Development of an in vitro microenvironment for maturing oocytes

The development of in vitro culture systems, comparable to the in vivo microenvironment in terms of effect on the oocyte growth and development could provide a valuable experimental tool for studying the mechanisms governing folliculogenesis. This tool might serve as well for practical clinical, agricultural, zoological, or biotechnological applications. This thesis reports on the importance of the microenvironment for the ovarian folliculogenesis process. The complexity of such a microenvironment was approached with a strategy based on functionalized PEG-hydrogels. The PEG matrix not only served as a scaffold, but it was also used a "reservoir" of immobilized cues. Tethered integrin-binding peptides in combination with other signaling factors aimed at better understanding the interactions of the oocyte and its surrounding granulosa cells that, most probably, determine the efficiency of the actual and the future in vitro mature oocyte production strategies. This work led also to investigating the lymph angiogenesis and the lymphatic transport in the context of oocyte maturation and their impact on mice fertility. In a first step, the mechanical properties of PEG-hydrogel were optimized for culturing secondary follicles. This report shows that the growth of the follicles was highly dependent on the mechanical properties of the surrounding environment. The optimal elastic modulus was found to be close to 900 Pa. In a second step, the effect of Arg-Gly-Asp (RGD) peptide, the minimal integrin-binding sequence, was studied. RGD presence did not influence the follicle growth rate but it significantly improved the quality of the produced oocytes. These findings demonstrated that approaching, biochemically and mechanically, the complexity of the ovarian extracellular matrix could be a winning strategy. The effect of key soluble factors was also investigated in order 1- to confirm their compatibility with the established 3-D culture system and 2- to further improve qualitatively and quantitatively the produced mature oocytes. Various combinations of gonadotropins such as the follicle-stimulating hormone (FSH) or the luteinizing hormone (LH) were tested. Interestingly, the effects of the gonadotropins in the 3-D PEG system were close to their known effects in vivo. Here the aberrant effects of these hormones in the used 2-D systems appeared clearly. c-Kit ligand (KL) is suspected to be one of the most important factors for the activation of primordial follicles and thus for controlling the exit from the resting pool. Previously, studying the effects of the two forms of KL (soluble and membrane-attached) had to cope with the lack of biologically-relevant immobilization strategies. For overcoming this problem, KL constructs were designed to include a substrate sequence for Factor XIIIa (NQEQVSPL or NQEQVSPLRCG). Thus the produced recombinant KL proteins could be enzymatically crosslinked to the PEG matrix. The different constructs of KL, including a wild type extracellular domain, were successfully cloned and produced from mammalian HEK-293 cells. The identity and the activity of the produced proteins were confirmed. Ovarian tissues from four days-old mice were cultured in PEG-hydrogels functionalized with KL. The results showed that the primordial follicles grew and were activated in the PEG-hydrogels where KL was immobilized but not when the soluble form of KL was preset in the medium. This experiment showed that the membrane-attached and the soluble form of KL play a different role in the rodent folliculogenesis. In parallel it was observed that blocking vascular endothelial growth factor receptor-3 (VEGFR-3) signaling had a critical but still unsuspected role in reproduction. This study demonstrates that variation in lymphangiogenesis is a regular, non-pathological event during folliculogenesis in the ovary; blocking lymphangiogenesis, might have an effect on hormone transport and thus on pregnancy. The reported results demonstrates that the blockade of lymphangiogenesis decreases the progesterone and estradioal levels during pregnancy and in fine results in failed fetal development. In conclusion, this study demonstrates the efficiency and the flexibility of a novel 3-D culture system. Circumventing problems inherent to the "on-plastic" standard culture, such as the loss of the granulosa-oocyte interactions, allowed the emergence of a culture system tailored for investigating fundamental folliculogenesis-related questions. Furthermore, the reported culture system might serve as a platform for developing clinical and biotechnology applications.

Murine ovarian follicle culture in PEG-hydrogel: Effects of mechanical properties and the hormones FSH and LH on development

Jeffrey Alan Hubbell, Jong Eun Ihm, Seung Tae Lee

The development of an in vitro culture system, comparable to the in vivo environment in terms of effects on oocyte growth and development, could provide a valuable experimental tool for studying the mechanisms governing oocyte development as well as practical clinical, agricultural, zoological, and biotechnological applications. This study reports on the importance of the microenvironment for the ovarian folliculogenesis process. The complexity of such a microenvironment was approached with a strategy based on functionalized PEG-hydrogels. The PEG matrix not only serves as a scaffold, but it is also used a reservoir of immobilized cues. Using tethered integrin-binding peptides in combination with other signaling factors, we aimed at better understanding the interactions of the oocyte and its surrounding granulosa that may determine the efficiency of the actual and the future in vitro mature oocyte production. In a first step, the mechanical properties of PEG-hydrogel were optimized for producing secondary follicles, in which the oocyte is surrounded by two layers of granulosa cells. Follicle growth was highly dependent on the mechanical properties of the surrounding environment, with the optimal elastic modulus being approx. 1 kPa. The effects of key soluble factors were also investigated to confirm their compatibility with the established 3-D culture system and to further qualitatively and quantitatively improve the produced mature oocytes. Accordingly, various combinations of the gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were tested. Interestingly, the effects of the gonadotropins in the 3-D PEG system were close to their known in vivo effects. In conclusion, this study demonstrates the efficiency and the flexibility of a novel 3-D culture system, PEG-hydrogel. Circumventing problems inherent to the "on-plastic" standard culture, such as the loss of the granulosa-oocyte interactions, allowed the emergence of a culture system tailored for investigating fundamental folliculogenesis-related questions. Furthermore, the reported culture system may serve as a platform for developing clinical and biotechnology applications.

Polymer Soc Korea2015

Hormonal regulation of Stat5 in the mammary gland - from signaling to morphogenesis

Sergio Klinke

The mammary gland development is regulated by steroid and peptide hormones and occurs mainly after birth, compared to the majority of the other organs in the body. The principal hormones responsible for the breast development are the estrogen, the progesterone, the growth hormone, the epidermal growth factor and the prolactin. The latter one is of specific interest since it is required for further development of the gland through pregnancy and lactation. During pregnancy and lactation, alveolar structures develop and differentiate into secretory cells. The signal transducer and activator of transcription 5 (Stat5) is known to be the main effector of prolactin signaling during pregnancy, inducing alveologenesis and differentiation of epithelial cells into secretory cells. I investigated if Stat5 has a role before pregnancy. Protein expression as well as mRNA expression analysis of glands from ovariectomized mice which were artificially stimulated with ovarian hormones (estrogen and progesterone) and control vehicle showed evidences that Stat5 expression can be stimulated by these two hormones. Contrary to the role established until now for stat5 in the mammary gland, I found by fluorescence visualization of Stat5-null epithelium as well as whole mount staining of the same glands that Stat5 is required even before pregnancy since null epithelium showed highly reduced growth already in 3 weeks-old virgin mice. This may indicate a new role for this protein in cell proliferation or survival in early stages of mouse mammary development. Furthermore, transplantation experiment of Stat5-null epithelium into wild-type recipient mice showed that pregnancy state was not able to rescue the normal phenotype. This may be due to complete loss of these cells which happened during the 5 weeks left for the transplanted epithelium to expand the cleared fat pad. This discovery opens a new debate on Stat5 role in the mammary gland development.

2008

Protein-binding microarray technology and ChIP-sequencing for the identification of novel targets of the human tumor suppressor AP2[alpha]

Jan Kerschgens

The mis-regulation of gene transcription in the living cell can be the cause of complex human diseases like cancer. One challenge of the post-genomic era of biomedical research is the development of in vitro techniques for the rapid and cost-effective analysis of regulatory transcriptional patterns of disease. Two crucial points for this endeavour have to be considered: (1) Sensitivity, since biologically active transcription factors are expressed at low amounts in the nucleus of the living cell and (2) the as close as feasible reconstitution of the nuclear microenvironment in order to guarantee as close to natural conditions for the performance of experiments as possible. The first part of this thesis reports on the application of protein-binding microarrays (PBM) bearing long-stranded DNA molecules for the analysis of a human disease-related protein, the human tumor suppressor activating enhancer-binding protein 2 alpha (AP2α). The second part of this thesis describes how chromatin-immunoprecipitation combined with high-throughput sequencing (ChIP-seq) and subsequent data analysis led to the discovery of a novel AP2α-binding motif in a cancer cell line model. We first developed a setup and protocol to perform on-chip protein-DNA molecule interaction analyses. Following this, recombinant AP2α protein was produced and purified using an E.coli host system. AP2α was subsequently assessed for its binding specificity to an array of ∼6000 human promoter and intergenic sequences. These experiments confirmed previously established AP2α DNA binding sequences and allowed the identification of novel ones. High and low binder sequences were selected from the PBM data and analyzed for specific AP2α binding in vivo utilizing reporter transfection and chromatin-immunoprecipitation (ChIP) assays. In contrast to the lowest AP2α-bound sequences, the highest PBM-bound sequences had significant effects on reporter gene expression. ChIP-qPCR revealed these sequences were bound by AP2α in vivo. From these results Kallikrein 5, a member of the kallikrein family of extracellular proteases that includes the prostate-specific antigen (PSA), which is currently emerging as one of the most prominent biomarkers of tumor progression for various types of cancers, and the growth-arrest specific 2 (GAS2) protein, which modulates cell susceptibility to p53-dependent apoptosis, were identified as novel AP2α targets. Finally, the binding patterns of AP2α from nuclear extracts of breast cancer tissue biopsies were assessed on the 6k human promoter arrays and compared to the binding patterns of 'healthy' tissue extracts. Overall these experiments confirmed known targets of AP2α. Interestingly a discrepancy of 26% was revealed when assessing the correlation of binding data from healthy and tumor tissue biopsies. This finding suggested that depending on the source of the protein, that is of either healthy or tumor tissue origin, the DNA-binding specificity of the protein factor under study might be altered and most importantly this change in binding specificity can be monitored by PBM. The second part of this thesis describes the analysis of the binding specificity of AP2α by combining chromatin-immunoprecipitation with high-throughput sequencing (ChIP-seq). The binding specificity of the human tumor suppressor protein AP2α in the living cell was assessed in a colon cancer cell model. Analysis of the ChIP-seq enriched sequences resulted in the discovery of a novel recognition motif for the in vivo binding of AP2α. Interestingly, this sequence motif resembles the motifs discovered from data sets with recombinant protein alone. Further analysis of a previously described in vitro and the new ChIP-seq discovered binding motifs were performed by electrophoretic mobility shift assays, which demonstrated that the newly discovered motif shows the same effects with colon cancer and breast cancer nuclear extracts, thereby suggesting a general relevance of the motif independent of the cell type. In these DNA-binding competition experiments (EMSA), the previously computed, in vitro derived DNA recognition sequences does not abolish native AP2α protein (obtained from crude nuclear cell extracts) interaction with the ChIP-seq discovered motif. This observation indicates for a complex situation in vivo that might require protein co-factors in order to allow specific AP2α-DNA interaction. Therefore additional experiments will be required in order to assess the novel motif further. In conclusion, this thesis work demonstrates that protein-binding microarrays (PBM) can be used for the analysis of binding patterns of a human disease-relevant protein. It could be shown that the PBM setup developed was capable of identifying novel cancer-linked target genes for AP2α. It was also shown to be sensitive enough to directly probe nuclear extracts from tissue biopsies for specific DNA-binding. Finally, a novel DNA-recognition motif for AP2α was discovered from cellular assays. Additional experiments are required to assess whether this motif can be utilized for the reliable assignment of novel probes that may subsequently be integrated onto protein-binding microarrays. Together with the analyses for additional protein markers of disease, this avenue might serve as a platform for developing PBM technology further towards biotechnological applications such as diagnostics of regulatory transcriptional markers and patterns of cancer.

EPFL2009