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Martin Bergmann

Publications and source records attributed to Martin Bergmann.

At least 19 recordsLinked to original sources
PubMed2006

DNMT1 and HDAC1 gene expression in impaired spermatogenesis and testicular cancer.

DNA methylation catalyzed by DNA methyltransferases (DNMTs) and histone deacetylation catalyzed by histone deacetylases (HDACs) play an important role for the regulation of gene expression during carcinogenesis and spermatogenesis. We therefore studied the cell-specific expression of DNMT1 and HDAC1 for the first time in human testicular cancer and impaired human spermatogenesis. During normal spermatogenesis, DNMT1 and HDAC1 were colocalized in nuclei of spermatogonia. While HDAC1 was additionally present in nuclei of Sertoli cells, DNMT1 was restricted to germ cells exhibiting a different expression pattern of mRNA (in pachytene spermatocytes and round spermatids) and protein (in round spermatids). Interestingly, in infertile patients revealing round spermatid maturation arrest, round spermatids lack DNMT1 protein, while pachytene spermatocytes became immunopositive for DNMT1. In contrast, no changes in the expression pattern could be observed for HDAC1. This holds true also in testicular tumors, where HDAC1 has been demonstrated in embryonal carcinoma, seminoma and teratoma. Interestingly, DNMT1 was not expressed in seminoma, but upregulated in embryonal carcinoma.

DNA (Cytosine-5-)-Methyltransferase 1↗
PubMed2006

Functional characterization of male germ cell-specific CREM isoforms.

Due to alternative promoter usage, splicing, and translational initiation, expression of the cAMP-responsive element modulator (CREM) gene results in the production of functionally different CREM proteins with either activating or repressing potential on target gene expression. Recently, we demonstrated 2 novel isoforms (CREM-2-F-G-H-Ib and CREM-2-G-H-Ib) in various germ cell types during normal and impaired human spermatogenesis. In contrast to known isoforms, these exhibit a transactivation domain but lack a kinase-inducible domain (KID) domain resulting in a disruption of the open reading frame. In the present study, we functionally analyzed these isoforms. Investigation of both in vitro and in vivo expressed proteins from human testis RNA suggests that a novel upstream open reading frame in exon 2 is translated from isoform CREM-2-F-G-H-Ib, giving rise to a full-length protein. Furthermore, in both isoforms, usage of downstream adeninethymine-guanines (ATGs) for translation initiation could be observed. Sequence-specific DNA binding of CREM isoforms was confirmed by electrophoretic mobility shift assays. Luciferase reporter gene assays in cells transfected with novel CREM cDNAs demonstrated that protein kinase A dependent stimulation was inhibited by coexpression of CREM-2-F-G-H-Ib but not of CREM-2-G-H-Ib.

Amino Acid Sequence↗
PubMed2006

Effects of estradiol infusion in GnRH immunized boars on spermatogenesis.

Active immunization of boars against gonadotropin-releasing hormone (GnRH) inhibits luteinizing hormone (LH) and testicular steroids, so that mitosis of spermatogonia is reduced and apoptosis increased. To clarify whether high amounts of estrogens which are synthesized in the boar testis support spermatogenesis, a group of 6 boars was immunized against GnRH and then infused for 7 weeks with estradiol (E2-17beta). For comparison, intact boars and immunized boars were infused with saline only. Testicular tissue was then analyzed by immunocytochemistry for apoptosis (TUNEL, EM), mitosis (Ki67), and estrogen receptor alpha (ER alpha). The specificity of ER alpha staining was confirmed by RT-PCR and Western blot. Immunization decreased LH and testosterone to minimal concentrations in immunized and E(2)17beta-infused immunized boars, whereas follicle-stimulating hormone (FSH) was not significantly altered. Estradiol decreased to base levels after immunization. Infusion increased E2-17beta in peripheral blood plasma of the immunized boars to physiological levels. Except for A-spermatogonia, all spermatogenic cells decreased after immunization by about 60%. After estradiol infusion, cell counts increased again and were intermediate between control and immunized boars. Mitosis of spermatogonia was reduced by nearly 50% due to immunization but was partly restored by E2-17beta infusion. Expression of ER alpha was localized in spermatogonia, suggesting stimulation of mitosis, which was further confirmed due to its predominant occurrence in stage I of the seminiferous epithelial cycle (main stage of cell division). Apoptosis was minimal in boars but elevated in the other 2 groups. Data showed that estrogens in physiological concentrations supported mitosis but were not sufficient to normalize sperm production because apoptosis was still high.

Animals↗
PubMed2006

Genetic imprinting during impaired spermatogenesis.

Disorders in genetic imprinting are discussed as potential genetic risk in assisted reproduction technology (ART), where most of the natural selection mechanisms are bypassed. As currently only limited information about genomic imprinting in disruptive spermatogenesis is available, we analysed the imprinting state of the paternally methylated gene H19 in various germ cell populations derived from seminiferous tubules exhibiting impaired spermatogenesis. Different germ cell types were isolated by laser microdissection from human testicular paraffin sections. Although the methylation state of the maternally imprinted gene SNRPN was investigated by methylation-specific PCR (M-PCR) to establish the isolation method, methylation of H19 was analysed by a single-strand conformation-based method. Contamination by somatic Sertoli cells was excluded because of Sertoli cell-specific vimentin immunohistochemistry before germ cell laser microdissection. We demonstrate correct genetic imprints for H19 even in spermatogonia selected from seminiferous tubules exhibiting spermatogenic arrest at the level of spermatogonia, providing no evidence for incorrect genomic imprinting in spermatozoa from infertile men used for ICSI.

Autoantigens↗
PubMed2006

Structure of mammalian spermatozoa in respect to viability, fertility and cryopreservation.

Morphological assessment of spermatozoa has a long history and it is generally accepted that specific morphologic structural deviations correlate with male sub- and infertility. Although many different and also new methods are used in semen analysis, light microscopy is still used for routine morphological evaluation. This paper gives an overview about the detailed structure of physiological mammalian spermatozoa as well as the most common morphological deviations in correlation to fertility. This should be the basis for explanation of problems resulting from semen cryopreservation. General aspects of semen cryopreservation should be regarded before to facilitate the understanding of methods and mechanisms.

Animals↗
PubMed2006

Mitotic activity of Sertoli cells in adult human testis: an immunohistochemical study to characterize Sertoli cells in testicular cords from patients showing testicular dysgenesis syndrome.

During puberty, normal somatic Sertoli cells undergo dramatic morphological changes due to the differentiation of immature pre-Sertoli cells in functionally active adult Sertoli cells. Sertoli cell maturation is accompanied with loss of their mitotic activity before onset of spermatogenesis and loss of pre-pubertal and occurrence of adult immunohistochemical Sertoli cell differentiation markers. Testes of infertile adult patients often exhibit numerous histological signs of testicular dysgenesis syndrome (TDS) such as microliths, Sertoli cell only (SCO) tubules, tubules containing carcinoma in situ and immature seminiferous tubules (Sertoli cell nodules). Sertoli cell tumours, however, are very rare neoplasms possibly due to the fact that the mechanism and temporal origin of neoplastic Sertoli cells underlying Sertoli cell tumourigenesis still remain unknown. To clarify the state of Sertoli cell differentiation in both immature seminiferous tubules of adult patients with TDS and Sertoli cell tumour, we compared the expression of the Sertoli cell differentiation markers vimentin, inhibin-alpha, anti-Muellerian-hormone, cytokeratin 18, M2A-antigen, androgen receptor and connexin43 with that of SCO tubules with hyperplasia. In addition, we demonstrated for the first time the existence of proliferating Sertoli cells by Ki67- and PCNA-immunostaining in Sertoli cell nodules of the adult human testis. Our data indicate that mitotically active Sertoli cells in Sertoli cell nodules will be arrested prior to puberty and, contrary to dogma, do not represent foetal or neonatal cells. Since all markers in Sertoli cell nodules revealed a staining pattern identical to that in neoplastic Sertoli cells, but different to that in Sertoli cells of SCO tubules with hyperplasia, it may be speculated that Sertoli cell tumours in adult men may originate from Sertoli cell nodules.

Adult↗
PubMed2006

Determination of some enzymes and macro- and microelements in stallion seminal plasma and their correlations to semen quality.

Seminal plasma is very important for sperm metabolism as well as sperm function and survival and transport in the female genital tract. Analysis of enzyme activities and concentrations of elements can estimate integrity and function of sperm cell membranes. In man much data are available about biochemical analyses of seminal plasma. However, not many studies have been conducted in horses yet. We collected ejaculates from 72 stallions, measured the volume, obtained seminal plasma by centrifugation and examined spermatozoa with light microscopy for motility, concentration, for dead sperm and morphology. Of seminal plasma fluid, we measured activities of aspartate-amino-transferase (AST), gamma-glutamyl-transferase (GGT), alkaline phosphatase (AlP), acid phosphatase (AcP) and lactate-dehydrogenase (LDH) as well as concentrations of sodium (Na(+)), potassium (K(+)), total and ionised calcium (Ca(TOTAL)/Ca(2+)), magnesium (Mg(2+)), phosphate (P), chloride (Cl), copper (Cu), iron (Fe) and zinc (Zn). In addition, correlations among different parameters in light microscopy and seminal plasma were statistically examined by using the Spearman rank correlation coefficient. Median enzyme activities for AST, GGT, AlP, AcP and LDH were 80.0, 7,500, 30,200, 20.0, 81.0 IU/L, respectively. Concentrations of Na(+), K(+), Ca(TOTAL), Ca(2+), Mg(2+), P, Cl were 110.5, 22.1, 2.9, 1.7, 3.1, 1.1 and 114.5 mmol/L, and of microelements Cu, Fe and Zn were 17.8, 1.9 and 13.2 micromol/L, respectively. Furthermore, we found significant correlations between semen volume as well as sperm concentration and AST, GGT, AlP, AcP and LDH as well as Fe and Zn. This made us propose a primary testicular and epididymal origin of these parameters. Significant correlation between GGT and motility may be a sign for its function for cell protection against free radicals. LDH activity significantly correlates with motility and progressive motility, live:dead-ratio and pathomorphology. In our study, LDH seems to be the most predictive enzyme for semen quality. This is the first report about GGT, AcP and LDH activities as well as iron in equine seminal plasma.

Acid Phosphatase↗
PubMed2006

Transition from preinvasive carcinoma in situ to seminoma is accompanied by a reduction of connexin 43 expression in Sertoli cells and germ cells.

Carcinoma in situ (CIS) represents the preinvasive stage of human germ cell tumors, but the mechanism leading to pubertal proliferation and invasive malignancy remains unknown. Among testicular gap junctional proteins, connexin 43 (Cx43) represents the predominant Cx, and, previously, an inverse correlation between synthesis of Cx43 protein and progression of tumor development was detected. In the present study, using cDNA microarray analysis, in situ hybridization, semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) from tissue homogenates, RT-PCR from microdissected tubules with normal spermatogenesis and CIS, and seminoma cells from invasive seminoma, we asked whether reduction of Cx43 protein is accompanied by a change of Cx43 transcripts. We detected a significant downregulation of Cx43 at mRNA level in Sertoli and germ cells starting in seminiferous tubules infiltrated with CIS and resulting in a complete loss in seminoma cells. It was demonstrated, that downregulation of Cx43 expression in neoplastic human testis takes place at the transcriptional level and starts in CIS. This reduction of Cx43 expression further suggests that early intratubular derangement in Cx43 gene expression and disruption of intercellular communication between Sertoli cells and/or Sertoli and preinvasive tumor cells may play a role in the progression phase of human seminoma development.

Adult↗
PubMed2006

Altered expression of ZO-1 and ZO-2 in Sertoli cells and loss of blood-testis barrier integrity in testicular carcinoma in situ.

Carcinoma in situ (CIS) is the noninvasive precursor of most human testicular germ cell tumors. In normal seminiferous epithelium, specialized tight junctions between Sertoli cells constitute the major component of the blood-testis barrier. Sertoli cells associated with CIS exhibit impaired maturation status, but their functional significance remains unknown. The aim was to determine whether the blood-testis barrier is morphologically and/or functionally altered. We investigated the expression and distribution pattern of the tight junction proteins zonula occludens (ZO) 1 and 2 in normal seminiferous tubules compared to tubules showing CIS. In normal tubules, ZO-1 and ZO-2 immunostaining was observed at the blood-testis barrier region of adjacent Sertoli cells. Within CIS tubules, ZO-1 and ZO-2 immunoreactivity was reduced at the blood-testis barrier region, but spread to stain the Sertoli cell cytoplasm. Western blot analysis confirmed ZO-1 and ZO-2, and their respective mRNA were shown by RT-PCR. Additionally, we assessed the functional integrity of the blood-testis barrier by lanthanum tracer study. Lanthanum permeated tight junctions in CIS tubules, indicating disruption of the blood-testis barrier. In conclusion, Sertoli cells associated with CIS show an altered distribution of ZO-1 and ZO-2 and lose their blood-testis barrier function.

Adult↗
PubMed2005

Specific localisation of gap junction protein connexin 32 in the gastric mucosa of horses.

In the glandular stomach, gap junctional intercellular communication (GJIC) plays an important role in the gastric mucosal defense system, and loss of GJIC is associated with ulcer formation. In spite of the high incidence of gastric ulcers in horses, particularly at pars nonglandularis, the presence of gap junctions in the equine stomach has not yet been studied. The objective was to obtain basic data on the distribution of gap junction protein connexin 32 (Cx32) in the different regions of normal equine gastric mucosa. Samples of mucosa were taken from seven horses at cardiac, fundic, and pyloric region and pars nonglandularis. To detect Cx32, immunohistochemical staining and Western blot analysis were performed. Corresponding mRNA was shown by RT-PCR and localised in tissue sections by in situ hybridisation. Cx32 was found in the glandular regions, whereas it was not detectable in squamous mucosa. Within the glandular epithelium, Cx32 was abundant in surface and foveolar cells and decreased towards the proliferative zone of the glands. These results suggest that gap junctions develop during the maturation of surface cells. Whether the lack of Cx32 at pars nonglandularis contributes to its susceptibility for developing ulcers, has to be further elucidated.

Animals↗
PubMed2005

Advanced fertility diagnosis in stallion semen using transmission electron microscopy.

Routine semen analysis of stallions is based on light microscopy (LM). However, there are still a number of animals that are subfertile or even infertile not being identified with conventional semen analysis. The objective of this study was to investigate the suitability of transmission electron microscopy (TEM) for advanced fertility diagnosis in stallion. We examined ejaculates of 46 stallions with known fertility. Animals were divided into three different groups: group 1, fertile stallions (pregnant mares> or =70%, n=29); group 2, subfertile stallions (pregnant mares 10-69%, n=14); group 3, infertile stallions (pregnant mares<10%, n=3). Ejaculates were collected in spring 2002. Conventional semen analysis (volume, sperm concentration, motility, live:dead ratio and percentage of morphologically normal sperm) was immediately performed after semen collection. Ultrastructural analysis included the evaluation of 200 acrosomes, heads, midpieces and cross-sections of tails as well as 100 longitudinal sections of tails from every ejaculate. Using LM, we found a significant increase of morphological deviations from 24.5% (x ) in group 1 to 34.5% in group 2 and 73.5% in group 3. Using TEM, we found a significant increase of detached acrosomes from 6.1% in group 1 to 7.6% in group 2 and 21.4% in group 3. Deviations in tubule pattern were also increased (but not significant) from 2.7% in fertile and 2.8% in subfertile to 11.4% in infertile stallions as well as multiple tails from 1.9% in fertile to 2.0% in subfertile and 8.9% in infertile. Our data indicate that TEM is suitable for advanced fertility diagnostic in stallions, giving a connection between fertility and morphology. It suggests that the most likely reason for sub- and infertility in stallion in case of increased LM pathomorphology of semen are acrosomal alterations, especially detached acrosomes.

Acrosome↗
PubMed2005

Standardization strategy for quantitative PCR in human seminoma and normal testis.

Housekeeping genes are commonly used as endogenous references in quantitative RT-PCR. Ideally these genes are constitutionally expressed by all cell types and do not vary under experimental conditions. Tissues of 9 normal testes and 22 classical pure seminoma were obtained for RNA-extraction. Real-time RT-PCR was used to examine the mRNA-expression of ubiquitin C, beta-actin, GAPDH, 18S ribosomal RNA (18S rRNA) and porphobilinogen-deaminase (PBGD). Additionally, 3 normal testicular tissues and 39 seminoma, including 1 normal testis and 17 seminoma of the RT-PCR group, were utilized for microarray analyses. Ubiquitin C (protein degradation) was down-regulated, GAPDH (carbohydrate metabolism), beta-actin (cytoskeleton), 18S rRNA (ribosome) and PBGD (porphyrin metabolism) were up-regulated in seminoma. A normalization of the target gene data with up-regulated housekeeping genes would equalize or underestimate up-regulated data and overestimate down-regulated data. We demonstrate that none of the investigated housekeeping genes is suitable for normalization of the target gene RT-PCR data, but may be essential for tumor metabolism in human seminoma. Further, we developed a standardization strategy, which is applicable to many experimental investigations.

Biomarkers, Tumor↗
PubMed2005

CREM activator and repressor isoform expression in human male germ cells.

The transcription factor cAMP-responsive element modulator (CREM) is known to play a vital role for male fertility as it has been demonstrated that male mice lacking a functional CREM gene are infertile. The CREM gene consists of 14 exons. Owing to alternative exon splicing, CREM gene expression results in the production of functionally different CREM proteins with either activating or repressing potential on target gene expression. Infertile men have been reported to reveal a substantial reduction of CREM activators and additional inaccurately spliced CREM transcripts. In the present study, we analysed the expression of CREM transcripts with the recently reported leader exons theta1 and theta2 and identified a new putative CREM repressor, namely theta1-F-H, in patients with impaired spermatogenesis. In addition, we applied single cell microdissection followed by RT-PCR with leader exons B, theta1 and theta2 to assign the expressed CREM activator and repressor isoforms to specific germ cell types within the seminiferous epithelium. Contrary to dogma, we demonstrated CREM activator and repressor isoforms in all germ cell types, but not in Sertoli cells. However, the percentage of germ cell samples that revealed positive RT-PCR signals for these CREM activators was higher in spermatocytes and round spermatids than in spermatogonia and elongated spermatids. It remains unknown whether these activator transcripts are physiologically active. Our data suggest a fine-tuning between CREM activator and repressor isoforms in normal germ cells that might be disturbed during impaired spermatogenesis.

Adult↗
PubMed2004

Differentiation markers of Sertoli cells and germ cells in fetal and early postnatal human testis.

The definition of the temporal sequence of appearance of fetal markers during prenatal and early postnatal development in Sertoli and germ cells may be important for understanding the mechanisms underlying their reexpression in disorders of the adult testis. For this reason, we studied the expression of Sertoli and germ cell markers in 25 human testes spanning a period from 8 gestational weeks to 4 years. Well-characterized antibodies were employed to anti-Müllerian hormone (AMH), cytokeratin 18 (CK18), vimentin (VIM), M2A-antigen (M2A), germ cell alkaline phosphatase (GCAP), and somatic angiotensin-converting enzyme (sACE) on formalin-fixed and microwave-pretreated paraffin sections. In Sertoli cells, AMH and VIM were consistently present. While VIM and CK18 were coexpressed in embryonic testes, CK18 was progressively downregulated and completely absent from the 20th gestational week. M2A was absent or moderately expressed in fetal Sertoli cells but increased during further development. In germ cells, M2A was consistently found in primordial germ cells (PGCs) as well as in M- and T1-prespermatogonia. In contrast, sACE and GCAP were absent from PGCs but were a distinct feature of late M- and early T1-prespermatogonia and appeared predominantly between the 18th and the 22nd gestational weeks. Both T2-prespermatogonia and postnatal prespermatogonia were devoid of any marker. While CK18 represents a differentiation marker for fetal Sertoli cells, M2A, GCAP, and sACE can be used as differentiation markers for the discrimination of different germ cell types during human prespermatogenesis. Because various immunophenotypes reflect distinct differentiation stages, this knowledge may be important for understanding adult testicular pathology.

Aging↗
PubMed2004

Expression of activator of CREM in the testis (ACT) during normal and impaired spermatogenesis: correlation with CREM expression.

The cAMP-responsive element modulator (CREM) is involved in regulating gene expression in haploid spermatids. Transcriptional activity of the CREM protein is thought to be regulated by activator of CREM in the testis (ACT). Applying RT-PCR and in situ hybridization, cell-specific gene expression of ACT was demonstrated in man, cynomolgus monkey and mouse. During normal spermatogenesis, RT-PCR revealed a strong signal in all three species. We sequenced monkey ACT cDNA and demonstrated that the putative amino acid sequence is highly conserved between these species. In situ hybridization demonstrated ACT mRNA in mid and late pachytene spermatocytes and in round spermatids. Among four infertile men with round spermatid maturation arrest (RSMA), only one patient revealed a strong signal for ACT, while three patients displayed a weak signal for both RT-PCR and in situ hybridization, although germ cells normally expressing ACT were present in these patients. In addition, CREM knockout mice known to be infertile due to RSMA also exhibited only a weak amplification product for ACT cDNA. ACT mRNA was barely detectable in some round spermatids, but was completely absent in pachytene spermatocytes. Database search revealed two and one CRE within the putative human and mouse ACT promoters respectively. Our findings indicate a conserved function of ACT during the evolution of mammalian spermatogenesis and suggest a role for CREM in ACT transcriptional regulation.

Adult↗
PubMed2004

From carcinoma in situ to testicular germ cell tumour.

The mechanisms of invasive tumour development from pre-invasive CIS are unknown. We examined changes in functional parameters of the tubular wall according to the increase in CIS cells and tubular size. Immunohistochemistry was performed on 37 testicular specimens from 25 patients with carcinoma in situ and/or malignant germ cell tumour for the detection of actin/myosin in myocytes, and laminin/integrin alpha 6 in the basement membrane of seminiferous tubules. Tumour cells were detected by PlAP, Sertoli cells by inhibin alpha and vimentin and by cytokeratin 18/connexin 26 immunoreactivity, which is selectively expressed together with CIS. Areas showing clusters of tumour cells surrounded by a fibrous sheet could be identified as enlarged tubules because of focal Sertoli cell-specific co-expression of inhibin alpha, vimentin, cytokeratin 18, and connexin 26 immunoreaction. These clusters exhibited an intact basement membrane shown by a persistent laminin/integrin alpha 6 immunoreactivity, but myocytes had lost their contractility indicated by the loss of myosin/actin immunoreactivity. They often showed septa originating from the fibrous sheet containing numerous capillaries. Focal areas of syncytiotrophoblastic cells within classical seminoma also expressing inhibin alpha, cytokeratin 18, and connexin 26 could be differentiated from single Sertoli cells within tumor cell clusters by typical hCG but absence of vimentin immunoreactivity. In contrast to the current concept of CIS cells passing the tubular wall, these data provide evidence for an additional theory, i.e. that the switch from pre-invasive CIS to invasive tumour takes place in situ by tubular enlargement due to tumour cell proliferation followed by Sertoli cell degeneration and conversion of the tubular wall into connective tissue.

Actins↗
PubMed2003

Different CREM-isoform gene expression between equine and human normal and impaired spermatogenesis.

Histone-to-protamine exchange causes chromatin condensation ceasing gene expression in elongating spermatids. Gene expression of protamines is regulated by the transcription factor cAMP-responsive element modulator (CREM). Altered CREM expression results in male infertility, as shown by CREM-knock-out mice being sterile due to round spermatid maturation arrest and patients exhibiting round spermatid maturation arrest revealing a lack or substantial reduction of both CREM-mRNA and CREM-protein. Similar defects in histone-to-protamine exchange have been suggested in infertile stallions exhibiting enlarged sperm heads. The CREM-gene consists of 14 exons. Alternative exon splicing results in the production of both activator and repressor proteins. To further clarify the role of different CREM-isoforms for male infertility, the expression pattern of various CREM-isoforms during equine and human normal and impaired spermatogenesis was investigated by RT-PCR. Stallions with normal spermatogenesis expressed six activators and three repressors. In men three activators and seven different repressors were detected. In one stallion and patients with impaired spermatogenesis, only repressors were found. It is concluded that (i). stallion and man reveal a different CREM expression pattern, (ii). the expression of CREM activators is a prerequisite for normal spermatogenesis, and (iii). the lack of CREM activator expression results in male infertility.

Alternative Splicing↗