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R. C. JONES

Spermatozoa of domestic animals may vary considerably in response to handling in the laboratory (Mann, 1964) and during preparation for artificial insemination (Jones, 1971a), but it is not known why such structurally similar cells should behave so differently. The plasma membranes of spermatozoa from the various species may differ in physical properties, but because spermatozoa are structurally unsuitable for tests which are performed on other cells, such as erythrocytes, this is not an easy hypothesis to test.

Jones (1971b) found that when boar semen was fixed in solutions of glutaraldehyde containing from 175 mm- to 100 mm-sodium cacodylate, the decrease in buffer concentration caused the plasma membranes of the spermatozoa to swell and break; around the head, where the response was greatest, the amount of swelling and incidence of breakage was inversely related to the buffer concentration of the fixative. As these methods may provide a measure

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R. C. JONES

As a sample of semen contains spermatozoa of various types depending on their normality, stage of maturity and degree of cytolysis, a quantitative study of the structure of the sample must consider the frequency of the different morphological types in a representative sub-sample. The effects of treatments may be determined by comparing different sub-samples. Since the methods that have been described for preparing spermatozoa for electron microscopy (Birch-Andersen & Blom, 1963; Saacke & Almquist, 1964; Healey, 1969; Quinn, White & Cleland, 1969) have disadvantages which make them unsuitable for use in such a study, a number of different methods have been examined. The procedures described below were developed for speed and convenience. They have given consistent results when used to prepare samples of spermatozoa from various sources (e.g. epididymis, semen, diluted and stored semen) and from a variety of mammals including the domestic ungulates, carnivores and rodents. For electron microscopy,

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R. C. JONES

The sequence of structural changes in the head of mammalian spermatozoa which have been associated with ageing or death (see review by Hancock, 1966) were determined in bull spermatozoa by Saacke & Marshall (1968). Bedford (1970) considered this acrosomal disintegration to be a `nonspecific degeneration' or `false' acrosomal reaction, quite different from the `true' acrosomal reaction (Barros, Bedford, Franklin & Austin, 1967) which involves vesiculation of the opposing acrosomal and plasma membranes. These reactions do not, however, account for all morphological modifications of the acrosome and the subject has therefore been examined further. This report summarizes the results of a number of studies, and records for boar spermatozoa the degenerative changes already described in bull spermatozoa by Saacke & Marshall (1968), which involved vacuolation of the acrosome. Another sequence of changes, not previously reported and which involves vesiculation of the acrosome, is also described.

Experiment 1 involved further examination of

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R. C. Jones

Summary. The mean spermatocrit and sodium, potassium and protein concentrations of fluid collected from the rete testis of the elephant were similar to values described for the ram. Sperm maturation, as assessed by the location of the cytoplasmic droplet in the middle piece, occurred in the distal head and the isthmus of the epididymis (middle segment). Spermatocrit determinations indicated that 96% of the fluid leaving the testis was reabsorbed by the efferent ducts and proximal initial segment, and 53% of the remainder was reabsorbed in the more distal parts of the head of the epididymis (initial and proximal middle segments). Sodium was reabsorbed in the same concentration as luminal fluid and not in amounts equimolar with potassium. The potassium concentration increased from 12·1 mequiv./1 in rete testis plasma to 64·8 mequiv./1 in the proximal head of the epididymis. About two thirds of the protein in rete testis fluid (3·8 mg/ml) was reabsorbed by the efferent ducts and more was absorbed by the head of the epididymis.

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R. C. Jones

Summary. Micropuncture samples of luminal fluid were collected from the rete testis and along the epididymis. Quantitative analyses showed that the ductuli efferentes reabsorb about half the protein leaving the testis. Considerable protein is secreted by the caput epididymidis (initial segment) and there is a net loss of protein from the corpus and cauda epididymidis.

Denatured, polyacrylamide gel electrophoresis showed that there are 5 proteins in rete testis fluid which are not present in blood (M r of 14 700, 22 800, 24 100, 43 200 and 44 800). One of these proteins (M r 14 700) is lost from plasma in the ductuli efferentes and 2 (M r 43 200 and 44 800) are lost in the corpus epididymidis. Twelve proteins appear in the epididymal plasma and are not present in rete testis fluid or blood: 6 appear in the caput epididymidis (M r 30 000, 31 000, 32 300, 17 400, 18 700 and 21 400), 3 in the corpus epididymidis (M r 12 800, 39 800 and 90 600) and 3 in the cauda epididymidis (M r 10 900, 56 300 and 63 000). A protein with the same molecular weight as a blood protein (149 500) accumulates in the corpus and cauda epididymidis.

None of the samples of luminal fluid contained particulate matter other than spermatozoa, indicating that the tammar is a useful animal for micropuncture studies.

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M. Lin and R. C. Jones

Summary. The spatial arrangement of the stages of the cycle of the seminiferous epithelium of the Japanese quail was investigated by preparing three-dimensional reconstructions of a seminiferous tubule from each of 3 quails. It was found that the stages were not distributed at random, but were arranged in a wave which spiralled helically along a seminiferous tubule. Adjacent stages in space were always adjacent numbers in the cycle of the seminiferous epithelium. Complete spermatogenetic waves were found in which all 10 stages of the cycle were in sequential order. However, in most waves the sequential order of stages was disturbed by the occurrence of modulations. The area of a cellular association varied from 4600 to 41 600 μm2 with a mean ± s.e.m. (3 animals) of 17 902 ± 2614 μm2. The number of Sertoli cells involved in an association ranged from 4 to 35, with a mean ± s.e.m. (3 animals) of 13·5 ± 2·8

The findings support our earlier suggestion that the kinetics of spermatogenesis in the quail are fundamentally similar to the pattern which has been described for mammals.

Keywords: spermatogenesis; cellular associations; Japanese quail

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D. Djakiew and R. C. Jones

Summary. Extratesticular sperm maturation in the echidna mainly occurs in the initial segment of the ductus epididymidis. The process involves the development of motility, migration and loss of the cytoplasmic droplet, a decrease in permeability to Congo red and the formation of sperm bundles. The spermatozoa are supported in the bundles by a matrix of electron-dense material; the bundles are very motile in undiluted samples of luminal fluids.

Micropuncture studies of anaesthetized echidnas showed that the ductuli efferentes absorb 74% of the fluid and 46% of the soluble protein that enters them. The initial segment of the ductus epididymidis absorbs 83% of the fluid which enters it, and its secretions increase the concentration of protein in luminal fluid by 107%. Denatured, linear-gradient polyacrylamide gel electrophoresis of micropuncture samples showed that 1 protein (apparent Mr = 100 500) which is not present in blood plasma is present in rete testis fluid, and a glycoprotein which is present in rete testis fluid (apparent Mr = 78 500) is absorbed by the ductuli efferentes. Six proteins which are not present in blood plasma are secreted into the initial segment of the ductus epididymidis; 5 are glycoproteins (apparent Mr = 48 500, 39 000, 32 000, 20 500 and 19 000) and one (apparent Mr = 82 500) is not. The most prominent electrophoresis bands corresponded to the glycoproteins with apparent molecular weights of 48 500, 20 500 and 19 000.

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R. C. Jones and J. Clulow

Summary. Micropuncture, microanalytical and microelectrode techniques were used to study electrochemical aspects of 7 elements and fluid in the ductuli efferentes and ductus epididymidis of the tammar. Rete testis fluid was isosmotic with blood and had a lower pH. It also contained lower concentrations of bicarbonate, sodium, calcium, magnesium, phosphorus and sulphur and higher concentrations of potassium and chloride than blood. The luminal fluid was acidified further during passage through the sperm ducts and all of the elements which were studied moved in or out of the lumen, usually against an electrochemical gradient. The ductuli efferentes reabsorbed 87% of the fluid leaving the testis without changing the intraluminal concentrations of sodium, potassium and calcium, but the concentrations of magnesium, phosphorus and sulphur increased. The caput epididymidis reabsorbed about half the fluid entering it: sodium concentrations decreased and those of potassium and phosphorus increased. There was also some fluid reabsorption and an increase in the values of potassium and phosphorus in the corpus epididymidis. There was little net transport of fluid in the cauda epididymidis; sodium, chloride, magnesium and phosphorus concentrations decreased and potassium values increased.

Studies involving filtration through a dialysis membrane of blood and fluid from the rete testis and cauda epididymidis showed that, whilst some of the calcium, magnesium, phosphorus and sulphur was associated with high molecular weight compounds in blood, the association was not significant in the reproductive fluids.

It is concluded that, except for chloride, the concentrations of the elements vary along the epididymis of the tammar in a way similar to (but different in magnitude) the pattern described for eutherians like the rat. However, the tammar does not accumulate as much organic material in the duct lumen as does the rat and little is present in the cauda epididymidis.

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J. Clulow and R. C. Jones

Summary. Male Japanese quail have relatively large testes (2·26% of body weight), a rapid rate of spermatogenesis (14·4–15·8 days) and an efficient production of spermatozoa (92·5 × 106/g testis per day). The daily output of spermatozoa is high (308 × 106 per bird, 2·08 × 106 per g body weight). The total number of extragonadal spermatozoa was 308 ± 22 × 106 per bird. Spermatozoa were transported through the genital ducts in about 1 day, maturing quickly in the epididymal region and stored briefly in the ductus deferens. Spermatozoa isolated in the ductus deferens by ligatures around the duct rapidly lost the capacity for motility after 3 days. It is concluded that, compared to mammals such as the rat, the reproductive strategy of the quail involves the rapid production, maturation and transport of spermatozoa through the reproductive tract, in association with a limited capacity to store spermatozoa for long periods within the male genital ducts.

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R. C. JONES and I. C. A. MARTIN

Summary.

In a factorial experiment skim milk, egg-yolk-citrate and synthetic diluents composed of fructose or lactose, sodium chloride and phosphate buffer containing 3·0% w/v of a lyophilized preparation of non-dialysable solids from (cow) milk were used as diluents for deepfreezing ram spermatozoa and for incubating spermatozoa at 37° C after thawing. All samples of semen were diluted forty-fold before freezing. Egg-yolk-citrate was inferior to skim milk as a diluent for freezing spermatozoa and for incubating spermatozoa after thawing. Of the two synthetic diluents, lactose synthetic was better for freezing spermatozoa whilst fructose synthetic was better for incubating spermatozoa after thawing. Only spermatozoa frozen in the lactose synthetic diluent and resuspended after thawing in the fructose synthetic survived incubation at 37° C for 2 hr as well as spermatozoa frozen and incubated in skim milk diluents.

Two factorial experiments compared egg-yolk-citrate and skim milk as diluents for freezing semen diluted from ten- to forty-fold. At ten-fold dilution, revival was much the same after freezing in milk or egg-yolk-citrate. A twenty- or forty-fold dilution was better than a tenfold dilution in milk, but revival was depressed at these higher dilution rates after freezing in egg-yolk-citrate. When semen was frozen at a tenfold dilution it was advantageous to resuspend spermatozoa in a diluent free of glycerol after thawing. A diluent based on Krebs—Henseleit— Ringer solution containing 0·5% w/v of non-dialysable milk solids was better for incubating spermatozoa after thawing than egg-yolk-citrate or milk.

A period of 5 hr equilibration at 5° C before freezing was better than 30 min equilibration.