Stewart M. Rhind

The effects of undernutrition, in utero, on reproductive function in adult male and female sheep

The aim of this study was to determine the effects of maternal undernutrition during pregnancy on adult reproductive function in male and female offspring. Groups of ewes were fed rations providing either 100% (High, H) or 50% (Low, L) of estimated metabolisable energy (ME) requirements for pregnancy, from mating until day 95 of gestation, and thereafter were conventionally managed. At 20 months of age, LH and FSH profiles, and LH responses to exogenous GnRH were measured in male and female offspring and, in males, testicular responses to exogenous LH (as measured by testosterone concentrations) were also measured. Undernutrition had no effect on the mean birth weights of lambs of either sex, or on testicular size in male animals at either 6 weeks or 20 months of age. L males exhibited significantly higher FSH concentrations than H males (P < 0.05) but there were no differences with treatment in FSH profiles in females, basal LH profiles or gonadotrophin responses to GnRH in offspring of either sex, and no difference in basal testosterone concentrations or in the testosterone response to exogenous LH administration in males. Semen quality at 20 months of age was unaffected by pre-natal undernutrition but ovulation rate was significantly reduced in L compared to H female offspring (P < 0.05). It is concluded that pre-natal undernutrition had no effect on male reproductive development and adult function, but reduced ovulation rate in female progeny. This effect was not associated with a change in gonadotrophin profiles or pituitary responsiveness.

Homozygosity for a single base-pair mutation in the oocyte-specific GDF9 gene results in sterility in Thoka sheep

The control of fecundity is critical in determining mammalian offspring survival. It is regulated principally by the ovulation rate, so that primates and large farm species commonly have a single offspring. Previously, several mutations have been identified in sheep which increase the naturally low ovulation rate; although in some cases homozygous ewes are infertile. In the present study we present a detailed characterization of a novel mutation in growth differentiation factor 9 (GDF9), found in Icelandic Thoka sheep. This mutation is a single base change (A1279C) resulting in a nonconservative amino acid change (S109R) in the C-terminus of the mature GDF9 protein, which is normally expressed in oocytes at all stages of development. Genotyping all animals for which reproductive records were available confirmed this mutation to be associated with increased fecundity in heterozygous ewes and infertility in homozygotes. Analysis of homozygote ovarian morphology and a number of genes normally activated in growing follicles showed that GDF9 was not involved in oocyte activation, but in subsequent development of the follicle. This study highlights the importance of oocyte factors in regulating fertility and provides new information for structural analysis and investigation of the potentially important sites of dimerization or translational modifications required to produce biologically active GDF9. It also provides the basis for the utilization of these animals to enhance sheep production.

Exposure to a complex cocktail of environmental endocrine-disrupting compounds disturbs the kisspeptin/GPR54 system in ovine hypothalamus and pituitary gland.

Background Ubiquitous environmental chemicals, including endocrine-disrupting chemicals (EDCs), are associated with declining human reproductive health, as well as an increasing incidence of cancers of the reproductive system. Verifying such links requires animal models exposed to “real-life,” environmentally relevant concentrations/mixtures of EDC, particularly in utero, when sensitivity to EDC exposure is maximal. Objectives We evaluated the effects of maternal exposure to a pollutant cocktail (sewage sludge) on the ovine fetal reproductive neuroendocrine axes, particularly the kisspeptin (KiSS-1)/GPR54 (G-protein–coupled receptor 54) system. Methods KiSS-1, GPR54, and ERα (estrogen receptor α) mRNA expression was quantified in control (C) and treated (T) maternal and fetal (110-day) hypothalami and pituitary glands using semiquantitative reverse transcription polymerase chain reaction, and colocalization of kisspeptin with LHβ (luteinizing hormone β) and ERα in C and T fetal pituitary glands quantified using dual-labeling immunohistochemistry. Results Fetuses exposed in utero to the EDC mixture showed reduced KiSS-1 mRNA expression across three hypothalamic regions examined (rostral, mid, and caudal) and had fewer kisspetin immunopositive cells colocalized with both LHβ and ERα in the pituitary gland. In contrast, treatment had no effect on parameters measured in the adult ewe hypothalamus or pituitary. Conclusions This study demonstrates that the developing fetus is sensitive to real-world mixtures of environmental chemicals, which cause significant neuroendocrine alterations. The important role of kisspeptin/GPR54 in regulating puberty and adult reproduction means that in utero disruption of this system is likely to have long-term consequences in adulthood and represents a novel, additional pathway through which environmental chemicals perturb human reproduction.

In utero exposure to low doses of environmental pollutants disrupts fetal ovarian development in sheep

Epidemiological studies of the impact of environmental chemicals on reproductive health demonstrate consequences of exposure but establishing causative links requires animal models using ‘real life’ in utero exposures. We aimed to determine whether prolonged, low-dose, exposure of pregnant sheep to a mixture of environmental chemicals affects fetal ovarian development. Exposure of treated ewes (n = 7) to pollutants was maximized by surface application of processed sewage sludge to pasture. Control ewes (n = 10) were reared on pasture treated with inorganic fertilizer. Ovaries and blood were collected from fetuses (n = 15 control and n = 8 treated) on Day 110 of gestation for investigation of fetal endocrinology, ovarian follicle/oocyte numbers and ovarian proteome. Treated fetuses were 14% lighter than controls but fetal ovary weights were unchanged. Prolactin (48% lower) was the only measured hormone significantly affected by treatment. Treatment reduced numbers of growth differentiation factor (GDF9) and induced myeloid leukaemia cell differentiation protein (MCL1) positive oocytes by 25–26% and increased pro-apoptotic BAX by 65% and 42% of protein spots in the treated ovarian proteome were differently expressed compared with controls. Nineteen spots were identified and included proteins involved in gene expression/transcription, protein synthesis, phosphorylation and receptor activity. Fetal exposure to environmental chemicals, via the mother, significantly perturbs fetal ovarian development. If such effects are replicated in humans, premature menopause could be an outcome.

Effects of prenatal undernutrition on emotional reactivity and cognitive flexibility in adult sheep

The experiment reported was designed to test two hypotheses: that prenatal undernutrition (a) increases emotional reactivity and (b) impairs cognitive flexibility in sheep. The mothers of one group were fed live weight maintenance requirements throughout pregnancy (control, C) while those of another group were fed 50% of that amount from days 1 to 95 of pregnancy and 100% from then onwards (prenatal undernutrition, PU). At 18 months of age, PU sheep were more active during restraint (P < 0.05) and approached a novel stimulus more slowly (P = 0.02). In response to a sudden stimulus, PU males and C females showed a higher initial level of locomotion compared to C males, which only gradually declined, while PU females started at a high initial rate, changing rapidly to immobility. In a T-maze, PU resulted in a shift of side preferences (laterality) from a general right-bias to neutrality in males and to a left-bias in females (P < 0.05). In the two reversal tasks, C males and PU females had a preference for one side over the other, while PU males showed no preference. In contrast to C males, PU males failed to improve their learning speed from the first to the second reversal (P < 0.05). It is concluded that PU can lead to increased emotional reactivity and changes in side preference in both sexes and impaired cognitive flexibility in males. Undernutrition during pregnancy, therefore, not only affects the welfare of the dam, but also the personality of her offspring.

Maternal undernutrition and the ovine acute phase response to vaccination

BackgroundThe acute phase response is the immediate host response to infection, inflammation and trauma and can be monitored by measuring the acute phase proteins (APP) such as haptoglobin (Hp) or serum amyloid A (SAA). The plane of nutrition during pregnancy is known to affect many mechanisms including the neuroendocrine and neuroimmune systems in neonatal animals but effects on the APP are unknown. To investigate this phenomenon the serum concentration of Hp and SAA was initially determined in non-stimulated lambs from 3 groups (n = 10/group). The dams of the lambs of the respective groups were fed 100% of requirements throughout gestation (High/High; HH); 100% of requirements for the first 65 d of gestation followed by 70% of requirements until 125 d from when they were fed 100% of requirements (High/Low; HL); 65% of liveweight maintenance requirements for the first 65 d gestation followed by 100% of requirements for the remainder of pregnancy (Low/High; LH). The dynamic APP response in the lambs was estimated by measuring the concentration of Hp and SAA following routine vaccination with a multivalent clostridial vaccine with a Pasteurella component, Heptavac P™ following primary and secondary vaccination.ResultsThe Hp and SAA concentrations were significantly lower at the time of vaccination (day 8–14) than on the day of birth. Vaccination stimulated the acute phase response in lambs with increases found in both Hp and SAA. Maternal undernutrition led to the SAA response to vaccination being significantly lower in the HL group than in the HH group. The LH group did not differ significantly from either the HH or HL groups. No significant effects of maternal undernutrition were found on the Hp concentrations. A significant reduction was found in all groups in the response of SAA following the second vaccination compared to the response after the primary vaccination but no change occurred in the Hp response.ConclusionDecreased SAA concentrations, post-vaccination, in lambs born to ewes on the HL diet shows that maternal undernutrition prior to parturition affects the innate immune system of the offspring. The differences in response of Hp and SAA to primary and secondary vaccinations indicate that the cytokine driven APP response mechanisms vary with individual APP.

Appetite regulatory neuropeptides are expressed in the sheep hypothalamus before birth

In the adult, a hypothalamic neural network acts to maintain energy balance in response to nutritional feedback from the periphery. Although there is an immediate requirement for this system to be functional at birth, it is unknown whether the components of this central neural network are expressed in the developing brain before birth. We therefore examined in the fetal sheep hypothalamus during late gestation gene expression for leptin receptor (OB‐Rb) and neuropeptides that regulate energy balance in the adult. Brains were collected from fetal sheep at 110 days (n = 12) and 140 days of gestation (n = 5) (term = 150 days) and gene expression was detected in all hypothalami using in situ hybridization with radiolabelled riboprobes for OB‐Rb, neuropeptide Y (NPY), agouti‐related peptide, pro‐opiomelanocortin and cocaine‐ and amphetamine‐regulated transcript (CART). All mRNAs were expressed in the arcuate nucleus of fetuses at both time points. Additional sites of mRNA expression were the dorsomedial hypothalamus (DMH) for NPY, the paraventricular nucleus (PVN), ventromedial hypothalamus (VMH) and lateral hypothalamic area for CART, and the DMH, PVN and VMH for OB‐Rb. We have therefore demonstrated that adult‐like localization of gene expression for OB‐Rb and key appetite regulatory neuropeptides is established in the ovine hypothalamus before birth. Thus, the fetus possesses a central appetite regulatory neural network with the potential to respond to changes in nutrient supply, which could impact on energy balance regulation both before and after birth.

Anthropogenic pollutants: a threat to ecosystem sustainability?

Pollutants, including synthetic organic materials and heavy metals, are known to adversely affect physiological systems in all animal species studied to date. While many individual chemicals can perturb normal functions, the combined actions of multiple pollutants are of particular concern because they can exert effects even when each individual chemical is present at concentrations too low to be individually effective. The biological effects of pollutants differ greatly between species reflecting differences in the pattern of exposure, routes of uptake, metabolism following uptake, rates of accumulation and sensitivity of the target organs. Thus, understanding of the effects of pollutants on wildlife and ecosystems will require detailed study of many different species, representing a wide range of taxa. However, such studies can be informed by knowledge obtained in more controlled conditions which may indicate likely mechanisms of action and suitable endpoint measurements. Responses may be exacerbated by interactions between the effects of pollutants and environmental stressors, such as under-nutrition or osmotic stresses and so changes in such variables associated with climatic changes may exacerbate physiological responses to pollutant burdens.

Cellular and hormonal disruption of fetal testis development in sheep reared on pasture treated with sewage sludge

The purpose of this study was to evaluate whether experimental exposure of pregnant sheep to a mixture of environmental chemicals added to pasture as sewage sludge (n = 9 treated animals) exerted effects on fetal testis development or function; application of sewage sludge was undertaken so as to maximize exposure of the ewes to its contents. Control ewes (n = 9) were reared on pasture treated with an equivalent amount of inorganic nitrogenous fertilizer. Treatment had no effect on body weight of ewes, but it reduced body weight by 12–15% in male (n = 12) and female (n = 8) fetuses on gestation day 110. In treated male fetuses (n = 11), testis weight was significantly reduced (32%), as were the numbers of Sertoli cells (34% reduction), Leydig cells (37% reduction), and gonocytes (44% reduction), compared with control fetuses (n = 8). Fetal blood levels of testosterone and inhibin A were also reduced (36% and 38%, respectively) in treated compared with control fetuses, whereas blood levels of luteinizing hormone and follicle-stimulating hormone were unchanged. Based on immunoexpression of anti-Müllerian hormone, cytochrome P450 side chain cleavage enzyme, and Leydig cell cytoplasmic volume, we conclude that the hormone changes in treated male fetuses probably result from the reduction in somatic cell numbers. This reduction could result from fetal growth restriction in male fetuses and/or from the lowered testosterone action; reduced immunoexpression of α-smooth muscle actin in peritubular cells and of androgen receptor in testes of treated animals supports the latter possibility. These findings indicate that exposure of the developing male sheep fetus to real-world mixtures of environmental chemicals can result in major attenuation of testicular development and hormonal function, which may have consequences in adulthood.

Alkyl phenols and diethylhexyl phthalate in tissues of sheep grazing pastures fertilized with sewage sludge or inorganic fertilizer.

We studied selected tissues from ewes and their lambs that were grazing pastures fertilized with either sewage sludge (treated) or inorganic fertilizer (control) and determined concentrations of alkylphenols and phthalates in these tissues. Mean tissue concentrations of alkylphenols were relatively low (< 10–400 μg/kg) in all animals and tissues. Phthalates were detected in tissues of both control and treated animals at relatively high concentrations (> 20,000 μg/kg in many tissue samples). The use of sludge as a fertilizer was not associated with consistently increased concentrations of either alkylphenols or phthalates in the tissues of animals grazing treated pastures relative to levels in control animal tissues. Concentrations of the two classes of chemicals differed but were of a similar order of magnitude in liver and muscle as well as in fat. Concentrations of each class of compound were broadly similar in tissues derived from ewes and lambs. Although there were significant differences (p < 0.01 or p < 0.001) between years (cohorts) in mean tissue concentrations of both nonylphenol (NP) and phthalate in each of the tissues from both ewes and lambs, the differences were not attributable to either the age (6 months or 5 years) of the animal or the duration of exposure to treatments. Octylphenol concentrations were generally undetectable. There was no consistent cumulative outcome of prolonged exposure on the tissue concentrations of either class of pollutant in any ewe tissue. Mean tissue concentrations of phthalate were higher (p < 0.001) in the liver and kidney fat of male compared with female lambs. We suggest that the addition of sewage sludge to pasture is unlikely to cause large increases in tissue concentrations of NP and phthalates in sheep and other animals with broadly similar diets and digestive systems (i.e., domestic ruminants) grazing such pasture.