Dorota Fiszer

Major histocompatibility complex expression on human, male germ cells: a review.

PROBLEM: The male reproductive compartment is an immunologically privileged site. The expression pattern of human leukocyte antigens (HLAs) may play an important role in the maintenance of immune tolerance toward differentiating gametogenic cells. This review presents current knowledge about HLA gene expression on human, male germ cells, on mRNA and protein levels, and on their methylation status.

ORIGINAL ARTICLE: The Role of IL-6, IL-10, TNF-α and its Receptors TNFR1 and TNFR2 in the Local Regulatory System of Normal and Impaired Human Spermatogenesis

Problem  To investigate the expression of genes coding for selected cytokines with antagonistic functions (IL‐6, IL‐10, TNF‐α) as well as TNF‐α receptors (TNFR1 and TNFR2) in correct spermatogenesis (normal proliferation), maturation arrest (proliferation inhibited) and testicular tumors (overgrowth).

The role of IL-6, IL-10, TNF-alpha and its receptors TNFR1 and TNFR2 in the local regulatory system of normal and impaired human spermatogenesis.

Problem  To investigate the expression of genes coding for selected cytokines with antagonistic functions (IL‐6, IL‐10, TNF‐α) as well as TNF‐α receptors (TNFR1 and TNFR2) in correct spermatogenesis (normal proliferation), maturation arrest (proliferation inhibited) and testicular tumors (overgrowth).

Interleukin-1 system in testis — quantitative analysis

There is a growing body of evidence suggesting that interleukin-1 and other cytokines may influence mammalian testis function. IL-la activity seems to be tissue specific, and it appears in literature as a testicular interleukin-1 (tIL-1)1. Its activity was detected both in testicular interstitium and seminiferous epithelium. Being secreted in para-or autocrine manner, IL-1 inhibits steroidogenesis in Leydig cells, what suggests the modulatory role of this cytokine in LH-dependent testosterone synthesis. Sertoli cells are also capable to produce IL-1. The level of interleukin-1 expression in these cells is distinctly connected with the intensity of DNA synthesis in gametogenic cells. Thus IL-1 could be a nutritional factor, important for activity of seminiferous epithelium. Its expression both in Sertoli and Leydig cells depends on the local concentrations of hCG, FSH and LH and the other cytokines (e.g. TNFa) or growth factors in testis. Hence, IL-1 appears as another element of regulatory network essential for maintenance of testis physiology.2

SCF and c-kit expression profiles in male individuals with normal and impaired spermatogenesis

The transcription levels of stem cell factor (SCF) and c‐kit were examined using real‐time RT PCR in interstitial and intratubular cell fractions, as well as in tissue homogenates from normal, azoospermic and neoplasmic patients. Peripheral blood mononuclear cells (PBMC) were used as a systemic control. The observed level of c‐kit expression in all investigated groups was generally higher than the expression of SCF. The highest (statistically significant) level of c‐kit was noted in testicular tumours (the greater part of which were represented by seminomas) in contrast to SCF mRNA, which may indicate an association between c‐kit overexpression and seminoma development. In Sertoli cell only syndrome, almost equal levels of SCF and c‐kit transcripts were noted. These results may indicate Leydig cells as the alternative source of c‐kit gene transcription. SCF transcript values were low and comparable among the analysed subgroups except that in maturation arrest at spermatocyte stage, the SCF gene expression was statistically higher than in testicular tumours. It appears from the study that c‐kit has been a dynamic gene, changing its activity in a variety of testicular pathologies while being expressed in all testicular compartments but clearly overexpressed in testicular tumours of seminomatous origin.

Analysis of mRNA expression for interleukin-1 genes on human testicular cells

We have investigated mRNA expression for IL-1 alpha and IL-1 beta gene on fractionated human testicular cells. Using RT-PCR and Northern blot hybridization technique we detected the presence of IL-1 alpha transcripts, predominantly in the intratubular compartment of the testis, comprising gametogenic and Sertoli cells. We were also able to detect mRNA for IL-1 alpha on the testicular interstitium, but at significantly lower levels. The intertubular compartment of the testis, mainly consisting of macrophages and Leydig cells, appeared however, to be a site for IL-1 beta gene expression. Our experimental data confirm previous results obtained in animal models indicating that the testis is capable of producing interleukin-1 under physiological conditions. Testicular IL-1 may function as a tissue-specific factor modulating both spermato- and steroidogenic activity of human testis.

Quantitative mRNA Analysis of IL‐1 Gene System in Human Testis

Problem: There is a growing body of evidence that interleukins exhibit modulatory activity on development of reproductive cells. In this context, there appears to be a role for IL‐1, which is also produced in human testis. We have analysed transcripts of IL‐1 gene system (IL‐1α, IL‐1β, IL‐1RI, IL‐1RII and IL‐1RA) to evaluate the possible link between the level of gene(s) transcription and their function.

Specific Fab fragments recovered by phage display technique recognizing human spermatozoa

Human hybridoma cell lines are often unstable and loose ability for antibody production. Sometimes, they show low and varying levels of heavy and light chains synthesis. Therefore it is reasonable to preserve generated specificities of light and heavy chains by cloning them to phagemid vector and creating phage display library. The aim of this study was to construct phage display library of Fab fragments recognizing sperm surface antigens. The source of mRNA constituted seven hybridoma cell lines producing antisperm antibodies which was proved by ELISA, and agglutination test as well as by inhibition of sperm to penetrate hamster oocytes. Fragments of cDNA encoding kappa/lambda and gamma chains were cloned into pComb3HSS phagemid vector and amplified in XL-1Blue. The library was panned against whole unfixed sperm cells. Three positive clones selected after fourth round of panning showed heavy chain belonging to VH4 family, two of them (G28, K61) possessed lambda chain from VL2 family and one (H43) kappa chain from VK1 family. As these Fabs revealed similarities to antibodies against some proteins involved in sperm motility and cell fusion it can be suggested that these Fabs may be a cause of infertility. Finally, we proved that it is feasible to preserve specificities produced by human hybridomas using phage display technique and we recovered some Fabs which may be of diagnostic and research value, and may also have some value for contraceptive vaccine.

Crem Activator Isoforms in Normal and Impaired Human Spermatogenesis Analyzed by Real Time RT-PCR

cAMP responsive element modulator (CREM) activator isoforms are involved in mammalian spermatogenesis and spermiogenesis. CREM proteins are highly expressed in postmeiotic germ cells of rodents and primates. Homozygous CREM inactivated mice exhibit round spermatid maturation arrest. The lack of CREM expression at both the mRNA and protein levels is associated with spermatid maturation arrest in infertile patients. Using real-time RT-PCR, we have examined the levels of CREM activator isoform mRNAs: CREMθ1, CREMθ2 and CREMt2 + Ex-γ in gametogenic and interstitial cell fractions from normal human testis, in homogenized tissue samples from spermatogenic arrest and from testicular tumors. We have shown for the first time the presence of CREM activator isoform containing exon γ (CREMτ2 + Exγ) in normal human spermatogenesis. Among the three CREM isoforms, CREMθ1 was expressed in its highest level in the male gonads. In comparison, CREMθ2 mRNA was significantly less suggesting that the P3 promoter is much more active in human testis than the P4 promoter. Minimal-nill levels of mRNA for either of the CREM activator isoforms were detected in lymphocytes or in gonadal tissues from patients with SCOS (Sertoli Cell Only Syndrome). This data underlines the significance of CREMθ1 isoform in the regulation of transcription during post-meiotic germ cell differentiation.

Complex nature of the human antisperm antibody response in SCID mice

Human peripheral blood mononuclear (PBMs) cells were introduced into the peritoneal cavity of severely‐combined immunodeficient (SCID) mice in concentrations of 2.5–4.0 × 107 cells per mouse. Whole mononuclear cell suspensions were used either unstimulated or following primary in vitro culture with human spermatozoa. In some experiments, immunodepletion of CD8+ cells was carried out prior to grafting. Lymphocytes were obtained from nonsensitized (to antigen) human subjects or from individuals who were primed in vivo (vasectomized individuals in case of sperm antigens). An enzyme‐linked immunosorbent assay was employed to assess total human immunoglobulin (G or M) levels as well as the specificity of the antibodies generated. We have been successful by generating primary and secondary immune responses with ‘naïve’ human lymphocytes, challenged with chlamydia or ovalbumin but without adjuvant or CD8+ immunodepletion; however, we were unable to induce specific antibodies to spermatozoa under this regime in SCID male mice. We then employed female SCID mice, treated with sperm antigen extracts (glycosylated or deglycosylated) encapsulated in liposomes and human lymphocytes obtained from ‘naïve’ or pre‐sensitized in vivo subjects. It was found that the most pronounced humoral response to sperm antigens was obtained with deglycosylated antigens and PBMs from vasectomized (in vivo pre‐primed to spermatozoa) individuals. A presented SCID mice model can be helpful at understanding of antisperm antibody development and the molecular nature of generated antibodies to modified sperm antigenic entities.