Mol. Hum. Reprod. Advance Access originally published online on April 22, 2005
Molecular Human Reproduction 2005 11(5):373-379; doi:10.1093/molehr/gah169
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Cellular expression of protamine 1 and 2 transcripts in testicular spermatids from azoospermic men submitted to TESE–ICSI
1Spermiologie-Biologie de la Reproduction, et unité 422 INSERM, hôpital A. Calmette, Boulevard du Professeur Jules Leclercq, CHRU-Faculté de Médecine, F-59037, 2Klinik für Urologie und Kinderurologie, Rudolf-Buchheim-Strasse 7, D-35385 Giessen, Germany, 3Biologie de la Reproduction, hôpital Jeanne de Flandre, CHRU, F-59037, 4Biolille, 17 rue de la Digue, BP 117, F-59016, 5Unité de Biostatistiques, Faculté de Médecine, Pôle Recherche and 6Service d'Andrologie, hôpital A. Calmette, Boulevard du Professeur Jules Leclercq, CHRU, F-59037 Lille cedex, France
7 To whom correspondence should be addressed at: Laboratoire de Spermiologie, hôpital A. Calmette, Boulevard du Professeur Jules Leclercq, CHRU, F-59037 Lille cedex, France. Email: mitchell{at}lille.inserm.fr
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Abstract |
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Testicular sperm extraction (TESE) combined with ICSI is used to treat azoospermia. However, the factors that influence the outcome of ICSI in this situation are ill-defined. We sought to investigate the expression of protamine 1 (PRM1) and protamine 2 (PRM2) transcripts in testicular spermatids from obstructive and non-obstructive azoospermic men with impaired spermatogenesis. The relationship between PRM1 and PRM2 transcript levels and the TESE–ICSI outcome was evaluated. The cellular expression of PRM1 and PRM2 mRNAs in single testicular spermatids from 41 azoospermic patients (in whom testicular spermatozoa were subsequently recovered and submitted for TESE–ICSI) was determined by radioactive in situ hybridization. Group I contained seven men with congenital, obstructive azoospermia and whose testicular biopsies indicated quantitatively normal spermatogenesis. Group II consisted of 18 azoospermic men with moderately impaired spermatogenesis. Sixteen men with non-obstructive azoospermia and severely deranged spermatogenesis (i.e. mixed atrophy with small foci of spermatids and spermatozoa) constituted group III. The spermatids of men with severely deranged spermatogenesis exhibited significant lower PRM1 mRNA expression than in the other patient groups. There were no significant inter-group differences in PRM2 mRNA expression. Spermatid PRM1 expression was lower in non-pregnant couples than in pregnant couples. The low number of spermatids in cases of mixed atrophy with small spermatogenic foci is associated with significantly lower PRM1 expression and a lower pregnancy rate. These results emphasize the role of PRM1 as a potentially critical factor in post-ICSI embryonic development.
Key words: azoospermia/in situ hybridization/protamine/testis
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Introduction |
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The DNA in human sperm chromatin is partitioned into both a nucleohistone and a nucleoprotamine fraction, with 15% of the DNA bound by histones and 85% of the DNA bound by protamines (Tanphaichitr et al., 1978
; Gatewood et al., 1990). During spermiogenesis, the protamine proteins play an integral role in spermatid chromatin compaction. Thus, protamines are responsible for the high condensation level seen in mature spermatozoa chromatin (Hecht, 1989; Oliva and Dixon, 1991; Dadoune, 1995; Steger, 1999). The resulting highly condensed chromatin may facilitate sperm motility and protect sperm DNA from damage and may even imprint the male genome (Oliva and Dixon, 1991; Balhorn et al., 1999). The question of a possible relationship between the state of condensation of spermatozoal nuclear chromatin and reduced male fertility was raised for the first time by Bedford et al. (1973); and, indeed, subsequent studies have confirmed this relationship (Bedford and Calvin, 1974; Zamboni, 1987; Francavilla et al., 1996). The level of decondensation is inversely related with sperm fertilizing ability (Carrell et al., 1998). Incomplete chromatin condensation has been observed in testicular spermatozoa of men affected by non-obstructive azoospermia (Hammadeh et al., 1999). Abnormal chromatin packaging in ejaculated spermatozoa is associated with infertility or early miscarriage (Evenson et al., 1999) and with low oocyte fertilization after ICSI (Sakkas et al., 1999). In the two decades (and following the elucidation of the protamine protein sequences by McKay et al. (1985, 1986)), numerous studies have focused on these sperm nuclear proteins (reviewed in Aoki and Carrell, 2003): we are thus now beginning to understand the link between the protamines and male fertility. A positive relationship between alterations in protamine protein content of human spermatozoa and male fertility has been demonstrated. An abnormal protamine 1/protamine 2 protein (PRM1/PRM2) ratio has been reported in the sperm of infertile human males, indicating that the relative amount of each protamine is important for proper spermatid differentiation (Balhorn et al., 1988; Khara et al., 1997; Carrell and Liu, 2001). De Yebra et al. (1998) have suggested a relationship between increased levels of PRM2 precursors and incomplete processing of PRM2. In addition to the above-cited observations concerning the protamine status of ejaculated spermatozoa, studies have also been carried out to evaluate the protamine transcript levels in testicular tissues, in order to clarify whether there might be a correlation with the spermatids' fertilizing capacity. The number of PRM1 and PRM2 mRNA positive spermatids is lower than normal in men with impaired spermatogenesis (Steger et al., 2001). A correlation has been demonstrated between successful fertilization and the ratio of the percentage of PRM1/PRM2 expressing spermatids. In fact, the PRM1/PRM2 protein and PRM1/PRM2 mRNA ratios could be more important than the absolute amount of protamine for fertility in humans (and also in mice (Cho et al., 2001)).
In addition to the relationship between the absolute numbers of PRM1- and PRM2-expressing cells and male fertility, an important question remains concerning the importance in fertility of the amounts of PRM1 and PRM2 transcripts per single spermatid. On the basis of real-time quantitative PCR results, Steger et al. (2003) recently suggested that the amount of PRM1 mRNA in spermatids was lower than normal in infertile patients undergoing testicular sperm extraction (TESE)–ICSI. The present study is the first to investigate levels of PRM1 and PRM2 transcripts in testicular biopsies from azoospermic men at the cellular (i.e. single spermatid) level. A quantitative radioactive in situ hybridization (ISH) method was used to evaluate the optical density of the hybridization signal for PRM1 and PRM2 mRNAs in spermatids from Bouin-fixed and paraffin-embedded testicular tissues from azoospermic patients in whom testicular sperm were present. The resulting data were compared to the ICSI outcome in order to explore the relationship between this parameter and the levels of PRM1 and PRM2 transcripts.
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Materials and methods |
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Testicular tissue and morphological evaluation
Testicular biopsies were taken from 41 azoospermic men aged 26–54 years in whom testicular spermatozoa were present. Group I consisted of seven men with congenital, obstructive azoospermia and complete, active spermatogenesis. FSH levels ranged from 2 to 8 IU/l (normal values: 1–10 IU/l). On the basis of histological observations, group II was made up of azoospermic patients whose testicular biopsies revealed moderately impaired spermatogenesis, i.e. a low number of spermatids (about 5–10 in each seminiferous tubule cross-section). Group III consisted of non-obstructive, azoospermic patients whose biopsies revealed severely deranged spermatogenesis—the seminiferous epithelium displayed Sertoli cells in most tubules and just a few tubules showing a tiny spermatogenic focus progressing through rare spermatids and spermatozoa. These biopsies were termed ‘mixed atrophy’. Individuals showing Sertoli cell-only or complete, early arrest of spermatogenesis without a positive TESE were not included in this investigation.
A large biopsy (200–700 mg, depending on testicular volume) was recovered from a single testicular site in order to minimize the possible devascularization reported after multiple biopsies in the same testis (Schlegel and Su, 1997). The largest part of the biopsy was immediately placed in 1 ml of IVF medium (Scandinavian, Gothenburg, Sweden). The second smallest part was fixed by immersion in Bouin's fixative. Tissues were dehydrated and embedded in paraffin. The histological evaluation was performed on 5 µm sections stained with haematoxylin–eosin.
Testicular sperm extraction
The testicular biopsies were transported rapidly to the IVF laboratory, where they were weighed and transferred into a tissue culture dish (Falcon 1006; Becton Dickinson Co., Meylan, France) in 200 µl of IVF medium. The tubule contents were extracted with two sterile scalpels. The resulting suspension from the tissue dispersion was carefully aspirated, placed into a 1.5 ml centrifuge tube and homogenized in 1 ml of IVF medium. The spermatozoa were extracted by passing the suspension several times through a 21-gauge needle. After centrifugation at 1400 g for 10 min, the presence of spermatozoa was examined by placing a microdroplet of the solution in a chamber under an inverted microscope at 400x magnification.
Cryopreservation of testicular spermatozoa
First-attempt IVF–ICSI cycles with fresh spermatozoa were performed for four couples in group I, six in group II and six in group III (Table I). For frozen–thawed spermatozoa, the samples were diluted with cryopreservation medium (Irvine Scientific, Santa Ana, CA, USA) and aspirated in straws which were automatically frozen in a minicool (Nicool LM 10, Air Liquide, Marne la Vallée, France) before being plunged into liquid nitrogen. The straws were placed in a 37°C chamber for 5 min for thawing. The frozen-thawed spermatozoa were placed into 0.6 ml of IVF for 10 min at 37°C with 5% CO2 and centrifuged at 2000 g for 10 min. The pellet was resuspended into 400 µl of culture medium and kept in an oven at 37°C with 5% CO2 until the start of the ICSI procedure.
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ICSI procedure
The cumulus-corona cells were removed by incubation for about 20 s in hyaluronidase 80 IU/10 ml (Fertipro N.V., Beerneem, Belgium) and then aspirated with a glass pipette with an opening of between 150 and 300 µm. The oocytes were rinsed several times in IVF medium and were then observed under an inverted microscope at 400x magnification. The presence or absence of the first polar body was noted. ICSI was performed in a Petri dish (Falcon 100G Beckton–Dickinson, France) containing 5 µl droplets of IVF medium for spermatozoa, two 2 µl droplets of PVP (Medicult) in the centre of the dish, and eight 5 µl droplets of IVF oocyte medium. The Petri dish was surrounded with lightweight, detoxified paraffin oil. A single spermatozoon was selected, aspirated tail first into the injection pipette (JCD International, France) and transferred to the PVP droplet. Even if the spermatozoa were immotile, their tails were damaged by pressing with the injection pipette against the bottom of the dish prior to injection into the oocyte. Embryo cleavage and quality were evaluated 20 and 44 h after injection.
Synthesis of cRNA probes
The plasmid vectors pGEM-T containing 153 and 294 bp fragments of the human PRM1 and PRM2 genes, respectively, were linearized by cutting at a single site with Nco I for the antisense probe and Not I for the sense probe. In vitro transcription was performed with SP6 RNA polymerase or T7 polymerase (for the antisense and sense probes, respectively) and [35S]CTP (Amersham Pharmacia Biotech, Orsay, France).
Tissue preparation and ISH
To determine the expression of PRM1 and PRM2 mRNAs in testicular biopsies, paraffin 5 µm serial sections were collected, mounted onto gelatin-coated slides and stored at 4°C. Tissue sections were subsequently deparaffinized and rehydrated. ISH was performed according to our routine, well-characterized procedure (Mitchell et al., 1997, 1999). Briefly, after proteinase K permeabilization, processed sections were hybridized with the hybridization buffer mix containing [35S]-labelled PRM1 or PRM2 cRNA probes (20 000 cpm/µl). Overnight hybridization at 54°C was followed by RNase treatment and a series of stringent washes, including a high-stringency wash at 60°C. Hybridized slides were dehydrated in 70 and 100% ethanol in ammonium acetate and then dipped in K5 emulsion (Integra Biosciences; Eaubonne, France). After a 10-day exposure, slides were developed. Following autoradiographic development, the tissue sections were counterstained using a weak haematoxylin stain. Probe specificity checks included incubation of sections of each testicular biopsy with 35S-labelled sense probes, pretreatment with ribonuclease and co-incubation with a 100-fold excess of unlabelled antisense probes. None of these control experiments resulted in specific labelling.
Serum FSH and inhibin B
Serum FSH and inhibin B were measured by immunoassay, as described in detail elsewhere (Pigny et al., 1997, 2000).
Quantitative analysis
Representative microscopic fields were selected at 400x magnification. At least four serial sections per biopsy were quantified. Up to 10 seminiferous tubules containing round spermatids were counted on each biopsy section. For both protamines, quantification of the signal intensity was performed by light microscopy under darkfield illumination. Quantification of PRM1 and PRM2 grain density was performed using an image analysis program (Densirag, Biocom, Les Ulis, France) and an Axiophot microscope (x60 epi-illumination darkfield objective, Zeiss, Göttingen, Germany) interfaced to a PC computer with a high-resolution camera. The relative signal intensity was assessed for all labelled spermatids above background for PRM1 and PRM2 antisense transcripts. The mean grain density per single spermatid (±SD) was calculated.
Statistical analysis
Statistical analysis was performed using SAS software, version 8.2. Due to the small sample sizes, inter-group comparisons were assessed using the Kruskall–Wallis non-parametric test. Post-hoc tests were performed using a Bonferroni correction. Correlations were assessed with the Spearman rank correlation test. The Wilcoxon non-parametric test was used to relate PRM1 and PRM2 expression to successful pregnancy. Statistical significance was considered as corresponding to a P value <0.05.
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Results |
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Localization of PRM1 and PRM2 transcripts
The human testicular samples used in this study were Bouin-fixed paraffin-embedded tissues which provided excellent histological detail and preservation of cellular RNA. For both PRM1 and PRM2 mRNA expressions, clusters of silver grains were observed predominantly in the adluminal region of the seminiferous epithelium (Figure 1). All testicular tissues showed a similar tubular distribution of silver grains. Silver grains indicating the presence of PRM1 and PRM2 transcripts were numerous and were localized in spermatids, in accordance with the reported expression patterns for these transcripts. The silver grains were not evenly distributed across all spermatids: ISH revealed a weak signal in some spermatids and a strong signal in other (neighbouring) cells, showing an intrinsic variability in the level of PRM1 and PRM2 transcripts. In cases with small spermatogenic foci (group III), those tubules displaying active spermatogenesis possessed a low number of post-meiotic cells expressing PRM1 (Figure 1C) and PRM2 (Figure 1F) transcripts.
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Quantitative PRM1 and PRM2 mRNA expression
Table I gives an overview of data grouped according to the individuals' azoospermia status and testicular histological examination. The mean optical density per single spermatid related to PRM1 in the 41 testicular samples was 76.47, with an SD of 34.33 (and minimum and maximum quantiles of 29.23 and 170.74, respectively). For PRM2, it was 109.25±44.52 (with minimum and maximum quantiles of 49.9 and 195, respectively).
PRM1 mRNA expression
In men with congenital, obstructive azoospermia (group I), the mean silver grain density for PRM1 expression per single spermatid was 81.97±16.92. The hybridization signal for PRM1 was 91.88±43.85 in spermatids of azoospermic patients characterized by moderately impaired spermatogenesis (group II). Finally, in cases of non-obstructive azoospermia with small spermatogenic foci (group III), the mean optical density related to PRM1 was 56.73±11.62. The Kruskal–Wallis non-parametric test revealed that the expression of PRM1 mRNA differed significantly (P=0.0076) between groups I and III. Post-hoc pairwise analysis showed a statistically significant, lower level of PRM1 expression (P<0.05) in group III compared to both groups I and II (Table I, Figure 1B and C).
PRM2 mRNA expression
In men in group I, the mean silver grain density for PRM2 expression was 124.20±57.41. The hybridization signal for PRM2 was 91.68±35.75 in spermatids of patients from group II. In patients with severely impaired spermatogenesis (group III), the mean optical density related to PRM2 was 127.65±38.96. The Kruskal–Wallis non-parametric test revealed that there was no significant difference in PRM2 mRNA expression between groups I, II and III (P=0.07).
Serum FSH and inhibin B
Men with congenital obstructive azoospermia (group I) and azoospermic men with moderately impaired spermatogenesis (group II) exhibited mean FSH values of 4.72±0.91 IU/l and 7.66±0.79 IU/l, respectively (normal range: 1–10 IU/l). The Kruskal–Wallis non-parametric test revealed a significant difference in FSH values between groups I and III (P=0.0007). Post-hoc pairwise analyses revealed significantly higher levels of FSH in group III compared to group I and to group II (Table I).
Mean serum inhibin B values ranged from 192.71±43.90 pg/ml (mean for group I) to 115.68±13.21 pg/ml (mean for group II). Low but detectable levels of inhibin B (mean value of 54.87±20.71 pg/ml) were observed in group III. The Kruskal–Wallis non-parametric test revealed a significant difference in inhibin B values between groups I and III (P=0.0004). Post-hoc pairwise analyses showed significantly lower levels of inhibin B in group III compared to both group I and group II (Table I).
A negative correlation could be demonstrated between serum FSH and inhibin B for groups I and III (r=–0.776, P<0.001).
ICSI outcome
Table I shows the outcome of ICSI after grouping the cycles together on the basis of obstructive azoospermia status (group I) and testicular histology (groups II and III). There were no differences between groups I and III in terms of the mean number of oocytes (retrieved, injected and fertilized), the embryo transfer rate and the mean number of embryos obtained and transferred. Pregnancy rates and delivery rates were influenced by the testicular histology: pregnancies and deliveries were mostly obtained in cases with normal (group I: five clinical pregnancies and seven live babies for 15 ICSI cycles) and moderately impaired spermatogenesis (group II, eight clinical pregnancies and 10 live babies for 30 ICSI cycles). Finally, in the group with severely impaired spermatogenesis (23 ICSI cycles), two pregnancies and four live babies were obtained. These results emphasize that testicular spermatozoa retrieved from azoospermic men are able to fertilize oocytes after ICSI. Testicular spermatozoa retrieved in cases of non-obstructive azoospermia and severely impaired spermatogenesis exhibited a poor ability to produce pregnancy.
PRM1 and PRM2 expression and ICSI outcome
There was a significant relationship between PRM1 expression and the pregnancy rate (Table II). Pregnancies were obtained in cases where the mean PRM1 expression per spermatid was 94.43±37.93, while no pregnancy occurred when the mean PRM1 expression was significantly lower, at 68.62±28.35 (P=0.0179). This finding suggests that the success of fertilization was related to the amount of PRM1 transcripts in testicular spermatids. In contrast, the pregnancy rate was not statistically related to the PRM2 expression.
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Discussion |
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This study is the first to investigate the levels of PRM1 and PRM2 mRNA per single spermatid from testicular biopsies of azoospermic men, and the possible relationship of these parameters to the ICSI outcome. The results showed that severely deranged spermatogenesis in non-obstructive azoospermia were associated with a significantly lower expression of PRM1 per single spermatid, when compared with obstructive azoospermia. In contrast, PRM2 expression did not differ between obstructive and non-obstructive azoospermic men. We also showed that the success of fertilization was related to the amount of PRM1 transcript in testicular spermatids.
Radioactive in-situ hybridization (followed by quantitative cellular analysis of PRM1 and PRM2 mRNA expression per single spermatid) has given us new insights into the ability of spermatids to express PRM1 and PRM2 mRNAs. The ISH approach indicated that there are high, intratubular variations in PRM1 and PRM2 expression. In comparison with other molecular biology methods, the advantage of ISH is that it assesses mRNA expression in individual cells. In line with reports from other authors (Wykes et al., 1995; Siffroi et al., 1998; Steger et al., 2000), the human protamine genes are expressed post-meiotically in spermatids. We noted that most of the labelled spermatids exhibited high levels of PRM1 and PRM2 transcripts. Both PRM1 and PRM2 require only a short exposure, reflecting the fact that these genes are expressed at a high level. However, we did observe intratubular variability in staining intensity, which was of particular interest for PRM1 expression in spermatids from testicular tissues displaying moderately impaired spermatogenesis. Similar variability in PRM2 expression was also observed within the three patient groups. In fact, it is well known that during normal spermatogenesis, the PRM1 and PRM2 hybridization signals vary according to the spermatids' differentiation step (Steger et al., 2000); at an early stage of maturation (steps 1 and 2), spermatids express weaker hybridization signals than in steps 3 and 4. Thus, the variability of PRM1 and PRM2 expression might be related (at least in part) to the stages of spermatid maturation. Furthermore, the level of PRM2 was higher than the level of PRM1 in normal human testis (Wykes et al., 1995; Steger et al., 2000). In our observation, PRM2 transcript levels were high in the group of obstructive azoospermic men, since transcripts for PRM1 corresponded to about 75% of the amount measured for PRM2 mRNA.
The most important result is that the PRM1 mRNA expression level was significantly lower in spermatids from testicular tubules displaying small spermatogenic foci when compared with tubules displaying normal or moderately impaired spermatogenesis. In case of severely altered spermatogenesis in non-obstructive azoospermia, it can be hypothesized that the dramatically reduced number of spermatids is associated with defective differentiation. Thus, male fertility can be related both to the number of spermatids expressing protamines (Steger et al., 2001) and the total amount of protamine expressed. These observations emphasize PRM1's key role in the quality of chromatin condensation in male fertility. An ultrastructural study performed in mature spermatids has shown certain alterations in chromatin condensation in cases of impaired spermatogenesis (Francavilla et al., 2001), and altered chromatin condensation represented one of the defects encountered in spermatozoa from infertile men (Zamboni, 1987; Francavilla et al., 1996).
In contrast, we did not observe a significant difference in the PRM2 mRNA expression level when comparing obstructive and non-obstructive azoospermia. This result is in line with data from Steger et al. (2003). As suggested by Bench et al. (1996) and Corzett et al. (2002), PRM2 might be more important in the context of mammalian speciation than in fertility. However, the chimeras resulting from disruption of the expression of the PRM1 or PRM2 genes were infertile and displayed nuclear condensation abnormalities (Cho et al., 2001). These observations show that both protamines are essential for normal sperm development.
In the present study, spermatid PRM1 expression was significantly lower in non-pregnant couples, in comparison with pregnant couples. These observations suggest that testicular spermatozoa with low PRM1 expression (such as those in tubules from severely deranged spermatogenesis) do not have the same ability to result in pregnancy as spermatozoa from testes with normal or moderately impaired spermatogenesis, where PRM1 expression is high. The low number of spermatids, combined with their defective differentiation, may have a negative effect on post-ICSI embryonic development. Our results emphasize PRM1's probable role as a critical factor in embryo implantation. TESE followed by ICSI is an effective treatment in patients with non-obstructive azoospermia. Fertilization and pregnancy were achieved when testicular spermatozoa were used for ICSI (Ng et al., 2000). With TESE–ICSI, fertilization and cleavage rates have been reported to be similar to those in men with normal spermatogenesis (Devroey et al., 1995, 1996; Silber et al., 1995, 1996; Van Steirteghem et al., 1998; Palermo et al., 1999). In cases of severely deranged spermatogenesis, it is generally admitted that the few testicular spermatozoa present are nevertheless potentially competent for syngamy and embryonic and fetal development. However, compared to ejaculated and epididymal spermatozoa, reduced fertilization and implantation rates have been reported when testicular spermatozoa were used for ICSI (Tournaye et al., 1996). The factors that influence the outcome of ICSI are numerous and ill-defined. At present, it is not clear whether disturbed chromatin condensation has an influence on ICSI results. In particular, many literature results argue in favour of a strong relationship between the protamine content of spermatozoa and spermatids on one hand and their fertilizing capacity on the other. The state of condensation of nuclear chromatin in spermatozoa is probably of major importance. In infertile men, the complete absence of PRM1 and PRM2 proteins in ejaculated spermatozoa (Silvestroni et al., 1976) and of PRM2 mRNA in round spermatids (Ziyyat et al., 1999) has been reported. More recently, Steger et al. (2001, 2003) have shown that PRM1 expression and the PRM1/PRM2 mRNA ratio in round spermatids from testicular biopsies may serve as potential predictive factors for the outcome of ICSI. These authors emphasized interesting perspectives in the diagnosis of testicular biopsies using molecular biological techniques. Our data investigating the protamine mRNA content of spermatids in azoospermic men could, therefore, help clarify the role of protamines in fertility.
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Acknowledgements |
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The technical assistance of R.M.Siminski and M.H.Gevaert (Service Commun de Morphologie Cellulaire, Faculté de Médecine, Lille, France) in providing histological sections is gratefully acknowledged. Silver grain quantification was performed in INSERM Unit 159 Paris, France (Director: Dr J.Epelbaum). This study was sponsored by INSERM Unit 422 Lille, France (Director: Dr J.C.Beauvillain), and the interregional cooperation G4 Reproduction.
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Submitted on February 1, 2005; accepted on March 14, 2005.
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