Ultrasonographic measures of body fatness and their relationship with plasma levels and adipose tissue expression of four adipokines in Welsh pony mares.

Obesity is responsible for metabolic dysregulations that alter fertility and induce pathologies. The objectives of the present study were to validate a reliable method for the evaluation of body fatness in mares and to associate the body fat estimation data to metabolic changes, including adipokines at the plasma and adipose tissue levels. To reach this purpose, animals were subjected to two extreme breeding conditions to study the variation of morphological, ultrasound, and physiological parameters. Twenty Welsh mares were followed up monthly from April to October before and after animals were moved outdoors to grasslands. Body weight (BW), body length (BL), height at the withers (HW), thoracic perimeter (TP), 5-point body condition score (BCS), and subcutaneous fat thickness (SFT) at the level of the shoulder, the lumbar region, and the rump, measured by ultrasonography, and plasma and adipose tissue metabolic indicators were assessed in parallel. Statistical analysis was performed using a linear mixed-effects model, whereas Pearson tests were used for the analysis of the correlations between the different parameters. Although mean BW did not increase significantly (P = 0.0940), TP (P = 0.0002) and BCS (P < 0.0001) increased during the study period. Ultrasonographic examination of subcutaneous adipose tissue showed an increase in SFT at the level of the shoulder (P < 0.0001), lumbar region (P < 0.0001), and rump (P < 0.0001). Plasma concentrations of nonesterified fatty acids (P < 0.0001), phospholipids (P < 0.0001), and cholesterol (P < 0.0001) increased significantly, whereas triglycerides (P < 0.0001) decreased significantly during the study period. Although both plasma concentrations and adipose tissue expression of leptin (P < 0.0001) and resistin (P < 0.0001) increased significantly, adiponectin (P < 0.0001) significantly decreased and visfatin remained unchanged (P = 0.8401). Expression of adipokine receptors studied showed the opposite pattern compared with their ligand. Ultrasonographic measurements of subcutaneous adipose tissue thickness at the shoulder, lumbar region, and rump are relevant indicators of fatness related with adipokine plasma concentrations and expression of adipokine-related receptors in adipose tissue, and particularly highlight seasonal effects.

KIRREL is differentially expressed in adipose tissue from 'fertil+' and 'fertil-' cows: in vitro role in ovary?

We have previously shown that dairy cows carrying the 'fertil-' haplotype for one quantitative trait locus affecting female fertility located on the bovine chromosome three (QTL-F-Fert-BTA3) have a significantly lower conception rate and body weight after calving than cows carrying the 'fertil+' haplotype. Here, we compared by Tiling Array the expression of genes included in the QTL-F-Fert-BTA3 in 'fertil+' and 'fertil-' adipose tissue one week after calving when plasma non-esterified fatty acid concentrations were greater in 'fertil-' animals. We observed that thirty-one genes were overexpressed whereas twelve were under-expressed in 'fertil+' as compared to 'fertil-' cows (P < 0.05). By quantitative PCR and immunoblot we confirmed that adipose tissue KIRREL mRNA and protein were significantly greater expressed in 'fertil+' than in 'fertil-'. KIRREL mRNA is abundant in bovine kidney, adipose tissue, pituitary, and ovary and detectable in hypothalamus and mammary gland. Its expression (mRNA and protein) is greater in kidney of 'fertil+' than 'fertil-' cows (P < 0.05). KIRREL (mRNA and protein) is also present in the different ovarian cells with a greater expression in granulosa cells of 'fertil+' than 'fertil-' cows. In cultured granulosa cells, recombinant KIRREL halved steroid secretion in basal state (P < 0.05). It also decreased cell proliferation (P < 0.05) and in vitro oocyte maturation (P < 0.05). These results were associated to a rapid increase in MAPK1/3 and MAPK14 phosphorylation in granulosa cells and to a decrease in MAPK1/3 phosphorylation in oocyte. Thus, KIRREL could be a potential metabolic messenger linking body composition and fertility.

Involvement of plasma adipokines in metabolic and reproductive parameters in Holstein dairy cows fed with diets with differing energy levels.

This study aimed to investigate the association between plasma adipokine concentrations and metabolic and reproductive parameters in Holstein dairy cows fed diets with different energy levels during the peripartum period. The experiment started 1 mo before first calving and was maintained for 2 lactations. Dry matter intake and energy balance in animals fed a low-energy (LE) diet were significantly lower than that of animals fed a high-energy (HE) diet in the first lactation. Body weight, milk production, back fat thickness, and plasma concentrations of fatty acids, glucose, and insulin were not affected by diet, whereas plasma leptin and adiponectin concentrations were lower and plasma resistin concentrations higher in animals fed the LE diet. Unlike concentrations of adiponectin, plasma resistin concentrations were positively correlated with back fat thickness and plasma fatty acids concentrations and negatively correlated with dry matter intake and plasma leptin concentrations. No effect of diet was found on reproductive variables; that is, pregnancy rates at 35 or 90 d after artificial insemination (AI); numbers of small (3-5 mm), medium (>5 and ≤7 mm), and large (>7 mm) follicles; calving-to-AI and calving-to-calving intervals; and magnitude and duration of the LH surge. However, the commencement of luteal activity after first calving occurred sooner and the frequency of LH pulses was higher in the HE group than in the LE group. A significant positive correlation was found between the number of follicles (of any size) and the area under the curve of plasma resistin concentrations. The number of small follicles was also positively correlated with the nadir of plasma resistin concentrations. Taken together, these results suggest that dietary energy content in the range applied here can alter the resumption of ovarian activity and LH pulsatility without affecting fat mobilization. Plasma adipokine profiles (leptin, resistin, and adiponectin) were significantly altered by diet and negative energy balance but relationships with reproductive variables were limited to follicular growth characteristics and plasma resistin concentrations.

Apelin (APLN) regulates progesterone secretion and oocyte maturation in bovine ovarian cells.

APLN and its G-protein coupled receptor APLNR are expressed in the bovine ovary. However their role in granulosa cells and oocytes is unknown. Here, we studied their expression in bovine ovarian cells and investigated their regulation in cultured luteinizing granulosa cells in response to IGF1 and FSH. We determined the effect and the molecular mechanism of APLN (isoforms 17 and 13) on bovine granulosa cell progesterone secretion and on oocyte maturation. By RT-qPCR and immunoblot, we showed that the expression of both APLN and APLNR in granulosa and oocytes significantly increased with ovarian follicles size whereas it was similar in theca interstitial cells. In vitro, in unstimulated luteinizing bovine granulosa cells and in response to IGF1 (10-8 M) but not to FSH (10-8 M), we observed that APLN (-17 and -13) (10-9 M) increased progesterone production; this was abolished in response to the APLNR antagonist ML221. These latter effects were dependent on the MAPK ERK1/2 kinase. Furthermore, we showed that APLN (-17 and -13) (10-9 M) increased cell proliferation through AKT signaling. Conversely, the addition of APLN-13 and APLN-17 to in vitro maturation medium containing IGF1 (10-8 M) but not FSH (10-8 M) arrested most oocytes at the germinal vesicle stage, which was associated with a decrease in progesterone secretion, an inhibition in MAPK ERK1/2 phosphorylation and an increase in PRKA phosphorylation in oocytes. Thus, APLN can increase progesterone secretion and cell proliferation in bovine luteinizing granulosa cells in vitro, while it blocks meiotic progression at the germinal vesicle stage during bovine oocyte in vitro maturation.

Expression of adenosine 5'-monophosphate-Activated protein kinase (AMPK) in ovine testis (Ovis aries): In vivo regulation by nutritional state.

In the present study, we identified AMPK and investigated its potential role in steroidogenesis in vivo in the ovine testis in response to variation in nutritional status (fed control vs. restricted). We performed immunoblotting to show that both active and non-active forms of AMPK exist in ovine testis and liver. In testis, we confirmed these results by immunohistochemistry. We found a correlation between ATP (Adenosine-Triphosphate) levels and the expression of AMPK in liver. Also, low and high caloric diets induce isoform-dependent AMPK expression, with an increase in α2, ß1ß2 and γ1 activity levels. Although the restricted group exhibited an increase in lipid balance, only the triglyceride and HC-VLDL (Cholesterol-Very low density lipoprotein) fractions showed significant differences between groups, suggesting an adaptive mechanism. Moreover, the relatively low rate of non-esterified fatty acid released into the circulation implies re-esterification to compensate for the physiological need. In the fed control group, AMPK activates the production of testosterone in Leydig cells; this is, in turn, associated with an increase in the expression of 3ß-HSD (3 beta hydroxy steroid deshydrogenase), p450scc (Cholesterol side-chain cleavage enzyme) and StAR (Steroidogenic acute regulatory protein) proteins induced by decreased MAPK ERK½ (Extracellular signal-regulated kinase -Mitogen-activated protein kinase) phosphorylation. In contrast, in the restricted group, testosterone secretion was reduced but intracellular cholesterol concentration was not. Furthermore, the combination of high levels of lipoproteins and emergence of the p38 MAP kinase pathway suggest the involvement of pro-inflammatory cytokines, as confirmed by transcriptional repression of the StAR protein. Taken together, these results suggest that AMPK expression is tissue dependent.

The insulin sensitiser metformin regulates chicken Sertoli and germ cell populations.

Metformin, an insulin sensitiser from the biguanide family of molecules, is used for the treatment of insulin resistance in type 2 diabetes individuals. It increases peripheral glucose uptake and may reduce food intake. Based on the tight link between metabolism and fertility, we investigated the role of metformin on testicular function using in vitro culture of Sertoli cells and seminiferous tubules, complemented by in vivo data obtained following metformin administration to prepubertal chickens. In vitro, metformin treatment reduced Sertoli cell proliferation without inducing apoptosis and morphological changes. The metabolism of Sertoli cells was affected because lactate secretion by Sertoli cells increased approximately twofold and intracellular free ATP was negatively impacted. Two important pathways regulating proliferation and metabolism in Sertoli cells were assayed. Metformin exposure was not associated with an increased phosphorylation of AKT or ERK. There was a 90% reduction in the proportion of proliferating germ cells after a 96-h exposure of seminiferous tubule cultures to metformin. In vivo, 6-week-old chickens treated with metformin for 3 weeks exhibited reduced testicular weight and a 50% decrease in testosterone levels. The expression of a marker of undifferentiated germ cells was unchanged in contrast to the decrease in expression of 'protamine', a marker of differentiated germ cells. In conclusion, these results suggest that metformin affects the testicular energy content and the proliferative ability of Sertoli and germ cells.

Significant differences in fertility between dairy cows selected for one QTL located on bovine chromosome 3 are not attributable to energy balance, although eating behaviour is affected.

Improvement of reproduction in dairy cows has become a major challenge in dairy production. We have recently shown that dairy cows carrying the 'fertil-' haplotype for one quantitative trait locus (QTL), affecting female fertility and located on the bovine chromosome 3, had a significantly lower conception rate after the first artificial insemination than cows carrying the 'fertil+' haplotype. The objective of this paper was to study other phenotypic modifications linked to this QTL. In the present study, 23 'fertil+' and 18 'fertil-' cows were characterized for live weight, milk production, food intake, eating behaviour and plasma metabolites. These parameters were measured during the first lactation, from calving to 40 weeks postpartum (wkpp). In the first 7 weeks of lactation, 'fertil+' primiparous cows had a significantly higher live BW and milk production than 'fertil-' cows. Dry matter intake tended to be slightly higher for 'fertil+' than for 'fertil-' primiparous cows in this period. However, energy balance was similar for the two haplotypes in the whole lactation, except in the first wkpp, and consequently, could not explain their different fertility. The major observation concerned the eating behaviour. 'Fertil+' primiparous cows had a significantly lower eating rate than 'fertil-' cows during the 40 weeks of lactation. In parallel, 'fertil+' cows spent significantly more time at the feeder for a similar number of visits than 'fertil-' cows. Furthermore, no differences in plasma concentrations of non-esterified fatty acids and insulin were observed between the two haplotypes. Plasma glucose was significantly lower in 'fertil+' than in 'fertil-' cows in the second wkpp. Taken together, our results show that 'fertil+' and 'fertil-' dairy cows, with different fertility, have also different eating behaviour without any variation in energy balance, except in the first week of lactation.

Data-derived reference profiles with corepresentation of progesterone, estradiol, LH, and FSH dynamics during the bovine estrous cycle.

Subfertility in cows is often associated with alterations in the hormonal patterns involved in the regulation of the estrous cycle. Reference profiles are needed to ground modeling projects aimed at describing these alterations and to develop tools for detecting abnormal dynamics. Various schematic views of LH, FSH, progesterone (P4) and estradiol (E2) patterns have been published but with no clear indication of the extent to which they are derived from real data. The objective of this study was to generate standard profiles for the main reproductive hormones that can be proposed as reliable references to represent the normal dynamics of these hormones over the estrous cycle. A database of hormonal profiles was compiled with 40, 23, 33, and 34 profiles for LH, FSH, E2, and P4, respectively, derived from publications in which changes over time of at least three of these four hormones, including LH, were reported. These profiles were digitalized and standardized over the time throughout the estrous cycle, considering the interval between two successive LH surges to be 21 days. After this standardization on the x-axis, a transformation on the y-axis was performed to center the profiles around their common dynamics. For each hormone, the reference profile was then considered to be the median of the adjusted profiles. Quartiles were reported to account for the time evolution of the variability around each reference profile. The reference profiles obtained showed that the procedure used was satisfactory for extracting the overall changes over time of LH, P4, and E2. Results were less satisfactory for FSH, because of a higher variability observed between the original profiles in our database. The corepresentation of the reference profiles, i.e., when depicted together on the same scale, emphasizes the interplay between these hormones more precisely than most of the schematic views available in literature. These data-derived profiles can be considered to be generic and useful for benchmarking the normal dynamics of gonadotrophins and steroid hormones over the estrous cycle in cow.

Analysis of in vivo oocyte maturation, in vitro embryo development and gene expression in cumulus cells of dairy cows and heifers selected for one fertility quantitative trait loci (QTL) located on BTA3.

We have previously shown that Holstein cows selected for their homozygous favorable ("fertil+") or unfavorable ("fertil-") haplotype at one quantitative trait loci (QTL) of female fertility located on chromosome 3 (QTL-F-Fert-BTA3) had a different success rate 35 and 90 days after the first artificial insemination. To determine whether the lower fertility in "fertil-" animals could be related to oocyte quality, we analyzed the embryo development rate in vitro and the oocyte meiotic maturation in vivo in "fertil+" and "fertil-" heifers. In vitro maturation and fertilization of immature oocytes recovered by ovum pick-up from "fertil+" and "fertil-" heifers resulted in similar cleavage and blastocyst rates in the two haplotypes. However the percentage of expanded blastocysts and the number of cells per blastocyst were significantly higher in "fertil+". Oocytes from presumptive preovulatory follicles were analyzed after ovarian stimulation. A similar rate of immature (from prophase to metaphase-I) and mature oocytes (metaphase-II) was obtained in the two haplotypes, whereas a significantly higher percentage of oocytes from metaphase-I to metaphase-II was observed in "fertil+" compared to "fertil-" heifers. Since cumulus cells (CCs) could reflect the developmental competence of oocytes, we analyzed the expression of seven genes included in the QTL-F-Fert-BTA3 using real-time PCR in bovine CCs after in vivo or in vitro maturation, as a model of higher and lower competence, respectively. Transcript levels of TAGLN2, EEF1A1 and PIGM were higher in CCs after in vitro maturation (IVM) compared to in vivo maturation, whereas no difference was observed for IFI16, KIRREL, SPTA1 and PEX19 expression. The expression levels of all these genes in in preovulatory CCs were not significantly different between "fertil+" and "fertil-" heifers. In conclusion, the lower fertility of "fertil-" females could be partially due to a lowest quality of the oocytes and consequently of preimplantation embryo development.

Ovarian parameters and fertility of dairy cows selected for one QTL located on BTA3.

Recently, one Quantitative Trait Locus (QTL) of female fertility located on Bos Taurus chromosome 3 (BTA3), QTL-F-Fert-BTA3, has been identified in Holstein breed. It is implied in the success rate after the first AI (AI1) in cow. The failure of pregnancy can be due to several factors involved in the different steps of the reproductive process. The aim of our study was to finely phenotype heifers and primiparous cows selected for their haplotype at the QTL-F-Fert-BTA3. We specifically studied the ovarian follicular dynamic and several fertility parameters. Females carrying the favourable haplotype "fertil+" or unfavourable haplotype "fertil-" were monitored by transrectal ultrasonography during their cycle before the first AI (AI1). Follicular dynamic was similar between the two groups. However, the length of the estrus cycle was shorter in heifers than in primiparous cows and two-wave cycles were shorter than three-wave cycles, regardless of the age and the haplotype. The concentration of plasma anti-Müllerian hormone was correlated with the number of small antral follicles. It was higher in heifers than in primiparous cows, independently of their haplotype. The success rate at the AI1 was significantly higher in "fertil+" than in "fertil-" primiparous cows, 35 d after the AI1 (70% vs 39%). In both haplotypes, pregnancy failure occurred mainly before 21 d after AI1. The commencement of luteal activity after calving was significantly earlier in "fertil+" than in "fertil-" primiparous cows. Calving-AI1 and calving-calving intervals were similar between "fertil+" and "fertil-" primiparous cows. Taken together, "fertil+" and "fertil-" primiparous cows present a difference in the success rate after AI1 that is not explained by variations of ovarian dynamics.

Effects of unsaturated fatty acids on progesterone secretion and selected protein kinases in goat granulosa cells.

Previous studies in cattle have shown influences of dietary unsaturated fatty acid (UFA) supplementation on ovarian function. However, it is unclear whether these UFA exert direct or indirect effects on ovarian steroid production or their mechanisms of action. We have recently shown that 5'AMP-activated protein kinase (AMPK) regulates progesterone secretion through mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MAPK ERK1/2) in rodent granulosa cells. Here, we investigated the effects of 3 UFAs, oleic acid (OA), linoleic acid (LA), and alpha-linolenic acid (ALA) on progesterone secretion in goat granulosa cells. Finally, we examined the effects of UFAs on MAPK ERK1/2 and AMPK phosphorylation in these granulosa cells. Oleic acid and LA (10 microM each), but not ALA (100 microM), increased progesterone secretion (P<0.05) in the presence or absence of insulin-like growth factor (IGF)-1 (10(-8) M) or FSH (5 x 10(-8)M). The different AMPK subunits, except for gamma3, are present in the goat ovary. Treatment with metformin (10mM), an activator of AMPK, increased AMPK phosphorylation (P<0.05) and reduced progesterone secretion by 50% (P<0.05) in the basal state and in response to IGF-1 or FSH in goat granulosa cells. Oleic acid and LA had no effect on AMPK phosphorylation, whereas they rapidly increased MAPK ERK1/2 phosphorylation (P<0.05). Finally, U0126, a MAPK ERK1/2 inhibitor, decreased OA- and LA-induced progesterone secretion (P<0.05), suggesting that these UFAs could stimulate progesterone secretion partly through MAPK ERK1/2 in the absence of IGF-1 and FSH in goat granulosa cells. The involvement of AMPK in this process remains to be demonstrated. Taken together, some fatty acids could improve ovarian steroidogenesis through the MAPK ERK1/2 signaling pathway and, consequently, have beneficial effects on goat fertility.

Role of adiponectin receptors, AdipoR1 and AdipoR2, in the steroidogenesis of the human granulosa tumor cell line, KGN.

BACKGROUND: Adiponectin is involved in the regulation of energy homeostasis and more recently in the reproductive functions. We have previously shown that adiponectin receptors (AdipoR1 and AdipoR2) are expressed in human granulosa cells. However, it remains to be investigated whether both AdipoR1 and AdipoR2 or only one of these receptors serve as the major receptor(s) for adiponectin in human granulosa cells. METHODS: The RNA interference (RNAi) technology was used to specifically knockdown the expression of either AdipoR1 or AdipoR2. Progesterone and estradiol levels in the conditioned media were measured by radioimmunoassay, and determination of cell proliferation by tritiated thymidine incorporation. The levels of adiponectin receptors and proteins involved in the steroidogenesis and in the signalling pathways were examined by western blot. RESULTS: We generated AdipoR1 (R1) and AdipoR2 (R2) knockdown KGN cell lines. R1 cells were apoptotic and had increased expression levels of cleaved caspase 3 and decreased levels of BAD phosphorylation and PCNA as compared with control or parental KGN cells. R2 cells had similar morphology to control or KGN cells. However, they produced less progesterone and estradiol and expressed lower levels of StAR protein in response to FSH or IGF-1 stimulation compared with control cells. Furthermore, the increase of MAPK ERK1/2 phosphorylation in response to human recombinant adiponectin and FSH was lower in R2 than control cells. CONCLUSIONS: In the human granulosa KGN cell-line, AdipoR1 seems to be involved in the cell survival whereas AdipoR2, through MAPK ERK1/2 activation, may be implicated in the regulation of steroid production.

[Role of the fatty acids in ovarian functions: involvement of peroxisome proliferator-activated receptors (PPAR) and adipokines]. / Rôle des acides gras sur les fonctions ovariennes : implications des Peroxisome Proliferator-Activated Receptors (PPAR) et des adipocytokines.

The impact of nutrition and energy reserves on the reproductive functions is known for a very long time. However, the metabolic factors involved in the interactions between nutrition and reproduction are still poorly understood. These factors may be hormones or nutrients (glucose, protein and fatty acids). However, it remains to determine whether these factors act directly or indirectly on the reproductive tissues. In this issue, we briefly summarize the impact of fatty acids on the development of ovarian follicles, oocyte and embryo. We then discuss the current hypotheses about the mechanisms of action of these fatty acids on the ovarian functions. We describe more particularly the role of some receptors of fatty acids, Peroxisome Proliferator-Activated Receptors (PPAR) and Liver X Receptors (LXR) and two adipokines, leptin and adiponectin on ovarian cells.

The effect of AMP-activated kinase activation on gonadotrophin-releasing hormone secretion in GT1-7 cells and its potential role in hypothalamic regulation of the oestrous cyclicity in rats.

Hypothalamic AMP-activated kinase (AMPK) is a key regulator of food intake in mammals. Its role in reproduction at the central level and, more precisely, in gonadotrophin-releasing hormone (GnRH) release has never been investigated. We showed that each subunit of AMPK is present in immortalised GnRH neurones (GT1-7 cells). Treatment with 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR) and metformin, two activators of AMPK, increased dose-dependent and time-dependent phosphorylation of AMPKalpha atThr172 in GT1-7 cells. Phosphorylation of acetyl-coenzyme A carboxylase at ser79 also increased. Treatment with AICAR (5 mM) or metformin (5 mM) for 4 h inhibited GnRH release in the presence or absence of GnRH (10(-8) M). Specific AMPK inhibitor compound C completely eliminated the effects of AICAR or metformin on GnRH release. Finally, we determined the central effects of AICAR in vivo on food intake and oestrous cyclicity. Ten-week-old female rats received a 50 microg AICAR or a saline i.c.v. injection. We detected increased AMPK and acetyl-CoA carboxylase phosphorylation, specifically in the hypothalamus, 30 min after AICAR injection. Food intake was significantly higher (P < 0.05) in animals treated with AICAR than in animals injected with saline, 24 h after injection. This effect was abolished after 1 week. Moreover, during the 4 weeks following injection, the interval between two oestrous stages was significantly lower in the AICAR group than in the saline group. Our findings suggest that AMPK activation may act directly at the hypothalamic level to affect fertility by modulating GnRH release and oestrous cyclicity.