Heterologous Spermatozoa M

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.

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