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Circadian rhythms are the endogenous cycling of hormones, activity patterns and gene expression that occur across 24 hours. Disruption of circadian rhythms has been associated with multiple health complications, including reduction of fertility. The bmal1 mouse provides an animal model for central and peripheral loss of rhythmicity. Herein the reproductive function and postnatal development in the bmal1 knockout mouse has been evaluated. The reproductive capability of the heterozygous breeding colony was investigated, with around 50% of the female breeder mice becoming pregnant within one estrus cycle. The offspring of the colony had a higher than expected level of perinatal mortality while the knockout and heterozygous genotype was under represented among the offspring surviving to weaning, suggesting high knockout embryo or perinatal losses. The circadian phenotype of this mouse model was confirmed, identifying the severe disruption of circadian behavioural rhythms. Further, the growth of the bmal1 knockout mice was retarded compared with their heterozygous and wild type littermates from weaning to 9 months of age. The reproductive function of the homozygous male bmal1 knockout mouse was evaluated. They showed poor fertility, poorly developed secondary sexual organs, reduced sperm count and reduced sperm motility. Female bmal1 knockout mice had delayed vaginal opening, delayed onset of first estrus, disrupted estrus cyclicity as well as impaired reproductive and mammary tissue development. Steroid hormone synthesis was compromised in both males (testosterone) and females (progesterone) and ovarian morphology revealed reduced corpora lutea formation and structural abnormalities. Female bmal1 knockout mice also evidenced profound infertility, which was caused by a continuum of reproductive insufficiencies including reduced ovulation of oocytes, poorer progression of the preimplantation embryo and failure to successfully implant in the uterus. While the ovaries of bmal1 knockout females were able to respond to exogenous stimulation, the number of ovulated oocytes was reduced, the fertilised oocytes were of reduced quality, progressed poorly to mature blastocyst and once again failed to implant. A bioinformatical evaluation of a panel of genes closely involved in reproduction and ovarian function was analysed for the presence of circadian enhancer regions (E-box sequences) or RORA response elements (RRE) in their promoter regions. It was revealed that many of the genes investigated contained one or more circadian E-box and RRE sequence, providing a mechanism for the disruption of circadian gene expression within the ovary to cause detrimental changes in gene expression. Further to this, the gene expression profile of these functional genes and clock genes were evaluated in ovarian tissues from wild type and knockout mice across the estrus cycles and across 24 hours. It was shown that the murine ovary rhythmically expressed the genes involved with the molecular clock across 24 hours, as well as several other genes previously associated with rhythmicity in peripheral tissues. Further, the loss of functional bmal1 gene expression resulted in up or down regulation of over 75% of the functional genes investigated, including steroidogenic acute regulatory protein (the rate limiting enzyme for progesterone synthesis). In conclusion, the bmal1 knockout mouse shows a significant multi-factorial loss in fertility in both males and females. This loss occurs across a range of tissues and results in heavily reduced fertility in the male and complete infertility in the female. Further research could identify in greater detail the precise molecular mechanisms underpinning of this disruption. / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

Abstract not available

"April 2002" Includes bibliographical references (leaves 200-249)

"April 2002" / Includes bibliographical references (leaves 200-249) / xv, 249 leaves : ill., plates (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Science, 2003

"April 2002" / Includes bibliographical references (leaves 200-249) / xv, 249 leaves : ill., plates (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Science, 2003

The TGFβ family of cytokines are potent signalling molecules that regulate tissue development, inflammation and immunity. Previous studies in mice with a null mutation in the Tgfb1 gene (TGFβ1-/- mice) implicate a key role for TGFβ1 in male reproductive function. These mice show profound infertility due to an inability to copulate successfully, associated with reduced testosterone and sperm production. The focus of this project was to 1) further characterize mechanisms underpinning reproductive deficiency in male TGFβ1-/- mice, 2) identify a reliable physiological marker of TGFβ1 availability in vivo, and 3) to determine whether exogenous TGFβ1 administration influences TGFβ1 availability and restores fertility. To investigate the causes of unsuccessful copulation by TGFβ1-/- mice, penis morphometry was examined. Penile organ structure, as assessed by scanning electron microscopy, was comparable between genotypes however a superfluous epidermal covering that impeded penile spine protrusion was evident in TGFβ1-/- mice. The epidermal covering was not due to increased epithelial cell proliferation, as measured by Brdu labelling and immunohistology. Behavioural observations of erectile activity showed that TGFβ1-/- mice achieved spontaneous erections albeit at reduced frequency compared to TGFβ1+/+ mice. The efficacy of exogenous TGFβ1 replacement was evaluated by first identifying measures of in vivo TGFβ1 availability and/or function and selecting an effective route of administration. Serum TGFβ1 and testosterone levels were reliable discriminators of TGFβ1 genotype. Gene expression and phagocytic function of peritoneal macrophages revealed no differences between genotypes. Exogenous sources of TGFβ1 for replacement studies included colostrum, naturally occurring in breast milk and recombinant human latent TGFβ1 (rhLTGFβ1). Colostrum did not increase circulating levels and rhTGFβ1 injection caused only transient elevation of serum levels. Thus mini-osmotic pumps were used to deliver a constant supply of cytokine to TGFβ1-/- mice. The fertility status of TGFβ1-/- mice receiving exogenous TGFβ1 was investigated. Reproductive behaviour in response to normal receptive female mice was assessed twice during treatment, on day 7 and day 14. Blood, liver and reproductive tissues were collected at sacrifice. Circulating TGFβ1 was increased in TGFβ1 treated TGFβ1-/- mice above TGFβ1-/- control levels, although this did not affect circulating testosterone. Erectile activity and sperm production were unchanged. Videotaping behaviour with estrous females revealed that the TGFβ1+/+ mice successfully mounted and intromitted, unlike the TGFβ1-/- controls. The TGFβ1-/- mice receiving exogenous TGFβ1 displayed moderately enhanced mounting and intromission behaviour although this remained less frequent than in the TGFβ1+/+ controls. Ejaculation behaviour was not observed in any TGFβ1-/- mice regardless of TGFβ1 replacement, compared to TGFβ1+/+ controls where >90% mice displayed ejaculated. Modest improvement in the copulation activity of the TGFβ1-/- mice receiving exogenous TGFβ1 suggests that systemic TGFβ1 availability can influence reproductive performance in male TGFβ1-/- mice. However since fertility was not restored, locally produced TGFβ1 in the reproductive tract and/or hypothalamic pituitary axis are also implicated in regulating fertility. These findings advance our knowledge of the role of the TGFβ1 cytokine in male reproductive physiology and may have relevance for devising new treatments for infertility and erectile dysfunction in men. / Thesis (Ph.D.) - University of Adelaide, School of Paediatrics and Reproductive Health, 2008