Toward in vitro fertilization in Brachiaria spp.

Brachiaria are forage grasses widely cultivated in tropical areas. In vitro pollination was applied to accessions of Brachiaria spp. by placing pollen of non-dehiscent anthers on a solid medium near isolated ovaries. Viability and in vitro germination were tested in order to establish good conditions for pollen development. Comparing sexual to apomictic plants, apomictic pollen has more abortion after meiosis during the microspore stage and a lower viability and, of both types, only some plants have sufficient germination in a high sugar concentration. Using in vitro pollination with the sexual plant, the pollen tube penetrates into the nucellus and micropyle, but the embryo sac degenerates and collapses. In the apomictic B. decumbens, in vitro pollination leads to the transfer of the sperm nuclei into the egg cell and the central cell. The results are discussed according to normal fertilization and barriers in sexual and apomictic plants

Involvement of sperm acetylated histones and the nuclear isoform of Glutathione peroxidase 4 in fertilization

We previously demonstrated that the nuclear form of Glutathione peroxidase 4 (nGPx4) has a peculiar distribution in sperm head, being localized to nuclear matrix and acrosome and that sperm lacking nGPx4 are more prone to decondensation in vitro. In this study we have hypothesized that sperm retained acetylated histones and nGPx4 are implicated in paternal chromatin decondensation and male pronucleus formation at fertilization. Indeed, significant higher amounts of acetylated histone H4 and acetylated histone H3 were observed by both immunofluorescence and western blotting in nGPx4-KO sperm vs WT ones. In vitro fertilization of zona pellucida- deprived oocytes by WT sperm in the presence of trichostatin (TSA) also demonstrated that paternal histone acetylation was inversely related to the timing of sperm nucleus decondensation at fertilization. In contrast, TSA had no effect on nGPx4-KO sperm, indicating they had a maximal level of histone acetylation. Moreover the paternally imprinted gene Igf2/H19 was hypomethylated in KO sperm compared to WT ones. The lack of nGPx4 negatively affected male fertility, causing a marked decrease in total pups and pregnancies with delivery, a significant reduction in pronuclei (PN) embryos in in vitro fertilization assays and an approximately 2 h delay in egg fertilization in vivo. Because the zona pellucida binding and fusion to oolemma of nGPx4-KO and WT sperm were similar, the subfertility of nGPx4 sperm reflected a decreased sperm progression through egg cumulus/zona pellucida, pinpointing a defective acrosome in line with acrosomal nGPx4 localization. We conclude that paternal acetylated histones and acrosomal nGPx4 are directly involved in fertilization

Effect of cortisol on bovine oocytes maturation and further embryonic development after in vitro fertilization

Dissertação de Mestrado, Engenharia Zootécnica, 07 de dezembro de 2018, Universidade dos Açores.A maturação meiótica dos ovócitos e o posterior desenvolvimento embrionário após a fertilização são importantes requisitos fisiológicos para a sobrevivência das espécies. Desta forma, o objetivo do presente estudo foi avaliar os efeitos da hormona relacionada com o stress, cortisol, na maturação nuclear e desenvolvimento embrionário de oócitos bovinos após fecundação in vitro. Esta hormona (C₂₁H₃₀O₅) é um corticosteroide da família de esteroides, produzido pela parte superior da glândula supra-renal libertada quando um organismo está sob stress. Vários estudos demonstraram que o cortisol desempenha um papel vital inibindo as quinases extracelulares reguladas por sinal, necessárias para a progressão da prófase meiótica, essenciais para o início de eventos iniciais de maturação do ovócito de maturação meiótica (retomada da meiose), ovulação e posterior desenvolvimento embrionário. No presente estudo, para avaliar o efeito do cortisol na maturação dos ovócitos bovinos e desenvolvimento embrionário, foram recolhidos um total de 1439 óculos de vacas e novilhas púberes, abatidas em matadouros e maturados in vitro durante 24 horas com diferentes concentrações de cortisol (0 (controlo); 50 μM; 150 μM; 250 μM). Posteriormente, 412 oócitos foram desnudados, corados com aceto-orceína, sendo avaliado o desenvolvimento meiótico. Os outros 1027 foram submetidos à fecundação in vitro (FIV) e cultivados durante 9 dias, sendo avaliados nos dias 2, 6 e 9, para clivagem, mórula e blastocisto, respetivamente. No controlo, 85% dos oócitos atingiram a metáfase II, diminuindo para 49, 32 e 15% para a concentração do cortisol (50, 150 e 250 μM, respetivamente). Para os embriões obtidos a partir dos oócitos submetidos à FIV, no grupo controlo, 28,3 ± 4,8% atingiram o estágio do blastocisto, enquanto que para as concentrações de cortisol esse valor diminuiu para 22,1 ± 5,4%, 15,4 ± 6,0% e 6,5 ± 2,1 % para 50, 150 e 250 μM de cortisol, respetivamente). Os resultados do presente estudo demonstraram claramente que o stress do animal e particularmente altas concentrações de cortisol prejudicam a maturação nuclear bovina, bem como o desenvolvimento embrionário posterior após a FIV.ABSTRACT: Oocyte meiotic maturation and further embryonic development after fertilization is the important physiological requirements for species survival. Herein, the aim of the study was to evaluate the effects of the stressful hormone, cortisol, on the nuclear maturation and embryo development of bovine oocytes after in vitro fertilization (IVF). This hormone (C₂₁H₃₀O₅) is a corticosteroid of the steroid family, produced by the upper part of the adrenal gland released when an organism is stressed. Therefore, several studies demonstrated that cortisol plays a vital role inhibiting the extracellular signal-regulated kinases, necessary for meiotic prophase progression, essential for onset of early events of meiotic maturation oocyte maturation (resumption of meiosis), ovulation and further embryo development. In the present study, to evaluate the effect of cortisol on bovine oocyte maturation and further embryonic development, a total of 1439 immature oocytes were collected from slaughtered cows and matured in vitro for 24 hours with different concentrations of cortisol (0 (control); 50 μM; 150 μM; 250 μM). Afterwards, 412 oocytes were denuded, dyed with aceto-orcein and evaluated for meiotic development. The other 1027 were submitted to IVF and cultured for 9 days, being evaluated on day 2, 6 and 9, for cleavage, morula and blastocyst, respectively. In the control, 85 % of oocytes reached Metaphase II, decreasing to 49, 32 and 15 % for the concentration of the cortisol (50, 150, and 250 μM, respectively). For the embryos, obtained from the oocytes submitted to IVF, in the control group, 28.3 ± 4.8% reached the stage of blastocyst, while for the concentrations of cortisol this value decreased to 22.1 ± 5.4%, 15.4 ± 6.0% and 6.5 ± 2.1% for 50, 150 and 250 μM of cortisol, respectively). Results of the present study clearly demonstrated that animal’s stress and particularly high concentrations of cortisol impair bovine nuclear maturation as well as the further embryonic development after IVF