Chorionic Gonadotropin Secretion Is Associated with an Inhibition of Follicular Growth and an Improvement in Oocyte Competence for In Vitro Maturation in the Mare1

Abstract

This study reports the follicular growth and oocyte competence for in vitro maturation and fertilization under the influence of circulating eCG. Three to 7 successive ultrasound-guided follicular punctures were performed on 4 pregnant mares from Day 23 until Day 75 of pregnancy and on 5 control mares whose embryonic vesicle was crushed on Day 22. All follicles larger than 5 mm were punctured 24 h after the largest follicle reached 18 mm. Expanded cumulus oocyte complexes (COCs) were stained at recovery to analyze the nuclear stage. Compact COCs were cultured in vitro for 46 h and either stained or processed for in vitro fertilization (IVF) and stained 26 h after IVF. In the control group, no mares showed an increase in eCG levels, whereas all the pregnant mares had concentrations higher than 100 ng/ml from Day 37. The number of follicles flushed during each puncture attempt significantly decreased with time for 3 of 4 pregnant mares. No significant change in this number was observed for the 5 control mares. The maturation rate of the oocytes from follicles 10-14 mm was significantly higher in the pregnant vs. the control group (14 of 17, 82%, vs. 13 of 30, 43%). The difference was not significant for the oocytes from follicles smaller than 9 mm or larger than 15 mm. After IVF, no oocyte was fertilized. The results led us to conclude that eCG is associated with an inhibition of follicular growth and an improvement in oocyte competence for in vitro maturation.

Organisation of the cytoskeleton during in vitro maturation of horse oocytes

“…Not only is the supply of equine ovaries from slaughterhouses limited, but the number of oocytes retrieved per ovary remains low (about 4 oocytes/ovary), compared to other domestic species (Hinrichs, 1991;Choi et al, 1993;Alm et al, 1997;Guignot et al, 1999). With regard to oocyte maturation in vitro, several culture systems have been adapted from those used successfully in other species but most have yielded disappointing rates of maturation to metaphase of the second meiotic division (40±70%, Willis et al, 1991;Dell'Aquilla et al, 1997;Goudet et al, 1998;Bru È ck et al, 2000). Furthermore, since in vitro matured equine oocytes show poor rates of fertilisation in vitro it seems likely that their developmental competence is also poor.…”

Comparative Evaluation of Nuclear Morphology of Equine Oocytes Aspirated in vivo and Stained with Hoechst and Orcein

“…Fluorescence microscopy of oocytes stained with the DNA-specific label Hoechst has repeatedly been described for different mammalian species [Mattson and Albertini, 1990;Viuff et al, 1991;Debey et al, 1993;Hinrichs et al, 1993aHinrichs et al, , b, 1995Schramm et al, 1993;Bézard et al, 1997;Goudet et al, 1998]. For visualization of the chromatin after Hoechst staining, oocytes are usually placed between slide and cover slide, which allows precise focusing on the DNA structures Goudet et al, 1998].…”

“…For visualization of the chromatin after Hoechst staining, oocytes are usually placed between slide and cover slide, which allows precise focusing on the DNA structures Goudet et al, 1998]. With this technique, GV stage chromatin configurations of equine oocytes have previously been characterized as different types of either so-called fluorescent nucleus or condensed chromatin.…”

“…In a more recent publication, Hinrichs and Williams [1997] suggested that 'fluorescent nucleus' re-presents the immature viable horse oocyte before acquisition of meiotic competence, while 'condensed chromatin' represents the chromatin of an oocyte resuming meiosis. Also Bézard et al [1997] and Goudet et al [1997Goudet et al [ , 1998] defined Hoechst-stained equine oocytes with dense chromatin as resuming meiosis, while oocytes with diplotene chromatin were characterized as GV stage. It appears that the chromatin patterns visualized by Hoechst and orcein staining generally agree, while the interpretation of the nuclear stages may differ, and a more harmonized definition of the meiotic stages is needed.…”

“…Only few morphological descriptions of the different chromatin configurations of equine oocytes are available. For the determination of the nuclear stages of oocytes by light and fluorescence microscopy, the DNA can be visualized by fluorescent staining with Hoechst [Hinrichs et al, 1993b;Goudet et al, 1997Goudet et al, , 1998], by staining with orcein after fixation with acetic alcohol [Fulka and Okolski, 1981;Okolski et al, 1991;Bézard and Palmer, 1992;Alm and Torner, 1994;Del Campo et al, 1995;Torner and Alm, 1995;Brück et al, 1996Brück et al, , 1997 or by staining with lacmoid after fixation in glutaraldehyde followed by acetic alcohol [Zhang et al, 1989;Willis et al, 1991Willis et al, , 1994. During fluorescence microscopy after Hoechst staining, only the chromatin is visible, while after orcein or lacmoid staining additional cellular structures in the nucleoplasm and cytoplasm can be taken into account for evaluation.…”