Patterns of Intracellular Calcium Oscillations in Horse Oocytes Fertilized by Intracytoplasmic Sperm Injection

In all species studied, fertilization induces intracellular Ca ([Ca]i) oscillations required for oocyte activation and embryonic development. This species-specific pattern has not been studied in the equine, partly due to the difficulties linked to in vitro fertilization in this species. Therefore, the objective of this study was to use intracytoplasmic sperm injection (ICSI) to investigate fertilization induced [Ca]i signaling and, possibly, ascertain problems linked to the success of this technology in the horse. In vivo and in vitro-matured mare oocytes were injected with a single motile stallion sperm. Few oocytes displayed [Ca]i responses regardless of oocyte source and we hypothesized that this may result from insufficient release of the sperm-borne active molecule (sperm factor) into the oocyte. However, permeabilization of sperm membranes with Triton-X or by sonication did not alleviate the deficient [Ca]i responses in mare oocytes. Thus we hypothesized that a step downstream of release, possibly required for sperm factor function, is not appropriately accomplished in horse oocytes. To test this, ICSI-fertilized horse oocytes were fused to unfertilized mouse oocytes, which are known to respond with [Ca]i oscillations to injection of stallion sperm, and [Ca]i monitoring was performed. Such pairs consistently displayed [Ca]i responses demonstrating that the sperm factor is appropriately released into the ooplasm of horse oocytes, but that these are unable to activate and/or provide the appropriate substrate that is required for the sperm factor delivered by ICSI to initiate oscillations. These findings may have implications to improve the success of ICSI in the equine and other livestock species. INTRODUCTION In oocytes from all mammalian species studied to date fertilization induces a series of species-specific intracellular Ca ([Ca]i) oscillations [1-3]. These transients are required not only to rescue the oocyte from meiotic arrest, but also for pronuclear formation, the initiation of DNA synthesis and normal progression of embryonic cleavage [2, 4]. Furthermore, recent research suggests that the frequency and duration of [Ca]i transients may impact subsequent embryonic development to preand post-implantation stages [5]. The exact mechanism(s) by which the sperm initiates [Ca]i oscillations in oocytes is not completely understood, but it involves production of inositol 1,4,5trisphosphate (IP3), which binds to its receptor in the endoplasmic reticulum, the major intracellular Ca store, thereby allowing Ca efflux into the cytosol [4]. It has been postulated that IP3 production may be initiated through G-proteins or tyrosine kinase signaling pathways linked to receptors present in the oolemma [6-9]. Nonetheless, other studies show that sperm-egg fusion precedes the initiation of [Ca]i transients, with a brief lag of time before the first [Ca]i spike is detected [10-11], which is consistent with the timing required for diffusion of a sperm-supplied molecule, therefore providing evidence for a sperm factor/fusion theory as responsible for the initiation of oscillations in mammals. Further support for a sperm factor delivered at the time of gamete fusion comes from findings demonstrating that injection of sperm extracts into oocytes of laboratory and livestock species or fertilization by intracytoplasmic sperm injection (ICSI) of human and mouse oocytes can induce fertilization-like [Ca]i responses [12-15]. Additionally, injection of sperm extracts into frog oocytes was shown to stimulate IP3 production [16], as reported for fertilization in this species [17]. In agreement with the sperm factor hypothesis, a sperm-specific phospholipase C enzyme (PLCζ) has been recently identified and proposed to be the sperm molecule responsible for IP3 production and initiation of [Ca]i oscillations during mammalian fertilization [18]. Nevertheless, the possibility that a membrane-linked signaling pathway initiated by the sperm at the time of gamete fusion, either acting upstream of or concomitantly with PLCζ, or that an altogether different mechanism(s) may be involved in oocyte activation in different mammalian species cannot be excluded with the information available so far. The pattern of fertilization-induced [Ca]i transients has not been studied in the equine, partly due to the low rates of sperm penetration that are achieved with in vitro fertilization (IVF) in this species [19-21]. Furthermore, ICSI has so far yielded inconsistent results in the horse, with variable oocyte activation and generally low embryonic development rates [19,20,22]. We have previously shown that equine oocytes display [Ca]i transients and become activated when injected with stallion sperm extracts (eSF) [23]. Interestingly, in the same study injection of fresh sperm into horse oocytes (ICSI) did not consistently initiate [Ca]i responses. We hypothesized that such failure may be due to inability of injected horse oocytes to promote release of the sperm-borne factor or, alternatively, to a deficient step in the activation of such factor. Therefore, our objective herein was to further investigate which of these possibilities may explain the failure for consistent initiation of [Ca]i oscillations following ICSI in horse oocytes. MATERIALS AND METHODS Animal Care and Welfare Experiments performed herein where live animals were used followed NRC animal care and welfare guidelines and were approved by the IACUC committee at the University of Massachusetts. Retrieval and Culture of Horse Oocytes Equine oocyte-cumulus complexes (OCCs) for in vitro maturation were obtained by scraping the follicular walls of abattoir-collected ovaries, at the equine reproduction laboratory in Texas A&M University (College Station, TX). Oocytes having expanded cumuli (meiotically competent oocytes) [24] were placed into 1 ml of equilibrated maturation medium (TCM 199 with Earle’s salts; Gibco Life Technologies, Inc., Grand Island, NY) supplemented with 5 μU/ml FSH (Sioux Biochemical Inc.; Sioux Center, IA), 10% fetal bovine serum, and 25 μg/ml gentamycin. The vial containing the oocytes was sealed and packaged into a commercial incubator (Minitube of America, Inc.; Verona, WI) for overnight shipment at 38C. Once in our laboratory, oocytes were placed in microdroplets of the same medium under light mineral oil in an incubator at 38C in 5% CO2 for a total incubation time of 40-42 h. Prior to experiments, oocytes were denuded from their cumulus cells by repeated pipetting in a hyaluronidase solution (0.1% in Dulbecco’s Phosphate Buffered Saline or DPBS; Sigma Chemical Co., St Louis, MO). In vivo matured horse oocytes were collected from preovulatory follicles in cycling mares 28-32 h post hCG injection, as previously described [25]. Briefly, mares were appropriately restrained in stocks and sedated with Detomedine hydrochloride (5 mg iv; Pfizer, Lees Summit, MO) and Butorphanol tartrate (5 mg iv; Fort Dodge Animal Health Co., Fort Dodge, IA). A 10 x 10 cm site was surgically prepared in the flank ipsilateral to the follicle to be aspirated, and a sterile trocar inserted after blocking the skin with Lidocaine (2 ml; The Butler Co., Columbus, OH). Then, a 14G disposable needle was inserted through the trocar and guided into the preovulatory follicle by holding the corresponding ovary against the abdominal wall with the gloved arm inserted per rectum. An assistant aspirated follicular contents with a 50-ml Air-Tite syringe attached to the aspiration needle through an intravenous extension set, until the follicular wall completely collapsed. Collected OCCs were placed in droplets of medium (as above but containing no FSH) and incubated up to 45-46 h post-hCG prior to ICSI, at 38C in 5% CO2 in humidified air. Prior to experiments, oocytes were denuded from cumulus cells as above. Following ICSI experiments and [Ca]i monitoring, all horse oocytes were further incubated in culture medium (no FSH) at 38C in 5% CO2 in humidified air for an additional 20-24 h prior to activation status evaluation. Retrieval and Culture of Mouse Oocytes Metaphase II (MII) mouse oocytes were obtained from the oviducts of 6-16 weekold B6D2F1 euthanized female mice after injection of 5 IU equine chorionic gonadotropin (eCG; Sigma), followed 48 h later by injection of 5 IU human chorionic gonadotropin (hCG; Sigma). Oocytes were retrieved 12-14 h after hCG injection and cumulus cells were removed by incubation in bovine testis hyaluronidase (0.1% in DPBS, 3 min; Sigma). Oocytes showing no evidence of degeneration and that had extruded the first polar body were used for experiments. Preparation of Stallion Sperm for ICSI Experiments Stallion semen was collected with an artificial vagina and frozen over liquid nitrogen vapor in a milk-based extender containing egg yolk and glycerol in 2.5 ml straws following conventional sperm freezing methods [26]. Frozen semen was stored in a liquid nitrogen tank until use. Individual straws were thawed in a water bath at 50C for 40 sec, as recommended for the particular freezing method. ICSI was performed with three different sperm treatments: Frozen-thawed motile sperm (untreated); Triton-X (Sigma) treated sperm; or, sonicated sperm heads. In all instances sperm were washed twice in DPBS (400 g, 12 min) after thawing, and then injected immediately when motile sperm were used, or processed accordingly for the other treatments. Triton-X treatment, which resulted in permeabilization of the membranes, was accomplished by incubating sperm in 5 ml of DPBS containing 0.1% (vol/vol) Triton X for 15 min. Triton-X treated sperm were then washed twice in injection buffer (75 mM KCl, 20 mM Hepes, pH=7.0) prior to ICSI. For injection experiments using sonicated sperm heads, the separation of heads and tails was carried out by a brief sonication (3 min; 4.5 Hz) in injection buffer containing EDTA submerged in ice water. These samples were then washed in injection buffer (no EDTA) and individual sperm heads used immediately for injection. ICSI Procedure ICSI was performed as previously described [27, 28]. Briefly, the corresponding sperm suspension (see sperm preparation) was mixed with an equal volume of 12% polyvinylpyrrolidone (PVP; Sigma) and a 50 μl drop of this sample placed on a plastic Petri dish, which served as the microinjection chamber. Manipulations were carried out on an inverted Nikon Diaphot microscope. Injections were performed with a piezomicropipette-driving unit (Piezodrill; Burleigh Instruments Inc.; Fisher, NY). The sperm was aspirated into the pipette (tail first if intact sperm was used) and several piezo pulses were applied to immobilize/permeabilize the sperm membrane. Then, the tip of the injection pipette containing the sperm/sperm head was moved until it reached the zona pellucida and several piezo-pulses were applied to advance the pipette through the zona. Once in the perivitelline space, the pipette was advanced further against the oolemma until the inner limit of its cortex, while applying light negative pressure. Penetration of the oolemma was accomplished by one or two piezo pulses of lower intensity, and the sperm/sperm head was injected into the ooplasm surrounded by a minimum amount of fluid. Fluorescence Recordings and [Ca]i Determination [Ca]i monitoring was carried out with fura-2 dextran (Fura-2D; Molecular probes; Eugene, OR) loaded horse oocytes as previously described [23,29,30]. In brief, denuded horse oocytes were microinjected using a Nikon Diaphot microscope (Nikon Inc., Garden City, NY) and Narishige manipulators (Medical Systems Corp., Great Neck, NY). Injection pipettes containing 0.5 mM fura-2D were advanced into the ooplasm of each individual oocyte and an appropriate volume of the reagent injected by pneumatic pressure (PLI-100, picoinjector, Harvard Apparatus, Cambridge, MA). The injection volume ranged from 15-20 pl. Mouse oocytes used in these experiments were loaded with 1 μmol/l of fura-2 acetoxymethylester (fura-2 AM; Molecular Probes) supplemented with 0.02% pluronic acid (Molecular Probes) by incubation at room temperature for 20