Signal transduction pathways regulate various aspects of mammalian sperm function. When human sperm were incubated in a medium supporting capacitation, proteins became tyrosine-phosphorylated in a time-dependent manner. This phosphorylation was inhibited by genistein, a protein tyrosine kinase inhibitor. Phosphorylation was also reduced when sperm were incubated either in the presence of increasing concentrations of extracellular Ca2+ or in a medium containing the Ca2+ ionophore A23187. This Ca2+-induced dephosphorylation was calmodulin-dependent, suggesting that calcineurin was involved. In this regard, the calcineurin inhibitor deltamethrin inhibited the Ca2+ ionophore-induced dephosphorylation. A limited number of Mr 80,000-105,000 polypeptides were the most prominent phosphotyrosine-containing proteins present in human sperm. Unlike mouse sperm, which contains a tyrosine-phosphorylated isoform of hexokinase, a phosphotyrosine-containing hexokinase in human sperm was not detected. Most of the tyrosine-phosphorylated proteins were Triton X-100-insoluble and were localized to the principal piece of the flagellum, the region where the cytoskeletal fibrous sheath is found. Prominent phosphotyrosine-containing proteins of Mr 82,000 and 97,000 were identified as the human homologues of mouse sperm AKAP82, the major fibrous sheath protein, and pro-AKAP82, its precursor polypeptide, respectively. These proteins are A Kinase Anchor Proteins, polypeptides that sequester protein kinase A to subcellular locations. Taken together, these results suggest that protein tyrosine phosphorylation may be part of a signal transduction cascade(s) regulating events pertaining to capacitation and/or motility in mammalian sperm and that an interrelationship between tyrosine kinase and cAMP signaling pathways exists in these cells.

Changes in the activities of the p34cdc2/cyclin B complex and mitogen-activated protein (MAP) kinase were analyzed after insemination of mouse eggs in vitro. Whereas histone H1 kinase activity (p34cdc2/cyclin B) fell to negligible levels by 90 min postinsemination, a decrease to negligible levels of myelin basic protein kinase activity (i.e., MAP kinase) was not observed until about 7 h postinsemination. The decrease in MAP kinase activity appeared to be linked to the prior decline in p34cdc2/cyclin B kinase activity, since inhibiting the fertilization-induced destruction of cyclin B by treating eggs with the microtubule inhibitor nocodazole prevented the decrease in each of these protein kinases; an intact spindle is required for cyclin destruction. Moreover, experimental elevation of MAP kinase activity by okadaic acid treatment under conditions that maintain negligible levels of p34cdc2/cyclin B kinase activity suggested that MAP kinase could be involved in pronuclear envelope dynamics. Specifically, preventing the fertilization-induced decrease in MAP kinase activity was correlated with inhibiting pronucleus formation, and elevating MAP kinase activity subsequent to pronucleus formation resulted in precocious pronuclear envelope breakdown prior to entry into M phase.

When western blots of human sperm proteins solubilized by acid extraction (presumably mainly acrosomal proteins) or by sodium dodecyl sulfate (SDS) were probed with biotin-conjugated Pisum sativum agglutinin (PSA), distinct sets of proteins were labelled in both preparations. When smears of human spermatozoa were treated with methanol either for 30 s or for 15 min and then exposed to FITC-conjugated PSA, the resulting fluorescence pattern essentially depended on the time of methanol treatment. With the longer treatment, fewer spermatozoa showed selective acrosomal labelling and more were labelled uniformly throughout, without a clear predilection for a single sperm region. With the shorter time of methanol treatment, the poorly topographically differentiated, whole-cell labelling was typical of dead spermatozoa as confirmed by a close correlation between the percentages of spermatozoa showing this type of labelling and of those stained supravitally with Hoechst 33258. The preferential whole-cell labelling of dead spermatozoa with PSA is considered to be due to increased availability of the nonacrosomal set of PSA-reactive sites in dead spermatozoa after a short treatment with methanol, whereas this treatment is probably not sufficient to expose most of these sites when applied to living spermatozoa. The simplicity of the staining protocol makes this method feasible in routine work in a number of clinical and research applications.

In vitro fertilization (IVF) is fraught with problems and currently proteomics approaches are being tried out to examine the microenvironment of the follicle in order to assess biological and immunological parameters that may affect its development. Additionally, better understanding of reproductive process may help increase IVF birth rate per embryo transfer and at the same time avoid spontaneous miscarriages or life threatening conditions such as ovarian hyperstimulation syndrome. The primary aim of this study was to search for specific differences in protein composition of human follicular fluid (HFF) and plasma in order to identify proteins that accumulate or are absent in HFF. Depletion of abundant proteins combined with multidimensional protein fractionation allowed the study of middle- and lower-abundance proteins. Paired comparison study examining HFF with plasma/serum from women undergoing successful IVF revealed important differences in the protein composition which may improve our knowledge of the follicular microenvironment and its biological role. This study showed involvement of innate immune function of complement cascade in HFF. Complement inhibition and the presence of C-terminal fragment of perlecan suggested possible links to angiogenesis which is a vital process in folliculogenesis and placental development. Differences in proteins associated with blood coagulation were also found in the follicular milieu. Several specific proteins were observed, many of which have not yet been associated with follicle/oocyte maturation. These proteins together with their regulatory pathways may play a vital role in the reproductive process.

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Mouse eggs arrested in metaphase II display high levels of cdc2/cyclin B1 and MAP protein kinase activities. Following fertilization there is a time-dependent decrease in the activity of each of these protein kinases. The decline in cdc2/cyclin B1 protein kinase correlates with the resumption of meiosis and the emission of the second polar body and precedes the decline in MAP kinase activity, which correlates temporally with the formation of the male and female pronuclear envelopes. These results suggest that high levels of MAP kinase activity are incompatible with the presence of a pronuclear envelope. To test this possibility, we expressed in mouse eggs a constitutively active form of MAP kinase kinase (MEK) whose only known target is p42/p44 MAP kinase. We show that following fertilization cdc2/cyclin B1 kinase activity declines and a second polar body is emitted. The endogenous MAP kinase remains active, however, and no pronuclear envelopes form. Thus, high levels of MAP kinase activity by itself in mouse eggs appear incompatible with the presence of a pronuclear envelope.

Mature human sperm initiate a rapid Ca2+ influx and the acrosomal exocytosis in response to progesterone. Recent evidence indicates that both events can be induced by antibody-mediated cross-linking of a sperm surface progesterone receptor. In many other systems in which signal is generated by receptor cross-linking, protein phosphorylation on tyrosine residues is involved in the signal transduction across the plasma membrane. In this study we examined whether tyrosine phosphorylation is implicated in the function of the sperm surface progesterone receptor, too. The effect of progesterone on the phosphorylation of proteins from a sperm membrane lysate was evaluated by in vitro kinase assay and by phosphoamino acid analysis using [gamma-32P]ATP as precursor. These experiments revealed a selective increase in the tyrosine phosphorylation of a 94-kilodalton phosphoprotein in the presence of progesterone. To decide whether the progesterone-induced increase in protein tyrosine phosphorylation is actually due to the hormone action on the cell surface, living sperm were treated with a cell-impermeant progesterone receptor agonist, and the resulting changes in the cellular level of phosphotyrosine proteins were examined. These experiments showed a clear relationship between the agonist binding and an increase in the phosphotyrosine concentration in the respective cells. This relationship was lost in the presence of genistein, which also efficiently inhibited the phosphorylation of the 94-kilodalton protein and the progesterone-induced acrosomal exocytosis. These results lead to the hypothesis that protein tyrosine phosphorylation is involved in signal transduction through the sperm surface progesterone receptor and may be implicated in nongenomic steroid effects in other cell types.