Cyclin A1 and gametogenesis in fertile and infertile patients: a potential new molecular diagnostic marker

Mark Schrader1,5, Carsten Müller-Tidow2, Stuart Ravnik3, Markus Müller1, Wolfgang Schulze4, Sven Diederichs2, Hubert Serve2 and Kurt Miller1

1 Department of Urology, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Berlin, Germany, 2 Department of Medicine, Hematology/Oncology, University of Münster, Münster, Germany, 3 Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas, USA and 4 Department of Andrology, University of Hamburg, Hamburg, Germany


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The study aim was to evaluate cyclin A1 mRNA expression levels as a potential molecular diagnostic parameter in the work-up of testicular tissue from fertile versus infertile patients. METHODS: Cyclin A1 expression was quantified in 55 cryopreserved testicular tissue specimens by fluorescence real-time RT–PCR. A conventional histological work-up was performed concomitantly in all tissue specimens with additional semi-thin sectioning in all cases of non-obstructive azoospermia (n = 12), maturation arrest (n = 17) and Sertoli cell-only syndrome (SCOS; n = 9). RESULTS: The mean (± SD) normalized cyclin A1 expression (NCyclinA1) was 3.82 ± 2.23 relative gene expression (RGE) in tissue specimens with normal spermatogenesis, and 0.625 ± 0.221 RGE in those with maturation arrest at the level of early spermatids. Only minimal NCyclinA1 was detected in tissue specimens with spermatogonia only or maturation arrest at the level of primary spermatocytes (0.005 ± 0.008). Cyclin A1 expression was absent in the majority of SCOS specimens (0.002 ± 0.002). CONCLUSIONS: These investigations suggested that cyclin A1 expression is altered in cases of spermatogenic disorders. Moreover, the level of cyclin A1 mRNA expression correlates with gametogenic disorders and seems well suited for a molecular-diagnostic classification supplementing the histopathological evaluation of spermatogenic disorders.

Key words: cyclin A1/cyclin-dependent kinase/male fertility/spermatogenesis


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Human spermatogenesis can be pathologically reduced or stopped at any stage, the result being andrologically determined infertility, for which IVF and ICSI might provide useful therapeutic options (Van Steirteghem, 2001Go). Since 1993, the treatment of choice in patients with non-obstructive azoospermia (NOA) has been testicular sperm extraction with subsequent assisted fertilization by microinjection of gametes into the cytoplasm of oocytes (Schoysman et al., 1993Go). One of the requirements for microinjection is the presence of a minimum number of mature testicular spermatids. Micromanipulation-assisted fertilization with round spermatids may be effective in some cases of post-meiotic arrest (Tesarik et al., 1996Go). Pregnancies have also been reported in single cases after fertilization with secondary spermatocytes (Sofikitis et al., 1998Go). Moreover, it was also demonstrated in 1999 that rapid transmeiotic and post-meiotic differentiation during in-vitro culture with subsequent intra-oocyte injection is a treatment option in patients with pre-meiotic spermatogenesis arrest (primary spermatocyte stage) (Tesarik et al., 1999Go). However, the two last-mentioned treatment options cannot be regarded as clinically established methods at present. Indeed, their success has not been confirmed by other laboratories, and there were even negative results for round spermatids (Urman et al., 2002Go). Pregnancy rates are extremely unsatisfactory for patients with spermatogenic arrest despite the current above-mentioned treatment modalities.

Knowledge regarding the causes of spermatogenesis disorders thus appears to be of decisive importance for improving current treatment approaches and establishing new approaches in patients with spermatogenic arrest.

Spermatogenesis is a highly complex process comprising mitotic proliferation, meiosis and the transformation of early spermatids into spermatozoa. Cyclins and cyclin-dependent kinases (Cdk) are important partners in the regulation of mitosis and meiosis (Wolgemuth et al., 1995Go, 2002Go). These serine/threonine protein kinases are complexes composed of a regulatory and a catalytic subunit designated as cyclin and cyclin-dependent kinases respectively (Pines, 1993Go; Solomon, 1993Go). Cdk activation is regulated by cyclin binding and post-translational modifications (Doree and Galas, 1994Go; Sherr, 1994Go). Nine classes of cyclins and at least 10 Cdk family members have been identified in vertebrates (Yang and Kornbluth, 1999Go; Ekholm and Reed, 2000Go).

The role of the cyclin/Cdk complex has been extensively studied in mitotic cells (Ekholm and Reed, 2000Go) and only recently has serious attention been paid to cells undergoing meiosis. Cyclin A1 is a meiosis-specific cyclin that binds Cdk2 and is required for meiosis in mice (Liu et al., 1998Go) and also in humans (Wolgemuth et al., 2002Go), and is highly expressed in spermatogenesis in both mice (Sweeney et al., 1996Go) and humans (Muller et al., 2000Go; Wolgemuth et al., 2002Go). It is also active with Cdk2 in both murine (Sweeney et al., 1996Go) and human cells (Yang et al., 1997Go; Wolgemuth et al., 2002Go). Cyclin A1/Cdk2 activity is probably partly regulated during meiosis by the cyclin H/Cdk7 kinase (Kim et al., 2001Go), a physiologically relevant activator of Cdks in general. Cyclin A1 is readily detectable in other cells only under pathological conditions, as in the case of leukaemia in humans (Muller et al., 2000Go) or murine overexpression leading to leukaemia (Liao et al., 2001Go). In contrast to cyclin A1, which is normally associated only with meiosis (Wolgemuth et al., 2002Go), as described above, the related cyclin A2 is ubiquitously expressed in adult tissues (Ravnik and Wolgemuth, 1996Go; Sweeney et al., 1996Go) but is not present at meiotic stages in the testis (Ravnik and Wolgemuth, 1999Go).

As mentioned earlier, cyclin A1 is required for normal spermatogenesis and meiosis in mice. The importance of cyclin A1 for a normal course of spermatogenesis was demonstrated in a knock-out mouse model (Liu et al., 1998Go). These authors showed by gene targeting that cyclin A1 (Ccna1)-/- mutants (Ccna1-/- mice will be designated as cyclin A1-/- in the text) were sterile due to a block of spermatogenesis before the first meiotic division. Meiotic arrest in cyclin A1-/- mice was associated with increased germ cell apoptosis and reduced Cdc2 kinase activation at the end of the meiotic prophase. In addition, the similarity between murine and human cyclin A1 expression has been confirmed using transgenic models (Muller et al., 2000Go).

Given the requirement of cyclin A1 for normal meiosis and the similarity between humans and mice, the first attempts to quantify cyclin A1 mRNA expression by fluorescence real-time RT–PCR in human testicular tissue from patients with infertility of varying aetiology are presented. The aim of this study was to examine whether abnormal levels of cyclin A1 expression are detectable in human testicular biopsies with spermatogenesis arrest.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients
Institutional Review Board approval was obtained for this study. All patients signed a consent form approved by the Committee on Human Rights in Research of the Free University of Berlin. In this prospective study, 55 testicular biopsies were taken from patients presenting with azoospermia-related infertility between May 1999 and December 2000. Most of these patients presented with NOA, the criteria of diagnosis for which were described previously (Jezek et al., 1998Go; Schulze et al., 1999Go). Briefly, the following criteria were applied for diagnosing NOA: (i) histological work-up disclosed tubules with a severe spermatogenesis disorder; (ii) history yielded no evidence of any operation, aplasia or inflammation leading to occlusion of the seminal ducts; and (iii) clinical and ultrasound examinations yielded no indication of an obstruction, atrophy or aplasia of the seminal ducts as the cause of the azoospermia, and two semen analyses were prepared according to World Health Organization guidelines (World Health Organization, 1993Go).

A testicular biopsy was performed in all cases (n = 55). A small incision was made in the tunica albuginea to remove tissue samples which comprised in total about 3 mm3.

Controls included 17 cases of azoospermia due to vasectomy for family planning. These patients later sought parenthood and thus underwent a vasovasostomy with cryopreservation of a biopsy specimen from one testicle in case the intervention failed. These specimens were placed at our disposal in conjunction with spermatocyte detection in the patients’ ejaculate.

Processing of testicular biopsy material
Within the framework of the study setting, tissue samples were divided after their removal. The largest part of the specimens was reserved for a possible ICSI attempt as well as for the histological work-up, while a small part was retained for the molecular-diagnostic examination. The tissue samples were subdivided into seven fractions and processed as follows.

The largest portion (fractions 1–3) was immediately placed in 1.0 ml of Sperm-Freeze solution (Medicult, Hamburg, Germany) and transferred to liquid nitrogen by a computer-guided system (Planer 10, Messer-Griesheim, Griesheim, Germany). Fraction 4 of the testicular tissue from each patient was placed in a Petri dish containing Sperm-Prep solution (Medicult) and examined within 10 min. Minced tissue was examined by phase-contrast microscopy at x400 magnification to detect cells of spermatogenesis, especially mature spermatids. Tissue samples found to contain mature spermatids were immediately cryopreserved, while those with negative findings were treated with collagenase type I (Sigma, Heidelberg, Germany) following a modified form of the protocol (Schulze and Knuth, 1998). This procedure was applied to achieve a slight optimization of sample assessment. The samples found to contain germ cells were likewise cryopreserved to ensure the availability of as much material as possible for future ICSI treatments. Wet preparation assessment performed in accordance with previous studies (Jezek et al., 1998Go) was taken into consideration for histological classification of the samples.

In fraction 5 of the sample, cyclin A1 expression was quantitatively determined by fluorescence real-time RT–PCR. The part of the biopsy material intended for this was shock-frozen immediately after removal (3–5 min) and then stored in liquid nitrogen.

Another part of the sample (fraction 6) was placed in Stieve’s solution [formaldehyde diaminobenzidine (DAB) 10 20.0 g, acetic acid 100% DAB 10 4.0 g, aqueous saturated 7% mercuric (II) chloride solution 76.0 g), paraffin-embedded and prepared in 5 µm slices. The slices were stained with haematoxylin and eosin (HE). The biopsy material was histologically evaluated according to a modified Johnsen score (Table IGo) (Johnsen, 1970Go; DeKretser and Holstein, 1976Go; Holstein and Breucker, 1994).


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Table I. Johnsen score of testicular biopsies modified according to Holstein and DeKretser (Johnsen, 1970Go; Holstein et al., 1971Go; DeKretser and Holstein, 1976Go)
 
In addition to assessment of the slices with HE staining and the wet preparation, tissue samples (fraction 7) were also prepared using the semi-thin sectioning technique (Holstein and Breucker, 1994). This procedure was performed in all samples with NOA, spermatogenic arrest and Sertoli cell-only syndrome (SCOS).

All tissue samples were examined by a single experienced investigator (W.S.).

RNA extraction
Total RNA was extracted using RNAzolBTM (WAK-Chemie Medical, Bad Homburg, Germany) according to the manufacturer’s instructions, and its quality assessed as described previously (Schrader et al., 2000Go). RNA was treated with DNase (Amersham Pharmacia Biotec, Freiburg, Germany); RNA yield was quantified using UV spectrophotometry. A portion (1 µg) of total RNA was subjected to 1% agarose gel electrophoresis. Preservation of 28S and 18S rRNA species was used to assess RNA integrity; samples without detection of 28S/18S RNA were excluded from further examination.

Quantitation of cyclin A1 gene expression by real-time quantitative RT–PCR
The quantitation of cyclin A1 mRNA expression levels was carried out using a real-time fluorescence detection method (Gibson et al., 1996Go; Heid et al., 1996Go). The cDNA was prepared and amplified by PCR in the ABI prism 7700 sequence detector (PE Biosystems, Foster City, CA, USA) as described previously (Muller et al., 2000Go). The cyclin A1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers and probes have been described elsewhere (Muller et al., 2000Go). Primer and probe combinations were positioned to span an exon–exon junction. When genomic DNA was used as a template, no bands were seen after PCR amplification. The probes were labelled at the 5' end with VIC (GAPDH probe) or FAM (cyclin A1), and at the 3' end with TAMRA, which served as a quencher. The 5'->3' nuclease activity of the Taq polymerase cleaved the probe and released the fluorescent dyes (VIC or FAM), which were detected by the laser detector of the sequence detector (Livak et al., 1995Go). When the detection threshold had been reached, the fluorescence signal was proportional to the amount of PCR product generated. The initial template concentration was calculated from the cycle number when the amount of PCR product passed a threshold set in the exponential phase of the PCR reaction. Relative gene expression (RGE) levels were calculated using standard curves generated by serial dilutions of U 937 leukaemia cell line cDNA. The relative amounts of gene expression were normalized (N) by using GAPDH expression as an internal standard as described previously (Muller et al., 2000Go). At least two independent analyses were performed for each sample. Figure 1Go illustrates a measuring step in gene quantification; inter-assay variability was 1.1–3.7%. Numerous other studies have pointed out that real-time fluorescence RT–PCR has the advantage of being significantly less variable than conventional RT–PCR procedures (Bustin, 2000Go). The coefficient of variation for Ct data was also shown to be 0.4–0.8% for real-time fluorescence RT–PCR, which is significantly better than values of 8–14% reported for conventional RT–PCR (Wittwer et al., 1997Go; Zhang and Byrne, 1997Go; Zhang et al., 1997Go).



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Figure 1. Detection of cyclin A1 in testicular tissue samples by real-time on-line RT–PCR analysis using the Taqman®. Concomitant detection of glyceraldehyde-3-phosphate dehydrogenase mRNA served as a reference for relative quantification. Relative gene expression levels were calculated using standard curves generated by serial dilutions of cyclin-A1-positive ML-1 leukaemia cell line cDNA ({blacksquare}) as described previously (Muller et al., 2000Go). Representative tissue specimens with JS 7–8 ({square}) 1.200 normalized RGE of cyclin A1.

 
Statistical analysis
Statistical analysis was performed using the Kruskal–Wallis test for non-parametric analysis of variance to compare the histological subgroups. A discriminant analysis of NCyclinA1 was also performed for the different histological subgroups. Values were expressed as follows: mean, SD, median, 25th percentile, 75th percentile and range. Statistical analysis was performed using SPSS Software Version 10.0.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Histopathology of the tissue samples
The histopathological examination of testicular biopsy material revealed SCOS in nine cases. In 17 other cases, biopsy findings showed partial tubular atrophy with maturation arrest (MA). These included four samples with spermatogonia only (Johnsen score 3; see Table IGo), five with spermatogenic arrest at the level of primary spermatocytes, corresponding to a Johnsen score of 4, and eight samples with a mixed picture of spermatogenic arrest at the level of secondary spermatocytes and early spermatids (Johnsen score 5–7).

Biopsy results in the remaining 12 cases showed the presence of mature spermatids (testicular spermatozoa) (Johnsen score >=8–10).

Moreover, all tissue samples had detection of full spermatogenesis in the control group of patients with proven fertility, in whom biopsies had been taken during reanastomosis (n = 17).

Testicular biopsies
Patients with full spermatogenesis and proven fertility (control group)
Tissue samples in these cases were characterized by high expression, with a mean (±SD) NCyclin A1 of 3.822 ± 2.232 relative gene expression (RGE) (range 1.412–7.341). The normalized RGE in the different subgroups are summarized in Table IIGo.


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Table II. Quantification of cyclin A1 mRNA in testicular biopsies by real-time fluorescence RT–PCR
 
Patients with normal spermatogenesis
Testicular biopsy yielded histological evidence of full spermatogenesis in 12 patients from the group with azoospermia of unclear origin. Tissue samples in all these cases were characterized by high expression, with a mean NCyclin A1 of 3.519 ± 3.141 RGE (range 0.935– 9.014). Eleven of 12 tissue samples with evidence of full spermatogenesis had an NCyclin A1 expression of >1.00 RGE. One tissue sample with only focally full spermatogenesis had an NCyclin A1 expression of 0.94 RGE, which was in the range of samples with maturation arrest.

Two specimens with an NCyclin A1 expression of 1.323 and 1.273 showed maturation arrest at the conventional histological work-up. Subsequent assessment by semi-thin sectioning disclosed focal islands with full spermatogenesis in both of these specimens.

Patients with maturation arrest
Seventeen testicular biopsies revealed partial tubular atrophy with maturation arrest. Among these were eight tissue samples in which semi-thin sectioning provided histological evidence of spermatogenesis arrest at the secondary spermatocyte and early spermatid level. In these cases, tissue samples showed a mean NCyclin A1 RGE of 0.625 ± 0.221 (range 0.381–0.967).

Nine tissue samples showed evidence of spermatogenesis arrest at the level of the spermatogonia (n = 4) and primary spermatocytes (n = 5). In these cases, tissue samples had a mean NCyclin A1 RGE of 0.005 ± 0.008 (range 0.000–0.027).

Cyclin A1 expression (NCyclin A1 range 0.617–0.438 RGE) was detected in three tissue specimens even though they showed SCOS at the histological work-up. The subsequent work-up by semi-thin sectioning revealed spermatocytes with minimal foci of early spermatids in one case (NCyclin A1 = 0.438 RGE) and maturation arrest at the level of early spermatids in two cases (NCyclin A1 = 0.555 and 0.617 RGE).

Patients with SCOS
Histology revealed germ cell aplasia in 12 patients. Tissue samples with SCOS showed only minimal NCyclin A1 expression (NCyclin A1 = 0.002 ± 0.002 RGE; range 0.000–0.006).

Discriminant analysis
The Kruskal–Wallis test showed significant differences between the NCyclin A1 copies expressed in the histological subgroups (P < 0.001). Discriminant analysis revealed that NCyclin A1 had a high predictive value for correct classification in the subgroup of tissue specimens with maturation arrest at the level of primary spermatocytes or spermatogonia versus maturation arrest at the level of secondary spermatocytes/early spermatids or full spermatogenesis.

The detection technique had a specificity of 100% [95% confidence interval (CI) 81.5–100%] and a sensitivity of 100% (95% CI 91.2–100%) with a selected cut-off value of NCyclin A1 0.08 RGE for differentiating tissue specimens in the subgroup with maturation arrest at the level of primary spermatocytes or spermatogonia versus maturation arrest at the level of secondary spermatocytes/early spermatids or full spermatogenesis.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Several recent reports have indicated that spermatogenesis is regulated to a large extent by the serine/threonine protein kinases complexes, which are composed of a regulatory subunit, cyclin, and the catalytic subunit designated as cyclin-dependent kinase (Cdk) (Liu et al., 1998Go; Ravnik and Wolgemuth, 1999Go; Liu et al., 2000Go).

The A-type cyclins appear to be particularly important in the control of spermatogenesis, and previous studies have suggested that a decisive role is played here by cyclin A1 (Sweeney et al., 1996Go; Liu et al., 1998Go; Ravnik and Wolgemuth, 1999Go; Liu et al., 2000Go; Wolgemuth et al., 2002Go). Cyclin A1 is expressed at highest levels in meiotic prophase germ cells (Sweeney et al., 1996Go) and at lower levels in certain leukaemic cells (Yang et al., 1997Go).

Gene targeting demonstrated a critical function for cyclin A1 in the progression of meiosis (Liu et al., 1998Go). These authors also established cyclin A1-/- mice that showed spermatogenesis arrest before the first meiotic division. Arrest of meiosis in the cyclin A1-/- mice was associated with increased germ cell apoptosis, synaptic abnormalities and reduced Cdc2 kinase activation at the end of the meiotic prophase. The cyclin A1-/- mice were developmentally normal, which shows that cyclin A1, in contrast to cyclin A 2 (Murphy et al., 1997Go), is not required for normal embryonic and post-natal somatic cell division.

In contrast to the infertile males, the female cyclin A1-/- mice showed no noticeable defects during oogenesis, which reflects the gender differences in the mechanisms underlying the regulation of meiosis (Wolgemuth et al., 1995Go).

In a more recent study (Liu et al., 2000Go), it was found that cyclin A1 may regulate the progression of meiosis by activating phosphorylation of Cdc25 phosphatases with subsequent amplification of the p34 Cdc2/cyclin B complex, termed ‘M-phase-promoting factor’ (MPF; (i.e., maturationpromoting factor).

Against the background of these previous studies, the present investigation was the first to quantify cyclin A1 mRNA expression by real-time fluorescence RT–PCR in testicular biopsy specimens from patients with spermatogenesis disorders. In contrast to the high level of average normalized cyclin A1 expression found in tissue specimens with full spermatogenesis, it was reduced in cases of maturation arrest at the level of secondary spermatocytes and early spermatids, minimal in specimens with spermatogonia only or maturation arrest at the level of primary spermatocytes, and absent in the majority of SCOS specimens. Thus, only a minimal level of cyclin A1 expression was seen in human testicular specimens with pre-meiotic gametogenesis arrest.

It is unclear, however, whether the lack of cyclin A1 expression is a causative, associated or resultant factor in the arrest of progression during meiosis. In analogy to the mouse model (Liu et al., 1998Go), the present study showed an association between cyclin A1 expression and meiosis during human spermatogenesis. Leaving aside speculation regarding causal connections between the lack of cyclin A1 expression and pre-meiotic maturation arrest, quantitative cyclin A1 determination shows high sensitivity and specificity in differentiating two groups of testicular tissue specimens: those with SCOS or pre-meiotic spermatogenic arrest (primary spermatocyte level) versus those with at least secondary spermatocytes, early spermatids or full spermatogenesis. Discriminant analysis showed that determination of NCyclin A1 in tissue samples had a high predictive value (98.6%) for correct classification in one of the two histological subgroups.

The results of the present study demonstrate that quantitative determination of cyclin A1 mRNA by real-time fluorescence RT–PCR enables a molecular-diagnostic classification of spermatogenic disorders.

Quantitative determination of cyclin A1 mRNA expression in testicular tissue is a potential diagnostic tool for assisted reproduction techniques, since it appears to predict the presence of secondary spermatocytes/early spermatids or mature spermatids in patients with NOA and might become a useful molecular-diagnostic parameter for supplementing histopathological diagnostics.

Of therapeutic interest is the question of whether disturbances in cyclin A1 expression in individual patients can contribute to the development of a spermatogenic disorder. In-vitro spermatogenesis may be a future treatment option for such patients, as it has been demonstrated that inhibition of protein phosphatases by okadaic acid in cultured male germ cells could overcome the cyclin A1 defect, thereby allowing Ccna1-/- mutant spermatocytes to enter metaphase I with cytologically normal chromosome configurations (Liu et al., 2000Go).


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors thank Dr Bernd Straub from the Department of Urology for his help and Ms Angelika Schneller, Ms Petra von Kwiatkowski and Ms Antonia Maas for their excellent technical support in assessing the samples. They are also grateful to Dr Werner Hopfenmüller (Department of Medical Statistics) for help in performing the statistical analysis. These studies were supported financially by the Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Berlin, Germany.


    Notes
 
5 To whom correspondence should be addressed at: Department of Urology, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Hindenburgdamm 30, 12200 Berlin, Germany. E-mail: schrader{at}medizin.fu-berlin.de Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on October 24, 2001; resubmitted on February 18, 2002; accepted on April 3, 2002.