Validation of reference genes in human testis and ejaculate.

Beta-actin (ACTB), glyceraldehyde-3-phosphate-dehydrogenase (GAPD), Heat Shock Protein 1, beta (HSPCB) and Adenosine Triphosphate subunit 5 beta (ATP5B) with distinct functional characteristics and expression patterns were investigated as suitable references for gene expression studies. We determined the expression stability of the four reference genes in ejaculates, cryopreserved as well as fixed and paraffin-embedded testicular tissue (from fertile and subfertile men) applying real-time qRT-PCR and statistical analysis. The mean gene expressions (mean Ct value) were compared for each gene between the fertile and subfertile donors by using the Wilcoxon-Mann-Whitney test. We did not observe significant statistical differences between variability of genes. To detect random effects, we used the two-way analysis of variance with a hierarchical model. The results show no significant statistical differences between proband and repetition within the probands. Taken together, we concluded that ACTB, GAPD, HSPCB and ATP5B have a variable expression within these samples, but this variability is not statistically significant. This finding demonstrated that all these genes could be appropriated for further studies on gene expression in ejaculate and testis tissue. Therefore, the selection of the suitable reference genes is highly specific for a particular experimental model and validation for each situation, on an individual basis, is a crucial requirement.

Protamine-1 represents a sperm specific gene transcript: a study in Callithrix jacchus and Bos taurus.

Spermatozoa are transcriptionally inactive cells, but contain acetylated histones, normally a characteristic of transcriptionally active cells. Acetylgroups are thought to represent epigenetic marks that are transmitted to the oocyte and are involved in starting gene expression in the zygote and in regulating gene expression during early embryogenesis. We performed reverse transcription polymerase chain reaction (RT-PCR) in the common marmoset monkey (Callithrix jacchus) and in bovine spermatozoa, oocytes, zygotes, two- and four-cell embryos to evaluate the presence of specific transcripts known to play a role during fertilisation and early embryo development, namely protamine-1 (PRM1), protamine-2 (PRM2), histone H1 (H1), histone H3 (H3), histone H4 (H4), cAMP-responsive element modulator (CREM), DNA methyltransferase-1 (DNMT1), TATA box-binding protein (TBP). All transcripts tested were present in spermatozoa of the common marmoset, while bull spermatozoa lack PRM2. Marmoset oocytes exhibited transcripts for H1, H3, H4 and TBP, whereas bovine oocytes revealed H1, H3, H4, CREM, DNMT and TBP mRNAs. In zygotes, we amplified H1, H4, TBP (marmoset) and PRM1, H1, H3, H4, CREM, DNMT1 and TBP (bovine). Two-cell embryos showed PCR products for H1, H3 and TBP in the marmoset. In the bovine, all transcripts could be observed except PRM2. In four-cell embryos, PCR signals were obtained for PRM1, H1, H3, H4 and TBP in the marmoset. In the bovine, all transcripts were detected except PRM2. Our data suggest that, in both C. jacchus and Bos taurus, PRM1 transcripts are delivered by the spermatozoon to the oocyte.

Apoptotic gene expression in potentially fertile and subfertile men.

Human sperm contain similar amounts of protamine-1 (P1) and protamine-2 (P2). Although an aberrant protamine ratio have been observed in subfertile men, functional evidence is provided by protamine knockout mice exhibiting male infertility. As sperm DNA integrity is known to be linked with DNA fragmentation and apoptosis, we investigated whether the DNA fragmentation factor 40 (DFF40) ratio or caspase (Casp4, Casp6) and tumor necrosis factor superfamily member 10 (TNFSF10) ratio together with the P1/P2 ratio represents a reliable biomarker to discriminate between fertile and subfertile men. Real-time quantitative RT-PCR was used for amplification of P1, P2 and DFF40 in 49 testicular biopsies. Casp4, Casp6 and TNFSF10 have been selected from a PCR apoptosis array and were further investigated in another group of testicular biopsies (22 subfertile men versus 11 potentially fertile men). Using Spearman's rank correlation coefficient analysis, we did not find a correlation between DFF40 and P1, P2, P1/P2, score, fertilization rate and age. In addition, logistic regression analysis demonstrated no statistically significant effect of the analyzed variables on pregnancy. A two-way analysis of variance with repeated measures of relative expression of Casp4, Casp6 and TNFSF10 versus P1 or P2 in potentially fertile men and subfertile patients demonstrated statistically significant differences between both groups, all tested gene combinations and the interaction between two genes and both groups in all cases analyzed. Furthermore, significant differences in the expression of Casp4 and TNFSF10 between the groups of potentially fertile and subfertile men could be demonstrated. In addition, the means of differences of selected gene combinations revealed that the protamine to apoptotic gene ratio is statistically different between both groups. Our data suggest that Casp4, Casp 6 and TNFSF10 are differentially expressed in potentially fertile and subfertile men and represent useful biomarkers for predicting male fertility in combination with P1 and P2.

Prognostic markers for competent human spermatozoa: fertilizing capacity and contribution to the embryo.

Management of male infertility largely depends on our understanding of cellular and molecular aspects of spermatogenesis as well as sperm function. Apart from standardized and comprehensive semen analysis, prognostic markers estimating the fertilizing capacity of either ejaculated spermatozoa or testicular spermatids are required. While there is general agreement that correct replacement of DNA-binding histones by protamines represents a prerequisite for achieving competent spermatozoa, especially in intracytoplasmic sperm injection (ICSI) where natural selection mechanisms are bypassed, the function of a variety of transcripts within the spermatozoa's cytoplasm and of remaining highly acetylated histones is still a matter of debate. Hence, this review brings the up-to-date research on mammalian spermatozoal chromatin composition into focus, which is discussed in conjunction with the paternal role on epigenetic reprogramming of the zygote following fertilization. As paternal transcripts have been demonstrated to be transmitted to the oocyte, it is now accepted that they represent more than solely remnants of previous transcriptional activity. Acetylation of histones, normaly a characteristic of transcriptional activity, was for a long time a miracle, as spermatozoa are transcriptionally inactive cells, but is now suggested to represent epigenetic marks that are transmitted to the oocyte and play an important role in the regulation of early gene expression in the developing embryo.

Aberrant mRNA expression of chromatin remodelling factors in round spermatid maturation arrest compared with normal human spermatogenesis.

During human spermiogenesis, chromatin condensation is associated with replacement of histones by protamines. This exchange is supported by acetylated histones and chromatin remodelling factors. Ten chromatin remodelling factor protein families are known. This study aims to analyse whether a different chromatin remodelling factor expression pattern exists between normal spermatogenesis and round spermatid maturation arrest as potential reason for impaired spermatogenesis and idiopathic male infertility. Laser capture microdissection was used to excise seminiferous tubules from testicular biopsies with normal spermatogenesis and round spermatid maturation arrest. RNA was isolated, first strand cDNA synthesis and pre-amplification were performed using Epigenetic Chromatin Remodelling Factors PCR arrays with 84 genes. Applying hierarchical cluster analysis, three gene expression clusters with six subgroups were identified. The expression pattern ranges from a few high expressed genes in round spermatid maturation arrest to a multitude of genes (74) which are more highly expressed in normal spermatogenesis than in maturation arrest. A total of 22 genes showed a significant difference between normal spermatogenesis and round spermatid maturation arrest (1 gene was up-regulated and 21 genes were down-regulated in the developmental arrest). The significantly different expression of chromatin remodelling factors between normal spermatogenesis and round spermatid maturation arrest may lead to impaired epigenetic information and aberrant transcription during sperm development representing one possible reason for developmental arrest of round spermatids.

Expression of histone 1 (H1) and testis-specific histone 1 (H1t) genes during stallion spermatogenesis.

In eukaryotic cells, the major protein constituents of the chromatin are histones, which can be divided into five classes, identified as H1, H2A, H2B, H3 and H4. During normal spermatogenesis, a testis-specific H1t is expressed in primary spermatocytes and believed to facilitate histone to protamine exchanges during spermiogenesis. In equine testes we detected the H1 protein at 22kDa by western blot analysis while H1t was detected at 29kDa. H1 protein was found to be expressed in all germ cells up to elongating spermatids (Sc) at stage IV. In peripubertal animals, there was a prolonged expression up to elongating spermatids (Sd1) at stage V. A fragment of the equine H1t gene was cloned (GenBank Accession No. AJ865320). The mRNA expression of H1t was found at the level in spermatogonia and in primary spermatocytes up to mid-pachytene at stage VIII/I, whereas H1t protein was found to be expressed up to round spermatides (Sa/Sb1) at stage VIII/I. In peripubertal animals, the H1t protein expression was detected up to elongating spermatids (Sb2) at stage II. Analysis of testes of different ages (< or =2 years) and (> or =3 years) by real-time RT-PCR revealed an increase of H1t mRNA expression, with a wide range of individual variety between 2 and 4 years old animals indicating a stable expression in animals older than 4 years old. This is the first study to show the testis-specific H1t in the stallion and gives evidence that the well-known peripubertal infertility in the stallion may be related to an insufficient histone to protamine exchange. The pattern of protamine gene expression, however, has still to be elucidated.