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E. P. Aalseth and R. G. Saacke

Summary. Storing cauda epididymal spermatozoa in seminal plasma or in defined media at 1 × 109 spermatozoa/ml for 24 h at 4°C caused swelling of the apical ridge on motile spermatozoa (SAR) provided concentrations of fructose in the range normally found in seminal plasma or comparable levels of glucose were present. Evaluation of these conditions indicated that, with glycolysable sugars in the media, pH dropped from 6·6–6·7 to 5·7–6·0. Most of the pH decrease occurred during the first 2 h of slow cooling from 37 to 4°C. pH decrease was undoubtedly due to sperm organic acid production which overwhelmed the relatively weak buffering capacity of the defined media and/or seminal plasma. Inducing pH decreases with HCl in fructose-free conditions, and using NaOH to prevent a pH decrease when fructose was included in media, demonstrated that exposing spermatozoa to pH values of 5·7–6·0 and not a specific response to fructose was the major cause of SAR.

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P. L. Senger and R. G. Saacke

Summary.

Addition of cow serum to diluted bull spermatozoa induced marked headto-head agglutination which declined during incubation for 9 hr at 37°C. Maintenance of the acrosomal cap during incubation was not adversely affected by serum. Agglutinating cells were those with intact acrosomal and cell membranes, as determined by differential interference-contrast and electron microscopy. Single cells in the same treatment groups experienced more rapid acrosomal deterioration than did untreated spermatozoa. Ultrastructurally, agglutinated cells were associated by a close apposition of the cell membranes in the acrosomal region. Structural integrity of the cell membrane and acrosome of agglutinated cells was the same after 9 hr of incubation as at the onset of the incubation period.

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W. NESBIT FLEMING and R. G. SAACKE

Summary.

Nineteen oocytes aspirated from mature Graafian follicles of normally cycling cows in standing oestrus were studied. Oocytes were fixed in phosphate-buffered glutaraldehyde followed by exposure to osmium tetroxide and embedding in Epon 812. Oocytes could be classified into four types based on characteristics of nuclear components and distribution of organelles and inclusions. These types represented late maturational changes occurring shortly before ovulation. The close association of granulosa-cell processes in Type-I maturing follicular oocytes showed progressive signs of deterioration in other oocyte types. In all oocytes, numerous mitochondria with 'hood-like' appendages were observed. Single cisternae of endoplasmic reticulum were closely associated with the surface of mitochondria and the cavity formed by the 'hood-like' appendage. Endoplasmic reticulum was also associated with lipid droplets and was observed to be continuous with the outer leaflet of the nuclear envelope. Infrequently, a more classical granular endoplasmic reticulum was apparent. The cytoplasmic matrix was moderately dense and contained scattered ribosomes and polysomes. Abundant vesicles containing small PAS-positive granules were evenly distributed throughout the ooplasm. Golgi dictyosomes, annulate lamellae, cortical granules and two types of nucleoli characterized these oocytes.

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E. P. Aalseth and R. G. Saacke

Summary. Swelling of the apical ridge and anterior acrosome of motile bovine spermatozoa was observed during in-vitro storage using differential interference-contrast optics. This morphological alteration is different from that described as the false acrosome reaction on immotile spermatozoa, apparent in ageing semen samples and which has been associated with cell death. In this study, transmission electron microscopy revealed that the apical ridge acrosomal matrix was extended into complex folds and/or projections. Acrosomal and plasma membrane integrity was retained. Storing spermatozoa (1500 × 106/ml) in seminal plasma at 4°C for 1 day was most conducive to the swelling of the apical ridge. Replacing seminal plasma with egg yolk—citrate inhibited swelling. However, incubating semen at 37°C in egg yolk—Tris—fructose extender (25 × 106 spermatozoa/ml) after storage in egg yolk—citrate at 4°C for ≥ 3 days restored the swelling characteristic.

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P. L. SENGER and R. G. SAACKE

The inability to define accurately the cytological characteristics associated with fertile mammalian ova is due, at least in part, to difficulty in obtaining and preparing adequate numbers of these cells for critical morphological studies. This communication describes a method which has proved useful in preparing single bovine ova for study using both light and electron microscopy. Bovine ova were recovered by follicular aspiration immediately following slaughter. Aspiration was accomplished with a 20-gauge hypodermic needle and a 2-ml syringe (Pl. 1, Fig. 1). The follicular fluid was placed in a watch-glass (Pl. 1, Fig. 2) and scanned, using a stereomicroscope in order to locate the ovum. Following location, the ovum was transferred with a micro-pipette from the follicular fluid to a fixation ampoule suspended in an ice water bath (Pl. 1, Fig. 3). The fixation ampoules were
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R. G. SAACKE and C. E. MARSHALL

The acrosomal cap of mammalian spermatozoa has received considerable attention from investigators using both light and electron microscopy. Recently, a comprehensive review of the subject has been published (Hancock, 1966). The structure of the acrosomal cap has been difficult to describe due to alterations which the cap undergoes during cell ageing, or after cell death (Hancock, 1953), and the labile nature of the acrosomal cap to chemical or physical processes involved in tissue preparation for microscopy (Bishop & Austin, 1957). The relationship of acrosome morphology and its alteration to the potentially fertile, motile, immotile or dead cell, as well as the sequence of alteration, must be more clearly understood before the role of this structure in fertilization can be fully evaluated.

This report summarizes our observations on freshly collected bovine spermatozoa examined by light and electron microscopy following fixation and by differential interference contrast without fixation. The use