Facilities and equipment

The group is currently located at IBILI (third floor) in Pólo III of the University, and at the Human Reproduction Service in the Coimbra University Hospital. Equipment includes flow hood and cell culture incubators, fluorescence microscopes (inverted and upright), osmometer and plate reader.

Collaborations

Areas of focus

The group focuses on several aspects of Reproductive Biology, from gametogenesis to pluripotent stem cells, with a common focus on metabolism, energy production and mitochondrial function.

Stem cell projects involve both spermatogonial stem cells, and the metabolic regulation of pluripotent stem cells (embryonic or induced), with a particular focus on mitochondrial function and how it regulates cell pluripotency, using strategies common also for some types of cancer cells.

Spermatogenesis, the way sperm are formed in the testis, is studied in rodents and cat, the latter as a model for endangered felids, using spermatogonial stem cells, whole mounts, cell and organ cultures. Special interest is paid to the regulation of spermatogenesis and the bioenergetics of male gamete formation, as well as to the effects of age and diabetes and to spermatogonial stem cell preservation, propagation and transplantation, using the cat as model for endangered species.

Sperm physiology is studied in several species, with emphasis on nuclear DNA status, Reactive Oxygen Species, energy production and metabolism. Mammalian ejaculates are very heterogeneous, and we are interested in isolating and characterizing subpopulations of sperm in an attempt to define the properties of the highest quality cells. This information is then used to try to predict the reproductive behavior of different samples, or to classify and select breeders in animal species.

On the clinical side, projects include the evaluation of sperm and oocyte quality for Assisted Reproduction and Human infertility management, including the development of novel tests. Recent projects focus on new assays for sperm and oocyte quality for Assisted Reproduction, ovarian tissue cryopreservation for women undergoing cancer treatment, the possible use of novel detergents as spermicides, and the deleterious environmental effects, such as those triggered by several substances (pollutants, pesticides, etc.), on sperm function and human fertility. 

Ongoing Projects

• Gamete Quality and Assisted Human Reproduction
  Ana Paula Sousa, Teresa Almeida-Santos

  The success of Assisted Reproduction Technologies is mainly affected by sperm and oocyte quality. Gamete quality is primarily evaluated based on parameters that give an approximate indication of functionality, but that seem to be inefficient in predicting fertility potential. Other characteristics seem better to evaluate gamete quality and fertility. Therefore, our aim is to determine which cellular and biochemical characteristics can be used to evaluate gametesquality as well as, to select good quality gametes for Assisted Reproduction.

   

  

   

  Collaborations:
  Artur Paiva, Centro de Histocompatibilidade do Centro
  Vasco Almeida, University of Oporto

   

• Oncofertility
  Alexandra Carvalho, Ana Paula Sousa, Teresa Almeida-Santos

  Ovarian tissue cryopreservation is an innovative approach to preserve fertility of patients undergoing oncological treatment, and that may be at risk of becoming infertile. We are developing novel strategies to both improve ovarian tissue cryopreservation and analysis following thawing, and improve tissue grafting and functionality.

  Collaborations: 
  Teresa Woodruff, Northwestern University, USA
  Merck

   

• The role of mitochondria in pluripotent stem cells
  Tânia Perestrelo, Maria Inês Sousa, Sandro Pereira, Ana Sofia Rodrigues

  Pluripotent stem cells, either derived from the inner cell mass of the early mammalian embryo (embryonic stem cells, ESCs) or reprogrammed from somatic cells (induced pluripotent stem cells, iPSCs) are great models for development and differentiation studies, as they can originate all cell types

  

  Our main goal is to characterize the metabolic and mitochondrial status of pluripotent stem cells, and determine how these processes can influence cell fate. In addition we have determined that many of the metabolic strategies used to maintain cellular pluripotency are also used in several types of cancer, thus linking stem cell research to oncobiology.

  Collaborations: 
  Denis Wirtz, Johns Hopkins University, EUA
  Miguel Ramalho-Santos, University of California San Francisco USA
  Paulo Oliveira, CNC

   

• Environmental Endocrine Disruptors and Male Fertility
  Renata Tavares 

  Increasing evidence has suggested the involvement of endocrine disruptors in the reduction of sperm quality worldwide, particularly in sperm counts. From these, organochlorine compounds such as dioxins, polychlorinated biphenyls (PCBs) and some pesticides are particularly relevant given their environmental widespread and persistence, high toxicity and accumulation in reproductive tissues and fluids. A multitasking approach to determine their possible effects on human male fertility and highlight their mechanisms of action thus seems urgent. We are performing an in vitro toxicological screening on a number of these compounds and further analyze their effects not only on several sperm functional parameters but also on the function of other cell types present in the testis, with emphasis on Ca2+ signaling and mitochondrial function. 

  Collaborations:
  Stephen Publicover, University of Birmingham, UK

   

• Modulation of human sperm function and contraception
  Renata Tavares, Andreia Silva 

  Avoiding unwanted pregnancies, reducing maternal and child mortality rates and preventing STD’s (Sexually Transmitted Diseases), are the main challenges for the development of new contraceptive compounds, given that existing options are either unavailable or inadequate, especially in developing countries. A novel strategy would be to look at compounds that may act on multiple targets, focusing on common features of both fertilization and cell infection by viruses (for example, HIV), such as membrane fusion.

  

  Collaborations:
  Stephen Publicover, University of Birmingham, UK
  INNOTECH International, France.

• Omics analyses of human sperm: from basic science to translational research
  Alexandra Amaral, Sara Rebelo

  Human sperm is a simple, compartmentalized cell, with a very specific function, thus constituting a proper model for omics studies. Still, unraveling what makes a “good sperm” (with high fertilization potential) constitutes a big challenge in andrology.

  

  We are using high throughput proteomic, metabolomics and epigenetic approaches, coupled to bioinformatic analyses, to answer two main questions: What characteristics define functional sperm? Can this information be used to modulate sperm function? The study focuses predominantly on the bioenergetic metabolism of the male gamete (an issue debated in the literature for decades). 

  Collaborations:
  Rafael Oliva, University of Barcelona

   

• Xenografting, in vitro Organ culture and Spermatogonial stem cells technologies as alternative approaches to gamete production and study 
  Paula Mota, Sara Amaral 

  Of the 37 wild species of felids existent, only the domestic cat and some other small felid species escape classifications such as threatened, vulnerable or endangered. We use the domestic cat as a model for endangered felines and follow three main guidelines in the field of genetic material recovery.
In Xenografting, grafting of tissues or cells across a species barrier, male gametogenesis is obtained by transplantation of testicular tissue from deceased immature animals to an immune-compromised recipient, which functions as a bioreactor for ex vivo spermatogenesis. 

  

  The organ culture model is based on the in vitro culture of small fragments of testis, for at least one round of spermatogenesis, to obtain sperm in vitro. This has been successfully achieved in immature mice and we are now developing it using the domestic cat. Lastly, and as a more general approach, as it can be used to rescue the genetic diversity of immature or adult animals, we are aiming to identify, sort, in vitro amplify and transplant spermatogonial stem cells from domestic cat. All these methods rely only on material obtained routinely in veterinary clinics and may all be used to explore more basic science questions in carnivores.

  

  Collaborations:
  Stefan Schlatt, CeRA and University of Muenster, Germany

   

• The effects of Diabetes on Male reproductive function: Bioenergetics and Metabolism
  Sandra Amaral

  Although diabetes has been known to have deleterious effects on male reproductive function for many years, the details remain unknown. In fact most studies attempt treatments overlooking the underlying cellular and molecular mechanisms, and have provided no specific solutions for diabetic patients. Furthermore, these studies do not take into account the different cell types in testis. Given that diabetes is a metabolic disease that hampers mitochondrial function, we aim to investigate the mechanisms involved in the reproductive dysfunction associated with diabetes focusing on mitochondrial (dys)function and related oxidative stress, as these are hallmark events in diabetes, centering this analysis simultaneously on two cell types:  Sertoli cells (SC) and sperm. We will also use the innovative non-invasive Raman spectroscopy technology to probe and characterize these cells under diabetic conditions. Additionally, we also plan to study the effects of Diabetes in spermatogenesis, through a 3D culture system and whole mount analysis. Our work includes therefore a multi-level approach involving immunocytochemistry and immuhistochemistry, bioenergetic, and metabolomic analysis. 

  Collaborations:
  Stefan Schlatt, CeRA and University of Muenster, Germany

   

Selected Publications

Different concentrations of kaempferol distinctly modulate murine embryonic stem cell function (2016). Marcelo Correia, Ana Sofia Rodrigues, Tânia Perestrelo, Sandro L. Pereira, Marcelo F. Ribeiro, Maria I. Sousa & João Ramalho-Santos. Food and Chemical Toxicology 87:148-156.
 
The non-genomic effects of endocrine-disrupting chemicals on mammalian sperm (2016) Renata S. Tavares, Sara Escada-Rebelo, Marcelo Correia & João Ramalho-Santos. Reproduction 151:R1-R13.
 
Identification of endogenous metabolites in human sperm cells using 1H-NMR and GC-MS. Carla Paiva, Alexandra Amaral, Miguel Rodriguez, Nicolau Canyellas, Xavier Correig, José Luís Ballescà, João Ramalho-Santos& Rafael Oliva (2015) Andrology 3: 496-505.
 
High glucose concentrations per se do not adversely affect human sperm function in vitro (2015)Joana Portela*, Renata S. Tavares*(*co-first authors), Paula Mota, João Ramalho-Santos & Sandra Amaral. Reproduction 150: 77-84.
 
Dichloroacetate, the pyruvate dehydrogenase complex and the modulation of mESC pluripotency. (2015) Ana Sofia Rodrigues, Marcelo Correia, Andreia Gomes, Sandro L. Pereira, Tânia Perestrelo, Maria Inês Sousa & João Ramalho-Santos. PLoS ONE 10(7):e0131663. doi: 10.1371/journal.pone.0131663.
 
Geography of follicle formation in the embryonic ovary impacts activation pattern during the first wave of follicular growth. (2015) Marília H. Cordeiro, So-Youn Kim, Katherine Ebbert, Francesca E. Duncan, João Ramalho-Santos & Teresa K. Woodruff. Biology of Reproduction 93(4):88. doi: 10.1095/biolreprod.115.131227. 2015.

The human sperm: a review of proteomic studies from basic science to clinical applications (2014) Alexandra Amaral, Judit Castillo, João Ramalho-Santos & Rafael Oliva. Human Reproduction Update 20: 40-62.

Inhibition of mitochondrial complex III blocks neuronal differentiation and maintains embryonic stem cell pluripotency (2013) Sandro L. Pereira, Mário Grãos, Ana Sofia Rodrigues, Sandra I. Anjo, Rui A. Carvalho, Paulo J. Oliveira, Ernest Arenas & João Ramalho-Santos. PLoS ONE 8:e82095.

p,p’-DDE activates CatSper and compromises human sperm function at environmentally-relevant concentrations (2013) Renata S. Tavares, Steven Mansell, Christopher L Barratt, Stuart M. Wilson, Stephen J. Publicover & João Ramalho-Santos.Human Reproduction 28: 3167-3177.

Evaluation of human sperm chromatin status after selection using a modified Diff-Quik stain indicates embryo quality and pregnancy outcomes following in vitro fertilization (2013). Renata S. Tavares, Andreia Filipa Silva, Bárbara Lourenço, Teresa Almeida-Santos, Ana Paula Sousa & João Ramalho-Santos.Andrology 1: 830-837.

Mitochondria functionality and sperm quality (2013) Alexandra Amaral, Bárbara Lourenço, Mónica Marques & João Ramalho-Santos. Reproduction 146: R163–R174.

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Brief Curriculum Vitae

PI Current Positions:
Associate Editor Human Reproduction 2001-2006; 2010-present.
Editorial Board Reproduction 2013-present
Member, National Council of Ethics for the Life Sciences 2012-2014.
FCT evaluator, 2012-present

PI Education and Previous Positions:
Fulbright Fellow, University of California San Francisco, USA, 2009.
Visiting Scientist at the Magee-Women’s Research Institute, University of Pittsburgh, USA, 2002-2006
Postdoctoral fellow at the Oregon National Primate Research Center (Oregon Health Sciences University), Portland, Oregon, USA, 1997-2000
Ph.D. in Cell Biology at the University of Coimbra, Portugal (1997). Research conducted at the Center for Neuroscience and Cell Biology
Visiting Graduate Student, University of Groningen, The Netherlands (1989)
B.Sc. in Biology at the University of Coimbra, Portugal (1988)