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FP7

GROWSPERM Report Summary

Project ID: 603568
Funded under: FP7-PEOPLE
Country: Netherlands

Periodic Report Summary 1 - GROWSPERM (Development of in vitro and in vivo strategies to avoid and cure male infertility)

Project reference: 603568
Funded under: FP7-PEOPLE-2013-ITN

Periodic Report Summary 1 - Development of in vitro and in vivo strategies to avoid and cure male infertility

In many European countries at least 20% of young men exhibit sperm parameters below the lower WHO reference level and this will affect their fertility.
Male infertility has a dramatic impact on the individual and/or couple’s psychological and social well-being and results in significant healthcare costs. Currently male patients that do not produce sperm have no therapeutic options to father children. Different therapeutic interventions for male infertility have to be developed depending on the type and severity of germ cell deficits in individual patients.
In cases where undifferentiated germ cells are present in the testis, strategies based on sperm development from spermatogonial stem cells (SSCs) in vitro or in vivo need to be established. However, if no germ cells are present in the testis, somatic cells of such patients will be the only option from which to develop their own genetic sperm. The development of these potential therapies for male infertility requires detailed understanding of the entire process of sperm production from stem cells through to functional sperm. This information is still incomplete and fragmented.

In this Network, nine early stage researcher (ESRs) and two experienced researchers (ERs) have been recruited. They are trained (WP4) in this GrowSperm network (www.Growsperm.eu) that joins together the complementary knowledge and expertise of several public and private EU partners from disciplines of physiology, cell biology, molecular biology, chemistry and medicine in the field of male reproduction to investigate three strategies for sperm development: (WP1) propagation of human SSCs in vitro followed by their transplantation, (WP2) sperm development in vitro from stem cells or early germ cells, (WP3) sperm development in human testis tissue grafts in vivo.

In this WP1, we further characterize the recently established human testicular culture systems that allow propagation of human spermatogonial stem cells (SSCs) and understand, control and optimize this process of SSC propagation in vitro on the molecular and cellular level. To achieve our goal, we are establishing a bioassay to measure proliferative activity of SSCs in a testicular cell culture. Furthermore, we are improving the cryopreservation protocol to allow storage of testicular cells. Techniques that are used include FACS analysis, slow freezing or vitrification, SSC transplantation, histology, immunohistochemistry and whole mount immunofluorescence. In addition, we are establishing age related reference values for testicular spermatogonial quantities in untreated prepubertal boys and will identify the effect on spermatogonial quantities in those that have already been exposed to gonadotoxic treatments.
In WP2, we aim to gain more insight into the process of in vitro differentiation of male germ cells. Therefore, in analogy to recently published mouse studies, we aim to establish in vitro spermatogenesis from human testicular biopsies in an organoid or 3D cell culture system. For that, we investigate the differentiation from spermatogonia to sperm and the balance between SSC self-renewal and differentiation as well as the influence of somatic cells on germ cell development. More precisely, we investigate the mechanism of the developmental switch from mitosis to meiosis, which is one of the most challenging steps to induce sperm development. Molecular mechanisms and factors involved in cord formation and interaction between different cell types, especially between somatic and germ cells of seminiferous tubules are investigated to get more insight on the control of germ cell development in various culture approaches using immunohistochemistry and whole mount immunofluorescence. Moreover, using generation of reporter animals, next-generation sequencing and (phospho)proteomics followed by in-depth bioinformatics analysis, we are aiming to identify biomarkers during the course of differentiation. In addition, computational tools that allow a system-biological approach are explored to identify meaningful pathways and networks, as well as interactions and interconnecting nodes that may serve as either drug targets for differentiation induction and/or platforms on which reporter assays can be developed. Furthermore, we are establishing in vitro differentiation of germ cells from human induced pluripotent stem cells (iPS). Human iPS cells have been established from fertile and infertile patients. In vitro derivation of PGCs from iPS is explored to allow future generation of sufficient cell numbers to perform robust biochemical analysis of protein-protein and protein-mRNA interactions during further differentiation of PGCs to more advanced germ cell stages.
In WP3, we are developing strategies to generate sperm by grafting fragments of intact testicular tissue and assessing the development of germ cells towards sperm. We use human prepubertal testis tissue grafting in conjunction with immunohistochemistry to identify factors that may be responsible for the establishment and maintenance of the SSC niche and to investigate subsequent differentiation of the spermatogonia. In addition, we have developed human fetal testis tissue grafting as a reliable method to recapitulate the prepubertal testis for use in these studies. Furthermore, we are developing a similar system in a primate model that closely resembles human germ cell development during the prepubertal stage. The knowledge gained from this work will help us to optimize the efficiency of the grafting system for germ cell development.

The expected outcome of the work from this project is to obtain essential knowledge about various aspects of male germ cell development, which can be used for development of potential therapeutic interventions for male infertility in the near future. The culture system for SSCs followed by transplantation that will be optimized in WP1 is being established for a future clinical transplantation therapy for cancer survivors that have become infertile due to their cancer therapy. This therapy would be of particular interest to male childhood cancer survivors as that have no option to cryopreserve sperm because sperm are not produced before puberty. Currently, several centres in Europe, including those of partner’s clinics, are cryopreserving testicular tissue from prepubertal boys diagnosed with cancer or other diseases requiring gonadotoxic therapies. The establishment of age related spermatogonial reference quantities in this project will serve as a clinical tool to determine the size and quality of the biopsy. Both, an optimized SSC culture system and an improved cryopreservation protocol for testicular cells to allow storage of cells during safety testing of an aliquot, are an important requirement for future clinical SSC transplantation therapy. At the same time, WP 2 and 3 will provide us with novel insights into crucial processes of sperm development that will help in establishing future in vitro and ex vivo sperm production for clinical purposes. Sperm production in vitro and ex vivo might represent an interesting alternative approach for SSC transplantation in patients for whom SSC transplantation is not an option, such as those with infiltration of cancer cells in the testis, sclerosis of the testis or those that lack SSCs. Sperm obtained using such alternative approaches could be used for IVF procedures to enable those couples of have their own biological children.

In this Network, all fellows, nine early stage researcher (ESRs) and two experienced researchers (ERs), have been recruited. By coaching these young scientists in this inter-sectorial and multidisciplinary network we aim to train the next generation of researchers within the EU. The researchers are trained in scientific and transferable skills within the network. They undertake many courses both inside and outside the network, and they participate in international conferences and workshops (WP4), They will be primed to become leaders in the field of Reproduction and will continue to investigate basic science and translational aspects leading to novel interventions and clinical applications in academic and/or private sectors.

Contact

Van Pelt, Ans (PhD, Associate Professor)
Tel: +31205667837
Email: a.m.vanpelt@amc.uva.nl
Website: www.growsperm.eu

Remark:
In this tekst box, it was not possible to include figure and logo in the publishable summary. Therefore, we have attached the publishable summary with logo and figure as a Word and PDF document.

Contact

Eugenie Quartier, (Controller European Grants)
Tel.: +31205666188
E-mail

Subjects

Life Sciences
Record Number: 191426 / Last updated on: 2016-11-17