Influence of equine growth hormone, insulin-like growth factor-I and its interaction with gonadotropins on in vitro maturation and cytoskeleton morphology in equine oocytes

Abstract

In horses, successful in vitro fertilization procedures are limited by our inability to consistently mature equine oocytes by in vitro methods. Growth hormone (GH) is an important regulator of female reproduction in mammals, playing an important role in ovarian function, follicular growth and steroidogenesis. The objectives of this research were to investigate: the effects of equine growth hormone (eGH) and insulin-like growth factor-I (IGF-I) on the in vitro maturation (IVM) of equine oocytes, and the effects of eGH in addition to estradiol (E 2 ), gonadotropins (FSH and LH) and fetal calf serum (FCS) on IVM. We also evaluated the cytoskeleton organization of equine oocytes after IVM with eGH. Equine oocytes were aspirated from follicles ,30 mm in diameter and matured for 30 h at 38.58C in air with 5% CO 2 . In experiment 1, selected cumulus-oocyte complexes (COCs) were randomly allocated as follows: (a) control (no additives); (b) 400 ng/ml eGH; (c) 200 ng/ml IGF-I; (d) eGH 1 IGF-I; and (e) eGH 1 IGF-I 1 200 ng/ml anti-IGF-I. In addition to these treatment groups, we also added 1 mg/ml E 2 , 5 IU/ml FSH, 10 IU/ml LH and 10% FCS in vitro (experiment 2). Oocytes were stained with markers for microtubules (anti-a-tubulin antibody), microfilaments (AlexaFluor 488 Phalloidin) and chromatin (TO-PRO 3 -iodide) and assessed via confocal microscopy. No difference was observed when eGH and IGF-I was added into our IVM system. However, following incubation with eGH alone (40%) and eGH, E 2 , gonadotropins and FCS (36.6%) oocytes were classified as mature v. 17.6% of oocytes in the control group (P , 0.05). Matured equine oocytes showed that a thin network of filaments concentrated within the oocyte cortex and microtubules at the metaphase spindle showed a symmetrical barrel-shaped structure, with chromosomes aligned along its midline. We conclude that the use of E 2 , gonadotropins and FCS in the presence of eGH increases the number of oocytes reaching oocyte competence.

Identificação imuno-histoquímica de VEGF e IGF-1 em ovários de cadelas no anestro e estro

“…(Shimizu et al, 2003;Rajkovic et al, 2006;Velazquez et al, 2008;Müller et al, 2009;Hernandez-Medrano et al, 2012;Lee et al, 2012;Oberlender et al, 2013;Pereira et al, 2013), VEGF e IGF-1 possivelmente tenham participação na dinâmica folicular e formação do corpo lúteo. Entretanto, não parece haver concentração diferenciada desses FCs entre anestro e estro, pelo menos para as estruturas avaliadas neste experimento.…”

“…FGF-2 e FGF-7, também angiogênicos, foram identificados nos ovócitos de cadelas (Songsasen et al, 2009) (Rajkovic et al, 2006;Pereira et al, 2013) e/ou sobre as células foliculares (Vendola et al, 1999). A utilização de FCs na maturação in vitro de ovócitos se baseia em suas prováveis atividades in vivo (Farsi et al, 2013), e dentre eles estão VEGF (Kere et al, 2014) e IGF-1 (Oberlender et al, 2013;Meiyu et al, 2014), este último também utilizado em meios de cultivo de ovócitos caninos (Rodrigues e Rodrigues, 2003;Chastant-Maillard et al, 2011).…”

“…Embora sua participação esteja evidenciada em diversas espécies, como seres humanos (Rajkovic et al, 2006;Lee et al, 2012), murinos (Rajkovic et al, 2006), ruminantes (Rajkovic et al, 2006;Velazquez et al, 2008), equinos (Müller et al, 2009;Pereira et al, 2013) e suínos (Shimizu et al, 2003;Oberlender et al, 2013), há poucos relatos em cães (Chastant-Maillard et al, 2011). Estudos indicam a presença de VEGF no corpo lúteo de cadelas no diestro e anestro inicial (Mariani et al, 2006) e a presença de IGF-1 no fluido folicular de cadelas (Reynaud et al, 2010), bem como a participação deles na maturação in vitro de ovócitos caninos Oliveira et al, 2009;ChastantMaillard et al, 2011).…”

Clinical Application of In Vitro Maturation of Oocytes

“…Some scholars believe that the effect of follicle-stimulating hormone is related to its concentration. When the concentration is 5 g/mL, a relatively high cleavage rate (79.1%) and blastocyst rate (16.1%) can be obtained [15]. The addition of LH or human chorionic gonadotropin to the IVM culture medium may promote protein synthesis, enhance oocyte metabolism, and facilitate oocyte maturation.…”

Insulin-Like Growth Factor-I Concentration in the Follicular Fluid of Bali Cattle and Its Role in the Oocyte Nuclear Maturation and Fertilization Rate

“…The European Union), 50 µg/ mL of gentamycin (Sigma-Aldrich, USA), without BSA supplementation (-BSA), supplemented with 0.3% BSA (+BSA), and 10% FF (v/v) from the follicle with diameter Ø<4 mm, 4≤Ø<6 mm, 6≤Ø<8 mm, and Ø≥8 mm during the luteal phase. Oocytes maturation was done in 100 µL droplets (approximately 10-15 COCs) in petridishes with diameter of 35 mm (Nunclon, Denmark) under mineral oil (Sigma-Aldrich, USA) and incubated for 24 h at 38.5 o C in 5% CO 2 in air (Modification of Pereira et al, 2013).…”