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
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Abstract |
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Key words: infertile men/in-situ hybridization/intracytoplasmic sperm injection/protamines/spermatogenesis
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Introduction |
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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., 1987, 1988
; LeLannic et al., 1993
; Lescoat et al., 1993
; Wykes et al., 1995
; Prigent et al., 1996
; Saunders et al., 1996
; Wykes et al., 1997
; Steger et al., 2000
).
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., 1988; Bench et al., 1996
). In contrast, human sperm nuclei contain significantly less protamine (Bench et al., 1996
) being consistent with the observation that, in man, histone-to-protamine exchange is only about 85% complete (Tanphaichitr et al., 1978
; Gatewood et al., 1987
; Prigent et al., 1996
). In transgenic mice, over-expression of PRM1 protein at its normal time of synthesis does not affect spermiogenesis (Peschon et al., 1987
; Zambrowicz et al., 1993
), while premature translation of PRM1-mRNA causes precocious chromatin condensation resulting in male sterility (Lee et al., 1995
). 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., 1995). All oocytes cleaved further to 4-cell embryos. Fishel et al. reported the implantation of such embryos after uterine transfer (Fishel et al., 1995
). 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., 1995
). 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., 1997
; Fishel et al., 1997
; Vanderzwalmen et al., 1997
; Yoshida et al., 1997
; Barak et al., 1998
; Bernabeu et al., 1998
; Kahraman et al., 1998
; Al-Hasani et al., 1999
; 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., 1999
). In contrast, higher fertilization and pregnancy rates have been achieved using spermatozoa obtained by TESE (Craft et al., 1993
; Devroey et al., 1995
; Silber et al., 1995
; Tournaye et al., 1995
). 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., 1995
; Yoshida et al., 1997
). 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., 1999
).
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.
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Materials and methods |
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For histological evaluation, 5 µm sections were stained with haematoxylineosin and scored, according to Holstein and Schirren (1983). In-situ hybridization was performed on 10 testes from 10 men (Table I, 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 I
, group II) exhibited at least qualitatively normal spermatogenesis (scores 108), while 27 (Table I
, group III) revealed impaired spermatogenesis (scores 71).
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Digoxigenin (DIG)-labelled cRNA-probes
Production of DIG-labelled cRNA-probes was performed as described previously (Steger et al., 1998, 2000
). 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., 1998, 2000
). 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 I). Group I (control) comprised patients 110 revealing obstructive azoospermia and quantitatively normal spermatogenesis (score 10). The 55 infertile men undergoing a combined TESE/ICSI treatment were divided into patients 1138 (group II) revealing at least qualitatively normal spermatogenesis (scores 108) and patients 3965 (group III) exhibiting impaired spermatogenesis (scores 71).
Since it is already known that PRM1-mRNA and PRM2-mRNA are present in round spermatids (Steger et al., 2000), 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, 1993). Differences between groups IIII 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.
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Results |
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PRM1-mRNA and PRM2-mRNA are known to be expressed in round spermatids (Steger et al., 2000). 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 I
, Figure 1A
). 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 I
, Figure 1B
). 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 31 (PRM1-mRNA: 30.3 ± 10.8%, PRM2-mRNA: 27.7 ± 10.1%) than in testes with scores 74 (PRM1-mRNA: 37.1 ± 9.4%, PRM2-mRNA: 39.7 ± 8.3%).
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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 IIII. 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 2). 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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Discussion |
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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., 1994; Martin-du-Pan and Bischof, 1995
). 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., 1994) and the genes for human PRM1 and PRM2 (Oliva and Dixon, 1991
) 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., 2000
). It is known that about 13% of azoospermic men exhibit a deletion on the long arm of their Y chromosome (Reijo et al., 1995
). 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., 1999a
,b
). 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., 1973). Subsequently, human male infertility has been related to both anomalous nucleoprotein composition (Silvestroni et al., 1976
; Chevaillier et al., 1987
) and aberrant nucleoprotein ratio (Balhorn et al., 1988
; Belokopytova et al., 1993
). 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., 1999
).
Phosphotungstic acid (PTA)-staining (Courtens and Loir, 1981) and aniline blue staining (Dadoune et al., 1988
) 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., 1996
, 1999
).
Chromomycin A3 (CAM3)-staining is an indicator of protamine deficiency in sperm chromatin. The range of CAM3-staining in fertilizing samples varies from 1520% (Bianchi et al., 1993) or 860% (Lolis et al., 1996
) in normospermic donors and was about 70% in infertile male factor donors (Bianchi et al., 1993
). 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., 1993
; Lolis et al., 1996
). 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., 1996
).
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., 1987). In infertile men, the complete absence of PRM1 and PRM2 protein in ejaculated spermatozoa (Silvestroni et al., 1976
) and of PRM2-mRNA in round spermatids (Ziyyat et al., 1999
) 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., 1990
).
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., 1993). 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., 1998
). The observation that Percoll-selected spermatozoa exhibit a high fertilizing capacity (Colleu et al., 1996
) 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 108; qualitatively normal spermatogenesis) and in about 30% (scores 71; 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., 1995; Schmiady et al., 1996
).
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.
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Acknowledgements |
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Notes |
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References |
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Submitted on August 25, 2000; accepted on December 11, 2000.