Biparental inheritance of gamma-tubulin during human fertilization: molecular reconstitution of functional zygotic centrosomes in inseminated human oocytes and in cell-free extracts nucleated by human sperm.

Human sperm centrosome reconstitution and the parental contributions to the zygotic centrosome are examined in mammalian zygotes and after exposure of spermatozoa to Xenopus laevis cell-free extracts. The presence and inheritance of the conserved centrosomal constituents gamma-tubulin, centrin, and MPM-2 (which detects phosphorylated epitopes) are traced, as is the sperm microtubule-nucleating capability on reconstituted centrosomes. gamma-Tubulin is biparentally inherited in humans (maternal >> than paternal): Western blots detect the presence of paternal gamma-tubulin. Recruitment of maternal gamma-tubulin to the sperm centrosome occurs after sperm incorporation in vivo or exposure to cell-free extract, especially after sperm "priming" induced by disulfide bond reduction. Centrin is found in the proximal sperm centrosomal region, demonstrates expected calcium sensitivity, but appears absent from the zygotic centrosome after sperm incorporation or exposure to extracts. Sperm centrosome phosphorylation is detected after exposure of primed sperm to egg extracts as well as during the early stages of sperm incorporation after fertilization. Finally, centrosome reconstitution in cell-free extracts permits sperm aster microtubule assembly in vitro. Collectively, these results support a model of a blended zygotic centrosome composed of maternal constituents attracted to an introduced paternal template after insemination.

Mitochondrial sheath movement and detachment in mammalian, but not nonmammalian, sperm induced by disulfide bond reduction.

The successful completion of the fertilization process requires the properly choreographed unsheathing of the tightly packaged sperm once it has been fully incorporated into the egg's cytoplasm. The nuclear and accessory structures of mammalian sperm become stabilized by disulfide bonds (S-S) during epididymal maturation. This stabilization is reversed during fertilization by the reduction of S-S cross-linking, but little is known about the effect of S-S reduction on individual disulfide-hardened structures such as the sperm's connecting piece, fibrous sheath, and mitochondria. Here, we demonstrate the action of the S-S-reducing environment on the mitochondrial sheath of mammalian sperm, visualized by the vital fluorescent probe MitoTracker and by electron microscopy. In both human and bull sperm, mitochondria form a compact helix (mitochondrial sheath) wrapped around the midpiece and connecting piece that can be fluorescently labelled by a short incubation with 100 nM MitoTracker. Exposure of bull sperm to 0.1-10 mM dithiothreitol (DTT; a disulfide bond-reducing agent) induced a time and dose-dependent sliding of the mitochondrial sheath down the axoneme, accompanied by the excision of the sperm tail and decondensation of the sperm nucleus. Increasing the concentration of DTT to 100 mM accelerated mitochondrial movement, causing a completed stripping of sperm mitochondria and partial disassembly of the connecting piece. Likewise, human sperm responded to DTT treatment by the sliding or removal of the mitochondrial sheath and decondensation of the sperm chromatin. These events were not observed in the sperm of lower vertebrates and invertebrates (Xenopus laevis and Lytechinus pictus, respectively) exposed to an excess of DTT. Thus the sensitivity of sperm mitochondria to the S-S reducing environment seems to be an exclusive feature of mammalian sperm. The movement of sperm mitochondria induced by S-S reduction may be an initial critical step in the disassembly of the mammalian sperm tail during fertilization.

Microtubule and chromatin dynamics during fertilization and early development in rhesus monkeys, and regulation by intracellular calcium ions.

To explore primate fertilization, oocytes and zygotes from fertile rhesus monkeys were imaged throughout fertilization, polyspermy, and artificial activation using confocal microscopy for microtubules and DNA, as well as ratiometric computer-enhanced video microscopy for intracellular calcium. Unfertilized oocytes displayed microtubules only in the radially oriented meiotic spindles. At insemination, a large calcium transient was followed by a series of smaller oscillations, and sperm astral microtubules had assembled from the sperm centrosome by 2.5 h after transient onset. This aster enlarged, and later duplicated, as the pronuclei converged near the cortex. Pronuclear apposition was prevented by microtubule inhibitors. At mitotic prophase, microtubules ensheathed both sets of condensing chromosomes. At metaphase, the spindle was barrel-shaped and eccentrically positioned with two small asters at the pole with the sperm tail. Microtubules emanating from the telophase spindle interacted with the adjacent cortex and displaced the spindle toward the cell center as first cytokinesis ensued. During polyspermy, each sperm nucleated an aster, and the frequency of calcium oscillations increased. Activation resulted initially in disarrayed microtubules that eventually organized into functional mitotic spindles. These kinetic results demonstrate that rhesus monkeys accomplish fertilization in a fashion nearly identical to that of humans and are, therefore, ideal models in which to investigate cytoskeletal events during human reproduction.