In mammals, gonadal steroids play an essential role in the process of sexual differentiation of the brain during embryonic development and early postnatal life, known as the critical period. During this period, testosterone secreted from the testes in males acts to masculinize and defeminize the developing brain after conversion to estradiol by the enzyme aromatase in the brain. Our previous research has identified granulin （grn） and p130 genes as sex steroid-regulated genes in neonatal rat hypothalamus, which are probably involved in sexual differentiation of the brain. According to our observations regarding the androgen- and estrogen-dependent regulation of grn and p130 genes and the sexually dimorphic patterns of their expression in the hypothalamus during the critical period, these genes could be good parameters for assessing the androgenic and/or estrogenic properties of potential environmental endocrine disrupting chemicals （EDCs） in the neonatal brain.

EDCs include a number of environmental chemicals that interact with an endocrine system, often due to their activity as a hormonal mimic. EDCs are supposed to act genomically with agonistic or antagonistic effects on sex steroid receptors, and alter reproductive function. For example, phthalate esters that are used as plasticizers and also found at low levels in foods such as dairy products are often mentioned as suspected endocrine disrupters having （anti）-estrogenic or （anti）-androgenic properties. Moreover, since phthalates have been shown to cross the placenta and pass into breast milk in animal studies, prenatal exposure and exposure from breastfeeding may occur in humans. Therefore, there is a risk that brain sexual differentiation is affected by exposure to EDCs during the critical period. The purpose of the present study is to elucidate whether perinatal exposure to di-n-butyl phthalate （DBP）, diisononyl phthalate （DINP） and di-2-ethylhexyl adipate （DEHA） affects several aspects of reproductive function in rats, especially sexual differentiation of the brain. To this end, the dams were provided with pulverized soy-free diet （low phytoestrogen diet） containing 20, 200, 2,000 and 10,000 ppm of DBP, 40, 400, 4,000 and 20,000 ppm of DINP, or 480, 2,400 and 12,000 ppm of DEHA from gestational day （GD） 15 to postnatal day （PDN） 21, the day of weaning, and anogenital distance （AGD）, serum sex steroid levels, hypothalamic gene expression of grn and p130 during neonatal period and serum gonadotropin levels and sexual behaviors after maturation were assessed.

Exposure to the high doses of DBP, DINP and DEHA during gestational period significantly decreased food consumption and body weight gain of dams, while a significant reduction of maternal food consumption during lactation period （PND 9-17） was observed in only 10,000 ppm DBP-exposed group. At PND 1, body weights of DBP-, DINP- or DEHA-exposed pups were also significantly decreased compared with those of control pups of corresponding sexes. When AGD was normalized by the cube root of body weight, exposure to DINP of all the doses used and the higher doses （2,400 and 12,000 ppm） of DEHA decreased AGD in male neonates, though that to DBP did not affect AGD in males. In female neonates, an increase in AGD was observed in DBP- and DINP-exposed animals at the highest doses. At PND 3 and 7, perinatal exposure to DBP, DINP and DEHA did not substantially affect serum concentrations of testosterone and estradiol as compared with the control group in both sexes. On the other hand, at PND 3, the expression of grn mRNA levels in males was decreased by DEHA, and that of p130 was decreased by DBP, DINP and DEHA, though the effects were not dose-dependent. At PND 7, the expression of grn gene in female pups was increased by higher doses （2,000 and 10,000 ppm） of DBP and all the doses, except for 4,000 ppm, of DINP, while that in male pups decreased by 480 and 12,000 ppm of DEHA. Hypothalamic expression of p130 mRNA in males was increased by lower doses （20 and 200 ppm） of DBP and all the doses of DINP, whereas that of females was decreased by 480 and 2,400 ppm of DEHA.

After maturation, serum LH, FSH and testosterone levels in male rats at postnatal week （PNW） 20 were not affected by perinatal exposure to any of the chemicals used. Although the numbers of both mount and intromission in 40 ppm DINP- and 480 ppm DEHA-exposed rats were significantly decreased, perinatal exposure to DBP and DINP at other doses and DEHA at any doses used did not affect the number of mount and intromission. On the other hand, a decrease in the number of ejaculation was observed in 200 and 2,000 ppm DBP-, 40 ppm DINP-, and 48 and 12,000 ppm DEHA-exposed rats, while in 10,000 ppm DBP-exposed rats, the number of ejaculation was increased. A decrease in post ejaculation interval was observed only in 10,000 ppm DBP-exposed rats. At both PNW 8-9 and 19-20, all the control and chemical-exposed females showed regular estrous cycles. Perinatal exposure to the chemicals also did not affect serum levels of LH, FSH and estradiol at both 1100 and 1600 h on the proestrous day at PNW 20. On the other hand, lordosis quotient examined on the proestrous day was significantly decreased in all the groups of animals perinatally exposed to DBP, DINP and DEHA at any doses.

The present results indicate that gestational exposure to these chemicals affects fetal development and AGD. Since AGD is commonly regarded as an androgen-sensitive developmental measure in rodents, the antiandrogenic effect of DINP and DEHA appears to be more potent than that of DBP. On the other hand, DBP and DINP at the highest dose used increased AGD in female neonates, suggesting a large amount of these phthalates may also have a weak androgenic property. Gene expression of grn and p130 in the hypothalamus was also affected by perinatal exposure to the chemicals. Although the effects of chemicals varied depending on the sexes of neonates, but not on the doses of chemicals, the increase in grn gene expression in female hypothalamus may be due to the estrogenic properties of the pthalates, and antiandorogenic properties of these compounds may account for the changes in p130 gene expression in males. Contrary to the pthalates, the adipate might exert antiestrogenic effects on males and androgenic effects on females, but further studies are needed to clarify the precise mechanisms underlying the actions of pthalate/adipate esters on sex steroid-dependent gene expression in the hypothalamus. After maturation, serum levels of sex steroids and gonadotropins in both male and female rats, as well as estrous cyclicity in females, were not changed by perinatal exposure to DBP, DINP and DEHA, indicating that these chemicals do not affect sexual differentiation of the brain controlling the endocrine system of hypothalamo-pituitary-gonadal （HPG） axis. On the other hand, inhibitory influences on sexual behaviors, especially on ejaculation in males and lordosis in females, were observed by perinatal exposure to these chemicals. Since it has been demonstrated that ejaculation-specific circuit exists within the larger circuitry for male sexual behavior, the chemicals may directly affect the ejaculation-specific circuit within the brain by altering the expression of grn and/or p130 genes. In females, the brain region responsible for inducing preovulatory GnRH surge is supposed to be the medial preoptic area （MPOA）, and that responsible for inducing lorsosis is the ventromedial nucleus of the hypothalamus （VMH）. The chemicals used in the present study may directly affect the organization of neuronal circuits in the VMH, but not MPOA, during the processes of the sexual differentiation of the brain.

In conclusion, the present study showed that inappropriate expression of grn and/or p130 genes in the hypothalamus of neonatal rats by perinatal exposure to DBP, DINP and DEHA may results in a decrease in sexual behaviors after maturation in both sexes without affecting the endocrine system of the HPG axis. The results also suggest that grn and p130 may be involved in the processes of not only masculinization （increase in male-type sexual behavior）, but also defeminization （decrease in female-type sexual behavior） of the rat. The phthalate/adipate esters may act directly on discrete regions of the hypothalamus regulating sexual behaviors, but not regulating gonadotropin secretion, thereby affect sexual differentiation of the brain with a resultant decrease in sex-specific behaviors in adulthood.

成熟した哺乳動物に見られる性行動の発現パターンや性腺刺激ホルモンの分泌パターンなどの雄雌差は、脳の機能的・形態的な雌雄差に基づいて発現する。このような脳の性分化は、発生過程の特定の時期（臨界期）に脳がアンドロジェンに暴露されることにより起こる。例えば、ラットの場合、出生後数日の特定の時期に精巣から分泌されるアンドロジェンが脳内で芳香化酵素によりエストロジェンへと代謝され、未分化な脳に作用して雄型に分化誘導すると考えられている。申請者の研究室の従来の研究により、グラニュリン遺伝子やp130遺伝子が新生子の視床下部において性ステロイドにより発現誘導され、脳の性分化に関与することが示唆されている。一方、近年プラスチックの可塑剤等に用いられているフタル酸／アジピン酸エステル類が、生殖毒性を持つことや性ステロイド作用を修飾することなど、内分泌撹乱化学物質として作用する可能性が報告されている。このことはフタル酸／アジピン酸エステル類が脳の性分化にも影響を及ぼす可能性を示唆している。本研究は、母体がフタル酸／アジピン酸エステル類を摂取した場合、新生子の脳の性分化関連遺伝子の発現や、性成熟後の性行動などにどのような影響を与えるかを解析し、これらの物質の脳の性分化過程に対する作用を解明すること目的としたものである。

実験にはWistar-Imamichi系ラットを用いた。妊娠15日目から産後21日目まで植物エストロジェンを含まない粉末飼料にdi-n-butyl phthalate （DBP）を20、200、2000、10000 ppm、diisononyl phthalate （DINP）を40、400、4000、20000 ppm、di-ethylhexyl adipate （DEHA）を480 ppm、2400 ppm、12000 ppm含有する餌を与えた。生後1日目に、アンドロジェン作用の指標となる肛門生殖突起間距離（AGD）を測定した結果、雄においてはDINP及びDEHAによって短縮が認められ、またDBP、DINPの最高用量では雌において延長が認められた。このことから、DINP及びDEHAが雄に対してアンチアンドロジェン作用を持つことが示唆され、高用量のDBP、DINPが雌に対してアンドロジェン作用を持つ可能性が考えられた。生後3日目、7日目の血中の性ステロイドレベルには大きな影響は見られなかった。生後3日目の雄においては、DINP、DEHAの一部の用量でグラニュリン遺伝子の発現の抑制が見られ、またp130の場合はいずれの物質によっても抑制が認められた。雌では、DBPの最低用量でグラニュリン遺伝子の増加、DEHAの最高用量でp130遺伝子の減少が見られた。生後7日目の視床下部におけるグラニュリン遺伝子発現は、雄においてはDEHA群で有意な抑制が、雌においてはDBP、DINP群で有意な増加が見られた。一方、p130遺伝子発現は、雄のDBP及びDINP群で増加が、雌のDEHA群で抑制が見られた。これらの効果に用量依存性は見られないものの、これらの物質は雄ではアンチアンドロジェン作用を、雌においてはアンドロジェン作用を持つことが示唆された。DBP、DINP及びDEHAの周産期暴露は性成熟後、一部の用量で雄の性行動を抑制し、全ての用量で雌の性行動を抑制した。しかし、雌雄ともに、性ステロイド及び性腺刺激ホルモンの分泌パターンには影響を与えなかった。また雌の性周期回帰にも影響を与えなかった。これのことから、これらの物質は視床下部―下垂体―性腺軸の内分泌系には影響を与えず、行動発現系の性分化に対して影響を及ぼす可能性が考えられた。

以上、AGDに対する効果、及び視床下部遺伝子発現に対する効果より、DBP、DINP及びDEHAの周産期暴露は雄に対してはアンチアンドロジェン作用、雌に対してはアンドロジェン作用を及ぼすことが示唆された。これらの物質の作用機序については、必ずしも用量依存性が認められないこと、あるいは雌雄で影響が異なることなどから現時点では一定の結論を出すことは困難であるが、それぞれの物質が性ステロイドの作用を修飾し、脳の性分化関連遺伝子の発現に影響を与え、性成熟後の性行動に影響を与える可能性が考えられた。しかし、ホルモンレベルには影響を与えなかったことから、行動発現系と内分泌系の性分化機構が異なること、そして研究で用いた物質は行動発現系の性分化に対して限局的な影響を及ぼすことが考えられた。本研究の結果は、脳の性分化機構の新しいメカニズムを提唱するとともにフタル酸／アジピン酸エステル類がその過程に影響を及ぼし得ることを示唆するもので、学術上、応用上貢献するところが少なくない。よって審査委員一同は本論文が博士（獣医学）の学位論文として価値あるものとして認めた。