Rock music improvisation shows increased activity in Broca's area and its right hemisphere homologue related to spontaneous creativity.

OBJECTIVE: The neural correlates of creativity are not well understood. Using an improvised guitar task, we investigated the role of Broca's area during spontaneous creativity, regardless of individual skills, experience, or subjective feelings. RESULTS: Twenty guitarists performed improvised and formulaic blues rock sequences while hemodynamic responses were recorded using functional near-infrared spectroscopy. We identified a new significant response in Broca's area (Brodmann area [BA] 45L) and its right hemisphere homologue during improvised playing but not during formulaic playing. Our results indicate that bilateral BA45 activity is common during creative processes that involve improvisation across all participants, regardless of subjective feelings, skill, age, difficulty, history, or amount of practice. While our previous results demonstrated that the modulation of the neural network according to the subjectively experienced level of creativity relied on the degree of deactivation in BA46L, our current results independently show a common concurrent activity in BA45 in all participants. We suggest that this is related to the sustained execution of improvisation in "motor control," analogous to motor planning in speech control.

Temporal and spatial dynamics of immune cells in spontaneous liver transplant tolerance.

The liver has long been deemed a tolerogenic organ. We employed high-dimensional mass cytometry and immunohistochemistry to depict the temporal and spatial dynamics of immune cells in the spleen and liver in a murine model of spontaneous liver allograft acceptance. We depicted the immune landscape of spontaneous liver tolerance throughout the rejection and acceptance stages after liver transplantation and highlighted several points of importance. Of note, the CD4+/CD8+ T cell ratio remained low, even in the tolerance phase. Furthermore, a PhenoGraph clustering analysis revealed that exhausted CD8+ T cells were the most dominant metacluster in graft-infiltrating lymphocytes (GILs), which highly expressed the costimulatory molecule CD86. The temporal and spatial dynamics of immune cells revealed by high-dimensional analyses enable a fine-grained analysis of GIL subsets, contribute to new insights for the discovery of immunological mechanisms of liver tolerance, and provide potential ways to achieve clinical operational tolerance after liver transplantation.

Septoclasts expressing epidermal fatty acid-binding protein (E-FABP, FABP5) in endochondral ossification.

BACKGROUND: Long-chain fatty acids (LCFAs) and retinoic acid (RA) are abundant in the growth plates (GPs) of long bones; however, their roles have not been elucidated. We observed that epidermal fatty acid-binding protein (E-FABP/FABP5) with a high affinity for both LCFAs and RA is exclusively expressed in the septoclasts located at the chondro-osseous junction (COJ) of the GP. HIGHLIGHTS: E-FABP expressed in septoclasts is involved in both LCFA metabolism and RA signaling as an intracellular transporter of both LCFAs and RA. Septoclasts with shortened cytoplasmic processes are associated with cartilage resorptive activity downregulation because of E-FABP deficiency or excess or deficiency of RA. In ontogeny, the septoclasts are differentiated from the pericytes and involved in the resorption of the uncalcified matrix of the cartilage templates in endochondral ossification. CONCLUSION: Septoclasts originate from pericytes and express E-FABP to play crucial roles in uncalcified matrix resorption by LCFA metabolism and RA signaling during endochondral ossification.

Selective involution of thymic medulla by cyclosporine A with a decrease of mature thymic epithelia, XCR1+ dendritic cells, and epithelium-free areas containing Foxp3+ thymic regulatory T cells.

Immunosuppressive drugs such as cyclosporine A (CSA) can disrupt thymic structure and functions, ultimately inducing syngeneic/autologous graft-versus-host disease together with involuted medullas. To elucidate the effects of CSA on the thymus more precisely, we analyzed the effects of CSA on the thymus and T cell system using rats. In addition to confirming the phenomena already reported, we newly found that the proportion of recent thymic emigrants also greatly decreased, suggesting impaired supply. Immunohistologically, the medullary thymic epithelial cells (mTECs) presented with a relative decrease in the subset with a competent phenotype and downregulation of class II major histocompatibility complex molecules. In control rats, thymic dendritic cells (DCs) comprised two subsets, XCR1+SIRP1α-CD4- and XCR1-SIRP1α+CD4+. The former had a tendency to selectively localize in the previously-reported epithelium-containing areas of the rat medullas, and the number was significantly reduced by CSA treatment. The epithelium-free areas, another unique domains in the rat medullas, contained significantly more Foxp3+ thymic Tregs. With CSA treatment, the epithelium-free areas presented strong involution, and the number and distribution of Tregs in the medulla were greatly reduced. These results suggest that CSA inhibits the production of single-positive thymocytes, including Tregs, and disturbs the microenvironment of the thymic medulla, with a decrease of the competent mTECs and disorganization of epithelium-free areas and DC subsets, leading to a generation of autoreactive T cells with selective medullary involution.

Expression and enhancement of FABP4 in septoclasts of the growth plate in FABP5-deficient mouse tibiae.

In our previous study, fatty acid-binding protein 5 (FABP5) was expressed in septoclasts with long processes which are considered to resorb uncalcified matrix of the growth plate (GP) cartilage, and no apparent abnormalities were detected in the histo-architecture of the GP of FABP5-deficient (FABP5-/-) mice. Those finding lead us to hypothesize that another FABP can compensate the deletion of FABP5 in septoclasts of its gene-mutant mice. Based on the hypothesis, the present study examined the expression levels of several other FABPs in septoclasts and their morphology in FABP5-/- mouse tibiae. Processes of FABP5-/- septoclasts tend to be shorter than wild septoclasts. FABP4-positive septoclasts in FABP5-/- mice were more numerous than those cells in wild mice.Peroxisome proliferator-activated receptor (PPAR) γ was expressed in FABP4-positive septoclasts of FABP5-/- mice as well as mice administered with GW1929, a PPARγ agonist, suggesting that the occurrence of PPARγ induces an increase of FABP4-positive septoclasts. The present finding suggests that the functional exertion of FABP5 in septoclasts is supplemented by FABP4 in normal and FABP5-/- mice, and that the expression of FABP4 is up-regulated in accompany with PPARγ in FABP5-/- for maintenance of resorptive activity in the GP.

Suppression of liver transplant rejection by anti-donor MHC antibodies via depletion of donor immunogenic dendritic cells.

BACKGROUND: We previously found two distinct passenger dendritic cell (DC) subsets in the rat liver that played a central role in the liver transplant rejection. In addition, a tolerance-inducing protocol, donor-specific transfusion (DST), triggered systemic polytopical production of depleting alloantibodies to donor class I MHC (MHCI) antigen (DST-antibodies). METHODS: We examined the role of DST-antibodies in the trafficking of graft DC subsets and the alloresponses in a rat model. We also examined an anti-donor class II MHC (MHCII) antibody that recognizes donor DCs more selectively. RESULTS: Preoperative transfer of DST-antibodies or DST pretreatment eliminated all passenger leukocytes, including both DC subsets and depleted the sessile DCs in the graft to ~20% of control. The CD172a+CD11b/c+ immunogenic subset was almost abolished. The intrahost direct or semi-direct allorecognition pathway was successfully blocked, leading to a significant suppression of the CD8+ T-cell response in the recipient lymphoid organs and the graft with delayed graft rejection. Anti-donor MHCII antibody had similar effects without temporary graft damage. Although DST pretreatment had a priming effect on the proliferative response of recipient regulatory T cells, DST-primed sera and the anti-donor MHCII antibody did not. CONCLUSION: DST-antibodies and anti-donor MHCII antibodies could suppress the CD8+ T-cell-mediated liver transplant rejection by depleting donor immunogenic DCs, blocking the direct or semi-direct pathways of allorecognition. Donor MHCII-specific antibodies may be applicable as a selective suppressant of anti-donor immunity for clinical liver transplantation without the cellular damage of donor MHCII- graft cells and recipient cells.

Monotherapy With Anti-CD70 Antibody Causes Long-Term Mouse Cardiac Allograft Acceptance With Induction of Tolerogenic Dendritic Cells.

Allograft rejection has been an obstacle for the long-term survival of patients. CD70, a tumor necrosis factor (TNF) family member critically expressed on antigen-presenting cells and strongly but transiently up-regulated during lymphocyte activation, represents an important co-stimulatory molecule that induces effective T cell responses. We used a mouse heterotopic cardiac transplantation model to evaluate the effects of monotherapy with the antibody targeting mouse CD70 (FR70) on transplantation tolerance and its immunoregulatory activity. FR70-treated C3H recipient mice permanently accepted B6 fully mismatched cardiac allografts. Consistent with the graft survival, the infiltration of CD8+ T cells in the graft was reduced, dendritic cells were differentiated into a tolerogenic status, and the number of regulatory T cells was elevated both in the graft and the recipient's spleen. In addition, naïve C3H given an adoptive transfer of spleen cells from the primary recipients with FR70 treatment accepted a heart graft from a matching B6 donor but not third-party BALB/c mice.　Our findings show that treatment with FR70 induced regulatory cells and inhibited cytotoxic T cell proliferation, which led to long-term acceptance of mouse cardiac allografts. These findings highlight the potential role of anti-CD70 antibodies as a clinically effective treatment for allograft rejection.

Novel Targeting to XCR1+ Dendritic Cells Using Allogeneic T Cells for Polytopical Antibody Responses in the Lymph Nodes.

Vaccination strategy that induce efficient antibody responses polytopically in most lymph nodes (LNs) against infections has not been established yet. Because donor-specific blood transfusion induces anti-donor class I MHC antibody production in splenectomized rats, we examined the mechanism and significance of this response. Among the donor blood components, T cells were the most efficient immunogens, inducing recipient T cell and B cell proliferative responses not only in the spleen, but also in the peripheral and gut LNs. Donor T cells soon migrated to the splenic T cell area and the LNs, with a temporary significant increase in recipient NK cells. XCR1+ resident dendritic cells (DCs), but not XCR1- DCs, selectively phagocytosed donor class I MHC+ fragments after 1 day. After 1.5 days, both DC subsets formed clusters with recipient CD4+ T cells, which proliferated within these clusters. Inhibition of donor T cell migration or depletion of NK cells by pretreatment with pertussis toxin or anti-asialoGM1 antibody, respectively, significantly suppressed DC phagocytosis and subsequent immune responses. Three allogeneic strains with different NK activities had the same response but with different intensity. Donor T cell proliferation was not required, indicating that the graft vs. host reaction is dispensable. Intravenous transfer of antigen-labeled and mitotic inhibitor-treated allogeneic, but not syngeneic, T cells induced a polytopical antibody response to labeled antigens in the LNs of splenectomized rats. These results demonstrate a novel mechanism of alloresponses polytopically in the secondary lymphoid organs (SLOs) induced by allogeneic T cells. Donor T cells behave as self-migratory antigen ferries to be delivered to resident XCR1+ DCs with negligible commitment of migratory DCs. Allogeneic T cells may be clinically applicable as vaccine vectors for polytopical prophylactic antibody production even in asplenic or hyposplenic individuals.

Psychological and physiological effects of laughter yoga sessions in Japan: A pilot study.

The purpose of this pilot study was to examine and evaluate the psychological and physiological effects of multiple sessions of laughter yoga on community members. Participants took part in a 45 min laughter yoga session once per month for 6 months. Before and after all sessions, participants completed the Profile of Mood States-Brief Japanese Version (J-POMS-B) questionnaire to assess their mood, and had blood drawn for the measurement of stress indicators and immune function. Serial changes in J-POMS-B scores were tested by three way analysis of variance, and changes in laboratory results per session were evaluated with a paired t-test. The results showed that repeated sessions of laughter yoga had psychologically beneficial effects, especially on the aspects of tension-anxiety, and vigor. Adrenocorticotropic hormone and cortisol values related to the participants' stress levels were significantly decreased after the fourth laughter yoga session. These results indicated that multiple laughter yoga sessions appeared to be effective in improving the psychological and physiological status of healthy adults.

Optimization of fixative solution for retinal morphology: a comparison with Davidson's fixative and other fixation solutions.

PURPOSE: Numerous fixative solutions are available but many are not amenable to the histomorphological preservation of retinae. The investigators specifically focused on retinal histological studies, which rather than 4% formaldehyde (FA), often use Davidson's fixative. However the latter has its limitations. The purpose of this study was to produce a new fixative which maintains retinae closer to the in vivo conditions. STUDY DESIGN: Experimental design. METHODS: Four fixative formulations (4% paraformaldehyde, Davidson's fixative, modified Davidson's fixative and an in-house fixative - TB-Fix) were tested on retinae and the outcomes on histomorphology and immunohistochemical staining for selected antigenic markers was compared. RESULTS: TB-Fix markedly improved morphological detail following hematoxylin and eosin staining, most importantly eliminating the spongiform appearance in the plexiform layer and the swelling of somata (including Müller cells), when compared to FA, Davidson's fixative and its modified version. Retinal samples fixed with TB-Fix or FA showed comparable results in immunohistological staining for neurons and glia in the retina. Importantly, while the whole eye fixed with FA collapsed in shape and induced artificial retinal detachment, the eye fixed with TB-Fix avoided deformation and detachment. Furthermore, we found that TB-Fix also prevented detachment from the culture plate when used to fix HEK293 cells, which are known to detach from the plate easily. CONCLUSION: It was demonstrated that TB-Fix provides an overall improvement in the preservation of retinal morphology and chemical composition.

Utility of Bartonella henselae IgM Western Blot Bands for Serodiagnosis of Cat Scratch Disease.

We evaluated the utility of Western blot (WB) bands of Bartonella henselae in detecting anti-B. henselae immunoglobulin M (IgM) for serodiagnosis of cat scratch disease (CSD). IgM band patterns were examined using sera from 92 patients clinically suspected of having CSD and from 130 healthy individuals. Positive WB bands were observed in 49 (53.5%) of the 92 patient sera. Three bands at 8 to 10, 31 to 35, and 70 kDa were regarded as relevant for B. henselae because all of the positive sera yielded at least one of the three bands, and none of the healthy control sera showed reactivity to any of them. In contrast, the positive rate of the patient sera by conventional indirect fluorescence antibody assay (IFA) for B. henselae IgM was 28.3% (26/92) among the patients. These finding suggest that the IgM-WB assay, although cumbersome to perform, can be used for confirmatory diagnosis of CSD with no false positivity in the control sera. Purification of proteins in the specific bands may contribute to the development of an IgM enzyme-linked immunosorbent assay (IgM-ELISA) with improved specificity and sensitivity.

Infection Programs Sustained Lymphoid Stromal Cell Responses and Shapes Lymph Node Remodeling upon Secondary Challenge.

Lymph nodes (LNs) are constructed of intricate networks of endothelial and mesenchymal stromal cells. How these lymphoid stromal cells (LSCs) regulate lymphoid tissue remodeling and contribute to immune responses remains poorly understood. We performed a comprehensive functional and transcriptional analysis of LSC responses to skin viral infection and found that LSC subsets responded robustly, with different kinetics for distinct pathogens. Recruitment of cells to inflamed LNs induced LSC expansion, while B cells sustained stromal responses in an antigen-independent manner. Infection induced rapid transcriptional responses in LSCs. This transcriptional program was transient, returning to homeostasis within 1 month of infection, yet expanded fibroblastic reticular cell networks persisted for more than 3 months after infection, and this altered LN composition reduced the magnitude of LSC responses to subsequent heterologous infection. Our results reveal the complexity of LSC responses during infection and suggest that amplified networks of LN stromal cells support successive immune responses.

The utility of a country-specific Bartonella henselae antigen in an IgM-indirect fluorescent antibody assay for the improved diagnosis of cat scratch disease.

Bartonella henselae strains genetically differ among nations. The utility of Japanese-specific YH-01 strain was investigated in developing indirect fluorescence antibody assay (IFA) for IgM in comparison with conventional IFA employing Houston-1 strain by testing 100 Japanese patients suspected of cat scratch disease. The country-specific IFA greatly improved the accuracy of diagnosis.

Changes in metabolic proteins in ex vivo rat retina during glutamate-induced neural progenitor cell induction.

Understanding how energy metabolism and related proteins influence neural progenitor cells in adult tissues is critical for developing new strategies in clinical tissue regeneration therapy. We have recently reported that a subtoxic concentration of glutamate-induced neural progenitor cells in the mature ex vivo rat retina. We herein explore changes in the metabolic pathways during the process. We firstly observed an increase in lactate and lactate dehydrogenase concentration in the glutamate-treated retina. We then investigated the levels of glycolytic enzymes and confirmed significant upregulation of pyruvate kinase M type (PKM), especially PKM2, enolase, phosphoglycerate mutase 1 (PGAM1), and inosine-5'-monophosphate dehydrogenase (IMPDH1) in the glutamate-treated retina compared to the untreated retina. An analysis of the subcellular localization of PKM2 revealed nuclear translocation in the treated retina, which has been reported to regulate cell cycle proliferation and glycolytic enzymes. Our findings indicate that the mature rat retina undergoes an increase in aerobic glycolysis. PKM2, both in the cytoplasm and in the nucleus, may thus play an important role during neural progenitor cell induction, as it does in other proliferating cells.

Development of a Highly Specific IgM Enzyme-Linked Immunosorbent Assay for Bartonella henselae Using Refined N-Lauroyl-Sarcosine-Insoluble Proteins for Serodiagnosis of Cat Scratch Disease.

The conventional anti-Bartonella henselaeIgM enzyme-linked immunosorbent assay (IgM-ELISA) methods for diagnosing cat scratch disease (CSD) remain poor in both sensitivity and specificity. We sought to develop an IgM-ELISA with improved accuracy in the serodiagnosis of CSD by exploring the antigens that are most suitable for an ELISA. We prepared 5 different protein antigens: antigen I (sonicatedB. henselaewhole-cell antigen), antigen II (N-lauroyl-sarcosine-insoluble antigen), antigen III (processed sarcosine-soluble antigen), and antigen IV and antigen V (sarcosine-insoluble and sarcosine-soluble antigens refined by DEAE-Sepharose Fast Flow ion-exchange chromatography). The IgM antibodies in the sera of 47 patients with clinically suspected CSD (24 definite, 23 suspected) and of 85 healthy individuals were examined by ELISAs using the 5 antigens, and the results were compared with those of an IgM indirect fluorescent antibody assay (IgM-IFA). In a reference panel, which consisted of 5 positive and 5 negative sera, antigen I and antigen III failed to distinguish between the two statuses, whereas the other three antigens succeeded in distinguishing between them. When the cutoff value was set at the 98th percentile of the ELISA index for healthy individuals, the sensitivity of IgM-IFA for the 24 cases of definite CSD was 54%, whereas the sensitivities of the IgM-ELISAs with antigen II, IV, and V were 75%, 83%, and 75%, respectively. The sensitivities of these three IgM-ELISAs for all 47 of the clinically suspected cases were 49%, 64%, and 51%, respectively. In contrast, the sensitivity of IgM-IFA was 28%. These results indicate that the refined sarcosine-insoluble proteins (antigen IV), which possessed the highest specificity among the 5 antigens, are the most appropriate for developing an IgM-ELISA for the highly specific serodiagnosis of CSD.

Up-regulation of DRP-3 long isoform during the induction of neural progenitor cells by glutamate treatment in the ex vivo rat retina.

Glutamate has been shown to induce neural progenitor cells in the adult vertebrate retina. However, protein dynamics during progenitor cell induction by glutamate are not fully understood. To identify specific proteins involved in the process, we employed two-dimensional electrophoresis-based proteomics on glutamate untreated and treated retinal ex vivo sections. Rat retinal tissues were incubated with 1 mM glutamate for 1 h, followed by incubation in glutamate-free media for a total of 24 h. Consistent with prior reports, it was found that mitotic cells appeared in the outer nuclear layer without any histological damage. Immunohistological evaluations and immunoblotting confirmed the emergence of neuronal progenitor cells in the mature retina treated with glutamate. Proteomic analysis revealed the up-regulation of dihydropyrimidinase-related protein 3 (DRP-3), DRP-2 and stress-induced-phosphoprotein 1 (STIP1) during neural progenitor cell induction by glutamate. Moreover, mRNA expression of DRP-3, especially, its long isoform, robustly increased in the treated retina compared to that in the untreated retina. These results may indicate that glutamate induces neural progenitor cells in the mature rat retina by up-regulating the proteins which mediate cell mitosis and neurite growth.

Fatty Acid Binding Protein 3 Is Involved in n-3 and n-6 PUFA transport in mouse trophoblasts.

BACKGROUND: Low placental fatty acid (FA) transport during the embryonic period has been suggested to result in fetal developmental disorders and various adult metabolic diseases, but the molecular mechanism by which FAs are transported through the placental unit remains largely unknown. OBJECTIVE: The aim of this study was to examine the distribution and functional relevance of FA binding protein (FABP), a cellular chaperone of FAs, in the mouse placenta. METHODS: We clarified the localization of FABPs and sought to examine their function in placental FA transport through the phenotypic analysis of Fabp3-knockout mice. RESULTS: Four FABPs (FABP3, FABP4, FABP5, and FABP7) were expressed with spatial heterogeneity in the placenta, and FABP3 was dominantly localized to the trophoblast cells. In placentas from the Fabp3-knockout mice (both sexes), the transport coefficients for linoleic acid (LA) were significantly reduced compared with those from wild-type mice by 25% and 44% at embryonic day (E) 15.5 and E18.5, respectively, whereas those for α-linolenic acid (ALA) were reduced by 19% and 17%, respectively. The accumulation of LA (18% and 27% at E15.5 and E18.5) and ALA (16% at E15.5) was also significantly less in the Fabp3-knockout fetuses than in wild-type fetuses. In contrast, transport and accumulation of palmitic acid (PA) were unaffected and glucose uptake significantly increased by 23% in the gene-ablated mice compared with wild-type mice at E18.5. Incorporation of LA (51% and 52% at 1 and 60 min, respectively) and ALA (23% at 60 min), but not PA, was significantly less in FABP3-knockdown BeWo cells than in controls, whereas glucose uptake was significantly upregulated by 51%, 50%, 31%, and 33% at 1, 20, 40, and 60 min, respectively. CONCLUSIONS: Collectively FABP3 regulates n-3 (ω-3) and n-6 (ω-6) polyunsaturated FA transport in trophoblasts and plays a pivotal role in fetal development.

Fatty acid binding protein 7 regulates phagocytosis and cytokine production in Kupffer cells during liver injury.

Kupffer cells (KCs) are involved in the progression of liver diseases such as hepatitis and liver cancer. Several members of the fatty acid binding proteins (FABPs) are expressed by tissue macrophages, and FABP7 is localized only in KCs. To clarify the role of FABP7 in the regulation of KC function, we evaluated pathological changes of Fabp7 knockout mice during carbon tetrachloride-induced liver injury. During liver injury in Fabp7 knockout mice, serum liver enzymes were increased, cytokine expression (tumor necrosis factor-α, monocyte chemoattractant protein-1, and transforming growth factor-ß) was decreased in the liver, and the number of KCs in the liver necrotic area was significantly decreased. Interestingly, in the FABP7-deficient KCs, phagocytosis of apoptotic cells was impaired, and expression of the scavenger receptor CD36 was markedly decreased. In chronic liver injury, Fabp7 knockout mice showed less fibrogenic response to carbon tetrachloride compared with wild-type mice. Taken together, FABP7 is involved in the liver injury process through its regulation of KC phagocytic activity and cytokine production. Such modulation of KC function by FABP7 may provide a novel therapeutic approach to the treatment of liver diseases.

Locally existing endothelial cells and pericytes in ovarian stroma, but not bone marrow-derived vascular progenitor cells, play a central role in neovascularization during follicular development in mice.

BACKGROUND: Neovascularization is necessary for follicular growth. Vascularization is first observed in preantral follicles, and thereafter the vasculature markedly increases in follicles undergoing development. Neovascularization includes angiogenesis and vasculogenesis. Vasculogenesis is the formation of new blood vessels by bone marrow-derived endothelial progenitor cells. It is unclear whether vasculogenesis occurs during follicular growth. Blood vessels must be mature to be functional blood vessels. Mature blood vessels are characterized by the recruitment of pericytes. However, it is unclear where pericytes come from and whether they contribute to neovascularization in the follicle during follicular growth. In this study, we investigated whether bone marrow-derived progenitor cells that differentiate into vascular endothelial cells or pericytes contribute to neovascularization during follicular growth. METHODS: A parabiosis model was used in this study. Six-week-old wild-type and transgenic female mice expressing green fluorescent protein (GFP) were conjoined between the lateral abdominal regions to create a shared circulatory system. After 6 weeks, the ovaries were obtained and immunostained for CD31/CD34 (a vascular endothelial cell marker), platelet-derived growth factor receptor-ß (PDGFR-ß) (a pericyte marker), and GFP (a bone marrow-derived cell marker). RESULTS: Cells that were positive for CD34 and PDGFR-ß were observed in the stroma adjacent to the primary or early preantral follicles and in the theca cell layer of the follicles from the late preantral stage to the preovulatory stage. CD31/CD34 and GFP double-positive cells were observed in the theca cell layer of the follicle from the antral stage to the preovulatory stage while the number of double-positive cells in the preovulatory follicles did not increase. PDGFR-ß and GFP double-positive cells were observed in the theca cell layer of the preovulatory follicle but not in the smaller follicle. CONCLUSIONS: Locally existing endothelial cells and pericytes in the stroma play a central role in the neovascularization during follicular growth, while bone marrow-derived endothelial cells and pericytes partially contribute to this process.