Proteomics, metabolomics and lipidomics in reproductive biotechnologies: the MS solutions

Background : A broader view of living systems complexity is bringing important contributions to biological sciences, since the genome expression is affected by other classes of molecules, which in their turn interact themselves in cellular metabolic pathways and biochemical networks. This level of information has been made possible by the emergence of the omic strategies, such as proteomics, metabolomics and lipidomics, that are mainly based on mass spectrometry (MS) platforms. MS has presented an incredible development over the last years, evolving to a powerful and universal analytical technique. Its ability to analyze proteins and small molecules such as lipids, sugars and metabolites at the structural level, with sensitivity and speed inconceivable a few years ago, is the major driving force in the omic fields. The development of electrospray and matrix-assisted laser desorption/ionization (MALDI) ionization techniques has decisively contributed to the many applications of this technology nowadays. Herein, we present and discuss omic concepts and strategies, as well as detail basic principles of MS. Applications and future perspectives of these approaches are focused in the reproductive medicine area. Review: The omic technologies propose global characterization of specific classes of target biomolecules of cellular systems as a strategy to achieve comprehensive understanding of biological functions. The genomics, aimed at performing the entire genetic sequencing of organisms, represented the seminal step towards the understanding of the complex logic that orchestrates the function of all organisms or the defects leading to diseases. But to express the phenotype, information needs to flow from DNA via carrier biomolecules through processes that are being addressed by new omic sciences such as the transcriptomics, proteomics, metabolomics, glycomics, lipidomics, and fluxomics. Mass spectrometry (MS) is nowadays the most powerful technique for the structural characterization of biomolecules, and has therefore become the central technique for the omic sciences. Using revolutionary ionization techniques such as electrospray (ESI) and matrix-assisted laser desorption ionization (MALDI), a wide range of biomolecules such as peptides, proteins, lipids and sugars are efficiently transferred in intact ionized forms to the gas phase for MS analysis. The development of ESI-MS and MALDI-MS has been awarded the Nobel Prize for Chemistry in 2002, rocketing the application of MS in the omic sciences. More recently, ambient ionization MS techniques, such as desorption electrospray ionization (DESI) and easy ambient sonic-spray ionization (EASI), have been developed for ionization in the open atmosphere, in a workup free and high throughput fashion directly from sample in their original environments. For the more complex samples, the coupling with separation techniques such as liquid chromatrography (LC) as well as the use of tandem MS (LC-MS/MS) has allowed comprehensive mixture characterization of major biomolecules. Conclusion: This manuscript describes recent advances of MS in the proteomics, metabolomics and lipidomics for biological sciences, and points out the relevant contributions that MS is likely to bring to fundamental and applied research in human and animal embryo biotechnologies.

Effect of angiotensin II with follicle cells and insulin-like growth factor-I or insulin on bovine oocyte maturation and embryo development.

The objective was to evaluate the effects of angiotensin II (Ang II), insulin-like growth factor-I (IGF-I) and insulin on the nuclear and cytoplasmic maturation of bovine oocytes in the presence of follicular cells. Cumulus-oocyte complexes (COCs) were cultured for 22h in the presence of follicular cells (control with cells) and Ang II, IGF-I or insulin (treatments), or in the absence of follicular cells (control without cells). Using these five groups, Experiment 1 was conducted with and without the addition of gonadotrophins. Only oocytes in the Ang II group resumed meiosis at rates (88.2+/-1.8% and 90.7+/-4.3% for oocytes cultured in the absence or presence of LH/FSH, respectively) similar to those observed for oocytes cultured in the absence of follicular cells (89.7+/-0.3% and 92.6+/-2.6%; P<0.01). In Experiment 2, the effect of Ang II alone and in combination with IGF-I or insulin on oocyte maturation for 7h (germinal vesicle breakdown), 12h (metaphase I) and 22h (metaphase II) was evaluated in a design similar to that of the first experiment. Ang II plus IGF-I or insulin induced the resumption of meiosis, irrespective of the presence of gonadotrophins (P<0.01). Experiment 3 used groups similar Experiment 2 to determine the rate of subsequent embryo development, using fetal calf serum (FCS) in the culture medium. The COCs were cultured in maturation medium for 1h (1+23h), 12h (12+12h) or 24h in the presence of follicular cells and the respective treatments and for the remaining period in the absence of follicular cells to complete 24h. In Experiment 4, BSA was used in lieu of serum in the maturation medium in a 12+12h maturation system. Oocytes matured using the 12+12h system with BSA or FCS in the presence of Ang II+IGF-I had higher rates of blastocyst formation than the other treatments (P<0.05). In conclusion, Ang II reversed the inhibitory effect of follicular cells on nuclear maturation of bovine oocytes, irrespective of the presence of gonadotrophins, IGF-I and insulin. However, oocyte cytoplasmatic maturation (i.e., subsequent embryo development), was higher when Ang II and IGF-I were present in the maturation medium containing follicular cells cultured for 12+12h.

Hormonal induction of ovulation and artificial insemination in suckled beef cows under nutritional stress.

The objective was to develop a program for inducing estrus (followed by insemination) of suckled beef cows under nutritional stress (poor body condition). A total of 123 cows, from 60 to 75 days postpartum, were classified according to their body condition score (BCS; range from 1 to 5, in increments of 0.5) and allocated into two groups. On Day 0 (without regard to stage of the estrous cycle), cows (n = 59) in the hormone induction (HI) treatment group were given an intravaginal device (IVD) containing 250 mg of medroxiprogesterone acetate (MAP) and an i.m. injection of 2.5 mg estradiol benzoate (EB). On Day 6, these cows were given 500 IU eCG i.m. and calves were weaned for 96 h. The IVD were removed on Day 7. Cows detected in estrus by 45 h after IVD removal were inseminated 12 h after standing estrus; cows not in estrus by 45 h after IVD removal received an i.m. injection of 100 microg gonadorelin (GnRH) and were inseminated 16-18 h later. In the control group (C), cows (n = 64) only had their calves weaned at Day 6 (for 96 h), with estrus detection and AI from Days 6 to 11. Overall, the BCS ranged from 2.0 to 3.0. In the treatment group, estrus and pregnancy rates in cows with BCS 2.0 (20 and 30%, respectively) was lower (P < 0.05) than those with BCS 3.0 (50 and 66.6%, respectively), but did not differ (P > 0.05) from BCS 2.5 (23.3 and 47.6%). In C group, only 2 of 66 cows were detected in estrus and bred (neither was pregnant). In conclusion, the program for induction of ovulation using MAP, EB, eCG and GnRH increased the pregnancy rate in beef cows in poor body condition, enabling AI to be done in a 63-h interval.