NADH and NADPH Dehydrogenase Activities in the Ovary of The

NADH and NADPH Dehydrogenase Activities in the Ovary of the

Crossbred Pig×Wild Boar

Hajime MIYAMOTO,Takehiko ISHIBASHI, Sakae NAKANO*and Toshihiro MIYAZAWA* Departmentof Animal Science, Faculty of Agriculture, Kyoto University,Kyoto-shi 606 *WakayamaLivestock Experimental Station , Susami-cho,Wakayama 649-31

(Received July 17, 1981)

Abstract The histochemistry of NADH and NADPH dehydrogenases has been studied in the ovary of the crossbred pig×wild boar, what is called "Inobuta", during the estrous cycle and at puberty. The surface epithelium and ovarian stroma showed strong NADH dehydrogenase and moderate NADPH dehydrogenase activities. In general, primary and growing preantra follicles showed moderate to strong activities of these enzymes. In antral follicles NADH dehydrogenase activity was moderate in the granulosa cells, very strong in the theca interna and very weak in the theca externa. The granulosa cells of follicles over 5mm in diameter showed more activity of this enzyme than those of smaller follicles. This enzyme activity in follicles varied little with respect to stages of the estrous cycle. The theca interna of antral follicles less than 2mm showed stronger NADPH dehydrogenase activity than the granulosa cells. In contrast, as observed in the pig ovary, this enzyme activity was stronger in the granulosa cells than in the theca interna of follicles over 2mm except at metestrus. The corpus luteum exhibited strong activities of these enzymes during metestrus and diestrus, but the corpus albicans during proestrus and estrus exhibited weak or moderate activities. There were little differences in these enzyme activities between the ovaries from cyclic animals and puberal animals. In general, the distribution and intensity of NADH dehydrogenase in the ovary of the crossbred pig×wild boar coincided to some extent with those of NAD-dependent dehydrogenases although the former had a wider distribution and higher intensity than the latter. Almost the same tendency was also observed between NADPH dehydrogenase and NADP-dependent dehydrogenase. Jpn. J. Zootech. Sci., 53 (3): 193-201, 1982

Both domestic pig (Sus scrofa domesticus) and wild boar (Sus scrofa) are non- ruminant ungulates of the Suidae but belong to different subspecies. The crossbred of wild boars on domestic pigs, however, can bring forth fertile young1). The crossbred

pig×wild boar, what is called "Inobuta", makes some contributions to meat production in some districts in Japan today and differs from the pig in several respects such as reproduction, growth rate or meat quality1). Although functions of the pig ovary have been studied extensively2-4), ovarian functions of the crossbred pig×wild boar are still unknown. Reduced nicotinamide-adenine dinucleotide (NADH) dehydrogenase and reduced nicotinamide-adenine dinucleotide phosphate (NADPH) dehydrogenase are necessary

Jpn. J. Zootech. Sci., 53 (3): 193-201 193 1982 MIYAMOTO, ISHIBASHI, NAKANO and MIYAZAWA to transfer hydrogen from the dehydrogenase reaction to tetrazolium resulting in formazan precipitation. Since NADH and NADPH dehydrogenases are prerequisite for the histochemical reaction of NAD-and NADP-dependent dehydrogenases, the knowl- edge on the distribution and intensity of NADH and NADPH dehydrogenase activities is necessary to ascertain the localization of dehydrogenases5). NADH dehydrogenas is an enzyme related to general metabolic activity of the cells, and NADPH dehydrogenase is one concerned with steroidogenesis. In the present work, we studied histochemically the distribution and intensity of NADH and NADPH dehydrogenase activities in the ovary of the crossbred domestic pig×wild boar during the estrous cycle and at puberty.

Materials and Methods

The crosses of male wild boar on female Landrace×Hampshire bring forth normal young, and these animals ("Inobuta") were used. The average length of the estrous cycle of these animals is 22 to 23 days1). For the determination of stages of the estrous cycle, the number, size, and conditions of visible antral follicles and corpora lutea in the ovary were examined according to the methods described previously for the cyclic ovarian changes in the pig2, 3,6, 7). The outward signs or symptoms of estrus were also examined. The estrous cycle was divided into estrus, metestrus, diestrus and proestrus.

The ovaries from eleven mature crossbred pig×wild boar weighing 90 to 130kg and from three at puberty were obtained at slaughter. They were immediately frozen in prechilled isopentane and stored in dry ice. They were sectioned at 15μm in a cryostat maintained at about -20℃ and the sections were placed on glass slides. The activities of NADH and NADPH dehydrogenases were detected histochemically by a slightly modified method of BARKA and ANDERSON8). The sections were placed in a solution containing 20mg of NADH or NADPH, 50mg of nitro-blue tetrazolium, 10 ml of 0.2M phosphate buffer at pH 7.2～7.4, 8ml of Ringer solution and 32ml of distilled water, and incubated at room temperature for 30min for NADH dehydrogenase and 1hr for NADPH dehydrogeoase. Following incubation, the sectiorns were fixed in 10% neutral formalin solution, dehydrated in the graded alcohol solution, and cleared in xylol. They were mounted with balsam. Control sections were incubated in a solution without the substrate and no reaction was observed in any of these controls. The enzyme activity was rated on an arbitrary scale of 0 to 5 according to the formazan deposits: 0, negative; 1, very weak; 2, week; 3, moderate; 4, strong; 5, very strong. Some sections from each ovary were stained with hematoxylin and eosin for the recognition of follicular stresia.

Results

Tables 1 and 2 summarize the distribution and intensity of NADH and NADPH dehydrogenase activities observed in the ovaries from the crossbred pig×wild boar

194 Ovarian Dehydrogenases in Pig×Wild Boar

Table 1. Histochemical activity of ovarian NADH dehydrogenase in the crossbred pig×wild boar, what is called "Inobuta", during the estrous cycle and puberty

The enzyme activity was rated on an arbitrary scale from 0 to 5:0, negative; 1, very weak; 2, weak; 3, moderate; 4, strong; 5, very strong. *Granulosa lutein cells. **Theca lutein cells.

Table 2. Histochemical activity of ovarian NADPH dehydrogenase in the crossbred pig×wild boar, what is called "Inobuta", during the estrous cycle and puberty

The enzyme activity was rated on an arbitrary scale from 0 to 5:0, negative; 1, very weak; 2, weak; 3, moderate; 4, strong; 5, very strong.

195 MIYAMOTO, ISHIBASHI, NAKANO and MIYAZAWA at different stages of the estrous cycle and at puberty. NADH dehydrogenase Surface epithelium A strong enzyme activity was observed in the surface epithelium throughout th cycle and at puberty. Follicle The granulosa cells of primary follicles possessed a strong enzyme activity, The granulosa cells of growing preantral follicles showed a moderate activity and the theca cells a strong activity during all the stages of the cycle and at puberty. The granulosa cells of antral follicles less than 5mm in diameter showed a moderate activity throughout the cycle and at puberty, whereas a very strong one was observed in the theca interna of these follicles and a very weak in the theca externa (Figs. 1, 2, 4, 5). Follicles over 5mm in diameter were present only during proestrus and estrus The granulosa cells of follicles over 5mm during these stages exhibited more activity than did those of smaller follicles. The granulosa cells of atretic follicles contained a less enzyme activity compared to those of normal follicles (Fig. 3). In general, a weak enzyme activity was present in the oocyte. Corpus luteum In recently ruptured follicles, i. e. the ones only a few days after ovulation, theca utein cells and granulosa lutein cells could be clearly distinguished. At early metes-l trus, a very strong enzyme activity was present in the theca lutein cells and a moderate one in the granulosa lutein cells (Fig. 6). The enzyme activity was strong during diestrus (Fig. 7) but weakened thereafter. The corpus albicans during proestrus and estrus exhibited a moderate activity (Fig. 8). No corpus luteum was present in the ovaries from the animals at puberty.

Explanation of Figures Histochemical demonstration of ovarian NADH and NADPH dehydrogenase activities in the crossbred pig×wild boar.

Fig. 1. An ovarian NADH dehydrogenase activity at estrus. ×2.7. Fig. 2. NADH dehydrogenase in an antral follicle with a diameter of about 2.5mm at estrus. A very strong activity is present in the theca interna, but a moderate activity in the granulosa cells and a very weak one in the theca externa. ×135. Fig. 3. NADH dehydrogenase in an atretic antral follicle over 5mm in diameter at estrus. A very weak activity is recognized in the granulosa cells (compare with Fig. 2). A very strong activity is present in the theca interna and a strong one in the ovarian stroma. ×54. Fig. 4. NADH dehydrogenase in an antral follicle with a diameter of about 4mm at diestrus. The activity is very strong in the theca interna, moderate in the granulosa cells and very weak in the theca externa. ×54. Fig. 5. NADH dehydrogenase in the antral follicle with a diameter of about 3mm at puberty. The enzyme is characteristically more abundant in the theca interna than in the granulosa cells as well as in the theca externa, as observed in Figs. 2 and 4. ×135. Fig. 6. NADH dehydrogenase in the corpus luteum at metestrus. The activity is stronger

in the theca lutein cells than in the granulosa lutein cells. ×135.

Fig. 7. A strong NADH dehydrogenase activity in the corpus luteum at diestrus. ×54. Fig. 8. A moderate NADH dehydrogenase activity in the corpus albicans at estrus. Note the decline in the activity. ×54.

196 Ovarian Dehydrogenases in Pig × Wild Boar

Ovarian stroma The ovarian stroma possessed a strong enzyme activity throughout the cycle and at puberty (Fig. 3). NADPH dehydrogenase Surface epithelium A moderate enzyme activity was observed in the surface epithelium throughout the cycle and at puberty.

197 Ovarian DeMydrogenases in Pig × Wild Boar

Follicle Primary and growing preantral follicles showed a weak to moderate enzyme activity. The theca interna of follicles less than 2 mm in diameter generally showed a strong enzyme activity which was stronger than that the granulosa cells showed (Figs. 10, 11). In contrast, the activity was very strong in the granulosa cells of the follicles over 2 mm, but less prominent in the theca interna (Figs. 9, 12, 13) except for the metestrus in which the theca interna showed a slightly higher activity than did

198 MIYAMOTO, ISHIBASHI, NAKANO and MIYAZAWA

the granulosa cells. Atretic follicles, especially granulosa cells, showed a lower activity than did normal follicles. A weak enzyme activity was observed in the oocyte. Corpus luteum There was no difference in the NADPH dehydrogenase activity between the theca lutein cells and the granulosa lutein cells. The corpus luteum exhibited a stron enzyme activity during metestrus and diestrus (Figs. 14, 15), but the corpus albicans during proestrus and estrus exhibited a weak activity (Fig. 16). Ovarian stroma A moderate enzyme activity was present in the ovarian stroma throughout the stages of the cycle and at puberty (Fig. 10).

Discussion

The results of the present study indicate that both NADH and NADPH dehydro-

genases are present in nearly all the ovarian cells of the crossbred pig×wild boar, suggesting the presence of enzymes concerned with steroidogenesis and general me- tabolism. In attempts to determine ovarian hydroxysteroid dehydrogenase activities in these animals, a weak Δ5-3β-hydroxysteroid dehydrogenase (3β-HSD) activity was found in the theca interna of follicles but no activity in the theca externa and granu-

losa cells9). Furthermore, a moderate to strong 3β-HSD activity was present in the corpus luteum during metestrus and diestrus, with a less activity during other stages, and a

weak activity in ovarian stroma9). A weak to very weak 17β-hydroxysteroid dehy-

drogenase (17β-HSD) activity was present in the granulosa cells, theca interna and corpus luteum throughout the cycle, while this enzyme was absent in the theca externa and ovarian stroma9). In the present study, a very strong activity of NADH dehydrogenase was found in the theca interna of antral follicles and a moderate activity in the granulosa cells, whereas only a very weak activity in the theca externa. The corpus luteum possessed

Explanation of Figures Fig. 9. NADPH dehydrogenase in an antral follicle over 5mm in diameter at estrus. The activity is stronger in the granulosa cells than either in the theca interna or in theca externa

(compare with Figs. 2-5). ×135. Fig. 10. NADPH dehydrogenase in an antral follicle less than 2mm in diameter at metestrus. The activity is stronger in the theca interna than either in the granulosa cells or in the theca

externa. A moderate activity is seen in the ovarian stroma. ×135. Fig. 11. NADPH dehydrogenase in an antral follicle less than 2mm in diameter at diestrus. The activity is strong in the theca interna and moderate in the granulosa cells. Similar features

are observed in Fig. 10, ×54. Figs. 12 and 13. NADPH dehydrogenase in the follicles with diameters of about 3.5mm and of about 2.5mm at diestrus (Fig. 12) and puberty (Fig. 13). The activity is stronger in the granulosa cells than in the theca interna. Note the inverse relationship with NADH dehydrogenase (compare with Figs. 2-5 and with Figs. 10, 11). ×135. Figs. 14 and 15. A strong NADPH dehydrogenase activity in the corpora lutea at metestrus

(Fig. 14, ×54) and diestrus (Fig. 15, ×135). Fig. 16. A weak NADPH dehydrogenase activity in the corpus albicans at estrus. Note the marked decline in the activity. ×54.

199 MIYAMOTO, ISHIBASHI, NAKANO and MIYAZAWA a strong activity of this enzyme during metestrus and diestrus, with a less activity during other stages, and the ovarian stroma also showed a strong activity. It seemed, therefore, in the ovary of the crossbred pig×wild boar the distribution and intensity of NADH dehydrogenase paralleled to some extent those of NAD-dependent dehydrogenases, 3β-HSD and 17β-HSD,although the former had a slightly wider distribution than the latter and the activity of the former was higher. The distribution and intensity of NADPH dehydrogenase resembled those of NADH dehydrogenase. In general, a NADPH dehydrogenase activity in follicles over 2mm in diameter was, however, inversely related to NADH dehydrogenase in that NADPH dehydrogenase activity was stronger in the granulosa cells than in the theca interna. These results in the ovary of the crossbred pig×wild boar are in close agreement with the findings in the pig ovary10)

In the preliminary experiments in the ovary of the crossbred pig×wild boar, the activity of glucose-6-phosphate dehydrogenase (G-6-PDH) (NADP-dependent dehydrogenase) was very strong in the theca interna of antral follicles and moderate in the granulosa cells except in the follicles over 5mm in which the granulosa cells showed a stronger activity than the theca interna. The G-6-PDH activity in the corpus luteum was strong or very strong during metestrus and diestrus but weak in other stages (MIYAMOTO,unpublished data). Hence, the distribution and intensity of NADPH dehydrogenase in the ovary of the crossbred pig×wild boar resembled to acertain extent those of G-6-PDH as observed in the goat ovary11, 12). In the rabbit ovary a consistent relationship did not exist between the activities of NADH dehydrogenase and 3β-HSD; the former had aconsiderably wider distribution than the latter5). However, when 3β-HSD was lacking in the ovarian cells, NADH dehydrogenase was lacking, too5). In the rat and hamster ovaries, an ap- proximately consistent relationship existed between the activities of NAD-dependent dehydrogenases (3β-HSD, 17β-HSD and lactate dehydrogenase) and NADH dehydro- genase13-15). The activities of 3β-HSD in the sheep corpus luteum closely paralleled NADH dehydrogenase pattern16). Parallelism was also observed between NADP- dependent dehydrogenase (G-6-PDH) and NADPH dehydrogenase in the rat ovary13). These results suggest that differences may exist among the species in the relationship between the patterns of NAD-dependent dehydrogenases and those of NADH dehy- drogenate in the ovary, and between the patterns of NADP-dependent dehydrogenase and those of NADPH dehydrogenase.

References 1) NAKANO,S., M. NAKAMURAand K. MINAGAMI,Animal Husbandry (Yokendo. Tokyo), 26: 324-326. 1972. 2) BODA, J. M., in Reproduction in Domestic Animals. (COLE, H. H. and P. T. CUPPS, eds.) 335-357. Academic Press. New York. 1959. 3) DZIUK, P. J., in Reproduction in Domestic Animals. 3rd ed. (COLE, H. H. and P. T. CUPPS, eds.) 455-474. Academic Press. New York. 1977. 4) BJERSING,L., in The Ovary. 2nd ed. (ZUCKERMAN,L. and B. J. WEIR, eds.) 303-391. Aca- demic Press. New York. 1977. 5) WATTENBERG,L. W., J. Histochem. Cytochem., 6: 225-232. 1958.

200 Ovarian Dehydrogenases in Pig×Wild Boar

6) DAY, B. N., in Reproduction in Farm Animals. (HAFEZ, E. S. E., ed.) 279-288. Lea and Febiger. Philadelphia. 1968. 7) CUPPS, P. T., L. L. ANDERSONand H. H. COLE, in Reproduction in Domestic Animals. 2nd ed. (COLE, H. H. and P. T. CUPPS, eds.) 217-250. Academic Press. New York. 1969. 8) BARKA, T. and P. J. ANDERSON,in Histochemistry. 296-333. Harper and Row Publishers. New York. 1963. 9) MIYAMOTO,H., T. ISHIBASHI, S. NAKANOand T. MIYAZAWA,Jpn. J. Zootech. Sci., (Suppl.) 52: 71. 1981. 10) BJERSING,L., Histochemie, 10: 295-304. 1967. 11) MIYAMOTO,H., M. IRIE, T. ISHIBASHIand K. UTSUMI, Jpn. J. Zootech. Sci., 50: 870-878. 1979. 12) MIYAMOTO,H., T. ISHIBASHIand K. UTSUMI, Jpn. J. Zootech. Sci., 51: 582-587. 1980. 13) PUPKIN, M., H. BRATT, J. WEISZ, C. W. LLOYDand K. BALOGH,JR., Endocrinology, 79: 316- 327. 1966. 14) BLAHA, G. C. and W. W. LEAVITT, Biol. Reprod., 3: 362-368. 1970. 15) SAIDAPUR,S. K. and G. S. GREENWALD,Am. J. Anat., 151: 71-86. 1978. 16) DEANE,H. W., M. F. HAY, R. M. MOOR, L. E. A. ROWSONand R. V. SHORT, Acta Endocr., 51: 245-263. 1966.

イノブタ卵巣のNADHおよびNADPH 脱水素酵素活性

宮本元・石橋武彦・中野栄*・宮澤寿広*

京都大学農学部,京都市606 *和歌山県畜産試験場 , 和歌山県周参見町649-31

イノシシとブタの交雑であるいわゆるイノブタは,ブるこの酵素活性は,これ以下の大きさの卵胞のものよりタに比べて繁殖能力,発育能力,肉質などに差異がある. 強かった.黄体以外の卵巣部位におけるNADH脱水素一般代謝やステロイド代謝に関与する脱水素酵素の組織酵素活性には,発情周期による変化はほとんど認められ化学的反応は,NADHまたはNADPH脱水素酵素に依なかった.一方NADPH脱水素酵素活性に関しては,2 存しているので,イノブタ卵巣の機能に関する研究の一 mm以下の胞状卵胞の卵胞膜内層は顆粒層より強い活性環として,卵巣中のこれらの脱水素酵素活性について組を示したが,2mm以上の卵胞では発情後期を除き顆粒織化学的に検討した.成熟イノブタ(雄イノシシ× 雌ブ層が非常に強い活性を示し,豚卵巣におけると同様にそタ)11頭と春機発動期の3頭から卵巣を採取し,BARKA の活性は卵胞膜内層より強かった,黄体におけるこれらとANDERSONの方法に準じて酵素を検出した.表在上皮の酵素活性は,発情後期と発情休止期に強く,発情前期と卵巣支質は,強いNADHおよび中等度のNADPH と発情期には弱くなった,成熟イノブタの卵巣と春機発脱水素酵素活性を示した.原始卵胞と卵胞腔形成前の発動期のものとの間には,酵素活性の差はほとんど認めら育卵胞は,一般に中等度または強い酵素活性を示した. れなかった.イノブタ卵巣中のNADHおよびNADPH 胞状卵胞のNADH脱水素酵素活性は,顆粒層で中等度脱水素酵素の分布と活性の強さは,NADおよびNADP であり,卵胞膜内層で非常に強く,外層では非常に弱か依存性脱水素酵素のそれとある程度一致する傾向がみらった.また,直径5mm以上の胞状卵胞の顆粒層におけれた.日畜会報,53(3):193-201,1982

日畜会報,53(3):193-201 201 1982