Relationships between hypothalamic gene expression and the resumption of ovulation in postpartum beef cows

The aim in this thesis was to gain an understanding of changes in gene expression in the hypothalamus of postpartum beef cows during the period of transition from suppressed ovarian follicular growth to increased follicular growth, and the resumption of ovulation. Beef cows tend to have an extended period of anoestrus after calving. This trait is particularly pronounced in tropically-adapted Zebu breeds. In addition to a genetic component, the postpartum anoestrous period can be influenced by age, body condition, the nutrient requirement of lactation, suckling stimulus, and maternal bonding. An extended postpartum anoestrous period is particularly evident in primiparous beef cows. This is understandable given that primiparous cows have yet to reach their mature body size which means there is a requirement to maintain maternal tissue growth whilst at the same time directing nutrients for milk production. Weaning removes maternal bonding, the suckling stimulus and nutrient requirement of milk production and, provided that nutrient supply and body condition are appropriate, primiparous cows show increased ovarian activity and resume ovulation after weaning. In the present thesis, groups of primiparous Zebu cows were weaned to promote increased ovarian follicular growth and hypothalamic gene expression was compared for weaned cows and contemporary cows that continued to lactate. Candidate genes were studied using quantitative real-time PCR (qRT-PCR) and a gene expression microarray was used to discover new genes and gene networks. Gene expression was examined in the anterior hypothalamic-preoptic area (sub-region H1) and posterior ventral hypothalamus (sub-region H2). The demarcation between H1 and H2 was a vertical line from the mid-point of the median eminence-pituitary stalk to the thalamus. Candidate genes studied by qRT-PCR included, gonadotrophin releasing hormone (GNRH1), kisspeptin (KISS1), neuropeptide Y (NPY), oestrogen receptor alpha (ESR1) and leptin receptor (LEPR). Marked regional expression was demonstrated for these genes. The expression of GNRH1 was greatest in the anterior hypothalamic region (sub-region H1) whilst the expression of KISS1 was greatest in the ventral posterior hypothalamic region (sub-region H2). Relative expression of LEPR, ESR1 and NPY was greater in H2 than H1. The regional gene expression patterns for GNRH1, KISS1, LEPR, ESR1 and NPY in the hypothalamus of cows were consistent with regional expression reported for other species. Weaning was associated with a decrease in the expression of LEPR, ESR1 and NPY. With regard to ovarian phenotype, there was a greater LEPR expression associated with ovarian phenotype 1 (OP1, follicles to 5mm) compared with ovarian phenotype 2 (OP2, follicles to 10mm) and ovarian phenotype 3 (OP3, recently ovulated) in sub-region H1. Relative expressions for ESR1, LEPR and NPY were highly correlated, particularly in sub-region H2. The evaluation of gene expression by microarray for cows with different ovarian phenotypes provided evidence of interactions between hormonal regulation and cell-cell signalling within the hypothalamus. Genes that were differentially expressed for different ovarian phenotypes were associated with reproduction, energy balance, the immune system and stress. Other genes that showed differential expression were involved with cell adhesion, synaptic transmission, ion signalling and neuronal development. The latter findings were interpreted to suggest that neuronal and glial cell plasticity is a feature of changes in reproductive functions of the hypothalamus. The evaluation of gene expression by microarray for weaned and suckled cows, irrespective of ovarian phenotype, identified differentially expressed genes associated with energy balance, fluid homeostasis, milk synthesis, stress, and oestrogen signalling. With regard the latter, thirty seven genes involved in oestrogen signalling through ESR1, or in other ways associated with oestrogen, were found to be differentially expressed between weaned and lactating cows. ESR1 occupied the central position of a primary gene network based on the present study. Six differentially expressed genes were shown by gene network analysis to be centred in nodes interacting closely with ESR1. Phospholipase-C-gamma (PLCG2), vitronectin (VTN) and endopin 1 (SERPINA3) are three genes associated with hypothalamic plasticity and neurotransmission that were differentially expressed between cows with OP1 and OP2, indicating a possible role in the shift to increased ovarian follicular growth and ovulation. The findings for ESR1 were consistent with the major role of oestrogen in female reproduction and in particular the known actions of oestrogen in regulating the hypothalamus during reproductive transition phases in females associated with puberty, seasonality and postpartum. Gonadotrophin inhibitory hormone (GnIH) is derived from Neuropeptide VF precursor (NPVF), which is encoded by NPVF gene transcripts. NPVF had reduced expression in cows that had ovulated (OP3) compared with OP1 and OP2. GnIH inhibits gonadotrophin secretion by directly acting on GnRH neurons as well as modulating the suppressive effects of oestrogen negative feedback. In addition, GnIH has been shown to play a role in seasonal regulation of reproduction in birds. The lesser expression of NPVF in cows that had resumed ovulation, particularly evident in sub-region H2, provides initial evidence that GnIH has an important role in maintaining the suppressive effects on reproduction during postpartum anoestrus in cattle. In summary, the studies in this thesis have identified hypothalamic genes and gene networks that potentially are important in the control of reproductive function in the postpartum cow. The thesis has also established that the postpartum cow can be used as an experimental model for fundamental studies that generate new knowledge on the reproductive biology of the postpartum period