Levels of sulfogalactosylglycerolipid in capacitated motile and immotile mouse spermatozoa

The purpose of this study was to determine whether sulfogalactosylglycerolipid (SGG) was desulfated during mouse sperm capacitation. Levels of [35S]SGG were determined in freshly retrieved caudal epididymal sperm, motile capacitated sperm, and immotile sperm, after feeding mature male mice with [35S]sulfate-laced chow for 32 days. Caudal epididymal sperm and coisolated epididymal cells were separated into pellet and interphase fractions by centrifugation through a two-step Percoll gradient (45 and 90%). Upon resuspension in Krebs-Ringer bicarbonate medium supplemented with 0.4% bovine serum albumin, the Percoll-gradient pellet fraction consisted mainly of motile capacitated sperm, whereas the interphase fraction comprised largely immotile sperm and fragmented epididymal epithelial cells. The level of [35S]SGG in the Percoll-gradient-pelleted sperm appeared to be much higher than that in the Percoll-gradient interphase sperm. Percoll-gradient-pelleted sperm were further incubated in the culture medium for 2 h. The level of [35S]SGG showed little or no change after 1 h, but was reduced appreciably after 2 h. At this time point, sperm motility was also decreased. Reduction of sperm SGG is correlated with sperm immotility and (or) senescence and may have no direct relation to the capacitation process.     

Direct effect of gossypol on TR-ST cells: perturbation of rhodamine 123 accumulation in mitochondria

Gossypol has deleterious effects directly on TR-ST cells originating from a rat testicular tumor. Exposure of TR-ST cells to gossypol (5 micrograms/ml) decreases their rate of protein synthesis approximately 30% within 1 h and 65% by greater than 10 h, causes intracellular vacuolation, changes cell shape from cobblestone to a rounded conformation and inhibits cell proliferation. Yet, these gossypol-treated cells remain viable, as assessed by their ability to hydrolyze fluorescein diacetate. Gossypol also perturbs mitochondrial transmembrane potential in TR-ST cells, as demonstrated by marked changes in rhodamine 123 staining. Mitochondria of control TR-ST cells avidly accumulate rhodamine 123, but those in cells exposed to gossypol (greater than or equal to 5 micrograms/ml) for greater than 1 h fail to sequester the fluorochrome. Instead, the cell cytoplasm shows a light and diffuse staining with rhodamine 123. Rat spermatozoa show a similar response. Conversely, at concentrations of 20 micrograms/ml, gossypol has minimal effects on rhodamine 123 accumulation by primary cultures of hepatocytes and by rat spermatogenic cells, including primary spermatocytes and spermatids (Steps 1-12). Moreover, TR-ST cells exhibit reduced mitochondrial staining with gossypol at an ED50 of 7.6 micrograms/ml, while those for the nontesticular Rat-1, AnAn, 3T3 and PtK2 cell lines are 13.1, 21.5, 28.5 and 26.4 micrograms/ml, respectively.     

Polymorphic phases of galactocerebrosides: spectroscopic evidence of lamellar crystalline structures

Fourier transform infrared spectroscopy was applied to study the structural and thermal properties of bovine brain galactocerebroside (GalCer) containing amide linked non-hydroxylated or alpha-hydroxy fatty acids (NFA- and HFA-GalCer, respectively). Over the temperature range 0-90 degrees C, both GalCer displayed complex thermal transitions, characteristic of polymorphic phase behavior. Upon heating, aqueous dispersions of NFA- and HFA-GalCer exhibited high order-disorder transition temperatures near 80 and 72 degrees C, respectively. En route to the chain melting transition, the patterns of the amide I band of NFA-GalCer were indicative of two different lamellar crystalline phases, whereas those of HFA-GalCer were suggestive of lamellar gel and crystalline bilayers. Cooling from the liquid-crystalline phase resulted in the formation of another crystalline phase of NFA-GalCer and a gel phase of HFA-GalCer, with a phase transition near 62 and 66 degrees C, respectively. Prolonged incubation of GalCer bilayers at 38 degrees C revealed conversions among lamellar crystalline phases (NFA-GalCer) or between lamellar gel and crystalline bilayer structures (HFA-GalCer). Spectral changes indicated that the temperature and/or time induced formation of the lamellar crystalline structures of NFA- and HFA-GalCer was accompanied by partial dehydration and by rearrangements of the hydrogen bonding network and bilayer packing mode of GalCer.     

Role of sperm surface arylsulfatase A in mouse sperm-zona pellucida binding

We have previously described the zonae pellucidae (ZP) binding ability of a pig sperm surface protein, P68. Our recent results on peptide sequencing of 3 P68 tryptic peptides and molecular cloning of pig testis arylsulfatase A (AS-A) revealed the identity of P68 as AS-A. In this report, we demonstrate the presence of AS-A on the mouse sperm surface and its role in ZP binding. Using anti-AS-A antibody, we have shown by immunoblotting that AS-A was present in a Triton X-100 extract of mouse sperm. The presence of AS-A on the sperm plasma membrane was conclusively demonstrated by indirect immunofluorescence, immunogold electron microscopy, and AS-A's desulfation activity on live mouse sperm. The AS-A remained on the head surface of in vivo capacitated sperm, as revealed by positive immunofluorescent staining of oviductal/uterine sperm. Significantly, the role of mouse sperm surface AS-A on ZP binding was demonstrated by dose-dependent decreases of sperm-ZP binding on sperm pretreatment with anti-AS-A IgG/Fab. Furthermore, Alexa-430 conjugated AS-A bound to mouse ZP of unfertilized eggs but not to fertilized ones, and this level of binding increased and approached saturation with increasing Alexa-430 AS-A concentrations. Moreover, in vivo fertilization was markedly decreased when mouse sperm pretreated with anti-AS-A IgG were artificially inseminated into females. All of these results designated a new function for AS-A in mouse gamete interaction.     

Role of sperm sulfogalactosylglycerolipid in mouse sperm-zona pellucida binding

Sulfogalactosylglycerolipid (SGG) is the major sulfoglycolipid of mammalian male germ cells. Like other sulfoglycolipids, SGG is believed to be involved in cell-cell/extracellular matrix adhesion. Specifically, we investigated whether sperm SGG played a role in sperm-egg interaction. Initially, we produced an affinity-purified, rabbit polyclonal immunoglobulin (Ig) G antibody that specifically recognized SGG (anti-SGG). Indirect immunofluorescence using anti-SGG IgG localized SGG to the convex and concave ridges and the postacrosome of the mouse sperm head. Pretreatment of sperm with anti-SGG IgG/Fab inhibited sperm-zona pellucida (ZP) binding in vitro in a concentration-dependent manner (to a maximum of 62%). This inhibition was observed at the level of primary binding. Sperm treated with anti-SGG IgG underwent the spontaneous and ZP-induced acrosome reaction at the same rate as control sperm treated with preimmune rabbit serum IgG. Fluorescently labeled SGG liposomes were shown to associate specifically with the egg ZP, whereas fluorescently labeled liposomes of galactosylglycerolipid (SGG's parental lipid) and phosphatidylserine (negatively charged like SGG) did not. Furthermore, coincubation of SGG liposomes with sperm and isolated ZP inhibited sperm-ZP binding in a concentration-dependent manner. These results strongly suggest an involvement of sperm SGG in direct binding to the ZP.     

Relationship between chromosome condensation and metaphase lysine-rich histone phosphorylation

Treatment of metaphase HTC cells with ZnCl2 inhibits histone phosphatase activity and leads to an increase in the hyperphosphorylated forms of the lysine-rich (F1) histone. Under normal conditions a massive phosphatase activity is triggered as the cells shift from M into G1 phase. In the presence of ZnCl2 this activity is abolished and thehyperphosphorylated form of F1 persists intact into G1. We have asked the simple question of whether the chromosome can still extend during the M-G1 transition even if the F1 histone is maintained in the hyperphosphorylated form. We observe an apparently normal extension os the chromosomal material under these conditions, though it is evident that high levels of ZnCl2 have rather substantial effects on other cell functions.     

Role of a germ cell-specific sulfolipid-immobilizing protein (SLIP1) in mouse in vivo fertilization.

Sulfolipid-immobilizing protein 1 (SLIP1) is a germ cell plasma membrane protein that binds specifically to sulfogalactosylglycerolipid, a sulfoglycolipid found preferentially in mammalian male germ cells (Lingwood, Can. J. Biochem. Cell. Biol. 63:1077-1085, 1985b). SLIP1 in mouse and rat sperm exists on the periacrosomal membrane, where sperm initially bind to eggs. Using the in vitro mouse sperm-egg binding assay with in vitro-capacitated sperm, we obtained results previously suggesting that sperm SLIP1 is involved in mouse sperm-zona pellucida interaction. In this study, using the in vitro sperm-egg binding assay, we showed that SLIP1 in uterine sperm was similarly engaged in this process. Involvement of mouse sperm SLIP1 was also shown to be important in the in vivo fertilization process. Superovulated females inseminated with caudal epidididymal and vas deferens sperm preexposed to anti-SLIP1 IgG yielded only 20% fertilized zygotes, while 80% fertilization was observed in females inseminated with sperm preincubated with preimmune serum IgG. The lower fertilization rate was not due to changes in the sperm capacitation rate as assessed by chlortetracycline staining.     

Energy metabolism of cultured TM4 cells and the action of gossypol

The energy metabolism of cultured TM4 cells, a cell line originally derived from mouse testicular cells, has been studied in relation to the action of gossypol. In the absence of externally added substrates, TM4 cells consumed oxygen at 37 +/- 5 nmoles O2 X mg protein-1 X h-1. Pyruvate stimulated oxygen consumption in a dose-dependent fashion up to 23%. Addition of glucose to the cells suspended in substrate-free medium inhibited oxygen consumption. At 5.5 mM glucose, the inhibition of oxygen consumption was 45 +/- 9%. The rate of aerobic lactate production from endogenous substrates was less than 7 nmoles lactate X mg protein-1 X h-1, even in the presence of optimal concentrations of the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone. The rate of aerobic lactate production was 920 +/- 197 nmoles X mg protein-1 X h-1 at external glucose concentrations of 2 mM or greater. The formation of aerobic glycolytic adenosine triphosphate (ATP) in 5 mM glucose comprised about 80% of the total ATP production. Gossypol stimulated both aerobic lactate production and oxygen consumption of the transformed testicular cells in a dose-dependent manner. The effect of gossypol on glucose transport, aerobic lactate production, and oxygen consumption is consistent with the hypothesis that gossypol modifies energy metabolism in these cells mainly by partially uncoupling mitochondrial oxidative phosphorylation. The possible impairment of cell and tissue function under gossypol treatment would depend on the metabolic properties of each specific differentiated cell.     

Modifications to histones immediately after synthesis

We have studied modifications occurring on histones immediately after synthesis. The labeling period was sufficiently brief such that the time interval between synthesis in the cytoplasm and deposition onto DNA can be easily detected. In this way it is possible to observe modifications occurring within seconds after arrival of the newly synthesized histone molecule in the nucleus. Histone F_2a1 is rapidly and extensively modified (in hepatoma tissue culture cells this is primarily by acetylation) so that the newly synthesized material appears primarily in the diacetyl modified form. The histone returns to a steady-state level of acetylation during the ensuing 30 minutes and this level is maintained throughout the lifetime of the cell. F₃ shows a somewhat similar pattern, except that it initially is less modified before it shifts to a steady-state level of modification. The lysinerich histone, F₁ is admitted to the nucleus with a modest level of phosphorylation. During the subsequent 60-minutes phosphorylation proceeds rapidly until the extent of phosphorylation typical of rapidly dividing cells is reached. This level of phosphorylation is then maintained in a steady-state against the action of a phosphatase. The rate of phosphorylation during the initial 60 minutes exceeds that at a later time by a factor of ~ ten. We argue that this observation accounts for previous observations of ³²P incorporation during G₁ and S phase and in the presence of inhibitors of DNA and histone synthesis.