DECREASE IN NUCLEAR FEULGEN-POSITIVE MATERIAL (DNA) UPON AGING IN IN VITRO STORAGE OF BOVINE SPERMATOZOA

Abstract

The Feulgcn-DNA content of sperm ceUs from 5 bulls was studied by means of microspectrophotomctry after storage at 5°C for 2, 3, 5, and l0 days in a yolk-citrate diluent permitting slow aerobic metabolism. A subsample of sperm cells from each bull was subjected to the Feulgen technique on each of the storage days selected. The cells sampled on each of these days received a standard 12 minute, 60°C hydrolysis. Absorption measurements at 546 m# of the individual cells indicated a marked progressive decrease in the Feulgen-DNA content of the stored spermatozoa. The loss of 30 per cent of the initial DNA at the end of 5 days' storage was highly significant statistically. This decrease approximately parallels the known decrease in fertility of stored sperm cells, as well as the increase in apparent embryonic mortality resulting from the use of similarly aged spermatozoa for artificial insemination.The relative constancy of the deoxyribonucleic acid (DNA) in the resting cell nucleus is a basic tenet of modern biology (8). This tenet for the male gametes is implicit in the recommendation that Feulgen-stained bull spermatozoa could provide a standard reference material for the microphotometric quantitation of DNA in other cell nuclei (14).The assumption of constancy in spermatozoa arises from a number of observations. Among these is the correlation, first established in the bovine (34), between the DNA content of haploid spermatozoan nuclei and the DNA content and the number of chromosome sets to be found in spermatogenic and somatic tissues (10,17,21,34).A second supporting fact is that in the absence of chromosome change the DNA content of cell nuclei appears to be relatively stable (1), for, except in the case of some secretory cells (16), it does not become metabolically so involved in the activity of cells as do other cellular components. This fact has lent support to the commonly held belief that the spermatozoon serves primarily as a convenient mechanism for transport of genetic information via DNA. During this transport it has been assumed that the DNA is wholly passive and is not involved in energy exchange mechanisms.No one seems to have seriously challenged the concept of constancy of the DNA in living spermatozoa, except Salisbury, who did so recently (22) on the basis of an increase in apparent early embryonic mortality in cattle resulting from the aging of spermatozoa by storage at 5°C before insemination (23). The evidence involving the aging of spermatozoa in the incidence of early embryonic mortality in cattle is supported by evidence on aging of spermatozoa in the female reproductive tract for other mammalian species--the rat and the guinea pig (28, 29)--and for poultry (9,20)

Dna Content of Human Spermatozoa After Storage at Low Temperatures

“…The difficulty does not, however, appear to be any function of faulty transmission of hereditary material in the spermatozoa. Salisbury et al (1961) endorsed the latter possibility in part on the basis of their finding that the use of aged bovine spermatozoa resulted in an apparent in¬ crease in early embryonic mortality (Salisbury, Bratton & Foote, 1952 Leuchtenberger (1958) points out that error due to non-homogeneous distribution of absorbing nuclear material is greatest at the absorption peak, 546 µ. It is possible that our results are, in some part, an artifact of this type of error.…”

“…Salisbury, Birge, De La Torre & Lodge (1961) reported that bovine sperm stored at 5°C for 10 days showed a progressive decrease in Feulgen-positive material, amounting to 30% by the 5th day. Chang (1959) found, also by means of Feulgen staining, that rabbit spermatozoa recovered from the female reproductive tract after insemination contained more DNA than freshly ejaculated spermatozoa.…”

Ageing of Rabbit Spermatozoa in the Male Tract and Its Effect on Fertility

“…These changes are distinct from, if additional to, presumed deterioration of the acrosome which will affect sperm penetration and, if widespread, will ultimately cause total failure offertilization. Ageing of bull spermatozoa in vitro has been shown to bring about a change in the reaction of the spermatozoa to Feulgen staining and to influence prenatal losses (Salisbury, Birge, de la Torre & Lodge, 1961 ;Salisbury, 1967) and there is evidence also that, in the rabbit, ageing in vivo of epididymal spermatozoa influences the DNA content of the spermatozoa (Bouters, Esnault, Salisbury & Ortavant, 1967). There is, thus, considerable support for the notion that the ageing of spermatozoa affects their DNA content.…”

Embryonic Survival Subsequent to Culture of Rabbit Spermatozoa at 38 and 40 C

“…When bull spermatozoa were aged in vitro at 4°C there was an increase in the embry¬ onic death rate associated with increased storage time (Salisbury & Flerchinger, 1961). These spermatozoa had a decreased dna content (Salisbury, Birge, De La Torre & Lodge, 1961). It is generally assumed that the chromatin of the mature spermatozoa is inert, that is to say, that it is not actively taking part in cellular metabolism; however, Graves & Salisbury (1963 have shown that labelled glycine, thymidine, glucose and fructose could be incorporated into the dna of the spermatozoa in vitro.…”