MOLECULAR REPRODUCTION AND DEVELOPMENT 26331336 (1990)

Immunocytochemical Studies of Hamster Oocyte Activation M.J.SINOSICH,’ S.E. LANZENDORF? AND G.D. HODGENS ‘Reproductive Biochemistry and Immunology, Department of Obstetrics and Gynecology, Royal North Shore Hospital, St. Leonards, New South Wales, Australia; 2Department of Obstetrics and Gynecology, Northwestern University Medical School, Chicago, Illinois; 3Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia

ABSTRACT By indirect immunofluorescence, using rabbit anti-heparin-binding placental protein (HBPP) antiserum, we studied HBPP expression by physiologically and non-physiologically (microsurgically)activated hamster gametes. Whereas mature gametes (sperm, metaphase II oocytes) were negative, in vivo conceived preimplantation embryos, from pronuclear to two-and four-cell stages, were HBPP positive. No HBPP was demonstrated in the zona pellucida, but HBPP-dependent immunofluorescence was localized in the perivitelline space. Oocytes incubated with hyaluronidase demonstrated variable responses from negative to positive. (Diluent or sperm) microinjected oocytes were all activated and HBPP positive within 4 h after stimulation. Thus neither activation by microinjection nor HBPP expression required paternal gametes. These kinetics suggest that HBPP may be a cortical granule secretogogue which can be applied to monitor oocyte responses during in vitro manipulations. Key Words: Heparin-binding placental protein, Irnrnunofluorescence, Microinjection, Nonphysiological oocyte activation, Fertilization

INTRODUCTION Diagnosis and management of human infertility have progressed in quantum leaps over the past 2 decades. In vitro fertilization (IVF) and embryo transfer (ET) had unified our clinical approaches to ovarian hyperstimulation with the more basic cell culture technology. However, IVF-ET has also provided the opportunity to study and apply this knowledge of spermoocyte interactions for management of male factor infertility patients, that is, males with suboptimal semen parameters who cannot be treated successfully by conventional IVF. This is a multifactorial defect including low sperm numbers and inability to penetrate the zona pellucida and/or fuse with oocyte plasma membrane. In these instances, microinjection of a spermatozoon directly into the ooplasm, bypassing the zona pellucida and plasma membrane, may be a means of

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treatment. Although this experimental technique has been established in many leading IVF-ET centers, the pregnancy rate in animals remains disappointingly poor (Iritani et al., 1988). During mammalian fertilization (fusion of a spermatozoon with the oolemma), the activated oocyte completes the second meiotic division and extrudes the second polar body. The fertilized (or activated) oocyte undergoes the cortical granule (CG) reaction, culminating in secretion of CG secretagogues into the perivitelline space (PVS). Then it enters into a programmed biochemical cascade, synthesizing and/or secreting embryo-derived antigens, such as early pregnancy factor (Morton et al., 19741,platelet activating factor (O’Neill et al., 1983,glycoconjugates (Lee et al., 19881,heparin-binding placental protein (HBPP; Sinosich et al., 19881, and chorionic gonadotrophin (Fishel et al., 1984).Oocyte activation may also be induced by nonphysiological stimuli, such as electric shock, hypothermia, glycolysis (hyaluronidase), and pricking (Gulyas, 1976; Tarkowski, 1975; Uehara and Yanagimachi, 1977). As microsurgical fertilization (by microinjection) includes many of these stimuli, we monitored oocyte responses by immunof luorescent detection of HBPP, an antigen expressed by fertilized, but not by resting, mature oocytes (Sinosich et al., 1988).

MATERIALS AND METHODS Gamete and Embryo Collection Female golden hamsters, Mesocricetus aunztus (Sasco-King, Omaha, NE), were superovulated by intraperitoneal (ip) injections of 25 IU pregnant mare serum gonadotropin (PMSG Sigma G-4877)between 0800 and 1000 h on the first day of the animals’ estrus cycle. Fifty-six hours after PMSG administration, the

Received December 1, 1989; accepted February 26, 1990. Address reprint requests to Michael J. Sinosich, Reproductive Biochemistry and Immunology, Department of Obstetrics and Gynecology, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia.

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Fig. 1. Principles of direct microinjection into ooplasm (see Materials and Methods for details).

animals were injected ip with 25 IU human chorionic gonadotropin (hCG; Sigma CG-2) to induce ovulation. The animals were sacrificed by cervical dislocation 1517 h post-hCG injection, and mature metaphase I1 oocytes were flushed from the excised oviducts into a modified Tyrode's solution containing Hepes buffer (TALP-Hepes;Bavister et al., 1983). Oocytes for microinjection were treated with 0.1% hyaluronidase in TALP-Hepes for 5-10 min to disperse cumulus cells, then washed three times in fresh media and incubated in 0.1 ml TALP-Hepes under heavy paraffin oil at 37°C in 5% C02/95% air until required for microinjection. Hamster embryos were obtained by mating the estrus female, on the evening of day 4 of estrus, with a proven-fertile male. The morning following mating was designated day 1 of pregnancy. Females were inspected for presence of spermatozoa in the vagina to ensure that mating had occurred. On the mornings of days 1 , 2 , and 3, they were sacrificed by cervical dislocation and pronuclear-stage, two-cell and four-cell embryos were flushed with 50mM sodium phosphate buffer, pH 7.4, containing 150mM NaCl (PBS) from the oviducts.

Sperm Microinjection Procedure Hamster spermatozoa were obtained from mature golden male hamsters (120-130 g) sacrificed by cervical dislocation. Cauda epididymides were removed and rinsed in saline to remove blood and debris. The cauda epididymides were held under 2.0 ml of saline, and the tubes were minced and sperm dispersed into buffer. Sperm suspension was sonicated (3 x 1 min a t 30 W power output) to dissociate sperm tails from heads. The nuclei were isolated by filtration through a column of glass wool and the suspension was divided into 100 pl aliquots and stored at -70°C. Prior to use, sperm nuclei were diluted 1/5 in 10% polyvinylpyrrolidone (90 kD)

in PBS to reduce surface tension and sperm nucleiglass attraction (Uehara and Yanagimachi, 1976). Light suction at the holding pipette (hp) anchors the spherical oocyte and zona pellucida (zp), allowing the micropipette (mp) to approach the gamete precisely (Fig. la). With an internal diameter of about 8 Fm, the mp is forced through the zp and into the ooplasm, into which either a single sperm head or diluent (sham control) was discharged (Fig. lb). The mp was gently withdrawn. The oocyte was released from the hp (Fig. lc), washed, and cultured for 4 h (37"C, 5% COz) in tissue culture medium 199 (Gibco, Grand Island, NY)supplemented with 10% fetal calf serum (Lanzendorf et al., 1988). All micromanipulations were carried out a t x 250 magnification on a Nikon Diaphot inverted microscope with phase-contrast optics.

Indirect Immunofluorescence Primary antibody, rabbit anti-guinea pig heparin binding placental protein (HBPP), was used at a 1/50 dilution. Secondary antibody (2nd Ab), swine antirabbit immunoglobulins (Dakopatts, Santa Barbara, CAI, was conjugated to fluorescein isothiocyanate (FITC; Sigma) in alkaline phosphate buffer (pH 8.6; Sinosich and Chard, 1979). The 2nd Ab-FITC conjugate was used a t 1/200 dilution. Gametes and embryos were fixed for 1 h at room temFig. 2. Dark-field (B) and transmission (A) microscopy of mature hamster oocytes and associated cumulus cells. After incubation with (D) and without (C) diluted primary antibody (rabbit anti-HBPP) and fluorescent-tagged second antibody, the oocytes were examined by fluorescence microscopy. Fig. 3. Transmission (A) and fluorescence (B-D) microscopy of hyaluronidase-treated oocytes after analysis for HBPP expression. Negative (antibody-free)control was indistinguishable from D.

Figs. 2 and 3.

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nantly localized at the oolemma, HBPP-dependent immunofluorescence was distributed throughout the cytoplasm of activated oocytes (Fig. 4Bii) and respective zonae (Fig. 4Aii-Cii). Morphology of in vivo fertilized oocytes remained spherical from pronuclear (Fig. 5A,B) to two- (Fig. 5C,D) and four-cell stages (Fig. 5E,F) of embryogenesis. Indeed, both blastomeres and zonae were regular and spherical. Independent of stage of development, all preimplantation hamster embryos expressed HBPP and fluoresced only when reacted with primary and secondary antibodies (Fig. 5A,C,E). In the absence of primary antibody, no fluorescence was observed. However, distribution of HBPP was limited to blastomere or zygote membrane. Zona pellucida (ZP) and tail of RESULTS fertilizing sperm ( S ) stained negative (Fig. 5A,C,E). After ovarian hyperstimulation with gonadotrophins The sperm head could not be visualized. (25 IU PMSG), numerous oocytes (n = 30-50) were recovered from the oviducts of each hamster. Twenty DISCUSSION metaphase I1 oocytes, collected from females (n = 21, HBPP was isolated from aqueous placental extracts were used for microsurgical studies. Sperm from three by positive affinity chromatography on heparinmales (n = 3)were included in this study. Similarly, 22 Sepharose. Preliminary studies indicated that this proovulated oocytes (n = 31, 17 pronuclear (n = 21, 33 tein inhibited granulocyte elastase activity (Sinosich et two-cell (n = 2), and seven four-cell (n = 1)embryos al., 1988). By immunohistochemistry, using a polywere analyzed. clonal rabbit anti-HBPP antiserum, it was possible to Under light (Fig. 2A) and dark-field (Fig. 2B) micros- study HBPP expression by preimplantation hamster copy, the morphology of formalin-fixed immunochemi- embryos. No HBPP-dependent immunof luorescence cally processed oocytes was not distinguishable from was demonstrated on mature, resting hamster gametes that of unfixed oocytes. Oocytes with respective cumu- (oocytes and sperm). By contrast, in vivo conceived emlus cells and zona radiata were readily identified, but bryos were HBPP positive from the pronuclear stage. glycerol-based mounting medium made the zona pellu- Thus it was now possible to distinguish immunologicida virtually invisible. After incubation in the pres- cally between activatedlfertilized and unfertilized ence or absence of diluted primary antibody (rabbit oocytes. The distribution of immunofluorescence suganti-HBPP) and 2nd Ab-FITC, no immunofluores- gested that HBPP was localized to the embryo surface cence was detected on either test (Fig. 2D) or negative or the perivitelline space. The mechanism for restrictcontrol (Fig. 2C) oocytes, respectively. Similarly, ham- ing HBPP distribution to the feto-maternal interface ster epididymal sperm were negative for HBPP. HBPP remains to be defined. immunofluorescence was demonstrated in cumulus Activation of mammalian oocytes can be achieved cells, but the proportion of reactive cells varied be- with many varied physiological and non-physiological tween oocytes. (chemical, electrical, thermal, physical) stimuli. GaIncubating oocytes and intact cumulus with hya- mete preparation for fertilization by microinjection exluronidase caused dispersion of the cumulus with re- poses both male and female gametes to such stimuli. lease of oocytes. HBPP-dependent immunof luorescence Whereas spermatozoa were sonicated to dissociate nuof treated oocytes varied from a strong reaction of the clei from tails, oocytes were exposed to negative and oocyte and zona (Fig. 3B,C) to undetectable reactivity positive pressure, glycolytic (hyaluronidase) activity, (Fig. 3D). Hyaluronidase-untreated oocytes, maintained in culture for 4-5 h, remained negative for HBPP. The influence of microinjection on oocyte physiology Fig. 4. Transmission (Ai-Di) and fluorescence(Ai-Dii) microscopy was evident a t two distinct levels. Microscopically, the of sperm (A,D)- or diluent (B,C)-injected oocytes. All oocytes were morphology of micromanipulated oocytes remained incubated in presence ( A X ) or absence (D) of primary antibody (antispherical (Fig. 4), with indentations occasionally ob- HBPP) before reaction with fluorescentlabeled swine anti-rabbit imserved at injection site (is). Biochemically, in the ab- munoglobulins antibody. sence of primary antibody (anti-HBPP), no immunoFig. 5. Light (B,D,F) and fluorescence (A,C,E) microscopy of in fluorescence was detected on any microsurgically vivo conceived preimplantation hamster embryos from pronuclear manipulated oocytes (Fig. 4Dii). By contrast, all (A,B) to two-cell (C,D) and four-cell (E,F)stage. All embryos were oocytes injected with sperm nuclei (Fig. 4Aii) or diluent incubated with anti-HBPP. No fluorescence was detected in negative (Fig. 4Bii,Cii) reacted positively. Although predomi- (antibody-free)controls. ZP, zona pellucida; S,spermatozoon.

perature (RT; 15-20°C) in 2% formalin-PBS, washed three times in PBS, and incubated for 1h at RT in diluted primary antibody (test), normal rabbit serum (NRS 1/50; serum control) or no primary antibody (PBS; negative control). After washing three times in PBS, sperm, oocytes, and embryos were separately incubated in 50 pl of 2nd Ab-FITC in the dark at RT for 1h. Gametes and embryos were washed three times in PBS and mounted in glycerol-based fluorescence-stabilizing medium (Johnson and Nogveix Araujo, 1981) and kept in the dark until examined for fluorescence on a Nikon Ortholux fluorescence microscope. Photography was done using Kodak 200 Ektachrome slide emulsion.

NONPHYSIOLOGICAL ACTIVATION OF OOCYTES

Figs. 4 and 5.

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and physical invasion of cytosolic compartment. Using HBPP expression as an indicator of oocyte fertilization or activation, the gentle suction and washing procedures did not activate oocytes. In addition, no HBPP was detected in untreated oocytes incubated in parallel with microinjected eggs. By contrast, all (diluent and sperm) injected eggs reacted positively, and hyaluronidase-exposed oocytes showed variable responses. In either situation, enzymatic or physical activation, HBPP immunoreactivity was detected within 4 h, and its distribution extended into the ZP, suggesting a breach of oocyte membrane integrity and, perhaps, function. Furthermore, HBPP expression does not require transcription of paternal DNA, indicating that HBPP is of maternal origin either translated from mRNA or secreted by cortical granules. Temporal kinetics support the latter. In conclusion, these data have established that in vitro and in vivo conceived hamster embryos (synthesize and/or) secrete HBPP, expression of which does not require transcription of paternal or maternal DNA. HBPP is, therefore, a maternal-derived antigen discharged by activated oocytes. Present-day microinjection technology may induce non-physiological activation and biochemically put the oocyte into the fertilized mode. Thus a potential fertilizing sperm may be introduced into an activated oocyte, clearly not the in vivo situation, thereby identifying a potential obstacle for conception. Perhaps the less traumatic procedure of zona drilling may be more appropriate (Gordon, 19881, but this technology also awaits critical biochemical appraisal. In either situation, using this hamster model and HBPP as an immunological marker of oocyte activation, it should be possible to develop microsurgical fertilization technology so that the fertilizing sperm is not introduced into an activated oocyte.

ACKNOWLEDGMENTS The authors acknowledge Mr. M. Bonifacio and Mr. A. Zakher for technical and Miss C. Tomaselli for clerical assistance. This study was funded by I.V.F. Ad-

vancement Foundation and National Health and Medical Research Council (grant 900006) grants.

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Immunocytochemical studies of hamster oocyte activation.

By indirect immunofluorescence, using rabbit anti-heparin-binding placental protein (HBPP) antiserum, we studied HBPP expression by physiologically an...
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