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 endangere....

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 ac....

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

List of materials used in this article

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	KMAR-5100
Incubator	Sanyo
Kanamycin sulfate from Streptomyces kanamyceticus	Sigma	K1377
Medium 199	Sigma	M3769	Powder for bicarbonate-buffered media
Medium 199	Sigma	M2520	Powder for HEPES-buffered media
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