Ursula Vitt

The TGF beta family member growth differentiation factor-9 (GDF-9) is an oocyte-derived factor that is essential for mammalian ovarian folliculogenesis. GDF-9 mRNAs have been shown to be expressed in the human ovarian follicle from the primary follicle stage onward, and recombinant GDF-9 has been shown to promote human ovarian follicle growth in vitro. In this study with primary cultures of human granulosa-luteal (hGL) cells, we investigated whether recombinant GDF-9 activates components of the Smad signaling pathways known to be differentially activated by TGF beta and the bone morphogenetic proteins (BMPs). As with TGF beta, GDF-9 treatment caused the phosphorylation of endogenous 53-kDa proteins detected in Western blots with antiphospho-Smad2 antibodies (alpha PS2). However, unlike BMP-2, GDF-9 did not activate the phosphorylation of antiphospho-Smad1 antibody (alphaPS1)-immunoreactive proteins in hGL cells. Infection of hGL cells with an adenovirus expressing Smad2 (Ad-Smad2) confirmed that GDF-9 activates specifically phosphorylation of the Smad2 protein. Infection of hGL cells with Ad-Smad7, which expresses the inhibitory Smad7 protein, suppressed the levels of both GDF-9-induced endogenous and adenoviral alpha PS2-reactive proteins. Furthermore, GDF-9 increased the steady state levels of inhibin beta(B)-subunit mRNAs in hGL cells and strongly stimulated the secretion of dimeric inhibin B. Again, Ad-Smad7 blocked GDF-9-stimulated inhibin B production in a concentration-dependent manner. We identify here for the first time distinct molecular components of the GDF-9 signaling pathway in the human ovary. Our data suggest that GDF-9 mediates its effect through the pathway commonly activated by TGF beta and activin, but not that activated by many BMPs. The results are also consistent with the suggestion that in addition to endocrine control of inhibin production by gonadotropins, a local paracrine control of inhibin production is likely to occur via oocyte-derived factors in the human ovary.

We aligned Incyte ESTs and publicly available sequences to the rat genome and analyzed rat chromosome 1q43-54, a region in which several quantitative trait loci (QTLs) have been identified, including renal disease, diabetes, hypertension, body weight, and encephalomyelitis. Within this region, which contains 255 Ensembl gene predictions, the aligned sequences clustered into 568 Incyte genes and gene fragments. Of the Incyte genes, 261 (46%) overlapped 184 (72%) of the Ensembl gene predictions, whereas 307 were unique to Incyte. The rat-to-human syntenic map displays rearrangement of this region on rat chr. 1 onto human chromosomes 9 and 10. The mapping of corresponding human disease phenotypes to either one of these chromosomes has allowed us to focus in on genes associated with disease phenotypes. As an example, we have used the syntenic information for the rat Rf-1 disease region and the orthologous human ESRD disease region to reduce the size of the original rat QTL to only 11.5 Mb. Using the syntenic information in combination with expression data from ESTs and microarrays, we have selected a set of 66 candidate disease genes for Rf-1. The combination of the results from these different analyses represents a powerful approach for narrowing the number of genes that could play a role in the development of complex diseases.

The first aim of this work is to elaborate an in vitro trial which can be used to estimate the fertility of cryopreserved semen. Five different ejaculates from each of 15 bulls are collected to assess the NRR. Three of these ejaculates per bull are individually examined in vitro. The main impact is laid on the assessment of the inducible acrosome reaction (AR) of vital spermatozoa after thawing. Additionally the motion characteristics and the ATP content of the sperm cells are studied. Fourteen of the bulls reveal NRR between 60 and 80% while one bull (No. 15) has a much lower NRR. In the 14 bulls with higher NRR the increase in true AR by stimulation with ionophore after heparin treatment is significantly correlated to the NRR. Moreover, the fertility of bulls is higher if the remaining number of vital spermatozoa with intact acrosome at the end of the trial is lower. On the other hand the total amount of true AR, which also includes the premature AR induced by heparin does not correlate to the NRR and is not predictive for fertility. This supports the thesis that it is not sufficient to have enough spontaneous or induced AR in a sperm sample but that the AR should occur only after an adequate stimulation. This in vitro trial represents a hard challenge for the spermatozoa missing regulating factors of the female genital tract. Bulls with a NRR of between 60% and 69% have only few or no sperm cells which are able to respond to calcium ionophore as is also confirmed by bull No. 15 (NRR = 43%). This functional spermatozoan parameter provides an approach to assess even slight differences in fertility between bulls of high NRR. The low fertility of bull No. 15 in this trial is already revealed by the low percentage of motile spermatozoa in the sample after thawing. Neither the motility parameters nor the ATP amount of the bulls with higher NRR disclose any possibilities to assess their fertility. The second aim of this work was to investigate the possibility of fertility improvement by exchanging seminal plasma. The seminal plasma of 8 ejaculates each of three bulls with low NRR was removed by centrifugation and replaced before cryopreservation by seminal plasma from bulls with higher NRR. This possibility of improving the fertilizing ability of bulls with low NRR cannot be confirmed by the obtained results. Differences between the specimens with and without seminal plasma can be percepted which lead to the conclusion of a protective function of the seminal plasma.