Zona-binding Ability Research Articles

Binding of caput epididymal mouse sperm to the zona pellucida

Immature sperm from the caput epididymis are immotile and infertile. It is thought that caput epididymal sperm are infertile due to their immotility, as well as to an inability to bind to the zona pellucida, suggesting the absence of a functional receptor for the zona. However, the sperm receptor for the zona pellucida has been identified previously as the enzyme galactosyltransferase (GalTase) ( L. C. Lopez et al. (1985 ) J. Cell Biol. 101, 1501–1510) and is present on the surface of caput as well as cauda epididymal sperm (N. F. Scully et al., (1987) Dev. Biol. 124, 111–124.). In this paper we examine this apparent conflict and show that immotile caput epididymal sperm are able to bind to the zona pellucida if they are first washed free of caput epididymal secretions, which contain factors that inhibit sperm-zona binding. Consistent with this finding are results that show that caput epididymal fluid is capable of inhibiting the binding of mature, cauda epididymal sperm to the zona pellucida. Caput epididymal fluid contains, among many other components, a soluble GalTase and an α-lactalbumin-like protein, both of which are capable of inhibiting mouse sperm-zona binding. Thus, caput epididymal sperm have the appropriate receptor, i.e., GalTase, for the zona pellucida, to which they can bind if removed from the inhibitory factors that mask their zona-binding ability.

Addition of androgens to cultured hamster epididymis increases zona recognition by immature spermatozoa.

The ability of spermatozoa recovered from the successive segments of the hamster epididymis to bind to the zona pellucida was studied and a major increase was found as spermatozoa passed from the proximal to the distal portion of the corpus epididymidis (1.95 compared with 20 spermatozoa bound/egg). Tubules from the proximal epididymis were cultured in conditions which preserved the motility of the contained spermatozoa for 48-72 h. Addition of 2 microM-5 alpha-DHT to the culture medium for 17 h stimulated the incorporation of 3H-labelled amino acids into several protein bands whose mobility in polyacrylamide gel electrophoresis was coincident with those of glycoproteins EP1-EP6, previously identified as androgen-dependent in the hamster epididymis in vivo. Examination of the material extracted from washed spermatozoa with 0.5 M-NaCl revealed the presence of radioactive proteins on spermatozoa. The zona-binding ability of spermatozoa from androgen-treated cultured proximal corpus tubules was significantly increased (P less than 0.001) as was the no. of spermatozoa/egg (5.51) compared with the value for control cultures (0.87 spermatozoa/egg). We suggest that androgen-dependent epididymal secretory proteins that associate with spermatozoa might participate in the formation or activation of a site for zona pellucida recognition in the sperm surface.

Hormonal regulation of zona‐binding ability and fertilizing ability of rat epididymal spermatozoa

AbstractRat spermatozoa from the proximal caput, the proximal corpus, the middle corpus, and the distal cauda epididymidis were examined for their ability to bind to the zona pellucida after a 1‐, 2.5‐, or 4.5‐h incubation at 34°C with rat eggs in cumulus. Caput spermatozoa did not bind to the zona after 1, 2.5, or 4.5 h of incubation. Corpus spermatozoa did bind to the zona, but the percentage of eggs with bound spermatozoa and number of bound spermatozoa per egg increased with the length of incubation. Cauda spermatozoa bound readily to the zona pellucida, and their zona binding ability did not change with longer incubations. It thus appears that rat spermatozoa gradually acquire the ability to bind to the zona pellucida in the corpus epididymidis.The zona‐binding capacity of cold immobilized cauda spermatozoa, defined as the percentage of eggs with bound spermatozoa, increased with the number of spermatozoa incubated and reached a plateau characteristic of the endocrine status of the animal. After castration, zona‐binding ability is progressively lost from day 3 until day 10 where it is nil. Testosterone supplementation maintains zona‐binding ability to control levels. Similarly, fertilizing ability declines from day 5 after castration until day 10. Testosterone prevents this loss of fertilizing ability. It thus appears that the development of zona‐binding ability during epididymal transit is, like the development of fertilizing ability, under androgen regulation.The close correlation between the onset of fertilizing ability and zona‐binding ability during maturation, the loss of fertilizing ability and zona‐binding ability after castration, and the recovery of both fertilizing ability and zona‐binding ability with testosterone treatment suggests that the androgen‐dependent development of zona‐binding ability is an important component of the acquisition of sperm fertilizing ability during epididymal transit.

Development of the ability to bind to zonae pellucidae during epididymal maturation: reversible immobilization of mouse-spermatozoa by lanthanum.

Mouse spermatozoa recovered from the caput, corpus, or cauda epididymidis were examined for their ability to bind the vitro to zonae pellucidae. Since spermatozoa from the caput epididymidis do not display progressive motility as compared with more mature spermatozoa, direct comparison of the different sperm populations may not measure zona binding ability validly. To equalize the motile properties of the spermatozoa, a method was developed to immobilize vigorously motile corpus and cauda spermatozoa. Reversible immobilization was achieved by incubation in 25 microM La3+ which resulted in a twitching, nonprogressive type of motility. La3+ incubation did not appear to affect the spermatozoa adversely, since vigorous motility (equivalent to the controls) of corpus and cauda sperm was displayed upon subsequent incubation in standard La3+-free culture medium. Moreover, cauda spermatozoa preincubated for 90 min in La3+ displayed levels of fertilization in vitro equivalent to their control counterparts. Using this La3+-immobilization technique, the zona binding ability of the different sperm population could be assayed. Gamete collision was insured under these conditions by shaking the gamete-containing dishes at 100 cycles/min. Regardless of the extent of sperm motility, a similar zona-binding pattern emerged: cauda sperm bound in high numbers, corpus sperm bound at some intermediate level (an average of 24% of cauda binding level), and caput sperm bound rarely (2% of cauda binding level). Thus it appears that, for mouse spermatozoa, the onset of fertility during epididymal transit parallels the ability to bind to zonae pellucidae. Unlike the interaction between spermatozoa and zonae, La3+ was unable to support sperm binding in to egg plasma membrane, supporting the view that mouse sperm may have different sites for interaction with the zonae pellucida and the egg plasma membrane.

Separation of calcium effects on motility and zona binding ability in mouse spermatozoa.

AbstractThe ionic specificity of Ca+2 requirements for binding to the zona pellucida and maintenance of motility were studied in mouse gametes by comparing the effects of four cations: Ca+2, Sr+2, La+3, and spermine cation (Sp+4). Cumulus‐free mouse eggs were inseminated in vitro with sperm which had been preincubated for 60 min in Tris‐NaCl buffer containing one or more of the above cations. Motility was assessed microscopically and zona binding, by the stop‐fix technique, utilizing centrifugation through a discontinuous density gradient. The sperm were motile upon release from the epididymis, and all sperm maintained their motility in complete culture medium. Sperm incubated in Tris‐NaCl buffer differed in motility among samples as follows: The majority of sperm samples remained motile in Tris‐NaCl containing 1.7 mM Ca+2, but lost motility if Ca+2 was absent. The remaining samples could be divided into two populations: those which maintained motility in the absence of Ca+2. Support of Zona binding required Ca+2 in all samples examined. Equimolar Sr+2 could substitute for Ca+2 in maintaining motility but not in supporting binding. Sp+4and La+3 did not support motility. If Sr+2 were present to maintain motility, both Sp+4 and La+3 supported binding as well as Ca+2. Sr+2 did not complete with any of the other cations for support of zona binding. Ca+2 and Sp+4 together in the medium produced a synergism in the number of sperm bound. La+3 would substitute for Ca+2 in this synergism. These results demonstrate for the first time that mouse sperm have Ca+2 requirement for the motility maintenance and that binding to the zona and maintenance of motility are two different Ca+2‐dependent processes whose active sites can be distinguished by cation substitution.