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Borgers M.,Medical Faculty | Wolter M.,Medical Faculty | Hentrich A.,Medical Faculty | Bergmann M.,Institute of Veterinary Anatomy | And 2 more authors.
Reproduction | Year: 2014

Disturbances of checkpoints in distinct stages of spermatogenesis (mitosis, meiosis, and spermiogenesis) contribute to impaired spermatogenesis; however, the efficiency of meiotic entry has not been investigated in more detail. In this study, we analyzed azoospermic patients with defined spermatogenic defects by the use of octamer-binding protein 2 for type A spermatogonia, sarcoma antigen 1 for mitosis-meiosis transition and SMAD3 for pachytene spermatocytes. Especially patients with maturation arrest (MA) at the level of primary spermatocytes showed significantly reduced numbers of spermatogonia compared with patients with histologically intact spermatogenesis or patients with hypospermatogenesis (Hyp). For a detailed individual classification of the patients, we distinguished between 'high efficiency of meiotic entry' (high numbers of pachytene spermatocytes) and 'low efficiency of meiotic entry' (low numbers of pachytene spermatocytes). Only patients with histologically normal spermatogenesis (Nsp) and patients with Hyp showed normal numbers of spermatogonia and a high efficiency of meiotic entry. Of note, only patients with histologically Nsp or patients with Hyp could compensate low numbers of spermatogonia with a high efficiency of meiotic entry. In contrast, patients with MA always showed a low efficiency of meiotic entry. This is the first report on patients with impaired spermatogenesis, showing that half of the patients with Hyp but all patients with MA cannot compensate reduced numbers in spermatogonia with a highly efficient meiosis. Thus, we suggest that compensatory meiosis mechanisms in human spermatogenesis exist. © 2014 Society for Reproduction and Fertility.


Dahlhoff M.,Ludwig Maximilians University of Munich | Grzech M.,Ludwig Maximilians University of Munich | Habermann F.A.,Ludwig Maximilians University of Munich | Habermann F.A.,Institute of Veterinary Anatomy | And 2 more authors.
Genesis | Year: 2012

Transgenic mouse lines expressing Cre recombinase in a cell-specific and tissue-specific manner are essential tools for studying gene function and for developing suitable models for human diseases. Here, we used an expression cassette containing the full 5′ untranslated region of the porcine insulin gene to generate a mouse line expressing Cre recombinase specifically in pancreatic β-cells by pronuclear DNA microinjection. We obtained a founder animal that transmitted the construct to its descendants in a Mendelian fashion and whose descendants showed a clear activation of β-galactosidase expression in pancreatic β-cells after crossing into the ROSA26 lacZ reporter mouse line. Cre expression in other organs was negative except for the kidney, intestine, and the cerebral pons where β-galactosidase activity was detected in a small percentage of the cells. This new mouse line is a valuable tool for recombination of floxed alleles in pancreatic β-cells in vivo. © 2011 Wiley Periodicals, Inc.


Kowalewski M.P.,Institute of Veterinary Anatomy | Fox B.,Institute of Veterinary Anatomy | Gram A.,Institute of Veterinary Anatomy | Boos A.,Institute of Veterinary Anatomy | Reichler I.,University of Zurich
Reproduction | Year: 2013

The luteal phase in dogs is governed by many poorly understood regulatory mechanisms. Functioning of the corpus luteum (CL) is unaffected by hysterectomy. Recently, the role of prostaglandins in regulating canine CL function was addressed suggesting a luteotrophic effect of prostaglandin E2 (PGE2) during the early luteal phase. However, compelling functional evidence was lacking. The potential of PGE2 to stimulate steroidogenesiswas tested in canine primary luteal cells isolated from developing CL of non-pregnant dogs. In addition, the luteal expression of prostaglandin transporter (PGT) and steroidogenic acute regulatory protein (STAR) was demonstrated and characterized in CL from non-pregnant bitches during the course of dioestrus as well as from pregnant animals during the preimplantation, post-implantation and mid-gestation periods of pregnancy and during luteolysis; the luteal expression of PGE2 receptors (EP2 and EP4) has been investigated at the protein level throughout pregnancy. Our findings show that PGE2 is an activator of STAR expression in canine luteal cells from early luteal phase, significantly up-regulating STAR promoter activity and protein expression resulting in increased steroidogenesis. The 3βHSD (HSD3B2) and P450scc (CYP11A1) expression remained unaffected by PGE2 treatment. The expression of PGT was confirmed in CL during both pregnancy and dioestrus and generally localized to the luteal cells. After initial up-regulation during the earlier stages of the CL phase, its expression declined towards the luteal regression. Together with the demonstration of EP2 and EP4 throughout pregnancy, and the decline in EP2 at prepartum, our findings further support our hypothesis that intra-luteal PGE2 may play an important role in regulating progesterone secretion in the canine CL. © 2013 Society for Reproduction and Fertility.


Langkabel N.,TU Berlin | Meyer S.,TU Berlin | Grosspietsch R.,TU Berlin | Brautigam L.,TU Berlin | And 3 more authors.
Archiv fur Lebensmittelhygiene | Year: 2012

In order to mince lymph nodes for MAIC PCR-analysis, the Mixer Mill MM200 (RETSCH GmbH, Haan, Germany) was used with reusable tungsten carbide grinding beads. Some of unexpected PCR results indicated the carryover of DNA contamination among different samples. Hence, several decontamination procedures were used and the surface of steel and tungsten carbide beads was examined for remaining and intact DNA. Physical methods (three washes with distilled water, autoclaving and UV treatment) did not eliminate the DNA from both type of surface; this was also true for a procedure with Exonuclease III and the commercial DNA-removing kit DNAaway® (only used for steel beads). Chemical methods 0.25 % peracetic acid (PAA) (pH 5 and pH 7) and sodium hypochlorite (NaCIO) (concentraction of active chlorine 4.7% or 5.4 %) removed DNA from tungsten carbide, but caused cracking of the surface of the beads. In conclusion, for grinding beads, the use of disposable material is suggested, as also employed for reagents and equipment in the PCR laboratory. © M. & H. Schaper GmbH & Co.


Jaroszynski L.,University of Heidelberg | Zimmer J.,University of Heidelberg | Fietz D.,Institute of Veterinary Anatomy | Bergmann M.,Institute of Veterinary Anatomy | And 2 more authors.
International Journal of Andrology | Year: 2011

The human DEAD-box Y (DBY) RNA helicase (aka DDX3Y) gene is thought to be the major azoospermia factor a (AZFa) gene in proximal Yq11. Although it is transcribed in many tissues, the protein is expressed only in spermatogonia. In this study, we demonstrate that this translational control mechanism is probably germ cell-specific because of its association with expression of a distinct class of DDX3Y testis transcripts present only in pre- and post-meiotic male germ cells. They are initiated from a second distal DDX3Y promoter domain at two distinct start sites in the gene's 5' untranslated region (UTR) exon-T sequence. With the aid of an EGFP-3xFLAG reporter cassette cloned downstream of DDX3Y minigenes containing exons 1-4 and two different exon-T extensions, we discovered that DDX3Y translation is influenced by the presence of several ATG triplets located in exon-T, thus upstream of the main translational ATG start codon in exon 1. Strong translational repression of the DDX3Y minigene transcripts was observed when they contained the longest exon-T sequence with five upstream ATG triplets (uATGs). The potential formation of complex distinct stem-loop structures serve here as additional repressor element. Only minor translational attenuation was seen for the DDX3Y minigene transcripts when containing the shortest exon-T sequence, that is, starting at first transcriptional start site (coined 'T-TSS-I'). It was completely released after its single uATG was abolished by mutation. As we found DDX3Y transcripts with the longest exon-T sequence predominantly in spermatids, our results suggest that the amount of DDX3Y protein in pre-meiotic germ cells and its absence in post-meiotic germ cells are tightly controlled by the different extensions of exon-T in this germ cell-specific DDX3Y transcript class. © 2010 The Authors. International Journal of Andrology © 2011 European Academy of Andrology.

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