Increased apoptosis and accelerated epithelial migration following inhibition of hedgehog signaling in adaptive small bowel postresection

The intestinal epithelium undergoes a marked adaptive response following loss of functional small bowel surface area characterized by increased crypt cell proliferation and increased enterocyte migration from crypt to villus tip, resulting in villus hyperplasia and enhanced nutrient absorption. Hedgehog (Hh) signaling plays a critical role in regulating epithelial-mesenchymal interactions during morphogenesis of the embryonic intestine. Our previous studies showed that blocking Hh signaling in neonatal mice results in increased small intestinal epithelial crypt cell proliferation and altered enterocyte fat absorption and morphology. Hh family members are also expressed in the adult intestine, but their role in the mature small bowel is unclear. With the use of a model of intestinal adaptation following partial small bowel resection, the role of Hh signaling in the adult gut was examined by determining the effects of blocking Hh signaling on the regenerative response following loss of functional surface area. Hh-inactivating monoclonal antibodies or control antibodies were administered to mice that sustained a 50% intestinal resection. mRNA analyses of the preoperative ileum by quantitative real-time PCR revealed that Indian hedgehog was the most abundant Hh family member. The Hh receptor Patched was more abundant than Patched 2. Analyses of downstream targets of Hh signaling demonstrated that Gli3 was twofold more abundant than Gli1 and Gli2 and that bone morphogenetic protein (BMP)2 was most highly expressed compared with BMP1, -4, and -7. Following intestinal resection, the expression of Hh, Patched, Gli, and most BMP genes was markedly downregulated in the remnant ileum, and, in anti-Hh antibody-treated mice, expression of Patched 2 and Gli 1 was further suppressed. In Hh antibody-treated mice following resection, the enterocyte migration rate from crypt to villus tip was increased, and by 2 wk postoperation, apoptosis was increased in the adaptive gut. However, crypt cell proliferation, villus height, and crypt depth were not augmented. These data indicate that Hh signaling plays a role in adult gut epithelial homeostasis by regulating epithelial cell migration from crypt to villus tip and by enhancing apoptosis.     

Analysis of the Sonic Hedgehog Signaling Pathway in Normal and Abnormal Bladder Development

In this study, we examined the expression of Sonic Hedgehog, Patched, Gli1, Gli2, Gli3 and Myocardin in the developing bladders of male and female normal and megabladder (mgb−/−) mutant mice at embryonic days 12 through 16 by in situ hybridization. This analysis indicated that each member of the Sonic Hedgehog signaling pathway as well as Myocardin displayed distinct temporal and spatial patterns of expression during normal bladder development. In contrast, mgb−/− bladders showed both temporal and spatial changes in the expression of Patched, Gli1 and Gli3 as well as a complete lack of Myocardin expression. These changes occurred primarily in the outer mesenchyme of developing mgb−/− bladders consistent with the development of an amuscular bladder phenotype in these animals. These results provide the first comprehensive analysis of the Sonic Hedgehog signaling pathway during normal bladder development and provide strong evidence that this key signaling cascade is critical in establishing radial patterning in the developing bladder. In addition, the lack of detrusor smooth muscle development observed in mgb−/− mice is associated with bladder-specific temporospatial changes in Sonic Hedgehog signaling coupled with a lack of Myocardin expression that appears to result in altered patterning of the outer mesenchyme and poor initiation and differentiation of smooth muscle cells within this region of the developing bladder.     

Development of antral follicles in cryopreserved cat ovarian tissue transplanted to immunodeficient mice

Ovarian cortex cryopreservation and xenotransplantation into immunodeficient mice represents a potential means for female germplasm conservation and an immediate model for investigation of folliculogenesis. The objectives of this study were to: (1) assess follicle survival after cryopreservation and transplantation of cat ovarian tissue into non-obese diabetic severely combined immunodeficient (NOD SCID) mice; and (2) evaluate the effects of gonadotropin treatments on follicular development in the transplanted tissue. Slices from the cat ovarian cortex were frozen and after thawing, transplanted under each kidney capsule of castrated male NOD SCID mice (eight xenografts in four mice). Sixty-two days after surgery, mice were randomly assigned (two per group) to gonadotropin-treated (eCG and hCG 88 h later) or control (saline-treated) groups. Twenty-four hours after the last injection, ovarian tissue was recovered and processed for histology. Fresh ovarian tissue from the same original source was similarly processed. Follicles were counted, measured, and classified as primordial, primary, secondary, or antral. Immunoreactive proliferating cell nuclear antigen (PCNA) stain was used to assess follicle viability. Microscopic examination revealed no evidence of necrosis or fibrosis. The grafts were well-vascularized, with follicles at all stages of development. Numbers of follicles in the transplanted tissue were markedly reduced compared to fresh tissue, with approximately 10% of follicles surviving freezing and transplantation procedures. Growing follicles positive for PCNA were found in all xenografts. Gonadotropin treatment did not alter the proportion of resting to growing follicles or mean follicle diameter by comparison with controls from untreated mice. By contrast, luteinization, but not ovulation, of antral follicles was observed only in grafts from treated mice. In summary, frozen-thawed cat ovarian cortex tissue not only survived xenotransplantation, it also contained follicles able to grow to antral stages. Exogenous gonadotropin treatment in this model resulted in luteinization of antral follicles but enhancement of follicular growth and ovulation did not occur.     

Developmental programming: impact of prenatal testosterone excess on ovarian cell proliferation and apoptotic factors in sheep

Prenatal testosterone (T) excess leads to reproductive dysfunctions in sheep, which include increased ovarian follicular recruitment and persistence. To test the hypothesis that follicular disruptions in T sheep stem from changes in the developmental ontogeny of ovarian proliferation and apoptotic factors, pregnant Suffolk sheep were injected twice weekly with T propionate or dihydrotestosterone propionate (DHT; a nonaromatizable androgen) from Days 30 to 90 of gestation. Changes in developmental expression of proliferating cell nuclear antigen (PCNA), BCL2, BAX, activated CASP3, and FAS/FASLG were determined at Fetal Days 90 and 140, 22 wk, 10 mo, and 21 mo of age by immunocytochemisty. Prenatal T treatment induced changes in expression of proliferative and apoptotic markers in a follicle-, age-, and steroid-specific manner. Changes in BAX were evident only during fetal life and PCNA, BCL2, and CASP3 only postnatally. Prenatal T and not DHT increased PCNA and decreased BCL2 in granulosa/theca cells of antral follicles at 10 and 21 mo but decreased CASP3 in granulosa/theca cells of antral follicles at 22 wk (prepubertal) and 10 and 21 mo. Both treatments decreased BAX immunostaining in granulosa cells of Fetal Day 90 primordial/primary follicles. Neither treatment affected FAS expression at any developmental time point in any follicular compartment. Effects on BAX appear to be programmed by androgenic actions and PCNA, BCL2, and CASP3 by estrogenic actions of T. Overall, the findings demonstrate that fetal exposure to excess T disrupts the ovarian proliferation/apoptosis balance, thus providing a basis for the follicular disruptions evidenced in these females.     

Immunohistochemical localization of proliferating cell nuclear antigen (PCNA) in the pig ovary

The aim of the study was to determine the expression of proliferating cell nuclear antigen protein (PCNA) in the pig ovary. The localization of PCNA was demonstrated in paraffin sections of pig ovarian tissue using primary mouse monoclonal anti-PCNA antibody. In primordial follicles, no remarkable staining for PCNA either in granulosa cells or in the oocytes was observed. In primary to secondary follicles, positive staining in oocytes and in some granulosa cells was detected. The advanced preantral and particularly actively growing small to large antral follicles showed extensive PCNA labeling in the layers of granulosa and theca cells and in the cumulus cells encircling the oocyte. PCNA labeling was expressed in nuclei of oocytes in preantral and small antral follicles. In atretic follicles, the level of PCNA protein expression was dependent on the stage of atresia. Follicles demonstrating advanced atresia showed only limited or no PCNA labeled granulosa and theca cells. The results of the study demonstrate that follicular growth and development in pig ovary may be effectively monitored by determining the granulosa cell expression of PCNA.     

The zebrafish mutants dre, uki, and lep encode negative regulators of the hedgehog signaling pathway

Proliferation is one of the basic processes that control embryogenesis. To identify factors involved in the regulation of proliferation, we performed a zebrafish genetic screen in which we used proliferating cell nuclear antigen (PCNA) expression as a readout. Two mutants, hu418B and hu540A, show increased PCNA expression. Morphologically both mutants resembled the dre (dreumes), uki (ukkie), and lep (leprechaun) mutant class and both are shown to be additional uki alleles. Surprisingly, although an increased size is detected of multiple structures in these mutant embryos, adults become dwarfs. We show that these mutations disrupt repressors of the Hedgehog (Hh) signaling pathway. The dre, uki, and lep loci encode Su(fu) (suppressor of fused), Hip (Hedgehog interacting protein), and Ptc2 (Patched2) proteins, respectively. This class of mutants is therefore unique compared to previously described Hh mutants from zebrafish genetic screens, which mainly show loss of Hh signaling. Furthermore, su(fu) and ptc2 mutants have not been described in vertebrate model systems before. Inhibiting Hh activity by cyclopamine rescues uki and lep mutants and confirms the overactivation of the Hh signaling pathway in these mutants. Triple uki/dre/lep mutants show neither an additive increase in PCNA expression nor enhanced embryonic phenotypes, suggesting that other negative regulators, possibly Ptc1, prevent further activation of the Hh signaling pathway. The effects of increased Hh signaling resulting from the genetic alterations in the uki, dre, and lep mutants differ from phenotypes described as a result of Hh overexpression and therefore provide additional insight into the role of Hh signaling during vertebrate development.     

Sonic hedgehog is produced by follicular dendritic cells and protects germinal center B cells from apoptosis

The Hedgehog (Hh) signaling pathway is involved in the development of many tissues during embryogenesis, but has also been described to function in adult self-renewing tissues. In the immune system, Sonic Hedgehog (Shh) regulates intrathymic T cell development and modulates the effector functions of peripheral CD4(+) T cells. In this study we investigate whether Shh signaling is involved in peripheral B cell differentiation in mice. Shh is produced by follicular dendritic cells, mainly in germinal centers (GCs), and GC B cells express both components of the Hh receptor, Patched and Smoothened. Blockade of the Hh signaling pathway reduces the survival, and consequently the proliferation and Ab secretion, of GC B cells. Furthermore, Shh rescues GC B cells from apoptosis induced by Fas ligation. Taken together, our data suggest that Shh is one of the survival signals provided by follicular dendritic cells to prevent apoptosis in GC B cells.     

Hedgehog参与西伯利亚鲟侧线机械和电感受器分化的初始证据

为揭示Hedgehog（Hh）信号与神经丘和壶腹器官分化的关系,研究以西伯利亚鲟（Acipenser baerii Brandt）为模型,首先对再生过程中的神经丘和壶腹器官的转录组进行比较分析,发现Hh信号通路关键基因（Shh、Patched 1）在两类感受器中差异表达,且它们的表达在再生过程中呈现动态性。然后用环巴胺（Cyclopamine,Hh信号抑制剂）处理西伯利亚鲟胚胎（st29）,用扫描电镜和FM1-43荧光染色对西伯利亚鲟仔鱼（st43-st44）分析发现环巴胺显著抑制了壶腹器官的发育。整体原位杂交表明,Shh、Patched1、Smoothened、Gli2在腹面侧线区域的表达受到了环巴胺的抑制。以上结果暗示Hh信号通路与神经丘和壶腹器官的发育有关,推测Hh信号在神经丘和壶腹器官的分化过程中起到了重要作用。     

Survival and growth of goat primordial follicles after in vitro culture of ovarian cortical slices in media containing coconut water

The development of culture systems to support the initiation of growth of primordial follicles is important to the study of the factors that control the earliest stages of folliculogenesis. We investigated the effectiveness of five culture media, two supplements and three culture periods on the survival and growth of goat primordial follicles after culturing ovarian cortex. The media were based on minimal essential minimum (MEM) and coconut water solution (CWS) added in the proportion of 0, 25, 50, 75 or 100%. The two supplements were none versus supplemented with insulin-transferrin-selenium, pyruvate, glutamine, hypoxanthine, and BSA. Pieces of goat ovarian cortex were cultured in the media for 1, 3 or 5 days and representative samples were evaluated at day 0 as non-cultured controls. The replicates were the two ovaries of five mixed breed goats. The number of primordial, intermediate, primary and secondary follicles at each period of culture and the number of degenerated follicles were evaluated. Mitotic activity of granulosa cells was studied by immunolocalization of proliferating cell nuclear antigen (PCNA). The number of follicles in each stage and degenerated follicles were statistically analyzed by ANOVA using a factorial design and the significance of differences assessed using Tukey test. Chi-square test was used to compare the percentage of follicles with PCNA positive granulosa cells. As the culture period progressed, the number of primordial follicles fell and there was a significant increase in the number of primary follicles. The fall in the number of primordial follicles was particularly marked after 1 day culture. No effect of media on the number of primordial and primary follicles was observed after culture, but MEM as well as supplements increased the number of intermediate follicles. Follicular degeneration was kept at the same level after culture in the media tested, except for pure CWS that increased the number of degenerated follicles. In contrast, addition of supplements to culture media reduced follicular degeneration. In non-cultured tissue, PCNA was expressed in granulosa cells of 31.6% of the growing follicles. This percentage had not significantly changed after 5 days culture in the various media, indicating the maintenance of proliferation activity of granulosa cells during culture. In conclusion, it is shown that goat primordial follicles may be successfully activated after in vitro culture in all media tested. However, when pure CWS is used the follicular degeneration is enhanced, but the addition of supplements to culture media decrease follicular degeneration.     

Involvement of cell proliferation in the process of follicular atresia in the guinea pig

Cell morphology and proliferation was investigated in the atretic follicles during estrous cycles in the guinea pig. Ovarian samples on days 1, 4, 8, 12 and 16 of the estrous cycle in the guinea pig were taken in the morning for histologic staining with hematoxylin and eosin (HE), and immunohistochemical staining of the protein proliferating cell nuclear antigen (PCNA). The results indicated that the granulosa cells degenerated and eliminated first in atretic follicles, while the fibroblast-like cells appeared in the innermost layer of theca interna cells. When the fibroblast-like cells migrated to the antrum, they proliferated and formed a new tissue in peripheral to the zona pellucida of the oocyte. Our results also revealed that the orientation of the theca interna cell arrangement changed twice during the process of atresia, and the loose connective tissue in the antrum was critical for follicular atresia. Therefore, follicular atresia was not a simple process of cell death and elimination, but coexisted with cell proliferation. To our knowledge, we have for the first time confirmed cell proliferation and the presence of new tissue in atretic follicles in guinea pigs.     

Role of nuclear factor-κB pathway in the transition of mouse secondary follicles to antral follicles

Nuclear factor-κB (NF-κB) signaling is involved in regulating a great number of normal and abnormal cellular events. However, little is known about its role in ovarian follicular development. In this study, we found NF-κB signaling is activated during the transition from secondary to antral follicles. We generated active NF-κB mice and found that antral follicular numbers were higher than wild-type ovaries. Activation of NF-κB signaling could enhance granulosa cell proliferation and regress granulosa cell apoptosis of mouse ovarian follicles. Higher follicle-stimulating hormone receptor (FSHR) and luteinizing hormone/choriogonadotropin receptor expressions were observed in active NF-κB ovaries compared to wild type. Furthermore, we confirmed that NF-κB signaling was indeed involved in the granulosa cell viability and proliferation through FSHR using COV434 cell line. This is the first experimental evidence that NF-κB signaling is implicated in the control of follicular development through FSHR and its corresponding target molecules, which might be achieved by targeting proliferation and apoptosis in follicular granulosa cells.     

Hedgehog induces opposite changes in turnover and subcellular localization of patched and smoothened

Secreted signaling proteins of the Hedgehog family organize spatial pattern during animal development. Two integral membrane proteins have been identified with distinct roles in Hedgehog signaling. Patched functions in Hedgehog binding, and Smoothened functions in transducing the signal. Current models view Patched and Smoothened as a preformed receptor complex that is activated by Hedgehog binding. Here we present evidence that Patched destabilizes Smoothened in the absence of Hedgehog. Hedgehog binding causes removal of Patched from the cell surface. In contrast, Hedgehog causes phosphorylation, stabilization, and accumulation of Smoothened at the cell surface. Comparable effects can be produced by removing Patched from cells by RNA-mediated interference. These findings raise the possibility that Patched acts indirectly to regulate Smoothened activity.     

Activation of Smurf E3 Ligase Promoted by Smoothened Regulates Hedgehog Signaling through Targeting Patched Turnover

Hedgehog signaling plays conserved roles in controlling embryonic development; its dysregulation has been implicated in many human diseases including cancers. Hedgehog signaling has an unusual reception system consisting of two transmembrane proteins, Patched receptor and Smoothened signal transducer. Although activation of Smoothened and its downstream signal transduction have been intensively studied, less is known about how Patched receptor is regulated, and particularly how this regulation contributes to appropriate Hedgehog signal transduction. Here we identified a novel role of Smurf E3 ligase in regulating Hedgehog signaling by controlling Patched ubiquitination and turnover. Moreover, we showed that Smurf-mediated Patched ubiquitination depends on Smo activity in wing discs. Mechanistically, we found that Smo interacts with Smurf and promotes it to mediate Patched ubiquitination by targeting the K1261 site in Ptc. The further mathematic modeling analysis reveals that a bidirectional control of activation of Smo involving Smurf and Patched is important for signal-receiving cells to precisely interpret external signals, thereby maintaining Hedgehog signaling reliability. Finally, our data revealed an evolutionarily conserved role of Smurf proteins in controlling Hh signaling by targeting Ptc during development.     

A positive role for patched-smoothened signaling in promoting cell proliferation during normal head development in Drosophila

The transmembrane receptor Patched regulates several developmental processes in both invertebrates and vertebrates. In vertebrates, Patched also acts as a tumor suppressor. The Patched pathway normally operates by negatively regulating Smoothened, a G-protein-coupled receptor; binding of Hedgehog ligand to Patched relieves this negative interaction and allows signaling by Smoothened. We show that Ptc regulates Drosophila head development by promoting cell proliferation in the eye-antennal disc. During head morphogenesis, Patched positively interacts with Smoothened, which leads to the activation of Activin type I receptor Baboon and stimulation of cell proliferation in the eye-antennal disc. Thus, loss of Ptc or Smoothened activity affects cell proliferation in the eye-antennal disc and results in adult head capsule defects. Similarly, reducing the dose of smoothened in a patched background enhances the head defects. Consistent with these results, gain-of-function Hedgehog interferes with the activation of Baboon by Patched and Smoothened, leading to a similar head capsule defect. Expression of an activated form of Baboon in the patched domain in a patched mutant background completely rescues the head defects. These results provide insight into head morphogenesis, a process we know very little about, and reveal an unexpected non-canonical positive signaling pathway in which Patched and Smoothened function to promote cell proliferation as opposed to repressing it.     

Evolution of prothymosin alpha and proliferating cell nuclear antigen (PCNA) immunoreactivity through the development of rat ovarian follicles

Summary The cellular distribution of prothymosin alpha (ProT) was studied in ovarian follicles of adult cycling rats. We found positive granulosa and theca cells throughout follicular maturation. When both ProT and proliferating cell nuclear antigen (PCNA) immunoreactivity was studied, we observed that both proteins were expressed in the same granulosa and theca cells, although sometimes ProT immunoreactivity was weak or absent in the mitotic (M) phase. Moreover, both peptides share the nuclear distribution, but ProT immunoreactivity was never seen in nucleoli. Therefore, we conclude that in mitotic cells ProT is expressed only in actively proliferating cells, since all ProT-positive cells were also positive for PCNA. ProT and PCNA immunoreactivities during the meiotic division were studied in oocytes. The presence of PCNA was, unlike ProT, constant throughout follicle development (except atretic oocytes). Oocytes expressed ProT from primordial follicles to the eighth generation, but more developed oocytes and atretic oocytes were not immunoreactive. In hypophysectomized rats, all oocytes were immunoreactive. Interestingly, in hypophysectomized rats treated with follicle stimulating hormone (FSH) that promoted follicle development, the more developed oocytes did not show ProT immunoreactivity. Since hypophysectomized rats were not treated with luteinizing hormone we conclude that ProT expression is not required to complete meiotic division I.