Effect of vitamin C deficiency on testicular structure in the guinea pig.
Vitamin C has been used in the management of male infertility on emperical grounds, particularly in the presence of non-specific seminal infections. Its presence in the seminal plasma of healthy adults in high concentration, ranging from 2.5 to12mg/dl, has been reported by various authors., However, the precise role of vitamin C in relation to male reproduction is not yet clear.
Chinoy observed that ascorbic acid was essential for the structural and functional integrity of androgen-dependent reproductive organs. Marked degenerative changes were seen in the testes, epididymis and vas deferens of scorbutic guinea pigs. Besides degeneration of the spermatogenic epithelium steroidogenesis and plasma testosterone level also showed a decline. On the other hand, excessive intake of vitamin C has been reported to cause reproductive failure in the male.
In view of these data, it was decided to study the structural changes in testes of scorbutic guinea pigs and correlate them with blood ascorbic acid levels and severity of scurvy.
The study was carried out on 25 adult male guinea pigs divided into two groups. The experimental group of 15 was kept on a scorbutic diet consisting of crushed barley, crushed gram, casein, calcium carbonate and common salt, supplemented with vitamin A and D twice a week. The control group of ten was given the same scorbutic diet but supplemented with 10 mg vitamin C every day. The animals were kept under daily observation for signs of scurvy and were weighed at weekly interval.
Animals were sacrificed at end of 6 weeks. Blood was collected after decapitation and serum ascorbic acid (SAA) estimated colorimetrically using dinitrophenyl-hydrazine. Testes were taken out, fixed in Bouin's fluid and paraffin sections prepared for H-E staining. Besides detailed histological examination, a semi-quantitative evaluation of the biopsy was done using Johnson's scoring method. Carl Zeiss ocular micrometer was used for histometry after necessary caliberation.
Student's 't' test was used for statistical analysis.
Control animals continued to gain weight throughout the period of observation. In contrast to this, vitamin C deprived animals rapidly lost weight from the second week onwards, [Fig. 1]. Lassitude, bleeding from gums, arthritis, loss of hair and anorexia were clearly evident by four weeks. By the 6th week, lassitude and difficulty in moving the hind limbs became so marked that the animals avoided moving altogether.
Out of ten control animals two died spontaneously within the six week follow up (20%), while the mortality was 40% in the scorbutic group within the same period. Thus, biochemical and histological observations are based on eight healthy and nine scorbutic animals. Although the animals dying spontaneously were also autopsied, their testes were not included for fear of variable autolytic changes.
The SAA values at the end of the 6th week are shown in [Table I]. There was a statistically significant decline in SAA (p<0.001) and it reached values below 0.1 mg/dl in four of the nine scorbutic animals, The lowest value in healthy guinea pigs was 0.55 mg/dl, with a mean of 0.73 mg/dl. While the two testes constituted 0.80 ± 0.02 per cent of the total body weight in healthy guinea pigs, it had fallen to 0.48 ± 0.01 per cent after six weeks of vitamin C deprivation.
At the end of 6th week, the scorbutic guinea pigs showed marked testicular degenerative changes [Fig. 2] and [Fig. 3]. Spermatogenesis had evidently ceased altogether and only a few actively dividing cells with mitotic figures could be made out. Only basal germ cells (spermatogonia) and Sertoli cells could be clearly made out. The rest was a diffuse, hyalinised, cytoplasmic mass. There was little change in the basement membrane which consisted of a minimal amount of fibrous tissue and fibroblast nuclei. Overall, the tubules appeared shrunken in diameter as compared to the control.
The interstitial tissue did not show any significant change. Leydig cells were apparently normal both in number and microscopic appearance.
The severity of testicular changes as measured on Johnson's 0-10 scale is also shown in [Table I]. The mean score in normal guinea pigs was 9.3. Score of 9 stands for 'many spermatozoa present, but somewhat disorganised', while 10 represents 'complete spermatogenesis and perfect tubule'. On the other hand, scorbutic guinea pigs had a mean score of 4.0 which implies 'only a few spermatocytes present'. The change was clearly significant (p< 0.001).
The tubular diameter and the average height of the germinal epithelium, from the basement membrane to the inner limit of the attached spermatids/spermatozoa, were measured in hundred circularly cut tubules from ten sections of each animal. In the case of scorbutic animals, the width of the germinal epithelium was taken from the basement membrane to the inner limit of the acellular cytoplasmic mass. The tubular diameter (mean ± S.E.) was 207.2 ± 6.7µ in the control and 164.3 ± 2.6µ in the scorbutic. Corresponding figures for the average height of the germinal epithelium were 60.5 ± 1.7µ and 46.7 ± 1.4µ in the control and scorbutic, respectively. The difference in both these parameters in the two groups was statistically significant.
Guinea pig, like man, does not synthesize vitamin C because of the absence of L-gulonolactone oxidase enzyme in the liver. A diet deficient in vitamin C produces acute scurvy in guinea pigs in a period of about 3-4 weeks. In the present study, the typical signs of scurvy like lassitude, anorexia, loss of weight, painful joints, difficulty in movement etc. became marked from the third to fourth week of starting a vitamin C deficient diet.
By the end of the 6th week, full blown scurvy was confirmed by the very low SAA level of 0.10 ± 0.01 mg/dl compared to 0.73 ± 0.03 in control guinea pigs. In acute scurvy in man, ascorbic acid may almost disappear from the serum. The control guinea pigs were in optimal vitamin C status and the mean ascorbic acid values found were almost comparable to those reported by Sharma in a larger number of Indian guinea pigs.
The sharp fall in ascorbic acid levels was associated with severe degenerative changes in the testes. Their weight had fallen from 0.80 ± 0.02 per cent of total body weight to 0.48 ± 0.01 per cent, indicating that the testicular atrophy was more severe than could be accounted for by the general weight loss. Histologically, active cell division and sperm formation had ceased and only the basal spermatogonia and Sertoli cells remained unaffected. On Johnson's scale the mean testicular biopsy score came down significantly from 9.3 in the control to 4.0 in the scorbutic (p<0.001).
Histometry confirmed the marked decline in spermatogenic activity. Kocen and Cavazos have also observed spermatogenic arrest in majority of scorbutic guinea pig testes. On the other hand, Mukherjee and Banerjee found that the guinea pig in I acute scurvy shows normal spermatogenesis, with minimal degenerative changes in the Leydig cells. This could possibly be due to the fact that they had observed their animals only for three weeks, by which time spermatogenesis was not affected. In the present study, six weeks of complete vitamin C deprivation has led to severe testicular degeneration and spermatogenic arrest. However, no consistent relationship could be made out between the SAA level and the severity of testicular damage.
The mechanism by which vitamin C deficiency inhibits spermatogenesis is still not clear. It could be an indirect consequence of the general cachexia and inability to utilise essential nutrients, particularly carbohydrates. Kocen and Cavazos have found a broad correlation between the degree of testicular degeneration and the loss in body weight. Another possibility is that vitamin C deficiency produces depressed steroido-genesis in the Leydig cells. Early degenerative changes in Leydig cells have been reported by Kocen and Cavazos and Mukherjee and Banerjee. Although in the present study with light microscope no definite changes could be made out in the Leydig cells within six weeks, it cannot be denied that their function may be depressed without any structural abnormality.
To what extent milder and more chronic forms of vitamin C deficiency can affect re- productive function in man needs to be examined in depth since man also depends solely on dietary vitamin C for his nutritional requirements.