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. 2024 Oct 10;103(12):104398. doi: 10.1016/j.psj.2024.104398

Chicken chemerin alone or in mixture with adiponectin-visfatin impairs progesterone secretion by primary hen granulosa cells

Ophélie Bernardi *,†, Mathias Fréville †, Christelle Ramé †, Maxime Reverchon *, Joëlle Dupont †,1
PMCID: PMC11539439  PMID: 39447332

Abstract

Adipokines including adiponectin (ADIPO), chemerin (CHEM) and visfatin (VISF) are involved in metabolism and reproductive functions. These 3 adipokines are present in ovarian cells in different preovulatory follicles in hens. We have previously shown that VISF and ADIPO are able to modulate in vitro steroid production by hen granulosa cells (GCs). It is, however, unclear whether CHEM acts on hen ovarian cells. In addition, no study has yet investigated the effect of a mixture of several adipokines such ADIPO, VISF, and CHEM on GCs from different preovulatory follicles. In this study, we investigated the effect of CHEM alone and in combination with ADIPO and VISF on cell viability, proliferation and progesterone secretion in cultured granulosa cells (GCs) from the largest follicles F1 and smaller ones (F3/F4) in the presence of gonadotropins (oLH and oFSH) or hIGF-1. First, various concentrations of chemerin were examined (0, 12, 25, 50, and 100 ng/mL) and then we determined the response to CHEM (at 25 ng/mL) in combination with ADIPO (10 µg/mL) and VISF (100 ng/mL). Chemerin exposure did not affect F1 and F3/F4 granulosa cell viability and proliferation whatever the concentation and in the presence of the mixture. However, it reduced progesterone secretion in dose dependent manner in both F1 and F3/F4 follicles. Furthermore, this CHEM inhibitory effect was significantly higher when CHEM was combined with ADIPO and VISF. Furthermore, CHEM reduced significantly oLH and oFSH- induced progesterone secretion in F1 GCs and oFSH and hIGF-1-induced progesterone secretion in F3/F4 GCs. Interestingly, this inhibitory effect of CHEM was similar in F1 GCs when CHEM was in mixture with ADIPO and VISF whereas it was significantly higher in F3/F4 GCs. Taken together, CHEM impairs progesterone secretion in cultured hen GCs and this inhibitory effect can be potentiated when it is in combination with other adipokines.

Key words: adipokine, ovarian cell, hen, cell culture, steroid

INTRODUCTION

Whether in mammals or birds, the nutrition modulates the body weight and composition and the level of plasma metabolites and metabolic hormones. Consequently, it regulates the female reproductive functions (folliculogenesis, ovulation and fertilization) due to the close inter relationships between metabolism and reproduction (Etches et al., 1983; Sirotkin and Grossmann, 2015). Indeed, the synchronisation of female reproductive functions is also dependent on nutritional status in birds (Mellouk et al., 2018a). Several metabolic hormones are known to be associated to the body weight variation and consequently to changes of the ovarian physiology in chicken (Hocking, 2004). Among the metabolic hormones already discussed in our previous review (Bernardi et al., 2021), we focus here on adiponectin (ADIPO), visfatin (VISF), and chemerin (CHEM) (Estienne et al., 2019; Bernardi et al., 2021). Indeed, their plasma concentrations are regulated in response to abdominal fat (Cai et al., 2021; Mellouk et al., 2018a) or muscle rate (Krzysik-Walker et al., 2008). Their plasma regulations could be associated to variations of plasma sexual steroids because ADIPO and CHEM receptors are expressed in ovarian cells (Bernardi et al., 2021) and in vitro studies showed that ADIPO and VISF modulates granulosa cell progesterone secretion (Chabrolle et al., 2007; Diot et al., 2015a).

ADIPO is the most adipokine abundant in adipose tissues of various animal species (human, pigs, rats, cows, and birds) with an important role in lipid and carbohydrate metabolism, insulin sensitivity and regulation of body weight (Kadowaki and Yamauchi, 2005; Barbe et al., 2019). Plasma VISF and CHEM are known to be associated with metabolic and immune disorders including obesity (Mattern et al., 2014; Carbone et al., 2017). Theses adipokines have been also characterized in reproductive axis in several animal species including birds (Barbe et al., 2019; Estienne et al., 2019; Bernardi et al., 2021). In human, ADIPO is absent in granulosa cells (GCs) while its receptors are present (Chabrolle et al., 2007, 2009). Visfatin is present in human ovarian follicles notably in GCs with a beneficial role in oestradiol and progesterone secretion induced by IGF-1 (Reverchon et al., 2014b). Thus, a supplementation of VISF during superovulation improves oocyte quality in aged female mice (Choi et al., 2012). Chemerin and its receptors have been also characterized in ovarian cells in several mammalian species. It reduces progesterone and oestradiol secretions in human and bovine GCs (Reverchon et al., 2012, 2014a).

Data about adipokines in ovary are also available in avian species. The mature hen ovary contains maturing preovulatory follicles showing a hierarchy according to their size (F5/6–F1) (Estienne et al., 2020). The largest follicle (F1), filled of yolk is first to ovulate, followed by the second largest follicle (F2) approximately 24 to 26 h later and this continues down the hierarchy. Granulosa cells are a main component of the hierarchical follicles that are very close to yolk and the whole is surrounded by theca cells layers (Estienne et al., 2020). Follicular growth and atresia depend on GCs development and apoptosis (Johnson, 2014). Thus, avian GCs from F1 follicles produce large amounts of progesterone but, unlike mammalian granulosa cells, no estrogen (Huang et al., 1979; Marrone and Hertelendy, 1983; Dinh et al., 2019). In birds, adipokines and their receptors are differentially expressed in ovarian follicle cells. Adiponectin is mainly expressed and secreted by theca cells (Chabrolle et al., 2007; Hadley et al., 2020). Adiponectin receptors have different locations with a lower expression in GCs for AdipoR1 while levels of AdipoR2 were unchanged between ovarian cells (Ramachandran et al., 2013). Moreover, ADIPO increased IGF-1 induced progesterone secretion in F2 and F3/F4 follicles while it halved progesterone secretion in response to FSH and LH in F3/F4 follicles (Maddineni et al., 2005; Chabrolle et al., 2007). Visfatin and CHEM are also present in ovarian cortex in particular in theca cells compared to GCs from hierchical follicles in hens (Ons et al., 2010; Diot et al., 2015a). However, Diot et al reported that VISF inhibits IGF-1 induced progesterone secretion in hen GCs in contrast to mammals (Diot et al., 2015a). In contrary to mammals, VISF is more characterized as a myokine due to a higher expression in skeletal muscle than adipose tissue (Krzysik-Walker et al., 2008; Mellouk et al., 2018c). However, to our knowledge, the effects of CHEM alone or in combination with other adipokines have not yet been investigated in chicken GCs.

Thus, the aims of the present study were to assess the impact of CHEM alone and in combination with ADIPO and VISF on cell viability, proliferation and progesterone secretion in cultured hen GCs from the largest follicles (F1) and smaller one (F3/F4).

MATERIAL AND METHODS

Animals and Ethical Issues

Fifty broiler breeders 26 weeks-old of the commercial ROSS 308 breed were reared at “Pôle Expérimental Avicole de Tours” (INRAe, Nouzilly, France DOI: 10.15454/1.5572326250887292E12) according to the standard breeding conditions. Ovarian follicles were collected during meat processing as local abattoir by-products by highly qualified and experienced laboratory staff. According to the ethical issues for the protection of animals, this project does not require the consent of the competent ethics committee for animal experiments. All experiments were performed in accordance with the European Communities Council Directive 2010/63/UE.

Isolation of Hen Granulosa Cells

We performed 4 independent trials and in each trial we collected 12 or 14 hens. After electronarcosis, the ovaries were immediately removed and placed in ice-cold sterile 1% NaCl saline solution for immediate use. Granulosa cells (GCs) from preovulatory follicles 1 (F1), and from preovulatory follicles 3 and 4 (F3/F4) were dissected as previously described (Tosca et al., 2006; Chabrolle et al., 2007). In order to obtain cells, GCs from F3 and F4 follicles were pooled into one single group, called F3/F4 group. Then, cells were prepared for culture by being dispersed in 0.3% collagenase type A (Roche Diagnostic, Meylan, France) in F12 medium containing 5% fetal bovine serum (FBS), at 37°C. Cells were pelleted by centrifugation (10 min, 800 g at room temperature), washed twice with fresh medium and counted in a haemocytometer. The viability of F1 and F3/F4 GCs was estimated by trypan blue exclusion. Cells were cultured in a medium composed of DMEM supplemented with 100 U/mL penicillin, 100 mg/L streptomycin, 3 mmol/L L-glutamine and 5% FBS. Cells were initially cultured for 24 h with no treatment. After overnight serum deprivation, granulosa cells were stimulated in fresh culture medium with or without test reagents for the appropriate time. All of the cultures were maintained in a water-saturated atmosphere of 95% air, 5% CO2 at 37 °C (Chabrolle et al., 2007; Fréville et al., 2024).

Hormones and Reagents

Granulosa cells from F1 and F3/F4 follicles were stimulated with CHEM (0, 12, 25, 50 and 100 ng/mL) or ADIPO (10 μg/mL) (Chabrolle et al., 2007) or VISF (100 ng/mL) (Diot et al., 2015a) or a mixture of these 3 adipokines associated or not with hIGF-1 (10−8 M) or oLH (10−8 M) or oFSH (10−8 M) for 24 h. Human recombinant IGF-1 was obtained from Sigma Aldrich (Saint Quentin Fallavier, France). Purified ovine luteinizing hormone LH (lot 26) and ovine follicle stimulating hormone FSH-20 (lot no.AFP-70228D) were obtained from Dr. A.F. Parlow and the National Hormone and Pituitary Program (Bethesda, MD). Indeed, purified chicken LH and FSH were not available. Human recombinant ADIPO full-length and human recombinant VISF were obtained from R&D systems (Lille, France). The chicken recombinant CHEM protein (full length, rRARRES2) was obtained from the Gallus gallus sequence (NM_001277476.1), produced in Escherichia coli and purified by a chromatography column-based on His-Tag under denaturing conditions (Agro-Bio, La Ferté Saint Aubin, France) (Bernardi et al., 2022, 2024b).

Cell Viability

Granulosa cells from F1 and F3/F4 follicles were treated in 96 wells plates for 24 h with different treatments as described previously. Cell viability was determined using a Cell Counting Kit-8 (CCK8) assay commercial kit (Sigma–Aldrich, Saint Quentin Fallavier, France). The assay was performed according to the manufacturer recommendations. Absorbances were measured at a 450 nm wavelength using a spectrophotometer.

Cell Proliferation

Granulosa cells from F1 and F3/F4 follicles were treated in 96 wells plates for 24 h with several treatments as described previously. Cell proliferation was determined using a BrdU (Bromodeoxyuridine) ELISA assay (Sigma-Aldrich, Saint Quentin Fallavier). Absorbances were measured at a 405 and 492 nm wavelength using a spectrophotometer. The assay was performed according to the manufacturer recommendations.

Measurement of Progesterone Secretion by Granulosa Cells

Progesterone (P4) concentrations in GCs from F1 and F3/F4 follicles were determined in serum-free media after culturing for 24 h in the absence (control group, CT) or in the presence of different concentrations of CHEM (0, 12, 25, 50, and 100 ng/mL) or ADIPO (10 μg/mL) or VISF (100 ng/mL) or a mix of these 3 adipokines with or without hIGF-1 (10−8 M) or oLH (10−8 M) or oFSH (10−8 M) for 24 h. The concentration of progesterone derived from hen granulosa cells, in the culture media was measured using an ELISA protocol as previously described (Canepa et al., 2008). The intra- and inter-assay coefficients of variation (CV) averaged <10 % and <4,3 %; respectively. The results are expressed as the amount (mean ± SEM) of steroid (ng/mL)/ 100 µg of total protein secreted per 24h.

Statistic Analysis

The GraphPad Prism software (version 6) was used for all analyses. For each experiment, 4 biological replicates were created for each concentration (n = 4) and the assay was performed 4 times to obtain 4 statistical replicates (N = 4). All data are represented as means ± standard error of mean (SEM) with a level of significance less or equal than 0.05 (p ≤ 0.05). One-way ANOVA followed by Dunn pair-wise comparison tests was performed to detect statistical differences between control and different concentrations of adipokines in the presence or absence of gonadotropins oLH and/or hIGF-1 and/or oFSH. Different asterisks indicate significant differences *p < 0.05, **p < 0.01, ***, p < 0.001, ****p < 0.0001 given by statistic tests.

RESULTS

Effect of Different Concentrations of Chicken CHEM on Cell Viability, Proliferation and Progesterone Secretion in Hen GCs From F1 and F3/F4 Follicles

Chicken CHEM treatment for 24 h did not affect cell viability (Figures 1A and 1B) (p = 0.1717 and p = 0.5293; respectively) and proliferation (Figures 1C and 1D) (p = 0.1231 and p = 0.1851; respectively) whatever the concentrations used (0–100 ng/mL) in preovulatory F1 (Figures 1A and 1C) and F3/F4 GCs (Figures 1B and 1D).

Figure 1.

Figure 1

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Viability (A and B) and proliferative (C and D) granulosa cells from F1 and F3/F4 follicles after chemerin exposure. Granulosa cells from hen F1 and F3/F4 follicles were cultured 24 h with growing doses of chemerin ranging from 0 ng/mL (control condition CT) to 100 ng/mL. Data are shown as the mean ± SEM with a level of significance less or equal than 0.05 (p ≤ 0.05).

We next determined whether the effect of chicken CHEM treatment on progesterone (P4) secretion by F1 and F3/F4 GCs. In both F1 and F3/F4 GCs, CHEM exposure reduced in a dose-dependant manner progesterone secretion from 12 to 100 ng/mL as compared to control condition (without CHEM supplementation, p < 0.0001 and p < 0.0001; respectively) (Figures 2A and 2B). Also, we confirmed that the amount of progesterone secreted by F1 GCs was significantly higher than those by F3/F4 GCs (19.04 ± 0.13 ng/mL vs. 9.06 ± 0.25 ng/mL, p < 0.0001) (Figure 2A and 2B) as several previous studies (Tosca et al., 2008; Rivas et al., 2016).

Figure 2.

Figure 2

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Progesterone secretion in culture medium by granulosa cells from F1 (A) and F3/F4 follicles (B) after chemerin exposure. Granulosa cells from hen F1 and F3/F4 follicles were cultured 24 h with growing doses of chemerin ranging from 0 ng/mL (control condition CT) to 100 ng/mL. Data are shown as the mean ± SEM with a level of significance less or equal than 0.05 (p ≤ 0.05). Different asterisks indicate significant differences as compared to the control CT at **** p < 0.0001.

Effect of chicken CHEM and the adipokines mixture on cell viability, proliferation and progesterone secretion in hen GCs from F1 and F3/F4 follicles in basal condition (without gonadotropin or hIGF-1)

We next investigated the effect of chicken CHEM at 25 ng/mL, a concentration found in the egg white during egg formation after follicle ovulation (Bernardi et al., 2024b), ADIPO (10 μg/mL) (Chabrolle et al., 2007) or VISF (100 ng/mL) (Diot et al., 2015a) or a mixture of these 3 adipokines.

In both F1 and F3/F4 GCs, any adipokines or mix exposure for 24 h did not affect cell viability as compared to control condition (p = 0.4666 and p = 0.5769; respectively) (Figures 3A and 3B). Similar data were obtained after supplementation of adipokines individually or in combination on F1 and F3/F4 GCs proliferation as compared to the control (p = 0.2101 and p = 0.4866; respectively) (Figures 3C and 3D).

Figure 3.

Figure 3:

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Viability (A and B) and proliferative (C and D) granulosa cells from F1 and F3/F4 follicles after adipokines exposure. Granulosa cells from hen F1 and F3/F4 follicles were cultured 24 h with different supplementation of chemerin CHEM (25 ng/mL), adiponectin ADIPO (10 µg/mL), visfatin VISF (100 ng/mL) or a mix of these 3 adipokines. Data are shown as the mean ± SEM with a level of significance less or equal than 0.05 (p ≤ 0.05).

In both F1 and F3/F4 GCs, progesterone secretion was significantly decreased after the addition of 25 ng/mL of CHEM, 100 ng/mL of VISF or a mixture of adipokines compared to the control condition CT (p < 0.0001 and p < 0.0001; respectively) (Figures 4A and 4B). As expected as (Chabrolle et al., 2007), ADIPO exposure (10 μg/mL) for 24 h did not affect the secretion of progesterone concentration as compared to control condition (Figures 4A and 4B). Interestingly, the progesterone release was significantly more decreased in response to the adipokines mixture than in the presence of CHEM alone in both F1 and F3/F4 GCs (p < 0.0001 and p < 0.0001; respectively) (Figures 4C and 4D).

Figure 4.

Figure 4:

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Progesterone secretion in culture medium by granulosa cells from F1 and F3/F4 follicles after adipokines exposure. Granulosa cells from hen F1 and F3/F4 follicles were cultured 24 h with different supplementation of chemerin CHEM (25 ng/mL), adiponectin ADIPO (10 µg/mL), visfatin VISF (100 ng/mL) or a mix of these 3 adipokines compared to control condition (A-B) and to CHEM addition (C-D). Data are shown as the mean ± SEM with a level of significance less or equal than 0.05 (p ≤ 0.05). Different asterisks indicate significant differences as compared to the control CT (A and B) or to CHEM condition (C and D) at **** p < 0.0001.

Effect of Chicken CHEM and the Adipokines Mixture on Progesterone Secretion in Hen GCs From F1 and F3/F4 Follicles in the Presence or Absence Gonadotropins or hIGF-1

We next examined the effect of chicken CHEM at (25 ng/mL), ADIPO (10 μg/mL) or VISF (100 ng/mL) or a mixture of these 3 adipokines on progesterone secretion in the presence or absence of oLH (10−8 M) or oFSH (10−8 M) for F1 GCs (Figure 5) and oFSH (10−8 M) or hIGF-1 (10−8 M) for F3/F4 GCs (Figure 6) for 24 h.

Figure 5.

Figure 5

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Progesterone secretion in culture medium by granulosa cells from F1 follicles after adipokines exposure with or without gonadotropins. Granulosa cells from hen F1 follicles were cultured 24 h with different supplementation of chemerin CHEM (25 ng/mL) (A), a mix of adipokines MIX (B), visfatin VISF (100 ng/mL) (C) or adiponectin ADIPO (10 µg/mL) (D) with or without LH (10−8 M) and FSH (10−8 M). Data are shown as the mean ± SEM with a level of significance less or equal than 0.05 (p ≤ 0.05). Asterisks indicate significant differences as compared to the control condition at * p < 0.05, ** p < 0.01, ***p < 0.001 and **** p < 0.0001. Comparaison of progesterone level in granulosa cells from F1 follicles between CHEM and MIX supplementation in response to FSH, LH and both together (E). Data are shown as the mean ± SEM with different letters indicating significant differences at p ≤ 0.05 between treatments.

Figure 6.

Figure 6

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Progesterone secretion in culture medium by granulosa cells from F3/F4 follicles after adipokines exposure with or without FSH or IGF-1. Granulosa cells from hen developing F3/F4 follicles were cultured 24 h with different supplementation of chemerin CHEM (25 ng/mL) (A), a mix of adipokines MIX (B), visfatin VISF (100 ng/mL) (C) or adiponectin ADIPO (10 µg/mL) (D) with or without IGF-1 (10−8 M) and FSH (10−8 M). Data are shown as the mean ± SEM with a level of significance less or equal than 0.05 (p ≤ 0.05). Asterisks indicate significant differences as compared to the control condition at * p < 0.05 and **** p < 0.0001. Comparaison of progesterone level in granulosa cells from F3/F4 follicles between CHEM and MIX supplementation in response to FSH, IGF-1 and both together (E). Data are shown as the mean ± SEM with different letters indicating significant differences at p ≤ 0.05 between treatments.

As previously shown in Figure 2, in basal conditions for both follicles F1 and F3/F4 GCs, we showed a significant decrease of progesterone secretion after the addition of chicken CHEM (Figure 5A and Figure 6A), mix of 3 adipokines (Figure 5B and Figure 6B) and VISF (Figure 5C and Figure 6C) (p < 0.0001). The supplementation of ADIPO did not change the progesterone secretion (p = 0.6421) (Figure 5D and Figure 6D).

In F1 GCs in response to oFSH or/and oLH, CHEM and the adipokines mixture supplementation significantly reduced progesterone secretion (p < 0.0001) (Figure 5A and 5B). However, ADIPO and VISF addition did not alter the secretion of progesterone in response too FSH or/and oLH (Figure 5C and 5D). Furthermore, we observed that the decrease of progesterone secretion in response to CHEM or the adipokine mixture in response to different gonadotropins was similar but significantly different to each gonadotropin (Figure 5E).

In F3/F4 GCs in presence or absence of oFSH and hIGF-1 supplementations, we also showed a significantly decrease in progesterone secretion after the supplementation of CHEM and the adipokines mixture as compared to the control condition (p < 0.0001) (Figures 6A and 6B). However, we observed a slight but significant decrease in progesterone secretion after addition of VISF only in response to oFSH (Figure 6C). We can also observe that the significant decrease of progesterone secretion in case of adiponectin supplementation in response to oFSH, hIGF-1 and both together (p < 0.0001) (Figure 6D). Interestingly, we have also shown that the inhibition of progesterone secretion in response to the adipokines mixture was significantly higher than those observed in presence to CHEM and gonadotropins alone (Figure 6E).

DISCUSSION

The present study provides the first evidence that chicken CHEM decreases in vitro progesterone secretion in basal state and in response to gonadotropins (LH and FSH) by F1 GCs and in response to oFSH and hIGF-1 in F3/F4 GCs. Furthermore, all these data were obtained without any effect on cell viability and proliferation. Interestingly, we showed that the decrease in progesterone secretion was significantly higher when CHEM was combined with VISF and ADIPO than it was incubated alone in F3/F4 GCs. However, this effet was not observed in F1 GCs.

In avian species, a mature ovary includes follicles of various size and stage of development. There are numerous pre-hierarchical follicles and the pre-ovulatory yellow follicles which are organized in a characteristic hierarchy (typically the largest 5 to 6 follicles). The pre-ovulatory follicle structure from the center to the periphery consists of a single yolk-filled oocyte, perivitelline cells, GCs, basement membrane, and theca cells (Schmierer et al., 2003). The growth, development, and function of follicles determine the egg-laying performance of chickens (Li et al., 2019). The proliferation and differentiation of GCs play an important role in follicle maturation. Considerable data exist about the molecular mechanisms involved in the process of gonadotropins-induced steroidogenesis in differentiated GCs (Calvo and Bahr, 1983; Johnson and Bridgham, 2001). However, much less is known in birds about additional factors such metabolic hormones that are crucial in the reproductive function in hen and particularly in broiler breeders (Eitan et al., 2014). Adipokines, notably ADIPO, VISF and CHEM, are one of the main families of hormones involved in the regulation of metabolic and reproductive functions in birds (Bernardi et al., 2021). Our laboratory has recently investigated the presence of CHEM in egg white and its role in the embryo development in broiler and layer hen and duck (Bernardi et al., 2022, 2023, 2024b, 2024a; Estienne et al., 2022).

In mammals, CHEM and its receptors are expressed in ovarian cells, including granulosa and theca cells, corpus luteum, and oocytes (Reverchon et al., 2012, 2014a). In avian species, they are found in the ovary of chicken (Mellouk et al., 2018b) and turkey, where their expression are higher in theca cells than granulosa cells (Diot et al., 2015b). Furthermore, a positive correlation between CHEM expression in GCs and the weight of F1 preovulatory follicle has also been highlighted (Mellouk et al., 2018b) suggesting a role of CHEM in the regulation of steroids production. However, the role of CHEM in basal state or in reponse to gonadotropins or hIGF-1 has never been reported in avian GCs from preovulatory follicles. In the present study, we have shown that chicken recombinant chemerin reduced in a dose dependent progesterone secretion in basal state and in response to oLH or oFSH in F1 GCs and in the presence of hIGF-1 and oFSH in F3 GCs without alteration of GCs viability and proliferation. Concerning the progesterone secretion, our results are in good agreement with data obtained in mammalian GCs. Indeed, in bovine granulosa cells, CHEM through CMKLR1 reduces steroidogenesis (progesterone and oestradiol secretion) in basal state and in response to FSH and IGF-1 (Reverchon et al., 2014a). In chicken, the involvement of CHEM receptors in the inhibitory effect of CHEM in the progesterone secretion remains to be determined. In mice, Wang et al., 2012 also observed that CHEM is a negative regulator of FSH-induced follicular steroidogenesis (Wang et al., 2012). However, in human GCs, the CHEM negative effect was present only in response to IGF-1 and not FSH or in basal state (Reverchon et al., 2012) and in porcine ovarian cells the CHEM effects were variable according to the different phases of the estrous cycle and the pregnancy (Rytelewska et al., 2021). Moreover, in human, CHEM through CMKLR1 could be involved in reproduction disorders like PolyCystic Ovary Syndrome (PCOS) (Tang et al., 2016; Estienne et al., 2021). Thus, the CHEM effect in GCs depends not only on the animal species but also on the physiological status.

Interestingly, we showed for the first time that not only CHEM alone but also a mixture of CHEM, ADIPO and VISF was able to reduce progesterone secretion in chicken GCs. Furthermore, if this negative effect of the mixture was similar to CHEM alone in F1 GCs, it was significantly higher in F3/F4 GCs suggesting additive effects of CHEM and ADIPO or/and VISF. In the present study we showed that in F1 GCs, ADIPO alone has no effect on progesterone secretion whereas VISF exerts only a significant negative effect in basal state (without gonadotropin addition). These results are in good agreement with our previous data for ADIPO (Chabrolle et al., 2007) and VISF (Diot et al., 2015a). In F3/F4 GCs, we observed that ADIPO alone reduced progesterone secretion not in basal state but in response to oFSH and/or hIGF-1 and VISF decreased progesterone release in basal state and in response to oFSH but not hIGF-1. Chabrolle et al., 2007 showed that ADIPO had no effect in basal state but increased IGF-1 induced progesterone secretion and reduced FSH-induced progesterone and Diot et al., 2015a observed that VISF decreased progesterone release in basal state and in response to FSH but not IGF-1. Thus, concerning the individual response of ADIPO and VISF on the F3/F4 GCs progesterone secretion, the different effects observed as compared to our previous studies could be explained by the breed of animals. Indeed, Diot et al., 2015a and Chabrolle et al., 2007 used ovarian follicles from laying breed hens whereas they were broiler hens in the present study. Furthermore, here the animals were younger than the other studies (26 weeks-old vs 52 and 70/80-weeks-old). In previous studies, we also found different expressions of adipokines and their receptors in the reproductive tract between laying and broiler animals (Bernardi et al., 2024b, 2024d, 2024c). Consequently, it could be interested to investigate effects of adipokines treatments in follicles from high and low performances hens. Moreover, progesterone secretion is regulated by steroidogenesis enzymes like StAR (Steroidogenic acute regulatory protein), P450scc (Cytochrome P450 side-chain cleavage) and 3β-HSD (3β-hydroxysteroid dehydrogenase) (Yamazaki et al., 2005). However, it remains to determine what are the molecular mechanism and signalling pathways involved after an exposure of CHEM alone and adipokines mix. It is already known that visfatin treatment reduced MAPK3/1 (Mitogen-activated protein kinase 3 and 1) phosphorylation and inhibited IGF-1 induced MAPK3/1 phosphorylation in chicken F1 GCs (Diot et al., 2015a). Futhermone, a treatment of ADIPO on F1 GCs elicited AMPK (AMP-activated protein kinase) phosphorylation (Chabrolle et al., 2007). To go further, since CHEM, ADIPO and VISF are already present in chicken granulosa cells, it would be interested to create a knockout cell line for each adipokine in order to investigate the effect after adipokines supplementation.

CONCLUSIONS

In conclusion, our study shows for the first time that CHEM alone reduces progesterone secretion without any alteration of cell viability and proliferation in GCs from hen preovulatory follicles. In addition, this effect was dependent on the differentiation status of the follicle (F1 vs F3/F4). Furthermore, it was significantly higher when CHEM was combined with ADIPO and VISF at concentration observed in the blood circulation. However, all in vitro results may be in contrast to in vivo suggesting to perform in vivo experiments. Taken together, these data suggest that adipokines play a role in chicken folliculogenesis and it is important to in vitro investigate not only the effect of one adipokine but a mixture of adipokines. However, it remains to better understand the molecular mechanisms involved in the inhibitory effect of CHEM alone and in mixture with other adipokines in the regulation of steroidogenesis in hen granulosa cells.

DISCLOSURES

The authors declare no conflicts of interest.

ACKNOWLEDGMENTS

The authors thank the ARNT for the Ophelie Bernardi's financial support. The authors are grateful to all the persons from the experimental unit (INRAE, PEAT, Centre Val de Loire DOI: 10.15454/1.5572326250887292E12) who take care of animals.

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