D. Claire Wathes

Polyunsaturated Fatty Acids in Male and Female Reproduction

Abstract In Westernized societies, average consumption of n-6 polyunsaturated fatty acids (PUFAs) far exceeds nutritional requirements. The ratio of n-6 to n-3 PUFAs is generally >10:1 whereas on a primitive human diet it was closer to 1:1. Diets fed to intensively farmed livestock have followed a similar trend. Both n-6 and n-3 PUFAs can influence reproductive processes through a variety of mechanisms. They provide the precursors for prostaglandin synthesis and can modulate the expression patterns of many key enzymes involved in both prostaglandin and steroid metabolism. They are essential components of all cell membranes. The proportions of different PUFAs in tissues of the reproductive tract reflect dietary consumption. PUFA supplements (particularly n-3 PUFAs in fish oil) are promoted for general health reasons. Fish oils may also benefit fertility in cattle and reduce the risk of preterm labor in women, but in both cases current evidence to support this is inconclusive. Gamma-linolenic acid containing oils can alter the types of prostaglandins produced by cells in vitro, but published data to support claims relating to effects on reproductive health are lacking. Spermatozoa require a high PUFA content to provide the plasma membrane with the fluidity essential at fertilization. However, this makes spermatozoa particularly vulnerable to attack by reactive oxygen species, and lifestyle factors promoting oxidative stress have clear associations with reduced fertility. Adequately powered trials that control for the ratios of different PUFAs consumed are required to determine the extent to which this aspect of our diets does influence our fertility.

Negative energy balance alters global gene expression and immune responses in the uterus of postpartum dairy cows

Most dairy cows suffer uterine microbial contamination postpartum. Persistent endometritis often develops, associated with reduced fertility. We used a model of differential feeding and milking regimes to produce cows in differing negative energy balance status in early lactation (mild or severe, MNEB or SNEB). Blood hematology was assessed preslaughter at 2 wk postpartum. RNA expression in endometrial samples was compared using bovine Affymetrix arrays. Data were mapped using Ingenuity Pathway Analysis. Circulating concentrations of IGF-I remained lower in the SNEB group, whereas blood nonesterified fatty acid and β-hydroxybutyrate concentrations were raised. White blood cell count and lymphocyte number were reduced in SNEB cows. Array analysis of endometrial samples identified 274 differentially expressed probes representing 197 recognized genes between the energy balance groups. The main canonical pathways affected related to immunological and inflammatory disease and connective tissue disorders. Inflammatory response genes with major upregulation in SNEB cows included matrix metalloproteinases, chemokines, cytokines, and calgranulins. Expression of several interferon-inducible genes including ISG20, IFIH1, MX1, and MX2 were also significantly increased in the SNEB cows. These results provide evidence that cows in SNEB were still undergoing an active uterine inflammatory response 2 wk postpartum, whereas MNEB cows had more fully recovered from their energy deficit, with their endometrium reaching a more advanced stage of repair. SNEB may therefore prevent cows from mounting an effective immune response to the microbial challenge experienced after calving, prolonging the time required for uterine recovery and compromising subsequent fertility.

The Effect of Linolenic Acid on Bovine Oocyte Maturation and Development

Dietary polyunsaturated fatty acids can influence reproductive performance. In dairy cattle, some high-fat diets resulted in higher blastocyst rates and improved embryo quality. These effects may partly be mediated by a direct action of fatty acids on oocyte development. The present study investigated the effect of linolenic acid (ALA; 18:3 n-3) supplementation on bovine oocyte maturation and early embryo development in vitro. Treatment of cumulus-oocyte complexes (COCs) with 50 μM ALA significantly increased the percentage of oocytes at the metaphase II (MII) stage compared with untreated controls (95% ± 2% vs. 84% ± 2%, respectively). Higher doses of ALA were detrimental. Treatment of COCs with 50 μM ALA compared with controls also resulted in a significantly higher percentage of cleaved embryos (77% ± 9% vs. 69% ± 9%, respectively) and blastocyst rate (36% ± 4% vs. 23% ± 5%, respectively) and better-quality embryos. Furthermore, COCs treated with ALA had significant increases compared with controls in: 1) prostaglandin E2 (PGE2) concentration (233% ± 41%) in the medium, 2) intracellular cAMP at 3 h of maturation, and 3) phosphorylation of the mitogen-activated protein kinases (MAPKs) during the first 6 h of maturation. Moreover, ALA overcame the suppressive effects of the prostaglandin-endoperoxide synthase 2 inhibitor (NS-398) on oocyte maturation and partially improved the maturation rate in the presence of the MAPK kinase inhibitor (U-0126). Linolenic acid could not, however, recover maturation in the presence of both inhibitors. In conclusion, treatment of bovine COCs with ALA during oocyte maturation affects the molecular mechanisms controlling oocyte nuclear maturation, leading to an increased number of MII-stage oocytes and improved subsequent early embryo development. This effect is mediated both directly through MAPK pathway and indirectly through PGE2 synthesis.

Metabolic profiles and progesterone cycles in first lactation dairy cows

This study investigated the ovarian function, metabolic profiles and fertility in first lactation Holstein-Friesian dairy cows (mean 305 day milk yield: 7417+/-191kg, n=37). Reproductive profiles obtained from milk progesterone analysis were categorized into normal (n=17) and four abnormal profiles (delayed ovulation, DOV1, n=9; DOV2, n=2; persistent corpus luteum, PCL1, n=6; PCL2, n=4; 1: immediately post-calving, 2: subsequent cycles). Fifty-five percent of cows had abnormal profiles with half of these being categorized as DOV1. Fertility of DOV1 and DOV2 cows was reduced whereas PCL1 and PCL2 cows had similar reproductive competence to normal profile cows. DOV1 animals had higher milk energy values, lower energy balances, lower dry matter intakes (DMI) and greater body weight and body condition score (BCS) losses post-calving than normal profile animals. DOV1 animals also had lower insulin-like growth factor-I (IGF-I) and higher betahydroxybutyrate (BHB) concentrations and tended to have the lower insulin and glucose concentrations in the pre-service period than normal profile cows. All PCL animals had vulval discharges postpartum. Despite this, the DMI, body weight and BCS changes, IGF-I concentrations and fertility of PCL1 animals was similar to normal profile cows. In conclusion, the high prevalence of delayed ovulation post-calving (DOV1) in primiparous high yielding cows lasted long enough (71+/-8.3 days) to have a detrimental impact on fertility and was associated with significant physiological changes. This study did not establish any detrimental effects of PCL profiles on fertility or production parameters.

Influence of energy balance on the somatotrophic axis and matrix metalloproteinase expression in the endometrium of the postpartum dairy cow

Postpartum dairy cows enter a period of negative energy balance (NEB) associated with low circulating IGF1, during which the uterus must undergo extensive repair following calving. This study investigated the effects of NEB on expression of IGF family members and related genes in the involuting uterus. Cows were allocated to two treatments using differential feeding and milking regimes to produce mild NEB or severe NEB (SNEB). Uterine endometrial samples collected 2 weeks post partum were analysed by quantitative PCR. The expression of IGF-binding protein 4 (IGFBP4) mRNA increased in the endometrium of SNEB cows, with trends towards increased IGFBP1 and reduced IGFBP6 expression. There were no significant differences between treatments in mRNA expression of IGF1, IGF2 or of any hormone receptor studied, but significant correlations across all cows in the expression levels of groups of receptors suggested common regulatory mechanisms: type 1 IGF receptor (IGF1R), IGF2R and insulin receptor (INSR); GHR with ESR1; and ESR2 with NR3C1. The expression of IGF1R and INSR also positively correlated with the circulating urea concentration. Matrix metalloproteinases (MMPs) are important in tissue remodelling and can affect IGF signalling via interaction with IGFBPs. The expression levels of MMP1, MMP3, MMP9 and MMP13 mRNAs all showed major upregulation in the endometrium of cows in SNEB and all except MMP9 were highly correlated with expression of IGFBP4. Alpha(2)-HS-glycoprotein (AHSG) and PDK4, two genes implicated in insulin resistance, were also highly expressed in SNEB. These results suggest that cows in SNEB experience alterations to the IGF and insulin signalling pathways in the postpartum endometrium. This may affect the rate of tissue repair with a possible negative impact on subsequent fertility.

Associations between lipid metabolism and fertility in the dairy cow

Dairy cows mobilise body tissues to support milk production and, because glucose supplies are limited, lipids are used preferentially for energy production. Lipogenic activity is switched off and lipolytic mechanisms in adipose tissue increase through changes in the expression of several key enzymes. This results in a loss of body condition, together with high circulating concentrations of non-esterified fatty acids. Changes in the synthesis, secretion and signalling pathways of somatotrophic hormones (insulin, growth hormone, insulin-like growth factor 1) and adipokines (e.g. leptin) are central to the regulation of these processes. A high reliance on fatty acids as an energy source in the peripartum period causes oxidative damage to mitochondria in metabolically active tissues, including the liver and reproductive tract. The expression of genes involved in insulin resistance (PDK4, AHSG) is increased, together with expression of TIEG1, a transcription factor that can induce apoptosis via the mitochondrial pathway. Polymorphisms in TFAM and UCP2, two autosomal mitochondrial genes, have been associated with longevity in dairy cows. Polymorphisms in many other genes that affect lipid metabolism also show some associations with fertility traits. These include DGAT1, SCD1, DECR1, CRH, CBFA2T1, GH, LEP and NPY. Excess lipid accumulation in oocytes and the regenerating endometrium reduces fertility via reductions in embryo survival and increased inflammatory changes, respectively.

Time Course of Defense Mechanisms in Bovine Endometrium in Response to Lipopolysaccharide

ABSTRACT Endometritis caused by uterine infection after calving reduces fertility and causes major economic losses to the dairy industry. This study investigated the time course of an inflammatory response in bovine endometrium triggered by exposure to bacterial endotoxin lipopolysaccharide (LPS). Mixed endometrial epithelial and stromal cells (9:1 ratio) were grown to confluence as a model system and treated with an optimized dose of 100 ng/ml LPS in vitro. Gene expression responses were measured using quantitative PCR, and gene products were investigated using assays of culture medium and Western blotting. Of 17 candidate genes tested initially, LPS treatment for 24 h up-regulated mRNA expression of TLR4 signaling (TLR4, CD14), cytokines (IL1B, TNF), chemokines (IL8, CXCL5), antimicrobial peptides (LAP, S100A8, S100A9, S100A12), and matrix metalloproteinases (MMP1, MMP13). A 48 h, LPS time course study showed that TNF increased first at 1 h, followed by peak expression of IL1B at 6 h, and those of S100A8, S100A12, and LAP at 12 h. The intracellular S100A8 protein content doubled at 12–24 h but with little excretion into the medium. Regarding prostaglandin biosynthesis, PTGES mRNA was slightly higher after LPS exposure, whereas expression of the PGF synthase AKR1B1 was inhibited. Despite this, LPS treatment stimulated the secretion of both PGE2 and PGF2alpha to a similar extent. These results suggest that the family of S100 Ca2+ binding proteins are released from damaged endometrial cells and may play a major antimicrobial role. Prostaglandin synthesis increased during the uterine infection, but we found no evidence that this was associated with a change in the PGE:PGF ratio.

Differential effects of linoleic and alpha-linolenic fatty acids on spatial and temporal mitochondrial distribution and activity in bovine oocytes.

Using specific stains and confocal microscope imaging, the patterns of mitochondrial distribution, mitochondrial inner membrane potential and reactive oxygen species (ROS) levels during bovine oocyte maturation were determined in the presence or absence of physiological concentrations of linoleic acid (LA; 100µM) or α-linolenic acid (ALA; 50µM). Mitochondrial distribution in control oocytes at 0h was mainly peripheral and changed to a diffused pattern after 1h of culture; this was maintained up to 24h. Mitochondrial clusters were observed during the early hours of maturation (1-4h); the majority of these were arranged in perinuclear fashion. LA supplementation resulted in: (1) delayed redistribution of the mitochondria from a peripheral to a diffuse pattern and a decreased percentages of oocytes showing perinuclear mitochondrial clusters, (2) decreased mitochondrial inner membrane potential at 1 and 24h compared with the control and (3) higher ROS levels, associated with a lower nuclear maturation rate. In contrast, ALA supplementation had no effect on mitochondrial distribution and activity and decreased ROS levels compared with the control; this was associated with an increased nuclear maturation rate. In conclusion, LA induced alterations in mitochondrial distribution and activity as well as increasing ROS levels, which mediate, at least in part, the inhibitory effect on oocyte maturation.

Peroxisome-proliferator-activated receptors and the control of levels of prostaglandin-endoperoxide synthase 2 by arachidonic acid in the bovine uterus.

Arachidonic acid is a potential paracrine agent released by the uterine endometrial epithelium to induce PTGS2 [PG (prostaglandin)-endoperoxide synthase 2] in the stroma. In the present study, bovine endometrial stromal cells were used to determine whether PTGS2 is induced by arachidonic acid in stromal cells, and to investigate the potential role of PPARs (peroxisome-proliferator-activated receptors) in this effect. Arachidonic acid increased PTGS2 levels up to 7.5-fold within 6 h. The cells expressed PPARalpha and PPARdelta (also known as PPARbeta) (but not PPARgamma). PTGS2 protein level was increased by PPAR agonists, including polyunsaturated fatty acids, synthetic PPAR ligands, PGA1 and NSAIDs (non-steroidal anti-inflammatory drugs) with a time course resembling that of arachidonic acid. Use of agonists and antagonists indicated PPARalpha (but not PPARdelta or PPARgamma) was responsible for PTGS2 induction. PTGS2 induction by arachidonic acid did not require PG synthesis. PTGS2 levels were increased by the PKC (protein kinase C) activators 4beta-PMA and PGF(2alpha), and the effects of arachidonic acid, NSAIDs, synthetic PPAR ligands and 4beta-PMA were blocked by PKC inhibitors. This is consistent with PPAR phosphorylation by PKC. Induction of PTGS2 protein by 4beta-PMA in the absence of a PPAR ligand was decreased by the NF-kappaB (nuclear factor kappaB) inhibitors MG132 and parthenolide, suggesting that PKC acted through NF-kappaB in addition to PPAR phosphorylation. Use of NF-kappaB inhibitors allowed the action of arachidonic acid as a PPAR agonist to be dissociated from an effect through PKC. The results are consistent with the hypothesis that arachidonic acid acts via PPARalpha to increase PTGS2 levels in bovine endometrial stromal cells.

Comparison of mRNA for IGFs and their binding proteins in the oviduct during the peri-oestrous period between dairy heifers and lactating cows

The oviduct provides the environment to support gamete maturation, fertilisation and early embryo development. As there is a high incidence of early embryonic death in lactating dairy cows, this study compared expression of IGF family members in the oviduct between lactating Holstein-Friesian dairy cows (n=16, 81±2.4 days in milk) and nulliparous heifers (n=16, age 1.6±0.07 years) at three stages of the oestrous cycle: A) newly selected dominant follicle in the luteal phase, B) follicular phase before the LH surge and C) pre-ovulatory phase after the LH surge. Expression of IGF1, IGF2, IGF binding protein 2 (IGFBP2), IGFBP3 and IGFBP6 mRNA was determined in the ampulla of the oviduct. Oviduct side (ipsilateral or contralateral) with respect to the dominant follicle did not affect gene expression. Expression of IGF1 and all three IGFBPs increased significantly between the luteal and the pre-ovulatory phases, with no further significant alteration post-LH surge. Concentrations of circulating IGF1 were higher in heifers than in cows, as was the mRNA expression of IGF1, IGFBP3 and IGFBP6. The pre-LH surge rise in IGFBP2 mRNA was only observed in heifers. IGF2 expression was not influenced by either age or stage of cycle. These three IGFBPs are generally considered to inhibit IGF action. These results indicate tight regulation of IGF bioavailability in the oviductal environment around oestrus, with pronounced differences between cows and heifers, which are likely to influence early embryonic development. Further studies are required to assess the implications for embryo survival.