Biomedical subjects

Martin Bergmann

Publications and source records attributed to Martin Bergmann.

27 records · Page 2Linked to original sources

PubMed2003

INSL3 ligand-receptor system in the equine testis.

We employed molecular and immunological techniques to investigate the expression of INSL3, a member of the insulin-like superfamily, in prepubertal testis, postpubertal testes exhibiting normal and disturbed spermatogenesis, and cryptorchid testes of male horses. In addition, the partial cDNA coding sequences of the equine homologue of the human relaxin/INSL3-receptor Lgr8 were determined. Nonradioactive in-situ hybridization with a cRNA probe for equine Insl3 and immunohistochemistry with a specific rabbit INSL3 antiserum localized Insl3 transcripts and immunoreactive INSL3 ligand to Leydig cells in all types of testes investigated. Quantitative polymerase chain reaction analysis revealed a down-regulation of Insl3 and an up-regulation of the relaxin/INSL3-receptor expression in unilateral cryptorchid versus descended testes. Western blot analysis of protein extracts from adult normal and cryptorchid testes and prepubertal testes showed a single immunoreactive band at 14.5 kDa, which correlates with the predicted size of equine proINSL3. Densitometric analysis of Western blot data of adult normal testes revealed significantly stronger expression of immunoreactive proINSL3 as compared to extracts derived from cryptorchid or prepubertal testes. Thus, decreased expression of immunoreactive INSL3 in cryptorchid and prepubertal equine testis is transcriptionally regulated. The detection of transcripts for equine Lgr8 in the testis has identified the testis as a potential target of INSL3.

Animals↗

PubMed2003

Update on the diagnostic safety for detection of testicular intraepithelial neoplasia (TIN).

Testicular intraepithelial neoplasia (TIN) of the testis is the noninvasive precursor of testicular germ cell tumours (GCT) and can be detected by a single random biopsy in 5% of patients with GCT in the contralateral testes. Although it is generally presumed that TIN is dispersed throughout the testis, we realize in about 60% of TIN bearing tissue close to testis tumours that its distribution is not homogenously diffuse, but may be focal. Thus we tested whether we can improve diagnostic safety in detecting TIN by increasing the number of biopsies. We could finally evaluate 295 men with proven testicular tumours. Three biopsies of contralateral testes were taken (each 5 mm length) from one surgical incision site and fixed in Bouin's solution or glutaraldehyde. TIN cells were histologically identified by their typical morphological characteristics and additionally by placental alkaline phophatase (PlAP) immunohistochemistry. Patients revealed testicular tumour without contralateral TIN in 271 cases and with contralateral TIN in 24 cases (8.1%). In 6 of these 24 men with contralateral TIN the cells could be detected in only one (n=5) or two (n=1) of the three specimen investigated. That means in these six patients TIN could have been missed if only one single random biopsy was taken. By increasing the number of biopsies (=increasing the number of investigated seminiferous tubules) the detection rate of contralateral TIN may be increased up to 8.1%. Thus we recommend multiple testicular biopsies to increase the diagnostic safety in detection of TIN. Biopsies may be taken from one randomly chosen surgical incision site.

Adult↗

PubMed2003

Decreased protamine-1 transcript levels in testes from infertile men.

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 (C(T)) representing a measure of the initial 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 C(T) difference (deltaC(T)) 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 C(T) 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.

Analysis of Variance↗

PubMed2002

Protamine-1 and -2 mRNA in round spermatids is associated with RNA-binding proteins.

RNA-binding proteins in round spermatids have previously been assigned to the coding sequence of Prm1- and Prm2-mRNA. To further characterize this protein-RNA interaction, prior to cDNA synthesis, microdissected cell profiles were digested with different proteases exhibiting a specific cleavage site followed by both conventional and real-time quantitative PCR. Best results were obtained with proteinase K and A followed by factor Xa protease, genenase I, and proteases V8. While enterokinase revealed PCR signals solely for Prm2, no amplification signal was obtained using chymotrypsin. These data suggest a protein segment rich in basic amino acids to be important for the binding to Prm1- and Prm2-mRNA. The fact that phenanthroline treatment instead of protease digestion also resulted in amplification signals suggests the involvement of zinc-finger-like protein-RNA interactions. Employing different primer pairs, RNA-binding proteins were shown to be localized at the 5' end of Prm1- and Prm2-mRNA. Since protein-RNA interactions are a common principle of posttranscriptional regulation of gene expression, the combination of microdissection, protease digestion, and real-time quantitative PCR provides a suitable tool for its investigation in a cell type-specific manner. Furthermore, the presence of RNA-binding proteins within the coding sequence of mRNAs demands proteinase K treatment prior to cDNA synthesis, a compelling necessity for the study of gene expression.

Cell Separation↗

PubMed2002

Altered expression of connexins 26 and 43 in Sertoli cells in seminiferous tubules infiltrated with carcinoma-in-situ or seminoma.

The expression of connexins (cx) 26 and 43 in testis infiltrated with carcinoma-in-situ (CIS) or seminoma was examined to gain insight into the relationship between aberrant gap junctional communication and spermatogenic impairment in the neoplastic testis. In uninvolved tubules with normal spermatogenesis, cx43 immunostaining was localized to the Sertoli-Sertoli junctional complex and cx26 was absent. In contrast, infiltrated tubules with spermatogonial arrest or CIS-only were negative for cx43, but displayed strong intracytoplasmic Sertoli cell staining for cx26. The Sertoli cells in these tubules re-expressed cytokeratin 18 (ck18), signifying a reversion to a less differentiated state. Western blot analysis for cx43 revealed a single immunoreactive band at 43 kD (normal spermatogenesis) and three bands at 43, 41, and 39 kD (impaired spermatogenesis with CIS or seminoma). For cx26, a doublet band at 26/28 kD (normal spermatogenesis) and an additional doublet band at 52/54 kD (impaired spermatogenesis with CIS or seminoma) were observed. The altered expression of cx26 and cx43 in Sertoli cells in testes infiltrated with CIS or seminoma suggests that a derangement in intercellular communication between Sertoli cells and between Sertoli cells and germ cells may play a role in the resulting spermatogenic impairment and possibly in the proliferation and neoplastic progression of CIS cells.

Adult↗

PubMedDate not supplied

In vivo effects of histone-deacetylase inhibitor trichostatin-A on murine spermatogenesis.

The acetylation state of core histones is controlled by two classes of enzymes, histone acetyl transferases (HATs) and histone deacetylases (HDACs). HDAC inhibitors, such as trichostatin-A (TSA), are able to induce cell cycle arrest by stimulating transcription of genes that negatively regulate cell growth and survival. However, little is known about the effect of HDAC inhibitors on spermatogenesis. TSA treatment of cultured murine germ cells from whole testes resulted in an increase of histone H4 acetylation in round spermatids, suggesting that a hypoacetylated state of these cells is important for their normal differentiation. In the present study, the in vivo effects of TSA on murine spermatogenesis were investigated. Subcutaneously applied TSA resulted in a dose-dependent decrease in relative testis weight due to impaired spermatogenesis. No obvious toxic effects of TSA treatment could be found. A second animal experiment confirmed that male mice receiving TSA under the same conditions as in the first experiment became infertile. This phenomenon was completely reversible. No evidence of histone H4 hyperacetylation in round spermatids could be found; however, the number of spermatids significantly decreased with increasing TSA concentrations. Additionally, a dramatic loss of pachytene-diplotene spermatocytes due to increased apoptosis was observed. This suggests that TSA was mainly effective at the level of meiosis. The other male reproductive organs showed no morphological changes compared to controls, suggesting that TSA action on the testis was not mediated by sex hormones.

Animals↗

PubMedDate not supplied

Effect of vasectomy on sperm nuclear chromatin condensation in the rabbit.

Histone-to-protamine exchange in haploid spermatids is known to play a central role for male fertility. The present study investigates, for the first time, the effects of vasectomy on the expression of protamines in the rabbit. During normal spermatogenesis, protamine-1 and protamine-2 mRNA were expressed from step 5 round spermatids to step 11 elongated spermatids. In unilaterally vasectomized animals, control testes revealed normal spermatogenesis with normal protamine expression, while vasectomized testes exhibited both normal spermatogenesis and spermatogenic arrest. Some testes with normal spermatogenesis revealed delayed expression of both protamine-1 and protamine-2. Furthermore, multinucleated round spermatids were a regular finding in these testes. In both treated and untreated animals, a higher percentage of spermatozoa from the cauda epididymis had highly condensed chromatin when compared with those from the testis. The percentage of spermatozoa with highly condensed chromatin from testes and epididymides from the vasectomized side of treated animals remained unchanged from controls. As the integrity of nuclear chromatin is important for oocyte fertilization, especially in intracytoplasmic sperm injection (ICSI), where most of the natural selection mechanisms are bypassed, our data add valuable information for the treatment of infertility by ICSI, showing that vasectomy may affect nuclear chromatin integrity of testicular spermatids but not epididymal spermatozoa. Microsurgical epididymal sperm aspiration (MESA), therefore, may be superior to testicular sperm extraction (TESE) in vasectomized patients.

Animals↗

PubMedDate not supplied

Connexin 33: a rodent-specific member of the gap junction protein family?

Gap junctional intercellular communication between Sertoli cells and between Sertoli cells and spermatogonia is considered to play a key role in the regulation of both proliferation and differentiation of germ cells. A member of the gap junction protein family, Connexin 33 (cx33), probably has an inhibitory effect on the formation of gap junctions and so far it is the only cx that has been exclusively found in rat and mouse testes. Thus, this connexin seems to be a special member of the cx family. Using immunohistochemistry, Western blot analysis, polymerase chain reaction, and reverse transcription (RT)-PCR (tissue homogenate and microdissected cells), we studied the possible occurrence of cx33 at the protein, the DNA, and the RNA level in human testis. Whereas immunohistochemistry using the only commercially available anti-cx33 antibody showed similar labeling to the rat within the seminiferous epithelium, we could not find any further evidence for the existence of cx33 using Western blot analysis, PCR, and RT-PCR in human testis. Based on the demonstration of the staining pattern of mitochondria in human germ cells and on preabsorption studies, we could demonstrate anti-cx33 antibody cross-reacting with mitochondrial ferritin, a protein localized in the mitochondria of human testicular spermatids. Therefore, we were not able to abide by the suspicion that cx33 is present in human testis. Additionally, it was not possible to demonstrate cx33 via PCR and immunohistochemistry in the testis of different mammals (dog, cattle, pig, horse, and marmoset monkey) with normal spermatogenesis. These data indicate that cx33 seems to be the first rodent-specific testicular cx.

Amino Acid Sequence↗

PubMedDate not supplied

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