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Fertilization markers

New techniques map the function of proteins, carbohydrates and lipids to obtain the highest quality of bovine embryos

1fALEXANDRE TELESFour groups of São Paulo researchers are attempting to map and correlate the function of molecular markers such as lipids, proteins and carbohydrates with the aid of non-invasive analytical techniques. To do this, they are using lasers and spectrometers to select the best bovine embryos from the in vitro fertilization performed in the laboratory. The goal is to ensure gestational success when transferring the embryos into the uteri of cows. Although Brazil currently holds the top spot worldwide for in vitro production—with 366,517 bovine embryos produced in 2013, representing 70% of the total—there is much room for improvement, because embryo losses amount to between 20% and 60%. The variation in these rates is due to, among other factors, the quality of the embryo and the receptivity of the endometrium, the membrane lining the uterus.

Advances in rapid techniques that are less invasive and affordable for the analysis and selection of embryos will help in biotechnological reproductive procedures both in animals and in humans in the future. An innovative method, which combines the production of images and the chemical mapping of molecular markers present in ova and embryos through mass spectrometry techniques, developed jointly by researchers at the University of Campinas (Unicamp) and the University of São Paulo (USP), could represent a significant advance in reproductive biotechnologies. “With this analytical tool it is possible to view specific parts of the bovine embryo, such as the zona pellucida, which covers the entire embryo and is responsible for connecting it to the uterus,” says Professor Rodrigo Catharino, coordinator of the research and of the Innovare Biomarkers Laboratory, affiliated with the Unicamp School of Pharmaceutical Sciences. The presence of biomarkers such as lipids, which act as indicators of biological processes, can improve this connection, resulting in pregnancies with a greater chance of success. In vitro fertilization involves ova collected from a donor cow, which are then matured and fertilized in the laboratory. After fertilization, the presumptive zygotes—diploid cells formed by the fusion of the egg with the sperm—remain in an incubator to develop for seven days, after which they are frozen or transferred to a receiving cow.

Bovine embryos ready to be transferred to recipients

In Vitro Brasil Bovine embryos ready to be transferred to recipientsIn Vitro Brasil

The research resulted in two scientific articles. In the first study, published in the journal Analytica Chimica Acta, the authors describe the use of Maldi mass spectrometry to view the various areas making up the bovine embryo. In Maldi, a matrix—usually an organic acid capable of absorbing light—is applied to the sample and then shot with laser beams, which result in the formation of ions (atoms that have lost electrons). The ionized molecules enter a vacuum tube (analyzer) and are carried to a detector. Smaller ions pass more quickly through the tube than larger ions. Thus, the substances of the samples form spectra, each having a different mass.

“The meeting of the points and the information indicating which molecule is part of the mass spectrum reveals which biomarker is most prevalent at each location of the sample,” says Catharino. “We can see the structures separately, not just the body of the embryo and oocyte—sex cell produced in the ovaries of animals—as shown by classic analytical techniques.” He points out that knowing the exact location of a chemical marker would represent an innovation. “Many markers described in the studies are unprecedented, which means that a lot of research is needed to find out what they mean within the in vitro embryo production process.”

Bovine embryos in blastocyst stage

Biology Laboratory of animal breeding/UFABC Bovine embryos in blastocyst stageBiology Laboratory of animal breeding/UFABC

The second article, published in the journal Reproduction, Fertility and Development, describes using the Maldi spectrometer to investigate lipid biomarker evolution in bovine ova and embryos of up to eight cells in size and in blastocysts, spherical structures formed in a later stage of embryonic development. “It was possible to chemically differentiate the lipids in each embryonic stage, because they act as chemical signatures of the stages of development.” A number of noteworthy individuals were involved in the Innovare research developments. They included Roseli Gonçalves, a researcher, and Professor José Visintin of the USP School of Veterinary Medicine and Animal Husbandry (FMVZ), Peter Bols of the University of Antwerp, Belgium, Gary Killian of Pennsylvania State University in the United States, and Dr. Mark Achilles, a veterinarian with Achilles Genetics, a company based in Garça, São Paulo State.

Gonçalves, who is working on a research project as part of FAPESP’s Young Investigators in Emerging Institutions program, says the fertilization process is not yet fully defined, so the results obtained in the research are important. While working on her doctorate at FMVZ under a FAPESP grant, she studied at the John Almquist Research Center at Pennsylvania State University, where she worked with fertility markers under Gary Killian as her adviser. After finishing her doctorate, she participated in a project coordinated by Killian, which resulted in the isolation of a protein called osteopontin, present in greater quantity in bulls of dairy breeds with high fertility. “We have seen the effect of this protein in the in vitro production of bovine embryos and semen freezing,” says Gonçalves, who worked on this project until the end of 2004.

Evaluation system for Raman spectroscopy

Biology Laboratory of animal breeding/UFABC Evaluation system for Raman spectroscopyBiology Laboratory of animal breeding/UFABC

At the Federal University of the ABC (UFABC), Santo André campus in the São Paulo Metropolitan Region, Professor Marcella Pecora Milazzotto has since 2010 been in the forefront of research on bovine embryos in collaboration with Herculano da Silva Martinho, a physicist by training. The first project was intended to evaluate the interaction of light from a low-intensity laser with biological materials. “We used the laser to stimulate sperm, oocytes and bovine embryos, and we got some interesting results with the female gamete (reproductive cells),” says Milazzotto. “We managed to activate important signaling pathways within the cells in the maturation process of this gamete. We realized that light affects both production and activation of proteins present in these oocytes.”

Besides this project, the group also works with Raman spectroscopy analysis, which uses a light beam to collect chemical and structural information from biological material. They conducted one such study to find out what was missing from the laboratory embryos to make them resemble those of the field. To do this, comparisons were made. “We use the same techniques used in human reproduction, such as embryogenic morphology, assessment of the speed of embryonic development in the laboratory, and Raman spectroscopy analysis of the laboratory embryos and those produced in the field,” says Milazzotto. And all the spectroscopic data were compared with data on energy metabolism, fat and stress to attempt to select the most viable. The discovery led to a patent filing for the university.

Embryo analysis using Maldi technique

Innovare Laboratory Embryo analysis using Maldi techniqueInnovare Laboratory

At the São Paulo State University School of Veterinary Medicine and Zootechny, Unesp Botucatu, Fernanda da Cruz Landim, of the Animal Reproduction and Veterinary Radiology Department, coordinates research projects to obtain information on the basic biology of bovine embryos; this research seeks to improve important techniques for marketing the product, such as increased in vitro embryo production and cryo-preservation, which is the freezing of excess embryos in the transfer process. One project compared gene expression—the process in which information encoded by a gene is decoded into a protein—between embryos of the Nelore breed (Bos taurus indicus) and the Simmental breed (Bos taurus taurus) produced in the laboratory and collected from cows. “We made a general pattern of gene expression and selected several different genes involved in lipid metabolism,” says Landim.

Maldi mass spectrometry was used to analyze the samples. “When we applied the Maldi technique to assess the lipid profile, to select the best embryos for cryo-preservation, we observed a different lipid profile for the Nelore and Simmental breeds produced in vivo and in vitro.” The embryos with the best qualities to withstand the freezing process were the Simmental produced in the field.

1. Analysis of the protein and lipid profile of pre-implanted bovine embryos obtained by in vitro fertilization, embryo transfer and nuclear transfer of somatic cells using mass spectrometry technique (No. 2010/01077-9); Grant mechanism: Young Investigators in Emerging Institutions; Principal investigator: Roseli Fernandes (USP); Investment: R$629,986.73 (FAPESP).
2. Metabolomics for non-invasive evaluation of in-vitro-produced bovine embryos (No. 2012/10351-2); Grant mechanism: Regular Research Grant; Principal investigator: Marcella Pecora Milazzotto (UFABC); Investment: R$174,631.10 (FAPESP).
3. Comparison of the global gene expression pattern of in-vitro-produced Bos taurus indicus and Bos taurus taurus embryos (No. 2010/09922-0); Grant mechanism: Regular Research Grant; Principal investigator: Fernanda Cruz Landim (Unesp), Investment: R$140,442.19 (FAPESP).

Scientific articles
Gonçalves, R. F. et al. Analysis and characterization of bovine oocyte and embryo biomarkers by matrix-assisted desorption ionization mass spectrometry imaging. Reproduction, Fertility and Development. July 11, 2014 (online).
Sudano, M. J. et al. Phosphatidylcholine and sphingomyelin profiles vary in Bos taurus indicus and Bos taurus taurus in-vitro– and in-vivo-produced blastocysts. Biology of Reproduction. V. 87 (6), p. 130. December 2012.