BIOLOGY OF REPRODUCTION 23, 189-205 (1980) Fertilization and Early Development of Cow Ova BENJAMIN G. BRACKETT,1’2’3 YON K. OH,2 JAMES F. EVANS2 and WILLIAM J. DONAWICK2 Department of Clinical Studies at New Bolton Center,2 University of Pennsylvania School of Veterinary Medicine, Kennett Square, Pennsylvania 19438 and D o Department of Obstetrics and Gynecology,3 w n University of Pennsylvania School of Medicine, lo a Philadelphia, Pennsylvania 191 74 de d fro m ABST RACT http microCscoowpy. ova Eanvedntsembraysososciatedwere witrhecovneorremdal infer1til-iz1a6ti-ocnell satnadgeseaarlnyd dsetvuedloipemdentby liginhtoathnedr elsepcetcrieosn s://a c were documented by observations of acrosome-reacted sperm cells embedded in the matrix of the a d zona pellucida, presence of sperm remnants within ooplasm and disappearance of cortical granules, e m appearance of centrioles at the 8-cell stage, and changes in mitochondria with development. Few ic sperm cells, apparently limited in penetration to one-third the thickness of the zona, along with .o u complete ovum penetration by only one sperm cell suggested a strong zona block to polyspermy. p Sperm remnants were occasionally found in blastomeric cytoplasm of 2-cell stage ova. Prominent .co m granules were seen in mitochondria of bovine ova before and after fertilization. /b Efforts to achieve fertilization in vitro by combining ova, recovered near the expected time of io ovulation from follicles or oviducts, with bull sperm treated with high ionic strength medium lre resulted in sperm penetration and development to 2- and 4-cell stage embryos judged normal by pro light and electron microscopy. Use of adifferent bull was associated with aberrant ovum activation d with retention of cortical granules, thus emphasizing a need for further definition of conditions /a compatible with bovine fertilization. rtic le -a b INTRODUCTION of follicular oocytes (Senger and Saacke, 1970; s Fleming and Saacke, 1972). Among the large tra attenCtioown ovasincaend inemitibarlyos obsehrvaavetionsreceivedreportsepdoradicby domestic species, ultrastructural aspects of ova, ct/23 fertilization and early development have been /1 Hartman et al. (1931). A better understanding /1 reported for the pig (Norberg, 1973a,b; Szollosi 8 of early embryonic development has followed 9 clinical application of embryo transfer in cattle and Hunter, 1973, 1978) and sheep (Calarco /27 production during recent years (Betteridge, and McLaren, 1976; Russe, 1975; Witenberger- 66 8 1977). Although the chronological develop- Torres and Flechon, 1974). Progress has been 59 ment of cow fertilization and cleavage is well made in efforts toward obtaining cow oocyte by known (Hamilton and Laing, 1946; Thibault, maturation in vitro (Edwards, 1965; Hunter et gu al., 1972; Jagiello et al., 1974; Iritani and Niwa, e 1966, 1967), attention to morphological s features has been limited. Blastocysts have been 1977; Leibfried and First, 1979; Newcomb et t on examined in the scanning electron microscope al., 1978; Shea et al., 1976; Sreenan, 1970; 08 (Flechon and Renard, 1978) and transmission Satoh et al., 1977; Thibault et al., 1975, 1976; A p electron microscopy has been applied in studies Trerpoourntssen ofet inal., vit1ro977). fertiOliznaltyion a few ofprelimcoinwary ova ril 20 1 appear in the literature, including earlier reports 9 from this laboratory (Brackett et al., 1977, 1978; Edwards, 1973; Iritani and Niwa, 1977; Accepted April 24, 1980. Von Bregulla et al., 1974). Received February 27, 1980. The purpose of work reported here was to ‘Address correspondence to: Dr. Benjamin G. investigate morphological and physiological Brackett, University of Pennsylvania School of Veterinary Medicine, New Bolton Center, 382 W. aspects of bovine fertilization and early devel- Street Road, Kennett Square, PA 19348. opment and to initiate experiments toward the 189 190 BRACKETT ET AL. goal of developing a repeatable method for in was located and, by placing umbilical tape through the vitro fertilization. broad ligament and around the body of the uterus, the reproductive tract was elevated and secured to a stainless steel bar positioned transversely across the MATERIALS AND METHODS incision. Ovaries were examined for corpora hemor- rhagica or corpora lutea, The fimbriated end of the Animals oviduct to be flushed was cannulated with poly- Sixteen young cows of Angus (A), Hereford (I-I), ethylene or Silastic tubing of appropriate diameter, Holstein (Hol), Jersey (J), and mixed breeds, includ- and the cannula was secured in place with fine silk or ing Angus X Guernsey (AXG) and Angus X Holstein by manual pressure of an assistant. Flushings were (AX Hol), were observed for regular, approximately retrograde with ‘\.‘50 ml of medium forced through a D 21-day, estrus cycles prior to use. Estrus behavior was blunt 18-gauge needle introduced into uterine horn ow detected during approximately 30-mm observation after clamping the horn near the cervix with forceps. nlo periods early in the morning, near mid-day and in the The medium was Ham’s F-10 (Grand Island Biological a d late afternoon or evening. When indicated by observa- Co., GIBCO, Grand Island, NY) supplemented with e d tlaiotnes inofthenervdoauys,nessan adodritionaatltempts obsertvoationride otfhoerr esctrouws,s a5n%d (v/va)ntibiohteicast,-treated 100fetalunitscalf speernuicmillin (HTFCSan,d G10IB0CO),(cid:1)g from i.e., standing heat, was made near midnight. Several streptomycin per ml (“Penicillin-Streptomycin Solu- h cows were used repeatedly in this study. Two young tion,” GIBCO). Flush ings were collected into large (50 ttp bsuupllsp,ly ofnreesh Jesresmeyen ainndthisonework.Holstein, were used to omvla) caonndic/oarl pelmasbtriycos testwerteubes.easilyAfterrecovaere3d0-mm byintearsvpailr,a- s://ac tion with a Pasteur pipette from the bottom of these a d Ovulation Induction and Artificial Breeding tubes, For identification and manipulation of ova, em Ova were ovulated in a normal cycle or following smuircrroousncdoepde bwyasflueidm,ployead.low pTohweer pos(1tm0-o7r0teXm) disfslueschtiinngg ic.o superovulation in response to treatments with follicle u was carried out similarly. p stimulating hormone and luteinizing hormone (FSH, .c o FSH-P” and LII, “PLH,” respectively, Armour-Bald- m win, Omaha, NE) or pregnant mare serumgonadotropin In vitro Fertilization Experiments /b p(KPraoMlasSmta,agzloaon,“dGinestyl,”Ml)F2(cid:1)asNVd(.ePsGcrFib2endO,rganonb“,PyrosEtinls,O”dsesn, HeUtolplajaonlh.dn)(1976C)a.on.d, time Effoofrtsovulawtioenre maasdeposstoiblerecovfoerr ferotivliazatioans nearintovitroth.e iolrepro Follicles greater than 8mm in diameter were aspirated, d The procedure adopted for superovulation in this and oviducts were flushed if ovulation had taken place /a work was initiated on cycle Days 9 to 11 with a single following treatment of donor cows with PMS and rtic ii..mm.. iinnjjeeccttiioonn ooff 225000or 3to0 m30g00PGFI2U0 PM4S8 hfollolawteerd, withby PGF20, Ovum donors were submitted to surgery for le-a ovum recovery between 60 and 96 h after the prostag- b the larger doses given to larger animals. Follicular s development was monitored by rectal palpation and lbaenhdainvior trebaytmentaround wh4ic0h to p4r8ecedhe.dFollicuolabrservedaspirateiosntrus trac occasionally cows were excluded from the study due was carried out by introducing a 20-gauge needle t/2 to inadequate responses. For in vivo fertilization, cows through the ovarian stroma and thus into the follicle 3/1 were artificially bred by deep cervical deposition of at a point distal to the surface. Gentle and repeated /1 “.(cid:1)1 ml of fresh semen diluted with Plus-X extender aspiration and distention of the follicle using a 5 ml 89 (‘\(cid:1)lO(cid:1) sperm cells) or by semen that had been stored syringe (containing ‘\‘l ml of medium) was carried out /27 frozen in one instance. The sperm cells involved in to dislodge the ovum and to allow its entry into the 66 observations of in vivo development were ejaculated syringe along with a gain in fluid volume of 0.5-4.0 85 by the same Jersey bull in each case. ml, The medium for follicular aspiration and for 9 b flushing the oviducts was either Ham’s F-b modified y Recovery of Ova and/or Embryos as described above or heparinized saline, 5 IU heparin gu For the in vivo studies, ova and/or embryos were (“Panheprin,” Abbott Laboratories, North Chicago, es recovered at intervals varying from 23 to 120 h after IL) per ml physiological saline (“Normal Saline, t o n artificial breeding. In one normally cycling cow (J) 0.9%,” Abbott). When the former medium was em- 0 inseminated with frozen stored semen, the embryo ployed for follicular aspiration, the syringe and needle 8 A was recovered at necropsy. All other ovum recoveries were first lined with heparin. Ovulated ova were p ewaecrhe acnaimrriaeld. out surgically to enable repeated use of arebocvoev,eredAll boyvafluwsheirneg heoldviduinctslargeretrvooglruamdees a(2s5-d5e0scribemdl) ril 20 1 Food was withheld for 36 to 48 h immediately of medium in conical 50 ml centrifuge tubes at 9 prior to surgery. Animals were treated preoperatively 37-38#{176C} for at least 1 h (1-2,7 h) following surgery. with atropmne and xylazine, and anesthesia was in- Upon recovery from the bottom of the large centri- duced with thiopental sodium followed by intubation fuge tubes in a 38#{176}Ctissue culture room, ova were and maintenance with halothane. Preparation of the transferred to defined medium routinely used for surgical site, draping, sterilization of instruments and rabbit in vitro fertilization (Brackett and Oliphant, other precautions for aseptic surgery were routine, 1975) and held in a small (30 mm diameter, 12 mm The cow was positioned in dorsal recumbency with deep), glass, sealable-type, tissue culture dish under hind quarters slightly elevated so that viscera inter- silicone oil (“200 Dielectric Fluid,” 50 cs. viscosity, fered less with manipulation of the genital tract. An Dow Corning Corp., Midland, MI) for not more than “25-cm midline incision afforded entry as far poster- 30 mm prior to insemination. ior as possible without damaging the udder. The uterus Semen, collected with an artificial vagina, was held FERTILIZATION AND EARLY DEVELOPMENT OF COW OVA 191 in a tightly stoppered test tube at 37-38#{176}C for 1-3.3 postfixed in 1.0% Os04, dehydrated by passage h in individual experiments. Sperm cells were then through a series of alcohols, and individually embed- treated in a way known to allow in vitro capacitation ded in Epon 812. All specimens were serially sectioned of rabbit sperm (Brackett and Oliphant, 1975), Semen with a diamond knife on an LKB ultramicrotome was diluted to 5.0 ml with defined medium prepared initially at 1 (cid:1)m thickness and stained with toluidine with 100 units penicillin and 100 (cid:1)g streptomycin/ml. blue for light microscopic orientation, followed by Sperm cells were washed by centrifugation (5 mm at thin sectioning (1 Mm). Approximately 50 grids were 734 X g), and diluted seminal plasma was removed by prepared from each ovum. The grids were stained with aspiration. Resulting packed sperm cells were resus- saturated uranyl acetate in 0.1% acetic acid, followed pended in(cid:1) 2.0 ml high ionic strength medium (defined by alkaline lead citrate (Reynolds, 1963). Samples medium with NaCI added to provide an osmolality of were examined and photographed in a Hitachi 12B D o “‘-‘380 mOsm/kg) and incubated for 5 mm. The cells electron microscope. w were recentrifuged for 5 mm and resuspended in 5.0 nlo ml defined medium after supernatant fluid was dis- a RESU LTS d carded, Sperm cells were subjectively assessed micro- e d sspceorpmically cells forwas modteilitteyr,mined and folltohweing concencotruantitoinng inofa In vivo Observations from hemocytometer. Approximately 106 spermatozoa in a Thirty-seven (71.2%) of 52 ovulated ova h small volume of fluid (usually “'vlO MI) were added to were recovered from five cows that were ttp 4e.x0perimmenlts,of definfreodzen mesdeiummen csotonrteadining in aomvpau.lesIn twwaos inseminated at variable intervals prior to ovum s://a c thawed according to directions (Atlantic Breeder’s recovery (Table 1). The onset of estrus (stand- ad Service, Lancaster, PA), and the sperm cells were ing heat) was observed 39-48 h after the em immediately treated and used for insemination as PGF2(cid:1) injection in the superovulation treat- ic described above. ments, and ovulation was well underway, if not .ou The ovum-containing solution was covered with completed, by 24 h after onset of estrus. These p.c silicone oil, and following insemination all air space o within the dish was completely eliminated by addi- treatments clearly shortened the normally m/b tional oil and placement of the dish top. The closed expected temporal relationship between onset io dish was wrapped in foil to exclude light and incu- of estrus and ovulation, Developmental stages lre bated in a small chamber (Anaerobic Culture Apparat- from 1-cell to morula were obtained for morph- pro us, small size, Arthur H. Thomas Co., Philadelphia, d P8%A).02Me:d8ia7% aNn2d poriiolr wteoreusee,quailnibdratetdhe samewithgas5m%ixtuCre0,2: odleovgeilcoaplment assessmwenast probab(Tlyable due1). toVavraiarbiaibliitlyity iinn /artic le moistened by passage through a washing bottle, was ovulation of individual ova, but hormonal -a continuously passed at a slow rate through the cham- influences on the temporal progression of bs bifneearrtt.iiloizna,tiAofnter somineitimaledoivuamobsweerrvea(tBiornactrkaentstferraetd 2a0n-d24froOmliphhanatt,fhteer deinf1isn9ee7md5-) fcearntinloiztation be comanpdletelyearly ruldeedvelopmoeunt.tal The foervmeenrts tract/23 into Ham’s F-10 modified as described above but with interpretation leads to the conclusion that /1 10% (v/v) heat-treated fetal calf serum, a medium ovulation in Exp. 34-66 (AXG) took place at /18 demonstrated to support early development for an least 20 h earlier for the 2-cell ovum, i.e., only 9/2 extended culture interval (Wright et al., 1976). Com- 7 3 h after insemination, and quite recently for 6 6 position of the atmosphere for embryo culture was 6 not altered. All glassware, media and tubing contacting ova still surrounded by cumulus cell masses. 85 9 ova and/or sperm cells were sterilized as is routine: Even more remarkable was the finding of b y dry heat for glassware, Millipore filtration (0.45 (cid:1).zm recently penetrated 1-cell stage and 8-cell stage g pore size) for media, chemical sterilization with u ethylene oxide for polyethylene or Silastic tubing. o3v4a-53, 64 Ahx aHfotel,r Taablesingle1). inTsheemseinationdata sug(Egexpst. est o n that bull sperm can undergo capacitation within 0 Examination of Ova 8 3 h and maintain fertilizing ability in the female A For morphological studies, ova were transferred to p microscope slides with small amounts of fluid and reproductive tract for ‘\,60 h. ril 2 examined under cover slips supported by four small From cow Ax Hol (in Table 1) ova in four 01 pillars of stopcock grease. The ova and any associated different (1-, 2-, 4-, and 8-cell) stages were 9 sperm cells were studied under phase contrast and found 64 h after artificial insemination. One of interference contrast microscopy (12.5-1250X) and the 1-cell ova was characterized by severely representatives were photographed in a Zeiss photo- microscope II. Ova were washed from microscope degenerated ooplasm (Fig. 1). Since recovery slides, collected and transferred for fixation in 1.0% was considered to be too soon after ovulation gluteraldehyde in 0,1 M phosphate buffer (prepared at for aging within the oviduct to account for the pH 7.2) for 30 mm, When further processing for condition of this ovum, it was concluded that electron microscopic examination was to be delayed, one of 17 ova released from the ovaries in ova were rinsed for 10 mm in fresh 0.1 M P04 buffer and then submerged in fresh buffer for storage at 5#{176}C response to the PMS and PGF2a treatment was for up to 10 days. For ultrastructural study, ova were incompetent prior to ovulation, An additional 192 BRACKETTETAL. ‘0 ‘‘ ‘-(cid:1) 0 E c .(cid:1) 0, ,(cid:1)0 0 C > (cid:1)- .9 ci 0(cid:1)(cid:1) -v o 00 cbie ci’-’ a - - I,’ a(cid:1) - -vLii Vci D :-(cid:1) .(cid:1) ow n ‘0 ‘0 * lo a d e d LI h fro m h N - - ttps ://a c a (cid:1)u(cid:1)LiV ‘>0#>v{176}(cid:1)‘ dem >(cid:1) - (cid:1)‘l - N (cid:1)0(cid:1)#{231} 00 ic.o u p - - - - (cid:1) H .c o ci 0 m (cid:1)C0(cid:1) L0i0O 0cC,.si.,(cid:1)..sc0 LCE0i,0.(cid:1). /biolre E.(cid:1) (cid:1)0 #{176}-(cid:1) 0’(cid:1) p a ‘(cid:1)0 -I (cid:1). (cid:1) -i .(cid:1)Li,4 ‘i>lo.C ro r-2 r.l * (cid:1) V d/a C0 ‘BLi ::c(cid:1)i(cid:1)-(cid:1) V.(cid:1) beOo0‘C(cid:1)J-’(cid:1)c’i rticle-abs - ‘(cid:1)i a(cid:1) r-i e’l ‘(cid:1)i- r.l “(cid:1)I (cid:1) .(cid:1) #{176}‘ ‘ V trac ,..,_r-4 (cid:1). * ‘C -- r-i 0uj,E...”C(cid:1). V(cid:1)‘c(cid:1) t/2 3 /1 (cid:1)Lvigo<:i-(cid:1) X.C /18 9 a 0(cid:1)0 2 aB’#{176} 0.0 /27 6 ,9-(cid:1) , (cid:1)I 0, 6 . 8 ocoi -(cid:1)9(cid:1)-v V 0‘O’ iN’i *‘0 N‘0 . *‘0 (cid:1)C0 ,LB>i(cid:1), 0 ‘(cid:1)..., N ‘0 59 by Li V g C ‘ (cid:1)5(cid:1)v u e .(cid:1) (cid:1) st o I (cid:1)‘‘(cid:1) a(cid:1) n 0 -(cid:1) h (cid:1) (cid:1) 8 (cid:1)0 B ii (cid:1) (cid:1) Ap .-(cid:1)(cid:1)CO (cid:1)V0’’(cid:1)v-‘v.0 0>00,.(cid:1).., ril 20 O 0, (cid:1) 2.’(cid:1). Xij.0 0C 19 0a uE 0+, + + 0+(cid:1) ..(cid:1)?E:‘ (cid:1)C(cid:1) <(cid:1) (cid:1)v ‘-‘.9 B U, vi v(cid:1) v(cid:1)i 0 (cid:1)iciQ (cid:1)‘#{176}E (cid:1).C Li 0, 0, Z (cid:1)a ., a I (cid:1)a (cid:1).0 - cia (cid:1) (cid:1) a (cid:1) X0,0 (cid:1) I ti.1 (cid:1) (cid:1) a(cid:1).o< ci(cid:1)’v(cid:1)I o(cid:1)E I- ,i(cid:1) --‘ *(cid:1) ‘..- *‘(cid:1)i< ‘(cid:1) *‘(cid:1) ‘(cid:1) *“i(cid:1) ‘. v0i 0. 2 FERTILIZATION AND EARLY DEVELOPMENT OF COW OVA 193 feature of this ovum in contrast to the others 23 h after insemination six 1-cell stage ova were was a total absence of any evidence for interac- recovered from oviducts of cow Ax G, and tion with sperm cells. l’he other uncleaved ova these recently ovulated ova were still surround- were mature as indicated by the presence of ed by cumulus oophorus cells, a feature not polar bodies within the perivitelline spaces of observed in other cows when ova were recov- each. Evidence of fertilization was not obvious ered at longer intervals after ovulation. Corona by light microscopy (see below), and the zonae radiata cell processes deeply embedded in the pellucidae were free of surrounding cellular matrices of zonae pellucidae were observed investments. When ovum recovery took place upon ultrastructural examination of the ova D o w n lo a d e d fro m h ttp s ://a c a d e m ic .o u p .c o m /b io lre p ro d /a rtic le -a b s tra c t/2 3 /1 /1 8 9 /2 7 6 6 8 5 9 b y g u e s t o n 0 8 A p ril 2 0 1 9 FIG. 1. Electron micrograph of an abnormal cow ovum ovulated in response to PMS and PGF20 treatment. Note indefinite perivitelline space due to degenerate ooplasm. PVS perivitelline space; V = vacuole (arrow) within degenerated vitellus; ZP = zona pellucida. X 2500. FIG. 2. Ultrastructural relationship of corona cell to zona pellucida of a normal cow ovum recovered soon after ovulation, 0 = ooplasm; CCP = corona cell process (arrow) embedded within the matrix of the zona pellu- cida; CC = corona cell outside the zona pellucida. X 5200. 194 BRACKETT ET AL. (Fig. 2), recovered within 36 h of the onset of pictured in Fig. 4, recovered from Ax Hol 64 h estrus. Sperm penetration of the cow ovum after artificial breeding, contained a male with intact cellular investments was document- pronucleus identifiable by its proximity to a ed by the additional ultrastructural observation sperm tail remnant within the ooplasm. Anoth- of an early stage of fertilization in one of these er ovum contained two well developed pro- ova (Fig. 3). From adjacent thin sections of this nuclei that had already assumed a central ovum, another part of the sperm tail (not position within the ooplasni. shown) was seen remaining in the perivitelline Usually no more than three sperm cells were space. The nine outer fibers and mitochondria seen in association with zonae pellucidae of D of the fertilizing sperm cell appeared somewhat cow ova exposed to sperm in vivo, Such sperm ow degenerated (Fig. 3), and activation of the cells were observed to have undergone the nlo ovum was apparently well underway. The acrosome reaction, leaving the inner acrosomal ad e so-called “hooded” mitochondria, characteristic membrane exposed. Penetration to approxi- d of oocytes of ungulates, represented a promi- mately one-third the thickness of the zona fro m nent ultrastructural feature. ‘I’he abundance of pellucida, but no further, was a frequent h mitochondria and the appearance of the mito- observation (Fig. 5), suggesting a strong block ttp s chondrial cristae suggested increased metabolic to polyspermy. Pronounced dissolution of the ://a activity within the ooplasm coincident with matrix of the zona pellucida by a penetrating ca d ovum activation. Ultrastructural features con- sperm cell was evidenced in the electron micro- e m sonant with release of cortical granules (Fig. 4), scope (not shown) by a clear area (penetration ic associated with ovum activation, were observed slit) approximately 10 times the size of the .ou p in cow ova found to be in the early pronuclear sperm head, .c o stage by electron microscopy, although as A representative 2-cell stage cow embryo is m mentioned above evidence for fertilization was shown in Fig. 6. This ovum was recovered from /bio not apparent by light microscopy. The ovum cow Ax I-Iol 64 h after artificial breeding. An lre p ro d /a _ rtic le #{14‘(cid:1)i9’} (cid:1). Ird(cid:1)I(cid:1) (cid:1)%. -a .(cid:1)t #{149}0 ‘. ‘f ,.‘ ._ bstrac #{149}#{149}(cid:1),(cid:1)2 .(cid:1). 0, t/2 I I’,”‘.(cid:1) - #{14‘9}i(cid:1).-,(cid:1) :(cid:1)r’ ,#{149}(cid:1)5 ‘.. -- 3/1/1 #{149} . 9., Ii 89 /2 7 (cid:1). 6 6 8 #{149} ‘ :‘ 59 b y g (cid:1) u e s t o n r 0 8 A p U ril 20 ‘ 1 9 ± FIG. 3. Electron micrograph of a cow ovum undergoing fertilization as evidenced by presence of sperm cell within ooplasm, many apparently active mitochondria, depleted complement of cortical granules at periphery of ooplasm and prominent perivitelline space. M = mitochondrion with characteristic “hooded” appearance; SR = sperm remnant (arrow) within ooplasm; PVS = perivitelline space; ZP = zona pellucida. X 10,500, FERTILIZATION AND EARLY DEVELOPMENT OF COW OVA 195 D o w n lo a d e d fro m h ttp s ://a c a d e m ic .o u p .c o m /b io lre p ro d /a rtic le -a b s tra c t/2 3 /1 /1 8 9 /2 7 6 6 8 5 9 I_ _(cid:1)_#{149}__(cid:1).#{149} - by FIG. 4. Cow ovum in the early pronuclear stage with ultrastructural evidence of cortical granule breakdown g u at the periphery of the ooplasm (arrow). 0 = ooplasm; PVS = perivitelline space. X 2400. e s FIG. 5. A representative electron micrograph showing an acrosome-reacted sperm cell that has apparently t o penetrated into the zona pellucida to the limit allowed by a strong block to polyspermy imposed by the fertil- n 0 ized cow ovum, SN = sperm nucleus; ZP = zona pellucida; PVS = perivitelline space; 0 = ooplasm. X 6000. 8 A p ril 2 0 1 unexpected finding was the presence of sperm two granules could be seen within the same 9 tail remnants within the blastomeric cytoplasm mitochondrion. Impressions from this work at this stage. This ultrastructural observation were that these well developed granules are was made in two of the ten 2-cell stage embryos present in a small percentage of mitochondria studied. Ooplasm of another 2-cell stage ovum and that the incidence increases soon after from the same cow is shown in Fig. 7 to demon- fertilization. strate typical intramitochondrial granules. These A representative 4-cell stage embryo, re- structures were occasionally observed in un- covered from cow Ax Hol 64 h after artificial fertilized as well as in fertilized ova. Usually breeding, is illustrated in Fig. 8. l’he periphery only one granule was present, but sometimes of blastomeric cytoplasm of each cell was 196 BRACKETT ET AL. D o w n lo a d e d fro m h ttp s ://a c a d e m ic .o u p .c o m /b io lre p FIG. 6. A 2-cell stage cow embryo as observed by interference contrast microscopy (inset, X 240). Electron rod microscopic examination revealed remnants of the fertilizing sperm cell (arrow) within blastometric cytoplasm. /a N = nucleus of blastomere; SR = sperm remnant. X 15,000. rtic le -a b s tra c devoid of cortical granules as expected for to become less electron dense. Also, mito- t/2 3 normally fertilized ova. An 8-cell stage ovum, chondria of the morula stage seemed to be /1 recovered from the same cow, afforded the first characterized by an increase in peripherally /18 9 occasion during early bovine development in arranged cristae. /2 7 which the centrioles were identifiable (Fig. 9). 6 6 8 Centrioles, found also in the morula stage, were 5 found near the blastomeric nuclei. In vitro Fertilization Experiments 9 b y Cortical granule breakdown appeared to be For in vitro fertilization, 12 cows were g u an ultrastructural feature of normal in vivo treated with PMS and PGF2a for ovulation es fertilization, and cortical granules were usually induction in 16 estrus cycles. In four treated t o n absent from the peripheral cytoplasm of each cycles of three different animals following 0 8 blastomere. Occasionally, associated with the repeated rectal palpation, the follicular re- A p Gbloaslgtoimereas,pparatuscleaveodr inovpaeriphapepraealred aretaos hoavfecertmaionre sspuorgnesrey. wSausrgery judgedwas peinrfaodrmeqeudate in to12 wcaasrerasnt at ril 20 1 than a few remaining cortical granules. The intervals of 60 to 96 h after prostaglandin 9 morula, resulting from in vivo fertilization of a treatment, and 32 follicular and 42 tubal ova single ovum ovulated in the course of a normal were recovered around the time of ovulation estrus cycle, was characterized by the presence for in vitro insemination. Results of these of a full complement of cortical granules in the experiments are shown in Table 2. peripheral ooplasm of a few of the approxi- Several leads emerge from the data resulting mately 16 blastomeres (Fig. 10). from four series of experiments (Table 2). The In the morula stage, mitochondrial structure superovulation procedure yielded recently ovu- was different when compared with earlier stages lated and preovulatory ova between 67 and 76 (Fig. 11). The mitochondrial matrix appeared h after the prostaglandin treatment, and best FERTILIZATION AND EARLY DEVELOPMENT OF COW OVA 197 D o w n lo a d e d fro m h ttp s ://a c a d e m ic .o u p .c o m /b io lre p ro d FIG. 7. Electron micrograph of part of a 2-cell cow embryo revealing intramitochondrial granules; these were /a seen in mitochondria of both unfertilized and fertilized cow ova. MG = mitochondrial granule; N = nucleus. rtic X 15,000. le -a b s tra c results were afforded when ova surrounded by a ery of ova from treated donors between 67 and t/2 3 healthy complement of follicular cells, obtained 76 h after prostaglandin treatment and the use /1 /1 within this interval, were inseminated in vitro. of freshly ejaculated sperm from the Jersey 8 9 In an initial series of experiments (Table 2) bull. In four experiments, 5 of 12 preovulatory /2 7 ova were recovered 86 to 96 h after prostag- ova recovered from follicles and 9 of 16 recent- 66 landin treatment, and the absence of develop- ly ovulated tubal ova were apparently fertilized 85 9 ment after in vitro insemination might be in vitro. The most convincing evidence of in b y attributable to excessive aging of oocytes prior vitro fertilization followed the demonstration g u to their recovery. This was suggested by an by electron microscopy of an absence of es inadequately responding ooplasm which facili- cortical granules at the periphery of blastomeric t o n tated the detection of a sperm cell within the cytoplasm in the 2-cell and 4-cell (Fig. 12) ova 0 8 perivitelline space of one ovum. Although resulting from experiment 34-62 (Table 2). A p pbeenenetratiosnlower othfan thenormzaoln,a pitellwucaisda conclumdeidght htahvaet out Awifthinal spesremries fro(m3 extpheerimeynotsu)ng Holwstaesin carribeudll. ril 20 1 sperm capacitation and the acrosome reaction The timing for ovum recovery seemed approp- 9 had taken place in vitro. Another series (two riate, but only 3 of 10 ova recovered from experiments) was carried out in which frozen follicles and 2 of 16 ova recovered from the semen was employed. Although ova were oviducts showed signs of fertilization as as- recovered at more appropriate intervals after sessed by light microscopy. Ultrastructural prostaglandin treatment, no signs of activation examination of the pronuclear ovum from Exp. or fertilization resulted. The most likely reason 40-50 (Table 2) revealed an absence of cortical was the inadequate condition of sperm cells granules at the periphery of the ooplasm but used. the presence of two or three clusters of cortical The most promising results followed recov- granules close to the vitelline membrane. Also, 198 BRACKETT ET AL. D o w n lo a d e d fro m h ttp s ://a c a d e m ic .o u p .c o m /b io $ (cid:1)“I #{1‘49},(cid:1) lrep FIG. 8. A 4-cell stage cow embryo as observed by interference contrast microscopy (inset, X 200). The elec- ro d tron micrograph reveals absence of cortical granules in the peripheral cytoplasm of each blastomere. B = blasto- /a mere. X 4200. rtic le -a b s tra c no sperm remnant could be demonstrated 1942). By comparison, the interval between t/2 3 within the cytoplasm, and by these more onset of estrus and onset of ovulation in treated /1 stringent criteria the ovum was judged not to be cows in the present study was usually shortened /18 9 fertilized, The pronuclear ovum from Exp. by several hours, The recovery of cow ova in /2 7 40-53 had many sperm attached to the zona pronuclear to 8-cell stages 64 h after artificial 6 6 pellucida, but cortical granules were present at insemination (Exp. 34-53, Table 1) apparently 85 9 the periphery of the ooplasm and no sperm tail reflects a prolonged interval over which ovula- b y remnant was seen within the cytoplasm. Al- tion takes place following PMS and PGF2a g u though the organelles appeared healthy, this treatment. The pronuclear ova were comparable e s ovum failed the test for fertilization according to those found 5 to 12 h after ovulation (Thi- t o n to the accepted ultrastructural criteria. The two bault, 1967),- and if the 8-cell stage was reached 0 8 ova that developed to the 2-cell stage in Exp. around 44 h after ovulation (Thibault, 1966; A 4tu0r-a5l6ly. (B‘foatbhle had2) cowretirceal examgrianneudles uplrtersaesntrtuc- at pthreesentAx dHaotal), cowthe fweertrielized ovulateodva recoovveerred a 3fr9om h pril 20 1 the periphery of blastomeric cytoplasm. One period. Also, sperm cells capable of fertilizing 9 appeared to be degenerated, while the other ova were present within the oviducts during this was apparently developing parthenogenetically. 39 h interval, The degenerating 1-cell unfertil- ized ovum recovered at the same time (Fig. 1) was most likely abnormal and not merely DISCUSSION degenerating as a result of aging following Most cows remain in estrus (standing heat) ovulation. The cow ovum is reportedly fertiliz- 18 h (Trimberger and Davis, 1943), and ovula- able for 24 h following ovulation (Thibault, tion usually occurs between 10 and 18 h after 1967). Since the cow was bred while in estrus, the end of estrus (Nalbandov and Casida, at least 12 h before ovulation, and the interval