Protein storage vacuoles (PSVs) from radicles and cotyledons of dry cotton seeds were isolated by differential centrifugation following homogenization in glycerol. Protein complement analysis of isolated PSVs with one dimensional SDS-PAGE gels revealed similar major storage proteins, viz. 53 and 48 kDa, with differences in lower molecular mass proteins. Radicle PSVs have apparently more 35-kDa and less 22-kDa storage protein than do cotyledon PSVs. The mineral composition of whole radicles, cotyledons and isolated PSVs from radicles and cotyledons was determined by atomic absorption spectroscopy and colorimetric elemental analyses. The concentration of calcium (Ca), magnesium (Mg), potassium (K) and phosphate (P) was lower in isolated PSVs from radicles than from cotyledons, resulting in a marked difference in the Mg/Ca and (Mg+Ca)/K ratios in PSVs from these two sources. Analysis of radicle and cotyledon tissue from dry seeds for mineral distribution with EDX and scanning electron microscopy revealed major concentrations of Mg, K and P in PSVs. These observations indicate that PSVs in radicles are similar in protein and mineral composition to PSVs in cotyledons. PSVs in radicles have the potential function as storage organelles to provide minerals and nutrients for radicle growth during imbibition and germination.

The soybean (Glycine max L. Merr.) seed coat is the terminal maternal tissue that supplies the developing embryo with nutrients. Between the embryo and seed coat, where this transfer presumably occurs, we have found (1,2) a specialized convex area (antipit) on the inside surface of the soybean seed coat that is opposed by a concave area (pit) on the surface of the cotyledon. The cells of this pit-antipit complex are larger than those of the surrounding tissue. Furthermore, the aleurone and cone cells of the antipit have many characteristics of secretory cells. Previous data about these structures has been cytological; very limited biochemical information is knownabout the area. This paper presents preliminary data about the elemental content of these surfaces in the mature seeds of domesticated soybean, G. max and a close relative, G. soja.The seed was dissected into right and left cotyledons; the right cotyledon was used for all microscopy and X-ray analysis. The seed coat was then separated from the cotyledon and excess tissue was removed.

AbstractThe morphological and ultrastructural changes that occur during preparation of porcine, bovine, and murine spermatozoa for flow cytometric quantification of the relative DNA content of the X‐ and Y‐chromosome‐bearing sperm populations were examined. Ejaculated spermatozoa from the boar and bull were washed using a series of dimethyl sulfoxide (DMSO) solutions prior to fixation, whereas the epididymal mouse spermatozoa were washed only in phosphate‐buffered saline (PBS). Spermatozoa from all three species were then fixed in ethanol and processed for fluorochrome staining by a treatment regimen consisting of sulfhydryl reduction and proteolysis. The processed sperm nuclei were stained for DNA with the fluorochrome, 4′‐6‐diamidino‐2‐phenylindole (DAPI) before quantification by flow cytometry. Scanning and transmission electron micrographs of sperm heads taken at various steps of the preparation and staining procedures show 1) that the rigorous washing procedure disrupted the plasma and outer acrosomal membranes, 2) that ethanol fixation resulted in removal of the outer membranes and disintegration of the nuclear envelope, and 3) that thiol and proteolysis treatment removed the remaining cellular organelles including the tail and rapidly induced partial decondensation of the tightly packed chromatin. Sequential micrographs showed that the nuclear matrix of all three species increased in thickness about twofold during the preparation and staining. Consequently, the harsh procedures currently used for quantitative staining of DNA for high‐resolution flow cytometric analyses destroy most cellular organelles and thereby prevent simultaneous characterization of DNA content and other sperm cell constituents.

Six procedures to extract acrosin from boar spermatozoa were compared. A procedure in which a combination of Hyamine 2389, glycerol, and freeze-thawing (HGF) was used, yielded an extract with significantly higher specific enzyme activity (Units/mg protein) than the other five procedures. The HGF procedure and two procedures in which Orvus ES Paste (OEP) and acetic acid were used as extractants, yielded extracts with the highest total enzyme activity. The amount of protein extracted by the procedures in which OEP and acetic acid were used was significantly greater than the amount of protein extracted by the HGF procedure. A procedure involving a simple acetic acid extraction was least effective in extracting acrosin from boar sperm. Storage of HGF acrosomal extract for 14 days at −196°C resulted in a higher enzyme activity than storage for 14 days at 5°C (P<0.05). In extracts from OEP-acetic acid procedures, enzyme activity was greater after storage for 14 days at 5°C than at −196°C. The correlation between enzyme activity (mU/106 sperm) and specific enzyme activity (Units/mg protein) within procedures was positive (P<0.001), when values were combined across length and temperature of storage.