j. nutr. sci. vitaminol., 38, 305-316, 1992

J. Nutr. Sci. Vitaminol., 38, 305-316, 1992

Effects of Vitamin B12-Deficiency on Testes Tissue in Rats

Tetsunori KAWATA,1 Tomomi TAKADA,1 Fusako MORIMOTO,1

Naoko FUJIMOTO,1 Nobuo TANAKA,2 Kazuhiro YAMADA,3

Masahiro WADA,3 Tadahiro TADOKORO,3 and Akio MAEKAWA3

1 Faculty of Education, Okayama University, Tsushimanaka, Okayama 700, Japan2 Department of Internal Medicine, The Jikei University of Medicine,

Katsushika-ku, Tokyo 124, Japan3 Department of Agricultural Chemistry, Tokyo University of Agriculture,

Setagaya-ku, Tokyo 156, Japan

(Received January 27, 1992)

Summary The state of vitamin B12-deficiency in rats was evaluated by determination of hepatic vitamin B12-dependent enzyme activities after

the animals had fed on a vitamin B12-deficient soybean protein diet for 150days. The effect of vitamin B12-deficiency on testicular tissue was also

studied by morphological observations. Growth of vitamin B12-deficient rats was retarded and marked increase in urinary methylmalonic acid was

observed. Vitamin B12 contents in the organs were depressed distinctly by the deficiency, especially in testes, vitamin B12 content decreased to 2.5

ng/g. Hepatic methionine synthase and methylmalonyl-CoA mutase activities showed striking depression to 5% of the control rats and extreme vitamin B12-deficiency was confirmed. Testes weight also showed marked

decrease together with their relative weight per 100g body weight. Mor

phological observations of testes of vitamin B12-deficient rats revealed atrophy of the seminiferous tubules and aplasia of sperms and spermatids. The above results proved that vitamin B12-deficiency affected rat testes,

and suggested that the rat could be the animal model for elucidation of the mechanism of B12 action on testicular functions.Key Words vitamin B12-deficient rats, methionine synthase, testicular

morphology

There are many reports on the result of B12 administration not only in cases with human B12-deficiency but also in some other diseases in the medical standpoint. Sharp and Witts (1) found in 1962 that B12 administration in the patients of

pernicious anemia improved sperm parameters and suggested that B12 would affect male reproductive function. Since then, results of B12 administration, in particular

Abbreviations used: B12, vitamin B12; CN-B12, cyanocobalamin.

305

306 T. KAWATA et al.

CH3-B12, in male infertility have been accumulated in Japan, and the therapeutic effects of B12 have been confirmed (2-4). Further, it was reported that CH3-B12 administration to the model mice with experimentally disturbed spermatogenesis by doxorubicin (adriamycin), an anticancer agent, improved the symptom (5, 6). Those reports have suggested that B12 may play an important role in maintaining normal testicular functions. However, the effects of B12-deficiency on the testicular function and its detailed mechanism of B12 have not been clarified.

While B12-deficient animal is useful for elucidating in vivo B12 functions, it is

generally said that it is difficult to keep severe dietary B12-deficient rats (7). The cases of dietary B12-deficiency in which changes in testicular morphology and sperm

parameters were observed have been reported only in Japan (8). This has caused a disturbance in the elucidation of the mechanism of B12 action on the testicular functions.

The authors observed the testicular morphology in B12-deficient rats in which B12-deficiency was judged from B12-dependent enzyme activities as a clue for investigating the effect and the mechanism of B12 action on the testicular functions. Moreover, the authors further discussed the possibilities of dietary B12-deficient rat used as the animal model for elucidation of the mechanism of B12 action.

MATERIALS AND METHODS

Animals and diets. Male Wistar weanling rats (about 25g) were used in

experiment. They were born from dams which were fed on a B12-deficient diet

during pregnancy and lactation. Newborn rats produced in this way were weaned

on the 23th day after birth, and divided into experimental groups. The B12-deficient

diet contained the following: (in %) soybean protein*1 18.0; glucose anhydrous*2

66.6; lard*3 10.0; salt mixture*4 5.0; vitamin mixture*4 0.25; choline chloride*5 0.15.

All rats were fed on the B12-deficient diet for 150days. B12-supplemented rats

received 1ƒÊg of CN-B12 per day by oral administration. Diet and water were given

ad libitum. Rats were individually housed in stainless steel screen-bottom cages

under the room conditions of constant temperature (22•}3•Ž), and a 12h cycle of

light (7 a. m.-7 p. m.) and dark (7 p. m.-7 a. m.).

Identification of vitamin B12-deficiency. Every 30th day during the experimen

tal period, the rats were transferred to metabolic cages and their urine was collected

for 24h. The amount of methylmalonic acid (MMA) was determined by the

method of Giorgio and Plaut (10). B12 contents in organs were determined by

microbiological assay with Lactobacillus leichmannii (ATCC 4797). Extraction

*1 "Ajipron SU" (crude protein content 85.4%).*2 Produced by Ajinomoto Co.*3 Produced by Nihon Yurou Yakuhin Co.*4 Formulated according to reference (9) . CN-B12 was omitted from vitamin mix

ture. All chemicals were purchased from Wako Pure Chemicals Industries Co.*5 Produced by Tokyo Kasei Industries Co .

J. Nutr. Sci. Vitaminol.

TESTES TISSUE IN B12-DEFICIENT RATS 307

procedures of B12 from rat organs were followed as previously described by Hayashi

et al. (11). After the rats had fed for 150days as the experimental period, hepatic

B12-dependent enzyme activities were determined. Their liver was removed after

perfusion with cold saline and homogenized with 4 volumes of 0.25M sucrose

containing 40mM potassium phosphate buffer, pH 7.5, per gram of liver. The

homogenate was centrifuged at 600•~g for 10min at 4•Ž; an aliquot of the

supernatant used for determination of methylmalonyl-CoA mutase (MMCoA

mutase) activity. Moreover, the supernatant fluid of 600•~g was centrifuged at

105,000•~g for 60min at 4•Ž. The supernatant fluid was collected and used for

determination of methionine synthase (Met synthase) activity. The determination

of Met synthase activity was carried out according to the method described by

Matsuno et al. (12), except that the authors used 0.2mM NADH instead of

FMNH2. The determination of MMCoA mutase activity was carried out according

to the method described by Kolhouse and Allen (13). Protein was determined by

the method of Lowry et al. with bovine serum albumin as standard (14).

Light micrographs of testes. The testes of rats were isolated and then the

testes' adipose tissue and blood were removed. The testes were fixed with 10%

formalin neutral buffer solution, pH 7.4, for 24h, at room temperature, and were

stained with hematoxylin and eosin.

RESULTS

State of vitamin B12-deficiencyGrowth curves of rats are shown in Fig. 1. Growth retardation of the

B12-deficient rats was already observed after 30-day feeding. Final body weight in the B12-supplemented control rats at the end of 150days was 45% of the B12-supplemented control rats. And decreases of food intake and food efficiency

Fig. 1. Growth curves of the vitamin B12-supplemented and -deficient rats. * Each

point presented the weight of the rats and showed mean•~SD of five rats. •›,

B12-supplemented; •œ, B12-deficient.

Vol. 38, No. 4, 1992


Fig. 2. Urinary methylmalonic acid contents of vitamin B12-deficient rats during

experimental period. * Each point presented the methylmalonic acid content of

B12-deficient rats and showed the mean•}SD of five rats. Methylmalonic acid in

urine of vitamin B12-supplemented rats was not detected throughout the

experimental period. •œ, B12-deficient.

Fig. 3. Vitamin B12 contents in various organs of vitamin B12-supplemented and

-deficient rats . * Each bar presented the vitamin B12 content in organ of rats and

showed mean•}SD of five rats.


TE

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Org

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A

B



Fig. 4. Light micrographs of testis in vitamin B12-supplemented and -deficient rats.

A, B12-supplemented •~40; B, B12-supplemented •~200; C, B12-deficient •~40; D,

B12-deficient •~200.

Vol. 38, No. 4, 1992


were observed in B12-deficient rats, compared to B12-supplemented rats.

Urinary MMA excretion by rats fed on the B12-deficient diet during the

experimental period increased markedly (Fig. 2). The amount of MMA in urine of

30days was significantly increased by B12-deficiency, and it continued to increase

throughout the experimental period of 150days. In the B12-supplemented rats,

urinary MMA was not detected.

The contents of B12 in the organs is shown in Fig. 3. The contents of B12 in the

brain, heart, liver, spleen, kidneys and testes of rats fed on the B12-deficient diet

decreased extremely, compared to the B12-supplemented rats given oral administra

tion of 1ƒÊg/day CN-B12. B12 content in the testes of B12-deficient rats declined to 2.5•}

0.5ng/g of wet tissues.

Then, the authors determined hepatic B12-dependent enzyme activities to

evaluate the B12-deficiency. Table 1 shows the activities of hepatic Met synthase

and MMCoA mutase in rats. Hapatic Met synthase and MMCoA mutase activities

in B12-deficient rats decreased to about 5% of the B12-supplemented rats.

Rat testicular morphology

Table 2 shows the wet weight and relative weight/100g body weight of the

organs. The relative weight/100g body weight of the brain, heart, liver, kidneys and

adrenals increased by B12-deficiency. However, the relative weight of testes of rats

fed on the B12-deficient diet decreased markedly to 56% of the control, i.e., the

values were 0.50•}0.05 and 0.89•}0.04, respectively. Similarly, it was shown that

B12-deficiency depressed testes weight in rats to about 26% of the control rats. The

testes weights in B12-deficient and B12-supplemented rats were 0.63•}0.05g and 2.43•}

0.06g, respectively.

The light microscopic observations of rat testicular morphology revealed

distinct changes due to B12-deficiency. The testicular morphology of the

B12-supplemented and B12-deficient rats is shown in Fig. 4. Production of sperms

and spermatids was observed in the B12-supplemented rats given oral administration

of 1ƒÊg/day CN-B12 (Photos A and B). However, contrary to the normal testicular

functions maintained in the control rats (Photos A and B), severe atrophy of the

seminiferous tubules, aplasia of sperms and spermatids as well as fibrotic change of

the seminiferous tubules were observed in the B12-deficient rats (Photos C and D).

DISCUSSION

Although rats are the most popular experimental animals for studies of B12, it is generally said that development of severe dietary B12-deficiency in rats is difficult to be induced (7). The following reasons are given for this: biological half-life of B12 is long and feeding of the B12-deficient rats extends over a long period; rats show a tendency of coprophagy; strict control is needed for breeding as well as preparation of B12-deficient diet. On the other hand, it was found that nitrous oxide would inactivate Cob(I)alamin and reduce Met synthase activity (15, 16). Kondo et al.



(17) reported that Met synthase activity decreased markedly after the start of exposure to nitrous oxide, reaching to about 15% of the control in the rats having been exposed to 50% nitrous oxide for 23days. From this, nitrous oxide-exposed rats are used as experimental B12-deficient animal in which B12-deficiency can be reproduced in a short time and relationship of B12 to folate and Met metabolism is being investigated.

In the present study, the state of B12-deficiency in dietary B12-deficient rats was evaluated. Weanling rats were used, which were weaned from the dams fed on the B12-deficient diet throughout the period of pregnancy and lactation to keep B12 transport through the placenta to the minimum. And B12-deficient rats were kept under the strict feeding conditions to minimize the risk of contamination of B12 during the experimental period. The B12-deficient rats produced under the strict feeding conditions showed remarkable growth retardation and extreme increase in urinary MMA excretion as well as exhaustion of in vivo B12 compared to previous reports (7, 18-23). Severe B12-deficiency was confirmed by the growth, urinary MMA excretion, and B12 contents in organs of rats. Moreover, the state of B12-deficiency in the rats was also assessed by hepatic B12-dependent enzyme activities. Hepatic MMCoA mutase and Met synthase activities in the B12-deficient rats decreased markedly to about 5% of the B12-supplemented rats. The activities in the rats fed on the B12-deficient diet for 150days were lower than the previous reports (24, 25). Thus, it was considered that dietary B12-deficient rats in the

present experiment were useful in elucidation of secondary metabolic changes induced by a marked decrease in B12-dependent enzyme activities.

Dryden and Hartman (26) reported that the relative weight/100g body weight of the heart, liver, kidneys, spleen, adrenals and testes increased in the B12-deficient rats fed on the 20% casein diet ad libitum for 189-301days. Although Dryden and Hartman (26) reported an increase in the relative weight of testes, B12-deficiency depressed distinctly the relative weight of testes in the present experiment. Similarly, the wet weight of the testes in B12-deficient rats decreased to 50% of the control rats. The authors already reported that decreases in the wet weight and relative weights of the testes were not observed in rats fed on the casein diet but only observed in the B12-deficient rats fed on the soybean protein diet (27). It is suspected that the decrease in testes weight has some correlations to amino acid composition, sulfur amino acid composition in particular, in dietary protein. Further investigation of Met metabolism in testes of B12-deficient rats is needed.

In relation to human reproductive functions, there are many reports on the effectiveness of B12 administration for the treatment of male infertility. Sharp et al.

(1) administered B12 to a male patient with pernicious anemia accompanied by oligozoospermia in 1962 and improved sperm parameters leading to conception 6months later. Then, Blair (28) reported in 1968 that B12 administration was effective even in the hematologically normal patient with oligozoospermia. At

present, B12, CH3-B12 in particular, is being used as a therapeutics for male infertility and its clinical effects have been proved (2-4). These reports indicate that B12

Vol. 38, No. 4, 1992


exhibits positive effects on male reproductive functions. Meanwhile, it was reported that doxorubicin, an anticancer agent, would inhibit nucleic acid synthesis in rat testes (29-31). Oshio et al. (5) and Ozaki et al. (6) reported that changes of the testicular morphology and of the parameter of sperms in the rats administered doxorubicin, such as decreases in the number of sperms and sperm motility, were alleviated by CH3-B12. From the results of studies with a model animal of experimentally disturbed spermatogenesis, it was assumed that B12 was participating in DNA synthesis in some way to maintain normal testicular functions. However, there are many unknown points in the mechanism of B12 action on testicular functions. To elucidate this, not only the animal model with experimentally disturbed spermatogenesis but also that of dietary B12-deficiency are considered extremely useful. The rats in the present study, which were judged as being severely B12-deficient from B12-dependent enzyme activities, showed distinct changes including extreme atrophy of the seminiferous tubules, aplasia of sperms and spermatids, and fibrotic change of the seminiferous tubules. Up to now, cases of dietary B12-deficient rats with changes in the testicular morphology and sperm parameters have been reported only in Japan. Namely, Umemura (32) reported in 1964 that decreased number of mature sperms and atrophic organization of the seminiferous tubules were observed in B12-deficient rats, although testicular morphology was not

given. In 1991, Oshio et al. (8) reported that reduced diameter of the seminiferous tubules, change in testicular morphology including decreased number of spermatids and their irregular arrangement, as well as decreased number of spermatids in the epididymides and active sperms were observed in mice fed on the B12-deficient diet containing 0.3ng/g diet of B12 for 10weeks. Together with the present results, the above findings not only clarified that B12-deficiency induces changes in the testicular morphology and sperm parameters in rats or mice but also suggested the possibility that dietary B12-deficient rats serve as the animal model for investigating the mechanisms of B12 action on testicular functions. Moreover, our rats showed remarkable changes in the testicular morphology and are considered useful as a more precise model animal.

The authors acknowledge the technical assistance of Yoshiko Ikawa, Akiko Haisa, Yuka Hirose, and Akiko Fujita. This work was supported by Vitamin B Research Committee and Nishikawa Chikusan Zaidan.

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j. nutr. sci. vitaminol., 38, 305-316, 1992

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