Microsc. Microanal. 23, 1197–1206, 2017 doi:10.1017/S1431927617012740


© MICROSCOPY SOCIETY OF AMERICA 2017


Use of Confocal Microscopy to Evaluate Equine Zygote Development After SpermInjection of Oocytes Matured In Vivo or In Vitro


Elena Ruggeri,1,2 Keith F. DeLuca,3 Cesare Galli,4,5 Giovanna Lazzari,4,5 Jennifer G. DeLuca,3 Joanne E. Stokes,1 and Elaine M. Carnevale1,*


1Department of Biomedical Sciences, Colorado State University, 1693 Campus Delivery, Fort Collins, CO 80523, USA 2Department of Obstetrics/Gynecology Reproductive Sciences, University of California, San Francisco, 513 Parnassus Avenue,


San Francisco, CA 94143, USA 3Department of Biochemistry andMolecular Biology, Colorado State University, 1870 Campus Delivery, Fort Collins, CO 80523, USA 4Laboratory of Reproductive Technologies, Avantea, Via Porcellasco 7f, 26100, Cremona, Italy 5Fondazione Avantea, Via Porcellasco 7f, 26100, Cremona, Italy


Abstract: Confocal microscopy was used to image stages of equine zygote development, at timed intervals, after intracytoplasmic sperm injection (ICSI) of oocytes that were matured in vivo or in vitro. After fixation for 4, 6, 8, 12, or 16 h after ICSI, zygotes were incubated with α/β tubulin antibodies and human anticentromere antibody (CREST/ACA), washed, incubated in secondary antibodies, conjugated to either Alexa 488 or Alexa 647, and incubated with 561-Phalloidin and Hoechst 33258. An Olympus IX81 spinning disk confocal microscope was used for imaging. Data were analyzed using χ2 and Fisher’s exact tests. Minor differences in developmental phases were observed for oocytes matured in vivo or in vitro. Oocytes formed pronuclei earlier when matured in vivo (67% at 6 h and 80% at 8 h) than in vitro (13% at 6 and 8 h); 80% of oocytes matured in vitro formed pronuclei by 12 h. More (p=0.04) zygotes had atypical phenotypes, indicative of a failure of normal zygote development, when oocyte maturation occurred in vitro versus in vivo (30 and 11%, respectively). Some potential zygotes from oocytes matured in vivo had normal phenotypes, although development appeared to be delayed or arrested. Confocal microscopy provided a feasible method to assess equine zygote development using limited samples.


Key words: zygote, confocal microscopy, equine, ICSI, cytoskeleton INTRODUCTION


Conventional in vitro fertilization has had poor success for horses (Squires et al., 2003; Leemans et al., 2016). However, assisted fertilization using intracytoplasmic sperm injection (ICSI) resulted in experimental pregnancies and limited offspring by the mid-1990s (Squires et al., 2003; Galli et al., 2007). Subsequently, ICSI was developed for clinical use in horses, with the production of pregnancies and offspring from subfertile mares and/or stallions with limited or poor sperm quality (Carnevale et al., 2007; Colleoni et al., 2007; Hinrichs, 2013). As the demand for ICSI increased in the equine industry, methods for maturing oocytes and culturing embryos in vitro were explored (Carnevale & Sessions, 2012; Hinrichs, 2013; Galli et al., 2014). However, our under- standing of equine fertilization and early embryo develop- ment is still limited. Oocytes can be matured in vivo or in vitro for equine


assisted reproduction. Oocyte maturation is induced in vivo by administration of compound(s) to the donor mare that initiate follicle and oocyte maturation within the dominant follicle during the follicular phase, and the oocyte can be


*Corresponding author. Elaine.Carnevale@colostate.edu Received September 7, 2017; accepted November 4, 2017


collected from the follicle before ovulation (Carnevale, 2016). Theoretically, the resulting oocytes should be of optimal quality, and collection, oviductal transfer, and fertilization in vivo of similar oocytes result in high pregnancy rates (Carnevale & Ginther, 1995). In vitro maturation of oocytes has been widely used in domestic animals and is of interest in human reproduction (Edwards, 1965; Hinrichs et al., 1993; Arlotto et al., 1996; Galli et al., 2007; Lonergan&Fair, 2016). In the horse, immature oocytes are collected from live mares or excised ovaries by collecting oocytes fromnumerous follicles of various sizes for in vitro maturation, fertilization, and foal production (Galli et al., 2013;Hinrichs, 2013; Carnevale, 2016). The extent to which the artificial environment, associated with in vitro maturation, affects the oocyte has not been fully determined. In addition, immature oocytes from small follicles are removed from their natural environment prior the development of conditions associated with follicle growth and hormonal stimulation. Consequently, oocytes from immature follicles are more variable in quality and developmental com- petency (Hinrichs,1991;Hytteletal.,1997). An understanding of differences in zygotes developing


from oocytes matured in vivo (IVO) and in vitro (IVM) would further our knowledge of the normal progression of postfertilization events and of potential alterations in cyto- skeletal and nuclear maturation prior to the first mitotic


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