Background
In vitro maturation (IVM), followed by
in vitro fertilization (IVF) and embryo transfer (ET) may restore fertility in humans, even in combination with cryopreservation [
1,
2]. IVM may be useful as rescue measure in conventional IVF protocols by maturing retrieved GV-stage oocytes
in vitro (
rescue IVM). IVF cycles often make available a mixed cohort of MII, metaphase I (MI), and post mature, including immature germinal vesicle (GV) oocytes. The effective competence of these retrieved immature oocytes is under debate [
3]. In IVM, maturation rates rarely exceed the 50-55% [
2]. Defective cytoplasmic maturation seems to be at the basis of the low maturation rates of IVM oocytes [
4].
IVM is applied with high yields in animal husbandry, not only to improve gamete preservation and to increase reproductive performance, but also to preserve endangered species or those of zootechnic interest [
5,
6].
Controlled ovarian stimulation, IVM and oocyte/embryo cryopreservation are not feasible for young cancer patients, where hormonal ovarian stimulation could increase the risk of cancer recurrence [
7]. In this case, the following strategies can be applied: i)
in vitro growth (IVG) and IVF of oocytes from cryopreserved cortical biopsies, mainly containing primordial follicles [
1,
8] or ii) the use of immature oocytes, cryopreserved at either the immature GV- or the mature MII-stage, i.e. before or after
in vitro maturation (IVM) [
9]. A chance for fertility preservation in prepubertals girls candidate to oncotherapy may be a pre-oncotreatment ovarian cortical strip, followed by cryopreservation and then, after disease recovery, a programmed IVG of thawed follicles, followed by IVM and IVF-ET of GV-stage oocytes [
1].
Several ultrastructural and clinical studies demonstrated that a morphological assessment is necessary for evaluating the outcome of these procedures on the oocyte quality [
10‐
14]. The scarce availability of human oocytes, especially in young patients, obviously limits morphological studies.
Indeed, animal models may be necessary to investigate the developmental competence of immature GV-stage oocytes subjected to IVM (to obtain MII-stage oocytes), and then to IVF. The ovine, a monovular species like human, could potentially represent an optimal animal model, being closer to human reproductive physiology than other species [
15]. However, ovine prepubertals oocytes (PO) may show a poor development competence that can be likely also accounted for a fine morphological impairment [
16,
17].
Therefore, we aimed to study by light and transmission electron microscopy the morphology of COCs obtained from prepubertal lambs before, during and at the end of IVM, in order to describe the timing of nuclear and cytoplasmic maturation, to evidence eventual alterations in respect to adult sheep, and ultimately to determine COCs quality. In this report, we account ultrastructural evidence of a prepubertal oocyte IVM impairment.
Methods
All chemicals in this study were obtained from Sigma Chemical CO. (St. Louis, MO, USA) unless stated otherwise.
Ovaries of slaughtered Sarda breed lambs (30-40 days old) and sheep (4-6 years old) were transported to the laboratory within 1-2 hours using Dulbecco Phosphate Buffered Saline solution (PBS) and antibiotics. Follicles with a diameter higher than 2 mm were sliced with a microblade and their content released in medium TCM199 (with Earle’s salts and bicarbonate) supplemented with 25 mmol HEPES, 0.1 g/L penicillin, 0.1 g/L streptomycin and 0.1% (w/v) polyvinylalcohol (PVA). COCs with 4-10 layers of granulosa cells, oocyte with uniform cytoplasm, homogenous distribution of lipid droplets and outer diameter of about 90 μm, were selected. Samples were washed 3 times in the same fresh medium and matured in vitro in TCM 199 supplemented with 10% heat-treated oestrus sheep serum (OSS), 1 IU/mL of FSH/LH and 100 μM cysteamine. 40-45 COCs were put in 500 μL of maturation medium in four-well culture dishes (Nunclon, Nalge Nunc International, Denmark), covered with 300 μL of mineral oil and cultured for 24 h in 5% CO2 in air at 39°C. In vitro maturation (IVM) experiments were performed at least 3 times.
Analysis of meiotic progression during IVM
IVM was performed as above described. At each time point (0, 7, 19 and 24 hours of in vitro culture), oocytes (n. 40 per each experimental groups) were decumulated by gentle pipetting using a narrow bore glass capillary, fixed in ice cold methanol, and incubated with 10 μg/mL Hoechst 33342 in ice-cold methanol for 15 min.
Stained oocytes were mounted into a small droplet of glycerol on a glass slide and examined under an epifluorescence inverted microscope (Nikon Diaphot, Japan).
Morphological evaluation by light and transmission electron microscopy (LM and TEM)
Prepubertal and adult COCs were fixed at retrieval (0 hour) and at different intervals during IVM (7, 19 and 24 hours) and then processed for LM and TEM. Per each experimental group were used 15 prepubertal and 20 adult COCs, obtained from at least three different animals. Methods of LM and TEM preparative were adapted from those previously described [
18,
19]. Briefly, COCs were fixed in 1.5% in Glutaraldehyde (SIC, Roma, Italia) in PBS solution for at least 2-5 days at 4°C. Samples were rinsed three times for about 10 minutes in PBS, post-fixed with 1% osmium tetroxide (Agar Scientific, Stansted, UK) in PBS and rinsed again in PBS. COCs were then embedded in small blocks of 1% of agar of about 5x5x1 mm in size and dehydrated in ascending series of ethanol (Carlo Erba Reagenti, Milano, Italia). Samples were immersed in propylene oxide (BDH Italia, Milano, Italia) for solvent substitution, embedded in epoxy resin (Electron Microscopy Sciences, Hatfield, PA, USA) and sectioned by a Reichert-Jung ultracut ultramicrotome. Semithin sections, of 1 μm thick, were stained with toluidine blue, examined by light microscopy (Zeiss Axioskop) and photographed by digital camera (Leica DFC 230). Ultrathin sections (60-80 nm) were cut by a diamond knife, mounted on copper grid and contrasted with saturated with Uranyl Acetate and Lead Citrate (Sic Roma, Italia). Finally, COCs were examined and photographed using Zeiss EM 10 and Philips MET CM 100 Electron Microscopes operating at 80 KV.
For the evaluation by LM and TEM, the following parameters were taken into consideration: general features (e.g. shape and dimension) of the oocyte and cumulus cells, shape and location of the nucleus, type and quality of organelles, integrity of the oolemma and the zona pellucida (ZP), appearance of the perivitelline space (PVS) (width, presence of fragments) [
18,
19].
Statistical analysis
Differences in the maturation progression were subjected to the Chi squared analysis. Statistical analysis was performed using the statistical software program Statgraphic Centurion XV (version15.2.06 for Windows; StatPoint, Inc., Herndon, VA, USA) and a probability of P ≤0.05 was considered the minimum level of significance.
Discussion
In this study, we described the fine morphology of COCs obtained from prepubertal lambs and adult sheep during IVM in standard conditions.
The precise and differentiated time setting of sampling - from the beginning (0 hours) to the end (24 hours) of IVM - used for collecting prepubertal and adult ovine COCs, allowed us to obtain original data on the ultrastructural changes of the oocytes during whole culture period.
This sampling model was fundamental to detect morphological differences between prepubertals and adults. Specifically, the LM and TEM evaluation of meiotic progression evidenced in prepubertal animals a delay in the nuclear maturation and alterations in the cytoplasmic maturation. In fact, in PO the GVBD-MI transition occurred with 12 hours of delay respect to AO (7 hours of IVM vs. 19 hours, respectively). The occurrence of similar percentages of meiotic progression rates after 7 hours of IVM, in both GV- and GVBD-stages, may also account for this delay. Differently, at this timing, the majority of AO normally reached GVBD/MII stage. Indeed, the MI-MII transition was faster in AO than PO (most of the AO were at MII-stage after 19 hours of IVM) even if, as in AO, nuclear maturation ended in 24 hours also in PO, with the achievement of the MII-stage.
These findings are likely due to an alteration of the specific and coordinated sequence of events between the oocytes and surrounding CCs. In the immature oocyte, CCs tightly connect to each other and to the oocyte by means of intercellular cytoplasmic processes presenting junctional complexes, including gap junctions that facilitate exchange of nutrients, small signalling molecules and ions. Oocytes depend on the cumulus cells for metabolism of glucose and supply of pyruvate for energy production [
20] and references herein cited]. Indeed, the GVBD depends on the uncoupling of CCs from the oocyte [
20]. Thus, the stage of nuclear maturation well correlates with a different CC arrangement, as also clearly shown
in vitro by our morphological data. Interestingly, in our data, the CC stage seemed to be associated to the nuclear phase, independently of the age of animals. When the nucleus was arrested at GV-stage, CCs were adherent to the ZP with numerous trans-zonal projections, responsible for the bi-directional communication between the oocyte and CCs. Differently, when meiosis resumed, CCs retracted by gradually detaching their trans-zonal projections from the ZP. This probably determined a tension on the oolemma that acquired the spiky shape. At MII-stage, when most of the CCs appeared already detached from the ZP, the oocyte had the recovery time necessary for restoring its original roundish shape beneath the ZP.
The heterocellular metabolic cooperation is so important to influence directly the bioenergetic requirements in the embryo after fertilization [
21]. According to this, the delay we found in the nuclear maturation of PO seemed associated to alterations of the oolemma and contacts with somatic cells. The alterations of latter structures could be responsible for the poor developmental competence of PO.
The association between nuclear stages and morphological changes of the oolemma shape and CC distribution well correlates to the changes of shape and position of the nucleus, irregular and flattened against the oolemma in adult GV-stage oocytes or roundish and slightly eccentric in prepubertal GV-stage oocytes. The above, in agreement with what previously reported in sheep [
22], could represent an ultrastructural parameter for the evaluation of the nuclear maturity, indicating the ability to resume meiosis
in vitro and progress up to MII-stage.
In vivo, the follicle-oocyte dialogue synchronizes the activation of oocyte growth and its developmental capacity and release. This process is coordinated by bi-directional signals received and transmitted through ovarian somatic cells, in particular the CCs [
20]. Oocytes enter a critical stage of development in response to diminishing cAMP (3’-5’-cyclic adenosine monophosphate) after the LH (luteinizing hormone) surge, resuming meiosis and progressing through a precisely synchronized nuclear and cytoplasmic maturation, to achieve full developmental competence [
21].
In vitro, the physical removal of COCs from ovarian follicles results in spontaneous resumption of meiosis (largely because of a decrease in cAMP concentrations via phosphodiesterase type 3, PDE3), causing asynchrony between cytoplasmic and nuclear maturation and decreased oocyte developmental competence. In this view, innovative approaches to IVM are currently proposed in order to modulate
in vitro cAMP concentrations within ovine COCs [
23] or to inhibit PDE3 [
24], to delay spontaneous nuclear maturation, and to improve developmental competence, with subsequent embryo viability.
Our ultrastructural analysis on prepubertal and adult ovine oocytes matured in vitro allowed not only to detect peculiar variations in PO, but also to individuate the timing of the delay in the nuclear maturation and the presumptive sequence of cytoplasmic alterations connected with it. Electron microscopy again resulted one of the techniques of choice for the evaluation of oocyte quality.
The ultrastructure of oocyte nucleus has been studied in lambs and sheep, mainly focusing on the nucleolus [
25]. Other authors found that the animal size corresponded to a different oocyte nucleolar activity. While small sized lambs showed by TEM a vacuolated oocyte nucleolus, with a fibrillar center located at the nucleolar periphery, in adult sized lambs and sheep, vacuoles disappeared and the nucleolus showed an electron-dense fibrillar sphere with a fibrillar centre attached to it in the form of a halo [
25]. Interestingly, our
in vitro matured lamb oocytes showed a similar nucleolar morphology to that found in adult sized lambs and sheep
in vivo[
22,
25], thus indicating that IVM conditions could sustain a
quasi physiological maturation.
Prepubertals showed modifications in the oocyte cytoplasmic maturation, mainly related to an altered CG distribution. In adult sheep oocytes, CGs relocate towards the ZP earlier than in prepubertal lamb during IVM, and finally aligned in a continuous monolayer under the oolemma, at MII stage. Prepubertal and adult goat oocytes subjected to IVM and IVF showed a similar CGs distribution [
26]. Differences in CG size and volume fraction were also present
in vitro in prepubertal and adult sheep oocytes [
16].
Concerning the morphology of other organelles, our data are generally in agreement with previous studies on ovine oocyte ultrastructure. In fact, the morphology of mitochondria, lipid droplets, Golgi complexes and endoplasmic reticulum, before and after maturation, were comparable with previous reports on prepubertal and adult sheep oocytes matured
in vitro[
16,
17,
22] or
in vivo[
27‐
29]. As well, we observed that the organelle redistribution during maturation was coincident with what described by others, both
in vitro and
in vivo[
16,
28].
The vacuole is a characteristic organelle that in the ovine oocyte is physiologically present in large numbers, differently from other species including humans. In humans, a large number of vacuoles appear only during degenerative process [
14,
19]. In ovine, vacuoles are usually numerous and distributed around the GV [
16,
28], even if they may be found even increased, in some pathological/experimental conditions [
27]. In our samples, we did not found a significant variation of the vacuole amount. However, differently from what previously described, we did not found a dense assemblage of vacuoles in the center of the oocyte ooplasm [
16,
27]. As well, we hardly found the localization of Golgi complexes around the GV [
28]. These minor ultrastructural differences can be likely related to the different breeding investigated previously and the different experimental conditions.
Acknowledgments
Funds for this work were provided by the University La Sapienza (University and Faculty grants) and by the Department of Life, Health and Environmental Sciences, University of L'Aquila.
The authors wish to acknowledge Dr. Marta Maione and Mr. Ezio Battaglione of the Laboratory for Electron Microscopy ‘Pietro M Motta’, Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, University La Sapienza, Rome and Elena Di Marco, Dept. of Life, Health and Environmental Sciences, University of L’Aquila for their contribution to the EM preparative. The Authors would also thank Prof. Maria Silvia Marottoli, Lecturer on the English language at the University of L’Aquila, for her English revision of the manuscript.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SAN and SN designed and directed the study. GGL, SS, FB and SN provided animal models, essential reagents and carried our in vitro maturation. MGP, SAN, YB and GM participated in LM and EM studies and evaluated Electron Microscopy micrographs. MGP, XB and GM wrote the manuscript. YB revised the draft paper. All the authors critically revised the manuscript and approved the final version of this article.