El oviductoanálisis transcriptómico, identificación de proteínas secretadas y aplicaciones en reproducción artificial

Résumé

The oviduct is the anatomical region of the female genital tract which connects the utherus with the ovaries. This organ plays a relevant role in gamete transport and storage, including the sperm reservoir. Moreover, fecundation and the first stages of embryo development take place in the oviduct. In the first part of this thesis, a technique on boar spermatozoa separation was developed on the bases of affinity for sugars involved in the sperm reservoir. A study on the ability of these spermatozoa to bind the zona pellucida (ZP) was done to evaluate a possible use on assisted reproduction techniques. In the second part of the thesis, a transcriptomics study of the oviduct of the rabbit doe was performed before and after ovulation, and during the preimplantational embryo development to identify key factors of each stage. Among the experimental techniques used, there was flow cytometry cell sorting to select the sperm subpopulations pre-incubated with fluorescent ovalbumin and the sugar Lewisa. Affinity for ZP was evaluated by incubating in capacitating medium each sperm subpopulation and its correspondent control with isolated ZPs from oocytes obtained from ovaries of a local abatoir. The quantity of sperm bound to the ZP was evaluated with Hoescht 33342 by fluorescence microscopy. The temporal differences in the transcriptome of the oviduct were evaluated by microarray analysis and RT-qPCR, doing the correspondent immunohistochemistry techniques. The analysis of secreted proteins to the oviductal fluid (OF) by western blot. Results show that it is possible to separate sperm subpopulations based on their affinity for sugar probes, and these subpopulations have a different affinity for the ZP, so they have a distinct fertilization potential. Microarray analysis show 602 transcripts differentially expressed between the analysed stages (pre-ovulatory, post-ovulatory, 8-cell containing oviduct and morulae containing oviduct). Furthermore, three oviductal proteins with a differential expression during the analysed stages were identified: osteoponting (SPP1), whose expression is highest in the post-ovulatory stage; neuropeptide Y (NPY), secreted before ovulation; metallopeptidase 7 (MMP7), whose expression is repressed during the last stages of pre-implantational development. To conclude with the first part of the thesis: 1) there are several sperm subpopulations with different affinities for sugars which have different affinities for the ZP, 2) it is feasible to separate these sperm subpopulations by flow cytometry cell sorting, but its affinity for the ZP is compromised, 3) it is mandatory to implement a method of sperm selection not involving sorting in order to avoid disminishing the affinity for the ZP. About the second part of the thesis: 1) rabbit doe oviductal transcriptome shows spatial and temporal differences through ovulation and pre-implantational development; 2) Several OF protein concentrations fluctuate along copulation and the end of pre-implantational development, among those: SPP1, NPY and MMP7; 3) Response to ovulation and copulation involves certain molecular mechanisms conserved across different mammalian species, favouring the expression of SERPINE1, SPP1 and PTGS2; 4) Response to embryo presence within the oviduct involves a local regulation of the immune system. However, it has not been discovered a clear universal mechanism of response due to discrepancy of results among species.