Effects of Growth Hormone on In Situ Culture of Bovine Preantral Follicles are Dose Dependent

Abstract

The objective of this study was to evaluate different concentrations of growth hormone (GH) on the development of bovine preantral follicles cultured included in the ovarian tissue (in situ) on the rates of morphologically normal, viable, primordial and developing follicles, as well as the oocyte and follicle diameter and ultrastructural analysis. Ovarian fragments collected from cows with no cross-breeds defined were cultured in situ for 1 and 7 days in minimal essential medium (α-MEM+) supplemented with different concentrations of recombinant human GH (0, 10, 25, 50 ng/ml). The ovarian fragments non-cultured (control) and cultured were processed for classic histology, mechanical isolation and electron transmission microscopy (MET). The parameters underwent anova (Tukey's and Dunnett's tests) and chi-square test (χ(2) ). After 7 days of culture, the treatment with 50 ng/ml GH showed no differences with fresh control (p > 0.05) and had greater effectiveness than in the 0, 10 and 25 ng/ml GH concentrations of the morphologically normal follicles. Regarding the primordial follicles, a reduction was observed in the 50 ng/ml GH concentration concomitant with the significant increase in developing follicles, differing from both the fresh control and the other GH concentrations tested. In addition, 50 ng/ml GH showed a larger follicle and oocyte diameter when compared to the other treatments cultured. Similar structures were ultrastructurally observed in the control group, 50 ng/ml GH. Follicles cultured in 10 ng/ml GH showed nuclear invagination, vacuoles and lesioned basal membrane. Hence, it is concluded that 50 ng/ml GH is the most effective concentration for the development of preantral follicles cultured in situ.

Effects of IGF-1 on the Three-Dimensional Culture of Ovarian Preantral Follicles and Superovulation Rates in Mice

“…Previous results showed that the ultrastructural features of follicles were normal in the presence of 30 ng/mL IGF-1 in vitro. In contrast, follicles displayed signals of degeneration at a 100 ng/mL concentration [ 29 ]. High concentrations of IGF-1 (100 ng/mL) also promoted bovine GCs apoptosis [ 15 ].…”

Sequential medium with GH and IGF‐1 improved in vitro development of bovine preantral follicles enclosed in ovarian tissue

“…The treatments were distributed in the following way: (a) αMEM + during the 14 days (αMEM + ), or αMEM + supplemented with (b) GH during 7 days, followed by 7 days with IGF‐1 (GH → IGF‐1), (c) IGF‐1 during 7 days, followed by 7 days with GH (IGF‐1 → GH) and GH plus IGF‐1 during 14 days of culture (GH + IGF‐1; Figure ). The concentrations used in this experiment were selected based on the best results previously obtained by the research team: 0 ng/ml IGF‐1 (Jimenez et al, ) and 50 ng/ml GH (Jimenez et al, ). The fragments were cultured during 1, 7 and 14 days at 37ºC, air humidified with 5% CO 2 and with total replacement of the medium every 2 days.…”

“…The material was contrasted with uranyl acetate 0.5%, dehydrated in increasing concentrations of acetone and included in Spurr's resin. Semithin sections (3 µm) were acquired in the ultramicrotome (RMC Products, Power Tome‐X, Arizona, USA) for a previous selection of the sections, and afterwards, the ultrathin cuts were made (60–70 nm) and positioned on copper meshes stained with uranyl and lead citrate (Jimenez et al,; ). They were then examined under the electron transmission microscope (Zeiss EM 900, Jena, Germany).…”

“…The concentrations used in this experiment were selected based on the best results previously obtained by the research team: 0 ng/ml IGF-1 (Jimenez et al, 2016a) and 50 ng/ml GH (Jimenez et al, 2016b). The fragments were cultured during 1, 7 and 14 days at 37ºC, air humidified with 5% CO 2 and with total replacement of the medium every 2 days.…”

“…Besides, GH stimulates both the protein synthesis and gluconeogenesis, and also participates in the ovulation (Barnett, Schilling, Greenfeld, Tomic, & Flaws, 2006;Gong, Baxter, Bramley, & Webb, 1997); moreover, in vivo experiments have shown that GH acts by promoting the development of bovine ovarian follicles, increasing the peripheral concentrations of insulin and/or IGF-1 in heifers (Gong et al, 1997). In vitro and in vivo studies reveal the importance of this hormone during the follicular development (Ferreira et al, 2016;Jimenez et al, 2016b;Magalhães-Padilha, Andrade, et al, 2012). In bovine ovaries, the mRNA of GHR has already been located in the oocyte of primordial and primary follicles and begins to be expressed in the granulosa cells of primary follicles, remaining during the secondary stage (Izadyar, Zhao, Tol, Colenbrander, & Bevers, 1999).…”