The redox status of human spermatozoa was found to have a profound influence on the fertilizing potential of these cells in association with qualitative and quantitative changes in the patterns of tyrosine phosphorylation. In general, oxidizing conditions enhanced tyrosine phosphorylation and stimulated sperm function, whereas reducing conditions had the opposite effect. Unstimulated human spermatozoa exhibited low levels of spontaneous acrosomal exocytosis and sperm-oocyte fusion and minimal reactive oxygen species generation, while phosphotyrosine expression was largely confined to a single protein of 116 kDa. However, if the spermatozoa were exposed to oxidizing conditions through the addition of exogenous H2O2, or the stimulation of endogenous NADPH-dependent reactive oxygen species generation, then a dramatic increase in tyrosine phosphorylation was observed (major phosphotyrosyl bands at 222 kDa, 200 kDa, 159 kDa, 133 kDa, 116 kDa and 82 kDa) in concert with the functional activation of the spermatozoa. A causal association between reactive oxygen species generation, tyrosine phosphorylation and sperm function was indicated by studies with the ionophore, A23187, which induced high rates of spermoocyte fusion together with enhanced rates of reactive oxygen species production and the increased expression of phosphotyrosyl proteins. This functional response to A23187 could be abrogated, without any concomitant change in sperm motility or viability, by using membrane permeant thiols or catalase to suppress the reactive oxygen species-induced increase in phosphotyrosine expression. The fact that the biological responses of human spermatozoa to biological agonists (recombinant human ZP3 and progesterone) could also be inhibited by catalase indicated the general relevance of these findings.(ABSTRACT TRUNCATED AT 250 WORDS)

Redox regulation of tyrosine phosphorylation in human spermatozoa and its role in the control of human sperm function

https://escholarship.org/content/qt8f67899s/qt8f67899s.pdf?t=ovhxkb

N-Acylethanolamines in human reproductive fluids.

To achieve fertilization, sperm and egg are equipped with specific molecules which mediate the steps of gamete interaction. In mammals, the first interaction between sperm and egg occurs at an egg-specific extracellular matrix, the zona pellucida (zp). The three glycoproteins, ZP1, ZP2, and ZP3, that comprise the zp have been characterized from many species and assigned different roles in gamete interaction. A large number of candidate-binding partners for the zp proteins have been described; a subset of these have been characterized structurally and functionally. Galactosyltransferase, sp56, zona receptor kinase, and spermadhesins are thought to participate in the primary binding between sperm and zp and may initiate the exocytotic release of hydrolytic enzymes in the sperm head, the acrosome reaction. Digestion of the zp by these enzymes enables sperm to traverse the zp, at which time the proteins PH20, proacrosin, sp38, and Sp17 are thought to participate in secondary binding between the acrosome-reacted sperm and zp. Once through the zp, sperm and egg plasma membranes meet and fuse in a process reported to involve the egg integrin alpha 6 beta 1 and the sperm proteins DE and fertilin. These molecules and the processes involved in gamete interaction are reviewed in this chapter within a physiological context.

Molecules involved in mammalian sperm-egg interaction.

Fertilization is a key step in the process of reproduction. In mammals, this begins with the entry of sperm into the fertile state by the process of capacitation, which occurs under instruction from the female reproductive tract, and is required for sperm access to and interaction with the egg. The cellular events of sperm–egg interaction result in gamete fusion and the initiation of the program of development and are further described in this chapter.

Fertilization in Mammals

Sperm-zona pellucida binding and penetration were assessed on the oocytes that failed to fertilize from couples with >/=3 oocytes treated by standard in-vitro fertilization (IVF). There were four groups: fertilization rate 0% (n = 369), 1-25% (n = 194), 26-50% (n = 81) and 51-95% (n = 100). Of the couples with zero fertilization rate 70% had </=5 spermatozoa bound per zona pellucida and 42% had no spermatozoa penetrating the zona pellucida of any oocyte. In contrast, in the 51-95% fertilization rate group, only 17% had </= 5 spermatozoa bound per zona pellucida and 6% had no spermatozoa penetrating the zona pellucida. There was a significantly higher frequency of poor sperm morphology (</= 5% normal) in couples with zero fertilization rate (36%) than in the fertilization rate group 51-95% (7%). Incubation of oocytes from 68 couples with zero fertilization rate and low sperm-zonae pellucidae binding with fertile donor spermatozoa resulted in normal sperm-zona pellucida binding and most zonae pellucidae being penetrated. In conclusion, defective sperm-zona pellucida interaction was the major cause for low fertilization rates in standard IVF. This was usually because of defects of the spermatozoa rather than defects of the oocytes. Sperm defects likely to cause failure of fertilization should be diagnosed before commencing IVF and the patients directed to intracytoplasmic sperm injection.

Defective sperm–zona pellucida interaction: a major cause of failure of fertilization in clinical in-vitro fertilization

Studies on the role of specific molecules in the human fertilization process and additional assessments of potential applications for these proteins are hampered by the limited amount of available biological material. However, this drawback might be circumvented by the recent cloning of several gamete-specific genes, which opens possibilities for the production of recombinant proteins. By use of cDNA and genomic DNA fragments of the human ZP3 gene, which encodes a major constituent of the zona pellucida surrounding the oocyte, a 2.7-kb minigene was constructed containing the natural third and fourth introns of the gene and a truncated intron between exons 2 and 3. This ZP3 DNA was transfected to Chinese hamster ovary cells, and a single-cell clone producing the recombinant ZP3 protein (recZP3) was generated. Western blot analysis of culture medium from these cells showed that recZP3 has a molecular mass + 5 kDa smaller than that of natural ZP3. Under reducing conditions, it migrates at an apparent molecular mass of 55-60 kDa. RecZP3 induced the sperm acrosome reaction and promoted fusion of human spermatozoa with zona-free hamster oocytes, indicating that the recombinant protein is biologically active. RecZP3 provides an attractive tool for studying the initial stage of the human fertilization process. Furthermore, it might have clinical applications in the development of diagnostic tests for male infertility and serve as target antigen in the design of contraceptive vaccines.

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Recombinant human zona pellucida protein zp3 produced by chinese hamster ovary cells induces the human sperm acrosome reaction and promotes sperm-egg fusion

R. J. Aitken

Papers

Recombinant human zona pellucida protein zp3 produced by chinese hamster ovary cells induces the human sperm acrosome reaction and promotes sperm-egg fusion