Isolation, Characterization, and in Vitro and in Vivo Differentiation of Putative Thecal Stem Cells

Isolation, characterization, and in vitro and in vivo differentiation of putative thecal stem cells

Arata Honda*, Michiko Hirose*, Kenshiro Hara†, Shogo Matoba†, Kimiko Inoue*, Himomi Miki*, Hitoshi Hiura‡, Mito Kanatsu-Shinohara§, Yoshiakira Kanai†, Tomohiro Kono‡, Takashi Shinohara§, and Atsuo Ogura*¶

*BioResource Center, RIKEN, Tsukuba, Ibaraki 305-0074, Japan; †Department of Veterinary Anatomy, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan; ‡Department of BioScience, Tokyo University of Agriculture, Setagaya-ku, Tokyo 156-8502, Japan; and §Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan

Edited by R. Michael Roberts, University of Missouri, Columbia, MO, and approved June 7, 2007 (received for review April 26, 2007) Although ovarian theca cells play an indispensable role in follicu- clues about the mechanisms of formation and maintenance of logenesis by providing follicular structural integrity and steroid the Leydig cell population and their biochemical characteristics. substrates for estrogen production, little information is available Initially, we undertook this study to isolate putative ‘‘female about their recruitment, growth, and differentiation because their germ-line stem (GS) cells,’’ the presence of which is still highly immature forms have not been identified. We have isolated puta- controversial among reproductive biologists and stem cell re- tive thecal stem cells with the ability to self-renew and differen- searchers (6–8). We initially expected that the isolation proce- tiate in vivo and in vitro. They are similar to fibroblasts in mor- dure optimized to male GS cells from neonatal testes (9, 10) phology and proliferate in vitro as round colonies with a might be applicable to putative female counterparts in neonatal homogenous cell population. They were induced to differentiate ovaries. Under our experimental conditions, we were able to into early precursors and steroidogenic cells in a stepwise manner generate round spherical colonies that produced a number of after treatment with serum, luteinizing hormone, and paracrine small primitive oocytes as if oogenesis had proceeded inside. factors from granulosa cells. At each differentiation step, these However, detailed biochemical analysis revealed that most of the cells displayed appropriate gene expression and morphological cell types comprising the colonies were somatic cells and not markers and later secreted androstenedione. The fully mature germ-line cells. These cells were later identified as putative morphology was achieved by coculture with isolated granulosa ovarian thecal stem cells. cells. When transplanted into the ovaries, the putative thecal stem A fundamental property of stem and progenitor cell division is cells colonized exclusively in the ovarian interstitium and the thecal the capacity to retain the proliferative state or to generate differ- layer of follicles as differentiated cells. Thus, thecal stem cells entiated daughter cells. These putative thecal stem cells were appear to be present in neonatal ovaries and can be isolated, purified easily in vitro, formed characteristic anchor-independent purified, and induced to differentiate in vitro. Thecal stem cells round colonies, and, after stimulation, started to differentiate and show characteristic signs of steroidogenesis. They also colonized the

could provide an invaluable in vitro experimental system to study BIOLOGY interactions among the oocytes, granulosa cells, and theca cells host ovarian tissue after transplantation in a manner similar to that DEVELOPMENTAL during normal folliculogenesis and to study ovarian pathology of Leydig stem cells in mice and rats (4, 5). Our study provides caused by theca cell dysfunction. evidence for the presence of putative thecal stem cells in the neonatal mouse ovary. Interestingly, unlike Leydig stem cells, follicle ͉ oocyte ͉ ovary putative thecal stem cells can be purified in vitro simply by opti- mizing the culture conditions without any cell-sorting procedures. Thus, these isolated putative thecal stem cells may provide an uring folliculogenesis in mammals, oocytes grow while invaluable and reproducible experimental system to study their surrounded by an increasing number of granulosa cell D biochemical characteristics. This system would include gene expres- layers. From the preantral stage onward, theca cells differentiate sion and signaling cascades, which are essential for normal follicu- as the outer layer of the follicle. These elements provide logenesis in mammals. structural integrity and the androgen substrate for granulosa cell estrogen production, thus playing an indispensable role in fol- Results licular development (1). Mature theca cells can be distinguished Isolation of Neonatal Ovarian Cells by Selective Culture. Because the by their morphology and location in situ and can be isolated original purpose of this study was to establish cell lines of female easily in a crude suspension for in vitro analysis. Much informa- GS cells (6), cells were cultured from newborn mouse ovaries tion has been reported about the factors and mechanisms according to a protocol for male GS cells with slight modifica- regulating theca cell steroidogenesis in several mammalian spe- tions (9, 10). The culture medium was essentially a serum-free cies (2). However, our knowledge about theca cell recruitment GS medium (GSM-K) containing growth factors [see Materials and growth is limited because of the lack of in vitro experimental systems to isolate, culture, and differentiate immature theca cells. Generally, it is difficult to distinguish immature theca cells Author contributions: A.H., M.H., T.K., and A.O. designed research; A.H., M.H., K.H., S.M., from ovarian stroma cells based on their morphology (3). K.I., H.M., H.H., M.K.-S., Y.K., T.K., T.S., and A.O. performed research; M.K.-S. and T.S. contributed new reagents/analytic tools; A.H., M.H., K.H., S.M., K.I., and Y.K. analyzed data; In contrast to theca cells, their male counterparts, Leydig cells, and A.H., Y.K., and A.O. wrote the paper. have been studied in detail because their putative stem cells can The authors declare no conﬂict of interest. be isolated from mouse and rat testes by flow cytometric sorting This article is a PNAS Direct Submission. (4, 5). Leydig cells can remain undifferentiated in vitro for Abbreviations: GS, germ-line stem; GSM, GS medium; GSM-K, serum-free GSM; GSM-S, months and can respond to growth factors to differentiate into serum-containing GSM; ER, endoplasmic reticulum; LH, luteinizing hormone; MVH, mouse steroidogenic precursor cells (5). After transplantation into the vasa homologue. testes of Leydig cell-deficient mice, these stem cells recolonize ¶To whom correspondence should be addressed. E-mail: [email protected]. normally in the host testicular interstitium and restore the serum This article contains supporting information online at www.pnas.org/cgi/content/full/ testosterone concentration (4). These in vitro and in vivo ap- 0703787104/DC1. proaches using isolated Leydig stem cells may provide further © 2007 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0703787104 PNAS ͉ July 24, 2007 ͉ vol. 104 ͉ no. 30 ͉ 12389–12394 Downloaded by guest on September 29, 2021 Fig. 2. Cytological and gene expression analysis of thecal stem cell colonies. (A) Paraffin-embedded section of colonies stained for BrdU (blue) and MVH (brown). Small cells were generally positive for BrdU but not for MVH. Grow- ing oocytes (arrows) were positive for MVH but not for BrdU, indicating that the oocytes came from preexisting cells in the colonies. (Scale bar: 50 ␮m.) (B) Gene expression analysis (RT-PCR) of the colonies. After the third passage (P3), oocytes disappeared from the colonies, as demonstrated by the absence of oocyte-specific markers (zona pellucida proteins Zp1, Zp2, and Zp3). MEF, Fig. 1. Development of thecal stem cell colonies in vitro.(A) The size of the mouse embryonic fibroblast. (C) Electron micrographs of the colonies. Each colonies increased rapidly during the first several days and gradually reached cell in the colonies had a relatively small cytoplasm that was rich in ribosomes the maximum size of Ϸ120 ␮m in diameter. Many oocytes (Ϸ20 ␮min and rough ER (arrowheads) (Upper). Cell-to-cell connections were sparse, and diameter) continued to protrude from the surface of the colonies (arrows). their intercellular space was filled with basement membrane (BM) and colla- (Scale bars: Left, 100 ␮m; Right,50␮m.) (B) The colonies are weakly positive for gen fibers (CF) (Upper). A thecal stem cell (T) directly surrounds the zona alkaline phosphatase staining. (C) Effect of growth factors on colony size pellucida (ZP) of an oocyte (O), but unlike typical granulosa cells, it does not during the first 14 days of culture. Single growth factors, or some combination protrude foot processes to the oocyte surface through the zona (Lower). thereof, were added to the basic GSM. , P Ͻ 0.05; , P Ͻ 0.01 [compared with * ** Arrowheads indicate microvilli from the oocyte. (Scale bars: 2 ␮m.) growth-factor-free GSM (three replicates, one-way ANOVA)].

and Methods and supporting information (SI) Table 1]. For the a 14-day culture period. Four growth factors, bFGF, EGF, LIF, primary cell preparation, fibroblast cells were allowed to attach and IGF1, exhibited significant effects on colony growth, al- onto the bottom of the gelatin-coated culture plate, but only though they did not seem to prolong the cell proliferation phase. PDGF and glial cell line-derived neurotrophic factor, which has a floating cells were passaged to the secondary culture plate. critical effect on male GS cell proliferation, had no effect These replated cells attached weakly on the bottom of the culture (Fig. 1C). wells during an overnight culture and formed round colonies comprising compact clusters of cells with unclear borders. Characterization of Isolated Ovarian Cells. During the first round of Colony formation was observed 34 times among 44 replicated culture (14 days), many oocytes (Ϸ20 ␮m in diameter) continued experiments (77% formation rate). The colonies were consis- to protrude from the surface of the colonies and were released tently positive for alkaline phosphatase staining (five replicated into the medium as single oocytes with or without a zona experiments), as are embryonic stem (ES) cells (11) and male GS pellucida (Fig. 1A). To determine whether these oocytes had cell colonies (9) (Fig. 1 A and B). However, the appearance of been derived from mitotically active cells (presumptive female these colonies differed from the established stem cell lines, and GS cells) or from preexisting prophase-I oocytes, we performed the intensity of alkaline phosphatase staining was apparently a cell proliferation assay by adding BrdU to the culture medium. weaker than that of ES cell colonies. The colonies were easily The BrdU-treated colonies were sectioned and analyzed immu- detached from the bottom of the plate by mechanical treatment, nochemically. Most of the cells comprising the colony were Ͻ10 and these freed colonies tended to aggregate with each other. ␮m in diameter and stained positively for BrdU, but none were The colonies were somewhat difficult to disperse to single cells positive for mouse vasa homologue (MVH, a germ-cell marker). by enzyme treatment, especially when they grew into large sizes In contrast, oocytes Ͼ15 ␮m in diameter that were positive for (Ͼ50 ␮m in diameter); therefore, we could not count the exact MVH were all negative for BrdU (Fig. 2A), indicating that they cell numbers in the colonies. When the first-appearing colonies were derived from preexisting small oocytes that had already (Ϸ10 ␮m in diameter) were allowed to grow singly in culture, entered prophase I. Consistent with this conclusion, the imprint- they rapidly increased in size for the first several days and then ing status of cells comprising the colonies was the somatic type gradually reached a maximum size of Ϸ120 ␮m in diameter (parent-of-origin-specific methylation), not the germ-cell type before the first passage at Ϸ14 days (Fig. 1A). Because the size (complete demethylation or biallelic methylation) (SI Fig. 6). of cells composing colonies did not change throughout the After a few passages, the colonies ceased producing oocytes, culture period, the cell proliferation rate is estimated to be probably because their precursor oocyte stock had been de- Ϸ1.7 ϫ 103-fold during the 14 days. Their active proliferation in pleted. This finding was confirmed by the disappearance of vitro is consistent with the intense incorporation of BrdU by the mRNA for oocyte-specific genes in the colonies after the third cells comprising the colonies in our cell proliferation assay passage (Fig. 2B). (below). We examined the effect of six growth factors in the basal Thus, the small cells in the colonies were most likely somatic GS medium (GSM) (SI Table 1) on the growth of colonies during cells, but we did not know which ovarian cell type(s) was being

12390 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0703787104 Honda et al. Downloaded by guest on September 29, 2021 Fig. 4. Light and electron micrographs of cells that differentiated from thecal stem cells in vitro. The lower images are the higher magniﬁcations of the rectangular areas of the middle images. Cells in GSM-S (A) retained the original undifferentiated features, such as the presence of rough ER (RER), ribosomes (R), and mitochondria with lamellar cristae (Mtl), whereas differentiating cells in GSM-SL (B) accumulated Golgi apparatuses (G), vacuoles (V), and mitochondria with cristae intermediate between the lamellar and tubular type (arrowheads). Cells in GSM conditioned with granulosa cells (C) and GSM-SLG (D) showed more differentiated steroidogenic features. Mtt, mitochondria with typical tubular cristae; SER, smooth ER; G, Golgi apparatuses; GSM-SL, GSM supplemented with LH, IGF1, and stem cell factor; GSM-SLC, GSM conditioned with granulosa cells. (Scale bars: 10 ␮m.) Fig. 3. The stepwise differentiation of thecal stem cells as indicated by gene expression patterns and cytoplasmic lipid droplet development. (A) Gene expression analysis (RT-PCR) of differentiating theca cells under different by their differentiation in vivo and in vitro in the subsequent culture conditions. The Lhr, Gli2, and Ptch2 genes were expressed as the thecal experiments, which are described below. cells differentiated in vitro. The Fshr and Ihh genes are markers for granulosa

cells. GSM-SL, GSM supplemented with LH, IGF1, and stem cell factor. (B) In Vitro Differentiation of Putative Thecal Stem Cells. The cells BIOLOGY Cytoplasmic lipid droplets stained with Oil red O. Initially, very few lipid

remained in their undifferentiated state and showed no signs of DEVELOPMENTAL droplets were visible in the thecal stem cell colonies (GSM-K), but these steroidogenic activity when the colonies were cultured in increased in number progressively as the theca cells differentiated to their fully mature state (GSM-SLG). (Scale bar: 50 ␮m.) (C) Thecal stem cells were GSM-K. Adding serum to the media (GSM-S) caused the cells cocultured with isolated granulosa cells (GSM-SLG). To identify the cells to spread rapidly as a monolayer onto the bottom of the dish (SI differentiating from the thecal stem cells correctly, granulosa cells were Fig. 7A and also see the cell shape in Figs. 3B and 4) and to prepared from EGFP-expressing mice. As expected, only ﬂuorescence- accumulate fine lipid droplets in their cytoplasm (Fig. 3B). The negative cells had lipid droplets (granular inclusions under Hoffman optics) cell numbers increased approximately four times faster when (black arrowheads). White arrowheads indicate EGFP-expressing granulosa cells were cultured in GSM-S than in GSM-K (estimated from cells. (Scale bar: 50 ␮m.) the size of the colonies for GSM-K). Although they did not stain intensely, the cells appeared to start steroidogenesis because many of the cells stained positively for 3␤-hydroxysteroid dehy- purified during culture and passage. Electron microscopic ob- drogenase (3␤-HSD), as shown by blue formazan precipitates in servations revealed that these cells had cytological features their cytoplasm (SI Fig. 7B). However, at the ultrastructural common to undifferentiated interstitial cells, and that their level, they generally kept their undifferentiated morphology and characteristics were essentially unchanged throughout the cul- showed few organelles specific to steroidogenesis (Fig. 4A). At ture period. They were oval or irregular in shape and had a this stage, they first showed very weak expression of the lutein- relatively small cytoplasm that was rich in rough endoplasmic izing hormone receptor gene, Lhr (Fig. 3A). reticulum (ER) and ribosomes, whereas smooth ER, Golgi Supplementing the medium with luteinizing hormone (LH), membranes, and lipid droplets were scarce (Fig. 2C). Cell-to-cell IGF1, and stem cell factor caused the theca cells to differentiate connections were sparse, and the intercellular space was filled further and to develop more cytoplasmic lipid droplets (Fig. 3B). with basement membranes and collagen fibers. Some cells Electron microscopy revealed that the cells developed low surrounded the zonae pellucidae of the oocytes directly, but electron-dense areas containing many Golgi apparatuses, small unlike typical granulosa cells, they never protruded foot pro- vacuoles, and few ribosomes (Fig. 4B). They also contained many cesses to the oocyte surface through the zona (Fig. 2C). Some of mitochondria with cristae intermediate between the lamellar the round colonies had a weak alkaline phosphatase reaction, and tubular types (Fig. 4B). The cells expressed the Gli2 gene, which usually localizes specifically in the thecal layers of growing a theca-specific effector gene of Hedgehog signaling in granulosa follicles and not in granulosa cells (11) (Fig. 1B). Gene expres- cells (13). The androstenedione concentration was significantly sion analysis demonstrated that the cells expressed markers for higher in the culture supernatant (87.6 and 80.8 pg/ml in two theca cells (Ptch1 and Gli3), but not for granulosa cells (Fshr and replicated experiments) than in the control medium (GSM Ihh) (Fig. 3A). Taken together, these findings suggest that the supplemented with LH, IGF1, and stem cell factor) containing cells isolated from the neonatal ovarian tissue with GSM-K were no cells (10.7 and 11.6 pg/ml in two replicated experiments). thecal stem-like cells (hereafter called ‘‘putative thecal stem Further differentiation of theca cells could be achieved by cells’’ or simply ‘‘thecal stem cells’’). This finding was supported supplementation with granulosa cell-conditioned medium.

Honda et al. PNAS ͉ July 24, 2007 ͉ vol. 104 ͉ no. 30 ͉ 12391 Downloaded by guest on September 29, 2021 Many cells showed characteristic features of steroidogenic cells: many large lipid droplets, smooth ER, Golgi apparatuses, and mitochondria with tubular cristae (Fig. 4C). They expressed all of the genes examined including Ptch2, which is thought to be expressed in mature theca cells (13) (Fig. 3A). A high concentration of androstenedione (116.6 and 150.2 pg/ml in two replicated experiments) was detected in the medium after 7 days of culture. Fully mature forms of theca cells were observed when they were cocultured with granulosa cells (GSM-SLG). To accurately identify the cells that differentiated from stem cells in vitro,a granulosa cell suspension was prepared from EGFP-expressing mice (‘‘green mice’’). As shown in Fig. 3C, only the fluorescence- negative, stem cell-originating cells had lipid droplets (granular cytoplasmic inclusions under Hoffman optics). Their shape was more oval than that of cells from the other culture groups. At the ultrastructural level, all lipid-including cells were rich in smooth ER and the mitochondria had tubular cristae. Eventually, no cells retained the features of the undifferentiated state (Fig. 4D). The androstenedione level in their medium reached 185.8 pg/ml.

Intraovarian Transplantation of Putative Thecal Stem Cells. Finally, to test whether the putative thecal stem cells isolated from the neonatal ovaries retained their stem cell-like properties through isolation and culture in vitro, we transplanted GSM-K-cultured stem cells into host ovarian tissues (Fig. 5A). After a 7-week culture period with four passages, stem cell colonies expressing green fluorescence were transplanted into the ovaries of two nontransgenic C57BL/6 strain female mice. Two weeks after transplantation, recipient females were stimulated with equine chorionic gonadotropin to induce follicular development, and their ovaries were removed for observation by fluorescence Fig. 5. Intraovarian transplantation of thecal stem cells (A) and ovaries 2 microscopy. The donor cells had proliferated actively and had weeks after transplantation of thecal stem cells, which colonized exclusively in surrounded fully developed follicles (Fig. 5B). In frozen sections, the ovarian interstitium and the thecal layer of follicles (B–D). (A) A host ovary EGFP-positive cells were distributed in the inner and outer theca removed from a mature female mouse (Left). The blood was wiped from the cell layers of large cells around fully grown follicles, or in the ovary on sterilized filter paper. Thecal stem cell colonies (arrows in Right) were cortical interstitial areas of small individual cells (Fig. 5C). A few transplanted into the ovary by a glass pipette. Arrowhead indicates an air small, probably less differentiated, theca cells were present bubble placed for controllable transfer. After transfer, the ovary was inserted ␮ around small follicles (Fig. 5D). No fluorescence-positive cells into the empty ovarian bursa of another female mouse. (Scale bar: 2 m.) (B) The donor thecal cells (EGFP-positive) surrounding two large follicles were were localized in the follicular epithelium. clearly visible by fluorescence microscopy (arrows). (C) A frozen section of a large follicular area. The donor thecal stem cells differentiated into large cells Discussion and were located in both the inner (I) and outer (O) thecal layers. They were Previously published experiments support the notion that inter- also present in the interstitial area of small cells (arrowheads). (Scale bar: 50 stitial pretheca cells are located within the ovarian stroma and ␮m.) (D) A frozen section of a small primary follicle. A few small, probably less may have stem cell properties (3), but theca stem cells have not differentiated, theca cells were present around the follicle (arrowheads). been identified to date. In this study, we provide a clear (Scale bar: 50 ␮m.) (B–D Right) Corresponding fields observed by fluorescent demonstration that putative thecal stem cells exist in neonatal microscopy are shown. ovaries, and that they can be isolated in vitro as round spherical colonies. By applying appropriate stimuli to the colonies in our addition, their proliferating ability in a nonanchored situation in vitro experimental system, we were able to fully reproduce the might have accelerated the purification. At the first cell prepa- differentiation of theca cells from undifferentiated stem cells to ration step and during subsequent passages, cells having the mature steroidogenic theca cells, in a stepwise manner. The differentiation steps represented in this study are illustrated in ability to spread onto the dish were eliminated gradually, and SI Fig. 8 together with the presumed corresponding sequence of finally only those proliferating in a nonanchoring manner re- follicular development. mained in the culture. The purity of the final cell suspension was confirmed by the RT-PCR assay and exclusive perifollicular and Purification of Putative Thecal Stem Cells in Selective Culture. One of interstitial colonization after intraovarian transplantation. Thus, the most intriguing findings was that putative thecal stem cells the unique nutritional requirements and nonanchoring prolifer- were readily isolated by culturing neonatal ovarian cells in GSM ation of thecal stem cells most likely contributed to their containing no serum. Generally, serum is added to the in vitro purification from the neonatal ovarian tissues. culture of a variety of cells including fibroblasts and granulosa The first aim of our study was to isolate and characterize cells (14), which are abundant in ovarian tissue. The require- putative oocyte stem cells (female GS cells), which were first ments of thecal stem cells to proliferate in vitro probably differ reported by Johnson et al. in 2004 (6). To address this aim, we from those of ovarian fibroblasts and granulosa cells. When we used the GS cell medium to culture the neonatal ovarian cells. examined the effects of several growth factors on the growth of During the first several passages, small oocytes could be iden- the colonies, only those thecal stem cells in colonies responded tified on the surface of colonies, as if they had been growing from and proliferated. This finding suggests that the serum-free the stem cells inside the colonies. However, our detailed histo- condition promoted selection of thecal stem cells in culture. In chemical examination identified no MVH-and-BrdU double-

12392 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0703787104 Honda et al. Downloaded by guest on September 29, 2021 positive oocytes, indicating that only preexisting postmeiotic ment membrane between the granulosa and the inner thecal oocytes grew out from the colonies. This result by itself does not layers in normal follicles. Instead, close proximity probably negate the presence of oocyte stem cells in the neonatal ovary, promoted a paracrine effect of the granulosa cells. The presence but these stem cells, if any, did not seem to respond to GS cell of such paracrine mechanisms has been shown experimentally in medium, which has been used in experiments on their male an in vivo assay in hypophysectomized rats (21). counterparts. A fundamental question that remains unanswered about the differentiation of the theca cell lineage is the mechanisms by Proliferation Ability of Putative Thecal Stem Cells in Vitro. bFGF, which the inner and outer thecal layers are formed from the EGF, LIF, and IGF1 significantly promoted the growth of stem common stem cells (3). The cells comprising the inner layer are cell colonies, but the culture conditions seemed to be suboptimal steroidogenic cells and blood vessel cells, whereas cells compris- because they did not proliferate indefinitely. Although the cells ing the outer layer are unidentified fibroblast-like cells and continued to divide for Ϸ2 months, as shown by the increases in smooth muscle cells (22, 23). Our transplantation experiments colony size, the proliferation rate decreased after Ϸ10 days, and revealed that the isolated thecal stem cells had the capacity to the cell number seemed to reach a plateau after 1 month of contribute to the formation of both inner and outer thecal layer culture. However, we cannot exclude the possibility that thecal cells, although the latter seemed to be more heterogeneous in stem cells have an inherently limited proliferation potential and composition. It is possible that the difference between cells in the that this feature was correctly reflected in vitro. Unlike male two thecal layers represents the different differentiation status of germ cells, which produce spermatozoa continuously throughout these theca cells’ lineage. This assumption agrees with the results life (15), the size of the primordial oocyte reserve appears to be of our in vitro differentiation experiments, in which coculture fixed during the prenatal period. Consistent with the nondividing with granulosa cells led to the terminal thecal differentiation character of oocytes, it is possible that thecal stem cells, which from the relatively undifferentiated, fibroblast-like cells. It is also localize in the ovarian interstitium, do not divide actively but interesting that, only 2 weeks after transplantation, the donor gradually acquire a proliferation ability as they differentiate to theca cells were observed in fully grown follicles. Because support the growing follicle. Generally, the tissue-specific stem follicular development in mature rodents is estimated to take Ϸ6 cells studied so far are thought to depend on specialized cellular weeks (24), it is probable that the transplanted thecal cells were microenvironments and the ex vivo expansion of pure popula- able to invade partially grown preexisting follicles. tions of tissue stem cells has proven elusive (16). We also As far as we know, this is the first demonstration of successful observed that induction of differentiation by serum simulta- intraovarian transplantation of cells isolated in vitro. We found neously promoted proliferation of differentiating theca cells in that direct transplantation of cells into intact ovaries in situ vitro. caused heavy bleeding, and these transplanted cells never survived. In combination with an ovary transfer (removal and Differentiation of Putative Thecal Stem Cells in Vitro and in Vivo. replacement) technique, the transplantation was very successful, Thecal stem cells in round colonies had morphological charac- and we could show that the putative thecal cells we purified were teristics common to undifferentiated interstitial cells such as transplantable, as are other tissue-specific stem cells (25). Our

small fibroblasts: they were oval and contained many organelles intraovarian transplantation technique should enable us to as- BIOLOGY

including mitochondria with laminar cristae, rough ER, and sess the ability of a variety of cells to differentiate and function DEVELOPMENTAL ribosomes. These thecal stem cells maintained these undiffer- in the ovarian tissue. entiated features for as long as they proliferated within round colonies. Stimulation with serum caused these cells to spread Theca Cell Culture as an in Vitro Model for Soma–Germ and Soma– onto the bottom of the dish and to start to accumulate small lipid Soma Interactions. In mice, assembly of oocytes and somatic cells droplets in their cytoplasm. Although they seemed to stay in a into follicles occurs within the first week after birth, and fully relatively undifferentiated state, they expressed a small amount expanded antral follicles appear shortly before puberty (26). The of Lhr mRNA. Treatment with LH, IGF1, and stem cell factor ovaries of 2-day-old mice are devoid of distinct follicular struc- or paracrine factors from granulosa cells caused further signif- tures, and when the follicles grow to enclose two to three layers icant differentiation of theca cells, leading to accumulation of of granulosa cells, theca cells start to surround the follicles and many lipid droplets, formation of smooth ER, and the emer- acquire the steroidogenic ability regulated primarily by LH. gence of mitochondria with tubular cristae. All these cytological Androstenedione, a steroid hormone produced by theca cells, is features are indicative of steroidogenic ability (17), a conclusion converted into estradiol by granulosa cells (1, 2). Thus, follicu- supported by the detection of androstenedione at a high con- logenesis is a complex process in which germ cells (oocytes) and centration in the supernatant. These differentiation patterns of two types of somatic cells (theca cells and granulosa cells) theca cells are consistent with previous findings showing that interact through paracrine or direct mechanisms. In this study, their proliferation and differentiation are independent of LH we first demonstrated that putative thecal stem cells can be receptors in the early stages, but become LH receptor- isolated and purified in vitro and induced to differentiate to dependent at a later stage (18), and that IGF1 synergistically acquire active steroidogenic ability. These isolated theca cells augments the LH stimulation of androstenedione production may provide invaluable experimental systems to study theca– (19). The stepwise emergence of several theca cell markers is granulosa and theca–oocyte interactions; e.g., kit–kit ligand (27) also consistent with a previous in situ hybridization study that and Hedgehog signaling systems (13). We have also recently suggested that granulosa-derived factors, Ihh and Dhh, are found that theca cells can promote the growth of granulosa-free candidate initiators of early thecal differentiation (13). We oocytes in a paracrine fashion, indicating the presence of theca- conclude that our culture system induced the differentiation of derived oocyte trophic factors (unpublished data). Other than theca cells from their stem cells in a stepwise manner similar to the factors involved in normal folliculogenesis, the mechanisms that occurring in vivo, without the presence of other cell types. underlying ovarian pathology caused by theca cell dysfunction, However, their full differentiation as shown by cells in the inner including hyperandrogenism and polycystic ovary syndrome (1), thecal layer of follicles required the presence of granulosa cells. could be unraveled by studying thecal stem cells in culture. Granulosa cells from adult bovine ovaries also stimulate androstenedione production by stromal cells (20). It is unlikely that Materials and Methods direct cell-to-cell contact with granulosa cells was required for Cell Preparation and Culture. To prepare thecal stem cells, ovaries such terminal differentiation because of the intervening base- were collected from newborn (2–4 days after birth) ICR or

Honda et al. PNAS ͉ July 24, 2007 ͉ vol. 104 ͉ no. 30 ͉ 12393 Downloaded by guest on September 29, 2021 C57BL/6-Tg(CAG-EGFP)C14-Y01-FM131Osb female mice. in the supernatant was measured by liquid chromatography- The latter are the so-called ‘‘green mice’’ that show green tandem mass spectrometric analysis (29). This analysis was fluorescence in the whole body (28). Cell culture was performed performed by Teikoku Hormone (Kawasaki, Japan). according to the protocol for establishment of male GS cells with slight modifications (9, 10). In brief, isolated ovaries were treated Intraovarian Transplantation. Thecal stem cells (3.5 ϫ 105) derived with 1 mg/ml collagenase in Hanks’s solution at 37°C for 15 min from C57BL/6-Tg(CAG-EGFP)C14-Y01-FM131Osb were cul- and then treated with 0.2% trypsin and 1.4 mg/ml DNase for 10 tured in GSM-K for 7 weeks with four passages. In a preliminary min. Loosened ovarian tissues were dissected into single cells by study, we found that direct transplantation of cells into intact gentle pipetting and were allocated into wells of a 0.1% (wt/vol) ovaries in situ caused heavy bleeding, and no cells survived after 5 gelatin-coated 24-well tissue-culture plate (2.0–2.5 ϫ 10 cells the operation. Therefore, we first removed the host ovaries from 2 per 2.0-cm well). After incubation for 4 h, floating cells were mature C57BL/6 females, and the blood was wiped from the recovered and placed into another culture well containing ovaries on sterilized filter paper (Fig. 5A). Approximately 20 GSM-K (SI Table 1). The cells were maintained at 37°C under colonies were picked up by a glass pipette (Ϸ100 ␮m inner 5% CO2 in air. A half-volume of the medium was changed every diameter) together with a minimal amount of medium. The tip 2–3 days. After 14 days of culture, colonies were dispersed by of the pipette was inserted into the host ovary along the longer trypsin treatment and replated into culture wells containing ϫ axis (Fig. 5A). The cell colonies were transplanted in the fresh medium (2 dilution). Colonies grew to the original size opposite side of the ovarian cortex by applying gentle positive in Ϸ10–14 days, and passages were repeated until the cells pressure, and the pipette was slowly pulled out of the ovary. Each stopped proliferating (at four to five passages). ovary that received donor cells was inserted into the empty Granulosa cells were prepared from follicles in the ovaries ovarian bursa of another host C57BL/6 female mouse under from 7.5 units of equine chorionic gonadotropin-stimulated TgX(act-EGFP)Osb#50 female mice (another green mouse anesthesia as described (30). Two weeks after transplantation, strain). To remove the thecal layers from the isolated follicles, the host females were administered 5 units of equine chorionic they were incubated in StemPro-34 serum-free medium contain- gonadotropin, and the ovaries were retrieved 48 h later. These ing 0.25% trypsin and 0.02% EDTA for 10 min at 37°C. Oocytes transplantation experiments were replicated twice. were washed and stripped of the enclosing follicular cells by pipetting. Groups of granulosa cells from 50 follicles were Cytological, Histological, and Gene Expression Analyses. Cytological, transferred to a four-well dish (Nunc, Roskilde, Denmark). histological, and gene expression analyses were performed ac- Cultures were carried out in GSM supplemented with 15% FBS cording to established protocols (see SI Materials and Methods). (GSM-S) (SI Table 1) at 37°C with 5% CO2 in air. Two days later, granulosa cell clumps were dispersed by trypsin treatment and Animals. All animals were maintained and used for experiments replated to a fresh culture plate (5ϫ dilution). The lack of in accordance with the guidelines of the RIKEN Institute. contamination by thecal cells in the granulosa cell suspension was confirmed by RT-PCR with specific primers (data not EGFP-expressing strains of mice (green mice) used in this study belong shown). Granulosa-conditioned medium was prepared from to Dr. M. Okabe (Osaka University, Osaka, Japan) and were provided wells after 24 h of incubation of 80–100% confluent granulosa by RIKEN BioResource Center with the support of the National cells. BioResources Project of the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The anti-MVH antibody was kindly provided by Dr. T. Noce (Mitsubishi Kagaku Institute of Life Science, ϫ 5 In Vitro Differentiation. Putative theca stem cells (1.1 10 cells) Tokyo, Japan). This research was supported by grants from the Ministry were cultured for 4–5 weeks with three passages and were then of Education, Culture, Sports, Science, and Technology (to A.H. and replated and cultured under one of the five conditions indicated A.O.); the Ministry of Health, Labour, and Welfare (A.O.); and Core in SI Table 1. After 6 days of culture, these cells were subjected Research for Evolutional Science and Technology (A.O.). A.H. is the to RT-PCR, Oil red O staining, or electron microscopic obser- recipient of a research fellowship from the RIKEN Special Postdoctoral vation. In some experiments, the androstenedione concentration Researchers Program.

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