The Ectodysplasin/Ectodysplasin receptor (Eda/Edar) signalling pathway is critical during development for the formation of skin appendages. However, its roles during adulthood are only recently being elucidated. Adult appendages, such as hair follicles (HFs), are known to become activated to respond to cutaneous injury. However, the HF houses distinct cell populations that display differing capacities to participate and persist in re-epithelialisation. We show, contrary to previous findings, that the best-characterised stem cell (SC) niche within the HF (the bulge) does not respond to injury during the earliest stages of wound healing. We propose that bulge SCs are prevented from participating in early repair as a protection mechanism against tumourigenesis. Despite the bulge niche not participating in early repair, we found the upper HF outer root sheath (ORS) to respond rapidly to injury. Our investigation into the role of Eda/Edar signalling during wound healing revealed that activation of the pathway was able to specifically induce proliferation within this portion of the HF. We further demonstrate a number of roles for the Eda/Edar pathway during adult wound healing, including, surprisingly, influencing several wound responses within the dermis. Specifically, an absence of Eda/Edar signalling in Tabby mice results in delayed wound healing, whereas acute activation of the pathway in wild-type (WT) mice can stimulate re-epithelialisation and enhance wound repair. These effects also translate to a model of human wound healing, where activation of Eda/Edar signalling accelerates re-epithelialisation and increases peri-wound proliferation. RNA-seq analysis reveals diverse gene regulation in the presence/absence of Eda/Edar signalling. Overall, these findings suggest that manipulation of the Eda/Edar pathway may represent an attractive potential therapeutic for enhancement of wound repair, potentially through maximising the natural growth capacity of peri-wound HFs.

In response to LH surge, the selected dominant follicle undergoes a series of complex events viz., meiotic maturation of the oocyte, terminal differentiation of follicular cells, cumulus expansion and follicular rupture leading to ovulation (release of fertilizable oocyte) and formation of corpus luteum. These complex set of events are mediated by dramatic changes in the temporo-spatial transcriptional regulation of a large number of genes in the preovularory follicle. In the present thesis, attempts have been made to delineate the transcriptional changes occurring in the periovulatory follicle in response to gonadotropin surge in monovulatory species (bonnet monkeys and buffalo cows). Further, attempts have also been made to investigate effects of increased circulating IGF-I and glucose on the periovulatory events. Chapter I provides a review of available literature on regulation and factors influencing various aspects of growth and development of ovarian follicles. Chapter II describes initial studies carried out in the bonnet monkeys to examine expression patterns of various genes considered as markers of ovulation and luteinization in granulosa cells of multiple preovulatory-like follicles before and after exposure to luteinizing dose of hCG. Chapter III describes the standardization and validation of an induced ovulation model system in buffalo cows involving determination of the presence of a large follicle by ultrasonography before administering exogenous PGF2αon day 7 of the cycle to induce luteolysis and administration of hCG 36 h post PGF2αto mimic LH surge-like stimulus. Attempts made to characterize the responsiveness of buffalo cows to a range of exogenous doses of GnRH in terms of LH secretion during summer and winter seasons and determination of the exogenous dose of GnRH necessary to elicit a surge-like increase in circulating LH levels during breeding season in buffalo cows are described in Chapter IV. Chapter V describes the global changes in the gene expression pattern in the periovulatory follicle of buffalo cows before and at various time points after the onset of gonadotropin surges. IGF-I is known to regulate the proliferation and survival of follicular cells and play an important role in the selection of dominant follicle however, its role during the periovulatory events remains to be delineated. Chapter VI describes the effects of increased circulating and intrafollicular IGF-I levels on the periovulatory events. An ongoing study to delineate the effects of transient increase in circulating glucose levels on the periovulatory events are described in Appendix I. Thus, this thesis aims at delineating (i) changes in the gene expression patterns regulating periovulatory events and (ii) effects of increased circulating IGF-I and glucose on the periovulatory events.

Polycystic ovary syndrome (PCOS) is an endocrine abnormality with multiple diagnostic criteria due to its heterogenic manifestations. One of the diagnostic criterion includes analysis of ultrasound images of ovaries for the detection of number, size, and distribution of follicles within the ovary. This involves manual tracing of follicles on the ultrasound images to determine the presence of a polycystic ovary (PCO). A novel method that automates PCO morphology detection is described. Our algorithm involves automatic segmentation of follicles from ultrasound images, quantifying the attributes of the segmented follicles using stereology, storing follicle attributes as feature vectors, and finally classification of the feature vector into two categories. The classification categories are PCO morphology present and PCO morphology absent. An automatic PCO diagnostic tool would save considerable time spent on manual tracing of follicles and measuring the length and width of every follicle. Our procedure was able to achieve classification accuracy of 92.86% using a linear discriminant classifier. Our classifier will improve the rapidity and accuracy of PCOS diagnosis, and reduce the chance of the severe health implications that can arise from delayed diagnosis.

In the ewe, one to three antral follicles emerge or grow from a pool of small antral follicles (1 to 3 mm in diameter) every 3 to 5 days and reach diameters of ¡Ý5 mm before regression or ovulation. Each follicular wave is triggered by a peak in serum concentrations of FSH. It is not clear what characteristics of an FSH peak cause follicular wave emergence and what aspects of development of a follicular wave are regulated by its preceding FSH peak.<p> In Experiment 1, we found that the amplitude of FSH peaks decreased, while basal serum FSH concentrations increased across the inter-ovulatory interval (P < 0.05). However, there were no associated changes in the growth, static or regression phases of follicular waves or the number and size of follicles in a wave. In Experiment 2, using computer-assisted quantitative echotextural analysis, we found that the numerical pixel value (NPV) for the wall of anovulatory follicles emerging in the third wave of the cycle was significantly higher than for waves 1 and 2 at the time of wave emergence but it decreased as follicles reached maximum follicular diameter (P < 0.05). A tendency for a similar pattern for the wall of follicles in the last wave of the cycle was also observed (P = 0.07).<p> In Experiment 3, treatment with ovine FSH (oFSH) increased the amplitude of an FSH peak by 5 to 6 fold. This treatment increased estradiol production (P < 0.05) but had little effect on other characteristics of the subsequent follicular wave. Daily injections of oFSH (Experiment 4) for four days, resulted in the occurrence of 4 discrete peaks in serum FSH concentrations. Each injection of oFSH resulted in the emergence of a new follicular wave.<p> In Experiment 5, six cyclic ewes received oFSH (0.1 ¦Ìg/kg, sc) every 6 h for 42 h, to try to give a gradual increase in the leading slope of an FSH peak. Serum FSH concentrations increased in oFSH treated ewes (P < 0.05) resulting in an additional peak between two endogenously driven FSH peaks and therefore, did not give the planned gradual leading slope to an FSH peak. Ovine FSH treatment occurred in the early growth phase of wave 1 of the inter-ovulatory interval and increased the growth rate of growing follicles in that wave, compared to control ewes (P < 0.05). This apparently induced dominance in follicles in wave 1, causing them to suppress wave emergence in response to the injected FSH. In Experiment 6, oFSH was infused constantly (1.98 ¦Ìg/ewe/h, iv, n = 6) for 60 h. Infusion of oFSH maintained serum FSH concentrations at a level similar to the zenith of a peak. This resulted in a superstimulatory effect with a peak in the mean number of large follicles on Day 2 after the start of FSH infusion (P < 0.001).<p> A hormonal milieu similar to low serum progesterone concentrations was created by treatment of ewes with prostaglandin and medroxyprogesterone acetate (MAP) sponges (Experiment 7). This treatment delayed regression of the penultimate follicular wave of a cycle. However, the delayed follicular atresia was accompanied by a greater degree of apoptosis in somatic cells of follicles growing in the penultimate wave compared to those in the final wave of the cycle, when collected one day before expected ovulation.<p> In conclusion, trends in basal serum concentrations of FSH and peaks in serum FSH concentrations, across the estrous cycle, are associated with changes in the image attributes of follicles emerging later in the estrous cycle, perhaps reflecting a greater readiness of those follicles for ovulation and formation of CL. The ovine ovary can respond to discrete peaks in serum FSH concentrations with the emergence of new follicular waves on a daily basis. This led us to conclude that follicular dominance is not evident in the ewe and peaks in serum FSH concentrations are likely to be driven by some endogenous rhythm that is unrelated to ovarian follicular secretory products. However, direct dominance can be induced by giving supplemented FSH during the growth phase of a follicle. Extended exposure of ovine ovaries to the serum concentrations of FSH found at the zenith of a peak overrides the mechanisms that recruit follicles into a wave and induces a superovulatory response in cyclic ewes. Finally, an increase in the incidence of apoptosis occurs in antral follicles in sheep that have an extended lifespan, prior to any morphological changes detectable by ultrasonography. This would seem to cause decreased follicular viability and lowered fertility of the oocytes that the follicles contain.

Polycystic ovary syndrome (PCOS) is an endocrine abnormality with multiple diagnostic criteria due to its heterogenic manifestations. One of the diagnostic criterion includes analysis of ultrasound images of ovaries for the detection of number, size, and distribution of follicles within the ovary. This involves manual tracing of follicles on the ultrasound images to determine the presence of a polycystic ovary (PCO). A novel method that automates PCO morphology detection is described. Our algorithm involves automatic segmentation of follicles from ultrasound images, quantifying the attributes of the segmented follicles using stereology, storing follicle attributes as feature vectors, and finally classification of the feature vector into two categories. The classification categories are PCO morphology present and PCO morphology absent. An automatic PCO diagnostic tool would save considerable time spent on manual tracing of follicles and measuring the length and width of every follicle. Our procedure was able to achieve classification accuracy of 92.86% using a linear discriminant classifier. Our classifier will improve the rapidity and accuracy of PCOS diagnosis, and reduce the chance of the severe health implications that can arise from delayed diagnosis.

In the ewe, one to three antral follicles emerge or grow from a pool of small antral follicles (1 to 3 mm in diameter) every 3 to 5 days and reach diameters of ¡Ý5 mm before regression or ovulation. Each follicular wave is triggered by a peak in serum concentrations of FSH. It is not clear what characteristics of an FSH peak cause follicular wave emergence and what aspects of development of a follicular wave are regulated by its preceding FSH peak.<p> In Experiment 1, we found that the amplitude of FSH peaks decreased, while basal serum FSH concentrations increased across the inter-ovulatory interval (P < 0.05). However, there were no associated changes in the growth, static or regression phases of follicular waves or the number and size of follicles in a wave. In Experiment 2, using computer-assisted quantitative echotextural analysis, we found that the numerical pixel value (NPV) for the wall of anovulatory follicles emerging in the third wave of the cycle was significantly higher than for waves 1 and 2 at the time of wave emergence but it decreased as follicles reached maximum follicular diameter (P < 0.05). A tendency for a similar pattern for the wall of follicles in the last wave of the cycle was also observed (P = 0.07).<p> In Experiment 3, treatment with ovine FSH (oFSH) increased the amplitude of an FSH peak by 5 to 6 fold. This treatment increased estradiol production (P < 0.05) but had little effect on other characteristics of the subsequent follicular wave. Daily injections of oFSH (Experiment 4) for four days, resulted in the occurrence of 4 discrete peaks in serum FSH concentrations. Each injection of oFSH resulted in the emergence of a new follicular wave.<p> In Experiment 5, six cyclic ewes received oFSH (0.1 ¦Ìg/kg, sc) every 6 h for 42 h, to try to give a gradual increase in the leading slope of an FSH peak. Serum FSH concentrations increased in oFSH treated ewes (P < 0.05) resulting in an additional peak between two endogenously driven FSH peaks and therefore, did not give the planned gradual leading slope to an FSH peak. Ovine FSH treatment occurred in the early growth phase of wave 1 of the inter-ovulatory interval and increased the growth rate of growing follicles in that wave, compared to control ewes (P < 0.05). This apparently induced dominance in follicles in wave 1, causing them to suppress wave emergence in response to the injected FSH. In Experiment 6, oFSH was infused constantly (1.98 ¦Ìg/ewe/h, iv, n = 6) for 60 h. Infusion of oFSH maintained serum FSH concentrations at a level similar to the zenith of a peak. This resulted in a superstimulatory effect with a peak in the mean number of large follicles on Day 2 after the start of FSH infusion (P < 0.001).<p> A hormonal milieu similar to low serum progesterone concentrations was created by treatment of ewes with prostaglandin and medroxyprogesterone acetate (MAP) sponges (Experiment 7). This treatment delayed regression of the penultimate follicular wave of a cycle. However, the delayed follicular atresia was accompanied by a greater degree of apoptosis in somatic cells of follicles growing in the penultimate wave compared to those in the final wave of the cycle, when collected one day before expected ovulation.<p> In conclusion, trends in basal serum concentrations of FSH and peaks in serum FSH concentrations, across the estrous cycle, are associated with changes in the image attributes of follicles emerging later in the estrous cycle, perhaps reflecting a greater readiness of those follicles for ovulation and formation of CL. The ovine ovary can respond to discrete peaks in serum FSH concentrations with the emergence of new follicular waves on a daily basis. This led us to conclude that follicular dominance is not evident in the ewe and peaks in serum FSH concentrations are likely to be driven by some endogenous rhythm that is unrelated to ovarian follicular secretory products. However, direct dominance can be induced by giving supplemented FSH during the growth phase of a follicle. Extended exposure of ovine ovaries to the serum concentrations of FSH found at the zenith of a peak overrides the mechanisms that recruit follicles into a wave and induces a superovulatory response in cyclic ewes. Finally, an increase in the incidence of apoptosis occurs in antral follicles in sheep that have an extended lifespan, prior to any morphological changes detectable by ultrasonography. This would seem to cause decreased follicular viability and lowered fertility of the oocytes that the follicles contain.

Prenatal mortality in the swine ranges from 30%~40%. Little is known about genes that involve in the preovulation events that the initiation of swine oocyte development. The main objective of this study was to utilize suppression subtractive hybridization¡]SSH¡^to delineate the differential gene expression between in vitro culture mature and antral-follicle oocytes of swine development. The knowledge of genes and their accumulated mRNA is essential to better understand the mechanisms involved in the oocyte maturation and the survival of the in vitro produced embryos. Porcine ovaries obtained from the slaughterhouse were used to collect oocytes from follicle with a diameter ≥ 3 mm. After in vitro mature for two days, oocytes with first polar body were subjected to as the testers and were lysed for mRNA extraction. Pools of 26 denuded oocytes without culture were submitted to suppressive subtraction hybridization (SSH) as the drivers. Forward and reverse subtractions were performed to identify candidate genes differentially expressed between in vitro culture mature and primordial-follicle oocytes. A total of one hundred and thirty-five differential expressed plasmid clones were sequenced, and each was analyzed by BLAST programs. Of these transcripts, 40 clones were subjected to differential screening by a dot blot cDNA array. We identified three genes like zona pellucida glycoprotein (ZP1), 1-aminocyclopropane-1-carboxylate synthase (ACS), and death associated protein 5¡]DAP 5¡^while other numerous clones remain novel. The in vivo functions of the genes remain further investigation.

Thesis (M.S.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains vii, 60 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 57-60).

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 141-162). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 141-162). Also available on the Internet.