Testicular cAMP responsive element modulator (CREM) protein is ...

Molecular Human Reproduction vol.4 no.1 pp. 9–15, 1998

Testicular cAMP responsive element modulator (CREM) protein is expressed in round spermatids but is absent or reduced in men with round spermatid maturation arrest G.F.Weinbauer1, R.Behr1, M.Bergmann2 and E.Nieschlag1,3 1Institute

of Reproductive Medicine of the University, Domagkstrasse 11, D-48129 Mu¨nster, and 2Institute of Anatomy and Cell Biology, Martin-Luther-University, D-06097 Halle, Germany

3To

whom correspondence should be addressed

Mice lacking the functional cAMP responsive element modulator (CREM) gene, a component of cAMPmediated signal transduction, exhibit a specific arrest of round spermatid development although follicle stimulating hormone (FSH) and androgen secretion are not impaired. We studied testicular expression of CREM protein by immunocytochemistry in four patients with complete spermatogenesis (obstructive azoospermia), in 20 infertile patients with round spermatid maturation arrest (n J 10) or mixed atrophy (n J 10) and in six prostate cancer patients undergoing orchidectomy. Concentrations of testosterone were below normal in three patients. Concentrations of luteinizing hormone (LH) were lowered in two patients and elevated in one patient. FSH concentrations were above normal in ten patients. During normal spermatogenesis, CREM was expressed in nuclei of round spermatids in stages I–III of spermatogenesis but not in elongating spermatids. Western blot analysis of testes from prostate cancer patients indicated a major CREM band of ~35 kDa. Among patients with predominant round spermatid maturation arrest, CREM expression was significantly reduced (P < 0.05) or undetectable as revealed by quantitative image analysis. CREM-negative spermatids failed to progress beyond stage III of spermatogenesis. Our observations suggest a role for CREM in human spermatid development and raise the possibility that altered CREM expression could be associated with spermatid maturation defects in some cases of idiopathic male infertility. Key words: CREM/human/infertility/spermatogenesis/testis

Introduction It is estimated that ~15% of couples suffer from fertility problems during reproductive age, and that infertility is distributed evenly across sexes (Nieschlag, 1997). Therapy for male infertility caused by hypothalamic or pituitary failure is most advanced since the prime regulatory hormones of spermatogenesis, gonadotrophin-releasing hormone (GnRH) and gonadotrophins, are well characterized (Weinbauer and Nieschlag, 1996). In ~30% of infertile men, however, oligozoospermia or azoospermia are present, despite unimpaired reproductive hormone secretion and lack of other known determinants of infertility (Nieschlag and Leifke, 1997). These men are currently categorized as suffering from idiopathic infertility, reflecting the fact that the underlying pathophysiology for the testicular defect has remained elusive until now. It has been suggested that impairment of Sertoli cell function (Bergmann and Kliesch, 1994; Steger et al., 1996) and germ cell function contributes to low germ cell production in oligozoospermic men; in particular, generations of spermatocytes and spermatids are often missing in men with low daily sperm production (Johnson et al., 1992). Complete arrest of spermatocyte and/ or round spermatid development is frequent in azoospermic men and has been, hypothetically, related to a genetic origin (Martin-du-Pan and Campana, 1993). Microdeletions of the Y chromosome have also been implicated in idiopathic infertility (Simoni et al., 1997; Pryor et al., 1997) but this assumption still awaits final proof. © European Society for Human Reproduction and Embryology

Recent studies have identified cAMP responsive element modulator (CREM) as a key component of spermatogenic development in the mouse (Blendy et al., 1996; Nantel et al., 1996). CREM binds to cAMP response elements, modulates the transcription of cAMP responsive genes, and regulates gene expression in spermatids (Lalli et al., 1996; Monaco et al., 1996; Walker and Habener, 1996). Inactivation of the CREM gene in mice provoked a highly specific lesion, i.e. round spermatid formation occurred, but these spermatids failed to elongate, resulting in infertility. Of particular importance was the observation that reproductive hormone secretion was not impaired (Blendy et al., 1996). Hence CREM deficiency in the mouse model is associated with a pattern of spermatogenic dysfunction similar to that in normo- or hypergonadotrophic men with spermatogenic arrest that may be clinically diagnosed as idiopathic infertility. Therefore we analysed the expression of CREM protein during complete spermatogenesis and the relationship between CREM expression and spermatid maturation arrest.

Materials and methods Patients Patients were selected from the Institute’s clientele and had been subjected to complete andrological workup including general health assessment, hormone and semen analysis (Table I). The patients had given informed consent for such an investigation to be performed.

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G.F.Weinbauer et al.

Table I. Clinical data of patients with obstructive azoospermia, round spermatid maturation arrest and mixed atrophy Patient

Age (years)

LH (IU/l)

Obstructive azoospermia 1 34 5.5 2 35 3.9 3 36 6.2 4 37 4.9 Round spermatid maturation arrest 5 38 5.5 6 28 6.5 7 30 4.7 8 28 2.3 9 27 4.1 10 33 1.8 11 17 10.3 12 27 3.4 13 38 3.4 14 29 3.1 Mixed atrophy 15 37 2.6 16 35 3.1 17 28 3.9 18 33 1.5 19 46 3.8 20 29 6.7 21 35 6.7 22 36 2.7 23 35 8.8 24 33 3.6

Testosterone (nmol/l)

FSH (IU/l)

Sperm concentration (3 106/ml)

14.5 5.7 23.0 20.7

3.3 4.2 9.0 3.8

0 0 0 0

9.2 17.5 17.4 18.2 15.5 18.2 27.6 20.3 19.2 24.7

2.8 9.9 4.9 7.1 4.4 3.0 14.0 2.4 2.1 7.4

0.1 0.6 1.0 2.5 2.3 0.1 0.1 0.1 0.4 2.0

17.6 17.4 17.0 6.3 12.2 20.6 23.5 21.8 12.4 10.4

2.2 6.1 13.8 5.0 3.4 13.2 8.9 7.1 25.9 5.9

2.1 2.1 1.2 1.0 4.0 0.6 1.4 5.6 2.4 2.4

Normal ranges are 2–10 IU/l for luteinizing hormone (LH), .12 nmol/l for testosterone and 1–7 IU/l for follicle stimulating hormone (FSH).

Ten infertile patients (mean age, 29.8 years; range, 17–38 years) were included based on the histological diagnosis of predominant round spermatid maturation arrest and 10 patients (mean age, 34.4 years; range, 29–46 years) with histological diagnosis of mixed atrophy, i.e. the presence of tubules with complete spermatogenesis along with tubules with various spermatogenic defects and tubules with complete atrophy were also included. Tissue from four men (mean age, 35.3 years; range, 34–37 years) with obstructive azoospermia but complete spermatogenesis as seen from biopsy material, served as control. Biopsies were available from both testes and were processed as previously described (Bergmann et al., 1994): one part was placed in Bouin’s fixative, the other part in 5% glutaraldehyde and postfixed in osmium tetroxide. Testis tissue obtained during orchidectomy from six prostate carcinoma patients (mean age, 67.5 years; range, 62–73 years) was both frozen on dry ice for Western blot analysis and fixed in Bouin’s solution for immunocytochemical analysis.

Immunocytochemistry Bouin-fixed specimens were dehydrated, embedded in paraffin and sectioned at 5 µm. A rabbit polyclonal antibody raised against recombinant mouse CREMτ (Delmas et al., 1993; Molina et al., 1993) (Upstate Biotechnology Incorporated, Lake Placid, NY, USA) was used at 1:600– 1:1200 dilution. This antibody recognizes the CREM isoforms α, β, γ, τ1, τ2 with similar affinity and inducible cAMP early repressor (ICER) with lower affinity. Another rabbit polyclonal antiserum raised against full-length polyhistidine-tagged human CREM protein was used at 1:50–1:200 (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA). Biotinylated anti-rabbit immunoglobulin (Ig)G from swine (1:400; Dako, Hamburg, Germany), alkaline phosphatase conjugated to extravidin (1:200, Sigma, St Louis, MO, USA) and New-Fuchsin (Dako) were used for visualization of bound primary antibody. Non-specific binding

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was blocked with 5% normal swine serum. Sections were counterstained for 5 s with Mayer’s haematoxylin. Control stains included replacement of the antibodies with 1% (w/v) bovine serum albumin (BSA) and preincubation with the human full-length polyhistidinetagged human CREM protein overnight at 4°C. To control for reactivity of the specimens, immunolocalization of α-smooth muscle actin, a structural protein unrelated to CREM, using a mouse monoclonal antibody (Schlatt et al., 1993) was also performed. As a further control for the specificity of the CREM antibodies, testicular sections from genetargetted mice lacking the CREM nuclear import signal (Blendy et al., 1996) were included in the analysis.

Histological analysis For immunocytochemical analysis, between six and nine sections from each biopsy specimen and one section from prostate cancer patient testis were evaluated. Spermatogenic stages were classified as described by Clermont (1963) from haematoxylin–eosin stained sections. Glutaraldehyde-fixed and osmium-tetroxide postfixed material, embedded in Epon 812, sectioned at 1 µm, and stained with Toluidine Blue (four or five sections per biopsy specimen) was used in the analysis of the spermatogenic status and this material was reanalysed for defining the most advanced stage of spermatid maturation based on the criteria described by Holstein and Roosen-Runge (1981). Quantitative analysis of CREM staining An image analysis system (KS400; Zeiss, Oberkochen, Germany) was used to quantify the staining of CREM on those sections used for immunocytochemical analysis. Interactive mode threshold setting was used for discrimination of CREM-positive spermatids from unstained cells based on the staining patterns seen in sections from the control group (obstructive azoospermia). The optimized sequence of com-

CREM and human spermatogenesis

Figure 1. Micrographs of human testicular tissue. Sections are counterstained with haematoxylin. (a) Obstructive azoospermia and complete spermatogenesis. Round spermatids in stages I–III of spermatogenesis are positive for cAMP responsive element modulator (CREM) (exemplified by large arrows). Elongating spermatids are CREM-negative (exemplified by bent arrows). (b) Mixed atrophy. Round spermatids contain CREM (large arrows) in the presence of elongating spermatids (bent arrows) in the seminiferous tubule on the left but are negative in the neighbouring tubule lacking spermatid elongation (single large arrow). (c) Complete spermatogenesis and localization of CREM in round spermatid nuclei (exemplified by large arrows). (d) Section from the same specimen probed with antibody preincubated with human recombinant CREM protein. CREM signal is absent. Original magnification 3625.

mands and settings was applied to all sections (again including those from the control group) at a defined microscope light intensity and all sections were analysed in one session. A preset measurement frame and objective 1:25 were used. Biopsy sections were completely scanned. Testis sections from prostate cancer patients were scanned using random stage movement until ù1000 CREM-positive cells had been analysed per each section. For each cell identified, i.e. surpassing the preset intensity threshold level, the integrated signal across the entire cell was determined automatically along a grey scale by an inbuilt densitomety algorithm. The median value for all measurements from one patient was used for statistical analysis. Our system is not equipped with calibration software. Hence, the values obtained are relative and expressed in arbitrary units of the equipment-inherent grey scale.

Western blotting Testis tissue from prostate cancer patients was homogenized in RIPAbuffer (Santa Cruz Biotechnology Inc.) and protein concentration was assessed by bicinchoninic assay method. A total of 400 µg protein of testicular extract were loaded onto 10% polyacrylamide gel electrophoresis (PAGE) and blotted onto nitrocellulose membrane. The blot was probed with CREM antibody at a dilution of 1:500, second antibody was used at 1:3000 and enhanced chemiluminescence for visualization. Unspecific binding was blocked with 0.5% Tween-20 and 5% non-fat milk powder. Full length polyhistidine-tagged human CREM protein

(Santa Cruz Biotechnology) was used as a positive control and for preincubation.

Hormone measurements and semen analysis Serum concentrations of luteinizing hormone (LH) and follicle stimulating hormone (FSH) were determined by an immunofluorometric assay (DELFIA; ADL Wallac, Freiburg, Germany). Concentrations of serum testosterone were measured by radioimmunoassay as previously described (Chandolia et al., 1991). Semen analysis was conducted in accordance with the guidelines provided by World Health Organization (WHO, 1992). Statistical analysis Sperm numbers between patients with round spermatid maturation arrest and mixed atrophy were compared by Mann–Whitney U-test. Data generated by image analysis were compared by Kruskal–Wallis’ non-parametric analysis of variance followed by Dunn’s test. P , 0.05 was considered to be significant.

Results Histological and immunocytochemical findings Comparable results were obtained with both antibodies. Testicular biopsies from patients with obstructive azoospermia 11


Figure 2. Micrographs of human testicular tissue. Sections are counterstained with haematoxylin. (a) Obstructive azoospermia and complete spermatogenesis. cAMP responsive element modulator (CREM) is expressed in nuclei of round spermatids (exemplified by large arrows). Elongating spermatid nuclei are CREM-negative (exemplified by bent arrows). (b) CREM antibody was replaced by buffer containing 1% bovine serum albumin (BSA) and CREM staining was abolished. (c) Round spermatid maturation arrest. Two seminiferous tubules are sectioned containing numerous round spermatids (exemplified by large arrows) but these spermatids lack CREM. (d) Same specimen as in (c), localization of α-smooth muscle actin in the wall of seminiferous tubules (large arrows). (e) Predominant spermatid maturation arrest. CREM protein is expressed in some round spermatid nuclei (exemplified by large arrows). Few elongating spermatids (exemplified by bent arrows) are also present in the tubules containing CREM-positive round spermatids. (f) Prostate carcinoma patient. CREM signal is seen in nuclei of round spermatids (exemplified by large arrows) whereas elongating spermatid nuclei are negative (exemplified by bent arrows). Original magnification 3625.

contained all stages of germ cell development, including the formation of testicular spermatozoa. In these men, CREM protein was strongly expressed in the nuclei of round spermatids (Figures 1a, 2a). This pattern of CREM expression was also seen in sections from prostate carcinoma patients with complete spermatogenesis (Figure 2f). Stage-related analysis revealed that the expression of CREM was confined to round spermatids 12

in stages I–III of spermatogenesis (Figures 1a, 1c, 2a, 2f). A faint signal was also seen in pachytene spermatocytes and Sertoli cells but not consistently. The CREM signal vanished following preincubation of antibody with human CREM protein (compare Figures 1c and d) and after replacement of the primary antibody by BSA (Figure 2b). In CREM-deficient mice lacking the nuclear import signal for CREM, no CREM

CREM and human spermatogenesis

Quantification of CREM staining The average staining intensity was similar between the control group and prostate cancer patients (Figure 3). Among patients with round spermatid maturation arrest, CREM signal was markedly reduced. In four of these men, no signal could be detected by the image analysis system and in the remaining six, computed values for CREM staining density ranged from 800–3731 compared with ù10000. In patients with mixed atrophy, CREM signals were reduced by ~50% but this effect did not attain statistical significance (P . 0.05). Figure 3. Quantification of cAMP responsive element modulator (CREM) signal in round spermatid nuclei from immunocytochemical sections by image analysis as described in detail in the method section. OAZ 5 obstructive azoospermia with complete spermatogenesis (n 5 4); SMA 5 round spermatid maturation arrest (n 5 10), MAT 5 mixed atrophy (n 5 10); PRC 5 specimens from prostate cancer patients with complete spermatogenesis (n 5 6). Values shown are means 6 SEM. *Significantly different when compared with OAZ group (P , 0.05).

Western blotting Western blotting of human testicular homogenates revealed a major band with an apparent molecular weight of 35 kDa. Preincubation of the antibody with human CREM protein abolished these bands (Figure 4). Hormonal and seminal parameters FSH concentrations were either within the normal range or elevated in ten patients (one with obstructive azoospermia, four with round spermatid maturation arrest, and five with mixed atrophy) (Table I). Subnormal concentrations were only seen for testosterone (three cases) and LH (two cases) and elevated LH concentrations were found in one patient. Ejaculates from patients with obstruction of the excurrent ducts were devoid of spermatozoa. Sperm concentrations in men with spermatid maturation arrest were extremely low in the range 0.1–2.53106/ml ejaculate (median value: 0.53106/ml). Among men with mixed atrophy, sperm numbers were 0.6–5.63106/ml ejaculate (median value: 2.13106/ml ejaculate, P , 0.05).

Discussion

Figure 4. Western blot from testicular tissue obtained from prostate carcinoma patients during orchidectomy. In all, 400 µg testicular protein was loaded. Lanes 1–3 and 6–8: testis extracts; lane 4: 0.5 µg cAMP responsive element modulator (CREM) full-length human protein with polyhistidine tag; lane 5: molecular weight marker. Blot A was probed with CREM antibody and blot B was probed with antibody preincubated with human recombinant protein.

signal was seen in the nuclei (not shown). α-smooth muscle actin expression was seen in all specimens and was localized to the cytoplasm of peritubular cells and cells surrounding the testicular blood vessels (Figure 2d). Among patients diagnosed with spermatid maturation arrest, round spermatid development was halted at stage III of spermatogenesis in 30–100% of seminiferous tubules (median value: 85%). This finding was confirmed from semithin sections. In such tubules, CREM expression was undetectable (Figures 1b and 2c). In the presence of elongated spermatids, however, CREM expression was detectable either in specimens with predominant round spermatid maturation arrest (Figure 2e) or with mixed atrophy (Figure 1b).

We investigated testicular expression of CREM in testis specimens with complete spermatogenesis and with various abnormalities of the spermatogenic process including round spermatid maturation arrest. During complete spermatogenesis, CREM was expressed in the nuclei of round spermatids in spermatogenic stages I–III. The signal vanished when spermatids started to elongate. The observed staining pattern for CREM in round spermatids is comparable, in a qualitative manner, to that reported for mice (Delmas et al., 1993) and rats (Walker and Habener, 1996). Such similarity of germ cellspecific CREM expression across species suggests a general relevance of this transcription factor for male gametogenesis and, in fact, a substantial degree of homology between human and mouse CREM genes has been shown (Masquilier et al., 1993). The continuing expression of CREM in round spermatids present until initiation of their elongation in the human testis could indicate that CREM is involved in initiating the elongation process in these spermatids. The arrest of round spermatid development in CREM-deficient mice (Blendy et al., 1996; Nantel et al., 1996) supports this view. Specific detection of human CREM by the CREM antibodies in the present study is evident from several findings: The observation that the nuclear CREM signal was absent in CREM-targetted mice demonstrates that this antibody does 13


not recognize the CREM-related cAMP responsive elementbinding (CREB) protein which is also expressed in round spermatid nuclei (Waeber et al., 1991) and in testis of CREMdeficient mice (Nantel et al., 1996). Preincubation of the antibodies with human CREM protein eliminated the CREM signals on sections and blots thus providing evidence that the antibodies used indeed recognize human CREM. Alternative exon splicing and alternative starting sites for translation result in CREM isoforms with either activating (τα, τ, τ1 and τ2) or repressing activity (α, β, γ, S-CREM) (Laoide et al., 1993; Delmas et al., 1993). Interestingly, CREM mRNA repressor isoforms are expressed at low levels in premeiotic cells (Foulkes et al., 1992), whereas during meiosis the CREMτ activator transcript is abundantly expressed and stabilized under the influence of FSH (Foulkes et al., 1993). In the postmeiotic haploid germ cells only activator forms of CREM are expressed (Delmas et al., 1993; Monaco et al., 1996). Hence, it is reasonable to assume that the CREM protein observed in human round spermatids represents a CREM activator isoform. The data obtained from Western blot analysis indicate a major band of 35 kDa detected by the same antibodies and preincubation with human CREM abolished this band. A faint CREM signal was also observed in pachytene spermatocytes and in Sertoli cells, although not consistently. Since the antibody used in these specimens also cross-reacts with all known isoforms of CREM, it is conceivable that these signals in spermatocytes represent CREM repressors. The observed signal in Sertoli cells could reflect the expression of the inducible cAMP early repressor (Molina et al., 1993). This isoform of CREM is present in Sertoli cells and has been claimed to influence the expression of the follicle-stimulating receptor expression in the rat (Monaco et al., 1995). In seminiferous tubules with maturational arrest at the level of round spermatids, CREM expression was absent or signficantly reduced. Analysis of the developmental stage of spermatids with altered CREM expression revealed that these spermatids failed to develop beyond stage III of spermatogenesis, i.e. the spermatid elongation process did not occur. In seminiferous tubules containing elongated spermatids, CREM expression was consistently detectable. A comparable lesion has been identified in the CREM-mutant mice in which spermatid elongation was also absent (Blendy et al., 1996; Nantel et al., 1996). These observations also suggest an assocation between spermatid maturation and CREM expression in the human testis. Whether altered CREM expression is a symptom of spermatid maturation arrest or is causally involved in the pathogenesis, thus providing a potential therapeutic clue, remains to be elucidated. α-smooth muscle actin was localized to the cytoplasm of peritubular cells and cells surrounding the testicular blood vessels and was expressed in all testicular specimens, thus eliminating the possibility that the lack or diminution of CREM expression was due to an intrinsic problem of specimen preservation and processing. It is extremely unlikely that the lack of detectable CREM protein in some patients was caused by FSH deficiency since FSH secretion was not impaired and FSH bioactivity was found to be maintained in these category of patients (Simoni and Nieschlag, 1995). FSH and testosterone 14

are potent stimulators of spermatogenesis in men (Kliesch et al., 1994) and in mice (Singh and Handelsman, 1996) but these vital reproductive hormones are apparently unable to uphold spermatid formation in CREM-deficient mice (Blendy et al., 1996). In that respect, absent or reduced CREM protein expression would provide an explanation why spermiogenesis did not proceed in these patients although they were endowed with the necessary reproductive hormones. In summary, the spermatid-related nuclear CREM signal in the human testis and the similarity of spermatogenic lesions in mice lacking a functional CREM gene and patients with altered testicular CREM expression indicate that CREM protein is relevant for spermatid maturation in the human. These findings further suggest that CREM might constitute a factor associated with a specific spermatid maturation defect in idiopathic male infertility.

Acknowledgements This work was supported by the Deutsche Forschungsgmeinschaft. The expert technical assistance of Agnes Ro¨sner, Heidrun Mrusek and Reinhild Sandowe is gratefully acknowledged. Testes from prostate carcinoma patients were kindly provided by Dr H.Schulze, Urology Clinic of the University at Bochum, Marienhospital Herne, Germany. This work is part of a doctoral thesis by R.Behr and we acknowledge the support of Prof. Dr. Clemen (Institute of Special Zoology and Comparative Embryology of the University Mu¨nster, Germany).

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