The establishment of cell associations and interactions is an important component of tissue remodeling, tissue morphogenesis and development. The ovary, probably more than other organs, exemplifies such events. Changes in cell associations and segregation of cell populations are characteristics of follicular development, ovulation, formation of corpus luteum (CL) and the demise of CL. The granulosa cells of the ovarian follicle have a number of options available to them, namely, to remain quiescent, to proliferate, to differentiate, or to be eliminated by programmed cell death (apoptosis). The molecular mechanisms that regulate apoptosis during follicular atresia has been extensively studied, and recent studies have suggested that the destiny of the developing follicles (continual growth and eventual ovulation or atresia) is dependent on the fate of the granulosa cell (survival vs. apoptosis), which is determined by the coordinated actions, balance of opposing activities and interactions of cell survival and death factors. It is widely accepted that gonadotropins either directly or indirectly regulate the expression of many proteins in the ovary, including growth factors, enzymes, and transcription factors that may impact multiple signaling cascades. Although the endocrinological regulatory mechanisms involved in follicular development and atresia have been characterized to a large extent, the precise temporal and molecular mechanisms underlying the regulation of these processes remain unknown.

C-Ski, a proto-oncogene, has been identified as the cellular homologue of v-Ski that was originally identified as the transforming gene of the avian Sloan-Kettering retroviruses, which transform chicken embryonic fibroblasts and lead to anchorage-independent growth. Ski protein is a nuclear transcriptional factor that does not bind DNA directly. Due to its unique binding properties with multiple factors, Ski could posses various roles in both the regulation of cellular proliferation and differentiation. Ovary is one of the tissues in which c-Ski expression has been identified but the role of this gene is unknown. Therefore, in the present thesis, the author aimed to locate Ski protein in rat ovaries to predict the possible involvement of Ski in follicular development, atresia and luteinization.

In Chapter I, the author examined the localization of Ski in the rat ovaries of the estrous cycles and having single generation of developing and atretic follicles in order to predict the possible involvement of Ski in follicular development and atresia. Ovaries obtained on the day of estrus were subjected to immunohistochemical analysis for Ski and proliferating cell nuclear antigen (PCNA) in combination with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). Ski was expressed in granulosa cells that were positive for TUNEL, but negative for PCNA in the follicles during the estrous cycle. Expression of Ski in TUNEL-positive granulosa cells, but not in PCNA-positive granulosa cells, was also verified in immature hypophysectomized rats having a single generation of developing and atretic follicles by treatment with 40 IU of equine chorionic gonadotropin (eCG). On day 2 after eCG administration, where preovulatory follicles with PCNA-positive granulosa cells were evident, neither Ski- nor TUNEL-positive granulosa cells were observed. On day 4 after eCG administration, a numerous number of follicles with TUNEL-positive granulosa cells appeared, indicating that these follicles were undergoing to atresia. TUNEL-positive granulosa cells in these atretic follicles were mostly positive for Ski, confirming the above findings that Ski is profoundly expressed in the granulosa cells of atretic follicles, but not in growing follicles. Taken together, these results suggest that Ski plays a role in apoptosis of granulosa cells during follicular atresia.

In response to the luteinizing hormone (LH) surge, a preovulatory follicle embarks on a terminal differentiation pathway (luteinization) that transforms granulosa and theca cells of a preovulatory follicle into luteal cells to form CL. Luteinizing follicular cells then undergo specific morphological changes as well as physiological alterations in their transition to luteal cells. In Chapter I, the author demonstrated the presence of Ski in granulosa cells of atretic follicles, suggesting the role of Ski as a proapoptotic factor. The preliminary experiments demonstrated c-Ski mRNA is abundantly expressed in luteal cells of the CL, suggesting the possible involvement of Ski in regulating luteinization. Thus, in ChapterII, the author examined Ski protein localization in the rat ovaries during ovulation and subsequent CL formation in order to predict the possible involvement of Ski in luteinization. Immunohistochemical analysis of Ski was performed on ovarian sections obtained from rats having single generation of CL. Follicular growth was induced in immature rats with eCG, then followed by 15 IU of human chorionic gonadotropin (hCG) to induce subsequent ovulation and luteinization. In this model, ovulatory rupture occurred 12 h after hCG injection. Ski was expressed in luteinizing granulosa cells at 6 and 12 h after hCG injection, and its expression was persisted after the formation of CL. No Ski-positive granulosa cells were observed in preovulatory follicles unless hCG was injected as was shown in Chapter I. Quantitative analysis revealed that the proportion of Ski-positive cells at 24 h after hCG injection was higher than that of the previous time points (before 12 h), suggesting that the number of Ski-positive cells increased after ovulation. Taken together, the results indicate that Ski expression is induced in granulosa cells by the effect of hCG (LH) and suggest that Ski may also play a role during luteinization of granulosa cells as well as apoptosis of granulosa cells during follicular atresia as shown in Chapter I.

Then, the author examined whether hCG (LH) directly induces gene expression of c-Ski in granulosa cells. Unexpectedly, c-Ski mRNA was expressed in granulosa cells regardless of hCG (LH) treatment in vivo and in vitro, and its expression level was unaffected by hCG (LH) treatment. The observation that c-Ski mRNA is expressed in granulosa cells before hCG injection, while Ski protein is absent, raises the possibility that the amount of Ski protein is regulated at the translational (including degradation), but not transcriptional, level during luteinization of granulosa cells. Of the factors reported so far, the author picked up Arkadia as a possible candidate that regulates Ski protein level, since Arkadia is shown to be responsible for the degradation of Ski protein. Thus, the author examined the expression level of Arkadia mRNA during luteinization of granulosa cells by real-time PCR. Contrary to the author's expectation, the level of Arkadia mRNA expression was unchanged during luteinization of granulosa cells, suggesting that contribution of Arkadia in the regulation of Ski protein level during luteinization might be little if any.

The results obtained in the present thesis clearly demonstrated that Ski protein is expressed in granulosa cells during follicular atresia and luteinization, but not in those in proliferating phase, suggesting that Ski plays multiple roles in apoptosis and differentiation of granulosa cells. In addition, the presence of the novel regulatory mechanism of Ski protein in response to hCG (LH) at translational or degradation level was also suggested. Further studies to explore the precise role of Ski as well as its regulatory mechanism must bring a new insight to understand the follicular atresia and postovulatory luteinization.

Skiはproto-oncogene、c-skiの翻訳産物で、核内転写調節因子として機能することが知られている。Skiは多くの組織で発現し、細胞の増殖や分化に伴い発現量が変化することから、細胞機能の調節に重要な役割を担っていることが推察されている。本研究では、卵胞発育や黄体形成などの卵巣機能の調節におけるSkiの役割を明らかにすることを目的とした。

第1章では、卵胞の発育および閉鎖過程におけるSkiの発現の解析を行った。まず正常性周期を回帰しているラット卵巣の凍結切片を作成し、Skiの局在を免疫染色により調べるとともに、proliferating cell nuclear antigen(PCNA)の免疫染色により増殖細胞を、TdT-mediated dUTP nick end labeling(TUNEL)染色により細胞死を観察した。その結果、Ski陽性の顆粒膜細胞を含む卵胞には多数のTUNEL陽性細胞が観察されたことから閉鎖卵胞であることが示唆され、また、Ski陰性の顆粒膜細胞を含む卵胞には多数のPCNA陽性細胞が観察されたことから発育卵胞であることが示唆された。そこで、SkiとTUNELの二重染色を行った結果、Ski陽性細胞とTUNEL陽性細胞は一致することが示された。これらの結果から、卵胞閉鎖に伴い顆粒膜細胞にSki発現が誘導されることが示唆された。次に、equine chorionic gonadotropin(eCG)処理した下垂体除去未成熟ラットを用いて、この可能性についてさらに検討した。eCG投与前にはSki、TUNELともに陽性細胞は観察されなかった。一方、eCG投与48時間後にはPCNA陽性の顆粒膜細胞が多数見られ、卵胞発育が起こっていることが示されたが、Ski、TUNELともに陽性細胞は見られなかった。しかし、96時間後になるとPCNA陽性の顆粒膜細胞は見られないものの、多数のTUNEL陽性細胞が見られたことから、卵胞閉鎖が起こっていることが示された。この時、同時にSkiも陽性細胞が多数出現していた。これらの結果から、卵胞閉鎖に伴い顆粒膜細胞にSkiの発現が誘導されることが明らかとなり、Skiは卵胞閉鎖に伴う顆粒膜細胞の細胞死に関与する因子であることが示唆された。

第2章では、黄体形成過程におけるSkiの発現の解析を行った。eCG投与により卵胞発育を誘導した未成熟雌ラットにhuman chorionic gonadotropin(hCG)を投与し、経時的に採取した卵巣の凍結切片を作成し、SkiとTUNELの染色を行った。その結果、hCG投与後3時間でc-skiおよびその翻訳産物であるSkiの発現が顆粒膜細胞にみられ、この発現は引き続き黄体細胞でも観察された。これらの結果より、hCGに暴露された卵巣ではSkiは顆粒膜細胞の黄体細胞への分化やその機能維持に関与していることが示唆された。このことは、生体内ではluteinizing hormone(LH)サージが顆粒膜細胞のSki発現を誘導する可能性を示している。そこで次に、eCG投与により卵胞発育を起こした未成熟ラットの卵巣から顆粒膜細胞を採取してLH添加、非添加の条件で培養し、c-ski mRNAの発現をRT-PCRにより調べた。その結果、in vitroでのLH添加によるc-ski mRNAの増加は観察されなかった。そこで、in vivoでhCGを投与し、経時的に採取した顆粒膜細胞におけるc-ski mRNAの発現を調べた結果、やはりc-ski mRNAの増加は観察されなかった。これらのことから、Skiは遺伝子レベルではなく、翻訳や分解のレベルで発現が調節されていることが考えられた。SkiについてはArkadiaと呼ばれるユビキチンリガーゼが分解に関わることが示されている。そこで、その遺伝子発現がLHにより変化するかどうかについて調べた。In vivoでhCGを投与し、採取した顆粒膜細胞ではArkadia mRNAが発現していたが、その発現量に経時的な変化は見られなかった。LHサージによるSkiの増加は遺伝子発現の変化を伴わないことからタンパク質レベルでの制御機構が存在する可能性が示されたが、少なくともArkadiaはその過程に関与していないことが示唆された。

以上、Skiは発育過程にある卵胞の顆粒膜細胞では発現が見られないが、卵胞閉鎖や 黄体形成の開始に伴い発現が誘導され、顆粒膜細胞の細胞死や黄体化に関与することが初めて示された。これらの知見は転写因子による卵巣機能の制御機構を考える上で特筆すべき結果であると考えられ、学術的、応用的意義は少なくない。よって、審査委員一同は本論文が博士(農学)の学位論文として価値あるものと認めた。