Sperm buffers

Epididymis Carnitine Inositol Phosphatidylcholine Cholesterol Glycoproteins Facihtates acetyl-CoA oxidation by spermatozoa (Chapter 9) Precursor for formation of phosphatidyhnositol bisphosphate Buffer to maintain pH and a source of chohne Stabilises membranes They coat the surface of the sperm to protect against IgA... 

Fig. 39. CD spectrum of Aplysia myoglobin compared to that of sperm whale myoglobin in 0.01 M phosphate buffer, pH 7.4 1071...

Hybridization buffer 50% deionized formamide, 10% dextran sulfate, 250 pg/mL sheared herring sperm DNA, 0.01% polyvinyl pyrrolidone and 0.1% sodium dode-cyl sulfate in 2X SET buffer... 

Add 10 mL hybridization buffer, (6X SSPE, 50% formamide, 0.5% SDS, 10% dextran, and 50 ug/ml. of sonicated salmon sperm or herring sperm DNA). Remove air and bubbles and seal by heating the open side. Incubate at 37°C for 1 h, open bag by cutting one comer, and drain. 

The abalone sperm AR can be artificially induced by raising the calcium ion concentration of seawater from the normal 10 mM to 50 mM in seawater buffered with 10 mM Tris at pH 8.2. Unlike other species used for fertilization studies, the abalone AR does not lead to the rapid death of the sperm. In abalone sperm, the acrosomal compartment is sealed off from the respiratory compartment acrosome-reacted, sperm will continue to swim for days if stored in the cold room at 4°C. The acrosomal exudate of these sperm is composed predominantly of soluble ly sin and 18K protein. Reducing and denaturing polyacrylamide gel electrophoresis of whole sperm, AV exudate, and purified lysin shows that abalone spermatocytes make a substantial investment in the synthesis of these two acrosomal proteins (Figure 6). 

Figure 8. The fusion of artificial phospholipid vesicles induced by 18K protein (a) and lysin (b) at the three concentrations indicated above (c, buffer alone). The upper panels are with sperm proteins from the red abalone (Hr H. refescens) the lower panels are with sperm proteins from the green abalone (Hf H. fulgens). The 18K proteins are more potent fusagens than lysin. Although the two 18K proteins are only 33.8% identical in primary structure, their five predicted amphipathic helices have similar hydrophobic moments (from Swanson and Vacquier, 1995a).

Figure 8. Infrared difference spectra of carbonyl sperm whale myoglobins reconstituted from different hemes vs. metmyoglobin in phosphate buffer, pH 6 4, Top protoheme. Middle deu-teroheme. Bottom mesoheme.

Franken DR, Oosthuizen WT, Cooper S, Kruger TF, Burkman LJ, Coodington CC, Hodgen CD. 1991. Electron microscopic evidence on the acrosomal status of bound sperm and their penetration into human hemizonae pellucida after storage in a buffered salt solution. Andrologia 23 205-208. 

Wash precipitates with 1 mL of freshly prepared IX RIPA-POL buffer with 100 Xg/mL herring sperm DNA and rock at room temperature for exactly 5 min. 

Example 1 If a frozen sperm sample normally used for artificial insemination was thawed and later refrozen in liquid nitrogen, the heads of the sperm cells may have been destroyed. In routine protocols for DNA extraction from sperm cells a special step is included to pretreat the heads. After this step the used buffer is not reused in later extraction steps because for a native sperm sample the DNA is in the pellet and not in the buffer. In contrast, for thawed and refrozen samples, most of the DNA can be in this buffer and therefore DNA extraction from the pellet would fail. 

In this assay, the spermatozoa in the well are suspended in a solution containing the chemoattractant. The capillary, containing either a control buffer or the chemoattractant, is immersed in the sperm suspension. When the capillary contains buffer only, the spermatozoa sense a descending gradient of the chemoattractant as they move from the well to the capillary (Figure 7). When the chemoattractant is in both the capillary and the well, they sense no gradient at all. A comparison is made between these two conditions. This assay thus measures the sperm tendency to leave the chemoattractant rather than to accumulate in it. In the case of sperm chemotaxis, sperm accumulation in the capillary is... 

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