Physiological and biochemical events related to stallion sperm capacitation in in vitro conditions

• Ano de defesa: 2020
• Autor(a) principal: Maitan, Paula Piccolo
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: eng
• Instituição de defesa: Universidade Federal de Viçosa
• Palavras-chave em Português: Espermatozoides; Garanhões; Capacitação; Adenilato ciclases; Reprodução Animal
• Link de acesso: https://locus.ufv.br//handle/123456789/28591

Only two foals have ever been born from conventional equine IVF (in vitro fertilization), both in the early 1990’s. The main reason for the conventional IVF failure is thought to be the stallion spermatozoa, which are not able to penetrate the zona pellucida, most likely due to incomplete activation (capacitation) under in vitro conditions. Capacitation is considered as a consecutive activation of different signaling pathways inducing physiological and biochemical modifications which primes the sperm for fertilization in vitro. To have the capacitation occur, the sperm must be under an environment (in vivo or in vitro) that contains bicarbonate (HCO 3- ), calcium (Ca 2+ ) and albumin. These elements will besides changes in membrane potential, provide changes in cyclic adenosine monophosphate (cAMP) levels, intracellular pH and intracellular Ca 2+ causing plasma membrane reorganization and cholesterol depletion leading the sperm to undergo the acrosome reaction and fertilize the oocyte. The aim of this work was to show by flow cytometry, fluorescence microscopy, cryo-electron microscopy and immunohistochemistry some capacitation steps in stallion spermatozoa in the presence of molecules that can trigger this process and also understand which molecules may be involve in some capacitation steps. The use of the probe Annexin-V in capacitating conditions showed a significant increase in the live, Annexin-V positive sperm population indicating a phosphatidylserine (PS) exposure in stallion sperm during this process (p≤0.05), but it was only in a small percentage of viable sperm. Stimulation of the sAC/cAMP/PKA pathway by caffeine and dibutyryl-cAMP indicated the relevance of this pathway for PS exposure. The observation of three different staining patterns for Annexin-V in live stallion sperm may warrant further investigation with respect to whether these represent sequential steps in membrane remodeling. Regarding the effects in membrane fluidity and induced acrosome reaction different concentrations of calcium did not interfere in the population live merocyanine and PNA-positive spermatozoa (p>0.05). Also, different concentrations of progesterone (P4) and bovine serum albumin (BSA) were not able to increase membrane fluidity or induced acrosome reaction (p≤0.05). However, calcium should be present in the medium since EGTA (ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; 2 mM) was able to block all this capacitation steps, but there is no need for higher concentrations (2 mM) to start capacitation in stallion sperm as is needed in other species. Regarding the adenilato cyclases (ACs), the presence of sAC (soluble) in the stallion sperm was shown to be responsible for the increase in membrane fluidity in the presence of bicarbonate. This finding was possible to demonstrate with the use of LRE1, a sAC specific inhibitor that showed no effect in other sperm physiological events (p>0.05). The immunostaining of sAC in stallion sperm indicated different localization compared to boar. In stallions, the enzyme was present as a dotted line (diadem-like pattern) distal from the acrosomal area, while in boars, the sAC was present in the acrosomal area and in the neck. The action of forskolin indicated that tmAC (transmembrane) may be present in the stallion sperm and may play a role in the acrosome reaction. However, the fact that a relatively high concentration (500 µM) was needed to show this effect compared to other reports, indicates that more studies like use of specific inhibitors of tmAC (as ddAdo) or immunostaining of the AC1 to 9 must be performed in stallion sperm to confirm the findings. In conclusion, the findings in this work elucidate several questions in stallion capacitation as PS exposure, role of different concentrations of calcium, progesterone and BSA in this process and the role of adenylyl cyclases in some stallion capacitation events showing that capacitation is a complex process that differs among species. Keywords: Adenylyl cyclases. Capacitation. Membrane reorganization. Stallion sperm