Isolation and characterization of the human ovarian cell population for transplantation into an artificial ovary

To support survival and growth of follicles, the transplantable artificial ovary should mimic the original organ, offering a physical (3D matrix) and biological support (cells). In order to replicate the ovarian cell populations, the aim of this study is to assess the proportions of stromal and endothelial cells in the ovarian cortex. To this end, ovarian biopsies were obtained from six women (mean age: 49 years). The epithelial layer and medulla were carefully removed. The cortex was finely minced and enzymatically digested and the isolated cells were fixed. For cell characterization, immunostaining for CD31 (for endothelial cells) and inhibin-α (for granulosa cells) was performed. Positive cells in each staining were counted and the proportion of the different cell populations was estimated from the total number of isolated cells. Since there is no specific marker for ovarian stromal cells, we estimated the proportion of these cells by performing a vimentin immunostaining and subtracting the proportions of CD31- and inhibin-α-positive cells. Immunostaining showed that 84% of isolated cells were vimentin-positive. From this pool, 3% were endothelial cells and 1% granulosa cells. Consequently, the population of ovarian stromal cells was 80%. In conclusion, our findings show that stromal cells represent the larger population of cells in the human ovarian cortex. While this ensures follicle survival and development in a normal ovary, we believe that the low proportion of endothelial cells could have a negative impact on the angiogenesis in the artificial ovary after the first days of transplantation.

Isolation and characterization of the human ovarian cell population for transplantation into an artificial ovary

To support survival and growth of follicles, the transplantable artificial ovary should mimic the original organ, offering a physical (3D matrix) and biological support (cells). In order to replicate the ovarian cell populations, the aim of this study is to assess the proportions of stromal and endothelial cells in the ovarian cortex. To this end, ovarian biopsies were obtained from six women (mean age: 49 years). The epithelial layer and medulla were carefully removed. The cortex was finely minced and enzymatically digested and the isolated cells were fixed. For cell characterization, immunostaining for CD31 (for endothelial cells) and inhibin-α (for granulosa cells) was performed. Positive cells in each staining were counted and the proportion of the different cell populations was estimated from the total number of isolated cells. Since there is no specific marker for ovarian stromal cells, we estimated the proportion of these cells by performing a vimentin immunostaining and subtracting the proportions of CD31- and inhibin-α-positive cells. Immunostaining showed that 84% of isolated cells were vimentin-positive. From this pool, 3% were endothelial cells and 1% granulosa cells. Consequently, the population of ovarian stromal cells was 80%. In conclusion, our findings show that stromal cells represent the larger population of cells in the human ovarian cortex. While this ensures follicle survival and development in a normal ovary, we believe that the low proportion of endothelial cells could have a negative impact on the angiogenesis in the artificial ovary after the first days of transplantation.(AU)

UTILIZAÇÃO DO PROCESSO BIOTECNOLÓGICO DO OVÁRIO ARTIFICIAL PARA AVALIAR A TOXICIDADE DO VITONOLIDO D E DA QUINOXALINA SOBRE OS FOLÍCULOS PRÉ-ANTRAIS CAPRINOS

Embora o número de novos casos de câncer venha aumentando anualmente, o número de sobreviventes também vem crescendo ao longo dos anos, em função do diagnostico precoce e de tratamentos quimioterápicos mais efetivos. Porém, dentre os grandes efeitos indesejáveis da quimioterapia, destaca-se a resistência a multi-drogas (RMD) e a falha ovariana que leva à infertilidade. Portanto, a presente estudo teve dois principais objetivos: 1) imunolocalizar membros da família de proteínas conhecidas como Transportadores ABC (ABCB1, ABCC2 e ABCG2) no tecido ovariano caprino (Fase 1) e 2) avaliar a toxicidade de dois novos compostos com potencial anticâncer, ou seja, o Vitanolido D - VD (Fase 2) e a Quinoxalina - QX (Fase 3) sobre os folículos pré-antrais (primordiais, primários e secundários) cultivados in vitro no interior do tecido ovariano. Na Fase 1, ovários de cabras foram processados e analisados por imuno-histoquímica, qPCR e Western Blottingg para a identificação do ABCB1, ABCC2 e ABCG2. Nas Fases 2 e 3, fragmentos do córtex ovariano foram cultivadas in vitro por 2 e 6 dias em -MEM+ ou -MEM+ adicionado de paclitaxel (PTX controle negativo) ou em diferentes concentrações de VD (1.5, 3.0 e 6.0 M) ou QX (1.5, 3.0 e 6.0 M), respectivamente. Nessas duas fases os folículos foram analisados antes (controle) e após o cultivo na presença dos compostos, considerando-se morfologia e ativação folicular; proliferação (Ki67) e apoptose (TUNEL) das células granulosa, a expressão proteica do ABCB1 e gênica dos fatores reguladores do ciclo celular ciclinas (A - CCNA, B1 - CCNB1, D1 - CCND1 e E1 - CCNE1) e quinases dependentes de ciclina (1 - CDK1; 2 - CDK2, 4 - CDK4 e 6 - CDK6)]. Os resultados mostraram que os três transportadores ABCs foram positivamente imunomarcadas em todas as categorias foliculares estudadas. No tocante aos compostos, a toxicidade do VD6.0 foi similar ao PTX e superior (P <0,05) às demais concentrações (1.5 e 3.0 M), após 2 e 6 dias. A QX também apresentou alta toxicidade aos folículos pré-antrais, especialmente na mais alta concentração (6.0 M), causando um bournout folicular e apoptose, superiores às concentrações de 1.5 e 3.0 M. Ao contrário do VD, a QX inibiu a expressão do ABCB1 nos folículos pré-antrais. Esse trabalho mostrou pela primeira vez que três membros da família dos ABC transportadores foram identificados no ovário caprino. Apesar dos valiosos resultados obtidos nesse estudo, os dois compostos, isto é, o VD e a QX, necessitam ser melhor investigados, sob o ponto de vista da função ovariana, visando auxiliar na sua utilização como agentes quimioterápicos com menor risco para a fertilidade feminina.

Although the number of new cases of cancer increases annually, the number of survivors has also increased over the years, due to the early diagnosis and more effective chemotherapy treatments. However, among the great undesirable effects of chemotherapy, the multidrug resistance (MDR) and the ovarian failure that lead to infertility. Therefore, the present thesis had two main objectives: 1) to immunolocalize members of the family of proteins known as ABC transporters (ABCB1, ABCC2 and ABCG2), responsible for the phenomenon of RMD in goat ovarian tissue (Phase 1) and 2) to evaluate the toxicity of two new compounds with anticancer potential, the Vitanolide D - VD (Phase 2) and Quinoxaline - QX (Phase 3) on the preantral follicles (primordial, primary and secondary) grown in vitro inside the ovarian tissue. In Phase 1, goat ovaries were processed and analyzed by immunohistochemistry, qPCR and Western Blotting for identification of ABCB1, ABCC2 and ABCG2. In Phases 2 and 3, fragments of the ovarian cortex were cultured in vitro for 2 or 6 days in -MEM+ or -MEM+ supplemented with paclitaxel (PTX - negative control) or at different RV concentrations (1.5, 3.0 and 6.0 M) or QX (1.5, 3.0 and 6.0 M), respectively. In these two phases the follicles were analyzed before (control) and after the culture in the presence of the compounds, considering morphology and follicular activation; proliferation (Ki67) and apoptosis (TUNEL) of the granulosa cells, the protein expression of ABCB1 and the gene of cyclin - cyclin regulatory factors (A - CCNA, B1 - CCNB1, D1 - CCND1 and E1 - CCNE1) and cyclin - dependent kinases 1-CDK1; 2-CDK2, 4-CDK4 and 6-CDK6)]. The results showed that the three ABCs transporters were positively immunolabelled in all follicular categories studied. Regarding the compounds, the toxicity of VD6.0 was similar to PTX and higher (P < 0.05) at the other concentrations (1.5 and 3.0 M) after 2 and 6 days. QX also showed high toxicity to the preantral follicles, especially in the highest concentration (6.0 M), causing a follicular burnout and apoptosis, higher than the concentrations of 1.5 and 3.0 M. Unlike VD, QX inhibited the expression of ABCB1 in preantral follicles. This work showed for the first time that three members of the family of ABC transporters were identified in ovary goat. Despite the valuable results obtained in this study, the two compounds, i.e., RV and QX, need to be better investigated, from the point of view of ovarian function, aiming to aid in their use as chemotherapeutic agents with lower risk for female fertility.

Desenvolvimento e aplicações do ovário artificial em caprinos / Development and applications of artificial ovary in goats

O presente artigo aborda de forma resumida as bases gerais da biotécnica de Manipulação de Oócitos Inclusos em Folículos Ovarianos Pré-antrais (MOIFOPA) com ênfase nos estudos relativos ao desenvolvimento de um ovário artificial na espécie caprina. A importância do ovário artificial para a elucidação da foliculogênese inicial, testes de drogas sobre a dinâmica folicular e a sua potencial utililização para a produção in vitro de embriões em larga escala respeitando-se o bem-estar animal é destacada. (AU)

The present paper summarizes the basic aspects of the biotechnology of manipulation of oocytes enclosed in ovarian preantral follicles emphasizing the studies to develop an artificial ovary in goat species.. The importance of the artificial ovary for the understanding of early folliculogenesis, evaluation the effects of different drugs on follicular dynamics and its potential use for the in vitro production of embryos in large scale ensuring the animal welfare is highlighted.(AU)

Desenvolvimento e aplicações do ovário artificial em caprinos / Development and applications of artificial ovary in goats

O presente artigo aborda de forma resumida as bases gerais da biotécnica de Manipulação de Oócitos Inclusos em Folículos Ovarianos Pré-antrais (MOIFOPA) com ênfase nos estudos relativos ao desenvolvimento de um ovário artificial na espécie caprina. A importância do ovário artificial para a elucidação da foliculogênese inicial, testes de drogas sobre a dinâmica folicular e a sua potencial utililização para a produção in vitro de embriões em larga escala respeitando-se o bem-estar animal é destacada.

The present paper summarizes the basic aspects of the biotechnology of manipulation of oocytes enclosed in ovarian preantral follicles emphasizing the studies to develop an artificial ovary in goat species.. The importance of the artificial ovary for the understanding of early folliculogenesis, evaluation the effects of different drugs on follicular dynamics and its potential use for the in vitro production of embryos in large scale ensuring the animal welfare is highlighted.