IVM Advances for Early Antral Follicle-Enclosed Oocytes Coupling Reproductive Tissue Engineering to Inductive Influences of Human Chorionic Gonadotropin and Ovarian Surface Epithelium Coculture

Abstract

In vitro maturation (IVM) is not a routine assisted reproductive technology (ART) for oocytes collected from early antral (EA) follicles, a large source of potentially available gametes. Despite substantial improvements in IVM in the past decade, the outcomes remain low for EA-derived oocytes due to their reduced developmental competences. To optimize IVM for ovine EA-derived oocytes, a three-dimensional (3D) scaffold-mediated follicle-enclosed oocytes (FEO) system was compared with a validated cumulus-oocyte complex (COC) protocol. Gonadotropin stimulation (eCG and/or hCG) and/or somatic cell coculture (ovarian vs. extraovarian-cell source) were supplied to both systems. The maturation rate and parthenogenetic activation were significantly improved by combining hCG stimulation with ovarian surface epithelium (OSE) cells coculture exclusively on the FEO system. Based on the data, the paracrine factors released specifically from OSE enhanced the hCG-triggering of oocyte maturation mechanisms by acting through the mural compartment (positive effect on FEO and not on COC) by stimulating the EGFR signaling. Overall, the FEO system performed on a developed reproductive scaffold proved feasible and reliable in promoting a synergic cytoplasmatic and nuclear maturation, offering a novel cultural strategy to widen the availability of mature gametes for ART.

Keywords: EGF signaling; PCL electrospun scaffold; early antral follicle; follicle-enclosed oocyte; human chorionic gonadotropin; in vitro oocyte maturation; ovarian surface epithelium; sheep.

Figure 1

Percentage of meiotic competent IVM EA-derived oocytes: role of chorionic gonadotropin stimulation. Three independent biological replicates were performed for each experimental group (total number of oocytes analyzed: 132, 134, 128, and 130, respectively, for Ctr (without CG), hCG, eCG, and hCG + eCG). Both CGs were used at 5 IU/mL. The significant data (from p < 0.05) analyzed vs. Ctr (without CG) or vs. 5 IU/mL hCG were indicated with * and ^# superscripts, respectively.

Figure 2

Influence of somatic cell cocultures during IVM of EA-derived oocytes. The IVMs were performed with hCG in the absence (Ctr) or presence of primary cell monolayers. The used ovine cell typologies were ovarian epithelial cells (OSE), granulosa cells (GC), or tenocytes (TC). Three independent biological replicates were performed for each experimental group (total number of oocytes analyzed: 129, 127, 129, and 126, respectively, for Ctr, OSE, GC, and TC. The significance of nuclear stage data (p < 0.05) was analyzed vs. Ctr (in the absence of somatic cells coculture) or vs. OSE and indicated with (*) or (#) superscripts, respectively.

Figure 3

An example of EA incubated as FEO for 24 h on PCL-Patterned electrospun scaffolds in the absence of any hormonal stimulation.

Figure 4

(A) Influence of hCG stimulation of FEO on the expression of steroidogenic-specific gene CYP19A1. The maturation protocols were optimized for hCG stimulation by carrying out the EA-FEO incubation on PCL scaffolds for 24 h and exposing them to increasing doses of hCG (5, 25, 50 IU/mL) or without any hormonal stimulation (Ctr). The responsiveness of FEO to hCG was functionally tested by comparing the CYP19A1 gene expression before (time 0, Ctr) and after hCG stimulation (at 1 and 24 h). Three independent biological replicates were performed. Ten follicular walls per group were processed for gene expression analysis. mRNA data were statistically analyzed vs. Ctr (Time 0) and indicated with (*) or (**) when significantly different for p < 0.05 or p < 0.01, respectively. (B) hCG influence of FEO meiotic resumption. The induction of maturation promoted by different doses of hCG (0, 5, 25, 50 IU/mL) was tested in EA-FEO after 24 h of incubation by analyzing the oocyte nuclear stages. Three independent biological replicates were performed for each experimental group (total number of oocytes analyzed: 126, 132, 127, and 133, respectively, for time 0, 5, 25, and 50 IU/mL). The significance of each nuclear stage data (GV, GVBD/MI, or MII) was statistically analyzed vs. Ctr (0 IU hCG) and indicated with (*) or (**) superscripts when p < 0.05 or p < 0.01, respectively.

Figure 5

Influence of eCG stimulation of FEO on oocyte meiotic resumption. The experiments were performed by exposing EA-FEO to increasing doses of eCG (0, 5, 25, 50 IU/mL) in the absence or presence of hCG (25 IU/mL). Three independent biological replicates were performed for each experimental group (total number of oocytes analyzed: 95, 95, 100, and 94 respectively for 0, 5, 25, and 50 IU/mL eCG and 99, 101, 101, and 94, respectively, for 25 IU/mL hCG, 25 hCG +5 eCG, 25 hCG +25 eCG, 25 hCG + 50 eCG IU/mL). The significance of each nuclear stage data (GV, GVBD/MI, or MII) collected in dose-response eCG stimulation was statistically analyzed vs. 0 eCG indicated with (*) superscript when p < 0.05. The significance of each nuclear stage data (GV, GVBD/MI, or MII) collected in dose-response eCG stimulation in the presence of 25 IU/mL hCG was statistically analyzed vs. 0 eCG + 25 hCG indicated with (#) superscript when p < 0.05.

Figure 6

Influence of somatic cell cocultures on EA-FEO maturation protocol. The EA-FEO were induced to mature with hCG (25 IU/mL) in the absence (Ctr) or presence of primary cell monolayers. The used ovine cell typologies were: ovarian surface epithelial cells (OSE), granulosa cells (GC), or tenocytes (TC). Three independent biological replicates were performed for each tested experimental group (total number of oocytes analyzed: 131, 126, 129, and 128, respectively, for Ctr, OSE, GC, and TC). The significance of nuclear stage data (p < 0.05) was analyzed vs. Ctr (hCG without cells) and indicated with (*) or (**) for p < 0.05 and p < 0.01 superscripts, respectively.

Figure 7

Role of somatic cell coculture on CYP19A1 expression. Three independent FEO maturation protocols were analyzed in the absence (Ctr: hCG alone) or in the presence of primary somatic cell cocultures. Ten follicular walls per group were processed for gene expression analysis. The significance of mRNA values (p < 0.05) was statistically analyzed vs. Ctr Time 0 or vs. Ctr 24 h of incubation and indicated with (**) or (#) superscripts for p < 0.05, respectively.

Figure 8

Time course of EGF signaling cascade in FEO cultured in the presence or absence of OSE and TC. Immuno-blot analysis of EGF signaling cascade phospho-activation in FEO with OSE and TC was performed at 0, 1, and 24 h. Three independent biological replicates were performed, and a representative blot is shown. Eight follicular walls per group were processed and collected at 0, 1, and 24 h of FEO. Protein quantification graphs report media of signals detected in the three tested biological replicates. The significance of protein expression values (p < 0.05) was statistically analyzed vs. 0 h of FEO and indicated with (**) for p < 0.01.

Figure 9

(A) Comparison between somatic cells’ coculture effect on FEO and IVM protocol. Four independent biological replicates were performed for each tested experimental group (total number of oocytes analyzed: 136 and 134 respectively for IVM and FEO Ctr without CG, 148 and 138 for IVM and FEO Ctr without cells coculture, and 143 and 144 for IVM and FEO with OSE, 140 and 142 for IVM and FEO with GC, and 135 and 137 for IVM and FEO with TC). The significance of MII data collected in different cell-coculture experimental groups was analyzed within each protocol (IVM or FEO) vs. EA-derived oocytes as negative Ctr (5 IU/mL hCG or 25 IU/mL hCG without cells, respectively, for IVM and FEO) and indicated with (*) or (**) superscript for p < 0.05 or p < 0.01. The significance of MII data collected in different cell-coculture experimental groups was analyzed within each protocol (IVM or FEO) vs. medium antral-derived oocytes as positive Ctr (5 IU/mL hCG without cells for IVM) and indicated with ($) superscript for p < 0.05. The significance of MII data between the two protocols (IVM vs. FEO) in each cell-coculture experimental group (OSE or GC or TC) was analyzed and indicated with (#) superscript for p < 0.05. (B) The developmental stage reached from parthenogenetic oocytes activation after 72 h of incubation. The parthenogenetic activation was performed on MII oocytes (extruded PB) obtained from IVM, and FEO optimized EA protocols (with OSE and hCG) and compared with that of IVM oocytes derived from medium antral follicles (positive Ctr). Three independent biological replicates were performed for each tested experimental group (total number of oocytes analyzed: 120, 134, and 128, respectively, derived from medium antral, IVM, and FEO protocols). The data were statistically analyzed, and the significant values for p < 0.05 were indicated with ^a vs. medium antral; ^b vs. EA IVM.