In dairy cows, lipid metabolism is crucial to maintain proper reproductive function, which is affected by negative energy balance (NEB) occurring during the early postpartum period, due to the elevated mobilization of fat reserves to liberate the energy that is required for milk production [1
]. Oocyte competence or capacity to support embryo development after fertilization is one of the key factors affected by misbalanced fatty acid (FA) metabolism during the NEB period [3
]. Indeed, high concentrations of free FAs in plasma and follicular fluid (FF) during NEB lead to lipotoxicity within the ovarian follicle and impair oocyte quality, thus reducing cow fertility [4
]. The impaired lipid composition of FF, which is particularly rich in saturated free FA (C18:0 stearic and C16:0 palmitic acids) in lactating cows, may explain their lower fertility as compared to heifers [5
], as well as the seasonal variations of oocyte developmental competence in dairy cows [6
Oocytes develop and acquire their developmental competence in the ovarian follicle through tight bidirectional communication with follicular somatic cells, with each compartment secreting specific regulation factors [7
]. Ovarian morphological and functional organization supports follicular development and oogenesis, protecting the growing oocyte. Inside the pre-antral follicle, epithelial-like granulosa cells (GC) and mesenchymal-like theca cells (TH) face each other across a basement membrane; these mural cells enclose the oocyte, which grows through folliculogenesis. TH cell layers are transpierced by blood microvessels, and since antrum appearance, the follicle is filled with a fluid that is rich in proteins, steroids, and lipids, coming from the blood and secretory activity of follicular somatic cells—TH, GC, and cumulus cells (CC), too [11
]. CC are the differentiated GC, which are tightly connected and metabolically coupled with an oocyte via gap junctions [13
] and form the oocyte cumulus complex (OCC). The interaction between TH and GC is essential for the maintenance of the original architecture and functions of ovarian cells, especially for the synthesis of steroids, which regulates reproductive function, in response to the follicle stimulation hormone (FSH) and luteinizing hormone (LH). The closest follicular environment of the oocyte, including FF, GC, and especially CC, has a crucial impact on the acquisition of oocyte developmental competence [12
] and possesses molecular factors that are predictive of oocyte developmental potential [17
Energy metabolism significantly varies between the different types of ovarian follicular cells, especially in oocytes and CC, notably relying on different energy sources, including amino acids, pyruvates over glucose, and fatty acids [20
]. To produce energy, oocytes use lipid reserves, besides the glucose derivate supplied by CC [22
Free FAs are the major blocks for the synthesis of glycerolipids, such as triacylglycerids (TG), which are mainly used for intracellular energy storage, forming together with cholesterol esters (CE) intracellular lipid droplets (LDs) as reservoirs of caloric reserves. LDs are also caches of FAs and sterol components that are needed for the biogenesis of the membranes [23
]. The cholesterol that is used by ovarian cells is derived from either the cellular uptake of plasma lipoproteins, which contain cholesterol, or de novo synthesis [24
]. Free FAs and cholesterol form the structural basis for the synthesis of paracrine hormones such as prostaglandins and steroids, which play crucial roles in female reproduction, with particular involvement in the ovulation process [25
] and the pre-ovulatory maturation of OCC in bovine [26
]. Three lipid families are crucial components of the biological membrane structure: glycerophospholipids containing diacylglycerols (DG) in their hydrophobic part; sphingolipids (SL) with a ceramide as the hydrophobic part, and sterols (ST)—the major non-polar lipids [27
]. Hydrolysis of the glycerolipids and SL produces messenger lipids such as DGs, ceramides, unsaturated FAs, and lysophosphatidic acid (LPA), and other membrane lipids may have signaling functions [28
In the ovary, circulating free FAs are transported from blood as complexes with albumin inside the ovarian follicle, and should be metabolized to form de novo membranes or used as energy sources for the oocyte [21
]. It is known that in somatic cells, captured FAs are esterified into TG and cholesterol esters (CE), and thus could be stored as neutral lipids in lipid droplets (LDs) for further use [23
]. Ovarian follicular somatic cells, in particular TH and GC, receive and metabolize nutritive molecules, including lipids in lipoprotein form from FF [20
], and transform them either for storage in LDs, or the building of a membrane bilayer, ATP production, or signaling molecules.
Although lipids are an integral part of all of the biological membranes and universal energy reserves, the distribution of lipids in mammalian ovaries is not homogenous, as was detected by mass spectrometry imaging (MSI). In porcine ovary, the MSI of lipids was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), which clearly discriminated stroma and ovarian follicles through ovarian sections with a particularly specific distribution of glycerophospholipids, sphingolipids, and cholesterol derivatives between somatic cells (GC/TH) and FF inside the follicles [30
]. In mice ovary, the localization and intensity of several lipids, including phosphatidylinositol (PI 38:4) and arachidonic acid (C20:4), varied between preovulated and post-ovulated stages [31
MALDI-TOF MS profiling is a technique that allows the direct detection of the endogenous molecules in a relatively wide mass range beginning from 100 Da to 25,000 Da (ions of lipids, or peptides and proteins), in scarce biological samples. In bovine, this technique was used to analyze lipid fingerprints in oocytes [32
], CC [35
], and FF [37
The enzymatic pathways involved in lipid transformations were studied preferentially in lipogenic organs such as adipose tissue [38
], liver [39
], and also in cancer cells [40
]. The proteins that are involved in FA metabolism may have tissue specific distribution, as shown for FA binding proteins encoded by numerous FABP
genes, with each one preferentially expressed in different tissue [41
]. The importance of triacylglycerol and FA metabolism in the oocyte–cumulus complexes and the crucial role of FA oxidation for oocyte quality in mouse was underlined in the review by Dunning et al. [42
]. In bovine, until now, the studies reporting on the expression of lipid metabolism-related genes in ovarian cells were focused on only a few key enzymes in oocytes, CC, and GC [35
]. Expression of the main FA transforming enzymes involved in lipogenesis, lipolysis, FA oxidation, and FA transport has revealed active lipid metabolism in the oocyte and surrounding CC and GC. Besides, both lipid synthesis and FA oxidation are involved in the regulation of GC proliferation and steroidogenesis [46
]. FA oxidation in CC was crucial for oocyte maturation in bovine [35
]. However, the mechanisms through which somatic ovarian follicular cells metabolize lipids for oocyte energy supply to promote oocyte quality, and genes regulating lipid metabolism in ovarian follicles, are still poorly elucidated.
The objective of this study was to investigate the lipid composition and transcriptional regulation of FA metabolism in bovine ovarian follicles. We focused our analysis on follicles between 3–8 mm in diameter, which are routinely used for recovering the oocytes for in vitro embryo production technologies in bovine [48
]. Lipid identification and their distribution in ovarian follicular compartments (FF, oocyte, CC, GC, and TH), coupled with the gene expression analysis of lipid metabolism-related genes in follicular cells, may enlighten these mechanisms.
In the present work, for the first time, using liquid chromatography coupled to high-resolution mass spectrometry, we identified several hundred lipids representing 21 lipid classes in cellular and fluid compartments of bovine ovarian follicles running the terminal part of the folliculogenesis. In addition, MALDI-MS profiling was, for the first time, used to compare lipid fingerprints in the different cell types of individual ovarian follicles. Then, 197 molecular species in the range of 250–1000 m
were formerly identified in bovine follicular cells, and these identifications implemented the list of lipids that were already annotated in bovine oocytes and cumulus cells [32
]. By these two approaches, we could identify the major lipids, which were differentially abundant among the somatic compartments: TH, GC, CC, and the oocyte. Although only 1/5 part of m
peaks detected in MALDI-MS spectra was precisely identified, these analyses coupled to MS imaging enabled localizing the most abundant lipids to ovarian follicular structures.
The majority of identified follicular lipids are known to be involved either in membrane synthesis or energy storage (CE, SL, GPL, GL). In fact, we have analyzed tertiary follicles where somatic follicular cells yet proliferate, and the oocytes, which were close to fully grown and potentially ready to resume meiosis, continued to acquire developmental potential by accumulating molecular reserves [51
]. At that stage, the follicles may have strong requirements in energy and building blocks for membranes biosynthesis. During follicular growth up to ovulation, constant bi-directional interplay occurs between the oocyte and surrounding follicular cells. Indeed, the oocyte secretes specific factors GDF9 and BMP15, and thus stimulates follicular cells to proliferate and secrete steroids and other factors, which, in turn, regulate oocyte growth [52
] and protect it from the harmful environment [54
]. Follicular fluid contains molecular factors that were both exuded from blood and follicular secretion [55
], and therefore acting as a buffer against adverse blood influence and as a medium of molecular exchanges between follicular cells. Significant proportions of the follicular fluid lipids that were identified here (FA, Cer, DG, lysophospholipids, LPI, LPE, LPC, ..) might exhibit mediator functions [28
]. FF extracellular vesicles such as exosomes [57
] have very specific lipid composition [58
], and may participate in these communications inside the follicle.
In order to analyze the lipid distribution through the ovarian follicle, molecular imaging and phenotyping technologies were performed. MSI on bovine ovarian sections allowed the ionization of numerous lipids, including PC, LPC, SM, and choline molecules. MSI segmentation map clearly discriminated FF from TH and GC layers, similarly to the MSI of porcine ovarian sections [30
]. This could be explained by different lipid compositions; for example SM, LPC, and choline molecules were preferentially detected in FF, but also by the different ionization capacity of lipids from either liquid or cellular compartments. The identified PC features were mainly localized to both follicular walls and fluid by MSI. Among the common identified lipids, PC represented 37%, and among the identified MALDI-MS m
peaks, PC represented more than 50%. PC are the major components of cellular membrane lipids with well-established roles in differentiation and cell proliferation [27
]. Therefore, PC localized to the follicular wall may be associated to membrane structures in TH and GC.
MALDI-MS lipid profiling confirmed the higher abundance of PC, as well as other membrane lipids such as PE and PS that feature in GC and TH cells as compared to FF. Overall, the FF lipid profile significantly differed from that of the follicular cells, as was expected from the non-cellular compartment. The TH and GC profiles were partially overlaid, as shown by the principal component analysis of MALDI fingerprints. In fact, although TH and GC are separated by the basal membrane, they are metabolically coupled; thus, many lipids were shared between these layers. However, the increased abundance in several LPC, LPE, SM, and TG in TH compared to GC were sufficient to discriminate these two cell types. A similar approach was performed on human lung where MSI confirmed the specific distribution of three phospholipids (PE, PI, and PG) in different regions of lung slice, illustrating the lipidome differences between alveolar macrophages, bronchial epithelial, and alveolar type II pulmonary cells, which were previously shown by LC-MS/MS [59
]. The specific lipid profiling of TH probably reflects its functional structure, which is rich in steroid-secreted cells, blood capillaries, and collagen [60
], compared to GC, which is more homogeneous and has smoother tissue.
MSI was not sufficiently resolute to discriminate GC and CC; however, MALDI-MS lipid fingerprints showed significant differences between these cells. Although CC was derived from the GC, the specific functions of CC are conditioned by their physical and metabolic coupling with the enclosed oocyte [61
]. Here, GC shared the most of the lipids with other compartments; however, in CC, several not-identified features were particularly abundant and formed a CC-specific cluster. In our previous study, a comparison of GC and CC lipid fingerprints revealed that two LPC, 12 PC, and 12 SM features were up-regulated in CC whereas several features including LPC, four PC, two PE, PS, and Cer were more abundant in GC [62
]. The differences between GC and CC lipid concentrations likely reflect the functional differences between these cells [63
Lipid concentrations in follicular compartments may be predictive for oocyte competence. Indeed, the concentration of several TG, PG, PI, PE and lysophospholipids LPE, LPI, and LPC in human FF demonstrated a strong potential association between the differentially elevated lipids and increased success rate of in vitro fertilization/intracytoplasmic sperm injection outcomes [64
]. While CC are physically and metabolically coupled with enclosed oocytes, the MALDI-TOF MS lipid profiling of these cells may also reflect oocyte competence. The differential abundance of several PC was observed in the bovine CC enclosing the oocytes with higher developmental potential after FA (22:6 n-3) supplementation [36
]. In human, the MALDI-TOF lipid profiles of CC showed a different abundance of PC species in the pregnant group and PE, PS, and PI features in the non-pregnant group after ICSI [65
]. Lipid composition also differed between the oocytes with different competence in bovine [33
], but it is difficult to thoroughly evaluate the roles of each feature in this difference.
Oocytes demonstrated very specific lipid profiles that were distinct from the other follicular compartments; nevertheless, the clustering of differential lipids revealed common features with CC or FF. Among the few identified lipids from oocyte-representative clusters, LPC(13:0) and LPC(18:0) were the most elevated in oocytes, LPC 22:4 was similarly abundant in oocytes and CC, and SM (d32:5) was detected at similar levels in oocytes and FF. Previously, we showed significant variations of several PC, SM, and free FA concentrations in the oocytes during meiotic maturation [34
]. This was in line with a decreasing of lipid reserves in mature oocytes as compared to the immature stage [43
], and highlighted the profound changes in both membrane composition and intracellular lipid storage during oocyte maturation.
A relative quantity of total lipids in cellular and fluid compartments was measured using Nile red fluorescent dye in situ targeting mainly the neutral lipids, TG, and cholesteryl esters, which are the main components of lipid droplets (LDs) [23
]. GC and TH layers demonstrated intensive fluorescence due to the accumulation of LDs, which may serve as an energy reserve and as precursors of the steroid hormones or secondary messengers produced by GC and TH.
Steroidogenesis in ovarian follicular cells is mediated by a number of well-known enzymes such as, for example, steroidogenic acute regulatory protein STAR, cytochrome P450 side chain cleavage CYP450scc, 3-beta-hydroxysteroid dehydrogenase HSD3β, and CYP450 aromatase in theca and granulosa cells [66
]. Here, transcriptome analysis confirmed the expression of genes involved in steroid biosynthesis in TH, GC, and CC, as expected. From the same analysis, expression of numerous genes involved in acyl-CoA, FA, and lipid biosynthesis, including the TG, SL, and GPL families, was evidenced in follicular cells, hypothesizing the existence of ovarian de novo lipogenesis in bovine ovary. It was previously reported that CC could produce cholesterol, and this process is regulated by the oocyte secretion of BMP15/GDF9 in mice [67
]. The expression of the key enzymes that are involved in FA synthesis was previously reported in porcine TH and GC [30
]. In bovine GC, CC, and oocytes, the expression of lipogenic enzymes FASN and ACC was evidenced at the protein level [35
]. Sterol regulatory element binding transcription factor 1 (SREBF1
) regulates the genes that were involved in cholesterol and FA biosynthesis [68
]. Here, it was detected as ubiquitously expressed in follicular cells, and thus may participate in the regulation of lipid metabolism-related genes.
The accumulation of lipids in LDs by follicular cells is regulated by a number of genes that are strongly expressed in GC, TH, and CC, and are involved in triglyceride biosynthesis (for example, GPAM
, and ACSBG1)
, lipid storage (SCARB1
, and PLIN5
), and glycerophospholipid metabolism (PLA2G16
, and GPD1
). Correspondent proteins participate in lipoprotein degradation and lipogenesis, leading to the formation of intracellular LDs. In humans, GC accumulate more LDs than CC in vivo [69
]. However, CC, which are specialized GC, are also able to rapidly accumulate LDs, especially when exposed to a high concentration of free FA [70
]. Such a rapid transfer of free FA from FF and their accumulation in the LDs of follicular cells protect the oocyte from lipotoxicity, as was shown in bovine CC [54
TG, CE, and free FA were also largely represented in FF, although there are no LDs. Indeed, total TG concentrations decreased, and total cholesterol increased in bovine FF along with follicular growth from small to large antral follicles [72
]. These lipids may be a part of high density lipoproteins (HDL), which are the smallest lipoproteins that can be transported from blood [24
]. Follicular cells may also synthetize lipoproteins, as human GC, which produced very low-density lipoproteins [73
]. Among the lipids identified in FF, there were many membrane lipids (PC, PE, PI, PS, and lysophospholipids). These neutral lipids may be a part of FF lipoproteins, similar to human plasma lipoproteins, as analyzed by MALDI-TOF MS [74
TH and GC cells received nutrients from circulating blood, including different lipoproteins and free FA coupled to albumins. In the present study, the expression of FA transporter CD36
] and scavenger receptors SCARB1
for HDL cholesterol esters uptake [76
], which was detected in both TH and GC, supports the exogenous origin of a part of FF neutral lipids. In addition, we showed that the lipoprotein lipase LPL
gene was highly expressed in TH cells, as well as other lipases in both TH and GC, suggesting that lipoproteins may be rapidly metabolized and liberate TG and CEs, which may be further used for energy storage and steroid synthesis. Follicular fluid also contain the debris of floating atretic GC and extracellular vesicles, which are released from cell membranes [77
] and are abundant in bovine FF [57
]. Therefore, the PC, as the other membrane phospholipids identified in FF, may originate from both lipoproteins and be membrane-derived, similar to extracellular vesicles, including exosomes [58
On the other hand, several genes coding for apolipoproteins A, E, M, L, and O were strongly expressed in GC, TH, and CC. At the protein level, ApoA1 was shown to be produced and secreted by chicken GC [79
]. ApoA1 protein was also evidenced by Western blot in the GC of healthy and polycystic ovary syndrome patients [80
]. By immunocytochemistry, the ApoA1 protein was revealed in luteal follicular cells in primates [81
]. In our previous work, using top–down proteomics, we have identified ApoA2 and ApoC3 proteins in bovine follicular cells (GC and CC) [82
]. ApoA1, ApoC3, ApoH, and ApoO were identified using differential detergent fractionation multidimensional protein identification in bovine oocytes and CC [84
]. Our present work reported the expression of corresponding genes in follicular cells and oocytes. Therefore, it seems that the bovine follicle is capable of not only degrading, but also synthetizing, the lipoproteins.
In addition, eight free FA, three SM, 28 LPC, and 13 cholesteryl ester features were identified in only FF by LC/HRMS. Around 10% of the total fatty acid concentration that is present in FF is complexed to albumin as free FA, and this pool is the most variable, and depends on the metabolic status of the cow [14
]. Some of the FF lipids, as LPA or unsaturated FA, can activate peroxisome proliferator-activated receptors PPARs, nuclear transcriptional factors that control the expression of genes that function in lipid and carbohydrate metabolism, vascular development, cell proliferation, and cell differentiation [85
]. Our study demonstrated that PPARG is strongly expressed in GC, CC, and oocytes, and may therefore regulate numerous lipid metabolism related genes in all of the compartments, as was here revealed by gene ontology analysis.
Sphingolipids are involved in membrane structure stabilization, cell-to-cell recognition, and cell signaling [86
]. The preferential location of SM and LPC to FF might be also related to extracellular vesicles, which contain these lipids [58
], and to lipoproteins, which contain both SM and lysophospholipids such as LPC [74
The circulating lysophospholipids are membrane components that are necessary to mediate the synthesis of various phospholipids and embed proteins into cell membranes; however, several lysophospholipids such as LPC and LPI were now recognized as hormone-like signaling molecules acting as the ligands of orphan G protein-coupled receptors, and thus considered as intercellular and intracellular lipid mediators [56
]. Therefore, LPC in FF might be involved in intrafollicular signaling and cell-to-cell communication.
LPCs that are derived from the PC are catalyzed either by lecithin-cholesterol acyltransferase LCAT with the formation of a cholesterol ester, or by phospholipases A2. According to our data, the genes LCAT
, and PLA2G1B
coding for the correspondent enzymes are expressed in TH, GC, and CC, whereas phospholipase A2-activating protein PLAA
is overexpressed in oocyte. Therefore, we hypothesize that LPC can be produced by follicular cells, and oocyte may influence this process. Integrated to lipoproteins, the LPC may be then secreted to FF, which therefore should contain the LPC originated from both plasma and follicular cells. Reciprocally, LPC and LPE are transformed to PC or PE by specific acyltransferases, and these enzymes (LPCAT2
, respectively) were also expressed in follicular cells, with significant overexpression in the oocytes. According to MALDI MS profiling, LPC (16:0) and LPC (18:0) were more detected in the oocyte than in somatic follicular cells, and LPC (22:4) were preferentially detected in oocytes and CC. These LPCs may be either imported from the follicular cells, or locally produced. In addition, oocyte expresses genes to transform LPC to PC (CEPT1
) or produce lipoproteins (APOO1
). Several lipid transporters are expressed in the oocyte–cumulus complex and indicate possible exchanges of different lipids between these two compartments, as was shown for FABP3 protein [88
], or between OCC and FF.
Within an OCC, a bovine full-grown oocyte exercises low transcriptional activity or is transcriptionally silent [89
]. Transcripts are accumulated during oocyte growth and then stored as long as protein synthesis is required. When polyadenylation occurred, mRNA could be translated. By our transcriptomic analysis, only polyadenylated mRNA levels were compared between the follicular cells by microarray; therefore, here, detected overexpressed mRNA could be translated in the oocyte at that stage. Although in this article we did not reported data on protein expression, fatty acyl synthase FASN, acetyl-CoA carboxylase ACC, FA binding protein FABP3, and lipid-droplet associated perilipin PLIN2 were detected at protein level in bovine immature oocytes recovered from 3–6 mm follicles [35
]. Moreover, among 811 proteins identified by proteomics in bovine oocyte, numerous ones are involved in lipid metabolism [84
], and the expression of correspondent genes was detected in the present study.
Full-grown bovine oocytes contain many lipid inclusions as LDs and ooplasm vesicles, which could be easily detected and quantified using specific fluorescent dyes such as NR or BODIPY [35
]. In cows, a number of intracellular LDs increased during oocyte growth in vivo [90
] and decreased during in vitro maturation [43
]. Significant correlation between the number of LDs in the oocytes and FA composition in FF was reported in bovine; however, no correlation was found between the number of LDs in the oocytes and the expression level of lipid biosynthesis genes SCD
in GC or CC [92
]. According to our transcriptomic data, a set of genes that are involved in the synthesis of glycerolipids (DGAT2
, sphingolipids (SPTLC2
), phospholipids PC and PE (CEPT1
), lipid storage (PLIN2
), and FA transport (FABP3
) are overexpressed in the oocytes compared to other follicular cells. Expression pattern corroborates with a presence of numerous LDs in the oocyte at that stage and suggest strong lipogenic activity, such as the FA elongation and synthesis of very long-chain fatty acyl-CoA, which is more pronounced in oocytes than in other follicular cells. Oocyte expression pattern also supports its capability to intensive lipolysis and FA oxidation activities, which corroborates with the presence, at protein level, of hormone-sensitive lipase (LIPE protein) in immature oocytes and an increase of LIPE phosphorylation and CPT1 protein abundance in bovine oocytes during maturation [35
In addition, transcriptome analysis revealed the role of the oocyte in PI3 kinase signaling and the synthesis of phosphatidylinositol phosphates (PIP) at the plasma membranes because of the overexpression of the genes that are involved in phosphatidylinositol metabolic processes (CDS1
Indeed, the hydrolysis of PIP2
and the production of inositol trisphosphate is involved in the triggering of oocyte activation after fertilization [93
]. In immature oocytes, which were here analyzed, mRNA may be stocked or partially translated. During oocyte maturation in bovine, protein de novo synthesis occurs in parallel with massive mRNA degradation [94
]. It is likely that the accumulation of lipid reserves, together with maternal mRNA and protein stocks, may serve to ensure oocyte maturation, fertilization, and zygote activation. The first rapid embryo cleavages require active machinery to produce cell structures from the maternal stock for at least 16 blastomers before relaying to the embryo genome that activates in 8/16-cells stage embryo in bovine [97
In summary, the provided detailed analysis of lipids and related transcriptional control in the bovine ovarian follicle implies the capacity of bovine follicular cells, especially theca and granulosa cells, to uptake lipoproteins and free FA, metabolize them, and transport lipids. From these blocks, the follicular cells may probably either produce new membrane lipids and lipoproteins or liberate energy through FA oxidation, or constitute lipid droplets as a reserve of energy and precursors of steroid hormones and secondary messengers. The lipid composition of follicular fluid and enclosed oocyte–cumulus complex coupled to a particular gene expression pattern in these cells presumes intensive lipid exchanges between these compartments, and the particular involvement of the oocyte in FA elongation, the synthesis of glycerophospholipids, glycerolipids, and lipid droplet formation. Oocytes thus accumulate lipids and also contain specific metabolic machinery that may be used for energy production, membrane synthesis, and signaling in order to assure meiotic maturation, fertilization, and provide necessary building blocks for the first embryo cells.
Although more investigations at the protein level are required, an analysis of the expression patterns of lipid metabolism-related genes in the follicular cells and the scrutiny of lipid composition in follicular compartments may imply that the ovarian follicle contains complex metabolic machinery for FA uptake and metabolism to assure intrafollicular homeostasis and prepare the oocyte for ovulation.