Annual Report of Activities CNC 2008 - Center for Neuroscience and ...

Biology of Reproduction and Human Fertility | Head: João RamalhoObjectivesThe main goal consists in determining what makesa good sperm, from a cellular, biochemical andmolecular standpoints, with focus on themitochondria and bioenergetics as it relates to cellfunction and homeostasis. Several animal modelsare used (horse, rat, cat, human), for differentpurposes. The horse has been used to bothcharacterize native Portuguese breeds, and as atool to improve animal management (reproduction,semen banking, artificial insemination) incollaboration with the Agricultural School ofCoimbra. Human work is carried out at theUniversity Hospitals of Coimbra where the groupis involved in quality control, gamete and embryoevaluations, gamete and tissue banking foroncology patients, and research aimed at directlyimproving the quality of service in the HumanReproduction Clinic. The rat has been used as amodel to characterize gametogenesis from abioenergetics standpoint, and to assess the effect ofdiabetics on reproductive parameters. The cat isused as a model for endangered felids, in terms ofpreservation of the germline and xenotransplantation.We are currently researching changes in sperm thatmay correlate with fertility (abnormal mitochondrialDNA replication, mitochondrial function, apoptosis,sperm chromatin status, ATP production,antioxidant defenses), as well as the effect ofdiabetes and age on testicular homeostasis, spermproduction, metabolism and physiology. Thesestudies are being carried out both in bulkpopulations of sperm from males with differentsemen characteristics, as well as in populations thathave been sorted by either classical methods orflow cytometry, and have relevance for thediagnosis and management of human and horse(in)fertility. Another goal is to develop simple teststo monitor sperm quality in an ejaculate in terms ofnuclear DNA status which can be applied both infield studies concerning the management ofendangered species, and in the clinic. Furthermore,the group’s expertise has recently led tocollaborations regarding the effect of mitochondrialbioenergetics on human embryonic stem cellpluripotency, and differentiation.Main Achievements1‐ Detailed analysis of ATP production,mitochondrial function, membrane stability,apoptosis, oxidative stress and antioxidantdefenses in equine sperm, and impact on stallionfertility in order to determine the best possibleindicators for stallion fertility, with relevance foranimal breeding and semen banking (part of thiswork has been published). Despite their economicimportance for decades this work also provided thefirst characterization of native breeds (Lusitanoand Sorraia) in terms of breed‐specific semenparameters (published).2‐Simultaneous use of several fluorescence‐basedassays to monitor human sperm quality and providea more accurate diagnosis of patient sperm qualityand male infertility (previously published).However, this work also clearly showed that routineclinical applications would have to be both cheaperand easier, which led to the development of a novelsimple assay to monitor human sperm DNA status,an important parameter that is not usuallyquantified. This assay was derived from previouswork carried out in the cat, and its usefulness inpredicting treatment outcomes has been validated ina multi‐center collaboration involving samples fromthe University Hospitals of Coimbra, two labsaffiliated with the University of Porto, and a lab inBrest (France). (This work is in press).3‐ Successful proof of principle that primate spermretains reproductive potential after freeze‐drying(paper published and on journal cover).4‐ Characterization of testicular bioenergetics as distinctfor that of other organs, and implication of testicularbioenergetics and uncoupling proteins in aging of themale reproductive tract (three papers in press)5‐ Discovery of a role for mitochondria inmaintaining human embryonic stem cellpluripotency. Mitochondrial inhibition usingantimycin A results in an up‐regulation ofpluripotency markers such as Nanog, in stem cells,while maintaing essential cellular characteristics. Infact, antimycin A in culture media can actuallyreplace the role of some growth factors, namelybFGF (work submitted).80