Activation of hamster zona-free oocytes by homologous and heterologous spermatozoa M. Maleszewski, D. Kline and R. Yanagimachi 1Department of Anatomy and Reproductive Biology, University of Hawaii School of Medicine, Honolulu, HI, USA; 2Department of Biological Science, Kent State University, Kent, OH, USA; and ^Department of Embryology, Institute of Zoology, University of Warsaw, Poland

Spermatozoa of a wide variety of species can fuse with zona-free hamster oocytes. Zona-free hamster oocytes were inseminated with spermatozoa of homologous (hamster) and other (mouse, guinea-pig and human) species, and their responses were closely examined to determine whether such interspecific sperm\p=n-\oocytefusion always induces normal oocyte activation. While guinea-pig and human spermatozoa could activate hamster oocytes as efficiently as hamster spermatozoa, mouse spermatozoa could not. Mouse spermatozoa fused readily with hamster oocytes, yet most oocytes remained inactivated at least during the first 1.5\p=n-\2h. The amount of M-phase (metaphase) promoting factor was reduced in hamster oocytes fused with one or several mouse spermatozoa; however, repetitive Ca2+ transients failed to occur unless oocytes were inseminated with a concentrated sperm suspension and penetrated by very many spermatozoa. These observations suggest that sperm\p=n-\oocytemembrane fusion per se is not sufficient to trigger oocyte activation, and that putative sperm-derived oocyte activating factors show some degree of species specificity.

Introduction interspecific sperm penetration causes normal oocyte activation in all cases. In the present study, we inseminated zona-free During normal fertilization, the membrane of the spermatozoon hamster eggs with homologous (hamster) as well as hetero- fuses with the oolemma before the sperm nucleus is incorpo¬ logous (human, guinea-pig and mouse) spermatozoa and rated into the ooplasm. This gamete membrane fusion is followed closely (i) meiotic stage of the oocyte, (ii) the degree followed by the resumption of meiosis in the oocyte, extrusion of cortical granule exocytosis, (iii) the activity of M-phase of cortical granules and the transformation of sperm and promoting factor (MPF), and (d) the intracellular free calcium oocyte nuclei into male and female pronuclei, respectively concentration ([Ca ]¡) in the oocyte. (Yanagimachi, 1994). All these post-fusion events are cumula¬ called activation. The oocyte activation mech¬ tively oocyte Materials and Methods anism is not fully understood. Penetration of the oocyte by the spermatozoon triggers an increase in intracellular free calcium Chemicals ([Ca y in the oocyte by initiating the repetitive release of Ca2+ from intracellular stores (Miyazaki et al, 1986; Kline and Unless otherwise stated, all chemicals were obtained from either Louis, or Matheson, Kline, 1992a). It is accepted that an increase in [Ca +\ is Sigma Chemical Company (St MO) and Bell necessary for activation of the oocyte; however, it is not Coleman Manufacturing Chemists (Norwood, OH). established how spermatozoa trigger Ca changes in oocytes. Sources of the following chemicals and biochemicals were as IL Activation of the oocyte may result from the membrane fusion follows: BSA fraction V (Miles Diagnostic, Kankakee, and BSA itself, interaction with the membrane receptor, or the introduc¬ Calbiochem, La Jolla, CA), crystalline (Sigma), trypsin NF ; ICN units ~ Biochemicals, tion of some sperm-derived activating substance (Foltz and (bovine pancreatic, 3000 mg Shilling, 1993; Swann, 1993; Swann et al, 1994; Taylor, 1994; Costa Mesa, CA), fura-2 AM (Molecular Probes, Eugene, OR), ICN Tesarik, 1994). hyaluronidase (bovine testicular, 300 units mg ~ ; Hamster oocytes freed from zonae pellucidae are unique in Biochemicals), fluorescein isothiocyanate (FITC) conjugated Lens and Limulus that they can fuse with a variety of heterologous spermatozoa lectins: culinaris agglutinin (LCA; Sigma) (Yanagimachi, 1984). However, it is not clear whether such polyphemus agglutinin (LPA; Sigma), paraphenylenediamine (Sigma), phytohaemagglutinin (Sigma), polyethylene glycol M. Maleszewski, of Anatomy and Correspondence: Department Reproductive molecular mass 1450 kDa; alcohol of Hawaii School of Medicine, Hawaii, (PEG, Sigma), polyvinyl Biology, University Honolulu, and HI 96822, USA. (PVA; Sigma), progesterone (Sigma), mineral oil (Squibb Received 8 March 1995. and Sons, Princeton, NJ). Downloaded from Bioscientifica.com at 09/27/2021 05:06:41PM via free access Media suspension (final concentration of spermatozoa was approxi¬ mately 6 10" spermatozoa ml~ ) and at 37.5°C. After M2 (medium 16 buffered with Hepes; Fulton and kept 10—20 min, when most oocytes had a few spermatozoa 1978) was used for collection and Whittingham, manipu¬ attached to the oolemma, the were washed lation of oocytes, M16 1971) for culture of firmly oocytes (Whittingham, gently in M2 and transferred to M16 for culture. and oocytes and for of mouse (Fraser Washing capacitation spermatozoa transfer of the were to avoid excessive and 1975), HCM (Wistrom and Meizel, for oocyte necessary Drury, 1993) of zona-free oocytes. of human mT (modified Tyrode's polyspermy capacitation spermatozoa, In some were examined for the release solution, and 1981) for experiments, oocytes Fleming Yanagimachi, capacitation of cortical granule material onto the surface of the oocytes. of guinea-pig spermatozoa, PBS-alb (Dulbecco's PBS con¬ Five to ten oocytes were in a 50 µ of m-TALP-8 taining 0.1% (w/v) BSA) for fixation and of placed drop washing under mineral oil in a Petri dish on the of an oocytes. For capacitation of hamster spermatozoa, placed stage inverted microscope. The temperature was kept at 37°C using modified Tyrode's solution (m-TALP-8) was used. The an air curtain incubator Instruments, White Plains, NY). was: 101.02 (Sage composition of this solution mmol NaCl \~ , Ten to 20 preincubated (acrosome-reacted) were 2.68 mmol KCl I-1, 1.80 mmol CaCl2 l"1, 0.49 mmol spermatozoa added to the medium and oocytes were continually examined MgCl2-6H20 l"1, 0.36 mmol NaH2P04 H20 l"1, · until they were fixed with 3% (v/v) formalin in PBS. 35.70 mmol NaHC03 I"1, 4.50 mmol D-glucose \ 1.0 mmol sodium pyruvate 1~ , 9.0 mmol sodium and in vitro. lactate 1 , 0.5 mmol 1 , 0.05 spermatozoa ~ of fertilization hypotaurine ~ mmol adrenaline Preparation guinea-pig I- \ 0.2 mmol sodium taurocholic acid 1_ \ 0.1 mmol EDTA Induction of the acrosome reaction of guinea-pig spermatozoa 1 ml~~ sodium G was carried out to the method of and ~ \ 0.01 mg bisulfite, 100 iu penicillin ml-1, according Yanagimachi Suzuki from the distal 50 µg streptomycin sulfate ml~ and 15 mg BSA ml- . (1985). Briefly, spermatozoa segment were suspended in Ca -deficient mT medium containing 85 µg lysolecithin ml" (Fleming and Yanagimachi, 1981). The con¬ Collection and hamster culture of golden oocytes centration of spermatozoa was 5—8 x 106ml_I. When they were incubated 1 2 Female were (37.5°C) for h and then exposed to mmol golden hamsters superovulated by i.p. injection + Ca 1 , the acrosome ~ the majority underwent reaction within of pregnant mares' serum gonadotrophin (30 iu per female) in 10—15 min. Ten to 20 of the the morning of day 1 and injection of hCG (30 iu per female) µ sperm suspension containing acrosome-reacted was added to 100 of M16 in the afternoon or of 3. Mature were spermatozoa µ evening day oocytes with hamster collected from the oviducts of superovulated females between zona-free oocytes (final concentration of sperma¬ tozoa was 8 IO5 ml 16 and 18 h after injection of hCG (Fleming and approximately spermatozoa ~ ). Yanagimachi, Between 10 and 20 min after when each 1980). were released into PBS 1 insemination, oocyte Oocytes containing mg had hyaluronidase ml After being rinsed with BSA-free M2, a few spermatozoa bound to the oolemma, the oocytes . were washed in M2 and then cultured in M16. oocytes were treated for 1 min with 1 mg trypsin ml~" in gently BSA-free M2 to remove the zonae pellucidae. Zona-free oocytes were washed in M2 containing 4 mg BSA mP1 and Preparation of mouse spermatozoa and fertilization in vitro. cultured in 50 µ drops of Mió under mineral oil (37.5°C under Spermatozoa from B6D2 Fl males were suspended in M16 5% C02 in air). containing 32 mg BSA ml" at a concentration of approxi¬

2x10 ml . were incubated mately spermatozoa ~ They at 37.5°C under 5% C02 in air for 2 h to allow capacitation and Preparation of spermatozoa acrosome reaction. The incidence of acrosome reaction in Preparation of hamster spermatozoa and fertilization in vitro. similar conditions was reported to be 20—30% (Barg et al, 1986). Insemination was carried on at 37.5°C A small drop (about 30 µ ) of dense spermatozoa mass was by adding 5-10 µ of to 100 M16 collected from the cauda epididymis of a mature male hamster; preincubated sperm suspension µ zona-free hamster concentration of it was put at the bottom of a short glass test tube (15 mm containing oocytes (final 50 mm) and covered with 2 ml of prewarmed (37.5°C) spermatozoa was approximately 1—2 x 10 spermatozoa ml each a ~ After 3-10 min, m-TALP-8 medium. After the tube was allowed to stand for ). when oocyte had few bound the were washed in M2 medium 2—5 min at 37.5°C, the upper 1 ml of medium was collected. spermatozoa, oocytes gently The medium thus obtained contained spermatozoa, 95-100% and subsequently cultured in M16. In the calcium measurement from of which were actively motile. The concentration of spermato¬ experiments, spermatozoa B6D2 Fl CF-1 were zoa was to 3 and males used, and were prepared adjusted approximately 106 spermatozoa ml ~ \ to the method described above. Aliquots (200 µ ) of the sperm suspension were covered with according mineral oil and incubated at 37.5°C under 5% C02 in air. After incubation for 4-5 h, spermatozoa were examined for the Preparation of human spermatozoa and fertilization in vitro. incidence of spontaneous acrosome reaction (Yanagimachi, Human semen samples were obtained from healthy donors and 1969). Sperm suspension was used for insemination when 50% centrifuged at 650 g for 5 min to sediment the spermatozoa. or more of the spermatozoa in the entire population were Aliquots (0.5—1.0 ml) of concentrated semen were placed under acrosome reacted. Zona-free hamster which had 2 ml HCM medium 26 BSA in a oocytes, containing mg ml ~ :, short been placed previously in a 100 µ m-TALP-8 droplet under glass test tube (15 mm 50 mm). The tube was placed at mineral oil, were inseminated by adding about 20 µ of sperm an angle of 45° and incubated at 37.5°C under 5% C02 in air. Downloaded from Bioscientifica.com at 09/27/2021 05:06:41PM via free access One hour later, the upper 1 ml of the medium was retrieved. manipulations, except for treatment with PEG, were performed The majority (90—95%) of spermatozoa in this medium was in agar-coated embryological watch-glasses at room tempera¬ actively motile. The sperm suspension was centrifuged at 650 g ture. Hamster oocytes and mouse two-cell blastomeres were for 5 min. The of was in HCM washed in BSA-free M2, and treated pellet spermatozoa resuspended thoroughly subsequently 1 was 10 a few with ml in such that the sperm concentration approximately for minutes phytohaemagglutinin (150 µg ~ spermatozoa ml-1. Aliquots (200 µ ) were placed in a Petri BSA-free M2). A single oocyte and a single blastomere were dish, covered with mineral oil and incubated at 37.5°C under aggregated until they firmly adhered to each other. Oocyte— 5% C02 in air. After incubation for 18—24 h, 2 µ of proges¬ blastomere pairs thus obtained were immersed in freshly ml in 10% 50% PEG in BSA-free M2 for 45-60 s, washed terone stock solution (100 µg ~ (v/v) dimethyl prepared (w/v) sulfoxide in 0.9% (w/v) NaCl) was added to a 200 µ drop of in M2 and cultured in M16. Fusion was usually completed by sperm suspension to induce acrosome reaction (Wistrom and 20-30 min after PEG treatment. Meizel, 1993). The incidence of acrosome reaction under these conditions was reported to be approximately 30% (Wistrom and Meizel, 1993). Ten minutes later, zona-free hamster Calcium measurements oocytes were introduced into the sperm suspension containing Zona-free hamster oocytes were loaded with fura-2 by Between and 30 min acrosome-reacted spermatozoa. 10 after incubation fura-2 AM 1 in M2 at 37°C 30 min in 33 µ ~ for insemination, when many were bound to each spermatozoa as described by Kline and Kline (1992b, 1994). Loaded oocytes oocyte, the oocytes were washed in M2, and then gently were transferred to 100 µ of BSA-free m-TALP-8 cultured in Ml6. droplets under mineral oil in a Petri dish and subsequently inseminated. A Deltascan system (Photon Technology International, South Brunswick, NJ) was used for fluorescence as cortical material on the recordings Staining of exocytosed granule surface of described Kline and Kline 1994). The fluorescence the oolemma by (1992b, signal was displayed as the ratio of fluorescence for the Oocytes were labelled with lectins according to the method 350 nm:385 nm excitation wavelengths after background sub¬ of Lee et al (1988). Oocytes were fixed with 3% (v/v) formalin traction. Emitted fluorescence was recorded at each excitation second. in PBS-alb for 30 min at room temperature. After fixation, they wavelength and the ratio calculated twice per After the were rinsed with 1 of fluorescence were labelled PBS-alb containing 100 mmol glycine ~ for completion recordings, oocytes 10 min to remove aldehyde, washed with PBS-alb and then with Hoechst 33258 or air-dried and stained with Giemsa to incubated for 30 min in PBS-alb containing 10 µg Hoechst estimate the number of fused spermatozoa. * 33258 ml" (Sigma) to label chromosomes. The oocytes were rinsed twice in PBS-alb, and then transferred to 100 µ of examination solution of FITC-conjugated lectins. Two lectins were used in Cytological of oocytes and embryos this LPA for terminal sialic study: (Limulus polyphemus) specific Cytological details of oocytes and embryos were examined acid residues, and LCA (Lens culinaris) for terminal specific moderately compressing them between a slide and cover- of two lectins by a-D-mannose residues. FITC-conjugates these slip, fixing with glutaraldehyde, staining with orcein and were dissolved in PBS, each at a concentration of 2 ml mg with a (Perreault and before lectin stock examining phase-contrast microscope and stored at 20°C. Immediately use, at II were — Zirkin, 1982). Oocytes remaining metaphase solutions were thawed, mixed and diluted with PBS-alb such recorded as unactivated, and those completing meiosis, as well that the concentration of each FITC-lectin was 200 mP µg as those at were considered activated. Some . pronuclear stage, were incubated with lectins for 30 min, rinsed thor¬ Oocytes oocytes were fixed, and air-dried preparations (Tarkowski, with PBS-alb and transferred to PBS 1 oughly containing mg 1966) were made. Slides were stained with Giemsa, allowing ml 1 ml were PVA ~ ~ and mg paraphenylenediamine They heads within the to be differentiated . spermatozoa ooplasm and and an mounted between a slide coverslip examined using from those outside of the oocyte; the former stained dark blue epifluorescence UV microscope. whereas the latter remained unstained (Miller and Masui, 1982). Fusion of hamster oocytes with blastomeres of two-cell mouse embryos Results Hamster oocytes (inseminated and uninseminated) were obtained as described above. Two-cell mouse embryos were Oocyte activation collected from the oviducts of superovulated B6D2 Fl females approximately 24 h after vaginal plugs were found. Zonae Zona-free hamster oocytes inseminated with hamster, pellucidae were removed by brief incubation in acidic Tyrode's human, guinea-pig and mouse spermatozoa were examined solution (Nicolson et al, 1975). Blastomeres were separated in between 1.5 and 2 h after insemination. During normal fertiliz¬ ~ needle. hamster formation takes PBS containing 4 mg BSA ml , using a thin glass ation of zona-free oocytes, pronuclei Collected blastomeres were kept in M2 before use. Experimen¬ place 1—1.5 h after insemination (Barros and Yanagimachi, tal oocytes 1 h after insemination, as well as control oocytes 1972). The results (summarized in Table 1) demonstrate that that were not inseminated, were fused with blastomeres using hamster oocytes penetrated by either human or guinea-pig PEG, according to the method of Czolowska et al (1984). All spermatozoa resume meiosis almost as efficiently as do the Downloaded from Bioscientifica.com at 09/27/2021 05:06:41PM via free access Table 1. Activation of hamster oocytes by homologous and heterologous spermatozoa determined 1.5-2 h after insemination

Total number Number (%) of Mean number + SD Percentage of activated Source of Number of of oocytes oocytes of spermatozoa per oocytes among spermatozoa experiments inseminated penetrated penetrated oocyte penetrated3

Hamster 35 28 (80.0) 2.92 ± 1.54 Man 99 45 (49.5) 1.51 + 0.82 84 Guinea-pig 49 31 (63.3) 1.68 + 0.84 87 Mouse 112 101 (90.2) 3.07 + 2.14 7

"All of these oocytes were at the pronuclear stage.

In the experiments in which hamster oocytes were in¬ seminated with mouse spermatozoa and cultured for 4 h, about 70% of penetrated oocytes were activated and were in the pronuclear stage (Table 2). About 30% of penetrated oocytes did not complete meiotic division and remained at metaphase II. The mean number of spermatozoa within activated oocytes was significantly higher than that in unactivated oocytes. Spontaneous activation was observed in 47% of control oocytes after 4 h of culture without insemination. The nuclei of spontaneously activated oocytes were either in the process of completion of meiosis or in the early stages of pronuclear development. In some spontaneously activated eggs, the female pronucleus was extruded within a polar body-like cytoplasmic bleb.

Cortical granule exocytosis from hamster oocytes penetrated by hamster, human and mouse spermatozoa Fig. 1. A hamster oocyte fused with a mouse spermatozoon, 2 h after When hamster were inseminated with acrosome- insemination; the are at two different focal oocytes photomicrographs planes reacted hamster after fixation and staining, (a) the side view of the metaphase plate of spermatozoa, spermatozoa quickly bound to the oolemma. the second meiosis; (b) a decondensed spermatozoon head (arrow) Spermatozoa were initially actively motile, within the ooplasm in addition to one spermatozoon on the oocyte but in 10—30 s, they abruptly ceased their tail movements surface. Scale bar represents 10 µ . (Yanagimachi, 1978). This seems to be a result of fusion of their membrane with the oolemma (Longo and Yanagimachi, 1993). When the oocytes were fixed between 10 and 15 s after cessation of sperm tail movement and analysed for fluorescent oocytes penetrated by hamster spermatozoa, whereas those labelling of the oolemma, 12 of 17 oocytes showed strong penetrated by mouse spermatozoa seldom activate within surface binding of FITC-lectins, indicating that cortical granule 1.5-2 h. Most oocytes remained at metaphase II, regardless of exocytosis occurred very rapidly after sperm—oocyte fusion the presence or absence of mouse spermatozoa within the (Fig. 2a). ooplasm (Fig. 1). Mouse spermatozoa within the hamster The moment of fusion of human and mouse spermatozoa ooplasm were either swollen or recondensed (Wright and with hamster oocytes was not determined precisely, partly Longo, 1988) at this time (Fig. 1). because of the difficulty in distinguishing acrosome-reacted Among oocytes penetrated by mouse spermatozoa, pen¬ from acrosome-intact spermatozoa in vivo. However, cortical etrated and activated oocytes contained about twice as many granule exocytosis occurred in hamster oocytes penetrated by spermatozoa as did penetrated and unactivated oocytes (mean human spermatozoa. Oocytes were fixed 1 h after insemination numbers ± SD spermatozoa per penetrated oocyte: 6.14 ± 4.74 and examined for cortical granule exúdate. All eight hamster and 2.84 ± 1.64, respectively; significantly different, < 0.005, oocytes containing decondensing human spermatozoa heads Student's unpaired f test). Two activated oocytes were heavily displayed punctate, strongly fluorescent cortical granule penetrated (10 and 15 spermatozoa in each oocyte). All material on their surfaces (Fig. 2b). Cortical granule exocytosis monospermic oocytes were unactivated. Among 94 oocytes did not occur in the five oocytes that were not penetrated by that failed to activate despite sperm penetration, seven were human spermatozoa. heavily penetrated, including one by ten spermatozoa and six The situation was different in the hamster oocytes by six spermatozoa. inseminated with mouse spermatozoa. When examined 1 h Downloaded from Bioscientifica.com at 09/27/2021 05:06:41PM via free access Table 2. States of hamster oocytes 4 h after insemination with mouse spermatozoa

Mean number ± SD spermatozoa per oocyte Number (%) of Percentage of Number of Total number penetrated activated Activated Unactivated Oocytes experiments of oocytes oocytes oocytes oocytes oocytes

Inseminated 55 38 (69.1) 71.1" 2.81±1.33b 1.81 + 0.75' Uninseminated 58 46.6 (control)d

'Tercentage of activated oocytes among penetrated oocytes. 'Significantly different (P<0.05, Student's unpaired t test). Oocytes left in the medium for 4 h without insemination. after insemination, although 22 of 88 oocytes fused with transients was not observed; the first Ca transient was similar spermatozoa, 14 (64%) of the penetrated oocytes showed no in duration to the following transients (Fig. 3c). In most cases, cortical granule exocytosis (Fig 2c), and only 4 (18%) exhibited the oocytes with repetitive Ca2+ transients, as well as those distinct cortical granule exocytosis. that had none, were highly polyspermic at the end of the calcium measurement (Table 4). M-phase promoting factor (MPF) activity in hamster oocytes with mouse fused spermatozoa Discussion By about 20 min after PEG treatment, more than 90% of fused in the of the oocyte-blastomere pairs resulting exposure Zona-free hamster oocytes are unique in that spermatozoa of a nucleus Table 3 blastomere to the cytoplasm of the oocyte. wide variety of other species can fuse with them (Yanagimachi, summarizes the incidence of premature chromosome conden¬ 1988). Extrusion of the second polar body and formation of fused sation of blastomere nuclei with uninseminated and female and male pronuclei were observed when hamster In of unin¬ penetrated hamster oocytes. control pairs (fusion oocytes were penetrated by guinea-pig (Yanagimachi, 1972), seminated oocyte with isolated two-cell blastomere), premature human (Yanagimachi et al, 1976) and mouse spermatozoa the of condensation of blastomere nucleus occurred in 81% (Hanada and Chang, 1972). However, it was not known pairs. In experimental pairs (fusion of penetrated oocytes with whether oocyte activation following interspecific sperm two-cell blastomere), only 7% of the fused pairs caused penetration is always entirely normal. the blastomere premature chromosome condensation of In the present study, mouse spermatozoa penetrated hamster the 93% in nucleus. In the ooplasm of remaining of fused pairs oocytes very efficiently, but could not activate the oocytes this group, the interphase blastomere nuclei were observed at least during the first 1.5-2 h. Most oocytes remained at 1.5 h after PEG treatment. metaphase II and cortical granules remained intact despite decreased MPF activity in the ooplasm. We observed that fail to induce intracellular Ca2+ Intracellular calcium ion concentration in hamster oocytes mouse spermatozoa repetitive ([Ca2+])¡ transients in hamster oocytes, possibly explaining their failure inseminated with mouse spermatozoa to activate hamster oocytes. Both human and guinea-pig In control hamster oocytes inseminated with hamster sper¬ spermatozoa could activate hamster oocytes as efficiently as matozoa, repetitive Ca2+ transients began within a few minutes hamster spermatozoa. after insemination (Fig. 3a). The initial Ca response consisted An aberrant reaction of hamster oocytes to the fusion with was described of two or three transients occurring at short intervals and a mouse spermatozoa by Igusa et al (1983), high basal Ca2+ concentration (Fig. 3a). The initial Ca2+ rise was who found that hyperpolarization of the oocyte membrane followed by later Ca2+ transients at constant intervals of potential, which occurs upon sperm—oocyte fusion, is delayed that more than 3—4 min, as reported by Miyazaki et al. (1993). by an average of 15 min. They found 10 Table 4 summarizes the changes of [Ca ]¡ in hamster mouse spermatozoa must penetrate a single hamster oocyte oocytes inseminated with mouse spermatozoa. No Ca2+ to induce activation in most of the oocytes. A similar transients were observed when hamster oocytes were insemi¬ observation was made by Fukuda and Chang (1978), who nated with diluted sperm suspension (the final concentration found that 90% of hamster oocytes were penetrated by more ~ was approximately 105 spermatozoa ml ; Fig. 3c). Only the than four mouse spermatozoa, but only 45% were activated. oocytes inseminated with concentrated sperm suspension In the present study, we tried to avoid heavy polyspermy. (approximately 1—2 IO6) showed repetitive Ca2+ transients Ninety-three per cent of hamster oocytes remained un¬ (Fig. 3b). The mean time of the initiation of the oscillatory Ca activated despite penetration by mouse spermatozoa. transients was approximately 17 min after insemination. The Highly polyspermic oocytes were more likely to become h after mouse typical initial response of a Ca rise with several rapid activated during the first 2 insemination with Downloaded from Bioscientifica.com at 09/27/2021 05:06:41PM via free access Table 3. Incidence of premature chromosome condensation (PCC) of the nucleus of a two-cell mouse embryo blastomere after polyethylene glycol (PEG)-mediated fusion with uninseminated or previously inseminated hamster oocytes

Number (%) Number of of oocytes Hamster oocytes fused pairs with PCC

Uninseminated8 16 13 (81.3) (control) Penetrated with 29 2 (6.9) mouse spermatozoa

"Fresh, uninseminated hamster oocytes were each fused to a single blastomere isolated from a two-cell mouse embryo and were examined 1-2 h after PEG treatment. Hamster oocytes were inseminated with mouse spermatozoa. One hour later they were each fused with a single blastomere isolated from a two-cell mouse embryo and they were examined 1.5 h after PEG treatment.

Under the same conditions, 71% of oocytes penetrated by mouse spermatozoa were activated (Table 2), indicating that the higher proportion of penetrated, activated oocytes seen at 4 h represents the additive effect of a small incidence of sperm induced activation plus the higher spontaneous activation incidence related to oocyte ageing. Mouse spermatozoa could reduce the concentration of MPF in hamster oocytes signifi¬ cantly. Probably shortly after penetration, the activity of MPF remains sufficiently high to prevent fast oocyte activation, but after several hours in culture, a reduction of MPF activity leads to the delayed release from the metaphase block. The present study revealed that mouse spermatozoa pen¬ etrating hamster oocytes commonly fail to induce repetitive intracellular Ca2+ transients. Even in some heavily polyspermic oocytes, calcium transients were not detected. A change in intracellular Ca was produced in some hamster oocytes inseminated with mouse spermatozoa, but only after insemi¬ nation with a concentrated sperm suspension, when many spermatozoa penetrated an oocyte nearly simultaneously. This observation is in agreement with that of Igusa et al (1983) on differences in hyperpolarizing responses during penetration of hamster oocytes by hamster and mouse spermatozoa. These responses reflect [Ca ]¡ changes and, in the present study, we that Fig. 2. fluorescein isothiocyanate (FITQ- demonstrate directly spermatozoa of different species have Photomicrographs showing hamster lectin (LCA and LPA) labelling on hamster oocytes inseminated with different effects on intracellular calcium in penetrated (a) hamster, (b) human, and (c) mouse spermatozoa. Oocytes were oocytes. fixed (a) 10—15 s, and (b, c) 1 h after insemination. The oocytes shown It is not known how activation of eggs is initiated during in (a) and (c) were compressed extensively under a coverslip before normal fertilization of the egg with homologous spermatozoa. a Scale being photographed. Arrows indicate fertilizing spermatozoon. Two major hypotheses have been proposed, one suggesting bar 10 represents µ . that the spermatozoon introduces an activating substance into the oocyte cytoplasm after fusion with the oolemma, and the other that the spermatozoon interacts with a transmembrane spermatozoa and none of 20 monospermic hamster oocytes receptor that produces second messengers in the oocyte. If the was activated. oocyte is activated by a cytosolic factor contributed by the Hamster oocytes commonly activate spontaneously during fertilizing spermatozoon (Swann, 1990), efficient activation of ageing in vivo (Austin, 1956; Yanagimachi and Chang, 1961; hamster oocytes by hamster, guinea-pig and human spermato¬ Longo, 1974) and in vitro (Igusa et al, 1983). Under the zoa may be explained by assuming that the oocyte-activating in vitro conditions used in the present study, 47% of hamster factors in spermatozoa are similar in nature and are almost oocytes had activated spontaneously after incubation for 4 h. equally potent. While the factor within a single mouse Downloaded from Bioscientifica.com at 09/27/2021 05:06:41PM via free access r (a) (b)

1.5

1.0 I

CO O 0.5 E c in 00 -I co uu Jw· 0.1

s ° o u_ co 10 20 0 10 20 o Time (min) s c (c) o I 4 h" CM çb 3 LL 1.5 3

-

1.0 §3,

te O 0.5

1 - I 0.1

0 20 40 60 Time (min) Fig. 3. Intracellular calcium changes in hamster oocytes: (a) after insemination with hamster spermatozoa; (b) after insemination with a concentrated (approximately 1—2 IO6 ml ') mouse spermatozoa ~ sperm suspension; and (c) after insemination with diluted 105 ml ') mouse (approximately spermatozoa ~ sperm suspension. Spermatozoa were added at the time indicated by the arrow. The left ordinates indicate the fura-2 fluorescence ratio and the right ordinates are the estimated [Ca2*^ obtained from the external calibration buffers (Kline and Kline, 1994). The oocyte shown in (c) contained 13 decondensed spermatozoa at the end of the record. The slow rise in [Ca2+]¡ was seen in some, but not all, similarly polyspermic oocytes that did not produce repetitive Ca2+ transients.

spermatozoon can activate an oocyte of the same species, its hamster egg and initiate calcium release and egg activation. In inability (or inefficiency) to induce activation of hamster contrast, mouse spermatozoa might not interact with the oocytes could be because it is present at a lower concentration receptor but, nevertheless, may fuse with the oocyte. The in than hamster, guinea-pig and human spermatozoa. Alterna¬ occurrence of activation and calcium oscillations in a few the tively, putative sperm-derived oocyte-activating factor oocytes may be produced by limited receptor interaction or is might exhibit some degree of species specificity. due to some perturbations of normal calcium homeostasis in Activation of oocytes may instead depend on a receptor- highly polyspermic eggs. mediated and signal transduction system (Foltz Shilling, 1993). Further cross-species fertilization experiments may provide If this that occurs, it is possible guinea-pig and human additional insight into the mechanisms of egg activation. These spermatozoa can interact with a surface receptor on the experiments demonstrate that sperm—oocyte membrane fusion Downloaded from Bioscientifica.com at 09/27/2021 05:06:41PM via free access Table 4. Summary of Ca transients caused by insemination of ten fura-2 loaded hamster oocytes with mouse spermatozoa

Final concentration Number of fused of spermatozoa Ca2+ transient Detection of spermatozoa3 + in insemination (time after fusion by - medium ( IO6 ml ') insemination, min.) (Giemsa/Hoechst) D G

0.2 G 13 25 0.2 - G 0 6 0.2 - H 5-10

- 0.4 G 13 —30 1.0 - G 11 1

- 1.0 - H I 2.0 + (15) G 11 —35 2.0 + (17) G 3 25 2.0 + (8) H > 10 2.0 + (23) G 1 —35

aD: decondensed sperm heads; G: Giemsa positive, but not decondensed sperm heads.

per se is not sufficient to stimulate the release of intracellular Kline JT and Kline D (1992b) Thapsigargin activates a calcium influx pathway calcium and to trigger oocyte activation. in the unfertilized mouse eggs and suppresses repetitive calcium transients in the fertilized egg Journal of Biological Chemistry 267 17624-17630 Kline JT and Kline D (1994) Regulation of intracellular calcium in the mouse This was from the National study supported by grants Institute egg: evidence for inositol triphosphate-induced calcium release, but not of Health HD-03402 (R. Yanagimachi) and HD-31683 (D. Kline). calcium-induced calcium release Biology of Reproduction 50 193—203 is a a M. 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