Diss. (sammanfattning). Uppsala : Sveriges lantbruksuniv., 2003. / Härtill 6 uppsatser.

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xv, 137 p.; also includes graphics. Includes bibliographical references (p. 122-137). Available online via OhioLINK's ETD Center

The mammalian ovary contains a vast reserve of oocytes, most of which are enclosed within preantral follicles. A Biopsy Pick-Up (BPU) method was tested to determine the feasibility of retrieving preantral follicles from mare ovaries in vivo. BPU fragments were collected from 5 to 21 yr old mares during the breeding season and processed using a tissue chopper, submitted to histological analysis or submitted to in vitro culture for 1 or 7 days. In conclusion, the BPU method is a reliable way to repeatedly harvest large numbers of viable, morphologically normal preantral follicles from living mares without affecting ovarian function or general reproductive health. Additionally, equine preantral follicles undergo development and growth when submitted to in vitro culture in α-MEM for 7 days, with some follicles maintaining morphological normality throughout. These technologies could be used in the future to enable the use of numerous oocytes present within the equine ovary to preserve genetic material or for large-scale embryo production.

Preserving viability of frozen gametes and reproductive tissues is crucial to understand and overcome species-specificities in respect to the diversity in cryobiological properties and requirements among cell types and tissues. The use of different animal models to study ovarian tissue cryopreservation will help to uncover several important factors related to germ cells preservation. Horses (Equus ferus caballus) have been proven to be an excellent model for reproductive biology studies with implications for humans. White-tailed deer (Odocoileus virginianus) are one of the most abundant wild species in the United States, but little information about their reproductive features are known. Therefore, five studies were conducted in this Dissertation with the following general objectives: (i) to develop ovarian tissue cryopreservation techniques for horses and white-tailed deer species; and (ii) to determine the effects of ovarian tissue cryopreservation techniques on morphological and molecular mechanisms related to folliculogenesis in horse and white-tailed deer species. In study one, equine ovarian tissue was used to determine the ideal ovarian fragment size for better cooling resistance under storage at 4°C. In study two, equine ovarian tissues were used to determine the toxicity effect of cryoprotective agents on ovarian tissue pre- and post-cryopreservation. In study three, equine ovarian tissues were used to compare slow-freezing versus vitrification; and to determine the best cryoprotective agents for each cryopreservation method. In study four, white-tailed deer reproductive tracts were used to characterize the age effect on reproductive features. In study five, white-tailed deer ovarian tissue was used to compare slow-freezing versus vitrification methods to preserve preantral follicles under in vitro culture. The main findings of the horse studies were: (i) equine ovarian tissue can be stored at 4°C for up to 24 h when biopsy ovarian fragments are used; (ii) ethylene glycol seems to be a less harmful cryoprotectant agent to equine preantral follicles; and (iii) both slow-freezing and vitrification methods similarly preserved the follicle morphology after time of culture. The main findings of the white-tailed deer studies were: (i) aging caused quantitative and qualitative effects on the ovarian reserve of white-tailed deer; (ii) fresh ovarian tissue can be cultured for up to seven days preserving the tissue integrity; and (iii) fragments cryopreserved by vitrification had higher follicle viability during in vitro culture than by the slow-freezing method. In conclusion, this work demonstrated the viability to cryopreserve equine and white-tailed deer ovarian tissue. Furthermore, the frozen-thawed equine and white-tailed deer ovarian tissue can be cultured for up to seven days.

Resumo: Objetivou-se testar diferentes protocolos de cultivo in vitro e in vivo de folículos ovarianos pré-antrais de fetos bovinos. Para tanto, um total de 41 ovários de fetos bovinos foram obtidos em matadouro, transportados e utilizados para o cultivo in vitro (n=20) e para o xenotransplante (n=21). Após processados no laboratório em fragmentos entre 0,5 e 1 mm3 foram encaminhados para os cultivos O cultivo in vitro baseou-se em protocolo bem sucedido de cultivo de FOPA em caprinos e testou diferentes fontes de macromoléculas e a utilização do azul de tripan na viabilidade dos tecidos cultivados a uma atmosfera controlada de 5% CO2 em ar, a 38,5°C e nutridos com dMEM (300 mOsm/L, pH 7,2) suplementado com antibióticos, ITS, piruvato de sódio, glutamina, hipoxantina, dAMPc, bFSH e IGF-I. A depender do tratamento, foi adicionado BSA (0,1%) SFB (10%) ou PVA (1%). O cultivo in vivo de FOPA foi executado por xenotransplante sob a cápsula renal num total de 65 camundongas imunodeficientes. Desenvolveu-se uma técnica de biopsia e verificou-se o efeito do tempo de transplante (30, 60 e 30 e 60 dias após o transplante) sobre a percentagem e a viabilidade de FOPA bem como a possível presença de folículos antrais. Num segundo momento, 32 receptoras receberam estímulo hormonal de 10 UI de eCG (n=18) e 10 UI r-hFSH (n=14). Os resultados mostraram que o cultivo com PVA apresentou FOPA normais em percentagem semelhante aos cultivos com BSA e PVA. Quanto ao cultivo por xenotransplante, observou-se o crescimento sucessivo de FOPA até estádios antrais ao longo do tempo de transplante (> 30 dias). O resultado das estimulações exógenas apresentou folículos antrais com oócitos que apresentaram o cumulus expandido em 2/5 (40%) dos oócitos selecionados para MIV de cada um dos tratamentos propostos. Concluindo, FOPA oriundos de fetos bovinos podem ser cultivados por pelo menos 8 dias em PVA... (Resumo completo clicar acesso eletrônico abaixo) / Abstract: This study aimed to evaluate different protocols of in vitro and in vivo preantral follicles (PFs) culture from bovine fetus. Thus, a total of 41 fetal bovine ovaries, from a slaughterhouse, were collected and transported at laboratory for in vitro culture (n=20) and for xenotransplantation (n=21), after processing into small cortical pieces measuring between 0,5 and 1mm3 the slices were cultured. The in vitro culture was based on well successful protocol of preantral follicles in caprine and tested different sources of macromolecules and trypan blue viability of cultured tissues cultured at controlled atmosphere (5%CO2 in air, 39°C). The culture medium used was dMEM (300 mOsm/L, pH 7, 2) supplemented with antibiotics, ITS, sodium pyruvate, glutamine, hypoxanthine, dAMPc, bFSH, and IGF-I. Depending of treatment, was added BSA (0,1%), FCS (10%) or PVA (1%). In vivo culture of preantral follicles was carried out by xenotransplantation under renal capsule of immunodeficient females mice (total of 65) that were submitted to a biopsy technique previously developed for tissue collection and to verify the effectiveness of time of transplantation (30, 60 and 30 and 60 days post surgery) under percentage and viability as well as putative growth of PFs to antral follicles. At second stage, 32 recipient mice were submitted to hormonal stimuli with 10 IU of eCG (n=18) and 10 IU of r-hFSH (n=14). The results showed that PVA culture presented normally PF in similar distribution when compared with BSA and PVA culture. Regarding xenotransplantation, successive growth of PF until antral stages was observed belong time of transplantation. Exogenous stimulation presented oocytes with expanded cumulus in 2/5 (40%) of selected oocytes for IVM from each treatment. Conclusively PFs from fetal bovine ovaries can be cultured at PVA at least 8 days and grows until antral stages after xenotransplantation procedures... (Complete abstract click electronic access below) / Orientador: Maria Rita Pacheco / Coorientador: Wilter Ricardo Russiano Vicente / Banca: José Ricardo de Figueiredo / Banca: José Antonio Visintin / Banca: Joaquim Mansano Garcia / Banca: Paulo Henrique Franceschini / Doutor

Over the past decade the concept that the hair follicle plays an important role in cutaneous wound repair has been established. Several elegant lineage tracing studies have demonstrated that hair follicle derived cells contribute to the long term maintenance of the epidermis following repair, while an absence of hair follicles is known to delay repair. The exact mechanisms surrounding hair follicle derived repair are unknown. Moreover, while multiple stem cell niches are present within the hair follicle, their relative importance during wound repair is still unclear. The hair follicle is also a regenerative mini-organ, undergoing regular cycles of growth and regression throughout life, yet surprisingly this has not been previously investigated with respect to wound repair. Data presented in this thesis reveals an unappreciated, yet fundamental link between the independent processes of hair cycle and wound repair, with a substantial acceleration in the rate of repair (~50%) observed in anagen phase. Importantly, the hair follicle appears to play a global role in repair, with differences in the contribution of multiple cell types to wound repair. In addition, this thesis addresses the early kinetics of hair follicle wound response for the first time. Anagen hair follicles are found predisposed to a more rapid and extensive response to injury, suggesting a higher overall percentage of repair derived from the hair follicle in anagen phase. Surprisingly, the bulge stem cell region, while critical for hair cycle appears to play little role in the events immediately following injury, and is not required for initiation of re-epithelialisation. Gene expression profiling reveals numerous genes associated with anagen accelerated repair, and identifies altered modulation of the immune system as a key mechanism. Further, anagen wounds are associated with an upregulation of developmental transcription factors, which may imply a more regenerative healing phenotype. These data reveal numerous targets with the potential to accelerate repair, which now require validation for their therapeutic potential. These targets could be of importance in promoting the repair of chronic wounds, an area of unmet clinical need. More generally, this thesis has established hair cycle as an important experimental variable, which must be controlled for in all future in vivo murine wounding studies.

While many studies have shown harmful effects of space flight on many tissues and systems of the human body, few studies have been done on the effects of space flight on the reproductive system. While the microgravity conditions of space flight are common knowledge, there is another component of space flight, that being higher than ambient (on Earth) levels of radiation. The purpose of this study was to examine the effects of simulated space flight on follicular development in the ovaries of mice, and to determine which component of spaceflight, i.e. microgravity, radiation, or a combination of the two, might be responsible for any changes in this follicular development. To simulate the environment of space, mice were exposed to higher levels of radiation by the use of cobalt plates and to simulated microgravity using a technique known as hind limb unloading. Four groups of mice-were used in this study; a control or untreated group, a group exposed to higher levels of radiation, a group exposed to simulated microgravity, and a group treated in both high radiation and simulated microgravity. The mice were further subdivided within these groups based on the amount of time they were kept alive after treatment/exposure (one, four, and nine months). The ovarian tissues were then analyzed to see the effects of these simulated conditions on the development of follicles. In all three treatment groups, development of follicles was restricted compared to the control group. Follicles from the various treatment groups appeared to be in the early stages of their development. It should be noted that these are preliminary results as the study is still in progress. One of the overarching questions that has been put forth by NASA over the last few decades is, can an organism, in this case a mammalian organism, complete an entire life cycle in space? This study may help to answer some of that question. If any of the components of space flight proves to be harmful to the female reproductive tissues human colonization of space would be problematic. If the damage incurred during space flight is irreversible, colonization of other worlds would also be problematic.

The porcine antral follicle, which consists of an oocyte and surrounding follicular components, including theca, granulosa, and cumulus cells and follicular fluid, is an essential microenvironment for oocyte development and maturation. Investigating cellular and molecular events in the context of the whole follicle will aid in our understanding of interactions between the oocyte and the follicular components. The objective of this dissertation was to develop a novel bioluminescent imaging model to visualize and measure cellular and molecular events in living intact ovarian follicles in vitro. Bioluminescence imaging was employed to facilitate noninvasive, dynamic, and real-time transgene analysis in living intact follicles. The time courses of luciferase-luciferin reactions, effective plasmid DNA and D-luciferin doses and their combinations were determined as the first step toward developing a new real-time bioluminescence imaging model. In addition, the efficient nonviral gene delivery methods: cationic lipid mediated gene transfer (chemical) and electroporation (physical) for the living intact follicles were determined. For the cationic lipid mediated gene transfer method, the 1:3 DNA lipid ratio was optimal. It was also found that the optimal condition of electroporation (4 electric pulses with 100 ms duration at field strength of 100 V/cm) resulted in 15 times higher luciferase activity and increased granulosa cell viability over the cationic lipid mediated gene transfer method. Moreover, increased granulosa cell viability, increased follicular fluid progesterone content, and oocytes with expanded cumulus cells were observed in intact follicles transfected by electroporation at a field strength of 100 V/cm. Finally, bioluminescence imaging was applied to quantify functional and ligand-activated estrogen receptor (ER) activity within living intact follicles. The functional ERs were differentially activated during the different stages of the estrous cycle in the mature sow; the levels of functional ER activity in cultured granulosa cells and intact follicles in vitro were increased from late luteal phase to early follicular phase and then significantly decreased at late follicular phase. The methodology developed herein can be applicable to further our understanding of oocyte and follicle development and oocyte maturation.

No description available.

No / Bone morphogenetic protein (BMP) signaling is involved in the regulation of a large variety of developmental programs, including those controlling organ sizes. Here, we show that transgenic (TG) mice overexpressing the BMP antagonist noggin (promoter, K5) are characterized by a marked increase in size of anagen hair follicles (HFs) and by the replacement of zig-zag and auchen hairs by awl-like hairs, compared with the age-matched WT controls. Markedly enlarged anagen HFs of TG mice show increased proliferation in the matrix and an increased number of hair cortex and medulla cells compared with WT HFs. Microarray and real-time PCR analyses of the laser-captured hair matrix cells show a strong decrease in expression of Cdk inhibitor p27(Kip1) and increased expression of selected cyclins in TG vs. WT mice. Similar to TG mice, p27(Kip1) knockout mice also show an increased size of anagen HFs associated with increased cell proliferation in the hair bulb. Primary epidermal keratinocytes (KC) from TG mice exhibit significantly increased proliferation and decreased p27(Kip1) expression, compared with WT KC. Alternatively, activation of BMP signaling in HaCaT KC induces growth arrest, stimulates p27(Kip1) expression, and positively regulates p27(Kip1) promoter activity, thus further supporting a role of p27(Kip1) in mediating the effects of BMP signaling on HF size. These data suggest that BMP signaling plays an important role in regulating cell proliferation and controls the size of anagen HFs by modulating the expression of cell-cycle-associated genes in hair matrix KC.