Molecular Human Reproduction, Vol. 9, No. 6, 331-336,
June 2003
© 2003 European Society of Human Reproduction and Embryology
Article |
Decreased protamine-1 transcript levels in testes from infertile men
Submitted on January 15, 2003; accepted on February 23, 2003
1 Institute of Veterinary Anatomy, Histology and Embryology, 2 Institute of Pathology, 3 Institute of Veterinary Physiology, Department of Biomathematics, 4 Urological Clinic of the University, University of Giessen and 5 Urological Clinic of the University, Muenster, Germany
6 To whom correspondence should be addressed at: Institut für Veterinär-Anatomie, -Histologie und -Embryologie, Frankfurter Strasse 98, 35392 Giessen, Germany. e-mail: Klaus.Steger{at}vetmed.uni-giessen.de
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ABSTRACT |
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Infertile men exhibit an aberrant protamine-1 (Prm1) to protamine-2 (Prm2) ratio at both the mRNA and protein level. We therefore investigated whether male infertility could be related to the amount of Prm1 and Prm2 mRNA by applying real time quantitative PCR following RNA extraction from routinely Bouin-fixed and paraffin-embedded testicular biopsies. Samples (n = 51) were normalized to the same amount and similar size of tissue sections. The threshold cycle (CT) representing a measure of the inital number of mRNA copies was significantly (P < 0.001) higher for Prm1, but not Prm2, and thus the amount of Prm1 mRNA was lower in men with at least qualitatively normal spermatogenesis (Prm1: 29.88 ± 2.99; Prm2: 34.28 ± 2.26) and impaired spermatogenesis (Prm1: 31.89 ± 2.54; Prm2: 35.59 ± 2.09) compared with men with obstructive azoospermia and quantitatively normal spermatogenesis (Prm1: 29.04 ± 1.02; Prm2: 34.91 ± 1.40). In addition, the Prm1 – Prm2 CT difference (
CT) was significantly (P < 0.001) decreased in these two groups. A negative correlation (r = –0.504; P < 0.001) was demonstrated between the score for efficiency of spermatogenesis and the CT for Prm1. These data suggest that the decreasing amount of Prm1 and, as a consequence, the aberrant Prm1:Prm2 mRNA ratio plays an important role for male infertility and may serve as a possible predictive factor for the outcome of ICSI. Key words: infertile men/protamines/real time quantitative PCR/spermatogenesis
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Introduction |
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Human male fertility is normally assessed on the basis of a semen profile reflecting the quality of the ejaculate, namely the total number of sperm and the percentage of morphologically normal and motile sperm (World Health Organization, 1992). In infertile men, however, these sperm parameters have been demonstrated to be not unrelated to successful fertilization in ICSI (Nagy et al., 1995; Novero et al., 1997). Since the selection of an unsuccessful sperm in ICSI has great emotional consequences for the couple, there is a need for an objective factor predicting the outcome of ICSI.
Correct histone-to-protamine exchange in haploid spermatids is known to play a vital role in the production of fertile sperm. Recently, male mice haplo-insufficient for protamine-1 (Prm1) and Prm2 have been reported to be infertile (Cho et al., 2001). While over-expression of protamine protein at its normal time of synthesis had no effect on spermatogenesis in transgenic mice (Zambrowicz et al., 1993), premature translation of protamine mRNA caused precocious chromatin condensation resulting in male sterility (Lee et al., 1995). Furthermore, it has been demonstrated that male mice lacking the gene for the cAMP-responsive element modulator (CREM), which is known to be involved in the regulation of protamine gene expression, are infertile due to round spermatid maturation arrest (Blendy et al., 1996; Nantel et al., 1996). In men with round spermatid maturation arrest, CREM expression is either drastically reduced or absent (Weinbauer et al., 1998; Steger et al., 1999). Male infertility has further been related to deficiencies in both the content of protamine protein in ejaculated sperm (Chevaillier et al., 1987; Balhorn et al., 1988; Bach et al., 1990; Blanchard et al., 1990; Belokopytova et al., 1993; Bench et al., 1998; deYebra et al., 1998) and the efficiency of protamine gene expression in spermatids of testicular biopsies (Steger et al., 2001).
To further clarify the role of protamine gene expression in male fertility, we have investigated Prm1 and Prm2 mRNA expression by real time quantitative PCR following RNA extraction from routinely Bouin-fixed and paraffin-embedded testicular biopsies. This novel technique supported and validated our previous findings (Steger et al., 2001) but, in addition, was more time-saving and more sensitive resulting in quantitative data. Furthermore, it was clearly demonstrated that the aberrant Prm1:Prm2 ratio in infertile patients is caused by a drastic reduction of Prm1, but not Prm2, gene expression.
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Materials and methods |
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Testicular tissue
In order to demonstrate the validity of the presented novel technique, testicular tissue samples used in this study were identical to those reported by Steger et al. (2001). Ethical approval and informed patient consent was obtained for the use of tissue samples in this study. In brief, testicular biopsies from 51 infertile men aged 23–54 years were analysed. In four patients with obstructive azoospermia after vasectomy, biopsies were carried out for diagnostic reasons during vasectomy reversal. These biopsies revealed normal spermatogenesis and served as controls. In 47 infertile patients with non-obstructive azoospermia, testicular tissue was obtained for diagnostic and therapeutic reasons at the same time. One part of the testicular specimen was immediately prepared and frozen for testicular sperm extraction (TESE), the other part 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 Bergmann and Kliesch (1998). The score is a measure of the efficiency of spermatogenesis describing the percentage of seminiferous tubules bearing elongated spermatids, i.e. score 10 means that 100% of the seminiferous tubules contain elongated spermatids. The study was performed on four testes (Table I, group I) with quantitatively normal spermatogenesis (score 10), as well as on 24 testes (Table I, group II) with at least qualitatively normal spermatogenesis (score 10–8) and 23 testes (Table I, group III) with impaired spermatogenesis (score 7–1). Infertile patients from groups II and III underwent TESE followed by ICSI.
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RNA extraction
RNA extraction was performed as detailed elsewhere (Fink and Bohle, 2002). In brief, the tissue sections (n = 5 each) were collected in a reaction tube and deparaffinized twice in 500 µl xylene for 10 min at 53°C. After centrifugation, the supernatant was discarded and the pellet resuspended in 200 µl 1 mol/l guanidine thiocyanate (GTC), 0.5% sarcosyl, 0.72% 2-mercaptoethanol, 20 mmol/l Tris–HCl, pH 7.5 (Stanta and Schneider, 1991). Adding proteinase K to a final concentration of 0.5 µg/µl, the samples were digested for 12–16 h (58°C). Note that digestion with proteinase K is indispensable for the liberation of RNA from crosslinking-fixed tissue (Fink et al., 2000) and RNA-binding proteins binding within the coding sequence of Prm1 and Prm2 mRNA in round spermatids (Steger et al., 2002). Afterwards, 20 µl 2 mol/l sodium acetate, 220 µl phenol (pH 4.3) and 60 µl chloroform/isoamylalcohol (24:1) were added, the samples were vortex-mixed and centrifuged at 12 000 g for 15 min at 4°C. The aqueous layer was collected, 1 µl glycogen (10 mg/ml) added, and precipitated with 200 µl isopropanol. Samples were frozen for 1 h at –20°C and centrifuged for 15 min at 12 000 g. The pellets were washed with 75% ethanol, air-dried and finally resuspended in 10 µl DEPC-treated H2O.
cDNA synthesis
For cDNA synthesis, 4 µl MgCl2 (5 mmol/l), 2 µl 10xPCR-buffer-II, 1 µl dNTP (10 mmol/l each), 1 µl random hexamers (50 µmol/l), 0.5 µl (10 IU) RNase inhibitor and 1 µl (50 IU) MuLV reverse transcriptase were added to 10 µl RNA. All products were purchased from Applied Biosystems (Weiterstadt, Germany). Samples were incubated at 20°C for 10 min and 43°C for 75 min. Reactions were stopped by heating to 95°C for 5 min. Subsequently, each sample was divided into two aliquots for Prm1 and Prm2 gene expression analysis.
Real time quantitative PCR
Real time quantitative PCR is based on the 5' nuclease activity of Taq polymerase for fragmentation of a dual-labelled fluorogenic hybridization probe. Using the Sequence Detection System 7700 (Applied Biosystems, Germany), RT–PCR for RNA quantification was performed as previously described (Fink et al., 1998). The threshold cycle (CT) indicates the fractional cycle number at which the amount of amplified target copies marks a fixed baseline and therefore is a measure for the initial target amount.
Based on the following equations, the ratio of Prm1 to Prm2 mRNA was calculated.
Prm10/Prm20 = K • 2 (CTPrm2 – CTPrm1)
and
log2 (K · Prm1/Prm2) = CT = CTPrm2 – CTPrm1
where Prm10 = initial number of protamine-1 mRNA copies; Prm20 = initial number of protamine-2 mRNA copies; E = efficiency of amplification; CTPrm1 = threshold cycle of protamine-1; CTPrm2 = threshold cycle of protamine-2; and K = constant.
After reverse transcription, 4 µl cDNA per sample were introduced for Prm1 and Prm2 mRNA analysis. 25 µl qPCRTM Mastermix (Eurogentec, Belgium), oligonucleotide primers (final concentration: 900 nmol/l, Table II), and hybridization probe (final concentration: 200 nmol/l, Table II) were added to an end volume of 50 µl. Cycling conditions were 95°C for 6 min, followed by 50 cycles of 95°C for 20 s, 59°C for 30 s, and 73°C for 30 s.
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Internal controls
Positive controls included the detection of amplification products for Prm1 and Prm2 mRNA in patients of the control group (Table I, group I). Negative controls included (i) samples containing only the reaction buffer without testicular tissue and (ii) samples lacking reverse transcriptase. Furthermore, intron-spanning primers were applied to avoid amplification of genomic DNA (Table II).
Statistical analysis
Statistical analyses were carried out applying the statistical program package BMDP (Dixon, 1993). Differences between groups I–III in the amount of Prm1 and Prm2, as well as in the Prm1:Prm2 mRNA ratio, have been analysed using one way analysis of variance (ANOVA). For the Prm1 – Prm2 CT difference (CT), a logarithmic transformation was applied, because the distribution of the difference was skewed to the right. If Levene’s test for equal variances showed different variances, the Welch test was performed for global mean comparison instead of ANOVA (both calculated with BMDP 7D). Subsequently, pairwise significance of mean differences between the groups has been checked with Tukey’s studentized range method. In addition, the correlation between the score, the CT of Prm1 mRNA, the CT of Prm2 mRNA, and the CT = CT Prm2 mRNA – CT Prm1 mRNA has been analysed (BMDP 6D) and represented graphically in the form of scatterplots. Logistic regression was employed to test whether successful fertilization is simultaneously related to the score, the CT of Prm1 mRNA, the CT of Prm2 mRNA, and the CT = CT Prm2 mRNA – CT Prm1 mRNA. P < 0.05 was considered to be significant.
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Results |
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In men with obstructive azoospermia and quantitatively normal spermatogenesis (Table I, group I), real time quantitative PCR resulted in threshold cycles for Prm1 and Prm2 cDNA of 29.04 ± 1.02 and 34.91 ± 1.40 respectively, the logarithm of the Prm1:Prm2 mRNA ratio was 5.95 ± 0.79 (Figure 1). The amount of Prm1 mRNA, but not Prm2 mRNA, was significantly reduced in infertile patients undergoing TESE/ICSI. In group II (Table I) representing patients with at least qualitatively normal spermatogenesis, the threshold cycles for Prm1 and Prm2 were 29.88 ± 2.99 and 34.28 ± 2.26 respectively, the logarithm of the Prm1:Prm2 mRNA ratio was 4.40 ± 1.67. In group III (Table I) representing patients with impaired spermatogenesis, the threshold cycles for Prm1 and Prm2 were 31.89 ± 2.54 and 35.59 ± 2.03 respectively, the logarithm of the Prm1:Prm2 mRNA ratio was 3.70 ± 1.76.
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One way ANOVA revealed that the amount of Prm1 mRNA, but not Prm2 mRNA, exhibits significant (P < 0.001) differences between groups I–III. In addition, significant (P < 0.001) differences between groups I–III could be demonstrated for the logarithm of the Prm1:Prm2 mRNA ratio. Applying the Tukey test, pairwise group comparisons showed statistically significant differences with P < 0.05. However, there was no significant difference in the amount of Prm2 mRNA between the three groups. A negative correlation could be demonstrated between the score for efficiency of spermatogenesis and the mean CT value of Prm1 mRNA (r = –0.504; P < 0.001), the mean CT value of Prm2 mRNA (r = –0.356; P < 0.008), and the logarithm of the Prm1:Prm2 mRNA ratio (r = 0.379; P < 0.004) (Figure 2).
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Infertile patients whose sperm were able to fertilize an egg applying ICSI revealed mean threshold cycles for Prm1 and Prm2 of 30.69 ± 2.93 and 34.87 ± 2.37 respectively, the logarithm of the Prm1:Prm2 mRNA ratio was 4.18 ± 1.69. Infertile patients whose sperm were unsuccessful in fertilizing an egg exhibited mean threshold cycles for Prm1 and Prm2 of 31.52 ± 3.00 and 35.10 ± 1.98 respectively, the logarithm of the Prm1:Prm2 mRNA ratio was 3.58 ± 1.95. Logistic regression showed no significant relationships between successful fertilization and the score, the CT of Prm1 mRNA, the CT of Prm2 mRNA, or the logarithm of the Prm1:Prm2 mRNA ratio.
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Discussion |
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During normal spermatogenesis, Prm1 and Prm2 proteins are present from step 4 to step 8 spermatids (Roux et al., 1987, 1988; LeLannic et al., 1993; Lescoat et al., 1993; Prigent et al., 1996) and are therefore expressed with temporal delay to their corresponding transcripts which have been demonstrated from step 1 to step 4 spermatids (Wykes et al., 1995; Saunders et al., 1996; Wykes et al., 1997; Steger et al., 2000, 2001). Correct histone-to-protamine exchange is a prerequisite for the complete differentiation of spermatids and the production of fertile sperm, since protamine–DNA interaction causes chromatin condensation, stopping gene expression in elongating spermatids (reviewed in Steger, 1999, 2001). This transcriptional stop prevents the addition of new genetic information in further differentiating spermatids. As a consequence, the protein equipment in sperm can be predicted by analysing the mRNA content in their predesessor cells, namely round spermatids in testicular biopsies. Since sperm obtained by TESE result in high fertilization rates (Craft et al., 1993; Devroey et al., 1995; Silber et al., 1995; Tournaye et al., 1995) and testes from patients with severely impaired spermatogenesis normally contain some small foci of spermatogenesis which allow TESE/ICSI to be carried out (Silber et al., 1995; Yoshida et al., 1997), the evaluation of testicular biopsies involving molecular biological analyses plays an increasing therapeutic role in the management of male infertility.
The present study has a direct bearing on this issue of determining the amount of Prm1 and Prm2 mRNA in testicular biopsies from men with normal and impaired spermatogenesis to further clarify the role of protamine gene expression for male fertility. Recently, it has been demonstrated that (i) RNA extraction from Bouin-fixed tissue results in successful RT–PCR (Robinson et al., 2001) and (ii) the threshold cycle obtained from formalin-fixed and paraffin-embedded tissue can directly be compared with data from cryomaterial (Godfrey et al., 2000; Specht et al., 2001; Cohen et al., 2002). Since both protamine transcripts are solely expressed in round spermatids, mRNA extraction from routinely Bouin-fixed and paraffin-embedded testicular biopsies followed by real time quantitative PCR is a suitable procedure for the study of quantitative gene expression in a special cell type and, in addition, represents a valuable tool for RNA extraction from archive material. Furthermore, this method is superior to in-situ hybridization followed by quantitative analysis of the percentage of positive round spermatids (Steger et al., 2001). Testicular biopsies can be analysed within 4–6 h. The method reported by Steger et al. (2001) takes 
1 week. Therefore, the technique presented in this study is more time-saving, tissue-saving and, in addition, more sensitive, resulting in quantitative data.
Prm1 mRNA, but not Prm2 mRNA, was significantly reduced in infertile men. Significant differences between fertile and infertile men were demonstrated for the difference in CT values (CT), that is the logarithm of the Prm1:Prm2 mRNA ratio. The latter result is in accordance with data from Balhorn et al. (1988) and Steger et al. (2001) reporting an aberrant Prm1:Prm2 ratio in infertile men at both the protein and mRNA level respectively. The present study, in addition, suggests Prm1 as the most critical factor for male fertility, since no significant differences between fertile and infertile men could be demonstrated for Prm2. This is in line with data obtained in Prm1 and Prm2 haplo-insufficient mice (Cho et al., 2001). Whereas only Prm1 was reduced in Prm1 chimeras, in Prm2 chimeras both Prm1 and Prm2 were reduced, the reduction being greater for Prm1 than for Prm2.
Recently, Corzett et al. (2002) determined the Prm1:Prm2 stoichiometry in the sperm of a variety of mammals. Data suggest substantial differences in the formation of the DNA–protamine complex in different mammalian species. While the Prm2 content of sperm chromatin is allowed to vary over a wide range between different species, the relative proportion of Prm1 and Prm2 is tightly regulated within a genus. The total protamine mass to DNA mass ratio is nearly identical (Bench et al., 1996). The variability in the Prm2 content suggests that the particular ratio of the two protamines may only be important in the context of speciation. Although changes in the expression of the two protamine genes are observed in distantly related species and different genera, variation does not appear to be tolerated within a species. Normal levels of Prm1 and Prm2, therefore, seem to be indispensable for the production of structurally and functionally intact sperm. Several studies (see below), including this one, demonstrated a positive relationship between alterations in the Prm1 and Prm2 content of human sperm and male infertility.
Patients with round-headed sperm have been shown to have an anomalous distribution of nuclear basic proteins containing more histones and less protamines than normal sperm (Blanchard et al., 1990). Silvestroni et al. (1976) reported the complete absence of protamine protein in ejaculates of infertile men. Balhorn et al. (1988) demonstrated that, in contrast to fertile men, sperm from infertile men display an aberrant Prm1:Prm2 protein ratio. The Prm2 protein, in addition, showed reduced affinity to DNA (Belokopytova et al., 1993). The reduction in the Prm2 protein content was concomitant with an increase in the amount of putative Prm2 protein precursors, suggesting incomplete processing of Prm2 protein (deYebra et al., 1998). Ziyyat et al. (1999) reported the complete absence of Prm2 mRNA in round spermatids. In contrast, Steger et al. (2001) demonstrated a reduction of the percentage of round spermatids expressing both Prm1 and Prm2 followed by an aberrant Prm1:Prm2 mRNA ratio. Furthermore, the focal reduction of Prm1 and Prm2 mRNA expression in seminiferous tubules with spermatogenic arrest at the level of round spermatids 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 sperm (Steger et al., 2001). Although genes for Prm1 and Prm2 are clustered on chromosome 16p13.3 (GeneBank, accession Z46940), data suggest that the expression of the Prm1 and Prm2 genes may actually be uncoupled in some developing spermatids of certain infertile men.
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Acknowledgements |
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The skilful technical assistance of J.Dern-Wieloch, G.Erhardt, A.Hax and A.Hild, Institute of Veterinary Anatomy, Histology, and Embryology, Giessen, as well as M.M.Stein, Institute of Pathology, Giessen, is gratefully acknowledged. Funding of this research programme was provided by DFG grant STE 892/1-3 and SFB 547, project Z1, Cardiopulmonary Vascular System.
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