Mammalian sperm capacitation and fertilization in vitro: a critique of methodology

Several methods have been devised for the isolation and labeling of structural components of spermatozoa. Rodent spermatozoa were cleaved rapidly and specifically at the junction of the heads and tails by treatment with various proteases, and the separate components were isolated by density-gradient centrifugation. Treatment with reducing agents released the mitochondrial membranes from the midpiece, exposing the underlying tail structures. Mouse spermatozoa were found to contain about 10(7) sites per cell that bind concanavalin A; most of the sites appear to be on the head, for fluorescein-labeled conjugates of concanavalin A were bound mainly to the acrosomal region. Binding of concanavalin A resulted in rapid agglutination of spermatozoa; mixed agglutinates could be formed with somatic cells, as well as with spermatozoa of other species. Fluorescent probes (naphthalenesulfonic acids) bound to the sperm plasma-membrane and caused an immediate loss of motility. In contrast, ethidium bromide bound to the nuclear structures, but did not cause immediate immobilization. These isolation and probing procedures should facilitate detailed chemical analysis of the major components of mammalian spermatozoa.

https://www.pnas.org/content/pnas/68/10/2436.full.pdf

Molecular probes of spermatozoan structures.

Acrosome reactions occurring in vitro in hamster sperm capacitated by bovine follicular fluid were severely inhibited by four synthetic trypsin inhibitors and by Zn2 +. Three polypeptide trypsin inhibitors and a synthetic chymotrypsin inhibitor did not inhibit the acrosome reaction, and Ca2 + overcame the inhibition by Zn2. These results suggest that a trypsin-like enzyme (possibly acrosin) plays a role in the acrosome reaction.

Evidence for the role of a trypsin-like enzyme in the hamster sperm acrosome reaction†

Summary Mouse eggs were fertilized in vitro, in the presence and absence of cumulus cells and the zona pellucida, confirming that cumulus cells are not necessary for the fertilization of mouse eggs with capacitated spermatozoa The findings suggest that cumulus cells produce a substance capable of inducing capacitation After lysis of the zona pellucida, the fertilization rate with capacitated (uterine) spermatozoa was higher than, and with non-capacitated (epididymal) spermatozoa was at least as high as, the fertilization rate of control eggs with an intact zona Washing and resuspension in a chemically defined medium almost entirely prevented fertilization with epididymal spermatozoa when the zona was present, but had no effect after its removal Capacitation of mouse spermatozoa appears to be required for the penetration of the zona pellucida only, not for entry into the vitellus When the eggs and spermatozoa were incubated in the same chymotrypsin solution which had induced lysis of the zona, the percentage of eggs penetrated by more than one spermatozoon was increased

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The rôle of cumulus cells and the zona pellucida in fertilization of mouse eggs in vitro.

THE ovum cannot be penetrated by a spermatozoon until the latter undergoes a change termed capacitation1 which, in the golden hamster, can be produced in vitro by incubation in various,body fluids2–4 including serum5 and (β-glucuronidase6. The process involves vesiculation of the plasma membrane with the outer acrosomal membrane7, which in turn seems to release egg-penetrating enzymes and exposes a surface which can fuse with the vitelline membrane of the egg8. As part of a series of investigations on the nature of these surface changes we have studied the effect of proteolytic enzymes on the adherence of sperm to eggs in vitro. Our results show that adherence of sperm to the zona pellucida is species specific both before and after capacitation. Capacitation alters the sperm surface, however, so that it will no longer adhere to trypsin-treated eggs.

Alteration of Sites on the Mammalian Sperm Surface following Capacitation

CAPACITATION, or the ability for immediate penetration of the ovum, occurs when epididymal sperm of the golden hamster (Mesocricetus auratus) are incubated in the presence of follicular fluid1–3. Yanagimachi has reported that sperm motility is maintained by a dialysate of detoxified bovine follicular fluid, but capacitation of the sperm does not occur unless a water-soluble non-dialysable fraction is also provided4. The chemical nature of these factors has not yet been determined. Because ovarian follicles, particularly those in atresia, are known to contain lysosomal enzymes5, we investigated the effect of a lysosomal preparation from beef liver on hamster epididymal sperm. Finding a striking stimulation of motility and capacitation of the sperm, we went on to test the lysosomal enzyme, β-glucuronidase, which is widely distributed in mammalian tissue including cells of the ovarian follicle6. We found that this enzyme duplicated the effect of the lysosomal preparation, as we shall now describe.

C. F. Hutchison

Papers

Capacitation of hamster spermatozoa by beta-glucuronidase.