high serum follicle stimulating hormone and azoospermia: correlation between testicular morphology, TESE results, semen analysis and serum hormone values in 103 infertile men. Hum Reprod 13, 12301234.[Abstract]
Kamp C, Huellen K, Fernandes S, Sousa M, Schlegel PN, Mielnik A, Kleiman S, Yavetz H, Krause W, Kupker W et al. (2001) High deletion frequency of the complete AZFa sequence in men with Sertoli-cell-only syndrome. Mol Hum Reprod 7, 987994.
Koshimizu U, Sawada K, Tajima Y, Watanabe D and Nishimune Y (1991) White-spotting mutations affect the regenerative differentiation of testicular germ cells: demonstration by experimental cryptorchidism and its surgical reversal. Biol Reprod 45, 642648.
Marchetti C, Hamdane M, Mitchell V, Mayo K, Devisme L, Rigot JM, Beauvillain JC, Hermand E and Defossez A (2003) Immunolocalization of inhibin and activin alpha and betaB subunits and expression of corresponding messenger RNAs in the human adult testis. Biol Reprod 68, 230235.
Meng X, Lindahl M, Hyvonen ME, Parvinen M, de Rooij DG, Hess MW, Raatikainen-Ahokas A, Sainio K, Rauvala H, Lakso M et al. (2000) Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science 287, 14891493.
Meng X, Pata I, Pedrono E, Popsueva A, de Rooij DG, Janne M, Rauvala H and Sariola H (2001) Transient disruption of spermatogenesis by deregulated expression of neurturin in testis. Mol Cell Endocrinol 184, 3339.[CrossRef][ISI][Medline]
Namiki M, Kondoh N, Matsumiya K, Sakoda S, Nishimune Y and Okuyama A (1993) Hormonal regulation of human testicular inhibin alpha and beta B subunit messenger RNAS. J Urol 150, 10071009.[ISI][Medline]
Okada H, Dobashi M, Yamazaki T, Hara I, Fujisawa M, Arakawa S and Kamidono S (2002) Conventional versus microdissection testicular sperm extraction for nonobstructive azoospermia. J Urol 168, 10631067.[CrossRef][ISI][Medline]
Parrott JA, Kim G and Skinner MK (2000) Expression and action of kit ligand/stem cell factor in normal human and bovine ovarian surface epithelium and ovarian cancer. Biol Reprod 62, 16001609.
Pichel JG, Shen L, Sheng HZ, Granholm AC, Drago J, Grinberg A, Lee EJ, Huang SP, Saarma M, Hoffer BJ et al. (1996a) Defects in enteric innervation and kidney development in mice lacking GDNF. Nature 382, 7376.[CrossRef][ISI][Medline]
Pichel JG, Shen L, Sheng HZ, Granholm AC, Drago J, Grinberg A, Lee EJ, Huang SP, Saarma M, Hoffer BJ et al. (1996b) GDNF is required for kidney development and enteric innervation. Cold Spring Harbor Symp Quant Biol 61, 445457.[ISI][Medline]
Pierik FH, Vreeburg JT, Stijnen T, De Jong FH and Weber RF (1998) Serum inhibin B as a marker of spermatogenesis. J Clin Endocrinol Metab 83, 31103114.
Pina Serra M, Quartu M, Ambu R, Follesa P and Del Fiacco M (2002) Immunohistochemical localization of GDNF in the human hippocampal formation from prenatal life to adulthood. Brain Res 928, 138146.[CrossRef][ISI][Medline]
Rossi P, Albanesi C, Grimaldi P and Geremia R (1991) Expression of the mRNA for the ligand of c-kit in mouse Sertoli cells. Biochem Biophys Res Commun 176, 910914.[ISI][Medline]
Rothman CM, Sims CA and Stotts CL (1982) Sertoli cell only syndrome 1982. Fertil Steril 38, 388390.[ISI][Medline]
Sandlow JI, Feng HL, Cohen MB and Sandra A (1996) Expression of c-KIT and its ligand, stem cell factor, in normal and subfertile human testicular tissue. J Androl 17, 403408.
Schlegel PN (1999) Testicular sperm extraction: microdissection improves sperm yield with minimal tissue excision. Hum Reprod 14, 131135.
Steinkamp M, Geerling I, Seufferlein T, von Boyen G, Egger B, Grossmann J, Ludwig L, Adler G and Reinshagen M (2003) Glial-derived neurotrophic factor regulates apoptosis in colonic epithelial cells. Gastroenterology 124, 17481757.[CrossRef][ISI][Medline]
Tajima Y, Onoue H, Kitamura Y and Nishimune Y (1991) Biologically active kit ligand growth factor is produced by mouse Sertoli cells and is defective in SId mutant mice. Development 113, 10311035.[Abstract]
Tash JA and Schlegel PN (2001) Histologic effects of testicular sperm extraction on the testicle in men with nonobstructive azoospermia. Urology 57, 334337.[CrossRef][ISI][Medline]
Tsujimura A, Matsumiya K, Miyagawa Y, Tohda A, Miura H, Nishimura K, Koga M, Takeyama M, Fujioka H and Okuyama A (2002) Conventional multiple or microdissection testicular sperm extraction: a comparative study. Hum Reprod 17, 29242929.
Vincent S, Segretain D, Nishikawa S, Nishikawa SI, Sage J, Cuzin F and Rassoulzadegan M (1998) Stage-specific expression of the Kit receptor and its ligand (KL) during male gametogenesis in the mouse: a Kit-KL interaction critical for meiosis. Development 125, 45854593.
von Eckardstein S, Simoni M, Bergmann M, Weinbauer GF, Gassner P, Schepers AG and Nieschlag E (1999) Serum inhibin B in combination with serum follicle-stimulating hormone (FSH) is a more sensitive marker than serum FSH alone for impaired spermatogenesis in men, but cannot predict the presence of sperm in testicular tissue samples. J Clin Endocrinol Metab 84, 24962501.
Widenfalk J, Parvinen M, Lindqvist E and Olson L (2000) Neurturin, RET, GFRalpha-1 and GFRalpha-2, but not GFRalpha-3, mRNA are expressed in mice gonads. Cell Tissue Res 299, 409415.[ISI][Medline]
Submitted on September 8, 2004; resubmitted on March 7, 2005; accepted on March 29, 2005.
|