Presented here are two protocols for the encapsulation of porcine oocytes in 3D culture conditions. In the first, cumulus-oocyte complexes (COCs) are encapsulated in fibrin-alginate beads. In the second, they are enclosed with fluorinated ethylene propylene powder particles (microbioreactors). Both systems ensure optimal conditions to maintain their 3D organization.
In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of assisted reproduction techniques such as oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and cloning of domestic animals by nuclear transfer from somatic cell. IVM is the method particularly of significance. It is the platform technology for the supply of mature, good quality oocytes for applications such as reduction of the generation interval in commercially important or endangered species, research concerning in vitro human reproduction, and production of transgenic animals for cell therapies. The term oocyte quality includes its competence to complete maturation, be fertilized, thereby resulting in healthy offspring. This means that oocytes of good quality are paramount for successful fertilization including IVF procedures. This poses many difficulties to develop a reliable culture method that would support growth not only of human oocytes but also of other large mammalian species. The first step in IVM is the in vitro culture of oocytes. This work describes two protocols for the 3D culture of porcine oocytes. In the first, 3D model cumulus-oocyte complexes (COCs) are encapsulated in a fibrin-alginate bead interpenetrating network, in which a mixture of fibrin and alginate are gelled simultaneously. In the second one, COCs are suspended in a drop of medium and encapsulated with fluorinated ethylene propylene (FEP; a copolymer of hexafluoropropylene and tetrafluoroethylene) powder particles to form microbioreactors defined as Liquid Marbles (LM). Both 3D systems maintain the gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, thereby preserving the functional relationship between the oocyte, and surrounding follicular cells.
The development of various culture systems, including three-dimensional (3D) ones, aims to provide optimal conditions for the growth and maturation of oocytes isolated from the follicles even at earliest stages of development. This is of great importance for assisted reproductive techniques (ART), especially in view of the increasing number of women who are struggling with infertility after cancer treatment1. Maturation of oocytes in in vitro conditions (IVM) is already a well-established technique mainly used for in vitro embryo generation for the purpose of livestock reproduction2. However, in most mammalian species, even if high rates of maturation of cumulus-oocyte complexes (COCs) can be achieved (range 60 to 90 %)3, their developmental competence is still inadequate to the needs. This is because the development of the zygotes obtained in such a way even up to the blastocyst stage is low and after the transfer into surrogate animals their viability to term is reduced. Consequently, there is a need to increase the developmental competence of embryos obtained from oocytes that were subjected to the IVM procedure4. Therefore, new maturation media5 are being devised and various periods of in vitro culture are tested6,7 along with supplementation of culture media with various growth factors and molecules8,9.
The first step of any complete IVM system is to create optimal conditions for sustainable growth of oocytes during in vitro culture. The oocyte growth is one of the specific indicators of the oocyte's ability to resume meiosis10,11. In addition, an appropriate oocyte in vitro culture system must be capable of supporting its nuclear maturation and cytoplasmic differentiation12. The morphology of the cumulus-oocyte complex is another important indicator used in ART clinics to select the best oocyte for subsequent steps of in vitro fertilization (IVF) procedure in humans and livestock12,13. Among morphological characteristics of COCs considered are: the oocyte diameter, its cytoplasm granulation and the first polar body integrity14,15. Besides, the oocyte developmental potential is correlated to the appearance and compaction of cumulus cells and number of their layers surrounding the oocyte. Very important for the appropriate oocyte in vitro culture system is also the maintenance of oocyte–cumulus cells proper interactions and cytoskeleton stability16,17,18,19. So far, in vitro oocyte growth within human COCs has been demonstrated20. The use of cow COCs also resulted with live births. These were isolated from immature ovarian follicles and then cultured for 14 days until the oocyte was sufficiently large to undergo the IVF procedure21. Similarly, COCs isolated from baboon antral follicles, subjected to IVM after in vitro culture yielded oocytes capable of reinitiating meiosis to the metaphase II stage with a normal appearing spindle structure22. However, in this study the authors did not try to fertilize them. Nevertheless such results indicate that a similar procedure could be applied not only to these particular mammalian species but also to human cumulus-oocyte complexes obtained from follicles what should allow to obtain oocytes of good quality suitable for a successful IVF technique.
The above described results were obtained with the application of conventional IVM protocols during which oocytes were cultured in two-dimensional (2D) systems. The routine procedure in 2D culture systems is covering oocytes, immersed in a drop of an appropriate culture media, with mineral oil23,24. It is assumed that an oil overlay during in vitro oocyte culture serves to prevent liquid evaporation, thus ensuring the maintenance of proper pH and osmotic pressure in the culture. Although such a 2D culture system allows to obtain, even up to 87% of mature pig oocytes25, it has been proven that the mineral oil overlay causes substantial diffusion of lipid soluble materials which are necessary for proper oocytes development26. Additionally, because of steroids (progesterone and estrogens) diffusion into the mineral oil during oocyte culture, a delay of nuclear maturation and reduction in developmental competence achievement of pig oocytes was observed. This may result in obtaining a small number of zygotes, which additionally are characterized by low developmental capacity to the stage of the blastocyst and by poor viability after transfer into recipient animals27. Therefore, attempts are being made to increase the developmental competence of embryos derived from oocytes received after IVM procedure, by creating optimal conditions for achieving both cytoplasmic and nuclear maturity of oocytes cultured together with CCs as complexes, especially using three-dimensional (3D) systems. Various innovative 3D in vitro culture systems have been developed in the last two decades28,29. These were designed to maintain the natural spatial organization of cells and to avoid their flattening in culture dishes what cannot be achieved in the traditional 2D cultures. The structural and functional activity of cultured COCs can be ensured by the maintenance of their proper architecture and undisturbed communication through gap-junctions between various compartments30. The suitability of bio-scaffolds for 3D in vitro culture of cumulus–oocyte complexes has been evaluated using natural biomaterials such as various components of the extra-cellular matrix (ECM; collagen and hyaluronic acid)31 or inert polymers (alginate)32. These attempts tested in several species brought promising results in terms of oocyte meiosis resumption and achievement of their full competence33,34,35. However so far, no 3D system suitable for COCs maturation isolated from large domestic animals, including pigs, has been developed.
This work describes two protocols that can be used for the 3D culture of porcine COCs. The first protocol describes encapsulation in fibrin-alginate beads (FAB). FAB can be formed by simultaneous mixing an alginate and fibrin solution, which undergo a synchronous gelation process. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity. A similar solution has been used previously for mouse ovarian follicle culture and maturation36. In the case of the presented protocol, to avoid premature degradation of the alginate-fibrin network, appropriately higher concentrations of calcium chloride solution are used, ensuring a fast and stable gelation process. The dynamic mechanical environment creates conditions similar to these in the natural intra-follicular environment in which COCs reside and increase in size. Additionally, the work shows representative results of COCs 3D culture systems, in which these are suspended in a drop of medium and encapsulated with fluorinated ethylene propylene (FEP; a copolymer of hexafluoropropylene and tetrafluoroethylene) powder particles, to form microbioreactors (Liquid Marbles, LM). LM are a form of 3D bioreactor that have been previously shown to support, among others, growth of living microorganisms37, tumor spheroids38 and embryonic stem cells39. LMs have been also successfully used for sheep oocyte culture40. In most experiments using LMs, bioreactors were prepared using polytetrafluoroethylene (PTFE) powder bed with particle size of 1 μm41. The presented protocol uses FEP, which is very similar in composition and properties to the fluoropolymers PTFE. But FEP is more easily formable and softer than PTFE and what is especially important, it is highly transparent.
Both 3D systems maintain the gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, preserving their functional relationship between the oocyte and surrounding follicular cells.
The following procedures were approved by the Animal Welfare Committee at the Institute of Zoology and Biomedical Research at Jagiellonian University.
1. Isolation of porcine cumulus-oocyte complexes
2. Encapsulation in fibrin-alginate hydrogel beads
3. Encapsulation in super-hydrophobic fluorinated ethylene propylene (FEP) microbioreactors.
NOTE: Make sure that all IVM procedures are carried out on thermostatically controlled table and COCs are maintained at 38 °C throughout their handling.
4. Characterization of COCs after 96-h IVM procedure using two 3D systems
NOTE: To determine the efficiency of the IVM systems used, score COCs morphologically under a light microscope. Additionally, use double fluorescent labeling with calcein AM and ethidium homodimer (EthD-1) dyes for analysis of COCs viability44.
In COCs using both IVM systems, the granulosa cells adhered tightly to each other, and most of the recovered COCs had intact layers of cumulus cells (Figure 1A,B). Additionally, substantial proportion of cumulus cells were retained.
The results obtained from the COCs viability analysis confirmed that both systems applied for the encapsulation of porcine oocytes in 3D in vitro conditions ensured optimal growth conditions (Figure 2). In both groups, only high-viability oocytes were observed, V1 = 13 %, V2 = 87 % for FAB and 10 % V1, and 90 % V2 for LM, respectively (Table 1).
Mitochondria, observed by transmission electron microscopy (TEM), were evenly distributed in oocytes, their shape after IVM were shell-like45. Only a few of them were elongated, moreover, their clustering was sporadically observed (Figure 1C,D). Endoplasmic reticulum was observed, either associated with mitochondria or free in the oocyte cytoplasm. Lipid droplets appeared as small dark round structures and Golgi apparatus were emerged with dilated cisternae (Figure 1C,D).
V1 | V2 | V3 | V4 | ||||
% | number | % | number | % | number | % | |
FAB | 13% | 7 | 87% | 48 | – | – | |
LM | 10% | 9 | 90% | 80 | – | – | |
COCs total number: 144 | 16a | 128b | 0 | 0 |
Table 1. Results on the viability of COCs after encapsulation using fibrin-alginate beads (FAB) and microbioreactors FEP, Liquid Marbles (LM). The results were given as average. Values with superscripts (a, b) differed statistically significantly (p <0.05).
Figure 1: Representative images showing the morphology and ultrastructure of COCs after a 96 h of encapsulation. (A) morphology of COCs inside FAB – light microscopy; (B) morphology of COCs inside LM – light microscopy; C: oocyte ultrastructure (TEM) after in vitro culture inside FAB; D: oocyte ultrastructure (TEM) after in vitro culture inside LM. O: oocyte; CCs: cumulus cells; ZP: zona pellucida; N: nucleus; M: mitochondria; G: Golgi apparatus; ER: endoplasmic reticulum; Lp: lipid droplet; FA: alginate and fibrin filaments; FEP: powder of copolymer of hexafluoropropylene and tetrafluoroethylene. Please click here to view a larger version of this figure.
Figure 2: Representative fluorescent images of COCs after 96 h of encapsulation. (A,B) The images obtained for the same COCs after in vitro culture using FAB with two filters to visualize green fluorescence or red fluorescence for live or dead cells respectively and (C) merge of both. (D,E) The images obtained for the same COCs after in vitro culture using LM with two filters to visualize green fluorescence or red fluorescence for live or dead cells, respectively and (F) merge of them. Scale bars = 50 µm. White asterisks indicate cells undergoing apoptosis. Please click here to view a larger version of this figure.
The ability to maintain in vitro growth not only of the oocyte but also of cumulus cells surrounding it and simultaneously supporting its maturation is exceedingly essential for the successful assisted reproductive technologies and for furthering the understanding of somatic cell/oocyte interactions especially in species undergoing prolonged follicular growth such as human beings or pigs. Continuously improved IVM techniques are becoming useful tool to preserve reproductive options in cases of polycystic ovarian syndrome (PCOS), premature ovarian failure, or definitive infertility (oncotherapy). In addition, since certain ovarian dysfunctions might be caused by dysregulated follicular growth, understanding the molecular and cellular mechanisms that control proper development of the oocyte may provide important insight into the pathophysiology and rational treatment of these conditions. In vitro culture systems that would support the maintenance of COCs 3D architecture during IVM, require a step of encapsulation in a matrix or inside a bioreactor. This work describes a protocol that can be applied for the culture of porcine oocytes. Both 3D in vitro culture systems presented here to induce growth and development of porcine COCs are user-friendly and allow for the effective control of both oocyte and cumulus cells survival.
The first in vitro culture method of COCs using their encapsulation in fibrin-alginate hydrogel beads (FAB) presented here allows for a 3D oocyte growth in an actively cell-responsive matrix environment. Previously published protocols based on alginate hydrogels were usually used for the investigation of the development of ovarian follicles isolated from numerous animal species with very promising results32. Interestingly, regardless of the final composition of the alginate hydrogel32 or the encapsulation method itself46, these studies showed that alginate bead-based 3D culture systems were able to support follicular growth and their survival.
Alginate hydrogels were gentle on the follicles, not affecting their further survival or development in vitro. Similarly, the in vitro culture protocol of COCs described for the first time here, is suitable to generate good quality pig oocytes, as it was documented at both morphological and ultrastructural levels. The presence of fibrinogen in an alginate solution and their simultaneous gelling, results in the formation of an interpenetrating network which creates and further stabilizes the three-dimensional environment. Due to the specific structural support inside such a matrix, cell-to-cell contacts and paracrine communication between oocytes and cumulus cells are maintained similarly to these present in vivo.
The two most critical steps in the maturation protocol described in this document are the transfer of FA capsules from incubation chambers into 96-well plates containing MM (step 2.10.) and the removal of COCs from the dissolved capsules after enzymatic degradation of the remaining alginate, using alginate lyase (step 2.11.). Both steps require significant manual skill and precision to avoid, in the first case the destruction of capsules, in the second – damage to COCs.
The main strength of this method, in addition to establishing optimal conditions for the oocyte maturation of a large domestic animal, is that during analysis using TEM, light microscopy or confocal microscopy, no capsule removal is necessary. Alginate is not a barrier when imaging. Leaving COCs in capsules facilitates manipulation during the staining procedure.
The second COC in vitro culture presented here is the one with the use of LM, which generally are nonstick droplets covered by micro- scaled particles and obtained by simply rolling small volumes of a liquid in a very hydrophobic powder47. Previous studies with the use of LM for culture, among others fibroblasts48, red blood cells49, organoid of a tumor38, pancreatic cells50 or sheep oocytes36 have been performed using polytetrafluoroethylene (PTFE) as a hydrophobic polymer for the preparation of the droplets. PTFE is widely used in the clinic (e.g., cardiovascular grafts).
In this work, we present, for the first time, a reliable method for microbioreactors made of FEP particles for in vitro culture of porcine COCs. The use of FEP in the presented protocol, which is very similar in composition and properties to the PTFE, makes preparation of LM much easier. This powder is softer than PTFE and what is especially important, it is transparent. This in turn simplifies the work during imaging of structures cultured with its use. The ease of forming LM using FEP is a definite advantage of this method.
The most critical step in the maturation protocol described in this document is picking up and transferring formed LM into 60 mm IVF Petri dish using a pipette tip (steps 3.3. and 3.4.). These steps require significant manual skill and precision to avoid destruction of LM.
Possibly, the main limitation of the 3D oocyte maturation induction protocols presented here is the relatively great difficulty in obtaining proper research material collected from animals not treated with antibiotics, anabolic steroids (i.e., nadrolone, boldenone) or melengestrol acetate which promote their rapid growth. These steroids, despite their use being banned in Europe, are still widely utilized in the industrial livestock breeding during the last two months of the fattening period, which causes, among others, disturbed sexual maturation.
In conclusion, both 3D systems presented here maintain the optimal gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, thus preserving the functional relationship between the oocyte, and surrounding follicular cells, what is crucial for proper oocyte maturation. Thus, both models are a valuable tool for basic research in reproductive biology and may also have clinical relevance, leading to improved infertility treatment. Additionally, they can also be applied for developing novel biotechnology methods, as well as for livestock improvement.
The authors have nothing to disclose.
The authors are very grateful to: dr Waclaw Tworzydlo (Department of Developmental Biology and Invertebrate Morphology, Institute of Zoology and Biomedical Research, Jagiellonian University) for technical facilities in TEM; to Ms. Beata Snakowska (Department of Endocrinology, Institute of Zoology and Biomedical Research, Jagiellonian University) for technical assistance; to the Department of Cell Biology and Imaging, Institute of Zoology and Biomedical Research, Jagiellonian University, JEOL JEM 2100HT (JEOL, Tokyo, Japan). This work was supported by grant 2018/29/N/NZ9/00983 from National Science Centre Poland.
General | |||
Antibiotic Antimycotic (100x) 100ml | Thermo Fisher | 15240062 | 2.5% final concentration for Handling Medium. 1% in PBS (step 1.2) |
DMEM/F12 (500ml) | Sigma-Aldrich | D8062 | Handling and Maturation Medium |
DPBS (w/o Ca, Mg), 1x, 500ml | Thermo Fisher | 14190144 | |
FCS (100 ml) | Thermo Fisher | 16140063 | 10% final concentration for both Handling Medium and Maturation Medium. (steps: 1.5. 2.6.) |
PBS (1x, pH 7.4) 500ml | Thermo Fisher | 10010023 | |
TBS Stock Solution (10x, pH 7.4) 500 ml | Cayman Chemicals | 600232 | 1x final concentration. Other brand can be use |
Maturation Medium | |||
hCG (1 VIAL of 10 000 U) | Sigma-Aldrich | CG10 | |
PMSG | BioVendor | RP1782721000 | |
Fibrin-alginate beads | |||
Alginate Lyase | Sigma-Aldrich | A1603 | (Step 2.11.1) |
Thrombin | Sigma-Aldrich | T9326-150UN | (Step 2.1) |
Calcium Chloride | Sigma-Aldrich | C5670 | (Step 2.1) |
Fibrinogen (250mg) | Sigma-Aldrich | F3879 | (Step 2.2) |
Sodium Alginate | Sigma-Aldrich | W201502 | (Step 2.3) use for alginate solution |
Liquid Marble | |||
FEP | Dyneon GmbH 3M AdMD | A-66670 | |
Morphological examination | |||
LIVE/DEAD Viability/Cytotoxicity Kit, for mammalian cells | Thermo Fisher | L3224 | (Step 4.1.) Emitted fluorescence: 494 nm for calcein, 528 nm for EthD-1; measure: 517 nm for calcein, 617 nm – EthD-1 |
VECTASHIELD Antifade Mounting Medium | Vector Laboratories | H-1000 | mounting medium |
Ultrastructure examination | |||
Glutaraldehyde solution | Sigma-Aldrich | G5882 | 2.5% final concentraion (Step 4.2.1.) |
LR White resin | Sigma-Aldrich | L9774 | (Step 4.2.4.) |
Methylene blue | Sigma-Aldrich | M9140 | (Step 4.2.5.) |
Osmium Tetroxide | Sigma-Aldrich | O5500 | (Step 4.2.3.) |
Sodium cacodylate trihydrate | Sigma-Aldrich | C0250 | Use for preparing 0.1M sodium cacodylate buffer (pH 7.2) |
Uranyl Acetate | POCH | 868540111 | (Step 4.2.4.) |
Specific instruments, tools | |||
30 mm Pteri dish | TPP | 93040 | |
60 mm IVF Petri dish | Falcon | 353653 | |
Ez-Grid Premium Cell Handling Pipettor | RI Life Sciences | 8-72-288 | |
Ez-Tip | RI Life Sciences | 8-72-4155/20 | |
Heating Table | SEMIC | Other brands can be used | |
Incubator | Panasonic | MCO-170AIC-PE | Other brands can be used |
Sterile petri dish (10 cm) | NEST Biotechnology | 704002 | |
Sterile syringe filters with 0.2 µm | GOOGLAB SCIENTIFIC | GB-30-022PES | |
Thermos | Quechua | 5602589 | Other brands can be used |