Critical Steps of Plasmodium falciparum Ookinete Maturation

The egress and fertilization of Plasmodium gametes and development of a motile ookinete are the first crucial steps that mediate the successful transmission of the malaria parasites from humans to the Anopheles vector. However, limited information exists about the cell biology and regulation of this process. Technical impediments in the establishment of in vitro conditions for ookinete maturation in Plasmodium falciparum and other human malaria parasites further constrain a detailed characterization of ookinete maturation. Here, using fluorescence microscopy and immunolabeling, we compared P. falciparum ookinete maturation in Anopheles coluzzii mosquitoes in vivo and in cell culture in vitro. Our results identified two critical steps in ookinete maturation that are regulated by distinct mosquito factors, thereby highlighting the role of the mosquito environment in the transmission efficiency of malaria parasites

Responsiveness of bovine cumulus-oocyte-complexes (COC) to porcine and recombinant human FSH, and the effect of COC quality on gonadotropin receptor and Cx43 marker gene mRNAs during maturation in vitro

Substantially less development to the blastocyst stage occurs in vitro than in vivo and this may be due to deficiencies in oocyte competence. Although a large proportion of bovine oocytes undergo spontaneous nuclear maturation, less is known about requirements for proper cytoplasmic maturation. Commonly, supraphysiological concentrations of FSH and LH are added to maturation media to improve cumulus expansion, fertilization and embryonic development. Therefore, various concentrations of porcine FSH (pFSH) and recombinant human FSH (rhFSH) were investigated for their effect on bovine cumulus expansion in vitro. Expression of FSHr, LHr and Cx43 mRNAs was determined in cumulus-oocyte complexes to determine whether they would be useful markers of oocyte competence. In serum-free media, only 1000 ng/ml pFSH induced marked cumulus expansion, but the effect of 100 ng/ml pFSH was amplified in the presence of 10% serum. In contrast, cumulus expansion occurred with 1 ng/ml rhFSH in the absence of serum. FSHr mRNA was highest at 0–6 h of maturation, then abundance decreased. Similarly, Cx43 mRNA expression was highest from 0–6 h but decreased by 24 h of maturation. However, the relative abundance of LHr mRNA did not change from 6–24 h of maturation. Decreased levels of FSHr, LHr and Cx43 mRNAs were detected in COCs of poorer quality. In conclusion, expansion of bovine cumulus occurred at low doses of rhFSH in serum-free media. In summary, FSHr, LHr and Cx43 mRNA abundance reflects COC quality and FSHr and Cx43 mRNA expression changes during in vitro maturation; these genes may be useful markers of oocyte developmental competence

Porcine oocyte maturation in vitro : role of cAMP and oocyte-secreted factors: a practical approach

Polyspermy or the penetration of more than one sperm cell remains a problem during porcine in vitro fertilization (IVF). After in vitro culture of porcine zygotes, only a low percentage of blastocysts develop and their quality is inferior to that of in vivo derived blastocysts. It is unknown whether the cytoplasmic maturation of the oocyte is sufficiently sustained in current in vitro maturation (IVM) procedures. The complex interplay between oocyte and cumulus cells during IVM is a key factor in this process. By focusing on this bidirectional communication, it is possible to control the coordination of cumulus expansion, and nuclear and cytoplasmic maturation during IVM to some extent. Therefore, this review focuses on the regulatory mechanisms between oocytes and cumulus cells to further the development of new in vitro embryo production (IVP) procedures, resulting in less polyspermy and improved oocyte developmental potential. Specifically, we focused on the involvement of cAMP in maturation regulation and function of oocyte-secreted factors (OSFs) in the bidirectional regulatory loop between oocyte and cumulus cells. Our studies suggest that maintaining high cAMP levels in the oocyte during the first half of IVM sustained improved oocyte maturation, resulting in an enhanced response after IVF and cumulus matrix disassembly. Recent research indicated that the addition of OSFs during IVM enhanced the developmental competence of small follicle-derived oocytes, which was stimulated by epidermal growth factor (EGF) via developing EGF-receptor signaling

Producción in vitro de embriones de bovino: influencia de la suplementación sérica y hormonal

On study the effect of different serum and hormonal supplements, when added to the maturation medium, on the in vitro maturation and subsequent fertilization and cleavage process of bovine oocytes. The oocyte maturation, fertilization and cleavage rates in the presence of ECS, FCS and hormonal supplements were significantly higher (p<0.001) than that obtained in the control group. No significant differences for maturation, fertilization and development were observed between the different supplements. The addition of serum and hormonal supplements to the maturation medium improved the in vitro maturation rates and subsequent in vitro fertilization and cleavage rates of bovine oocytes.El objetivo del presente estudio fue investigar si diferentes suplementos séricos y hormonales, cuando se adicionan al medio de maduración, mantienen los procesos de maduración, fecundación y división de ovocitos de bovino in vitro. Los índices de maduración ovocitaria, fecundación y división, en presencia de los suplementos ECS, FCS y hormonas fueron superiores significativamente (p<0,001) que aquellos observados en el grupo control. No se apreciaron diferencias significativas para los estadíos de maduración, fecundación y división entre los diferentes suplementos estudiados. Concluimos que la adición de suplementos séricos y hormonales al medio de maduración mejora los índices de maduración ovocitaria y subsiguientes índices de fecundación y división in vitro de ovocitos de bovino

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

Detection of intermediates and kinetic control during assembly of bacteriophage P22 procapsid

Bacteriophage P22 serves as a model for the assembly and maturation of other icosahedral double-stranded DNA viruses. P22 coat and scaffolding proteins assemble in vitro into an icosahedral procapsid, which then expands during DNA packaging (maturation). Efficient in vitro assembly makes this system suitable for design and production of monodisperse spherical nanoparticles (diameter ≈50 nm). In this work we explore the possibility of controlling the outcome of assembly by scaffolding protein engineering. The scaffolding protein exists in monomer-dimer-tetramer equilibrium. We address the role of monomers and dimers in assembly by using three different scaffolding proteins with altered monomer-dimer equilibrium (weak dimer, covalent dimer, monomer). The progress and outcome of assembly was monitored by time-resolved X-ray scattering which allowed us to distinguish between closed shells and incomplete assembly intermediates. Binding of scaffolding monomer activates the coat protein for assembly. Excess dimeric scaffolding protein resulted in rapid nucleation and kinetic trapping yielding incomplete shells. Addition of monomeric wild type scaffold with excess coat protein completed these metastable shells. Thus, the monomeric scaffolding protein plays an essential role in the elongation phase by activating the coat and effectively lowering its critical concentration for assembly

Vitrification of human immature oocytes before and after in vitro maturation: a review

The use of immature oocytes subjected to in vitro maturation (IVM) opens interesting perspectives for fertility preservation where ovarian reserves are damaged by pathologies or therapies, as in PCO/PCOS and cancer patients. Human oocyte cryopreservation may offer some advantages compared to embryo freezing, such as fertility preservation in women at risk of losing fertility due to oncological treatment or chronic disease, egg donation and postponing childbirth. It also eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In addition, a successful oocyte cryopreservation program could eliminate the need for donor and recipient menstrual cycle synchronization. Recent advances in vitrification technology have markedly improved the oocyte survival rate after warming, with fertilization and implantation rates comparable with those of fresh oocytes. Healthy live births can be achieved from the combination of IVM and vitrification, even if vitrification of in vivo matured oocytes is still more effective. Recently, attention is given to highlight whether vitrification procedures are more successful when performed before or after IVM, on immature GV-stage oocytes, or on in vitro matured MII-stage oocytes. In this review, we emphasize that, even if there are no differences in survival rates between oocytes vitrified prior to or post-IVM, reduced maturation rates of immature oocytes vitrified prior to IVM can be, at least in part, explained by underlying ultrastructural and biomolecular alterations

Modulation of the effectiveness of 17-alpha-hydroxy-20-beta-dihydroprogesterone or of a gonadotrophic extract on the in vitro intrafollicular maturation of oocytes of the rainbow trout Salmo gairdnerii by various non-maturing steroids [Translation from: Compte Rendu Hebdomadaire des Seances de l'Academie des Sciences, Paris, Series D 281, 811-814, 1975]

The effectiveness of 17 α-hydroxy-20 β-dihydroprogesterone (17 α-20 β Pg) or of a trout hypophyseal gonadotrophic extract on the in vitro intrafollicular maturation of trout oocytes can be modulated by steroids which do not have a direct maturing effect; the effectiveness of the gonadotrophic extract is lowered by oestradiol and oestrone and increased by testosterone. As these steroids have no significant effect on maturation induced by 17 α-20 β Pg, the site of their activity is probably in the follicular envelopes. Corticosteroids, and Cortisol and cortisone in particular increase the effectiveness of the gonadotrophic extract, but increase the effectiveness of 17 α-20 β Pg even more strongly, suggesting that this 'progestagen' has a direct effect on oocyte sensitivity

Effect of Oocyte Vitrification Before and After in Vitro Maturation Towards Bcl-2, Bax and Bcl-2/Bax Ratio Expression

Objectives: to compare the expression of Bcl-2, Bax and Bcl-2/Bax ratio in cumulus cell and oocyte between vitrified oocyte pre and post in vitro maturation.Materials and Methods: Maturation was operated in medium TC 100 µl for 24 hours. Vitrification begins with washing oocyte in PBS basic medium supplemented of 20% serum for 1-2 minutes, followed by equilibration medium PBS + 20% serum + 10% ethylene glycol for 10-14 minutes, then transferred to 20% serum + PBS + 0.5 M sucrose + 15% ethylene glycol + PROH 15% for 25-30 seconds. Thawing is processed by submerging the oocytes in the media: 1). PBS + 20% serum + 0.5 M sucrose, 2). PBS + 20% serum + 0.25 M sucrose, and 3). PBS + 20% serum + 0.1 M sucrose. Imunocytochemistry observed the expression of Bcl-2, bax and Bcl-2/bax ratio.Results: Bcl-2 expression on oocyte in control group differed significantly with treatment group, Bcl-2 expression on cumulus in control group differed significantly with treatment 1 group. Bax expression on oocyte in control group differed significantly with treatment group. Bax expression on cumulus in control group differed significantly with treatment group. Bcl-2/Bax expression ratio on oocyte and cumulus did not differ significantly in all groupConclusion: No difference Bcl-2/Bax expression ratio on oocyte and cumulus between vitrified oocyte pre and post in vitro maturation

A Requirement for Fatty Acid Oxidation in the Hormone-Induced Meiotic Maturation of Mouse Oocytes

We have previously shown that fatty acid oxidation (FAO) is required for AMP-activated protein kinase (PRKA)-induced maturation in vitro. In the present study, we have further investigated the role of this metabolic pathway in hormone-induced meiotic maturation. Incorporating an assay with 3H-palmitic acid as the substrate, we first examined the effect of PRKA activators on FAO levels. There was a significant stimulation of FAO in cumulus cell-enclosed oocytes (CEO) treated with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and RSVA405. In denuded oocytes (DO), AICAR stimulated FAO only in the presence of carnitine, the molecule that facilitates fatty acyl CoA entry into the mitochondria. The carnitine palmitoyltransferase 1 activator C75 successfully stimulated FAO in CEO. All three of these activators trigger germinal vesicle breakdown. Meiotic resumption induced by follicle-stimulating hormone (FSH) or amphiregulin was completely inhibited by the FAO inhibitors etomoxir, mercaptoacetate, and malonyl CoA. Importantly, FAO was increased in CEO stimulated by FSH and epidermal growth factor, and this increase was blocked by FAO inhibitors. Moreover, compound C, a PRKA inhibitor, prevented the FSH-induced increase in FAO. Both carnitine and palmitic acid augmented hormonal induction of maturation. In a more physiological setting, etomoxir eliminated human chorionic gonadotropin (hCG)-induced maturation in follicle-enclosed oocytes. In addition, CEO and DO from hCG-treated mice displayed an etomoxir-sensitive increase in FAO, indicating that this pathway was stimulated during in vivo meiotic resumption. Taken together, our data indicate that hormone-induced maturation in mice requires a PRKA-dependent increase in FAO