Frozen-thawed Epididymal Spermatozoa Research Articles

<I>In vitro</I> Fertilization in Pig: Studies on the Ability of <I>in vitro</I> Matured and Fertilized Oocytes to Form Male Pronucleus

The present study was carried out to establish in vitro fertilization system with high rates of sperm penetration and male pronucleus formation in pig. The major results were summarized as follows. 1) Preservation of ejaculated spermatozoa for 24 h at 15 C promoted the ability of the spermatozoa to penetrate oocytes matured in vitro. 2) Frozen-thawed epididymal spermatozoa can penetrate oocytes matured in vivo and in vitro. Piglets were obtained from in vitro fertilization of in vivo matured oocytes with frozen-thawed epididymal spermatozoa. 3) Numbers of follicle cells surrounding oocytes and concentrations of progesterone in the maturation medium significantly affect meiotic progression of oocytes to the second metaphase and male pronuclear formation after in vitro fertilization. 4) When oocytes were cocultured with follicle shells in a non-static culture system, both rates of maturation and male pronuclear formation were high irrespective of the number of follicle cells surrounding the oocyte. 5) In vitro matured oocytes with high and low rates of male pronuclear formation showed the same protein synthesis as in vivo matured oocytes both before and after in vitro fertilization. After 6 h of insemination, the decrease in the 46K and 25k bands and the increase in the 22k band were observed. 6) Glutathione levels in oocytes were related to rates of male pronuclear formation after in vitro fertilization. Piglets were born from in vitro fertilization of oocytes matued in the medium containing cysteine with ejaculated spermatozoa.These findings indicate that the number of cells surrounding oocytes, the concentration of progesterone in the maturation medium, the maturation culture system and glutathione levels in oocytes are important for nomal maturation of oocytes resulting in high rates of male pronuclear formation after in vitro fertilization.

A monoclonal antibody to a zona pellucida-proteinase inhibitor binding component on murine spermatozoa

A monoclonal antibody, J-23, was produced to an epitope of a binding (acceptor) component on the plasma membrane in the acrosomal cap region of the mouse sperm head. The component binds a proteinase inhibitor of seminal vesicle origin at ejaculation and participates in the in vitro binding of capacitated sperm to the zona pellucida. The antibody, an IgM molecule, recognizes affinity purified acceptor, crude acceptor and whole sperm as determined by ELISA methodology. The antibody reacts with a 15,000 molecular weight component, the size previously determined for the acceptor, found in the supernatants of frozen-thawed cauda epididymal sperm. In addition, it binds to a 21,000 molecular weight component generated by mixing an excess of purified inhibitor (6400 daltons) with a crude acceptor preparation. J-23 binds to an epitope in the same region of the sperm head as does the inhibitor. This epitope becomes fully expressed during epididymal maturation and is found only in the lumen of epididymal tissues. Pretreating sperm with J-23 inhibits their ability to bind the inhibitor as well as the zona pellucida. Pretreating sperm with the inhibitor has little effect on the binding of J-23. These data indicate that J-23 recognizes an epitope on the acceptor but the epitope is not directly involved with inhibitor binding.