Catherine D. Thaler

Microtubules and Microtubule Motors: Mechanisms of Regulation

Microtubule-based motility is precisely regulated, and the targets of regulation may be the motor proteins, the microtubules, or both components of this intricately controlled system. Regulation of microtubule behavior can be mediated by cell cycle-dependent changes in centrosomal microtubule nucleating ability and by cell-specific, microtubule-associated proteins (MAPs). Changes in microtubule organization and dynamics have been correlated with changes in phosphorylation. Regulation of motor proteins may be required both to initiate movement and to dictate its direction. Axonemal and cytoplasmic dyneins as well as kinesin can be phosphorylated and this modification may affect the motor activities of these enzymes or their ability to interact with organelles. A more complete understanding of how motors can be modulated by phosphorylation, either of the motor proteins or of other associated substrates, will be necessary in order to understand how bidirectional transport is regulated.

Defining oligosaccharide specificity for initial sperm‐zona pellucida adhesion in the mouse

The identity of the sperm surface protein(s) responsible for sperm‐zona pellucida binding in the mouse, as well as the characteristics of the oligosaccharide groups on zona pellucida glycoprotein 3 (ZP3) having ligand activity toward this receptor, remain controversial. Conflicting results from several groups have made interpretation of the current data difficult. By developing a quantitative binding assay to evaluate the molecular interactions between mammalian sperm and the zona pellucida during initial gamete interactions, we directly quantified sperm‐ZP binding interactions at the molecular level for the first time. The ZP binding assay demonstrated that live, capacitated mouse sperm bind solubilized 125I‐labeled ZP glycoproteins in a concentration‐dependent manner characterized by a rapid forward rate constant of 3.0 × 107 M−1 min−1. Following the initial characterization, the binding assay was used to examine the roles of the sperm surface enzymes galactosyltransferase (GalTase) and fucosyltransferase (FucTase) in sperm‐zone pellucida binding in the mouse. These data indicate that substrates for FucTase, but not for GalTase, inhibit sperm‐ZP binding, in contrast to earlier reports in which GalTase substrates significantly inhibited sperm binding to intact ZPs. A model is presented which resolves conflicting results between assays using intact ZPs and the results obtained here using soluble 125I‐ZPs. Assuming a complex binding/recognition site, monosaccharides that could occupy part of the binding site would have a dramatic effect on sperm‐ZP binding to the intact ZP, since they need only occupy the binding sites for a short time (∼ 100 msec) to disrupt binding. The current results suggest that the sperm ZP3 receptor binding site minimally recognizes the galβ1,3GlcNAc moiety also recognized by FucTases. The current data do not exclude the possibility that additional sugar residues form part of the ligand oligosaccharide group and are recognized by a yet‐to‐be‐identified sperm surface protein which serves as the ZP3 receptor.

Sonication of Mouse Sperm Membranes Reveals Distinct Protein Domains

Abstract Molecular interactions between sperm and zona pellucida (ZP) during mammalian fertilization are not well characterized. To begin to characterize sperm components that are involved in sperm-ZP interactions, we isolated and density fractionated sperm membranes. The membrane fractions recovered from a density fractionation protocol were characterized, and sonication was compared with vortexing for preparation of sperm membranes by examining the distribution of proteins in the membrane fractions obtained from these 2 protocols. Biochemical and microscopic analyses were used to determine the composition of the sonicated membrane fractions, and immunoblotting was used to identify fractions containing some of the previously suggested ZP3 receptors. Transmission electron microscopy revealed that bands 1–3 contained membrane vesicles and band 4 contained axonemal and midpiece fragments. SDS-PAGE revealed that bands 1 and 2 shared many proteins, but band 3 contained a number of unique proteins. Surface labeling with 125I demonstrated that bands 1 and 2 contained the majority of the sperm surface protein markers, whereas band 3 contained minor amounts of surface markers. Lectin-binding characteristics of sperm membrane glycoproteins were used to compare the relative distribution of glycosylated proteins in vortexed or sonicated membrane preparations. These characterizations indicate that sonication enhanced the differential distribution of sperm membrane proteins among the density fractions and suggests that this method is preferable for preparation of membrane fractions to be used for identification of proteins that mediate sperm-egg interactions.

Protease activation and the signal transduction pathway regulating motility in sperm from the water strider Aquarius remigis

Many motile processes are regulated such that movement occurs only upon activation of a signaling cascade. Sperm from a variety of species are initially quiescent and must be activated prior to beating. The signaling events leading to the activation and regulation of sperm motility are not well characterized. Mature seminal vesicle sperm from the water strider Aquarius remigis are immotile in vitro, but vigorous motility is activated by trypsin. Trypsin‐activated motility was blocked by pretreatment of the sperm with BAPTA‐AM to chelate intracellular Ca2+ and was partially rescued by subsequent addition of A23187 and Ca2+. Thapsigargin stimulated motility in the absence of trypsin, suggesting that intracellular Ca2+ stores are available. In addition, motility could be fully activated by the phosphatase inhibitor calyculin A, suggesting that the immotile state is maintained by an endogenous phosphatase and that kinase activity is required for motility. The MEK1/2 inhibitor U0126 significantly reduced trypsin activated motility, and MPM‐2, an antibody which recognizes proline‐directed phosphorylation by kinases such as MAPK, recognized components of the water strider sperm flagellum. Antibodies specific for the mouse protease activated receptor PAR2 recognized an antigen on the sperm flagellum. These results suggest that trypsin stimulates a Ca2+ and MAPK mediated signaling pathway and potentially implicate a PAR2‐like protein in regulating motility.

Distinct Membrane Fractions from Mouse Sperm Bind Different Zona Pellucida Glycoproteins

Abstract Interactions between sperm and zona pellucida (ZP) during mammalian fertilization are not well characterized at the molecular level. To identify sperm proteins that recognize ligand ZP3, we used sonicated sperm membrane fractions as competitors in a quantitative binding assay. Sonicated membranes were density fractionated into 4 fractions. Bands 1–3 contained membrane vesicles, and band 4 contained axonemal and midpiece fragments. In competitive binding assays, bands 1, 2, and 3 but not band 4 were able to compete with live, capacitated, intact sperm for soluble 125I-ZP binding. Affinity-purified ZP fractions consisting of a ZP3-enriched fraction (125I-ZP3) and a fraction enriched for ligands ZP1 and ZP2 and depleted of ZP3 (125I-ZP1/2) were obtained by antibody affinity purification of ZP3. In competitive binding assays, bands 2 and 3 competed for 125I-ZP3 binding, but band 1 did not interact with enriched 125I-ZP3. None of the membrane fractions competed for 125I-ZP1/2 binding. These results demonstrate that band 2 and band 3 contain sperm components that interact with ZP3 alone and that components in band 1 interact with ZP3 in conjunction with either ZP1 or ZP2. These data indicate that there must be at least 2 unique sperm plasma membrane components that mediate intact sperm interactions with ZP glycoproteins in mouse. Bands 2 and 3 are likely to contain a primary ZP-binding protein because they interacted directly with ZP3, whereas band 1 may contain sperm proteins involved in later interactions with the ZP, perhaps transitional interactions to maintain sperm contact with the ZP during acrosomal exocytosis.

Function of the Egg's Extracellular Matrix

Publisher Summary This chapter discusses function of the eggs extracellular matrix and focuses on the two egg-associated structures—that is, cumulus layer and zona pellucida, specifically in regard to their involvement in the process of fertilization. Before membrane fusion between mammalian gametes can occur, the sperm must first negotiate two major extracellular layers that surround the egg. These layers are the cumulus layer, which is made up of cumulus cells, hyaluronic acid, and a number of minor components; and the zona pellucida, which is a matrix made up of three to five glycoproteins that are secreted by the developing oocyte. The cumulus oophorus plays many essential roles in maturation, ovulation, and fertilization of the mammalian oocyte. The chapter reviews the functions of the cumulus as related to the overall success of fertilization and look specifically at three interrelated functions of the cumulus matrix: (1) production of soluble factors, (2) transport into the oviduct for fertilization, and (3) creation of a selective barrier or filter for sperm. Many studies have suggested that the cumulus matrix is a chemical and mechanical barrier to sperm progress toward the oocyte. The zona pellucida plays critical roles in sperm recognition and adhesion, initiation of acrosomal exocytosis, sperm penetration of the matrix, and, subsequently, protection of the fertilized egg and embryo. The structure of the zona pellucida, especially the carbohydrate composition of the individual glycoproteins and the precise arrangement of those molecules, is important in determining biological function. Following sperm-egg fusion and egg activation, the zona pellucida undergoes molecular changes, collectively known as the zona reaction, which function to block polyspermy.

Waveform Generation Is Controlled by Phosphorylation and Swimming Direction Is Controlled by Ca2+ in Sperm from the Mosquito Culex quinquefasciatus

ABSTRACT Most animal sperm are quiescent in the male reproductive tract and become activated after mixing with accessory secretions from the male and/or female reproductive tract. Sperm from the mosquito Culex quinquefasciatus initiate flagellar motility after mixing with male accessory gland components, and the sperm flagellum displays three distinct motility patterns over time: a low amplitude, a long wavelength form (Wave A), a double waveform consisting of two superimposed waveforms over the length of the flagellum (Wave B), and finally, a single helical waveform that propels the sperm at high velocity (Wave C). This flagellar behavior is replicated by treating quiescent sperm with trypsin. When exposed to either broad spectrum or tyrosine kinase inhibitors, sperm activated by accessory gland secretions exhibited motility through Wave B but were unable to progress to Wave C. The MEK1/2 inhibitor UO126 and the ERK1/2 inhibitor FR180204 each blocked the transition from Wave B to Wave C, indicating a role for MAPK activity in the control of waveform and, accordingly, progressive movement. Furthermore, a MAPK substrate antibody stained the flagellum of activated sperm. In the absence of extracellular Ca2+, a small fraction of sperm swam backwards, whereas most could not be activated by either accessory glands or trypsin and were immotile. However, the phosphatase inhibitor okadaic acid in the absence of extracellular Ca2+ induced all sperm to swim backwards with a flagellar waveform similar to Wave A. These results indicate that flagellar waveform generation and direction of motility are controlled by protein phosphorylation and Ca2+ levels, respectively.

Germ-cell hub position in a heteropteran testis correlates with the sequence and location of spermatogenesis and production of elaborate sperm bundles.

In insects, spermatogonial cells undergo several mitotic divisions with incomplete cytokinesis, and then proceed through meiosis and spermatogenesis in synchrony. The cells derived from a single spermatogonial cell are referred to as a cyst. In the water strider Aquarius remigis, spermiogenesis occurs within two bi‐lobed testes. In contrast to most insects, in which the germ‐cell hub is located apically and sequential stages of spermatogenesis can be seen moving toward the base of the testis, each lobe of the water strider testis contains a single germ‐cell hub located medially opposite to the efferent duct of the lobe; the developing cysts are displaced toward the distal ends of the lobe as spermiogenesis proceeds. Water strider sperm have both a long flagellum and an unusually long acrosome. The water strider spermatids elongate most of the flagellum prior to morphogenesis of the acrosome, and exhibit several stages of nuclear remodeling before the final, mature sperm nucleus is formed. The maturing sperm are aligned in register in the cyst, and the flagella fold into a coiled bundle while their acrosomes form a rigid helical process that extends from the cyst toward the efferent duct. Mol. Reprod. Dev. 82: 295–304, 2015.

Characterization of plasma membrane associated type II α-d-mannosidase and β-N-acetylglucosaminidase of Aquarius remigis sperm

For successful fertilization to occur, molecules on the surface of male and female gametes must recognize each other in a complementary manner. In some organisms, sperm possess a glycosidase on the plasma membrane overlying the head while eggs have glycoproteins that are recognized by those glycosidases resulting in sperm-egg recognition. In this study, two glycosidases, mannosidase and β-N-acetylglucosaminidase, were identified and biochemically characterized in Aquarius remigis sperm. The mannosidase had a Km of 2.36 ± 0.19 mM, a Vmax of 27.49 ± 0.88 pmol/min and a Hill coefficient of 0.94 ± 0.18 at its optimal pH of 7.0. The mannosidase was extracted most efficiently with CHAPSO but was also efficiently extracted with sodium chloride. Mannosidase activity was effectively inhibited by swainsonine, but not by kifunesine, and was significantly reduced in the presence of Mn(2+) and Mg(2+), but not Zn(2+). N-acetylglucosaminidase had a Km of 0.093 ± 0.01 mM, a Vmax of 153.80 ± 2.97 pmol/min and a Hill coefficient of 0.96 ± 0.63 at its optimal pH of 7.0. N-acetylglucosaminidase was extracted most efficiently with potassium iodide but was also efficiently extracted with Triton X-100 and Zn(2+), but not Ca(2+), Co(2+), Mn(2+) or Mg(2+), significantly inhibited its activity. Taken together, these results indicate that the A. remigis sperm surface contains at least two glycosidases that may recognize complementary glycoconjugates on the surface of water strider eggs.

Culex pipiens sperm motility is initiated by a trypsin-like protease from male accessory glands

In most animals, sperm are stored in a quiescent state in the male reproductive tract and only initiate motility when released into either the female reproductive tract, or, in the case of broadcast spawners, the external environment. Male accessory gland secretions transferred into the female reproductive tract may provide factors that modulate sperm viability and storage, or aid in sperm competition, as well as activate sperm motility. In several insects, serine proteases have been implicated in activating sperm motility. Our previous studies have shown that, in Culex quinquefasciatus, either a male accessory gland extract or purified trypsin is sufficient to initiate sperm motility in vitro. The objective of this study was to identify and characterize trypsin‐like enzymes produced in the Culex male accessory glands. Mass spectrometry was used to analyze accessory gland proteins and this preliminary proteomic analysis identified 4 trypsin‐like proteases (trypsin, trypsin4, and two trypsin7 isoforms). When measured with the chromogenic trypsin substrate Na‐benzoyl‐L‐arginine‐ethyl‐ester‐hydrochloride (BAEE), trypsin‐like protease activity in the accessory glands was robust, with a pH optimum of 8. The pH range for the Culex trypsin activity was substantially narrower than a mammalian homologue (porcine pancreatic trypsin). A soybean trypsin inhibitor (SBTI) ‐agarose affinity column was used to independently identify trypsin‐like accessory gland proteins. Several proteins were enriched in the eluate, as detected by silver staining of SDS–PAGE gels. Taken together, these data demonstrate the presence of trypsin‐like activity and several trypsin‐like proteins in the Culex male accessory glands.