In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle

Summary

We describe the procedures for isolation of growing oocytes from ovarian follicles at early stages of development, as well as the setup of an in vitro culture system which can support the growth and differentiation up to the fully-grown stage.  

Abstract

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that during the reproductive life span only about 1% of the oocytes in an ovary mature and ovulate, several techniques have been developed to increase the exploitation of the ovarian reserve to the growing population of non-ovulatory follicles. Such technologies have allowed interventions of fertility preservation, selection programs in livestock, and conservation of endangered species. However, the vast potential of the ovarian reserve is still largely unexploited. In cows, for instance, some attempts have been made to support in vitro culture of oocytes at specific developmental stages, but efficient and reliable protocols have not yet been developed. Here we describe a culture system that reproduce the physiological conditions of the corresponding follicular stage, defined to develop in vitro growing oocytes collected from bovine early antral follicles to the fully-grown stage, corresponding to the medium antral follicle in vivo. A combination of hormones and a phosphodiesterase 3 inhibitor was used to prevent untimely meiotic resumption and to guide oocyte's differentiation.

Introduction

During the reproductive life span, only a minimal fraction of the oocytes that are present in an ovary mature, are released in the fallopian tubes upon ovulation, and are available for being fertilized and develop into a viable embryo¹. On the other hand, most of the oocytes within an ovary undergo atresia and are never ovulated. In vitro embryo production (IVP) technologies have attempted to increase the exploitation of the ovarian reserve^2,3. Thus far, such technologies have allowed interventions of fertility preservation, selection programs in livestock, and conservation of endangered species. Nevertheless, most protocols use oocytes that have basically completed the growth phase within the antral ovarian follicle, and hence are referred to as fully-grown oocytes. In cattle, where IVP technologies are widely used, fully grown oocytes reach a final diameter of approximately 120 µm and are collected from follicles that span from 2 to 8 mm in diameter (medium antral follicles)¹. Upon isolation from the follicles, such oocytes are in vitro matured and fertilized. The zygotes are then cultured up to the blastocyst stage and either transferred into a recipient or cryopreserved. In cattle, as well as in many other species, despite the potential offered by IVP, the number of in vitro produced embryo per cow did not largely improve for the last 40 years. This is in part due to the limited number of fully grown oocytes that populate an ovary at a given time which can be retrieved and subjected to standard IVP techniques^4,5,6.

The oocytes enclosed within early antral follicles, i.e., those follicles that are less than 2 mm in diameter, represent a potential source to be used in fertility preservation programs⁷ , as an ovary roughly contains 10 times more early antral follicles than medium antral⁸. However, these oocytes are still in the growth phase and have not yet reached the fully-grown stage⁹. As such, they are still transcriptionally active, producing mRNAs that will be stored for later developmental steps, and have not yet undergone all the differentiation process required to confer the oocytes with the ability of spontaneously resuming and completing meiosis I once isolated from the follicular compartment^10,11. Therefore, they cannot be directly submitted to standard in vitro maturation (IVM) protocols, but they require an additional period of culture that would allow them to complete the growth phase and properly differentiate.

The transition from the growing to the fully-grown stage, which in cattle occurs when the follicle develops from the early antral to the medium antral stage, is one of the critical steps during oocyte development. In cattle, several studies attempted to recapitulate these events in vitro^{2,12,13,14,15,16,17,18,19}. However, to date no reliable protocols have been developed and only limited success has been reported. According to previous studies²⁰, these growing oocytes constitute a homogeneous population. Besides being transcriptionally active, their chromatin is dispersed in the germinal vesicle (GV), in a configuration that is named GV0^2,21. Conversely, the population of fully-grown oocytes obtained from medium antral follicles is more heterogeneous, a condition that is mirrored by the various degrees of chromatin compaction (GV1, GV2 and GV3) that can be observed²⁰. Among these, previous data have shown that GV2 and GV3 oocytes are overall characterized by a better quality and higher embryonic developmental competence^{20,21,22,23,24}.

Starting from the above observations, here we describe a 5-days long culture system of oocytes (L-IVCO) that allows the differentiation of oocytes isolated as cumulus-oocyte complexes (COCs) from early antral follicles. This culture strategy has evolved from 10 years long studies conducted in our lab and roots its ground on the previously developed 24-48 hours in vitro oocyte culture (IVCO)², prematuration systems^23,25 and zinc supplementation during oocyte culture . A combination of follicle stimulating hormone (FSH) and a phosphodiesterase-3 (PDE3) inhibitor, able to enhance cumulus-oocyte communication², prevent untimely meiotic resumption², and support oocyte growth² was used.

Protocol

Ovaries were collected from 4 to 8 years old  Holstein dairy cows recovered at the local abattoir (INALCA S.p.A., Ospedaletto Lodigiano, LO, IT 2270M CE, Italy).

1. Media preparation

NOTE: All media must be prepared at least four hours before use. Sodium bicarbonate buffered media are incubated at 38.5 °C and 5% CO₂ in air, maximum humidity. HEPES-buffered media are maintained at 38.5 °C in thermostatic oven.

1.  Long in vitro culture of oocytes (L-IVCO) medium
   1. Prepare 15 mL of the basic culture medium (M199-B): Supplement M199 with 2 mM glutamine, 0.4% fatty acid free bovine serum albumin (BSA), 0.2 mM sodium pyruvate, 25 mM sodium bicarbonate, 0.1 mM cysteamine, 21.3 µg/mL of phenol red, 75 μg/mL of kanamycin and 4% Polyvinylpyrrolidone (PVP; 360 k molecular weight).
   2. Prepare 3 mL of the holding medium (M199-H): To M199-B add 5 µM cilostamide and pour it in a 35 mm Petri dish.
   3. Prepare the L-IVCO medium (M199-L): Supplement M199-B with 0.15 μg/mL Zn sulphate, 10^-4 IU/mL FSH, 10 ng/mL estradiol, 50 ng/mL testosterone, 50 ng/mL progesterone and 5 µM Cilostamide.
   4. Place 200 µL of M199-L medium in each well of the 96 well coated plate. Fill the wells in the four edges of the plate with sterile culture water to compensate for evaporation and to maintain appropriate humidity during culture.
   5. Incubate the 96 well plate and the M199-H medium in the incubator at 38.5 °C and 5% CO₂ in air, maximum humidity.
2. Dissection medium
   1. Prepare the dissection medium (M199-D): Supplement M199 with 0.4% BSA fraction V, 0.164 mM penicillin, 0.048 mM streptomycin, 1790 units/L heparin. M199-D can be prepared in bulk, dispensed in 20 mL aliquots and stored at 4 °C for 6 months. When needed, warm and supplement 1 aliquot.
   2. Prepare 20 mL of M199-D supplemented with 5 µM cilostamide (M199-D cilostamide).

2. Ovary collection and processing

NOTE: All procedures are conducted at room temperature (26 °C) unless otherwise indicated.

1. Recover the ovaries at the abattoir from cows.
2. Place the ovaries in sterile saline (NaCl, 9 g/L) at 26 – 28 °C added with penicillin 100 U/mL and streptomycin 0.1 mg/mL.
3. Transport the organs to the laboratory in warm sterile saline within 4 h.
4. Wash the ovaries 4x in sterile saline maintained at 26 °C.
5. Remove all mid-to-large antral follicles by aspirating all follicles more than 2 mm in diameter using a 18 G needle connected to an aspiration pump with vacuum pressure set at -28 mmHg and place the aspirated ovaries in a beaker with sterile saline at 26 °C.
   NOTE: Removal of the content of follicles > 2 mm is a critical step to remove as much as possible the source of fully-grown oocytes that would ‘contaminate’ the experiment.
6. Under a horizontal laminar flow hood, place one ovary at the time on a sterile polytetrafluoroethylene cutting board. Using a surgical blade No. 22 mounted on a scalpel handle, cut slices of ovarian cortex (the outer portion of the ovary, which contains the follicles), 1.5 – 2 mm thick and parallel to the major axis of the organ.
7. Place the slices of ovarian cortex in a sterile glass Petri dish covered with dissecting medium on a warm plate at 38.5 °C.
   NOTE: From now on all the procedures are performed at 38.5 °C using a warm plate.

3. Selection and isolation of the follicles and retrieval of the COCs

1. Place one ovarian cortex slice in a 60 mm glass Petri dish with 2-3 mL of M199-D.
2. Using a dissection microscope, select the follicles between 0.5 – 2 mm using a micrometer-equipped eyepiece.
3. Identify the healthy, non-atretic follicles under the stereomicroscope. Assess follicle atresia by observing morphological parameters, such as very clear translucent appearance, with a dark COC inside. Discard the atretic follicles and process all the others.
4. Using a surgical blade  No. 22 mounted on a scalpel handle remove the ovarian tissue surrounding the follicle on one side until the follicle is exposed on one edge.
5. Using a 26G needle mounted on a syringe, carefully make a slit in the exposed follicle wall. This action will release the follicular content, comprising the COC, follicular fluid and clumps of cells.
6. Identify the COC under the microscope and examine for cumulus integrity, zona pellucida integrity and homogeneity of the cytoplasm. If these criteria are fulfilled, aspirate the COC using a P20 pipette.
7. Place the isolated COC in M199-D cilostamide.
8. Continue the isolation procedure for 30 min.

4. Selection of COCs to be subjected to in vitro culture

1. Under the dissection microscope, select healthy COCs based on the criteria in step 3.6.
2. In a 60 mm Petri dish, prepare 16 drops of 20 µL of M199-D cilostamide and place one healthy COC per drop (Figure 1A).
3. Using an inverted microscope attached to a camera measure the oocyte diameter, excluding the zona pellucida, using the software provided with the camera.
4. With a clear visualization of the oocyte, make two perpendicular measurements excluding the zona pellucida (Figure 1B).
5. Assure whether the mean of the two oocyte’s measurement, excluding the zona pellucida, is within a range of 100 – 110 µm. Discard COCs with not-rounded shaped oocyte or with oocytes that are not measurable.
6. Transfer the selected COCs in a 35 mm dish containing M199-H medium and keep them in the incubator at 38.5 °C and 5% CO₂ in air, maximum humidity until step 5.1.
7. Repeat steps 3 and 4 up to 4x. The overall working time must not exceed 2 h.

5. Long in vitro culture of the oocytes (L-IVCO)

1. Transfer one COC per well in the center of a well of the 96 well plate to be prepared in step 1.1.5.
2. Incubate the plate for 5 days at 38.5 °C and 5% CO₂ in air, maximum humidity.
3. Every other day (day 2 and day 4) prepare fresh M199-L as described in step 1.
4. Renew half of the medium by removing 100 µL of medium and replacing with 100 µL of freshly prepared M199-L. Perform the medium renewal under the stereomicroscope and avoid moving the COCs into the well.

6. COC classification after the culture

1. At the end of the L-IVCO, analyze the COCs’ morphology under the dissection microscope.
2. Classify as depicted in Figure 2.
   1. Classify as Class 1 if COCs show a compact cumulus cell investment with no sign of cumulus expansion and cell degeneration.
   2. Classify as Class 2 if COCs show a compact cumulus cell investment with no sign of cumulus expansion and cell degeneration and with antrum-like formation in the cumulus mass.
   3. Classify as Class 3 if COCs show several layers of cumulus cell with no sign of cumulus expansion and some disaggregated cells in the outer layer of cumulus cells and no antrum-like formation.
   4. Classify as Class 4 if COCs shows abundant loss of cumulus cells extending for more than 50% of the oocyte surface, and signs of cell degeneration and cell debris.

7. Evaluation of meiotic progression after culture

1. Oocyte denudation
   1. Place each COC in a single well of a four-well plate containing 400 µL of 199D per well. 
   2. Under a dissection microscope gently remove the cumulus cells mechanically by repeated pipetting using a pipette set at 130-140 µL.
   3. Once the oocytes are free of the cumulus investment, transfer them to another well containing 199D.
   4. Repeat the process until all the oocytes are completely denuded.
2. Oocyte nuclear staining
   NOTE: From now on all the procedures are performed at room temperature. Reagents are at room temperature.
   1. Fix the oocytes in paraformaldehyde 4% in phosphate buffer saline (PBS) for 1 h.
      CAUTION: Wear personal protective equipment when handling paraformaldehyde and dispose of contaminated materials in accordance with hazardous waste disposal guidelines.
   2. Wash the oocytes 3x for 5 min each in PBS containing 1% polyvinylalcohol (PVA).
      NOTE: The samples can be processed right away or stored at 4 °C for maximum one week.
   3. Place the oocytes in PBS containing 0.1% Triton X for 10 min.
   4. Wash the oocytes 3x for 5 min each in PBS containing 1% PVA.
   5. Place the oocytes singularly in drops of 5 µL of antifade medium supplemented with 4’,6-diamidino-2-phenylindole (DAPI) dilactate (1 µg/mL) over a slide.
   6. Place two strips of double-sided tape along the long sides of the slide, to avoid excessive flattening of the oocytes when putting the cover slip on top.
   7. Place the cover slip on top, make it adhere to the tape and keep in the dark while processing all the samples.
   8. Analyze the oocytes using a conventional epifluorescence microscope equipped with DAPI filters (Excitation/Emission: 358⁄461) to assess the meiotic progression of the oocytes.
   9. Classify the oocytes according to their meiotic progression: GV - oocytes with different degrees of chromatin condensation within the GV; MI – oocytes from GV breaking down to metaphase I; and degenerate – oocytes that could be not identified as being at any of the previous stages.

Results

At the end of the L-IVCO, the gross morphology of the COCs changed and 4 classes were identified based on the appearance of the cumulus cells, as shown in Figure 2. Based on the morphological criteria commonly adopted to select healthy COCs^11,26,27, the class 1, 2 and 3 were judged healthy, while the class 4, which showed clear signs of degeneration such as the absence of complete layers of cumulus ...

Discussion

Here we describe a culture system for growing oocytes that promotes oocyte development for 5 days by supporting their viability and preventing meiotic resumption. This latter aspect is of the outmost importance to allow the continued growth and differentiation necessary to confer the oocyte with meiotic and embryonic developmental competence^2,20, that would be otherwise blocked by a premature resumption of the meiotic division.

When de...

Materials

Name	Company	Catalog Number	Comments
4-well dishes	Nunclon	179830
96-well dish	Becton Dickinson Biosciences	356649	BioCoat™ Collagen I
Bovine Serum Albumin (Fatty acid free)	Sigma	A8806
Bovine Serum Albumin (Fraction V)	Sigma	A3311
Cell culture water	Sigma	W3500
Cilostamide	Sigma	C7971
Cysteamine	Sigma	M9768
Digital camera	Nikon Corp	Camera DS-5M
Disodium phosphate	Sigma	S5136
Estradiol	Sigma	E2758
Glutamax Supplement	Thermo Fisher Scientific	35050061
Gonal F	Merck Serono
Heparin	Sigma	H3149
Hepes	Sigma	H3784
Vacuum pump	Cook-IVF
Incubator	Sanyo
Kanamycin sulfate from Streptomyces kanamyceticus	Sigma	K1377
Medium 199	Sigma	M3769	Powder for hepes-buffered TCM199
Medium 199	Sigma	M2520	Powder for M199-D
Microscope	Nikon Corp	Nikon Diaphot
Microscope	Nikon Corp	Eclipse E 600
Monopotassium phosphate	Sigma	P5655
Paraformaldehyde	Sigma	158127
Penicilin	Sigma	P3032
Phenol Red	Sigma	P5530
Polyvinyl alcohol	Sigma	P8137
Polyvinylpyrrolidone	Sigma	P5288	360k molecular weight
Potassium chloride	Sigma	P5405
Progesterone	Sigma	P8783
Sodium bicarbonate	Sigma	S5761
Sodium choride	Sigma	P5886
Sodium pyruvate	Sigma	P4562
Streptomycin	Sigma	S9137
Testosterone	Sigma	86500
Triton X	Sigma	T9284
Vectashield with DAPI	Vector Laboratories	H1200
Water	Sigma	W3500
Zinc sulfate heptahydrate	Sigma	Z0251