Mark P. Green

Identification and functional characterization of UDP-glucuronosyltransferases UGT1A8*1, UGT1A8*2 and UGT1A8*3.

UDP-glucuronosyltransferase (UGT) 1A8 is part of the UGT1 locus and is expressed exclusively in extrahepatic tissues. Analysis of UGT1A8 exon 1 sequence has identified four genotypes from a population of 69 individuals. While there are four alleles, one of the single base pair changes leads to a silent mutation at T255, while the other mutations lead to amino acid substitutions at positions 173 and 277, creating three allelic variants. UGT1A8*1 (A173C277), UGT1A8*1a (T255A>G), UGT1A8*2 (G173C277) and UGT1A8*3 (A173Y277). The allelic frequencies of UGT1A8*1, UGT1A8*1a, UGT1A8*2 and UGT1A8*3 are 0.551, 0.282, 0.145 and 0.022, respectively. To examine the properties of the UGT1A8 proteins, UGT1A8*1 and UGT1A8*2 were cloned from a human colon cDNA library and UGT1A8*3 generated by mutagenesis using UGT1A8*1 as template. The cDNAs were expressed in HK293 cells to examine catalytic function as well as abundance as observed by analysis of UGT1A8-GFP (green fluorescent protein) expression. The single amino acid change that identifies UGT1A8*1 (A173) and UGT1A8*2 (G173) has little impact on function, while the UGT1A8*3 (Y277) is a conserved amino acid alteration represented by a dramatic reduction in catalytic activity. Protein abundance, as determined by Western blot analysis following transient transfection, is not altered. In addition, functional UGT1A8-GFP variants displayed staining in the cytoplasmic region, indicating that each protein is expressed in similar cellular compartments. Together, these data suggest that the null UGT1A8*3 results from structural changes and not a lack of protein expression. Allelic variation leading to singular codon changes could potentially alter drug metabolism in extrahepatic tissues.

Soluble ligands and their receptors in human embryo development and implantation.

Extensive evidence suggests that soluble ligands and their receptors mediate human preimplantation embryo development and implantation. Progress in this complex area has been ongoing since the 1980s, with an ever-increasing list of candidates. This article specifically reviews evidence of soluble ligands and their receptors in the human preimplantation stage embryo and female reproductive tract. The focus will be on candidates produced by the human preimplantation embryo and those eliciting developmental responses in vitro, as well as endometrial factors related to implantation and receptivity. Pathways to clinical translation, including innovative diagnostics and other technologies, are also highlighted, drawing from this collective evidence toward facilitating joint improvements in embryo quality and endometrial receptivity. This strategy could not only benefit clinical outcomes in reproductive medicine but also provide broader insights into the peri-implantation period of human development to improve fetal and neonatal health.

Effects of circulating progesterone and insulin on early embryo development in beef heifers

The aims of this study were to determine the effect on early embryo development of feeding a diet formulated to enhance circulating insulin concentrations and secondly to investigate the association between early embryo development and maternal progesterone concentrations in beef heifers. The study was carried out in 32 Simmental x Holstein Friesian heifers 22-25 months of age weighing 506+/-7kg and in condition score 3.1+/-0.1. Animals were fed two diets that were isoenergetic and isonitrogenous, but that would encourage either propionate (diet A) or acetate (diet B) production in the rumen. The rationale was that propionate would induce a greater insulin release in response to feeding. Animals were fed a 50:50 mix of the two diets for 14 days at 0.8x maintenance, with straw provided ad libitum. Animals were then fed one of the experimental diets for 3 weeks prior to synchronisation of oestrus and insemination and for a further 16 days following mating. All heifers were blood sampled daily from oestrus synchronisation and eight animals on each diet underwent daily transrectal real-time ultrasonography to determine the day of ovulation. All heifers were slaughtered at Day 16 after mating. While feeding of diet A (propionic) caused a significant (P<0.05) increase in the plasma insulin to glucagons ratio differences in insulin were not significantly different. This is probably due to the fact that insulin concentrations were quite high as the heifers used in the present study were in good body condition making further increases in insulin difficult to achieve. Diet did not affect size of ovulatory follicle (DIET A: 15.1+/-0.7mm; diet B: 14.6+/-0.7mm), day of ovulation (diet A: 3.5+/-0.2 days; diet B: 3.4+/-0.2 days), mean plasma progesterone concentration (diet A: 4.7+/-0.4ng/ml; diet B: 5.2+/-0.3ng/ml), corpus luteum weight (diet A: 6.0+/-0.2g; diet B: 6.0+/-0.2g) or pregnancy rate (diet A: 81.3%; diet B: 81.3%). However, the proportion of well-elongated (>10cm) embryos on Day 16 was higher in animals fed diet A than in those fed diet B (84.6% versus 38.5%; P<0.05). While progesterone concentration did not differ between pregnant and non-pregnant heifers, progesterone did show an earlier post-ovulatory rise in heifers with well-elongated (>10cm) embryos with levels in these animals significantly higher on Days 4 and 5 than in heifers with small (<10cm) embryos at slaughter. This study demonstrated an enhancement in early embryo development in animals fed a diet generating an increased insulin:glucagon ratio that was not related to circulating maternal progesterone concentrations. However, across diets, enhanced embryo development was associated with elevated plasma progesterone on Days 4 and 5 following mating.

Nutritional skewing of conceptus sex in sheep: effects of a maternal diet enriched in rumen-protected polyunsaturated fatty acids (PUFA)

BackgroundEvolutionary theory suggests that in polygynous mammalian species females in better body condition should produce more sons than daughters. Few controlled studies have however tested this hypothesis and controversy exists as to whether body condition score or maternal diet is in fact the determining factor of offspring sex. Here, we examined whether maternal diet, specifically increased n-6 polyunsaturated fatty acid (PUFA) intake, of ewes with a constant body condition score around the time of conception influenced sex ratio.MethodsEwes (n = 44) maintained in similar body condition throughout the study were assigned either a control (C) diet or one (F) enriched in rumen-protected PUFA, but otherwise essentially equivalent, from four weeks prior to breeding until d13 post-estrus. On d13, conceptuses were recovered, measured, cultured to assess their capacity for interferon-tau (IFNT) production and their sex determined. The experiment was repeated with all ewes being fed the F diet to remove any effects of parity order on sex ratio. Maternal body condition score (BCS), plasma hormone and metabolite concentrations were also assessed throughout the study and related to diet.ResultsIn total 129 conceptuses were recovered. Ewes on the F diet produced significantly more male than female conceptuses (proportion male = 0.69; deviation from expected ratio of 0.5, P < 0.001). Conceptus IFNT production was unaffected by diet (P > 0.1), but positively correlated with maternal body condition score (P < 0.05), and was higher (P < 0.05) in female than male conceptuses after 4 h culture. Maternal plasma hormone and metabolite concentrations, especially progesterone and fatty acid, were also modulated by diet.ConclusionThese results provide evidence that maternal diet, in the form of increased amounts of rumen-protected PUFA fed around conception, rather than maternal body condition, can skew the sex ratio towards males. These observations may have implications to the livestock industry and animal management policies when offspring of one sex may be preferred over the other.

Melatonin: a possible link between the presence of artificial light at night and reductions in biological fitness

The mechanisms underpinning the ecological impacts of the presence of artificial night lighting remain elusive. One suspected underlying cause is that the presence of light at night (LAN) supresses nocturnal production of melatonin, a key driver of biological rhythm and a potent antioxidant with a proposed role in immune function. Here, we briefly review the evidence for melatonin as the link between LAN and changes in behaviour and physiology. We then present preliminary data supporting the potential for melatonin to act as a recovery agent mitigating the negative effects of LAN in an invertebrate. Adult crickets (Teleogryllus commodus), exposed to constant illumination, were provided with dietary melatonin (concentrations: 0, 10 or 100 µg ml−1) in their drinking water. We then compared survival, lifetime fecundity and, over a 4-week period, immune function (haemocyte concentration, lysozyme-like and phenoloxidase (PO) activity). Melatonin supplementation was able only partially to mitigate the detrimental effects of LAN: it did not improve survival or fecundity or PO activity, but it had a largely dose-dependent positive effect on haemocyte concentration and lysozyme-like activity. We discuss the implications of these relationships, as well as the usefulness of invertebrates as model species for future studies that explore the effects of LAN.

A Comparison of the Anti-Luteolytic Activities of Recombinant Ovine Interferon-Alpha and -Tau in Sheep

Abstract Interferon tau (IFNT) is secreted by the trophectoderm of ruminant conceptuses during the peri-implantation period and serves an anti-luteolytic function. The question as to whether IFNT is superior as an anti-luteolytic agent to closely related Type I IFNs, such as IFN alpha (IFNA), which have a different function, remains unanswered. Thus, the aim of this study was to determine whether equivalent antiviral (AV) units of ovIFNA and ovIFNT are equipotent in extending estrous cycle length. Four distinct ovIFNA mRNA (ovIFNA1–4) were cloned from ovine lymphocytes. Recombinant ovine IFNs (ovIFNT4 and ovIFNA1) were prepared in the yeast Pichia pastoris. The AV activity of the purified IFNs was determined on a bovine cell line (MDBK) and on transformed ovine luminal uterine epithelial cells. Indwelling uterine catheters were fitted into crossbred ewes on Day 3 postestrus (Day 0 = estrus). Between Days 10 and 18 postestrus, ewes received twice-daily infusions of 0.7 × 107 IU of either ovIFNA1 or T4, plus serum albumin. Control ewes received serum albumin only. Daily blood samples were collected for progesterone determination, and ewes were monitored twice daily for estrus. Both ovIFNA (P = 0.04) and ovIFNT (P = 0.01) caused estrous cycle extension in nonpregnant ewes compared to controls when administered at equivalent AV doses. In conclusion, the uniqueness of IFNT as an anti-luteolytic agent most likely resides in its unique expression pattern rather than its special biopotency.

Maternal age and ovarian stimulation independently affect oocyte mtDNA copy number and cumulus cell gene expression in bovine clones

STUDY QUESTION Does maternal ageing and ovarian stimulation alter mitochondrial DNA (mtDNA) copy number and gene expression of oocytes and cumulus cells from a novel bovine model for human IVF? SUMMARY ANSWER Oocytes collected from females with identical nuclear genetics show decreased mtDNA copy number and increased expression of an endoplasmic reticulum (ER) stress gene with repect to ovarian stimulation, whilst differences in the expression of genes involved in mitochondrial function, antioxidant protection and apoptosis were evident in relation to maternal ageing and the degree of ovarian stimulation in cumulus cells. WHAT IS KNOWN ALREADY Oocyte quality declines with advancing maternal age; however, the underlying mechanism, as well as the effects of ovarian stimulation are poorly understood. Human studies investigating these effects are often limited by differences in age and ovarian stimulation regimens within a patient cohort, as well as genetic and environmental variability. STUDY DESIGN, SIZE, DURATION A novel bovine cross-sectional maternal age model for human IVF was undertaken. Follicles were aspirated from young (3 years of age; n = 7 females) and old (10 years of age; n = 5 females) Holstein Freisian clones following multiple unstimulated, mild and standard ovarian stimulation cycles. These bovine cloned females were generated by the process of somatic cell nuclear transfer (SCNT) from the same founder and represent a homogeneous population with reduced genetic and environmental variability. Maternal age and ovarian stimulation effects were investigated in relation to mtDNA copy number, and the expression of 19 genes involved in mitochondrial function, antioxidant protection, oocyte-cumulus cell signalling and follicle development in both oocytes and cumulus cells. MATERIALS, SETTING, METHODS Young (3 years of age; n = 7 females) and old (10 years of age; n = 5 females) Holstein Freisian bovine clones were maintained as one herd. Stimulation cycles were based on the long GnRH agonist down-regulation regimen used in human fertility clinics. Follicle growth rates, numbers and diameters were monitored by ultrasonography and aspirated when the lead follicles were >14 mm in diameter. Follicle characteristics were analysed using a mixed model procedure. Quantitative PCR (qPCR) was used to determine mtDNA copy number and reverse transcriptase-qPCR (RT-qPCR) was used to measure gene expression in oocytes and cumulus cells. MAIN RESULTS AND THE ROLE OF CHANCE Method of ovarian stimulation (P = 0.04), but not maternal age (P > 0.1), was associated with a lower mtDNA copy number in oocytes. Neither factor affected mtDNA copy number in cumulus cells. In oocytes, maternal age had no effect on gene expression; however, ovarian stimulation in older females increased the expression of GRP78 (P = 0.02), a gene involved in ER stress. In cumulus cells, increasing maternal age was associated with the higher expression of genes involved in mitochondrial maintenance (TXN2 P = 0.008 and TFAM P = 0.03), whereas ovarian stimulation decreased the expression of genes involved in mitochondrial oxidative stress and apoptosis (TXN2 P = 0.002, PRDX3 P = 0.03 and BAX P = 0.03). LIMITATIONS, REASON FOR CAUTION The low number of oocyte and cumulus cell samples collected from the unstimulated cycles limited the analysis. Fertilization and developmental potential of the oocytes was not assessed because these were used for mtDNA and gene expression quantification. WIDER IMPLICATIONS OF THE FINDINGS Delineation of the independent effects of maternal age and ovarian stimulation regimen on mtDNA copy number gene expression in oocytes and cumulus cells was enabled by the removal of genetic and environmental variability in this bovine model for human IVF. Therefore, these extend upon previous knowledge and findings provide relevant insights that are applicable for improving human ovarian stimulation regimens. STUDY FUNDING/COMPETING INTERESTS Funding was provided by Fertility Associates and the University of Auckland. J.C.P. is a shareholder of Fertility Associates and M.P.G. received a fellowship from Fertility Associates. The other authors of this manuscript declare no conflict of interest that could be perceived as prejudicing the impartiality of the reported research.

Long-term alteration of follicular steroid concentrations in relation to subclinical endometritis in postpartum dairy cows.

The focus of this study was to investigate the effect of subclinical endometritis (scEndo) on ovarian follicular steroid concentrations in early postpartum pasture-fed dairy cows. Mixed-age lactating dairy cows (n = 169) were examined to ascertain uterine health status on d 21 postpartum (±3 d). From this herd, a cohort of scEndo and uninfected cows (n = 47) were selected using uterine cytology to determine scEndo. To ensure cows with scEndo were selected for the study, a conservative threshold [>18% polymorphonuclear (PMN) cells among uterine nucleated cells] was chosen as a selection threshold. Ovarian follicular dynamics were assessed by ultrasonography on d 21, 42, and 63 postpartum. On the latter 2 d, all follicles >4 mm in diameter were ablated, and 4 d later, the largest (F1) and second largest (F2) follicles were measured and their follicular fluid aspirated. Hematological variables and plasma metabolites were measured also on these days to further characterize scEndo cows. On d 21, the prevalence of scEndo was approximately 9% in this herd; by d 42 infections had self-resolved in the majority (81%) of those cows classified as having scEndo on d 21. The scEndo cows had a delayed return to cyclicity; however, no effect was evident on ovarian follicle size or growth rate. Weeks after scEndo had self-resolved and cyclicity was restored, decreased (P = 0.07) testosterone and increased (P = 0.07) cortisol concentrations were evident in F1 follicles of scEndo compared with uninfected cows. Progesterone concentrations of F1 increased (P < 0.05) in 11- to 16-mm diameter follicles of scEndo cows, whereas estradiol, androstendione, and dehydroepiandrosterone concentrations were decreased (P < 0.05) in F1 8- to 10-mm diameter follicles of scEndo cows. These 3 steroids also differed (P < 0.05) between F1 follicle size categories of scEndo but not uninfected cows. On d 21, mean plasma albumin concentration was decreased (P = 0.02) in scEndo cows. In summary, early postpartum scEndo had surprisingly long-term influences on the steroid concentrations of ovarian follicles long after infections had self-resolved. This is likely to affect oocyte quality and may partially explain the reduced conception rates and longer interval between calving and conception that are often associated with scEndo, although more detailed investigations are required to substantiate this theory.

Identification and quantification of differentially represented transcripts in in vitro and in vivo derived preimplantation bovine embryos.

Identification of transcripts at key development stages of preimplantation embryos is critical for a better understanding of early embryogenesis. The current study had two aims. The first was to characterize the relative abundance of multiple transcripts during several developmental stages, namely, metaphase II‐stage oocytes (MPII), as well as 2‐cell, precompact morula (PCM) and in vitro‐produced blastocyst (IVTBL) stage embryos. The second was to characterize differences in the relative abundance of transcripts present in in vivo‐ (IVVBL), in vitro‐ (IVTBL), and nuclear transfer‐derived (NTBL) blastocysts. It was hypothesized that the identification of differentially represented transcripts from these embryos would reveal not only developmentally important genes, but also genes that might be aberrantly expressed due to embryo production techniques. Individual clusters from a large bovine expressed sequence tag (EST) library (http://genome.rnet.missouri.edu/bovine/) of female reproductive tissues and embryos were compared using Fishers Exact Test weighted by number of transcripts per tissue by gene. Of the 3,144 transcripts that were present during embryogenesis, 125 were found to be differentially represented (P ≤ 0.01) in at least one pairwise comparison. Fifteen of these transcripts were selected for further examination using quantitative real‐time PCR (qRTPCR) to determine differences in transcript abundance. Twelve of the 15 transcripts were differentially represented (n = 9, P ≤ 0.01; n = 3, P ≤ 0.05) in at least one pairwise comparison. In summary, identification of differentially represented transcripts in early embryo development, which are modulated by in vitro techniques, should provide markers to ensure the production of embryos closer to those developed in vivo. Mol. Reprod. Dev. 76: 48–60, 2009.

Ovarian stimulation leads to shorter stature in childhood

BACKGROUND We aimed to determine whether children conceived with ovarian stimulation alone (OS(A)) would differ phenotypically and biochemically from naturally conceived children of fertile and subfertile parents. METHODS Healthy pre-pubertal children aged 3-10 years, born at term, after singleton pregnancies were recruited in Auckland (New Zealand) and were allocated into three groups: (i) children conceived following OS(A) and naturally conceived children of (ii) subfertile and (iii) fertile parents. Anthropometric, endocrine and metabolic parameters were recorded. Childrens heights and body mass index (BMI) were expressed as standard deviation scores (SDS) and corrected for genetic potential (i.e. parental height or BMI). RESULTS Three hundred fifty-two children were studied: 84 OS(A) subjects and 268 naturally conceived controls consisting of 54 children of subfertile parents and 214 children of fertile parents. Children of subfertile and fertile parents did not differ in measured outcomes. Overall, OS(A) children were shorter than children of both subfertile (SDS: -0.08 ± 0.09 versus 0.32 ± 0.07; P= 0.001) and fertile (SDS: -0.08 ± 0.09 versus 0.45 ± 0.10; P= 0.004) parents when corrected for genetic height potential. OS(A) boys were shorter than boys of subfertile (SDS:-0.18 ± 0.14 versus 0.42 ± 0.16; P= 0.03) and fertile (SDS: -0.18 ± 0.14 versus 0.35 ± 0.08; P= 0.01) parents. There was also a trend towards OS(A) girls being shorter than girls of subfertile parents (P= 0.06), but not significantly shorter than those of fertile parents (P= 0.17). OS(A) children also had a lower corrected BMISDS than children of subfertile (SDS-0.90 ± 0.15 versus -0.37 ± 0.17; P= 0.06) and fertile (-0.90 ± 0.15 versus -0.34 ± 0.10; P= 0.008) parents. Among metabolic parameters, fasting glucose was lower in OS(A) children than that in children of fertile parents (4.62 ± 0.07 versus 4.81 ± 0.04; P= 0.006). CONCLUSIONS Conception after OS(A) was associated with shorter stature, particularly in boys, compared with naturally conceived children of fertile and subfertile parents.