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Free access

Mélodie Diot, Maxime Reverchon, Christelle Ramé, Yannick Baumard, and Joëlle Dupont

In mammals, nicotinamide phosphoribosyltransferase (NAMPT) is an adipokine produced by adipose tissue that is found in intracellular and extracellular compartments. The intracellular form of NAMPT is a nicotinamide phosphoribosyltransferase, whereas the extracellular form is considered an adipokine. In humans, NAMPT regulates energy metabolism and reproductive functions, such as ovarian steroidogenesis. To date, no study has investigated the role of NAMPT in hen ovaries. We investigated whether NAMPT is present in hen ovarian follicles and its role in granulosa cells. Using RT-PCR, western blotting and immunocytochemistry, we detected mRNA transcripts and proteins related to NAMPT in theca and granulosa cells from pre-ovulatory follicles. Using RT-PCR, we demonstrated that mRNA NAMPT levels were higher in granulosa cells than they were in theca cells and that during follicle development, theca cell levels decreased, whereas levels remained unchanged in granulosa cells. NAMPT protein quantities were significantly higher in theca cells than they were in granulosa cells, but they were unchanged during follicular development. Plasma NAMPT levels, as determined by ELISA and immunoblotting, were significantly lower in adult hens than they were in juveniles. In vitro, treatment with human recombinant NAMPT (100 ng/ml, 48 h) halved basal and IGF1-induced progesterone secretion, and this was associated with a reduction in STAR and HSD3B protein levels and MAPK3/1 phosphorylation levels in granulosa cells. These effects were abolished by the addition of FK866, a specific inhibitor of NAMPT enzymatic activity. Moreover, NAMPT had no effect on granulosa cell proliferation. In conclusion, NAMPT is present in hen ovarian cells and inhibits progesterone production in granulosa cells.

Free access

Lucie Tosca, Christelle Ramé, Christine Chabrolle, Sophie Tesseraud, and Joëlle Dupont

Although its mechanism of action is still unclear, metformin is an anti-diabetic drug effective to restore cyclicity and spontaneous ovulation in women with polycystic ovary syndrome. It may also reduce the risk of cancer. We have recently shown that metformin treatment decreases steroidogenesis through AMP-activated kinase (AMPK) in granulosa cells of various species. Here, we investigated the effects and the molecular mechanisms of metformin in IGF1-induced proliferation and protein synthesis in cultured bovine granulosa cells. Treatment with metformin (10 mM) for 24 h reduced cell proliferation and the levels of cyclin D2 and E, and increased the associations cyclin D2/p21 and cyclin D2/p27 without affecting cell viability in response to IGF1 (10−8 M). It also decreased IGF1-induced protein synthesis and phosphorylation of P70S6 kinase and ribosomal S6 protein. Interestingly, metformin treatment for 1 h decreased MAPK3/1 (ERK1/2) and P90RSK phosphorylation without affecting AKT phosphorylation in response to IGF1. Adenovirus-mediated expression of dominant-negative AMPK totally abolished the effects of metformin on cell proliferation and phosphorylation of P70S6K in response to IGF1. It also eliminated the inhibitory effects of metformin on MAPK3/1 and P90RSK phosphorylation. Taken together, our results strongly suggest that metformin reduces cell growth, protein synthesis, MAPK3/1, and P90RSK phosphorylation in response to IGF1 through an AMPK-dependent mechanism in cultured bovine granulosa cells.

Free access

Virginie Maillard, Pascal Froment, Christelle Ramé, Svetlana Uzbekova, Sébastien Elis, and Joëlle Dupont

Resistin, initially identified in adipose tissue and macrophages, was implicated in insulin resistance. Recently, its mRNA was found in hypothalamo–pituitary axis and rat testis, leading us to hypothesize that resistin may be expressed in ovary. In this study, we determined in rats and cows 1) the characterization of resistin in ovary by RT-PCR, immunoblotting, and immunohistochemistry and 2) the effects of recombinant resistin (10, 100, 333, and 667 ng/ml)±IGF1 (76 ng/ml) on steroidogenesis, proliferation, and signaling pathways of granulosa cells (GC) measured by enzyme immunoassay, [3H]thymidine incorporation, and immunoblotting respectively. We observed that resistin mRNA and protein were present in several bovine and rat ovarian cells. Nevertheless, only bovine GC abundantly expressed resistin mRNA and protein. Resistin treatment decreased basal but not IGF1-induced progesterone (P<0.05; whatever the dose) and estradiol (P<0.005; for 10 and 333 ng/ml) production by bovine GC. In rats, resistin (10 ng/ml) increased basal and IGF1-induced progesterone secretion (P<0.0001), without effect on estradiol release. We found no effect of resistin on rat GC proliferation. Conversely, in cows, resistin increased basal proliferation (P<0.0001; for 100–667 ng/ml) and decreased IGF1-induced proliferation of GC (P<0.0001; for 10–333 ng/ml) associated with a decrease in cyclin D2 protein level (P<0.0001). Finally, resistin stimulated AKT and p38-MAPK phosphorylation in both species, ERK1/2-MAPK phosphorylation in rats and had the opposite effect on the AMPK pathway (P<0.05). In conclusion, our results show that resistin is expressed in rat and bovine ovaries. Furthermore, it can modulate GC functions in basal state or in response to IGF1 in vitro.

Free access

Claude Fabre-Nys, Audrey Chanvallon, Nathalie Debus, Dominique François, Frédéric Bouvier, Joelle Dupont, Lionel Lardic, Didier Lomet, Christelle Ramé, and Rex J Scaramuzzi

The proportion of anoestrous ewes ovulating after exposure to a sexually active ram is variable mainly due to whether an LH surge is induced. The aim of this study was to determine the role of oestradiol (E2) in the ram-induced LH surge. In one study, we measured the plasma concentrations of E2 in ewes of different breeds before and after the ‘ram effect’ and related these patterns to the presence and latency of the LH surge, while another compared ovarian responses with the ‘ram effect’ following exposure to rams for 2 or 12 h. In all ewes, the concentration of E2 increased 2–4 h after rams were introduced and remained elevated for 14.5±0.86 h. The quantity of E2 secreted before the LH surge varied among breeds as did the mean concentration of E2. The granulosa cells of IF ewes collected after 12 h exposure to rams secreted more E2 and progesterone and had higher levels of StAR than the 2 h group but in MV ewes there was no differences between these groups for any of these parameters. These results demonstrate that the LH surge induced by the rams is a result of increased E2 secretion associated with increased levels of STAR in granulosa cells and that these responses varied among breeds. The results suggest that the variable occurrence of a LH surge and ovulation may be the result of variable ovarian responses to the ‘ram effect’ and insensitivity of the hypothalamus to the E2-positive feedback signal.

Free French abstract: A French translation of this abstract is freely available at http://www.reproduction-online.org/content/149/5/511/suppl/DC1.

Free access

Namya Mellouk, Christelle Ramé, Joël Delaveau, Christophe Rat, Maxime Marchand, Frédéric Mercerand, Angélique Travel, Aurélien Brionne, Pascal Chartrin, Linlin Ma, Pascal Froment, and Joëlle Dupont

Overfed hens selected for their rapid growth become fatter and develop reproductive disorders. Herein, we aimed to demonstrate that food restriction leading to a weight reduction and/or a supplementation with fish oil may be effective in preventing reproductive disorders through the regulation of adipokine expression in broiler hens. This study included four groups of food restricted (Rt) or ad libitum hens (Ad, feeding at a rate 1.7 times greater than Rt hens) supplemented or unsupplemented with fish oil (1%). The Rt diet significantly increased plasma chemerin (RARRES2) levels during the laying period, delayed sexual maturity by one week and improved egg quality and fertility. These effects were associated with higher progesterone production in response to IGF1 (or LH) in cultured granulosa cells and in vivo egg yolk, as compared with Ad hens. Fish oil supplementation had similar effects to the Rt diet on progesterone (P < 0.05), but without any effect on fertility. Using RT-PCR, we found that RARRES2 levels were lower in theca cells of Rt hens and NAMPT levels were increased by the fish oil supplementation. A significant positive correlation between RARRES2 expression in granulosa cells and the weight of F1 preovulatory follicle was observed, as well as a negative correlation of plasma RARRES2 levels with hatchability. Thus, food restriction but not fish oil supplementation improved fertility, and this was associated with variations in RARRES2 plasma and ovarian expression in hens.

Open access

Gonçalo Pereira, Ricardo Bexiga, João Chagas e Silva, Elisabete Silva, Christelle Ramé, Joëlle Dupont, Yongzhi Guo, Patrice Humblot, and Luís Lopes-da-Costa

Adipokines emerged as regulators of metabolism and inflammation in several scenarios. This study evaluated the relationship between adipokines (adiponectin, chemerin and visfatin) and cytological (subclinical) endometritis, by comparing healthy (without), transient (recovered by 45 days postpartum (DPP)) and persistent (until 45 DPP) endometritis cows (n = 49). Cows with persistent endometritis had higher adiponectin concentrations in plasma (at 21 DPP, P < 0.05 and at 45 DPP, P < 0.01) and in uterine fluid (at 45 DPP, P < 0.001), and higher chemerin concentrations in plasma (P < 0.05) and uterine fluid (P < 0.01) at 45 DPP than healthy cows. Cows with persistent endometritis had higher gene transcription in the cellular pellet of uterine fluid and protein expression in the endometrium of these adipokines and their receptors than healthy cows. Adiponectin plasma concentrations allowed to discriminate healthy from persistent endometritis cows, in 87% (21 DPP) and 98% (45 DPP) of cases, and adiponectin and chemerin uterine fluid concentrations at 45 DPP allowed for this discrimination in 100% of cases. Cows with concentrations above the cutoff were a minimum of 3.5 (plasma 21 DPP), 20.4 (plasma 45 DPP), and 33.3 (uterine fluid 45 DPP) times more at risk of evidencing persistent endometritis at 45 DPP than cows with concentrations below the cutoff. Overall, results indicate a relationship between adipokine signalling and the inflammatory status of the postpartum uterus of dairy cows, evidencing that adipokines represent suitable biomarkers of subclinical endometritis, able to predict the risk of persistence of inflammation.