Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

Summary

This manuscript describes an experimental approach to morphologically and biochemically characterize horse oocytes. Specifically, the present work illustrates how to collect immature and mature horse oocytes by ultrasound-guided ovum pick-up (OPU) and how to investigate chromosome segregation, spindle morphology, global histone acetylation, and mRNA expression.

Abstract

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previously developed in mice. Specifically, the chromosomes and the centromeres of metaphase II (MII) oocytes were fluorescently labeled and counted on sequential focal plans after confocal laser microscope scanning. This analysis revealed a higher incidence in the aneuploidy rate when immature oocytes were collected from the follicles and matured in vitro compared to in vivo. Immunostaining for tubulin and the acetylated form of histone four at specific lysine residues also revealed differences in the morphology of the meiotic spindle and in the global pattern of histone acetylation. Finally, the expression of mRNAs coding for histone deacetylases (HDACs) and acetyl-transferases (HATs) was investigated by reverse transcription and quantitative-PCR (q-PCR). No differences in the relative expression of transcripts were observed between in vitro and in vivo matured oocytes. In agreement with a general silencing of the transcriptional activity during oocyte maturation, the analysis of the total transcript amount can only reveal mRNA stability or degradation. Therefore, these findings indicate that other translational and post-translational regulations might be affected.

Overall, the present study describes an experimental approach to morphologically and biochemically characterize the horse oocyte, a cell type that is extremely challenging to study due to low sample availability. However, it can expand our knowledge on the reproductive biology and infertility in monovulatory species.

Introduction

A vast array of assisted reproduction techniques has been developed to treat infertility in women, companion animals, and endangered species. One of the most common procedures in clinical settings is the retrieval of metaphase II (MII)-stage oocytes from the ovarian follicles by ultrasound-guided transvaginal aspiration, ovum pick-up (OPU)¹. These oocytes are then fertilized in vitro (IVF), with the resulting embryo(s) implanted in a recipient uterus. The MII-stage (mature) oocytes are retrieved following the administration of exogenous gonadotropins. However, this treatment is associated, in some patients, with the development of ovar....

Protocol

All the procedures were approved by the Animal Care and Use Committee CEEA Val de Loire Number 19 and were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.

1. Oocyte Collection and In Vitro Maturation

1. Ovum pick-up
   1. Daily assess by ultrasound the diameter of ovarian follicles in a cohort of adult mares. At the emergence of a follicle ≥33 mm (dominant follicle), inject the mare with 1,500 IU of human chorionic gonadotrophin (hCG) in the upper part of the jugular vein (i.v.).
      1. Before performing the injection, swab the area with al....

Results

The original findings of these experiments were described in depth previously⁷ and are reported herein as an example of results that can be obtained using the described protocols.

Maturation rate

Of the 32 COCs retrieved by OPU from dominant follicles, 28 (88%) were at the MII stage. Fourteen of the 58 COCs collected from follicles 5-25 mm in size .......

Discussion

Even though IVM has been performed in horses for more than 20 years¹⁶, we do not yet know whether the oocyte can be the origin of embryonic aneuploidy, as has been proposed for humans¹⁷. The reason is probably that the preparation of oocyte spreads for chromosome counting results in considerable sample loss. With this in mind, a survey of the methods used for investigating errors in chromosome segregation was conducted to search for the most appropriate technique to apply t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ultrasound probe	Aloka	UST-5820-7,5
human chorionic gonadotrophin	centravet	CHO004	1,500 unit/animal IV
detomidine	centravet	MED010	9 - 15 µg/kg IV
butylscopolamine bromure	centravet	EST001	0.2 mg/kg IV
stereomicroscope	NIKON	SMZ-2B
butorphanol	centravet	DOL003	10µg/kg IV
benzyl-penicillin	centravet	DEP203 IM	15,000 UI/animal
TCM199	Sigma-Aldrich	M3769 10X1L	powder for hepes-buffered TCM199
Hepes sodium salt	Sigma-Aldrich	H3784-100G
bovine serum albumin	Sigma-Aldrich	A8806
heparin	Sigma-Aldrich	H3149-10KU
NaHCO3-buffered TCM199	Sigma-Aldrich	M2154-500ML	liquid for IVM medium
epidermal growth factor	Sigma-Aldrich	E4127
newborn calf serum	Sigma-Aldrich	N4762-500ML
4-well dishes	NUNCLON	144444
incubator	Heraeus	BB6060
monastrol	Sigma-Aldrich	M8515
hyaluronidase	Sigma-Aldrich	H3506
pronase	Sigma-Aldrich	P5147
paraformaldehyde	Sigma-Aldrich	P6148
polyvinyl alcohol	Sigma-Aldrich	341584
triton-X 100	Sigma-Aldrich	T8787
normal donkey serum	Sigma-Aldrich	D9663
rabbit anti-Aurora B phospho-Thr232	BioLegend	636102
TRITC-conjugated donkey anti-rabbit IgG	Vector Laboratories	711-025-152
Vecta-Shield	Vector Laboratories	H-1000
YOPRO1	Thermofisher Scientific	Y3603
confocal laser scanning microscope	LSM 780	Zeiss
confocal laser scanning microscope	LSM 700	Zeiss
ImageJ software	rsb.info. nih.gov/ij/download.html	free resource
mouse anti-alpha-tubulin	Sigma-Aldrich	T8203
rabbit anti-acH4K16	Upstate Biotechnology	07-329
AlexaFluor 488-coniugated donkey anti-mouse IgG	Life Technologies	A21202
4’,6-diamidino-2-phenylindole	Sigma-Aldrich	D8417	DAPI
centrifuge	Eppendorf	5417R
RNALater	Invitrogen	AM7020
Luciferase RNA	Promega	L4561
PicoPure RNA Isolation Kit	Applied Biosystems	12204-01
random hexamers	Thermofisher Scientific	N8080127
mouse Moloney leukaemia virus reverse transcriptase	Thermofisher Scientific	28025013
SYBR green supermix	BioRad	1708880
specific primers	Sigma-Aldrich		specific primers were designed using Primer3Plus software (free resource)
thermal-cycler	BioRad	MyiQ
mouse monoclonal anti-CENPA	Abcam	ab13939
mouse monoclonal anti-Aurora B	Abcam	ab3609