PubMed · 8929666
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A Trounson, A Bongso. 1996. Fertilization and development in humans.. https://doi.org/10.1016/s0070-2153(08)60425-1
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To identify a novel candidate(s) for acrosomal proteins that act on the sperm/egg interaction, a DNA fragment was PCR-amplified from a cDNA library of acrosin-deficient mouse testis and then used as a probe to screen a mouse testis cDNA library. Complementary DNA clones encoding each of two similar but different serine proteases, TESP1 and TESP2, have been identified. The nucleotide sequences of these clones indicate that mouse TESP1 and TESP2 are initially synthesized as preproproteins of 367 and 366 amino acids, respectively. Comparison of the two TESP sequences with those of typical serine proteases suggests that each TESP zymogen is probably converted into a two-chain mature enzyme consisting of light and heavy chains covalently linked by a single pre-existing disulfide bond. The conversion may be accomplished by another protease(s) with a trypsin-like cleavage specificity, since it is unlikely that the mature TESP1 and TESP2 are capable of splitting the Lys-Ile bond between the light and heavy chains. Northern blot analysis of total cellular RNA demonstrates that the TESP1 and TESP2 genes are expressed only in the testis, and the transcripts are abundantly present in the haploid round spermatids. Moreover, immunocytochemical analysis of mouse cauda epididymal sperm using affinity-purified antibodies reveals that these two TESPs are both localized in the sperm acrosome and are released during the acrosome reaction induced by calcium ionophore A23187. These findings provide additional clues for elucidating the mechanisms involved in the sperm/egg interactions, including penetration of the zona pellucida by sperm.
Zona pellucida protein 3, a protein of the egg's extracellular matrix, and progesterone secreted by granulosa cells surrounding the oocyte are regarded as physiological stimuli of sperm acrosome reaction. Signal transduction steps initiated by both stimuli result in influx of Ca2+ from the extracellular space. Herein, we propose a role for prostaglandin (PG) E as a physiological inducer of Ca2+ influx and acrosome reaction in human spermatozoa. PGE1 specifically binds to human sperm membranes (Kd = 20.4 nM; Bmax = 88 fmol/mg protein) and induces a pertussis toxin-insensitive, transient increase in intracellular Ca2+ concentrations, which can be blocked by microM concentrations of La3+, Gd3+, and Zn2+. The kinetic profile was similar to that observed after progesterone challenge. Sequential application of both agonists did not lead to cross-desensitization. E prostaglandins were found to be the only prostanoids with agonistic properties (EC50 values for PGE1 and PGE2: <10 nM and 300 nM, respectively). Pharmacological characteristics were not compatible with those of cloned prostanoid receptors indicating the expression of a distinct membrane receptor. Activation of the sperm E prostanoid receptor stimulates incorporation of [alpha-32P]GTP azidoanilide into immunoprecipitated Galphaq/11 subunits. Thus, in human sperm, PG induces Ca2+ influx and acrosome reaction via a Gq/11-coupled E prostanoid receptor. The block of PGE1-induced Ca2+ transients and acrosome reaction by physiological Zn2+ concentrations highlights a role of Zn2+ as an endogenous Ca2+ channel blocker present in seminal plasma protecting sperm from premature PGE1-evoked increases in intracellular Ca2+ concentrations.
Acrosome reaction inactivation (ARI) is a process that renders sperm irreversibly refractory to the egg jelly (the natural inducer of the acrosome reaction, AR). This process triggered by the egg jelly, is associated with an increase in [Ca2+]i. However, we show here that a rise in [Ca2+]i alone is not sufficient to induce ARI, since artificially increasing [Ca2+]i with either an ionophore or rising external pH, does not trigger ARI. Contrary to the AR which strictly requires Ca2+, ARI can be triggered almost equally well by Sr2+. On the other hand, Mn2+ inhibits ARI and, as we showed earlier, does not affect AR. These observations indicate that the mechanisms involved in ARI differ from those leading to AR. In addition, we report here that high external pH (a non-physiological inducer of AR) triggers the AR in previously inactivated sperm by opening the same Ca2+ channels activated by the egg jelly. Considering that the opening of Ca2+ channels is one of the earliest responses triggered by the egg jelly and that ARI requires the egg jelly receptor to be activated, we have concluded that ARI involves the uncoupling between the egg jelly receptor and Ca2+ channels. Furthermore, intracellular pH (pHi) determinations, in the presence or absence of ionomycin to substitute for the uncoupled Ca2+ channels, indicate that pHi regulation is also impaired in inactivated sperm. In conclusion, ARI is a manifestation of the uncoupling of the egg jelly receptor from the different ion transport systems required for the acrosome reaction.