Three-dimensional levitation culture improves in-vitro growth of secondary follicles in bovine model.

RESEARCH QUESTION: Does a three-dimensional culture system based on magnetic levitation with nanoparticles assembly maintain the follicular structure and viability with adequate growth rates leading to oocyte maturation after long-term culture? DESIGN: Randomized-controlled trial of treatments in a bovine model. Secondary follicles (n = 213) isolated from bovine ovaries were cultured in a two-dimensional system (two-dimensional control) or three-dimensional levitation system with different concentrations (three-dimensional 50 µl/ml, 100 µl/ml and 200 µl/ml) of magnetic nanoparticles. Follicular growth (diameter, daily growth and growth patterns), morphology (normal, degenerated and extruded follicles), antrum formation, oocyte viability and chromatin configuration were assessed. RESULTS: Secondary follicles of three-dimensional 200-µl/ml treatment showed higher viability, antrum formation and lower degeneration rates than two-dimensional control. Also, follicles cultured in the three-dimensional 200-µl/ml treatment presented a most homogenous daily growth rate as shown by the lowest variance and standard deviation. Compared with the two-dimensional control, the proportion of non-growing and slow-growing follicles were 3.8-fold lower and 1.6-fold higher, respectively, in the three-dimensional 200-µl/ml treatment. After in-vitro maturation, the three-dimensional 200-µl/ml had a greater proportion of viable oocytes (1.7-fold) and meiotic resumption rates (2.4-fold) than the two-dimensional control treatment. CONCLUSION: The three-dimensional levitation culture system improves the viability of in-vitro development of bovine secondary follicles, antrum formation and lower extrusion and degeneration rates and adequate growth rate leading to relevant oocyte viability and meiotic resumption after in-vitro maturation. This approach does not require a specific medium, and has the potential as an alternative method to in-vitro follicle culture in several species, including humans.

Toxicity effect of Auxemma oncocalyx fraction and its active principle oncocalyxone A on in vitro culture of caprine secondary follicles and in vitro oocyte maturation / Efeito da toxicidade da fração da Auxemma oncocalyx e seu princípio ativo oncocalyxona A no cultivo in vitro de folículos secundários e na maturação in vitro de oócitos de caprinos

The extract of Auxemma oncocalyx (A. oncocalyx) and its main component, oncocalyxone A (onco A), possess anti-tumoral activity that may affect fertility. There is limited literature on the action of these substances regarding caprine folliculogenesis. In this study, we evaluated the effect of A. oncocaly and onco A on the in vitro culture of isolated secondary follicles (Experiment 1) and on the in vitro maturation (IVM) of oocytes from caprine antral follicles grown in vivo (Experiment 2). Isolated secondary follicles were distributed in six groups: the non-cultured control was immediately fixed in 4% paraformaldehyde. The remaining follicles were cultured for 7 days in α-minimal essential medium (α-MEM+ ) alone (control) or with dimethyl sulfoxide (DMSO), doxorubicin (DXR), A. oncocalyx, or onco A. After culture, the follicles were evaluated for antrum formation, growth rate, apoptosis (TUNEL), cellular proliferation (PCNA), as well as the expression of BCL2 and BAX. In addition, cumulus oocyte complexes (COCs) were aspirated and allocated into five treatments for IVM: control, cultured only in maturation base medium (TCM 199+ ); or supplemented with DMSO; DXR; A. oncocalyx or onco A. After IVM, oocyte chromatin configuration and viability were assessed. After 7 days of culture, a reduction was noted in the percent of morphologically intact follicles, antrum formation, growth rate, and numbers of PCNApositive granulosa cells (P < 0.05). After culture, the DXR treatment showed a higher percent of TUNELpositive follicles and relative BAX:BCL2 mRNA ratios (P < 0.05). After IVM of the COCs, DXR, A. oncocalyx, and onco A treatments showed a greater percent (P < 0.05) of abnormal oocytes and a lower percent of viable oocytes as compared with the control group (P < 0.05)...(AU)

O extrato da Auxemma oncocalyx (A. oncocalyx) e seu componente, Oncocalyxona A (onco A), possui atividade antitumoral, podendo afetar a fertilidade. Entretanto, estudos sobre a ação dessas substâncias em relação à foliculogênese caprina são desconhecidos. O objetivo desse estudo foi avaliar o efeito da A. oncocalyx e onco A no cultivo in vitro de folículos secundários isolados (Experimento 1) e na maturação in vitro (MIV) de oócitos de folículos antrais caprinos crescidos in vivo (Experimento 2). Folículos secundários isolados foram distribuídos em seis grupos, em que o controle não-cultivado foi imediatamente fixado em paraformaldeído 4%. Os folículos restantes foram cultivados durante 7 dias em α-MEM+ sozinho (controle) ou suplementado com DMSO, doxorrubicina (DXR), A. oncocalyx ou onco A. Após o cultivo, os folículos foram avaliados quanto à formação de antro, taxa de crescimento, apoptose (TUNEL) e proliferação celular (PCNA), bem como a expressão dos genes BCL2 e BAX. Além disso, os complexos cumulus-oócitos (CCOs) foram aspirados e distribuídos em cinco tratamentos para MIV: o controle em meio de maturação (TCM 199+), e os demais tratamentos suplementados com DMSO, DXR, A. oncocalyx ou onco A. Depois da MIV, a configuração da cromatina e viabilidade oocitária foram avaliadas. Após 7 dias de cultivo, observou-se redução na percentagem de folículos morfologicamente intactos, na formação de antro, na taxa de crescimento e no número de células PCNA positivas (P < 0,05). Depois do cultivo, no tratamento DXR foi observada maior percentagem de folículos TUNEL positivos (P < 0,05) e também aumento na taxa de RNAm BAX: BCL2 (P < 0,05). Após MIV dos CCOs, nos tratamentos com DXR, A. oncocalyx e onco A, observou-se maior (P < 0,05) percentagem de oócitos anormais e menor de oócitos viáveis quando comparados ao grupo controle (P < 0,05)...(AU)

Toxicity effect of Auxemma oncocalyx fraction and its active principle oncocalyxone A on in vitro culture of caprine secondary follicles and in vitro oocyte maturation / Efeito da toxicidade da fração da Auxemma oncocalyx e seu princípio ativo oncocalyxona A no cultivo in vitro de folículos secundários e na maturação in vitro de oócitos de caprinos

The extract of Auxemma oncocalyx (A. oncocalyx) and its main component, oncocalyxone A (onco A), possess anti-tumoral activity that may affect fertility. There is limited literature on the action of these substances regarding caprine folliculogenesis. In this study, we evaluated the effect of A. oncocaly and onco A on the in vitro culture of isolated secondary follicles (Experiment 1) and on the in vitro maturation (IVM) of oocytes from caprine antral follicles grown in vivo (Experiment 2). Isolated secondary follicles were distributed in six groups: the non-cultured control was immediately fixed in 4% paraformaldehyde. The remaining follicles were cultured for 7 days in α-minimal essential medium (α-MEM+ ) alone (control) or with dimethyl sulfoxide (DMSO), doxorubicin (DXR), A. oncocalyx, or onco A. After culture, the follicles were evaluated for antrum formation, growth rate, apoptosis (TUNEL), cellular proliferation (PCNA), as well as the expression of BCL2 and BAX. In addition, cumulus oocyte complexes (COCs) were aspirated and allocated into five treatments for IVM: control, cultured only in maturation base medium (TCM 199+ ); or supplemented with DMSO; DXR; A. oncocalyx or onco A. After IVM, oocyte chromatin configuration and viability were assessed. After 7 days of culture, a reduction was noted in the percent of morphologically intact follicles, antrum formation, growth rate, and numbers of PCNApositive granulosa cells (P < 0.05). After culture, the DXR treatment showed a higher percent of TUNELpositive follicles and relative BAX:BCL2 mRNA ratio’s (P < 0.05). After IVM of the COCs, DXR, A. oncocalyx, and onco A treatments showed a greater percent (P < 0.05) of abnormal oocytes and a lower percent of viable oocytes as compared with the control group (P < 0.05)...

O extrato da Auxemma oncocalyx (A. oncocalyx) e seu componente, Oncocalyxona A (onco A), possui atividade antitumoral, podendo afetar a fertilidade. Entretanto, estudos sobre a ação dessas substâncias em relação à foliculogênese caprina são desconhecidos. O objetivo desse estudo foi avaliar o efeito da A. oncocalyx e onco A no cultivo in vitro de folículos secundários isolados (Experimento 1) e na maturação in vitro (MIV) de oócitos de folículos antrais caprinos crescidos in vivo (Experimento 2). Folículos secundários isolados foram distribuídos em seis grupos, em que o controle não-cultivado foi imediatamente fixado em paraformaldeído 4%. Os folículos restantes foram cultivados durante 7 dias em α-MEM+ sozinho (controle) ou suplementado com DMSO, doxorrubicina (DXR), A. oncocalyx ou onco A. Após o cultivo, os folículos foram avaliados quanto à formação de antro, taxa de crescimento, apoptose (TUNEL) e proliferação celular (PCNA), bem como a expressão dos genes BCL2 e BAX. Além disso, os complexos cumulus-oócitos (CCOs) foram aspirados e distribuídos em cinco tratamentos para MIV: o controle em meio de maturação (TCM 199+), e os demais tratamentos suplementados com DMSO, DXR, A. oncocalyx ou onco A. Depois da MIV, a configuração da cromatina e viabilidade oocitária foram avaliadas. Após 7 dias de cultivo, observou-se redução na percentagem de folículos morfologicamente intactos, na formação de antro, na taxa de crescimento e no número de células PCNA positivas (P < 0,05). Depois do cultivo, no tratamento DXR foi observada maior percentagem de folículos TUNEL positivos (P < 0,05) e também aumento na taxa de RNAm BAX: BCL2 (P < 0,05). Após MIV dos CCOs, nos tratamentos com DXR, A. oncocalyx e onco A, observou-se maior (P < 0,05) percentagem de oócitos anormais e menor de oócitos viáveis quando comparados ao grupo controle (P < 0,05)...

Reports on in vivo and in vitro contribution of medicinal plants to improve the female reproductive function / Relatos sobre a contribuição in vivo e in vitro de plantas medicinais na melhora da função reprodutiva feminina

Medicinal plants are known as a prolific source of secondary metabolites which have important function both in vivo and in vitro during the ovarian folliculogenesis and steroidogenesis in many animal species. Some secondary metabolites can act as antioxidants generally through their ability to scavenge reactive oxygen species (ROS) or can regulate ovarian hormonal production. In general, these properties are responsible for the medicinal functions to treat woman infertility disorder. Some plants are constituted of biological actives substances which have been used to treat reproductive dysfunction. However, until recently, little was known about the implication of plants and/or their secondary metabolites on in vitro folliculogenesis and steroidogenesis. With the development of the technology, there is an increase implication of those substances in assisted reproductive technology (ART). The present review highlights some medicinal plants used in the treatment of woman disorders related to infertility. In addition, it provides an in vivo and in vitro overview of herbs and their active compounds with claims for improvement of ovarian activity thus showing their implication in female reproductive health care.(AU)

Sabe-se que as plantas medicinais são uma fonte abundante de metabólitos secundários que têm função importante tanto in vivo quanto in vitro durante a foliculogênese e a esteroidogênese ovarianas em muitas espécies animais. Alguns metabólitos secundários podem atuar como antioxidantes, geralmente através de sua capacidade de eliminar espécies reativas de oxigênio (ROS) ou podem regular a produção hormonal ovariana. Em geral, essas propriedades são responsáveis pelas funções medicinais usadas para tratar distúrbios da infertilidade feminina. Algumas plantas contêm substâncias biológicas ativas que têm sido utilizadas para tratar a disfunção reprodutiva. No entanto, até recentemente, pouco se sabia sobre o efeito das plantas e/ou seus metabólitos secundários na foliculogênese e na esteroidogênese in vitro. Com o desenvolvimento da tecnologia, há uma implicação crescente dessas substâncias na tecnologia de reprodução assistida (TRA). A presente revisão destaca algumas plantas medicinais utilizadas no tratamento de distúrbios femininos relacionados à infertilidade. Além disso, fornece uma visão in vivo e in vitro de ervas e seus compostos ativos com alegações de melhora da atividade ovariana, mostrando assim seu envolvimento nos cuidados de saúde reprodutiva feminina.(AU)

Avaliação da maturação nuclear de oócitos bovinos submetidos a três diferentes sistemas de incubação / Evaluation of nuclear maturation of bovine oocytes submitted to three different incubation systems

This study aimed to evaluate three different incubation systems in nuclear maturation of bovine oocytes.Follicles of 2 to 8 mm in diameter were aspirated for obtaining cumulus-oocyte complexes (COCs). Thereafter,COCs were subjected to in vitro maturation (IVM) in TCM-199 medium supplemented at 38.5 °C for 23 h indifferent incubation systems: bench incubator with high oxygen (20% O2 in air), bench incubator with lowoxygen (5% O2) and portable incubator with low oxygen. After IVM, cumulus cells were removed and oocyteswere stained with Hoechst 33342. The slides were analyzed by a fluorescence microscope and the oocytes wereclassified into: degenerated (DG), metaphase I (MI) and metaphase II (MII). No difference (P > 0.05) wasobserved among the rates of DG, MI and MII for the different incubation systems. Therefore, the use of portableincubation system is a viable alternative for in vitro maturation of bovine oocytes.(AU)

Avaliação da maturação nuclear de oócitos bovinos submetidos a três diferentes sistemas de incubação / Evaluation of nuclear maturation of bovine oocytes submitted to three different incubation systems

This study aimed to evaluate three different incubation systems in nuclear maturation of bovine oocytes.Follicles of 2 to 8 mm in diameter were aspirated for obtaining cumulus-oocyte complexes (COCs). Thereafter,COCs were subjected to in vitro maturation (IVM) in TCM-199 medium supplemented at 38.5 °C for 23 h indifferent incubation systems: bench incubator with high oxygen (20% O2 in air), bench incubator with lowoxygen (5% O2) and portable incubator with low oxygen. After IVM, cumulus cells were removed and oocyteswere stained with Hoechst 33342. The slides were analyzed by a fluorescence microscope and the oocytes wereclassified into: degenerated (DG), metaphase I (MI) and metaphase II (MII). No difference (P > 0.05) wasobserved among the rates of DG, MI and MII for the different incubation systems. Therefore, the use of portableincubation system is a viable alternative for in vitro maturation of bovine oocytes.

Role of insulin-like growth factor-I and follicular fluid from ovarian follicles with different diameters on porcine oocyte maturation and fertilization in vitro.

The objective of this study was to determine the effects of insulin-like growth factor-I (IGF-I) (0, 60, 120, 180, and 240 ng/mL) and follicular fluid (FF) derived from 2 to 5 and 6 to 10 mm diameter follicles (SpFFs and LpFFs, respectively) added during in vitro maturation (IVM) of porcine oocytes on nuclear maturation and IVF. Cumulus-oocyte complexes (COCs) were matured in NCSU-37 medium supplemented with SpFFs or LpFFs and various IGF-I concentrations. The COCs were cultured for 44 hours, and then fertilized in vitro. Maturation and IVF results were recorded 18 hours after insemination. The IVM (%) was higher (P < 0.05) in the COCs matured in LpFFs than with SpFFs when 0 (90.0 ± 6.9 vs. 76.3 ± 10.7) or 60 ng/mL IGF-I (92.0 ± 8.1 vs. 81.8 ± 10.2) was added. In SpFFs media, there was a quadratic relationship (P < 0.01) between IGF-I concentration and IVM (peak results at IGF-I = 129 ng/mL). However, when the COCs were matured with LpFFs, there was a decreasing linear effect between IGF-I concentration and IVM. At all concentrations of IGF-I, the percentage of degenerated oocytes was higher in COCs matured in SpFFs than in LpFFs. Penetration (%) did not differ (P > 0.05) between COCs matured with SpFFs or LpFFs when 60 (66.8 ± 9.4 vs. 72.7 ± 11.3) or 180 ng/mL of IGF-I (75.7 ± 10.4 vs. 73.8 ± 13.2) were used. Monospermy (%) was similar between SpFFs and LpFFs only with addition of 120 ng/mL IGF-I. The IVF performance (%) did not differ between COCs matured with SpFFs or LpFFs when IGF-I concentrations of 120 (28.5 ± 8.8 vs. 38.5 ± 8.3) and 180 ng/mL (24.3 ± 10.2 vs. 30.12 ± 8.2) were used. There was no effect of IGF-I concentration or of FF type on the number of penetrated sperm per oocyte and on male pronuclear formation. For COCs matured with SpFFs, there was a quadratic relationship between IGF-I concentration and penetration, monospermy, and IVF performance (peak results at IGF-I = 179, 122, and 135 ng/mL, respectively). Thus, on the basis of the observed quadratic relationships, we inferred that when using SpFFs, the addition of IGF-I (122-179 ng/mL) to the IVM medium produced results similar to those obtained with LpFFs without adding IGF-I. In conclusion, the addition of IGF-I to the IVM medium supplemented with SpFFs increased maturation and improved IVF results. Alternatively, IGF-I had no effect on IVM or IVF when used with LpFFs.