Assay for Penetrability of Frozen-thawed Stallion Spermatozoa into Zona-free Hamster Oocytes.

Abstract

Abstract. In Japan, low availability of equine oocytes makes it very difficult to improve the in vitro fertilization (IVF) system of horses. In this situation, an assay for sperm penetrability into zona-free hamster oocytes could be useful as a simulator of the IVF system. The aim of this study was to clarify the optimum conditions of the pretreatment of frozen-thawed stallion spermatozoa with caffeine and ionophore A23187 (IA) in the penetration assay. Frozen-thawed stallion spermatozoa were washed and then pretreated with IA in modified BO solution (mBO) supplemented with caffeine. After the pretreatment, the spermatozoa were used for the penetration assay with zona-free hamster oocytes. At first, effect of increasing concentrations of caffeine (0-10 mM) were examined on the penetration of spermatozoa pretreated with 1 µM IA for 60 sec. The highest penetration rate after 10 h co-culture with oocytes was obtained for spermatozoa penetrated in the presence of 1 mM caffeine (49.0%). Next, when spermatozoa were pretreated with 0-1.0 µM IA for 0-180 sec in the presence of 1 mM caffeine, significantly higher penetration rates after 10 h co-culture with oocytes were obtained under the condition of 0.1 µM IA for 120 sec (52.2%) and 1.0 µM IA for 60 sec (40.9%). When oocytes were fixed 2, 4, 6, 8 and 10 h after IVF using spermatozoa treated with 0.1 µM IA for 120 sec in mBO containing 1 mM caffeine, the penetration rates increased significantly from 2 h (10.4%) to 4 h (21.7%) and from 6 h (27.5%) to 8 h (49.0%). These results indicate that pretreatment with 0.1 µM IA for 120 sec in the presence of 1 mM caffeine effectively promotes the penetration of frozen-thawed stallion spermatozoa into zona-free hamster oocytes, and that under this condition, spermatozoa might complete the acrosome reaction during 6 to 8 h of incubation after IA treatment.

New Insights into the Mechanisms of Fertilization: Comparison of the Fertilization Steps, Composition, and Structure of the Zona Pellucida Between Horses and Pigs1

“…Similar results were obtained with equine spermatozoa and bovine or hamster zona-free oocytes, with bovine spermatozoa and equine or porcine zona-free oocytes, and with rat spermatozoa and porcine zona-free oocytes [62][63][64][65]. Sartini and Berger [16] suggest that ligand-receptor conservation mediates the spermatozoa-oolemma binding between porcine and bovine species.…”

Effect of human tubal fluid medium and hyperactivation inducers on stallion sperm capacitation and hyperactivation

“…Caffeine has been shown to induce hyperactivation in bovine sperm and to increase tyrosine phosphorylation and acrosomal exocytosis after induction with lysophosphatidylcholine (LPC) treatment (Marquez & Suarez, ). In stallion sperm, caffeine has been used as a sperm capacitation treatment in IVF studies with 11% (Del Campo, Donoso, Parrish, & Ginther, ) and 4.3% (Morato, Soares, Orero, Mogas, & Miro, ) of fertilized oocytes being reported and up to 49% in hamster zona pellucida‐free oocytes (Matsukawa, Takahashi, & Hanada, ). Caffeine also increased tyrosine phosphorylation levels and acrosomal exocytosis induced by LPC (Hochi, Korosue, Choi, & Oguri, ; Pommer, Rutllant, & Meyers, ).…”

In Vitro Development of Equine Oocytes from Preserved Ovaries after Intracytoplasmic Sperm Injection

“…Sperm pretreatment improves the pronuclear formation rate for ICSI in mice and cattle [40,41]. We previously demonstrated that pretreatment of stallion sperm with ionophore A23187 and caffeine is effective for increasing penetration of zona-free hamster oocytes [21] and the pronuclear formation rate after ICSI [22]. During fertilization, a sperm cytoplastic factor is r eleased an d calcium oscillations are generated in mammalian oocytes [42].…”

“…The spermatozoa were then exposed to i o n o p h o r e A 2 3 1 8 7 ( 0 . 1 µ M ; C a l b i o c h e mNovabiochem, La Jolla, CA, USA) for 2 min and resuspended in Hepes-buffered Hanks' medium s u p p l e m e n t e d w i t h 3 m g / m l B S A ( i n i t i a l fractionation by cold alcohol precipitation; Sigma) and 1 mM caffeine (Sigma) for 4-6 h at 37 C [21,22]. Five to ten oocytes with a first polar body were loaded into 1.5 ml microcentrifuge tubes containing 500 µl of M2 medium supplemented with 3 mg/ml BSA and centrifuged at 12,000 g for 3 min to facilitate sperm injection.…”