George G. Brown

An analysis of the pH-dependent chemical-shift behavior of phosphorus-containing metabolites

Abstract The pH-dependent chemical-shift behavior of phosphorus-containing metabolites was studied. Metabolites of interest included inorganic phosphate, methyl phosphonate, phosphomonoesters, phosphodiesters, phosphonates, phosphagens, and nucleotide phosphates. The data are documented in tabular form in terms of limiting chemical shifts and appropriate pKa values. Rearranged Henderson-Hasselbalch equations are presented, for both the one- and the two-pKa cases.

Sperm motility in the horseshoe crab, Limulus polyphemus L: I. Sperm behavior near eggs and motility initiation by egg extracts☆

Abstract Limulus spermatozoa are nonmotile when spawned and become motile only after encountering a sperm motility initiating factor (SMI) exuded by the egg. SMI extracts (produced by washing intact eggs with distilled water, lyophilizing the supernatant to dryness, and redissolving the dried extract in artificial seawater, ASW) initiate sperm motility in the absence of eggs. Utilizing such SMI extracts, sperm motility initiation was found to be unaffected by changes in temperature from 16 to 30°C, pH from 6.3 to 8.6, and salinity from 85 to 125% ASW. Within these ranges, sperm motility initiation was an “all-or-nothing” response, with greater than 99% of the spermatozoa becoming motile. Also, each sperm swam with apparently the same speed (at a given temperature) until spontaneously stopping within 10 min after the addition of SMI extracts. Evidence was found that SMI may bind irreversibly to a receptor, which is inactivated within a few seconds or minutes, leading to the observed cessation of motility. Observations of sperm behavior near intact eggs showed no evidence of chemotaxis. Spermatozoa observed to swim toward intact eggs progressed with a uniform speed and were motile less than 5 sec from initiation of motility until attaching to the egg. The presence of an all-or-nothing response to SMI, the independence of sperm motility to experimental parameters, and several other characteristics of the animal and its spermatozoa make Limulus a potentially excellent model animal for examination of sperm motility control mechanisms.

Effect of lectins and sugars on primary sperm attachment in the horseshoe crab, Limulus polyphemus L☆

The apical regions of motile Limulus spermatozoa readily adhere to the outer layer of the egg envelope. Shortly after this adherence or primary attachment, the sperm acrosome reaction occurs, resulting in a stronger adhesion (secondary attachment). A sperm attachment assay that quantified the number of spermatozoa attaching to egg sections was utilized to identify components involved in primary attachment. The number of spermatozoa attached was examined after treatment of either egg sections or spermatozoa with various compounds. Egg sections treated with asparagus pea lectin (250 micrograms/ml) bound significantly fewer spermatozoa as compared to those exposed to wheat germ agglutinin, concanavalin A, and garden pea lectin. Furthermore, sperm attachment was also greatly reduced when egg sections were first incubated with the glycosidase, alpha-L-fucosidase (less than or equal to 5% of controls). Treatment of spermatozoa with alpha-L-fucose, fucoidan, or p-aminophenyl fucoside also reduced sperm attachment when compared to Millipore-filtered artificial seawater controls. Egg sections were treated with fluorescein-conjugated lectins to confirm that the lectins actually bound to portions of the egg envelope and that various sugars are present in the egg envelope. Evidence suggests that the methylpentose, alpha-L-fucose, plays an important role in primary sperm attachment in Limulus.

Vesicle involvement in the egg cortical reaction of the horseshoe crab, Limulus polyphemus L.

Ultrastructural observations (TEM) of the cortical reaction in Limulus polyphemus have been difficult to obtain due to the relative impermeability of the transparent egg envelope to standard fixatives. With the application of trialdehyde fixation techniques [Kalt, M. R., and Tandler, B. (1971). J. Ultrastruct. Res. 36, 633–645], the cortical reaction has now been examined and the role of cortical vesicles has been determined. The size of these vesicles in uninseminated eggs is heterogeneous, with small vesicles (0.5 μm) being apposed to the plasmalemma and with large vesicles (4 μm) located in a lower layer of the egg cortex. The contents of the small vesicles are translucent under the electron beam. With the onset of egg activation these vesicles fuse with the overlying plasmalemma. The contents of the large vesicles appear electron dense and exhibit distinctly different morphologies. Shortly after insemination these large vesicles begin to enlarge by fusing together. By 9 min after insemination some enlarged vesicles fuse with the plasmalemma to form pits on the egg surface. The remaining enlarged vesicles continue to fuse with the plasmalemma until approximately 60 min after insemination when few vesicles are remaining.

Storage, Transportation, and Fertility of Undiluted and Diluted Paddlefish Milt

Abstract Milt samples from paddlefish (Polyodon spathula) were collected from specimens obtained from the Missouri River near Chamberland, South Dakota, in 1991 and the Opelika City Reservoir System near Auburn, Alabama, in 1992. Ice-chilled milt samples either were left undiluted or were diluted with one of three chilled extenders, transported overnight on ice, and analyzed for sperm concentration, extracellular pH, electron microscopical characters, sperm motility percentage, and duration of sperm motility. Paddlefish milt had relatively low sperm counts compared with milt of other fish species, averaging 1.8 × 109 spermatozoa/mL. Extracellular pH averaged 8.22. A sperm acrosome was demonstrated by electron microscopy. Dechlorinated tap water and 10% artificial sea water (ASW) activated and sustained sperm motility better than 25% ASW Transportation of milt had no apparent negative effect on fertility. For storage 1–5 d after collection, undiluted milt provided an average fertilization rate (93%) that w...

Phosphorus-31 nuclear magnetic resonance studies of spermatozoa from the boar, ram, goat and bull

1. Phosphorus-31 nuclear magnetic resonance spectroscopy (31P-NMR) was utilized to examine semen and spermatozoa isolated from boars, rams, goats and bulls. 2. Whole semen was found to contain high but variable levels of glycerylphosphocholine, most of which was located in the seminal plasma rather than in the spermatozoa. 3. Spermatozoa concentrated into a pellet were found to contain resonances for inorganic phosphate, phosphomonoesters including fructose-1,6-bisphosphate and glucose-6-phosphate, and free nucleotide triphosphate peaks. 4. A broad resonance was observed from 30 to -25 ppm which originated partly from nucleotide tri- and diphosphates. 5. No high energy molecules such as phosphoarginine or phosphocreatine which could act as energy shuttles were observed.

Cortical reaction in inseminated eggs of the horseshoe crab, Limulus polyphemus L☆

Abstract The egg cortical reaction in Limulus polyphemus is described in four events. Approximately 10 min after insemination, small “pits” are visible in the cortex of the inseminated Limulus egg. These pits progressively enlarge, eventually coalesce, and finally disappear, leaving a smooth-appearing surface approximately 60–90 min after insemination. Based on these visible changes, the following four events comprise the cortical reaction: (I) uninseminated egg—smooth surface, (II) inseminated egg—appearance and growth of pits, (III) inseminated egg—coalescence of pits, and (IV) inseminated egg—appearance of smooth surface. Preparation of these events for SEM studies demonstrated morphological characteristics and sequential development of the pits. Also numerous microvilli are found on the surface of uninseminated and inseminated eggs during the cortical reaction. An increase in their diameter during the reaction is particularly unique.

Sperm motility in the horseshoe crab, Limulus polyphemus L: II. Partial characterization of a motility initiating factor from eggs and the effects of inorganic cations on motility initiation

Abstract Egg extracts (obtained by washing intact Limulus eggs with either distilled water or artificial seawater, ASW) contain a sperm motility initiating factor (SMI). The SMI is heat stable (withstands boiling to dryness), passes through a dialysis membrane, and is retained by G-10 Sephadex (indicating a molecular weight of less than 700). Qualitative analysis (by X-ray fluorescence spectroscopy) and quantitative analysis (by atomic absorption spectroscopy) of SMI extracts revealed the presence of four divalent cations (Ca, Mg, Ni, and Cu) and one monovalent cation (K) that affect sperm motility. When assayed individually at high concentrations, all of the divalent cations initiate sperm motility and K + inhibits motility initiation by the divalent cations. However, none of the divalent cations are present at concentrations high enough to produce the observed SMI activity, and since K + is present when motility is initiated by SMI, K + must not normally be an inhibitor. Therefore, if inorganic cations are involved in normal sperm motility initiation in Limulus , they are acting in conjunction with some other low molecular weight factor.

Viability of cryopreserved spermatozoa of the horseshoe crab, Limulus polyphemus L.

Summary Semen of the horseshoe crab, Limulus polyphemus L., was frozen and stored at −74°C for periods up to 50 days in a glycerol/sea water medium (phosphate buffer). After collecting in glass centrifuge tubes, untreated semen was cooled between 15 and 5°C at a rate of −0.5°C/min. After 30 min of equilibration, semen was transferred to 2-ml glass ampoules and diluted with three volumes of diluent (25 ml of 0.4 M NaCl/0.1 M glycine, 4 ml of 0.028 M NaH2P04/0.072 M Na2HP04(pH. 7.2), and 5 ml of glycerol). Each sample was then cooled−6°C/min between 5°C to −35°C and quick-frozen to −79°C by immersion in a solid C02/acetone bath. After varying storage periods, cryopreserved semen samples were thawed under running tap water (20°C) and their viability tested. Compared to unfrozen control semen, viability tests (control = 100%) indicated that 73% of the spermatozoa showed eosin dye exclusion, 40% showed sperm-egg attachment, and 17% of fertilized eggs showed embryonic development.

ULTRASTRUCTURAL CHARACTERISTICS OF THE NON-EXPANDED AND EXPANDED EXTRA-EMBRYONIC SHELL OF THE HORSESHOE CRAB, LIMULUS POLYPHEMUS L.

During embryonic development of the horseshoe crab, Limulus polyphemus, a structural layer, the extra-embryonic shell (EES), develops in the periembryonic space. Late in development the egg envelope breaks away and the EES expands slowly (over a period of several days) to approximately twice its original diameter. The EES is composed of non-cellular material organized into three distinct layers. The outermost (layer 1) is electron translucent and exhibits numerous hairlike projections. The middle layer (layer 2) is differentiated from layer 1 by its greater density under the electron beam. The innermost layer (layer 3) comprises 99% of the mass of the EES and is intermediate in electron density between layers 1 and 2. The inside surface of the non-expanded EES is smooth, and easily distinguished from the outside surface, which is rough and accentuated by polygonal structures. The deep indentations separating these structures are probable sources of preformed surface area utilized during EES expansion. The...