The gonad, or genital ridge, is unique among all organs, because of its bipotential ability to differentiate into either a testis or an ovary. For this reason, the gonadal sex differentiation is a particularly interesting model that affords opportunities for comparative analyses in organogenesis. In mammals, sex differentiation of gonads, which governs the sex of individual's whole body, is genetically determined depending on the presence or absence of Sry (sex-determining gene on Y chromosome) encoding a putative transcription factor. The male-specific expression of Sry in mice is transiently activated for a short period from 11.0 days post coitus (dpc) to 12.0 dpc in gonadal somatic cells (pre-Seroli cells). In pre-Sertoli cells, Sry directly initiates the transcription of Sox9, a member of Sry-related gene family, which is also necessary and sufficient for testicular differentiation as well as Sry. Unlike Sry, however, Sox9 continues to be expressed in pre-Sertoli cells throughout testis development, suggesting that the maintenance of sufficiently high-levels of Sox9 expression is crucial for the subsequent testis formation. In male gonads, Sry/Sox9 initiates several male-specific morphogenetic events such as coelomic cell proliferation, mesonephric cell migration into gonad, vasculogenesis just beneath coelomic epithelia and cord formation until 12.5 dpc. In female gonads, on the other hand, no clearly-defined morphogenesis is detected in ovarian differentiation at this period (Fig. 1). These testis-specific morphogenetic events during sex differentiation period suggest that male gonads have a higher energy metabolism than female ones. However, the regulatory mechanisms of energy metabolism remain to be elucidated in not only gonadal sex differentiation but also general organogenesis.

In general, glucose is widely known as the major cellular energy source and forms an energy reserve as glycogen, a polymer of glucose. Previous study has shown that embryonic testis is one of glycogen-rich tissues in mouse organogenic embryos, and that glycogen accumulation predominantly occurs in the differentiating Sertoli cells within newly-formed testicular cords at 12.5 dpc (Kanai et al., 1989). Moreover, the glycogen deposits in pre-Sertoli cells rapidly disappear shortly after the testicular cord formation, suggesting that the glycogen granules in pre-Sertoli cells act as an energy source in the dynamic morphogenesis of testis. However, no further information concerning the molecular mechanisms initiating glycogen accumulation and its functional significance in developing XY gonads is available at present.

In chapter 1, to reveal the mechanisms regulating glycogen accumulation in male gonads, I performed detailed histological and genetic analyses, and in vitro organ culture experiments. In developing XY gonads, glycogen accumulation starts to occur in pre-Sertoli cells from around 11.2 dpc in a center-to-pole pattern, similar to the spatio-temporal profile of Sox9 expression. Glycogen accumulation was also found in XX male gonads of Sry-transgenic embryos, but not in XX female gonads of wildtype embryos at any developmental stages. These results imply that glycogenesis in pre-Sertoli cells is one of the earliest cellular events direct downstream of Sry action. Moreover, glycogen accumulation in pre-Sertoli cells was significantly inhibited by PI3K inhibitor LY294002, but not by MEK inhibitor PD98059 in vitro. In addition, active phospho-AKT (PI3K effector) showed a high degree of accumulation in gonadal somatic cells of genital ridges in a testis-specific manner, both in vitro and in vivo. Therefore, these findings suggest that immediately after the onset of Sry expression, the activation of PI3K-AKT pathway promotes testis-specific glycogen storage in pre-Sertoli cells.

In chapter 2, in order to investigate the functional significance of glycogen or high-glucose condition on gonadal sex differentiation, I performed a series of glucose-deprivation (GD) experiments in vitro, and conducted detailed histological and molecular analyses. My data demonstrated that, of the various somatic cell types in XY and XX gonads, pre-Sertoli cells are the most sensitive to glucose starvation. Although GD did not affect the initiation of Sox9 expression in XY genital ridges, it resulted in a markedly defective maintenance of SOX9 expression in pre-Sertoli cells, leading to the failure of testis cord formation and severely reduced expression of several extracellular matrix (ECM) components. The addition of FGF9 (SOX9-maintenance factor)/ECM gel (a mediator of FGF signal) restored SOX9 expression and the subsequent cord formation in XY genital ridges of GD. However, Fgf9 expression was not altered in GD XY explants, and the addition of FGF9 alone did not rescue the defective SOX9 expression or cord formation in GD. These findings indicate that the establishment of SOX9 maintenance mechanism via the ECM-mediated positive feedforward pathway in pre-Sertoli cells is a metabolically active process with high-energy requirements. This further suggests the importance of the high-glucose condition assured by glycogen in the establishment of SOX9 maintenance mechanism in testis differentiation.

In summary, here I propose a novel model of molecular mechanism regulating the energy metabolism governed by Sry from both supply and demand aspects in pre-Sertoli cells, in gonadal sex differentiation (Fig. 2). In this model, Sry induces glycogenesis [energy supply] through the activation of PI3K-AKT pathway in pre-Sertoli cells. Simultaneously, Sry also initiates a downstream cascade requiring high-energy metabolism [energy demand], involving the establishment of SOX9 expression-maintenance mechanism through an ECM-mediated feedforward pathway. The glycogen deposits in pre-Sertoli cells induced by Sry are likely to act as a backup energy source for the subsequent establishment of SOX9 maintenance mechanism. Since transient glycogen accumulation is observed in various aspects of organogenesis, such cumulative glycogen may assure sustainable energy supply for the proper completion of embryogenesis. Taken together, this study not only reveals a novel role of Sry regulating the energy metabolism in gonadal sex differentiation but also opens up the new field of mammalian embryogenesis dealing with organogenesis and energy metabolism together.

Fig. 1. Schematic representation of testis-specific dynamic morphogenesis in gonadal sex differentiation in mice

Fig. 2. A possible model showing the regulation of energy metabolism by Sry

「器官形成」とは未分化な細胞群がその運命を決定され、分化し、さらにダイナミックな形態形成を伴って、特定の器官へ分化する過程を指す。ここ20年ほどの遺伝子解析技術の発達により、器官形成に関わる遺伝子が次々に明らかになってきた。しかし一方で、遺伝子以外にも、温度や圧力など遺伝子と相互作用をする「環境的因子」が器官形成を含む個体発生に関与することが近年示唆されている。申請者はこれら環境的因子の一つとして「エネルギー代謝」に着目した。ダイナミックな器官形成過程ではエネルギー代謝も厳密に制御されていることが想像されるが、器官形成とエネルギー代謝との関連性についてはほとんど明らかになっていない。

生殖腺は唯一、一つの未分化な原基が二つの異なる器官、すなわち精巣か卵巣に分化するという非常に特殊な過程を経る。哺乳類において転写因子Sryとその直接標的因子Sox9はどちらも精巣決定に必要かつ十分な機能を持つ。マウスのSryは未分化生殖腺のセルトリ前駆細胞においてのみ胎齢11.0日(days post coitus[dpc])から12.0dpcの一過性発現を示す一方、Sox9の発現は同じくセルトリ前駆細胞において11.2dpcから始まり生後まで維持されることから、Sox9の発現が維持されることで精巣分化に必要な様々な因子が誘導されると考えられる。Sryの発現以降、体腔上皮細胞の増殖、中腎細胞の生殖腺への移入、血管形成が促され、12.5dpcまでに精巣索形成を伴う精巣への形態形成が引き起こされる。一方で卵巣では形態的な変化は起こらない。このような精巣/卵巣の初期分化における形態形成の顕著な性差から、卵巣と比べ、精巣への初期分化にはより多くのエネルギー供給を必要とすると推測されるが、これまで性分化過程でエネルギー代謝の雌雄差等については全く不明であった。そこで、本研究はマウスの生殖腺の性分化過程をモデルに、器官形成とエネルギー代謝の関係について遺伝子レベルで解析したものである。

第一章では、エネルギー供給を検討した。11.5dpcの生殖腺を組織学的に観察したところ、二次的なバックアップエネルギー源として知られるグリコーゲンがXY特異的に蓄積していた。グリコーゲンの蓄積はSox9陽性のセルトリ前駆細胞においてのみ11.2dpcから開始するというSox9と同じパターンを示した。また、XXsry性転換雄マウスにおいてグリコーゲン蓄積が誘導されたことから、Sryの直下でグリコーゲンの蓄積が制御されていることが示唆された。さらに、器官培養系に、各種シグナル伝達因子阻害剤を添加した結果、Insulinおよびその下流因子であるPI3Kの阻害剤が特異的にグリコーゲン蓄積を抑制した。また、Insulin receptorの強制活性化[bpv(phen)]やPI3Kの下流因子Aktの強制活性化により、XX生殖腺でもグリコーゲンの蓄積が誘導された。以上の結果から、Sryはセルトリ前駆細胞においてInsulin-PI3K-AKT経路を雄特異的に活性化させることでグリコーゲン蓄積を誘導していることが強く示唆された。

第二章では、グルコース飢餓培養系(Glucose Deprived:GD)を用いて、性分化期の性/細胞/イベント特異的なエネルギー要求性を比較検討した。未分化生殖腺をGD条件にて器官培養したところ、XYの精巣索形成が特異的に阻害されており、セルトリ前駆細胞特異的にGDに応じた小胞体の拡張が起きていた。セルトリ前駆細胞において、GD条件でもSryおよびSox9の初期発現誘導は正常に行われるものの、その後Sox9発現が維持出来ないため精巣索形成が異常になることが明らかになった。Sox9下流のCol9a3やLamininなどの細胞外基質(ECM)の発現がGDで著しく低下していたことから、MatrigelをGDに加えるとSox9の発現及び精巣索形成が劇的に回復した。以上の結果から、性分化期の生殖腺においてセルトリ前駆細胞での「ECMを介したSox9の発現維持機構の確立」が最もエネルギー要求性の高いmolecular eventであることが示唆された。

以上の結果から、精巣決定遺伝子Sryはエネルギー要求性の高い「ECMを介したSox9発現維持」を促して精巣索形成を誘導する一方で、事前にInsulin-PI3K-AKT経路を介して「グリコーゲン蓄積」を誘導することでエネルギー供給をも同時に制御することが明らかになった。本研究は哺乳類の器官形成とエネルギー代謝の関係を遺伝子レベルで結び付けた新しい視点を提供しており、これらの研究成果は、獣医学学術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のあるものと認めた。