Effects of the addition of insulin-transferrin-selenium (ITS) and/or metformin to the in vitro maturation of porcine oocytes on cytoplasmic maturation and embryo development.

CONTEXT: One of the main problems of porcine in vitro maturation (IVM) is incomplete cytoplasmatic maturation. Nuclear and cytoplasmic maturation will determine the future success of fertilisation and embryo development. Insulin-transferrin-selenium (ITS) has insulin-like and antioxidant effects, and metformin (M) is an insulin-sensitiser and antioxidant drug. AIMS: To assess the effects of adding ITS and/or M in porcine IVM media on cytoplasmic maturation and early embryo development. METHODS: Cumulus -oocyte complexes (COC) were IVM with M (10-4 M), ITS (0.1% v/v), M+ITS or no adding (Control). KEY RESULTS: ITS increased glucose consumption compared to Control and M (P <0.01), and M+ITS did not differ from ITS or Control. Redox balance: M, ITS and M+ITS increased glutathione (P <0.01) and decreased lipid peroxidation (P <0.005). The viability of cumulus cells by flow cytometry increased with M (P <0.005) and decreased with ITS (P <0.001); M+ITS did not differ from Control. After IVF, M increased penetration and decreased male pronucleus (P <0.05). Embryo development: cleavage increased with M (P <0.05), and blastocysts increased with ITS and M+ITS (P <0.05). The number of blastocyst cells increased with ITS (P <0.05). CONCLUSIONS: Adding ITS and M+ITS to porcine IVM media benefits embryo development to blastocysts, but ITS alone has better effects than M+ITS. IMPLICATIONS: ITS is an excellent tool to improve IVM and embryo development after IVF in pigs.

DMSO supplementation during in vitro maturation of bovine oocytes improves blastocyst rate and quality.

The molecule Dimethyl sulfoxide is widely used as drug solvent. However, its antioxidant property was poorly explored. In this study, we evaluated the effect of DMSO supplementation during oocyte in vitro maturation (IVM) on embryo development and quality. Bovine oocytes were matured with different DMSO concentrations (0, 0.1, 0.25, 0.5, 0.75, 1 and 10% v:v) followed by in vitro fertilization. Subsequently, quality indicators such as gene expression of SOX2, OCT4, CDX2, SOD1, oocyte and embryo redox status and DNA damage were evaluated. Polar body extrusion and blastocyst rates increased with 0.5% v:v DMSO. Moreover, first polar body extrusion and blastocyst rates did not increase with 1%, and 10% of DMSO reduced polar body extrusion and did not produce blastocyst. Optimal concentration of DMSO for the use on the maturation was estimated at around 0.45% v:v. Supplementation with 0.5% v:v DMSO has not affected mRNA abundance of genes key in blastocyst, however 0.75% increased gene expression of OCT4 and SOX2. Oocytes matured with 0.5% v:v DMSO and blastocyst from DMSO group showed reduced lipid peroxidation respect control. Total Glutathione concentrations increased in blastocyst stage in DMSO group. DMSO increased the total cell number of blastocysts but not TUNEL positive cells. In conclusion, our results suggest that low DMSO concentrations used during bovine oocytes in vitro maturation increases the maturation, as well as the blastocyst rate and its quality, without demonstrating deleterious effect on embryo cells.

Apoptosis in porcine cumulus-oocyte complexes: Relationship with their morphology and the developmental competence.

The aim of this study was to assess the presence and distribution of apoptosis in porcine cumulus-oocyte complexes (COCs) and its relations with COC morphology and developmental competence. The COCs were obtained from slaughterhouse ovaries, classified into A1 (top category), A2, B1, B2, C, and D based on their morphology. A1, A2, and B1 were matured and fertilized in vitro, and blastocyst rate was compared among them. Before and after in vitro maturation (IVM), annexin-V staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were performed to assess early and late apoptosis, respectively. There was a significant increase in both annexin-V (+) oocytes and TUNEL (+) cumulus cells as morphology further deteriorated. There were no statistical differences regarding annexin-V (+) oocytes within immature and post-IVM COCs, but TUNEL (+) oocytes were only observed in post-IVM COCs. Early and late apoptosis was detected in cumulus cells of all categories of immature and post-IVM COCs. However, the difference was only significant for annexin-V (+). There were no significant differences in embryo development. Therefore, apoptosis increases as the morphological features of the immature COCs decrease. In conclusion, the selection of COCs from Categories A1, A2, and B1 may be used as a selection criterion for in vitro development.

Effects of EPA on bovine oocytes matured in vitro with antioxidants: Impact on the lipid content of oocytes and early embryo development.

The eicosapentaenoic acid (EPA) is an n-3 polyunsaturated fatty acid (PUFA) present in the lipid composition of bovine oocytes. Little is known about the importance of EPA in bovine oocyte maturation and embryo development in vitro. Although previous work suggest that n-3 PUFAs may inhibit oocyte maturation, the available data are inconsistent. In this study, we evaluated the effect of EPA (1, 10, 100 nM) during in vitro maturation (IVM) of bovine oocytes, alone and in combination with vitamin E (VE) or cysteamine (CYS). EPA treatment in IVM decreased oocyte lipid content and affected lipid droplets pattern (P < 0.05). EPA 100 nM reduced oocytes maturation rate (P < 0.05), without affecting cumulus expansion. At the concentrations tested, EPA did not modify embryo development. However, the addition of antioxidants during IVM reduced the levels of reactive oxygen species in the culture system by increasing intracellular glutathione content (P < 0.05). Besides, the combination of EPA with VE or CYS reduced the percentages of MI oocytes after 24 h of IVM (P < 0.05). EPA reduced oocyte lipid content without any detrimental for embryo development.

Role of the N, N'-dimethylbiguanide metformin in the treatment of female prepuberal BALB/c mice hyperandrogenized with dehydroepiandrosterone.

The present study investigated the role of the N, N{'}-dimethylbiguanide metformin (50 mg/100 g body weight in 0.05 ml water, given orally with a canulla) in the prevention of endocrine and immune disorders provoked by the hyperandrogenization with dehydroepiandrosterone (DHEA) in prepuberal BALB/c mice. The treatment with DHEA (6 mg/100 g body weight in 0.1 ml oil) for 20 consecutive days, recreates a mouse model that resembles some aspects of the human polycystic ovary syndrome (PCOS). The treatment with DHEA did not modify either body mass index (BMI) or blood glucose levels, but did increase fasting insulin levels when compared with controls. Markers of ovarian function - serum estradiol (E), progesterone (P) and ovarian prostaglandin E (PGE) - were evaluated. The treatment with DHEA increased serum E and P levels while ovarian PGE diminished. When metformin was administered together with DHEA, serum insulin, E and P levels, and ovarian PGE values did not differ when compared with controls. Using flow cytometry assays we found that the treatment with DHEA diminished the percentage of the CD4 + T lymphocyte population and increased the percentage of the CD8 + T lymphocyte population from both ovarian tissue and retroperitoneal lymph nodes. However, when metformin was administered together with DHEA, the percentages of CD4 + and CD8 + T lymphocyte populations from both ovarian tissue and retroperitoneal lymph nodes were similar to those observed in controls. Finally, when DHEA was administered alone it increased the serum tumor necrosis factor-alpha (TNF-alpha ) levels when compared with controls; however, when metformin was administered together with DHEA, serum TNF-alpha levels were similar to controls. These results indicate that metformin is able, directly or indirectly, to avoid the endocrine and immune alterations produced when mice are hyperandrogenized with DHEA.

The influence of dehydroepiandrosterone on early pregnancy in mice.

The aim of the present report was to study the role of high levels of dehydroepiandrosterone (DHEA) on the ovarian function and embryonic resorption during early pregnancy in BALB/c mice. Pregnant animals were injected with DHEA following both the post-implantatory (DHEA-2) and peri-implantatory (DHEA-6) models. Morphological studies of implantation sites showed 40% of embryonic resorption in the DHEA-2 group while 100% of resorption was observed in the DHEA-6 group. Serum samples of both DHEA-2 and DHEA-6 groups showed higher estradiol levels and a lower progesterone concentration than those of control groups. Ovarian prostaglandin E levels after both DHEA-2 and DHEA-6 treatments increased when compared to control groups. The antioxidant metabolite glutathione diminished during both DHEA treatments. In summary, the data presented here suggest that DHEA treatment during early pregnancy modulates the ovarian function and is responsible for embryonic resorption with different degrees depending on when it is administered.

Effects of dehydroepiandrosterone on ovarian cystogenesis and immune function.

The purpose of the present report was to study the possible relationship between ovarian functionality and the immune response during cystogenesis induced by androgenization with dehydroepiandrosterone (DHEA). Daily injection of DHEA (6 mg/kg body weight) for 20 consecutive days induced ovarian cysts in BALB/c mice. As markers of ovarian function, serum estradiol (E) and progesterone (P) and the ovarian inmunomodulator prostaglandin E (PGE) were analyzed. In order to know how the integrity of the tissue was altered after induction of cystogenesis, the oxidative status was also evaluated. Serum E and P levels, and ovarian PGE concentration, were increased in animals with cysts compared with healthy controls. The oxidant status (quantified by malondialdehyde (MDA) formed after the breakdown of the cellular membrane by free radical mechanisms) was augmented, meanwhile the antioxidant (evaluated by the glutathione (GSH) content) diminished during the induction of cystogenesis. Both immunohistochemical and flow cytometry assays demonstrated that DHEA treatment increased the number of T lymphocytes infiltrating ovarian tissue. Therefore, while ovarian controls showed equivalent expression of CD4+ and CD8+ T cell subsets, injection of DHEA yielded a selective ovarian T cell infiltration as demonstrated by enhanced CD8+ and diminished CD4+ T lymphocyte expression. These results show that the development of cysts involves changes in ovarian function and an imbalance in the oxidant-antioxidant equilibrium. We observed also both an increased and selective T lymphocyte infiltration.

[Effect of leptin in the production of progesterone and estradiol by ovarian rat tissue]. / Efecto de la leptina en la producción de progesterona y estradiol por el ovario de rata.

An alteration in the balance of autocrine/paracrine ovarian factors may lead to different diseases. The aim of the present report was to evaluate the relationship between insulin growth factor 1 (IGF-1) and leptin (Lp) in the progesterone (P) and estradiol (E) production after ovarian rat FSH stimulation. Thus, ovarian explants were incubated with FSH, IGF-1 and Lp, and P and E were evaluated by means of specific radioimmunoassays. FSH increased ovarian P and E production. IGF-1 synergized with FSH in P as well as in ovarian E production. Lp had not shown any effect on the FSH capacity to increase P and E in the ovarian tissue; nevertheless it reverted the synergistic effect of IGF-1 on FSH for P as well as for E. These data raise the possibility that abnormal Lp levels may contribute to infertility in women with polycystic ovarian syndrome by counteracting the sensitizing effects of IGF-1 in ovarian tissue.

Efecto de la leptina en la produccion de progesterona y estradiol por el ovario de rata / Effect of leptin in the production of progesterone and estradiol by ovarian rat tissue

EI desequilibrio de factores autocrinos/paracrinos en el ovario conduce a diversas enfermedades. EI objetivo dei presente trabajo fue evaluar Ia relación entre el factor de crecimiento insulínico 1 (IGF 1) y Ia leptina (Lp) en Ia producción de progesterona (P) y estradiol (E) por el ovario de rata estimulado cort hormona folículo estimulante (FSH). Para ello, se incubaron explantes ováricos con FSH, IGF 1 y Lp, evaluándose P y E en el sobrenadante por radioinmunoensayo. La FSH estimuló Ia producción de P y E por el ovario. EI IGF 1 produjo un importante efecto sinérgico con FSH tanto en Ia síntesis de P como de E. La Lp no tuvo efecto sobre Ia acción de FSH en el aumento de P y E por el ovario, sin embargo revirtió el efecto sinérgico de IGF 1 sobre FSH tanto para P como para E. Podemos concluir que Ia Lp podría actuar directamente disminuyendo el efecto sinérgico de IGF 1 sobre FSH. Estos resultados podrían sugerir que niveles anormales de Lp encontrados en mujeres con síndrome de ovario poliquístico provocarían una desensibilización ovárica al IGF 1 que afectaría Ia producción de P y E.

Efecto de la leptina en la produccion de progesterona y estradiol por el ovario de rata / Effect of leptin in the production of progesterone and estradiol by ovarian rat tissue

EI desequilibrio de factores autocrinos/paracrinos en el ovario conduce a diversas enfermedades. EI objetivo dei presente trabajo fue evaluar Ia relación entre el factor de crecimiento insulínico 1 (IGF 1) y Ia leptina (Lp) en Ia producción de progesterona (P) y estradiol (E) por el ovario de rata estimulado cort hormona folículo estimulante (FSH). Para ello, se incubaron explantes ováricos con FSH, IGF 1 y Lp, evaluándose P y E en el sobrenadante por radioinmunoensayo. La FSH estimuló Ia producción de P y E por el ovario. EI IGF 1 produjo un importante efecto sinérgico con FSH tanto en Ia síntesis de P como de E. La Lp no tuvo efecto sobre Ia acción de FSH en el aumento de P y E por el ovario, sin embargo revirtió el efecto sinérgico de IGF 1 sobre FSH tanto para P como para E. Podemos concluir que Ia Lp podría actuar directamente disminuyendo el efecto sinérgico de IGF 1 sobre FSH. Estos resultados podrían sugerir que niveles anormales de Lp encontrados en mujeres con síndrome de ovario poliquístico provocarían una desensibilización ovárica al IGF 1 que afectaría Ia producción de P y E.(AU)

Efecto de la leptina en la producción de progesterona y estradiol por el ovario de rata. / [Effect of leptin in the production of progesterone and estradiol by ovarian rat tissue]

An alteration in the balance of autocrine/paracrine ovarian factors may lead to different diseases. The aim of the present report was to evaluate the relationship between insulin growth factor 1 (IGF-1) and leptin (Lp) in the progesterone (P) and estradiol (E) production after ovarian rat FSH stimulation. Thus, ovarian explants were incubated with FSH, IGF-1 and Lp, and P and E were evaluated by means of specific radioimmunoassays. FSH increased ovarian P and E production. IGF-1 synergized with FSH in P as well as in ovarian E production. Lp had not shown any effect on the FSH capacity to increase P and E in the ovarian tissue; nevertheless it reverted the synergistic effect of IGF-1 on FSH for P as well as for E. These data raise the possibility that abnormal Lp levels may contribute to infertility in women with polycystic ovarian syndrome by counteracting the sensitizing effects of IGF-1 in ovarian tissue.