Round spermatids from infertile men exhibit decreased protamine-1 and -2 mRNA

Klaus Steger1,7, Klaus Failing2, Thomas Klonisch3, Hermann M. Behre4, Martina Manning5, Wolfgang Weidner5, Lothar Hertle6, Martin Bergmann1 and Sabine Kliesch6

1 Institute of Veterinary Anatomy, 2 Department of Biomathematics, Institute of Veterinary Physiology, University of Giessen, 3 Institute of Anatomy and Cell Biology, 4 Andrology Unit, Department of Urology, University of Halle, 5 Department of Urology, University of Giessen and 6 Department of Urology, University of Münster, Germany


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
During spermiogenesis, histone-to-protamine exchange causes chromatin condensation. Spermatozoa from infertile men are known to exhibit an increased protamine-1 (PRM1) to protamine-2 (PRM2) protein ratio. Since patients undergoing testicular sperm extraction (TESE) followed by intracytoplasmic sperm injection (ICSI) reveal low fertilization rates, whether the outcome of ICSI could be related to the percentage of round spermatids expressing PRM1-mRNA and PRM2-mRNA was investigated. Applying in-situ hybridization, 55 testicular biopsies from men undergoing TESE/ICSI were investigated. The percentage of PRM1-mRNA and PRM2-mRNA positive spermatids was significantly (P < 0.0001) decreased in men with at least qualitatively normal spermatogenesis (PRM1-mRNA: 58.4 ± 13.8%; PRM2-mRNA: 56.4 ± 11.3%) and impaired spermatogenesis (PRM1-mRNA: 32.6 ± 10.8%; PRM2-mRNA: 31.7 ± 11.1%) compared with men with obstructive azoospermia and quantitatively normal spermatogenesis (PRM1-mRNA: 79.9 ± 4.6%; PRM2-mRNA: 78.1 ± 5.7%). A positive correlation (rPRM1 = 0.733; rPRM2 = 0.784; P < 0.001) was demonstrated between the score and the percentage of PRM1-mRNA and PRM2-mRNA positive spermatids. While successful fertilization was neither related to the score, nor to the percentage of PRM1-mRNA and PRM2-mRNA positive spermatids, a significant (P < 0.05) relationship was demonstrated between successful fertilization and the PRM1-mRNA to PRM2-mRNA ratio. Therefore, the PRM1-mRNA to PRM2-mRNA ratio in round spermatids may serve as a possible predictive factor for the outcome of ICSI.

Key words: infertile men/in-situ hybridization/intracytoplasmic sperm injection/protamines/spermatogenesis


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Human male fertility is normally assessed on the basis of a semen profile reflecting the quality of the ejaculate, namely the sperm concentration, the total number of spermatozoa, the percentage of morphologically normal spermatozoa, and the percentage of motile spermatozoa [WHO (World Health Organization), 1992Go]. In patients with severely impaired spermatogenesis, where techniques of assisted reproduction, namely intracytoplasmic sperm injection (ICSI), are necessary for the treatment of infertility, these basic sperm parameters are not related to the outcome of ICSI (Nagy et al., 1995Go; Novero et al., 1997Go). In azoospermic men, it is even more difficult to estimate their chances to father a child by modern ICSI therapy in combination with testicular sperm extraction (TESE). Therefore, a prognostic parameter to estimate their chances for successful fertility treatment could help doctors and patients in counselling and treatment. Possibly, additional parameters, such as the sperm chromatin organization, may be decisive for male fertility.

During spermiogenesis, haploid spermatids undergo profound changes in both the composition and the compaction state of their nuclear chromatin. Whereas in round spermatids, DNA-binding histones are replaced by transition proteins, in elongating spermatids, transition proteins are removed from the condensing chromatin and are replaced by protamines. Protamine-1 (PRM1) was present in all mammalian spermatozoa analysed so far. In addition, protamine-2 (PRM2) has been detected in spermatozoa of mouse, hamster, stallion, and some primates, including man (reviewed in Hecht, 1989 , 1990; Oliva and Dixon, 1991 ; Dadoune, 1995 ; Wouters-Tyrou et al ., 1998; Steger, 1999). In man, transcripts for both PRM1 and PRM2 have been demonstrated from late step 1 round spermatids to early step 4 elongating spermatids. The corresponding proteins were present from step 4 elongating spermatids to step 8 elongated spermatids (Roux et al., 1987Go, 1988Go; LeLannic et al., 1993Go; Lescoat et al., 1993Go; Wykes et al., 1995Go; Prigent et al., 1996Go; Saunders et al., 1996Go; Wykes et al., 1997Go; Steger et al., 2000Go).

Although the relative proportion of PRM1 and PRM2 is highly variable between different species (1:2 in mouse, 1:1 in man, and 2:1 in hamster), the total protamine mass to DNA mass ratio is nearly identical between different mammals (Balhorn et al., 1988Go; Bench et al., 1996Go). In contrast, human sperm nuclei contain significantly less protamine (Bench et al., 1996Go) being consistent with the observation that, in man, histone-to-protamine exchange is only about 85% complete (Tanphaichitr et al., 1978Go; Gatewood et al., 1987Go; Prigent et al., 1996Go). In transgenic mice, over-expression of PRM1 protein at its normal time of synthesis does not affect spermiogenesis (Peschon et al., 1987Go; Zambrowicz et al., 1993Go), while premature translation of PRM1-mRNA causes precocious chromatin condensation resulting in male sterility (Lee et al., 1995Go). This implies that stringent temporal and stage-specific gene expression is of pivotal importance for correct nucleoprotein exchange and complete differentiation of round spermatids into mature spermatozoa.

Vanderzwalmen et al. were the first to succeed in fertilizing a human oocyte by an elongated spermatid (Vanderzwalmen et al., 1995Go). All oocytes cleaved further to 4-cell embryos. Fishel et al. reported the implantation of such embryos after uterine transfer (Fishel et al., 1995Go). The first birth of a child after round spermatid injection into human oocytes confirmed the feasibility of this novel approach and demonstrated that there is no genetic barrier to fertilization by round spermatids (Tesarik et al., 1995Go). However, injection of round spermatids resulted in a significantly lower fertilization rate and a higher developmental arrest resulting in only a few, if any, pregnancies (Antinori et al., 1997Go; Fishel et al., 1997Go; Vanderzwalmen et al., 1997Go; Yoshida et al., 1997Go; Barak et al., 1998Go; Bernabeu et al., 1998Go; Kahraman et al., 1998Go; Al-Hasani et al., 1999Go; Ghazzawi et al., 1999). Low fertilization and pregnancy rates, in addition, have been achieved using megalohead spermatozoa exhibiting poorly condensed nuclear chromatin (Kahraman et al., 1999Go). In contrast, higher fertilization and pregnancy rates have been achieved using spermatozoa obtained by TESE (Craft et al., 1993Go; Devroey et al., 1995Go; Silber et al., 1995Go; Tournaye et al., 1995Go). Thus, testicular biopsies may play an important therapeutic role in the management of male infertility, since testes from patients with severely impaired spermatogenesis and maturation arrest resulting in azoospermia may contain some small foci of spermatogenesis which allow TESE/ICSI to be carried out (Silber et al., 1995Go; Yoshida et al., 1997Go). However, high rates of DNA fragmentation have been reported in round spermatids retrieved from testicular biopsies of men with non-obstructive azoospermia suggesting that fragmented DNA in haploid spermatids may be one of the features of the pathology associated with azoospermia (Jurisicova et al., 1999Go).

Since the selection of an unsuccessful spermatozoon has both emotional and financial consequences for the couple, a realistic estimation of the outcome of ICSI based on objective predictive factors is of pivotal importance. Balhorn et al. (1988) demonstrated that infertile men exhibit ejaculated sperm with an increased PRM1 to PRM2 protein ratio caused by a lower PRM2 protein content as compared to fertile men. The present study aims at an answer as to whether a correlation exists between the percentage of testicular round spermatids expressing PRM1-mRNA and PRM2-mRNA and the fertilizing capacity of these spermatids. For this purpose, testicular biopsy specimens from infertile patients undergoing a combined TESE/ICSI treatment were investigated applying in-situ hybridization with digoxigenin (DIG)-labelled cRNA probes against PRM1-mRNA and PRM2-mRNA. The percentage of round spermatids exhibiting a positive hybridization signal was determined. Furthermore, whether the outcome of TESE/ICSI, namely successful fertilization followed by embryo transfer, pregnancy and healthy birth, was related to the histological score, the percentage of PRM1-mRNA and PRM2-mRNA positive spermatids and the PRM1-mRNA to PRM2-mRNA ratio was analysed.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Testicular tissue
After written informed consent, testicular biopsies were obtained from 65 infertile men, aged 23–54 years. In 10 patients with obstructive azoospermia (after vasectomy), biopsies were carried out for diagnostic reasons during vasectomy reversal and served as normal controls. In 55 infertile patients with non-obstructive azoospermia, testicular tissue was obtained for diagnostic and therapeutic reasons at the same time. One part of the testicular tissue was immediately prepared and frozen for TESE, the other part of the testicular biopsy specimen was fixed by immersion in Bouin's fixative and embedded in paraffin using standard techniques.

For histological evaluation, 5 µm sections were stained with haematoxylin–eosin and scored, according to Holstein and Schirren (1983). In-situ hybridization was performed on 10 testes from 10 men (Table IGo, group I) with obstructive azoospermia and quantitatively normal spermatogenesis (score 10) and on 55 testes from 55 infertile patients undergoing TESE followed by intracytoplasmic sperm injection (ICSI). From these patients, 28 (Table IGo, group II) exhibited at least qualitatively normal spermatogenesis (scores 10–8), while 27 (Table IGo, group III) revealed impaired spermatogenesis (scores 7–1).


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Table I. Characterization of patient groups I–III
 
Serum FSH
For each patient, serum FSH concentrations were measured by time-resolved fluorometric assay. Serum FSH concentrations of >7 IU/l were regarded as elevated compared with men with proven fertility (Cooper et al., 1991Go).

Digoxigenin (DIG)-labelled cRNA-probes
Production of DIG-labelled cRNA-probes was performed as described previously (Steger et al., 1998Go, 2000Go). Briefly, a 156 nt and a 294 nt polymerase chain reaction (PCR)-product of the human PRM1 and PRM2 gene respectively was subcloned in pGEM-T (Promega, Heidelberg, Germany). Plasmids were transformed in the XL1-Blue E. coli strain (Stratagene, Heidelberg, Germany) and extracted by column purification (Qiagen, Hilden, Germany). In-vitro transcription of DIG-labelled PRM1-cRNA and PRM2-cRNA was performed using the x10 RNA-DIG labelling mix (Boehringer Mannheim, Mannheim, Germany) and RNA-polymerases T3 and SP6. Vectors containing the PRM1 and PRM2 inserts were digested with NcoI and NotI (New England Biolabs, Frankfurt, Germany) for the production of sense-cRNA (NcoI) and antisense-cRNA (NotI) respectively.

In-situ hybridization
In-situ hybridization was performed as described previously (Steger et al., 1998Go, 2000Go). Briefly, 5µm sections were partially digested with proteinase K, postfixed in 4% paraformaldehyde, and exposed to 20% acetic acid. After prehybridization in 20% glycerol, sections were covered with the DIG-labelled sense or antisense cRNA-probes. Both cRNA were used at a dilution of 1:100 in hybridization-buffer containing 50% deionized formamide, 10% dextran sulphate, x2 saline sodium citrate (SSC), x1 Denhardt's solution, 10 µg/ml salmon sperm DNA, and 10 µg/ml yeast t-RNA. Hybridization was performed overnight at 37°C in a humidified chamber containing 50% formamide in x2 SSC. Post-hybridization washes were performed, according to Lewis and Wells (1992). After blocking with 3% bovine serum albumin, sections were incubated with the anti-DIG Fab-antibody conjugated to alkaline phosphatase (Boehringer Mannheim) overnight at 4°C. Staining was visualized by developing sections with nitroblue-tetrazolium/5-bromo-4-chloro-3-indolylphosphate in a humidified chamber protected from light. For each test, negative controls were performed using DIG-labelled cRNA sense-probes.

Statistical analyses
Patients have been arranged into three groups (Table IGo). Group I (control) comprised patients 1–10 revealing obstructive azoospermia and quantitatively normal spermatogenesis (score 10). The 55 infertile men undergoing a combined TESE/ICSI treatment were divided into patients 11–38 (group II) revealing at least qualitatively normal spermatogenesis (scores 10–8) and patients 39–65 (group III) exhibiting impaired spermatogenesis (scores 7–1).

Since it is already known that PRM1-mRNA and PRM2-mRNA are present in round spermatids (Steger et al., 2000Go), 10 seminiferous tubules containing round spermatids were counted from each biopsy. For both protamines, the ratio of labelled to unlabelled cells was determined.

Statistical analyses were carried out applying the statistical program package BMDP (Dixon, 1993Go). Differences between groups I–III in the percentages of PRM1 and PRM2 positive spermatids as well as in the PRM1-mRNA to PRM2-mRNA ratio have been exposed using one way analysis of variance (BMDP 7D). For the PRM1-mRNA to PRM2-mRNA ratio, a logarithmic transformation was applied, because the distribution of the ratio was skewed to the right. Subsequently, pairwise significance of mean differences between the groups was checked with the Tukey studentized range method. The t-test for independent samples was used (BMDP 3D) to reveal correlations between score data and successful fertilization, percentage of PRM1 and PRM2 positive spermatids, and the logarithm of the PRM1-mRNA to PRM2-mRNA ratio.

In addition, the correlation between the score, the percentage of PRM1 and PRM2 positive spermatids, and the logarithm of the PRM1-mRNA to PRM2-mRNA ratio was analysed (BMDP 6D). Logistic regression was employed to test whether successful fertilization, pregnancy or healthy birth was simultaneously related to the score, the percentage of PRM1 and PRM2 positive spermatids, and the logarithm of the PRM1-mRNA to PRM2-mRNA ratio. P < 0.05 was considered to be significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Men with obstructive azoospermia and normal spermatogenesis (score 10; group I) revealed normal serum FSH concentrations (4.7 ± 1.4 IU/l). While infertile men with at least qualitatively normal spermatogenesis (scores 10–8; group II) exhibited normal serum FSH values (4.7 ± 3.2 IU/l), patients with impaired spermatogenesis (scores 7–1; group III) displayed elevated serum FSH values (17.5 ± 10.6) (Table IGo). However, no relationship was found between serum FSH concentrations and successful fertilization, pregnancy or healthy birth.

PRM1-mRNA and PRM2-mRNA are known to be expressed in round spermatids (Steger et al., 2000Go). In men with obstructive azoospermia and normal spermatogenesis (group I), 79.9 ± 4.6% and 78.1 ± 5.7% of round spermatids contained PRM1-mRNA and PRM2-mRNA respectively, exhibiting a mean PRM1-mRNA to PRM2-mRNA ratio of 1.02 (Table IGo, Figure 1AGo). The percentage of round spermatids revealing a positive hybridization signal for PRM1 and PRM2 was significantly reduced in patients who underwent TESE/ICSI treatment (Table IGo, Figure 1BGo). In group II, 58.4 ± 13.8% and 56.4 ± 11.3% of round spermatids contained transcripts for PRM1 and PRM2 respectively. The mean PRM1-mRNA to PRM2-mRNA ratio was 1.03. In group III, 32.6 ± 10.8% and 31.7 ± 11.1% of round spermatids contained transcripts for PRM1 and PRM2 respectively. The mean PRM1-mRNA to PRM2-mRNA ratio was 1.03. Within the latter group, the number of round spermatids revealing a positive hybridization signal for PRM1-mRNA and PRM2-mRNA was lower in testes with scores 3–1 (PRM1-mRNA: 30.3 ± 10.8%, PRM2-mRNA: 27.7 ± 10.1%) than in testes with scores 7–4 (PRM1-mRNA: 37.1 ± 9.4%, PRM2-mRNA: 39.7 ± 8.3%).



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Figure 1. In-situ hybridization showing the distribution pattern of protamine (PRM)1-mRNA in a seminiferous tubule with normal spermatogenesis (A) and impaired spermatogenesis (B). Arrows indicate positive spermatids, arrowheads indicate negative spermatids. Note that seminiferous tubules with impaired spermatogenesis contain fewer positive spermatids than tubules with normal spermatogenesis. Bar = 20 µm.

 
Testes from patients with impaired spermatogenesis and scores ranging from 7 to 1 always contained some seminiferous tubules exhibiting an arrest of spermatogenesis at the level of round spermatids. Within these tubules only a small number of round spermatids was present and many of these cells were multinucleated. In-situ hybridization revealed a rather weak signal within these cells. A total of 14.3 ± 10.9% and 14.2 ± 11.4% of round spermatids contained transcripts for PRM1 and PRM2 respectively. Therefore, the percentage of round spermatids containing PRM1-mRNA and PRM2-mRNA was significantly lower in seminiferous tubules with an arrest of spermatogenesis at the level of round spermatids than in tubules with complete spermatogenesis.

One-way analysis of variance revealed that the percentage of round spermatids expressing PRM1-mRNA and PRM2-mRNA exhibited highly significant (P < 0.0001) differences between groups I–III. Applying the Tukey test, all pairwise group comparisons showed statistically significant differences with P < 0.01. In contrast, no group differences could be demonstrated for the logarithm of the PRM1-mRNA to PRM2-mRNA ratio. Furthermore, a positive correlation (rPRM1 = 0.733; rPRM2 = 0.784; P < 0.001) could be demonstrated for the score as determined by the percentage of seminiferous tubules containing elongated spermatids and the percentage of PRM1-mRNA and PRM2-mRNA positive round spermatids (Figure 2Go). Fertilization, pregnancy and birth were not related to the score, the percentage of PRM1-mRNA and PRM2-mRNA positive spermatids, and the logarithm of the PRM1-mRNA to PRM2-mRNA ratio using logistic regression. However, when less than 30% of round spermatids expressed PRM1-mRNA, no pregnancy except one out of 13 had been achieved. A significant (P < 0.05) relationship existed between successful fertilization followed by embryo transfer and the logarithm of the PRM1-mRNA to PRM2-mRNA ratio. When embryo transfer was carried out, the mean PRM1-mRNA to PRM2-mRNA ratio was 1.00, while in the case of no embryo transfer the mean PRM1-mRNA to PRM2-mRNA ratio was increased to 1.35.



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Figure 2. Scatterplot of the percentage of round spermatids which are positive for PRM1-mRNA (left) and PRM2-mRNA (right) and the score. r = correlation coefficient; ***P < 0.001.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
To date, TESE followed by ICSI exhibits fertilization and pregnancy rates that are comparable to other IVF techniques (Van Steirteghem et al., 1998Go; Palermo et al., 1999Go). To improve further the success of TESE/ICSI treatment, especially in patients with non-obstructive azoospermia, there is a strong need for reliable predictive factors supporting the selection of a successful spermatozoon.

While infertile men with at least qualitatively normal spermatogenesis display normal serum FSH concentrations, patients with non-obstructive azoospermia or severe oligozoospermia exhibit elevated serum FSH concentrations due to an increasing number of seminiferous tubules with Sertoli cell only characteristic (Bergmann et al., 1994Go; Martin-du-Pan and Bischof, 1995Go). However, in this study, serum FSH concentrations were neither related to the percentage of PRM1-mRNA and PRM2-mRNA positive spermatids, nor to the overall outcome of ICSI. This is consistent with the data reported by Novero et al. (1997) demonstrating that the serum FSH concentration in the male has no influence on fertilization, cleavage, implantation, pregnancy, and the outcome of ICSI.

In testicular biopsies with scores ranging from 7 to 1, various patterns of spermatogenic impairment could be observed including seminiferous tubules with spermatogenic arrest at the level of round spermatids (SARS). Here, only a small number of spermatids were present with most of them being multinucleated. In-situ hybridization revealed a weak signal for both PRM1-mRNA and PRM2-mRNA in about 14% of round spermatids. Thus, in tubules with SARS, round spermatids contained significantly less transcripts for both PRM1 and PRM2 than in tubules with at least qualitatively normal spermatogenesis, but, similar to normal spermatids, the PRM1-mRNA to PRM2-mRNA ratio was 1:1. This focal reduction of PRM1-mRNA and PRM2-mRNA expression in tubules with SARS adjacent to tubules with at least qualitatively normal spermatogenesis suggests local differences in the presence of regulating factors responsible for the correct differentiation of round spermatids into mature spermatozoa.

Both the gene for human transition protein 1 (TNP1) (Kistler et al., 1994Go) and the genes for human PRM1 and PRM2 (Oliva and Dixon, 1991Go) are known to contain a cAMP-responsive element (CRE) in their promoter regions which serves as binding-site for cAMP-responsive element modulator (CREM). In men with round spermatid maturation arrest, round spermatids showed reduced CREM and TNP1 expression probably due to an underlying gene defect in CREM gene expression (Steger et al., 1999). Recently, male mice with a targeted deletion of the Ca2+/calmodulin-dependent protein kinase IV (Camk4) gene have been shown to be infertile. This infertility has been assigned to a disrupted histone-to-protamine exchange revealing a specific loss of PRM2 and a prolonged retention of TNP2 in haploid spermatids (Wu et al., 2000Go). It is known that about 13% of azoospermic men exhibit a deletion on the long arm of their Y chromosome (Reijo et al., 1995Go). Furthermore, DNA strand breaks were found significantly more often in sperm samples from men with oligospermia compared to sperm samples of normal fertile men (Host et al., 1999aGo,bGo). While in infertile men Schlicker et al. (1994) failed to detect any mutation in the genes encoding for TNP1, PRM1, and PRM2, Kramer et al. (1997) identified a mutation in the PRM1 -> PRM2 -> TNP2 domain in two out of five oligozoospermic infertile men using PCR-based mutation scanning analysis.

Bedford et al. were the first to raise the question of a possible relationship between the state of condensation of sperm nuclear chromatin in spermatozoa of ejaculates and reduced fertility (Bedford et al., 1973Go). Subsequently, human male infertility has been related to both anomalous nucleoprotein composition (Silvestroni et al., 1976Go; Chevaillier et al., 1987Go) and aberrant nucleoprotein ratio (Balhorn et al., 1988Go; Belokopytova et al., 1993Go). Recently, Filatov et al. reported a relationship between disordered chromatin packing in spermatozoa of ejaculates determined from the accessibility of DNA to ethidium bromide and the rates of embryo cleavage in an IVF programme (Filatov et al., 1999Go).

Phosphotungstic acid (PTA)-staining (Courtens and Loir, 1981Go) and aniline blue staining (Dadoune et al., 1988Go) revealed abnormal chromatin condensation in about 20% of morphologically normal human spermatozoa. The percentage of aniline blue stained spermatozoa was significantly correlated with sperm vitality and morphology and was higher in the population of morphologically normal than in abnormal spermatozoa (79.1 and 49.4% respectively). Although the percentage of chromatin condensation assessed by aniline blue staining was significantly higher in ejaculated spermatozoa than in those spermatozoa derived from testis biopsies, the outcome of ICSI was the same. Thus, chromatin condensation of testis extracted sperm is believed not to be a reliable indicator of successful fertilization, implantation, and pregnancy rate in TESE/ICSI programmes (Hammadeh et al., 1996Go, 1999Go).

Chromomycin A3 (CAM3)-staining is an indicator of protamine deficiency in sperm chromatin. The range of CAM3-staining in fertilizing samples varies from 15–20% (Bianchi et al., 1993Go) or 8–60% (Lolis et al., 1996Go) in normospermic donors and was about 70% in infertile male factor donors (Bianchi et al., 1993Go). The frequency of CAM3-staining spermatozoa is related to the frequency of spermatozoa responding positively to the nick-translating action of DNA-polymerase I. Normal males present sperm parameters with a CAM3-staining of <30% and endogenous nicks of <10% of their spermatozoa. Spermatozoa in samples with low CAM3-staining and low endogenous nicks had significantly higher fertilization rates, in vitro, than did samples with high CAM3-staining and high endogenous nicks (Bianchi et al., 1993Go; Lolis et al., 1996Go). However, spermatozoa from patients with high CAM3-staining and high endogenous nicks are not limited in their ability to achieve fertilization using ICSI (Sakkas et al., 1996Go).

A comparative study on nuclear proteins in ejaculated human semen with both normal and defective routine parameters comprised no significant differences in the relative percentage of histones, transition proteins, and protamines (Lescoat et al., 1987Go). In infertile men, the complete absence of PRM1 and PRM2 protein in ejaculated spermatozoa (Silvestroni et al., 1976Go) and of PRM2-mRNA in round spermatids (Ziyyat et al., 1999Go) has been reported. An additional study on two patients with round-headed spermatozoa revealed an anomalous distribution of nuclear basic proteins containing more histones (13.1%), more transition proteins (9.9%), and less protamines (PRM1: 29.4%, PRM2: 6.6%) than normal spermatozoa (1.5, 3.4, 36.5 and 24.1% respectively) (Blanchard et al., 1990Go).

Balhorn et al. (1988) and Belokopytova et al. (1993) demonstrated that, in contrast to fertile men, sperm from infertile men display an increased PRM1 to PRM2 protein ratio caused by a lower PRM2 protein content (PRM1: 38%; PRM2: 28%). The PRM2 protein, in addition, showed reduced affinity to DNA (Belokopytova et al., 1993Go). The reduction in the PRM2 protein content occurred concomitant with an increase in the amount of putative PRM2 protein precursors, suggesting incomplete processing of PRM2 protein (deYebra et al., 1998Go). The observation that Percoll-selected spermatozoa exhibit a high fertilizing capacity (Colleu et al., 1996Go) may be due to the fact that these spermatozoa contain more PRM2 protein. Since PRM2 contains less cysteine than PRM1, the PRM2 protein may be less efficiently cross-linked by disulphide bonds and, therefore, to be decondensed more readily in the oocyte.

Data from Balhorn et al. (1988) and Belokopytova et al. (1993) are in accordance with the results of this study, suggesting that the expression of the PRM1 and PRM2 genes may actually be uncoupled in some developing spermatids of certain infertile men. In contrast to previous studies investigating the nuclear protein equipment of spermatozoa in ejaculates, we were interested in predictive factors for azoospermic men undergoing TESE and, therefore, analysed the percentage of round spermatids expressing PRM1-mRNA and PRM2-mRNA in testicular biopsies. It was found that about 80% of round spermatids in testicular biopsies from men with obstructive azoospermia and normal spermatogenesis contained transcripts for PRM1 and PRM2. The PRM1-mRNA to PRM2-mRNA ratio was 1:1. The percentage of round spermatids expressing PRM1-mRNA and PRM2-mRNA was significantly reduced in infertile men. Here, positive hybridization signals were obtained in about 60% (scores 10–8; qualitatively normal spermatogenesis) and in about 30% (scores 7–1; severe spermatogenic impairment) of round spermatids.

However, the successful introduction of a spermatozoon into the cytoplasm of a metaphase II oocyte is not by itself a sufficient condition to achieve fertilization. Poor chromatin packaging and damaged DNA may also contribute to failure of sperm decondensation after ICSI and result in failure of fertilization. Therefore, not the ejection of the spermatozoon, but the failure of oocyte activation may be the principle cause of failed fertilization after ICSI (Flaherty et al., 1995Go; Schmiady et al., 1996Go).

In summary, the percentage of round spermatids expressing PRM1-mRNA and PRM2-mRNA was found to be significantly reduced in infertile men compared to men with obstructive azoospermia and normal spermatogenesis. Furthermore, a positive correlation was demonstrated between the score and the percentage of round spermatids containing transcripts for PRM1 and PRM2. Fertilization, pregnancy, and birth were not found to be related to the serum FSH concentration, the score, or the percentage of PRM1-mRNA and PRM2-mRNA positive spermatids. Instead, successful fertilization showed a significant relationship with the logarithm of the PRM1-mRNA to PRM2-mRNA ratio in round spermatids. Therefore, this ratio may serve as a possible predictive factor for couples undergoing a combined TESE/ICSI treatment. Moreover, the chance for induction of pregnancy was very low, if <30% of round spermatids expressed PRM1-mRNA and PRM2-mRNA.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The skilful technical assistance of G.Erhardt, A.Hax and A.Hild, Institute of Veterinary Anatomy, Giessen, as well as F.Flammang and N.Wolf, Department of Urology, Münster, is gratefully acknowledged. Funding of this research programme was provided by DFG grant STE 892/1–2.


    Notes
 
7 To whom correspondence should be addressed at: Institut für Veterinär-Anatomie, Frankfurter Strasse 98, D-35392 Giessen, Germany. E-mail: Klaus.Steger{at}vetmed.uni-giessen.de Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Al-Hasani, S., Schopper, B., Kupker, W. et al. (1999) Intracytoplasmic injection of round and elongated spermatids in patients with spermatogenic maturation arrest. Geburtshilfe Frauenheilkunde, 59, 220–224.[ISI]

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Barak, Y., Kogosowski, A., Goldman, S. et al. (1998) Pregnancy and birth after transfer of embryos that developed from single-nucleated zygotes obtained by injection of round spermatids into oocytes. Fertil. Steril., 70, 67–70.[ISI][Medline]

Bedford, J.M., Bent, M.J. and Calvin, H.J. (1973) Variation in the structural character and stability of the nuclear chromatin in morphologically normal spermatozoa. J. Reprod. Fertil., 33, 19–29.[Medline]

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Bench, G.S., Friz, A.M., Corzett, M.H. et al. (1996) DNA and total protamine masses in individual sperm from fertile mammalian subjects. Cytometry, 23, 263–271.[ISI][Medline]

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Submitted on August 25, 2000; accepted on December 11, 2000.