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

Human Reproduction Round spermatids from infertile men exhibit decreased protamine-1 and -2 mRNA Klaus Steger 2 Klaus Failing 1 Thomas Klonisch 0 Hermann M.Behre 5 Martina Manning 4 Wolfgang Weidner 4 Lothar Hertle 3 Martin Bergmann 2 Sabine Kliesch 3 0 Institute of Anatomy and Cell Biology 1 Department of Biomathematics, Institute of Veterinary Physiology, University of Giessen 2 Institute of Veterinary Anatomy 3 Department of Urology, University of Mu nster , Germany 4 Department of Urology, University of Giessen 5 Andrology Unit, Department of Urology, University of Halle 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%; PRM2mRNA: 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. infertile men/in-situ hybridization/intracytoplasmic sperm injection/protamines/spermatogenesis - 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), 1992]. 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., 1995; Novero et al., 1997). 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., 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 (...truncated)