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Michael K Skinner - One of the best experts on this subject based on the ideXlab platform.
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induction of ovarian primordial Follicle assembly by connective tissue growth factor ctgf
PLOS ONE, 2010Co-Authors: Ryan Schindler, Eric E Nilsson, Michael K SkinnerAbstract:Primordial Follicle assembly is a process that occurs when oocyte nests break down to form individual primordial Follicles. The size of this initial pool of primordial Follicles in part determines the reproductive lifespan of the female. Connective tissue growth factor (CTGF) was identified as a potential regulatory candidate for this process in a previous microarray analysis of Follicle development. The current study examines the effects of CTGF and associated transforming growth factor beta 1 (TGFβ-1) on Follicle assembly. Ovaries were removed from newborn rat pups and placed in an organ culture system. The ovaries treated with CTGF for two days were found to have an increased proportion of assembled Follicles. CTGF was found to regulate the ovarian transcriptome during primordial Follicle assembly and an integrative network of genes was identified. TGFβ-1 had no effect on primordial Follicle assembly and in combination with CTGF decreased oocyte number in the ovary after two days of culture. Over ten days of treatment only the combined treatment of CTGF and TGFβ-1 was found to cause an increase in the proportion of assembled Follicles. Interestingly, treatment with TGFβ-1 alone resulted in fewer total oocytes in the ovary and decreased the primordial Follicle pool size after ten days of culture. Observations indicate that CTGF alone or in combination with TGFβ-1 stimulates primordial Follicle assembly and TGFβ-1 can decrease the primordial Follicle pool size. These observations suggest the possibility of manipulating primordial Follicle pool size and influencing female reproductive lifespan.
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platelet derived growth factor modulates the primordial to primary Follicle transition
Reproduction, 2006Co-Authors: Eric E Nilsson, Chris Detzel, Michael K SkinnerAbstract:Primordial Follicles steadily leave the arrested pool and undergo a primordial to primary Follicle transition during the female reproductive lifespan. When the available pool of primordial Follicles is depleted reproduction ceases and humans enter menopause. The present study was designed to investigate the actions of several growth factors previously identified as candidate regulatory factors for the primordial to primary Follicle transition with a microarray analysis. Ovaries from 4-day-old rats were placed into culture and treated for 2 weeks with platelet-derived growth factor (PDGF), anti-PDGF neutralizing antibody, vascular endothelial growth factor (VEGF), neuregulin (NRG), or kit ligand (KITL) as a positive control. PDGF-treatment resulted in a significant decrease in the percentage of primordial Follicles and a concomitant increase in the percentage of developing primary Follicles compared to controls. In contrast, ovaries treated with an anti-PDGF neutralizing antibody had a significant increase in the percentage of primordial Follicles demonstrating an inhibition of endogenous Follicle development. Ovaries incubated in the presence of VEGF or NRG had no change in Follicle development. Observations indicate that PDGF, but not VEGF or NRG, promotes the primordial to primary Follicle transition. Immunohistochemical localization indicated that the PDGF protein was present in the oocytes of both primordial and developing Follicles. PDGF-treatment of cultured ovaries resulted in an increase in KITL mRNA expression. KITL has been previously shown to promote the primordial to primary Follicle transition. KITL-treatment of ovaries had no effect on expression of Pdgf or any PDGF homologs or receptors. Therefore, PDGF appears to be produced by the oocyte and acts as one of several extracellular signaling factors that regulate the primordial to primary Follicle transition. These observations provide insight into the cell-cell interactions involved in the regulation of primordial Follicle development and can be used in the future development of therapies for some forms of infertility.
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alterations in the ovarian transcriptome during primordial Follicle assembly and development
Biology of Reproduction, 2005Co-Authors: Phillip Kezele, Eric E Nilsson, Jacquelyn M Ague, Michael K SkinnerAbstract:The assembly of the developmentally arrested primordial Follicle and subsequent transition to the primary Follicle are poorly understood processes critical to ovarian biology. Abnormal primordial Follicle development can lead to pathologies such as premature ovarian failure. The current study used a genome-wide expression profile to investigate primordial Follicle assembly and development. Rat ovaries with predominantly unassembled, primordial, or primary Follicles were obtained. RNA from these ovaries was hybridized to rat microarray gene chips, and the gene expression (i.e., ovarian transcriptome) was compared between the developmental stages. Analysis of the ovarian transcriptome demonstrated 148 genes up-regulated and 50 genes down-regulated between the unassembled and primordial Follicle stages. Observations demonstrate 80 genes up-regulated and 44 genes down-regulated between the primordial and primary Follicle stages. The analysis demonstrated 2332 genes common among the three developmental stages, 146 genes specific for the unassembled Follicles, 94 genes specific for the primordial Follicles, and 151 genes specific for the primary Follicles. Steroidogenic genes are up-regulated between unassembled and primordial Follicles, and then many are again down-regulated between primordial and primary Follicles. The hormones inhibin and Mullerian inhibitory substance (MIS) display a similar pattern of expression with the highest levels of mRNA in the primordial Follicles. Several novel unknown genes that had dramatic changes in expression during primordial Follicle development were also identified. Gene families/clusters identified that were up-regulated from unassembled to primordial Follicles include growth factors and signal transduction gene clusters, whereas a down-regulated gene family was the synaptonemal complex genes associated with meiosis. Gene families/clusters that were up-regulated between primordial and primary Follicles included immune response genes, metabolic enzymes, and proteases, whereas down-regulated gene families include the globulin genes and some steroidogenic genes. The expression of several growth factors changed during primordial Follicle development, including vascular endothelial growth factor and insulin-like growth factor II. Elucidation of how these changes in gene expression coordinate primordial Follicle assembly and the primordial to primary Follicle transition provides a better understanding of these critical biological processes and allows selection of candidate regulatory factors for further investigation.
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regulation of ovarian primordial Follicle assembly and development by estrogen and progesterone endocrine model of Follicle assembly
Endocrinology, 2003Co-Authors: Phillip Kezele, Michael K SkinnerAbstract:The assembly of the developmentally arrested primordial Follicle and the subsequent transition of the primordial Follicle to the primary Follicle are critical processes in normal ovarian physiology that remain to be elucidated. Ovarian Follicles do not proliferate and the primordial Follicles present in the neonate represent the total number of gametes available to a female throughout her reproductive life. The primordial Follicles are oocytes surrounded by less differentiated squamous granulosa cells and are derived from oocyte nests, and primary Follicles are oocytes surrounded by a single layer of cuboidal granulosa cells that have initiated Follicle development. Abnormalities in primordial Follicle assembly, arrest, and development (i.e. primordial to primary Follicle transition) can cause pathological conditions such as premature ovarian failure. In this study newborn rat ovaries were cultured for 7 d. The rate of primordial Follicle assembly in vivo was identical with the rate in vitro. Interestingly, the rate of primordial Follicle transition to the primary Follicle was found to be 3 times greater in culture. This abnormal rate of primary Follicle development in culture suggests the primordial Follicle does not arrest in development as observed in vivo. To investigate this phenomena newborn rat ovaries were cultured in the presence of progesterone, estradiol or calf serum. Estradiol, progesterone, or calf serum significantly reduced the level of initial primordial to primary Follicle transition. Approximately 60% of Follicles make the primordial to primary Follicle transition in control ovaries and about 30% in treated ovaries. Steroids and calf serum had no effect on the primordial to primary Follicle transition in ovaries collected and cultured from postnatal 4-d-old rats, suggesting the effects observed are restricted to the initial wave of primordial to primary Follicle transition. Interestingly, progesterone was also found to significantly reduce the rate of primordial Follicle assembly. All viable oocytes assembled into primordial Follicles in control ovaries and approximately 40% remained unassembled in progesterone-treated ovaries. Progesterone was also found to reduce primordial Follicle assembly in vivo with 10% of the total Follicles remaining unassembled in progesterone injected neonatal animals. Analysis of cellular apoptosis demonstrated that progesterone inhibited the coordinated oocyte apoptosis required for primordial Follicle assembly. The hypothesis developed is that high levels of maternal and fetal steroids prevent premature primordial Follicle assembly and primordial to primary Follicle transition in the embryo. After birth steroid levels fall dramatically and the primordial Follicles are free to assemble and initiate development. These observations suggest a novel role for steroids and the maternal-fetal endocrine unit in the control of ovarian primordial Follicle assembly and early follicular development.
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regulation of ovarian primordial Follicle assembly and development by estrogen and progesterone endocrine model of Follicle assembly
Endocrinology, 2003Co-Authors: Phillip Kezele, Michael K SkinnerAbstract:The assembly of the developmentally arrested primordial Follicle and the subsequent transition of the primordial Follicle to the primary Follicle are critical processes in normal ovarian physiology that remain to be elucidated. Ovarian Follicles do not proliferate and the primordial Follicles present in the neonate represent the total number of gametes available to a female throughout her reproductive life. The primordial Follicles are oocytes surrounded by less differentiated squamous granulosa cells and are derived from oocyte nests, and primary Follicles are oocytes surrounded by a single layer of cuboidal granulosa cells that have initiated Follicle development. Abnormalities in primordial Follicle assembly, arrest, and development (i.e. primordial to primary Follicle transition) can cause pathological conditions such as premature ovarian failure. In this study newborn rat ovaries were cultured for 7 d. The rate of primordial Follicle assembly in vivo was identical with the rate in vitro. Interestingly, the rate of primordial Follicle transition to the primary Follicle was found to be 3 times greater in culture. This abnormal rate of primary Follicle development in culture suggests the primordial Follicle does not arrest in development as observed in vivo. To investigate this phenomena newborn rat ovaries were cultured in the presence of progesterone, estradiol or calf serum. Estradiol, progesterone, or calf serum significantly reduced the level of initial primordial to primary Follicle transition. Approximately 60% of Follicles make the primordial to primary Follicle transition in control ovaries and about 30% in treated ovaries. Steroids and calf serum had no effect on the primordial to primary Follicle transition in ovaries collected and cultured from postnatal 4-d-old rats, suggesting the effects observed are restricted to the initial wave of primordial to primary Follicle transition. Interestingly, progesterone was also found to significantly reduce the rate of primordial Follicle assembly. All viable oocytes assembled into primordial Follicles in control ovaries and approximately 40% remained unassembled in progesterone-treated ovaries. Progesterone was also found to reduce primordial Follicle assembly in vivo with 10% of the total Follicles remaining unassembled in progesterone injected neonatal animals. Analysis of cellular apoptosis demonstrated that progesterone inhibited the coordinated oocyte apoptosis required for primordial Follicle assembly. The hypothesis developed is that high levels of maternal and fetal steroids prevent premature primordial Follicle assembly and primordial to primary Follicle transition in the embryo. After birth steroid levels fall dramatically and the primordial Follicles are free to assemble and initiate development. These observations suggest a novel role for steroids and the maternal-fetal endocrine unit in the control of ovarian primordial Follicle assembly and early follicular development.
George Cotsarelis - One of the best experts on this subject based on the ideXlab platform.
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wnt dependent de novo hair Follicle regeneration in adult mouse skin after wounding
Nature, 2007Co-Authors: Zaixin Yang, Thomas Andl, Sarah E Millar, George CotsarelisAbstract:The mammalian hair Follicle is thought to form anew only during development, and loss of an adult Follicle is generally considered permanent. Fifty years ago in Nature, Billingham and Russel reported 'hair neogenesis' in rabbit skin, but this was later discounted. Now it is back, with the discovery that hair Follicle regeneration is triggered by wounding the skin of adult mice. This suggests that mammalian skin responds to wounding with greater plasticity and regenerative capacity than was previously believed, and has implications for those studying wound healing, tissue regeneration and stem cell function. The mammalian hair Follicle was thought to form only during development, so loss of an adult Follicle was considered permanent. Here Cotsarelis and colleagues solve a fifty-year-old debate, and show that wounding the skin of adult mice triggers de novo hair Follicle regeneration. The mammalian hair Follicle is a complex ‘mini-organ’ thought to form only during development1; loss of an adult Follicle is considered permanent. However, the possibility that hair Follicles develop de novo following wounding was raised in studies on rabbits2,3, mice4 and even humans fifty years ago5. Subsequently, these observations were generally discounted because definitive evidence for follicular neogenesis was not presented6. Here we show that, after wounding, hair Follicles form de novo in genetically normal adult mice. The regenerated hair Follicles establish a stem cell population, express known molecular markers of Follicle differentiation, produce a hair shaft and progress through all stages of the hair Follicle cycle. Lineage analysis demonstrated that the nascent Follicles arise from epithelial cells outside of the hair Follicle stem cell niche, suggesting that epidermal cells in the wound assume a hair Follicle stem cell phenotype. Inhibition of Wnt signalling after re-epithelialization completely abrogates this wounding-induced folliculogenesis, whereas overexpression of Wnt ligand in the epidermis increases the number of regenerated hair Follicles. These remarkable regenerative capabilities of the adult support the notion that wounding induces an embryonic phenotype in skin, and that this provides a window for manipulation of hair Follicle neogenesis by Wnt proteins. These findings suggest treatments for wounds, hair loss and other degenerative skin disorders.
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wnt dependent de novo hair Follicle regeneration in adult mouse skin after wounding
Nature, 2007Co-Authors: Mayumi Ito, Sarah E Millar, Thomas Andl, Zaixin Yang, Chunhua Cui, Noori Kim, George CotsarelisAbstract:The mammalian hair Follicle is a complex 'mini-organ' thought to form only during development; loss of an adult Follicle is considered permanent. However, the possibility that hair Follicles develop de novo following wounding was raised in studies on rabbits, mice and even humans fifty years ago. Subsequently, these observations were generally discounted because definitive evidence for follicular neogenesis was not presented. Here we show that, after wounding, hair Follicles form de novo in genetically normal adult mice. The regenerated hair Follicles establish a stem cell population, express known molecular markers of Follicle differentiation, produce a hair shaft and progress through all stages of the hair Follicle cycle. Lineage analysis demonstrated that the nascent Follicles arise from epithelial cells outside of the hair Follicle stem cell niche, suggesting that epidermal cells in the wound assume a hair Follicle stem cell phenotype. Inhibition of Wnt signalling after re-epithelialization completely abrogates this wounding-induced folliculogenesis, whereas overexpression of Wnt ligand in the epidermis increases the number of regenerated hair Follicles. These remarkable regenerative capabilities of the adult support the notion that wounding induces an embryonic phenotype in skin, and that this provides a window for manipulation of hair Follicle neogenesis by Wnt proteins. These findings suggest treatments for wounds, hair loss and other degenerative skin disorders.
Teresa K Woodruff - One of the best experts on this subject based on the ideXlab platform.
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size specific Follicle selection improves mouse oocyte reproductive outcomes
Reproduction, 2015Co-Authors: Shuo Xiao, Francesca E Duncan, Lu Bai, Catherine T Nguyen, Lonnie D Shea, Teresa K WoodruffAbstract:Encapsulated in vitro Follicle growth (eIVFG) has great potential to provide an additional fertility preservation option for young women and girls with cancer or other reproductive health threatening diseases. Currently, Follicles are cultured for a defined period of time and analyzed as a cohort. However, Follicle growth is not synchronous, and culturing Follicles for insufficient or excessive times can result in compromised gamete quality. Our objective is to determine whether the selection of Follicles based on size, rather than absolute culture time, better predict Follicle maturity and oocyte quality. Multilayer secondary mouse Follicles were isolated and encapsulated in 0.25% alginate. Follicles were cultured individually either for defined time periods or up to specific Follicle diameter ranges, at which point several reproductive endpoints were analyzed. The metaphase II (MII) percentage after oocyte maturation on day 6 was the highest (85%) when Follicles were cultured for specific days. However, if Follicles were cultured to a terminal diameter of 300-350 μm irrespective of absolute time in culture, 93% of the oocytes reached MII. More than 90% of MII oocytes matured from Follicles with diameters of 300-350 μm showed normal spindle morphology and chromosome alignment, 85% of oocytes showed two pronuclei after IVF, 81% developed into the two-cell embryo stage and 38% developed to the blastocyst stage, all significantly higher than the percentages in the other Follicle size groups. Our study demonstrates that size-specific Follicle selection can be used as a non-invasive marker to identify high-quality oocytes and improve reproductive outcomes during eIVFG.
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Multiple Follicle culture supports primary Follicle growth through paracrine-acting signals
Reproduction (Cambridge England), 2013Co-Authors: Jessica E. Hornick, Francesca E Duncan, Lonnie D Shea, Teresa K WoodruffAbstract:In vitro Follicle growth in alginate hydrogels is a unique and versatile method for studying ovarian and Follicle biology that may also have implications for fertility preservation. Current culture systems support the development of isolated mouse Follicles from the secondary stage onward. However, it has been a challenge to grow smaller Follicles in vitro due to the dissociation of the oocyte from companion somatic cells. Recent work has demonstrated that coculturing primary Follicles with mouse embryonic fibroblasts or ovarian stromal cells supports Follicle survival and growth. In this study, we demonstrate that Follicles themselves can exert a beneficial coculture effect. When primary Follicles were cultured in groups of five or ten (multiple Follicle culture), there was increased growth and survival. The multiple Follicle culture approach maintained Follicle integrity and resulted in the formation of antral stage Follicles containing meiotically competent gametes. The growth and survival of primary Follicles were highly number dependent, with the most significant enhancement observed when the largest number of Follicles was grown together. Our data suggest that the Follicle unit is necessary to produce the secreted factors responsible for the supportive effects of multiple Follicle culture, as neither denuded oocytes, oocyte-secreted factors, nor granulosa cells alone were sufficient to support early Follicle growth in vitro. Therefore, there may be signaling from both the oocyte and the Follicle that enhances growth but requires both components in a feedback mechanism. This work is consistent with current in vivo models for Follicle growth and thus advances the movement to recapitulate the ovarian environment in vitro.
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isolated primate primordial Follicles require a rigid physical environment to survive and grow in vitro
Human Reproduction, 2012Co-Authors: Jessica E. Hornick, Francesca E Duncan, Lonnie D Shea, Teresa K WoodruffAbstract:background: In vitro Follicle growth is a promising fertility preservation strategy in which ovarian Follicles are cultured to produce mature and fertilization-competent oocytes. However, in primates, there has been limited success with in vitro Follicle growth starting from primordial and primary Follicles because adequate isolation methods and culture strategies have not been established. Understanding how to use primordial Follicles for fertility preservation has significant implications because these Follicles are the most abundant in the ovary, are found in all females and are fairly resistant to cryopreservation and chemotherapeutics. methods: In the primate ovary, primordial Follicles are concentrated near the collagen-rich ovarian cortex. To obtain these Follicles, we separated the ovarian cortex prior to enzymatic digestion and enriched the primordial Follicle concentration by using a novel double filtration system. To test the hypothesis that a rigid physical environment, as found in vivo, is optimal for survival, primordial Follicles were cultured in different concentrations of alginate for up to 6 days. Follicle survival and morphology were monitored throughout the culture. results: We found that primate ovarian tissue can be maintained for up to 24 h at 48C without compromising tissue or Follicle health. Hundreds of intact and viable primordial Follicles were isolated from each ovary independent of animal age. Follicle survival and morphology were more optimal when Follicles were cultured in 2% alginate compared with 0.5% alginate.
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A new hypothesis regarding ovarian Follicle development: ovarian rigidity as a regulator of selection and health
Journal of Assisted Reproduction and Genetics, 2011Co-Authors: Teresa K Woodruff, Lonnie D SheaAbstract:The mammalian ovary consists of a large number of dormant immature Follicles, each containing a single oocyte and located on the periphery of the ovary. With each reproductive cycle, a group of immature Follicles is sequentially activated to resume growth, and pituitary gonadotropins and ovarian steroid and peptide hormones cooperate to ensure further growth and development. A single dominant Follicle eventually emerges, ovulates, and then involutes to allow the selection of the next group of Follicles. While hormones are known to control the later stages of folliculogenesis, little is known about the pathways that activate individual immature primordial Follicles in the dormant Follicle pool. We advance a new hypothesis: that Follicle activation is dependent on the physical environment of the ovary in addition to well-established hormonal cues. This novel perspective on ovarian function may provide new avenues to study Follicle dynamics and identify therapeutic targets for ovarian dysfunction.
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a novel two step strategy for in vitro culture of early stage ovarian Follicles in the mouse
Fertility and Sterility, 2010Co-Authors: Shi Ying Jin, Lonnie D Shea, Ariella Shikanov, Lei Lei, Teresa K WoodruffAbstract:Objective To develop an in vitro strategy to support the growth of early-stage Follicles and produce mature oocytes competent for fertilization. Design Whole ovaries from 8-day-old mice were cultured for 4 days, and then secondary Follicles were isolated and cultured for 12 days in a three-dimensional alginate or fibrin-alginate (FA) hydrogel matrix. Setting University-affiliated laboratory. Animals Mice. Intervention(s) None. Main Outcome Measures Histologic evaluation of Follicle development, steroid hormone production, and rates of oocyte maturation, oocyte fertilization, and embryo formation. Result(s) Culture of 8-day-old mouse ovaries for 4 days resulted in transition of the Follicle population from primordial and primary Follicles to secondary Follicles, similar to that seen in a 12-day-old ovary. Isolated secondary Follicles cultured for 12 days showed larger increases in oocyte diameter and more frequent antrum formation and theca cell differentiation in the FA-hydrogel matrix compared with the alginate matrix. Steroid hormone secretion patterns were consistent with the changes in Follicle morphology and cell differentiation observed in the cultured Follicles. Compared with oocytes from alginate Follicle cultures, a greater number of oocytes retrieved from the FA-based Follicle cultures progressed to metaphase I, reached metaphase II, and could be fertilized and cleaved to two-cell embryos. The organ culture plus FA-hydrogel Follicle culture strategy produced a very high rate of oocyte progression to metaphase II (88 ± 8.7% [mean ± SEM]) and formation of two-cell embryos (54 ± 4%). Conclusion(s) A strategy combining whole ovary culture of early-stage Follicles and subsequent FA hydrogel in vitro Follicle culture produced a high percentage of oocytes competent for fertilization; this might provide new options for fertility preservation in women and prepubertal girls facing fertility-threatening diseases or treatments.
Zaixin Yang - One of the best experts on this subject based on the ideXlab platform.
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wnt dependent de novo hair Follicle regeneration in adult mouse skin after wounding
Nature, 2007Co-Authors: Zaixin Yang, Thomas Andl, Sarah E Millar, George CotsarelisAbstract:The mammalian hair Follicle is thought to form anew only during development, and loss of an adult Follicle is generally considered permanent. Fifty years ago in Nature, Billingham and Russel reported 'hair neogenesis' in rabbit skin, but this was later discounted. Now it is back, with the discovery that hair Follicle regeneration is triggered by wounding the skin of adult mice. This suggests that mammalian skin responds to wounding with greater plasticity and regenerative capacity than was previously believed, and has implications for those studying wound healing, tissue regeneration and stem cell function. The mammalian hair Follicle was thought to form only during development, so loss of an adult Follicle was considered permanent. Here Cotsarelis and colleagues solve a fifty-year-old debate, and show that wounding the skin of adult mice triggers de novo hair Follicle regeneration. The mammalian hair Follicle is a complex ‘mini-organ’ thought to form only during development1; loss of an adult Follicle is considered permanent. However, the possibility that hair Follicles develop de novo following wounding was raised in studies on rabbits2,3, mice4 and even humans fifty years ago5. Subsequently, these observations were generally discounted because definitive evidence for follicular neogenesis was not presented6. Here we show that, after wounding, hair Follicles form de novo in genetically normal adult mice. The regenerated hair Follicles establish a stem cell population, express known molecular markers of Follicle differentiation, produce a hair shaft and progress through all stages of the hair Follicle cycle. Lineage analysis demonstrated that the nascent Follicles arise from epithelial cells outside of the hair Follicle stem cell niche, suggesting that epidermal cells in the wound assume a hair Follicle stem cell phenotype. Inhibition of Wnt signalling after re-epithelialization completely abrogates this wounding-induced folliculogenesis, whereas overexpression of Wnt ligand in the epidermis increases the number of regenerated hair Follicles. These remarkable regenerative capabilities of the adult support the notion that wounding induces an embryonic phenotype in skin, and that this provides a window for manipulation of hair Follicle neogenesis by Wnt proteins. These findings suggest treatments for wounds, hair loss and other degenerative skin disorders.
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wnt dependent de novo hair Follicle regeneration in adult mouse skin after wounding
Nature, 2007Co-Authors: Mayumi Ito, Sarah E Millar, Thomas Andl, Zaixin Yang, Chunhua Cui, Noori Kim, George CotsarelisAbstract:The mammalian hair Follicle is a complex 'mini-organ' thought to form only during development; loss of an adult Follicle is considered permanent. However, the possibility that hair Follicles develop de novo following wounding was raised in studies on rabbits, mice and even humans fifty years ago. Subsequently, these observations were generally discounted because definitive evidence for follicular neogenesis was not presented. Here we show that, after wounding, hair Follicles form de novo in genetically normal adult mice. The regenerated hair Follicles establish a stem cell population, express known molecular markers of Follicle differentiation, produce a hair shaft and progress through all stages of the hair Follicle cycle. Lineage analysis demonstrated that the nascent Follicles arise from epithelial cells outside of the hair Follicle stem cell niche, suggesting that epidermal cells in the wound assume a hair Follicle stem cell phenotype. Inhibition of Wnt signalling after re-epithelialization completely abrogates this wounding-induced folliculogenesis, whereas overexpression of Wnt ligand in the epidermis increases the number of regenerated hair Follicles. These remarkable regenerative capabilities of the adult support the notion that wounding induces an embryonic phenotype in skin, and that this provides a window for manipulation of hair Follicle neogenesis by Wnt proteins. These findings suggest treatments for wounds, hair loss and other degenerative skin disorders.
Lonnie D Shea - One of the best experts on this subject based on the ideXlab platform.
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size specific Follicle selection improves mouse oocyte reproductive outcomes
Reproduction, 2015Co-Authors: Shuo Xiao, Francesca E Duncan, Lu Bai, Catherine T Nguyen, Lonnie D Shea, Teresa K WoodruffAbstract:Encapsulated in vitro Follicle growth (eIVFG) has great potential to provide an additional fertility preservation option for young women and girls with cancer or other reproductive health threatening diseases. Currently, Follicles are cultured for a defined period of time and analyzed as a cohort. However, Follicle growth is not synchronous, and culturing Follicles for insufficient or excessive times can result in compromised gamete quality. Our objective is to determine whether the selection of Follicles based on size, rather than absolute culture time, better predict Follicle maturity and oocyte quality. Multilayer secondary mouse Follicles were isolated and encapsulated in 0.25% alginate. Follicles were cultured individually either for defined time periods or up to specific Follicle diameter ranges, at which point several reproductive endpoints were analyzed. The metaphase II (MII) percentage after oocyte maturation on day 6 was the highest (85%) when Follicles were cultured for specific days. However, if Follicles were cultured to a terminal diameter of 300-350 μm irrespective of absolute time in culture, 93% of the oocytes reached MII. More than 90% of MII oocytes matured from Follicles with diameters of 300-350 μm showed normal spindle morphology and chromosome alignment, 85% of oocytes showed two pronuclei after IVF, 81% developed into the two-cell embryo stage and 38% developed to the blastocyst stage, all significantly higher than the percentages in the other Follicle size groups. Our study demonstrates that size-specific Follicle selection can be used as a non-invasive marker to identify high-quality oocytes and improve reproductive outcomes during eIVFG.
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Multiple Follicle culture supports primary Follicle growth through paracrine-acting signals
Reproduction (Cambridge England), 2013Co-Authors: Jessica E. Hornick, Francesca E Duncan, Lonnie D Shea, Teresa K WoodruffAbstract:In vitro Follicle growth in alginate hydrogels is a unique and versatile method for studying ovarian and Follicle biology that may also have implications for fertility preservation. Current culture systems support the development of isolated mouse Follicles from the secondary stage onward. However, it has been a challenge to grow smaller Follicles in vitro due to the dissociation of the oocyte from companion somatic cells. Recent work has demonstrated that coculturing primary Follicles with mouse embryonic fibroblasts or ovarian stromal cells supports Follicle survival and growth. In this study, we demonstrate that Follicles themselves can exert a beneficial coculture effect. When primary Follicles were cultured in groups of five or ten (multiple Follicle culture), there was increased growth and survival. The multiple Follicle culture approach maintained Follicle integrity and resulted in the formation of antral stage Follicles containing meiotically competent gametes. The growth and survival of primary Follicles were highly number dependent, with the most significant enhancement observed when the largest number of Follicles was grown together. Our data suggest that the Follicle unit is necessary to produce the secreted factors responsible for the supportive effects of multiple Follicle culture, as neither denuded oocytes, oocyte-secreted factors, nor granulosa cells alone were sufficient to support early Follicle growth in vitro. Therefore, there may be signaling from both the oocyte and the Follicle that enhances growth but requires both components in a feedback mechanism. This work is consistent with current in vivo models for Follicle growth and thus advances the movement to recapitulate the ovarian environment in vitro.
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isolated primate primordial Follicles require a rigid physical environment to survive and grow in vitro
Human Reproduction, 2012Co-Authors: Jessica E. Hornick, Francesca E Duncan, Lonnie D Shea, Teresa K WoodruffAbstract:background: In vitro Follicle growth is a promising fertility preservation strategy in which ovarian Follicles are cultured to produce mature and fertilization-competent oocytes. However, in primates, there has been limited success with in vitro Follicle growth starting from primordial and primary Follicles because adequate isolation methods and culture strategies have not been established. Understanding how to use primordial Follicles for fertility preservation has significant implications because these Follicles are the most abundant in the ovary, are found in all females and are fairly resistant to cryopreservation and chemotherapeutics. methods: In the primate ovary, primordial Follicles are concentrated near the collagen-rich ovarian cortex. To obtain these Follicles, we separated the ovarian cortex prior to enzymatic digestion and enriched the primordial Follicle concentration by using a novel double filtration system. To test the hypothesis that a rigid physical environment, as found in vivo, is optimal for survival, primordial Follicles were cultured in different concentrations of alginate for up to 6 days. Follicle survival and morphology were monitored throughout the culture. results: We found that primate ovarian tissue can be maintained for up to 24 h at 48C without compromising tissue or Follicle health. Hundreds of intact and viable primordial Follicles were isolated from each ovary independent of animal age. Follicle survival and morphology were more optimal when Follicles were cultured in 2% alginate compared with 0.5% alginate.
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A new hypothesis regarding ovarian Follicle development: ovarian rigidity as a regulator of selection and health
Journal of Assisted Reproduction and Genetics, 2011Co-Authors: Teresa K Woodruff, Lonnie D SheaAbstract:The mammalian ovary consists of a large number of dormant immature Follicles, each containing a single oocyte and located on the periphery of the ovary. With each reproductive cycle, a group of immature Follicles is sequentially activated to resume growth, and pituitary gonadotropins and ovarian steroid and peptide hormones cooperate to ensure further growth and development. A single dominant Follicle eventually emerges, ovulates, and then involutes to allow the selection of the next group of Follicles. While hormones are known to control the later stages of folliculogenesis, little is known about the pathways that activate individual immature primordial Follicles in the dormant Follicle pool. We advance a new hypothesis: that Follicle activation is dependent on the physical environment of the ovary in addition to well-established hormonal cues. This novel perspective on ovarian function may provide new avenues to study Follicle dynamics and identify therapeutic targets for ovarian dysfunction.
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a novel two step strategy for in vitro culture of early stage ovarian Follicles in the mouse
Fertility and Sterility, 2010Co-Authors: Shi Ying Jin, Lonnie D Shea, Ariella Shikanov, Lei Lei, Teresa K WoodruffAbstract:Objective To develop an in vitro strategy to support the growth of early-stage Follicles and produce mature oocytes competent for fertilization. Design Whole ovaries from 8-day-old mice were cultured for 4 days, and then secondary Follicles were isolated and cultured for 12 days in a three-dimensional alginate or fibrin-alginate (FA) hydrogel matrix. Setting University-affiliated laboratory. Animals Mice. Intervention(s) None. Main Outcome Measures Histologic evaluation of Follicle development, steroid hormone production, and rates of oocyte maturation, oocyte fertilization, and embryo formation. Result(s) Culture of 8-day-old mouse ovaries for 4 days resulted in transition of the Follicle population from primordial and primary Follicles to secondary Follicles, similar to that seen in a 12-day-old ovary. Isolated secondary Follicles cultured for 12 days showed larger increases in oocyte diameter and more frequent antrum formation and theca cell differentiation in the FA-hydrogel matrix compared with the alginate matrix. Steroid hormone secretion patterns were consistent with the changes in Follicle morphology and cell differentiation observed in the cultured Follicles. Compared with oocytes from alginate Follicle cultures, a greater number of oocytes retrieved from the FA-based Follicle cultures progressed to metaphase I, reached metaphase II, and could be fertilized and cleaved to two-cell embryos. The organ culture plus FA-hydrogel Follicle culture strategy produced a very high rate of oocyte progression to metaphase II (88 ± 8.7% [mean ± SEM]) and formation of two-cell embryos (54 ± 4%). Conclusion(s) A strategy combining whole ovary culture of early-stage Follicles and subsequent FA hydrogel in vitro Follicle culture produced a high percentage of oocytes competent for fertilization; this might provide new options for fertility preservation in women and prepubertal girls facing fertility-threatening diseases or treatments.