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Association of Meiotic Arrest with Lack of BOULE Protein Expression in Infertile Men

Institute of Reproductive Medicine (C.M.L., A.K., E.N., J.G.), Westphalian Wilhelms-University, D-48129 Muenster, Germany; and Departments of Obstetrics, Gynecology, and Reproductive Sciences, Physiology, and Urology (E.Y.X., R.A.R.P.), Program in Development and Stem Cell Biology, University of California, San Francisco, California 94143-0556

Address all correspondence and requests for reprints to: Prof. Dr. J. Gromoll, Institute for Reproductive Medicine, Domagkstrasse 11, D-48149 Münster, Germany. E-mail: gromolj{at}uni-muenster.de.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Spermatogenesis is a complex developmental process of mitotic and meiotic cell divisions that ultimately results in production of haploid spermatozoa. Recent studies in flies demonstrate that the BOULE gene encodes a key factor of meiosis in male germ cells, regulating the expression of twine, a cdc25 phosphatase, which promotes progression through meiosis. In this study, we investigated whether a common mechanism underlies the block of germ cell maturation observed in idiopathic and nonidiopathic azoospermic patients with meiotic arrest. We examined, by immunohistochemistry, BOULE and CDC25A phosphatase protein, the human homolog of twine, expression in 47 men with meiotic arrest, mixed atrophy, or normal spermatogenesis. The presence of genetic alterations within the BOULE gene was investigated by single-stranded conformation polymorphism. BOULE protein expression in men with complete spermatogenesis can be restricted to stages from leptotene up to stages of late spermatocytes, whereas CDC25A expression ranges from leptotene spermatocytes to elongating spermatids. Although spermatocytes were present in all testicular biopsies with meiotic arrest (28 testes), BOULE protein expression was completely lacking. In addition, in nearly all biopsies in which BOULE was absent, CDC25A was concomitantly lacking. However, no mutations or polymorphisms in the BOULE gene were identified, which could explain the lack of BOULE or CDC25A expression. These results indicate that a major group of infertile men with meiotic arrest lack BOULE protein and its putative target, CDC25A expression. The spermatogenic failure seems to arise from factor(s) upstream of BOULE, which are possibly involved in regulating transcription and/or translation of BOULE. Thus, the spermatogenic damage leading to meiotic arrest is independent of the etiology and indicates that BOULE is a possible fundamental mediator of meiotic transition in the human.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
MEIOSIS IS THE process of one DNA replication followed by two cell cleavages, with divisions of the chromosomal content leading to haploid cells, that takes place during gamete maturation. The regulation of meiosis at the molecular level is still poorly understood. Recently some candidate meiotic genes have been identified in animal models (1). Among them, the highly conserved BOULE gene was shown to be a key regulator of meiosis in Drosophila, and boule-deficient fruitflies are infertile and display meiotic arrest in their male germ cells (2). BOULE belongs to the DAZ gene family, which consists of the autosomal BOULE and deleted in azoospermia-like and the Y-chromosomal DAZ gene (3, 4). In males, the expression of all family members is highly germ cell specific, and they all share an RNA binding domain, suggesting their possible function in the translational control of genes involved in germ cell maturation. In fruitflies, boule is expressed in the prophase of the first meiotic division and regulates the expression of twine, a cdc25 phosphatase required to activate the maturation promoting factor, consisting of the cdc2/cyclin B complex, which is crucial for entering the G₂/M transition phase to complete the first meiotic division (5, 6). Recently it was shown that the human BOULE might have the same or a very similar function as the Drosophila homolog as a regulator of meiosis because spermatogenesis was restored in boule mutant flies made transgenic for the human BOULE gene (6). This suggests that the expression of BOULE might be important for meiotic transition in the human as well.

Spermatogenic arrest during meiosis as a cause of infertility affects 6% of azoospermic men in our patient population. The histological picture of meiotic arrest is rather constant. Meiotic arrest is characterized by germ cells that enter meiosis and undergo the first chromosomal reduction from 4n to 2n but that are then unable to proceed further. This results in tubules containing spermatocytes as the latest developmental stage of germ cells (7). Meiotically arrested spermatocytes accumulate in the tubules, degenerate, and are easily distinguishable from normal spermatocytes by their partially condensed chromosomes. Although the cause of infertility in patients with meiotic arrest often remains unidentified, this histological picture can be observed in patients with nonidiopathic infertility as well, such as in case of microdeletions of the Y-chromosome, chromosomal abnormalities, and cryptorchidism (8), suggesting that different causal factors can result in the same effect.

In this study, we investigated whether a common molecular mechanism underlies the block of germ cell maturation observed in idiopathic and nonidiopathic azoospermic patients with meiotic arrest. To this end, we compared the expression of BOULE, as an example of a factor involved in the meiotic transition, and its putative target gene, CDC25A, in testicular biopsies of men with normal spermatogenesis and of patients with either complete meiotic arrest in all of the tubuli or with a variable picture of spermatogenic damage in their seminiferous tubuli (mixed atrophy).

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
Patients and biopsies

We analyzed testicular biopsy specimens obtained from patients with complete meiotic arrest (n = 18) or mixed atrophy (n = 16). Vasectomized men with complete spermatogenesis (n = 17) acted as the control group (Table 1). All testicular samples were obtained by open biopsy procedures performed in the attempt to obtain testicular sperm for intracytoplasmic sperm injection. All patients gave written informed consent to the procedures and to further studies with their unneeded biopsies. This general consent was approved by the local ethics committee.

Four patients had bilateral testis biopsies with varying histological appearances, with one testis having a complete meiotic arrest and the contralateral testis having mixed atrophy. In 14 patients, only one testis biopsy could be obtained, of which four were diagnosed with meiotic arrest, six were diagnosed with mixed atrophy, and four were from the vasectomized patients with normal spermatogenesis (Table 1). The average age of patients with meiotic arrest was 31.9 ± 5.1 yr, the average age of patients with mixed atrophy was 32.5 ± 4.6 yr, and the average age of patients with complete spermatogenesis was 43.2 ± 5.9 yr.

Hormones

Peripheral blood was obtained for analysis of hormone values. FSH and LH were analyzed by immunofluorometric assays (Autodelfia; Wallac, Freiburg, Germany), and serum testosterone was measured by RIA (Diagnostic Systems Laboratories, Sinsheim, Germany).

Genomic DNA isolation

EDTA blood samples were used for genomic DNA analysis. PCR for the different exons of the BOULE gene was performed using the forward and reverse primer combinations indicated in Table 2. Starting from genomic DNA and isolated from blood samples of the 18 patients with meiotic arrest, each exon was amplified by PCR. The different amplicons were subjected to single-stranded conformation polymorphism analysis, and migration patterns were visualized by silver staining (9).

Bouin’s-fixed, paraffin-embedded specimens of testes from patients with meiotic arrest were sectioned at 4 µm. Control sections were made from patient biopsies diagnosed with complete spermatogenesis and mixed atrophy. All sections were treated equally. After deparaffinization and rehydration, primary antibodies against two different meiosis-specific proteins were applied. Sections were analyzed by a peptide-specific polyclonal antibody previously shown to recognize exclusively the human BOULE protein (7) and by a polyclonal antiserum against CDC25A (sc-97; Santa Cruz Biotechnology, CA). Both were applied for 60 min at room temperature in blocking buffer. After washing, Dako-LSAB 2 System (Dako Diagnostika, Hamburg, Germany) was added for 30 min, followed by a washing step and incubation with DAB (Dako Diagnostika) for 20 min. Controls for all immunohistochemical staining procedures were performed by omitting the primary antibody on adjacent sections. The sections were counterstained with hematoxylin for 10 sec and mounted under cover slips with Dako Faramount (Dako Diagnostika) before observation using an upright microscope (Axioskop; Zeiss, Oberkochen, Germany) at different magnifications (objectives, x25 and x40). The noncounterstained sections were observed with differential interference contrast (Nomarski Optics; Zeiss) at x40. Digital images of equal exposure were acquired with a CCD camera (Axiocam; Zeiss) controlled by image software (Axiovision; Zeiss).

Statistics

The results of the bilateral testes evaluation are shown per patient. Data are given as means ± SD. For statistical comparison, t test or one-way ANOVA followed by Dunn’s Method or Kruskal-Wallis one-way ANOVA on ranks were used. P < 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
Clinical parameters

The clinical parameters of the patients are shown in Tables 3, 4, and 5. All patients were azoospermic; however, four patients (patients 7, 14, 18, and 29) displayed meiotic arrest in one testes, whereas in the contralateral testis, mixed atrophy was found. Therefore, the testes were grouped according to their histological diagnosis (Tables 1, 3, and 4). In the group with complete meiotic arrest (n = 18), 10 patients had a diagnosis of idiopathic infertility, whereas in eight patients, azoospermia could be related to some known causal factor, e.g. Y-chromosomal microdeletions, ring chromosome, and Klinefelter syndrome (Table 3). Similarly, among the patients with mixed atrophy (n = 16), the azoospermia was idiopathic in eight patients, and in eight patients, it could be related to known causal factors (Table 4). Testis volume was significantly smaller in the two groups with spermatogenic defects compared with the vasectomized patient group. FSH levels differed significantly, with azoospermic patients displaying meiotic arrest (14.1 ± 11.8 IU/liter) and patients with mixed atrophy (15.1 ± 9.1 IU/liter) having significantly higher FSH levels compared with the group of vasectomized patients (3.9 ± 3.0 IU/liter; Tables 3–5).

In all biopsies with complete spermatogenesis (vasectomized patients), expression of BOULE protein was detectable in germ cells of the first meiotic division (prometaphase) beginning at the leptotene spermatocyte stage, was intensified in zygotene spermatocytes, and reached its highest level in pachytene spermatocytes (Fig. 1, A–C). The BOULE protein was exclusively localized in the cytoplasm but never in the nucleus or in close proximity of the meiotic chromosomes. Staining intensities decreased from the diplotene stage to undetectable levels in more advanced spermatogenic stages, such as round and elongated spermatids. Nonspecific staining was frequently observed in peritubular cells, some Leydig cells, and blood capillaries. Two different patterns of BOULE expression were observed in the patients with meiotic arrest and mixed atrophy. All testes with complete meiotic arrest contained spermatocytes in seminiferous tubules that completely lacked expression of BOULE. In addition, some of these spermatocytes displayed a degenerate morphology and were loosely attached to the seminiferous epithelium (Fig. 1, D and E; Table 3). Twelve of the 22 biopsies displaying mixed atrophy showed moderate BOULE spermatocyte staining in the majority of the tubules. In three biopsies, BOULE expression was completely absent, although spermatogenic stages more advanced than spermatocytes were detected (Table 4). The remaining seven biopsies showed strong staining of BOULE in the spermatocytes.

View larger version (131K): [in this window] [in a new window]   FIG. 1. Immunohistochemical staining of BOULE (A–E) and CDC25A phosphatase (F–H) of sequentially sectioned biopsies and a schematic drawing of the expression pattern of both proteins during spermatogenesis (right column). The upper six images represent sections of testis biopsies from patients with complete spermatogenesis at different magnifications counterstained with hematoxylin (A, B, D, E, F, H); or Nomarski optics of sections not counterstained (C and G). BOULE is exclusively expressed in the spermatocytes (A–C, small arrow), and as a result, CDC25A is also expressed in round spermatids and early elongating spermatids (F and G). The expression pattern in the more mature germ cells shifts from the cytoplasm into the nucleus (G, large arrow), whereas the most condensed spermatids lack CDC25A expression (G). Patients with complete spermatogenic arrest lack BOULE in the spermatocytes (D, small arrow) and mostly CDC25A phosphatase expression (H, small arrow). The schematic drawing (right column) showing all spermatogenic cell types displays the coexpression of BOULE and CDC25A phosphatase starting in zygotene spermatocytes (Z) until the late pachytene stage (LP). Beginning with spermiation, CDC25A expression shifts from the cytoplasm into the nucleus, where it remains up to the elongation phase. Ad, Spermatogonia A dark; Ap, spermatogonia A pale; B, spermatogonia B; Pl, preleptotene; L, leptotene; Z, zygotene; EP, early pachytene; MP, mid pachytene; LP, late pachytene; II, secondary spermatocyte; rS, round spermatids; eS, elongating spermatids. Scale bars, 50 µm (C and G); 20 µm (all others).

In vasectomized patients with normal spermatogenesis, the onset of the CDC25A phosphatase protein expression during the prometaphase of the first meiotic cleavage paralleled the BOULE expression pattern (Fig. 1, B, C, F, and G), but, unlike BOULE, it extended to later stages. The high CDC25A expression levels remained cytoplasmic throughout completion of both meiotic cleavages (Fig. 1F) and shifted with the onset of spermiogenesis to the nucleus, where it remained up to the spermatid elongation. Finally, it translocated into the remaining spermatid cytoplasmic body and was no longer detected before shedding of the cytoplasmic droplet (Fig. 1G). Frequently a weak staining of CDC25A immunopositive reaction in Sertoli cells was found (Fig. 1, F–H). In all testes biopsies of vasectomized patients with normal spermatogenesis, a similar CDC25A expression pattern was observed.

CDC25A protein expression could not be detected in most testes of patients with complete meiotic arrest, thus paralleling the lack of BOULE expression in those patients (Fig. 1, D and H). In only four testes biopsies (Table 3), some CDC25A staining was observed. The diagnoses in these four patients were deletion of the azoospermia factor region b and c, idiopathic azoospermia, Klinefelter syndrome with maldescended testes, and testicular maldescent (Table 3).

In patients with mixed atrophy, all biopsies except one showed moderate (n = 7) or strong (n = 14) CDC25A phosphatase expression. The one testicular biopsy with germ cells more mature then spermatocytes was negative for both CDC25A and BOULE protein.

Screening of the BOULE gene

To investigate whether mutations or genetic polymorphisms of the BOULE gene contributed to the observed phenotype of absent BOULE expression, the coding region of the BOULE gene (exon 2–10) was investigated by single-stranded conformation polymorphism. Neither mutations nor polymorphisms could be identified in patients and controls.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
In this study, we characterized the BOULE and CDC25A protein expression in human spermatogenesis and showed for the first time that the spermatogenic damage in azoospermic patients with meiotic arrest is associated with lack of BOULE expression. BOULE protein-specific staining was precisely found in the prometaphase of the first meiotic division in patients with normal spermatogenesis. This finding is in agreement with earlier studies in flies, mice, and men (3, 5). Unlike in mice, in which Boule persists up to the early spermatids, human BOULE is only detectable up to diplotene spermatocytes. In human spermatogenesis, BOULE is localized only in the cytoplasm, which differs from the fruitflies, in which boule translocates from a premeiotically perinuclear position to the cytoplasm of the growing spermatocytes at the onset of meiosis (10). This shuttling of boule between nucleus and cytoplasm, however, does not seem to be essential for the function of the protein because the meiotic defect of boule knockout flies can be rescued by the introduction of the human BOULE gene, which does not exhibit the observed protein translocation (5).

The onset of M phase during meiosis is controlled by the M phase-promoting factor, which consists of cyclin B and the cell cycle division gene cdc2. This complex is essential for resumption of meiosis, and its activity is regulated by reversible phosphorylation, in which Wee1 and Myt1 kinases phosphorylate and, as a result, inactivate the complex, whereas CDC25 phosphatase inactivates the M phase complex (11). Several CDC25 genes have been identified, of which CDC25A is predominantly expressed in the testis and is crucial for spermatogenesis (12, 13). In the present study, CDC25A expression was detectable in primary and secondary spermatocytes, stages that correspond well with the onset of BOULE protein expression (Fig. 1). In mouse testis, Cdc25A protein expression is confined to the nucleus and cytoplasm of pachytene and diplotene spermatocytes up to round spermatids (14). In rats, the Cdc25A protein remains in the cytoplasm throughout the entire meiosis (15). In our study, we could observe CDC25A staining in pachytene spermatocytes, with cytoplasmic localization up to the end of meiosis. CDC25A then translocates from the cytoplasm to the nucleus and can be detected in the round spermatids before being shed out again before the end of spermiation (Fig. 1).

The lack of BOULE protein expression in all biopsy samples with complete meiotic arrest seems to be independent of the etiology of the spermatogenic damage and identifies this factor as a possible fundamental mediator of meiotic transition in the human as well. The lack of BOULE expression and, possibly, of other important regulators of meiosis not investigated in the present study might represent a common key molecular mechanism involved in meiotic arrest. If this is true, it might be possible to rescue meiosis in vitro in testis tissue culture by addition of recombinant BOULE protein.

The factors involved in the regulation of BOULE expression are not known. At present, we do not know whether the lack of BOULE protein coincides with the lack of BOULE mRNA. Future studies will have to clarify whether BOULE itself is regulated by another RNA-binding protein at the translational level or whether transcription factors are involved in the regulation of BOULE mRNA expression.

Factors downstream of BOULE are crucial for meiosis as well. From fruitflies, we know that the cdc25 homolog twine is under strict translational control of boule. twine and boule interact genetically, and as a result, protein expression from twine mRNA correlates with the cytoplasmic accumulation of boule. If boule is deleted, twine protein expression is drastically reduced, and mRNA expression is increased (5). The resulting phenotype of a twine mutant fly displaying a meiotic arrest very much resembles that of the boule mutant fly (16). Heterologous expression of twine in boule mutant flies could rescue the phenotype by restoring the meiotic entry, indicating its crucial function for meiosis. Transposed to our finding in the human, one can assume a similar scenario, in which BOULE regulates translation of CDC25A phosphatase mRNA. In patients lacking BOULE, CDC25A phosphatase mRNA might still be present or even up-regulated but not translated (Fig. 2). The lack of BOULE in the three biopsies and the presence of CDC25A protein expression (Table 4) could be due to trace amounts of BOULE present in the biopsies but not detectable with the staining system being used or to illegitimate translation of the CDC25A mRNA independent of BOULE. The lack of CDC25A phosphatase presumably leads to hyperphosphorylation of the M phase-promoting factor complex, resulting in a completely inactive form. This could be the main reason for the spermatogenic failure observed in the patients. In any case, the concomitant lack of BOULE and its putative target, CDC25A, results in a meiotic arrest.

View larger version (13K): [in this window] [in a new window]   FIG. 2. A model for BOULE function during human spermatogenesis. An unknown factor, presumably a RNA-binding protein or transcriptional factor is required for the translational or transcriptional induction of BOULE. BOULE protein itself regulates the translation of CDC25A, which in turn, activates the M phase-promoting factor, which is required for the G2/M transition. If the BOULE stimulating factor is missing (B), BOULE protein is lacking, and as a result, CDC25A mRNA is not translated. Therefore, spermatogenesis is halted at the spermatocyte stage, and the germ cells undergo necrosis.

This study demonstrates that a major subgroup of patients with meiotic arrest lacks expression of BOULE protein. It is possible that regulating factors upstream from BOULE are either nonfunctional or not expressed. From detailed studies in the fruitfly, it is evident that genes such as can and aly are required for earlier steps of meiosis and may regulate expression of boule (19). If human homologs of such factors exist, our results could be explained by mutations or polymorphisms of these factors that may render them nonfunctional. Otherwise, there is a rigid control of the resumption of meiosis, which could be affected by several factors, e.g. the quality of germ cells or integrity of DNA. This could lead to the histological phenotype of one testis with complete meiotic arrest, with the other one exhibiting some degree of progressing spermatogenesis as observed in our patients (Tables 3 and 4). The observed tubule-to-tubule variation in one testis could reflect similar regulatory steps. Independent of the mechanism required for BOULE expression, we conclude that lack of BOULE expression has the potential to be a cause of meiotic arrest in a subset of infertile men. Future work should now focus on identifying the factors that regulate BOULE expression and the mRNA targets of BOULE protein.

	Acknowledgments

 
We are grateful to Prof. Dr. M. Bergmann at the University of Giessen for contributing biopsy material. We thank J. Salzig, M. Kloth, S. Altikriti, and N. Terworth for technical assistance. Prof. Manuela Simoni and Susan Nieschlag, M.A., are acknowledged for language editing of the manuscript.

	Footnotes

 
This work was supported by the Fritz-Thyssen and Alexander von Humboldt Foundations and by a grant of the Innovative Medical Research of the University (IMF-GR110311).

Received July 8, 2003.

Accepted December 23, 2003.

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