Thesis (Ph. D.)--University of Virginia, 1998. / Includes bibliographical references (130-147). Also available online through Digital Dissertations.

No description available.

The RNA binding protein DAZL (Deleted in Azoospermia) is essential for germ cell survival and subsequent fertility. The transgenic mouse DAZL model has confirmed that knockout (KO) females are infertile as a direct consequence of complete postnatal oocyte ablation. Interestingly, the heterozygous (Het) DAZL females have increased fertility giving rise to significantly more viable offspring, accompanied by significantly reduced plasma FSH and increased inhibin B compared to levels observed in the wildtype (Wt) females. Recent studies to identify putative DAZL mRNA targets suggest that DAZL may have multiple functions and mRNA targets throughout germ cell development. However, how this protein functions within the oocyte and how functional copy number gives rise to increased fertility remains to be fully elucidated. The studies in this thesis sought to identify putative DAZL mRNA targets in addition to molecular mechanisms which may be either affected direct or indirectly as a result of the functional copy number of DAZL (Wt or Het) within the oocyte or follicular unit. Oocytes from Wt and Het were evaluated for their expression of selected oocyte genes and comparative analysis suggests that oocyte gene expression is significantly altered between the genotypes. Genes of interest include Oosp1 and H1foo, both of which are down-regulated in mRNA expression in Het d21 oocytes and d10 ovaries compared to the Wt. Furthermore, an in silico bioinformatics approach was utilised to identify putative DAZL mRNA targets using a consensus DAZL binding sequence. One candidate target, PDCD4, previously identified as a tumour suppressor gene was selected for further investigation. Despite PDCD4 mRNA and protein being highly expressed within the ovary, no difference in mRNA levels between Het and Wt was observed. However, although not ruling out the possibility of being a DAZL target we now have evidence that PDCD4 can function within the steroidogenic cells of the corpus luteum in relation to functional luteolysis. Indirect actions of DAZL upon local regulation and response of follicle growth in culture were evaluated to investigate follicles at the gonadotrophin dependent stage of growth. Individual follicles from Wt and Het d21 mice were cultured in the presence of FSH at 1iu, 0.5iu, 0.1iu and 0.01iu for a six day period. Final follicle size/morphology did not differ between genotypes at 1iu, 0.5iu and 0.1iu of FSH, but by d3 at 0.01iu FSH growth of Wt follicles was significantly (P<0.001) perturbed compared to the Het. Despite no difference in final size between 1iu, 0.5iu, 0.1iu FSH treatments, mRNA analysis of individual follicles demonstrated a significant up-regulation of FSH receptor (P<0.05), aromatase (P<0.05) and inhibin βA (P<0.01) and a significant down-regulation in inhibin βB (P<0.01) expression in the Het follicles compared to the Wt, suggesting an increase in follicle maturity, sensitivity and hence suitability for selection as viable pre-ovulatory follicles. Furthermore, atresia rates from cultured follicles were significantly lower (P<0.05 (1iu, 0.1iu FSH); P<0.01(0.01iu FSH)) in the Het compared to the Wt. These studies provide strong evidence that multiple mechanisms within the oocyte/follicle are directly and indirectly affected as a result of functional copy number of DAZL. Although direct in vivo targets remain to be identified it is clear that DAZL protein potentially targets multiple mRNAs at different stages of development, pre-programming the oocyte to increase the sensitivity of follicle and/or the functioning within a transcription complex regulating development. In conclusion, the beneficial consequences of increased fertility in the Het females is accompanied by a possible acceleration in oocyte and follicle maturation, an increased sensitivity to FSH in vitro with evidence of advanced stages of growth and, a reduction in follicle atresia. These differences support the suggestion that DAZL is having systemic effects on the paracrine communication within the follicle unit between the oocyte and somatic cells altering regulation and subsequent selection, and affecting final ovulation rate and litter size.

Low fertility is a major cause of lost productivity in the cattle industry. In addition, cattle provide a convenient model to study ovarian physiology in monovular species including humans. Our previous microarray studies in the bovine ovary showed the upregulation of two clusters, miR-212-132 and miR-183-96-182, in luteal relative to follicular tissues. The studies in this thesis were aimed at establishing the functional involvement of these miRNAs during the follicle-luteal transition using a bovine model as well as human tissues. The aim of the first study was to characterise the expression of miR-132 and miR-96 within luteal tissue using ISH and FACS. The expression of miR-132 was detected in most luteal compartments while miR-96 was not detectable using ISH. Further examination using FACS showed that miR-212-132 expression remained unchanged in sorted endothelia (CD144+) and steroidogenic (CD144-Nile Red (NR) +) cell fractions. In contrast, expression of miR-183 and miR-96 was significantly increased in CD144+ compared to CD144-NR+ fractions. To elucidate potential roles of these miRNAs in the CL, I used existing online databases to identify putative miRNA targets. I identified 3042 predicted bovine gene targets of these miRNAs as well as 174 miRNA targets that had been experimentally validated in human, mouse and/or rat. I also identified putatively targeted signalling pathways primarily involved in cell survival, proliferation and differentiation. For further investigation, I narrowed my list of targets to FOXO1 and ADCY6, the expression of which was naturally down- regulated during luteinisation. The second study used an in vitro model of bovine granulosa cell luteinisation. Levels of miR-183-96-182 and miR-212-132 increased significantly (P < 0.05) during the first 4 days of luteinisation in vitro. The function of miR-132 and miR-96 during luteinisation in vitro was studied. Transfection of bovine granulosa cells with specific miRNA inhibitors or mimics of miR-132 and miR-96 led, respectively, to abolished expression and a significant increase in the levels of these miRNAs (P < 0.01) within 4 days. These changes in miRNA levels did not have any effect on transcript levels of the predicted mRNA targets, FOXO1 and ADCY6, during luteinisation. However, progesterone production by luteinising granulosa cells decreased (P < 0.05) on day 2 after transfection with miR-132 inhibitor. The results demonstrated that putative miRNA target genes remained unchanged during in vitro luteinisation which was not consistent with in vivo results. The third study aimed to elucidate the effect of miRNA inhibition in bovine luteal cells in culture. The loss of miR-132 led to an increase (P < 0.05) in FOXO1 transcript but not protein levels. In contrast, inhibition of miR-96 increased protein but not transcript levels of FOXO1. Moreover, miR-96 inhibition induced an increase in the caspase 3/7 response of luteal cells to serum deprivation indicating an anti-apoptotic effect of this miRNA on these cells. In the fourth study, I investigated the role of miR-132 and miR-96 in human luteinised granulosa cells obtained from IVF patients. The levels of FOXO1 protein were significantly increased following depletion of miR-132 and miR-96, whereas caspase3/7 increased in response to miR-96 inhibition, regardless of whether cells had been serum deprived or not. Similarly, using Annexin V and Trypan blue staining an increase in numbers of apoptotic cells was observed in response to miR- 96 inhibition. In addition, reduction of FOXO1 with the siRNA inhibited the apoptotic effect of miR-96 inhibition. Interestingly, inhibition of pooled miR-132 and miR-96 reduced progesterone secretion. However, this effect was prevented by transfecting cells with FOXO1 siRNA. These results suggest that the effects of these miRNAs on cell survival and progesterone production are mediated through targeting FOXO1. In summary, my results identify miR-96 and miR-132 as potentially critical factors in ensuring luteal cell survival and steroidogenesis in both cattle and human.

The main themes of the present thesis was the regulation of ovarian antral follicle growth, the manipulation of follicular dynamics and ovulation rate, as well as the characterization of the ovine corpus luteum (CL). Two treatments with ovine follicle stimulating hormone (oFSH) were used to assess the responsiveness of small antral follicles during different times in a follicular wave. Follicular dynamics were monitored by transrectal ultrasonography and serum FSH concentrations were measured. Two experiments were performed on anestrous Western White Face (WWF) ewes to independently examine whether or not the ovulations during treatment with a medroxyprogesterone acetate (MAP)-containing sponge and prostaglandin F2á (PGF2á), were due to the direct effects of PGF2á on the ovary or the effects of a rapid decline in progesterone at PGF2á-induced luteolysis. Non prolific Suffolk ewes were used to assess the effectiveness of treatment with medroxyprogesterone acetate (MAP)-containing sponge and prostaglandin F2á (PGF2á) to increase lambing rate. Re-introduction of rams to pre-isolated, mid-anestrous, WWF ewes was used to look at the effect of increased pulsatile secretion of LH on ovarian antral follicular dynamics at different stages of follicular wave development. We also used ovarian transrectal ultrasonography and computer assisted image analysis as non-invasive techniques to investigate whether or not there were correlations between ultrasound image attributes of the ovine CL and changing progesterone concentrations over time, in prolific and non prolific ewes. The results of the present studies showed that, in the ewe, small antral follicles can respond to the injection of FSH to yield a follicular wave more frequently than seen in a normal cycle and in the presence of a large growing antral follicle. Non induced waves can emerge during the growth phase of a wave induced by injection of oFSH. These results bring into question the presence of functional follicular dominance in the ewe. Ovulations occurred after PGF2á injection but during continuous treatment with MAP, but those ewes experiencing a decline in serum progesterone concentrations in the presence of MAP did not ovulate any follicles. We concluded that ovulations occurring after PGF2á injection, in the presence of a MAP sponge could be due to a direct effect of PGF2á at the ovarian level rather than a sudden decline in circulating progesterone concentrations. Treatment of Suffolk ewes with MAP-containing sponges and injection of PGF2á did not increase lambing rate, perhaps due to asynchrony of ovulations. Re-introduction of rams to previously isolated ewes resulted in a subtle increase in LH pulse frequency on the day of ram introduction in ewes in the static phase of a follicular wave. However, there were no consistent changes in follicular dynamics or estradiol secretion in response to this increase in LH pulse frequency. We concluded that changes in LH pulse frequency do not dramatically change ovarian antral follicular dynamics in the anestrous ewe. Both total luteal area and mean spot pixel values for the CL were correlated with the pattern of serum concentrations of progesterone from day 3 to day 15 after ovulation in WWF ewes and from day 3 to day 14 in Finn ewes. There were no significant correlations between progesterone concentrations and spot pixel heterogeneity for either WWF or Finn ewes. We concluded that pixel heterogeneity is a poor indicator of progesterone secretory ability of the CL when compared to mean pixel values. However, luteal area and mean spot pixel values are better but not strong indicators of the functional status of the CL in cyclic ewes.

The main themes of the present thesis was the regulation of ovarian antral follicle growth, the manipulation of follicular dynamics and ovulation rate, as well as the characterization of the ovine corpus luteum (CL). Two treatments with ovine follicle stimulating hormone (oFSH) were used to assess the responsiveness of small antral follicles during different times in a follicular wave. Follicular dynamics were monitored by transrectal ultrasonography and serum FSH concentrations were measured. Two experiments were performed on anestrous Western White Face (WWF) ewes to independently examine whether or not the ovulations during treatment with a medroxyprogesterone acetate (MAP)-containing sponge and prostaglandin F2á (PGF2á), were due to the direct effects of PGF2á on the ovary or the effects of a rapid decline in progesterone at PGF2á-induced luteolysis. Non prolific Suffolk ewes were used to assess the effectiveness of treatment with medroxyprogesterone acetate (MAP)-containing sponge and prostaglandin F2á (PGF2á) to increase lambing rate. Re-introduction of rams to pre-isolated, mid-anestrous, WWF ewes was used to look at the effect of increased pulsatile secretion of LH on ovarian antral follicular dynamics at different stages of follicular wave development. We also used ovarian transrectal ultrasonography and computer assisted image analysis as non-invasive techniques to investigate whether or not there were correlations between ultrasound image attributes of the ovine CL and changing progesterone concentrations over time, in prolific and non prolific ewes. The results of the present studies showed that, in the ewe, small antral follicles can respond to the injection of FSH to yield a follicular wave more frequently than seen in a normal cycle and in the presence of a large growing antral follicle. Non induced waves can emerge during the growth phase of a wave induced by injection of oFSH. These results bring into question the presence of functional follicular dominance in the ewe. Ovulations occurred after PGF2á injection but during continuous treatment with MAP, but those ewes experiencing a decline in serum progesterone concentrations in the presence of MAP did not ovulate any follicles. We concluded that ovulations occurring after PGF2á injection, in the presence of a MAP sponge could be due to a direct effect of PGF2á at the ovarian level rather than a sudden decline in circulating progesterone concentrations. Treatment of Suffolk ewes with MAP-containing sponges and injection of PGF2á did not increase lambing rate, perhaps due to asynchrony of ovulations. Re-introduction of rams to previously isolated ewes resulted in a subtle increase in LH pulse frequency on the day of ram introduction in ewes in the static phase of a follicular wave. However, there were no consistent changes in follicular dynamics or estradiol secretion in response to this increase in LH pulse frequency. We concluded that changes in LH pulse frequency do not dramatically change ovarian antral follicular dynamics in the anestrous ewe. Both total luteal area and mean spot pixel values for the CL were correlated with the pattern of serum concentrations of progesterone from day 3 to day 15 after ovulation in WWF ewes and from day 3 to day 14 in Finn ewes. There were no significant correlations between progesterone concentrations and spot pixel heterogeneity for either WWF or Finn ewes. We concluded that pixel heterogeneity is a poor indicator of progesterone secretory ability of the CL when compared to mean pixel values. However, luteal area and mean spot pixel values are better but not strong indicators of the functional status of the CL in cyclic ewes.

The ovary provides a niche environment where female germ cells (or oocytes) are generated, stored within follicles and later matured in preparation for use during reproductive life. Following an extensive period of quiescence, which in human, may be up to forty years; the follicle surrounding the oocyte begins to grow, promoting maturation of the oocyte, and culminating in the expulsion of a fully mature oocyte in preparation for fertilisation. These events occur cyclically as part of the menstrual (or estrus) cycle and involve extensive remodelling of both the follicle and its surrounding extracellular tissue. Cyclic follicle activation and growth is also associated with concurrent remodelling of the blood vasculature within the ovary, specifically the vessels surrounding the growing follicle. These blood vascular remodelling changes are well explored and have been demonstrated to be necessary for follicle growth, hormone synthesis, ovulation and for the development and function of the corpus luteum. Physiologically, the lymphatic vasculature is known to closely interact with the blood vasculature and plays a number of important physiological roles including the return of extra-vascular fluid to the blood circulation, and in turn, maintenance of systemic fluid homeostasis. Additionally the lymphatic network is required for the trafficking of immune cells from the periphery to lymph nodes; during the initiation of an immune response and for the absorption of lipids and lipid soluble vitamins in the gastrointestinal tract. The lymphatic vasculature develops and functions concomitantly with the blood vasculature; however unlike the blood vasculature, the aetiology of lymphatic vasculature within the ovary is unknown. It is unclear whether lymphatic remodelling events occur in association with folliculogenesis and are necessary for fertility, as is seen with the blood vasculature. To elucidate the mechanisms involved in the establishment and remodelling of the lymphatic vasculature within the ovary, I undertook expansive characterisation of its development and hormonal regulation. I exploited both hormonal manipulation and a known model of disrupted ovarian lymphatic development, the Adamts1 null mouse line, to examine the mechanisms controlling ovarian lymphangiogenesis. Quantitative morphometric analysis of vessel size and number in postnatal mouse ovary revealed that the ovarian lymphatic vasculature develops postnatally between day 8.5 and 12.5, and in synchrony with induction of ovarian CYP19a1 (Aromatase); the time when secondary follicles become FSH-responsive and estrogenic. The establishment of the lymphatic vasculature was also associated with the induction of pro-lymphangiogenic growth factors Vegfc and Vegfd, and their receptor, Vegfr3. Formation of ovarian lymphatics required the matrix-remodelling protease Adamts1, since ovaries from Adamts1⁻/⁻ mice failed to undergo normal lymphatic vascular development. FSH promoted remodelling of the existing lymphatic vascular maturation by increasing lymphatic vessel size in normal (Adamts1⁺/⁻) ovaries, and promoted the expansion of a new lymphatic vascular network by increasing vessel number and size in Adamts1⁻/⁻ ovaries. These vessel changes were also associated with the induction of prolymphangiogenic factors, Vegfc and Vegfd, as well as their receptor, Vegfr3 providing a mechanistic explanation for the hormonal mediated lymphangiogenesis. The concurrent establishment of the lymphatic vasculature with the first postnatal induction of ovarian Aromatase, and the hormone-regulated lymphangiogenesis suggests that a hormonal communication, likely estrogen, may synchronise lymphangiogenesis with folliculogenesis. Like FSH, exogenous estradiol was able to promote the expansion of a new lymphatic vascular network by increasing vessel number and size in Adamts1⁻/⁻ ovaries. Additionally, FSH-analog eCG was able to enhance ovarian lymphangiogenesis during the window of lymphatic establishment (postnatal development of Adamts1 null), whilst inhibition of the production of estradiol using the Aromatase inhibitor Letrozole, during this same window failed to have any effect. This study is the first to investigate the relationship between ovarian lymphatic development and remodelling and folliculogenesis. The present studies reveal that the ovary undergoes lymphatic vascular remodelling, described elsewhere as adult or secondary lymphangiogenesis and that this process involves hormonal contributions from FSH and estradiol, as well as the extracellular matrix protease, Adamts1. This work provides the first evidence of a malleable lymphatic system and a model for regulation of normal adult lymphangiogenesis, and may one day be used to explore ways in which to regenerate damaged vessels to cure lymphatic diseases and disorders. These results also significantly advanced the understanding of the tightly regulated processes controlling fluid dynamics within the ovary. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1454847 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2009

The ovary provides a niche environment where female germ cells (or oocytes) are generated, stored within follicles and later matured in preparation for use during reproductive life. Following an extensive period of quiescence, which in human, may be up to forty years; the follicle surrounding the oocyte begins to grow, promoting maturation of the oocyte, and culminating in the expulsion of a fully mature oocyte in preparation for fertilisation. These events occur cyclically as part of the menstrual (or estrus) cycle and involve extensive remodelling of both the follicle and its surrounding extracellular tissue. Cyclic follicle activation and growth is also associated with concurrent remodelling of the blood vasculature within the ovary, specifically the vessels surrounding the growing follicle. These blood vascular remodelling changes are well explored and have been demonstrated to be necessary for follicle growth, hormone synthesis, ovulation and for the development and function of the corpus luteum. Physiologically, the lymphatic vasculature is known to closely interact with the blood vasculature and plays a number of important physiological roles including the return of extra-vascular fluid to the blood circulation, and in turn, maintenance of systemic fluid homeostasis. Additionally the lymphatic network is required for the trafficking of immune cells from the periphery to lymph nodes; during the initiation of an immune response and for the absorption of lipids and lipid soluble vitamins in the gastrointestinal tract. The lymphatic vasculature develops and functions concomitantly with the blood vasculature; however unlike the blood vasculature, the aetiology of lymphatic vasculature within the ovary is unknown. It is unclear whether lymphatic remodelling events occur in association with folliculogenesis and are necessary for fertility, as is seen with the blood vasculature. To elucidate the mechanisms involved in the establishment and remodelling of the lymphatic vasculature within the ovary, I undertook expansive characterisation of its development and hormonal regulation. I exploited both hormonal manipulation and a known model of disrupted ovarian lymphatic development, the Adamts1 null mouse line, to examine the mechanisms controlling ovarian lymphangiogenesis. Quantitative morphometric analysis of vessel size and number in postnatal mouse ovary revealed that the ovarian lymphatic vasculature develops postnatally between day 8.5 and 12.5, and in synchrony with induction of ovarian CYP19a1 (Aromatase); the time when secondary follicles become FSH-responsive and estrogenic. The establishment of the lymphatic vasculature was also associated with the induction of pro-lymphangiogenic growth factors Vegfc and Vegfd, and their receptor, Vegfr3. Formation of ovarian lymphatics required the matrix-remodelling protease Adamts1, since ovaries from Adamts1⁻/⁻ mice failed to undergo normal lymphatic vascular development. FSH promoted remodelling of the existing lymphatic vascular maturation by increasing lymphatic vessel size in normal (Adamts1⁺/⁻) ovaries, and promoted the expansion of a new lymphatic vascular network by increasing vessel number and size in Adamts1⁻/⁻ ovaries. These vessel changes were also associated with the induction of prolymphangiogenic factors, Vegfc and Vegfd, as well as their receptor, Vegfr3 providing a mechanistic explanation for the hormonal mediated lymphangiogenesis. The concurrent establishment of the lymphatic vasculature with the first postnatal induction of ovarian Aromatase, and the hormone-regulated lymphangiogenesis suggests that a hormonal communication, likely estrogen, may synchronise lymphangiogenesis with folliculogenesis. Like FSH, exogenous estradiol was able to promote the expansion of a new lymphatic vascular network by increasing vessel number and size in Adamts1⁻/⁻ ovaries. Additionally, FSH-analog eCG was able to enhance ovarian lymphangiogenesis during the window of lymphatic establishment (postnatal development of Adamts1 null), whilst inhibition of the production of estradiol using the Aromatase inhibitor Letrozole, during this same window failed to have any effect. This study is the first to investigate the relationship between ovarian lymphatic development and remodelling and folliculogenesis. The present studies reveal that the ovary undergoes lymphatic vascular remodelling, described elsewhere as adult or secondary lymphangiogenesis and that this process involves hormonal contributions from FSH and estradiol, as well as the extracellular matrix protease, Adamts1. This work provides the first evidence of a malleable lymphatic system and a model for regulation of normal adult lymphangiogenesis, and may one day be used to explore ways in which to regenerate damaged vessels to cure lymphatic diseases and disorders. These results also significantly advanced the understanding of the tightly regulated processes controlling fluid dynamics within the ovary. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1454847 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2009

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Virginia, 2000. / Includes bibliographical references (leaves 162-178). Also available online through Digital Dissertations.