Unraveling the role of lipid droplets and perilipin 2 in bovine luteal cells

Steroidogenic tissues contain cytosolic lipid droplets that are important for steroidogenesis. Perilipin 2 (PLIN2), a structural coat protein located on the surface of lipid droplets in mammalian cells, plays a crucial role in regulating lipid droplet formation and contributing to various cellular processes such as lipid storage and energy homeostasis. Herein, we examine the role that PLIN2 plays in regulating progesterone synthesis in the bovine corpus luteum. Utilizing gene array databases and Western blotting, we have delineated the expression pattern of PLIN2 throughout the follicular to luteal transition. Our findings reveal the presence of PLIN2 in both ovarian follicular and steroidogenic luteal cells, demonstrating an increase in its levels as follicular cells transition into the luteal phase. Moreover, the depletion of PLIN2 via siRNA enhanced progesterone production in small luteal cells, whereas adenovirus‐mediated overexpression of both PLIN2 and Perilipin 3 (PLIN3) induced an increase in cytosolic lipid droplet accumulation and decreased hormone‐induced progesterone synthesis in these cells. Lastly, in vivo administration of the luteolytic hormone prostaglandin F2α resulted in an upregulation of PLIN2 mRNA and protein expression, accompanied by a decline in serum progesterone. Our findings highlight the pivotal role of PLIN2 in regulating progesterone synthesis in the bovine corpus luteum, as supported by its dynamic expression pattern during the follicular to luteal transition and its responsiveness to luteotropic and luteolytic hormones. We suggest PLIN2 as a potential therapeutic target for modulating luteal function.


| INTRODUCTION
2][3] These organelles store neutral lipids and are encapsulated by a phospholipid monolayer embedded with lipid droplet-associated proteins, notably perilipins (PLIN1-5).These proteins are crucial for stabilizing the lipid droplet structure and facilitating protein complex assembly on the surface. 4In the corpus luteum, PLIN2 (also known as adipose differentiation-related protein, ADRP) is notably expressed in steroidogenic luteal cells. 5This structural coat protein is implicated in regulating fatty acid mobilization and lipid droplet formation across various mammalian cells 6 and plays a key role in ovarian processes such as follicular maturation, 7 oocyte development, 8 angiogenesis, 9 and steroidogenesis. 2 Although lipid droplets are ubiquitous across tissues and have been extensively studied in adipocytes, those in luteal cells exhibit a distinctive composition, being enriched not only with triglycerides but also with cholesteryl esters.This unique composition underscores their vital role in both energy provision and as substrates for progesterone synthesis. 5,10he ovary is a dynamic organ that undergoes remarkable structural and functional changes. 11The functional unit of the ovary is the ovarian follicle, which comprises oocytes, theca cells, and granulosa cells.Post-ovulation, these follicles experience significant transformations, a process initiated by luteinizing hormone (LH), which is synthesized and secreted by the anterior pituitary gland.LH induces follicle rupture to release the ovum and stimulates the transition of theca and granulosa cells from follicular to luteal, forming small and large luteal cells in the corpus luteum. 12Subsequently, LH stimulates progesterone production in luteal cells through the cAMP/Protein Kinase A (PKA) pathway, 10,13 a crucial process for establishing and maintaining pregnancy.Previous studies indicate that LH regulates pathways that promote the activation of hormone-sensitive lipase (HSL, also known as LIPE) and the mobilization of cholesterol, a process that is required for progesterone synthesis. 2,10,14In this process, lipid droplets play a crucial role. 2 Both small and large luteal cells react to LH stimulation; however, small luteal cells show a notably higher responsiveness to LH and to activators of the cAMP/PKA signaling pathway compared to large luteal cells. 6Furthermore, under basal conditions, large luteal cells have a higher capacity to synthesize progesterone compared to small luteal cells. 15uteolysis is a natural developmental process essential for regulating the female reproductive cycle. 16t the onset of luteolysis, there is a precipitous decline in serum progesterone concentrations, followed by the structural demise of the gland. 17Uterine-derived Prostaglandin F2α (PGF2α) initiates luteolysis in a variety of species, including domestic animals, [18][19][20][21] rodents, 22,23 guinea pigs, 24 rabbits, 25 and primates, with both endogenous PGF2α and estrogen playing roles in primates. 26PGF2α exerts its effects via the phospholipase C-intracellular calcium-protein kinase C (PKC) pathway.In the ovine, luteal regression is marked by a sudden increase in free cholesterol and triglyceride levels and a notable alteration in fatty acid composition. 27regnancy, on the other hand, was reported to inhibit or reverse these changes. 27While PGF2α is well known to be involved in the regression of the CL, its role in lipid synthesis and other processes within the corpus luteum remains unclear.
Given the unique enrichment of lipid droplets in ovarian luteal cells, 2,5,[28][29][30] our study aims to explore the role of PLIN2 in regulating lipid metabolism and steroidogenesis in the corpus luteum.We characterize PLIN2 expression in follicular and luteal cells and examined changes during in vitro differentiation of theca and granulosa cells.Employing genetic knockdown and overexpression techniques in small luteal cells, we investigate PLIN2's impacts on lipid droplet abundance and progesterone synthesis.Additionally, we assess PLIN2 expression in vivo following administration of a luteolytic dose of PGF2α.This comprehensive approach provides insights into the regulation, function, and significance of lipid droplets in follicular and luteal cells, shedding light on the complex interplay between these organelles and essential processes in the corpus luteum.

| Follicular cell isolation
All procedures were approved by the Animal Care and Use Committee at the University of Nebraska-Lincoln.The University of Nebraska-Lincoln is AAA-LAC-certified.Non-lactating, composite beef cows [25% MARC III (1/4 ) and theca cells (n = 6) were isolated from the largest and second largest follicles.In brief, follicular granulosa cells from dominant antral follicles were suspended in DMEM/F12 culture media.After the granulosa cells were removed, the theca interna was removed with fine forceps.Granulosa cells were washed by centrifugation three times at 150 × g for 5-10 min and filtration through a 70 μm nylon mesh.The theca interna were suspended in collagenase 2 (103 IU/mL, Atlanta Biologicals) in DMEM/ F12 and dispersed using constant agitation at 37°C for 1 h.Dispersed theca cells were removed from the undigested tissue by filtration through a 70 μm mesh, then washed by centrifugation three times at 150 × g for 5-10 min.

| Luteal cell isolation
For luteal cell isolation, bovine ovaries were collected at a local slaughterhouse, and mid-cycle non-pregnant corpora lutea were staged as described. 32Uteri were checked for the presence of a fetus or visible gross abnormalities.The ovaries were immersed in 70% ethanol and then transported to the laboratory at 4°C in PBS.Using sterile technique, the corpus luteum was surgically dissected from the ovary and finely minced and dissociated using collagenase (103 U/ mL) in basal medium [M199 supplemented with antibiotics (100 U/mL penicillin G-sodium, 100 μg/mL streptomycin sulfate, and 10 μg/mL gentamicin sulfate)] for 45 min in spinner flasks at 35°C.The supernatant was transferred to a sterile 15 mL culture tube, washed three times with sterile PBS, and re-suspended in 10 mL of elutriation medium (calcium-free DMEM medium, 4.0 g/L glucose, antibiotics, 25 mM HEPES, 0.1% BSA, and 0.02 mg/mL deoxyribonuclease l; pH 7.2) on ice.Fresh dissociation medium was added to the remaining undigested tissue, incubated with agitation for an additional 45 min and processed as described above.The viability of cells was determined using trypan blue and cell concentration was estimated using a hemocytometer prior to cell elutriation.Freshly dissociated cells were re-suspended in 15 mL of elutriation medium (calcium-free DMEM, 25 mM HEPES, 0.1% BSA, 0.02 mg/mL deoxyribonuclease, 3.89 g/L sodium bicarbonate, 3 mg/mL glucose, and antibiotics).Dispersed luteal cells were enriched for small and large luteal cells using a Beckman Coulter Avanti J-20 XP centrifuge equipped with a Beckman JE-5.0 elutriator rotor.The eluates were collected through continuous flow as previously described. 33In brief, a 100-mL fraction (F1) composed of erythrocytes and endothelial cells was obtained using a flow rate of 16 mL/min at 1800 RPM.Subsequently, the next 100 mL fraction (F2) was collected at a flow rate of 16 mL/min at 1400 RPM, which contained small luteal cells.A third fraction (F3) was collected using a flow rate of 24 mL/min at 1200 RPM.The remaining fraction (F4) was obtained at a flow rate of 30 mL/min at 680 RMP and was characterized by a high enrichment of large luteal cells.Cells were pelleted and resuspended in M199.Cells from fractions F2 and F4 were utilized in the experiments described.The viability, concentration, size of cell, and purity (%) in each fraction were determined using a hemocytomer and the trypan blue exclusion test.Cells with a diameter of 15-25 μm were classified as small luteal cells (purity of >90% enriched small luteal cells), and cells with a diameter >30 μm were classified as large luteal cells (purity of 70%-90% enriched large luteal cells). 34,35

| Cell culture preparation and treatment with luteinizing hormone (LH)
Luteal cell cultures were plated in 12-well culture dishes at 5 × 10 5 cells/well or 24-well culture dishes at 2.5 × 10 5 cells/well.Cells were cultured overnight in culture media [M199 supplemented with 5% FBS, 0.1% BSA, and antibiotics] at 37°C in an atmosphere of 95% humidified air and 5% CO 2 .
Before treatments, cells were rinsed with PBS, and fresh serum-free culture medium was placed on cells and equilibrated at 37°C in an atmosphere of 95% humidified air and 5% CO 2 for 2 h.Cells were treated with culture medium alone or LH (10 ng/mL) for 4 h at 37°C in an atmosphere of 95% humidified air and 5% CO 2 .

| Microarray
We mined bovine gene expression arrays from the NCBI GEO repository (GSE83524) to analyze the expression of the lipid droplet coat proteins PLINs 1-5s in freshly isolated bovine granulosa (GC, n = 4) and theca (TC, n = 3) cells from large follicles and from purified preparations of small (SLC, n = 3) and large (LLC, n = 3) bovine luteal cells from mature corpora lutea.Details of the isolation and analysis were previously published. 36,37Significant differences were identified as changes greater than 1.5-fold between GC and LLC or between TC and SLC, which were supported by unpaired t-tests with p < .01.Levels of ACTB mRNA were not significantly different among cell types.When all cell types were combined as a group, the relative expression of ACTB mRNA was 7845 ± 164, mean ± SEM.

| siRNA knockdown of PLIN2
PLIN2 was knocked down using silencing RNA (siRNA) to determine the effects of PLIN2 on progesterone production.In brief, enriched small luteal cell populations were transfected with siControl or PLIN2 siRNA (75 nM) for 6 h using Lipofectamine RNAimax in an opti-MEM1 culture medium.Following transfection, 5% FBS was added to the culture medium, and incubations were continued for 48 h.Successful knockdown of PLIN2 was confirmed by Western blot for each experiment.Following knockdown of PLIN2, the medium was changed, and cells were equilibrated for 2 h before treatment with LH (10 ng/mL) for 4 h.Conditioned medium and cell lysates were immediately collected and stored at −20°C until further analysis.

| Treatment with adenoviruses
9][40] In brief, enriched small luteal cells were seeded into 12-well culture dishes and maintained at 37°C in an atmosphere of 95% humidified air and 5% CO 2 for 24 h before adenoviral infection.Ad.βGal, Ad.PLIN2, or Ad.PLIN3 were added to cell cultures in a serum-free culture medium.After 2 h, the media was replaced with M199 enriched with 5% FBS and maintained for an additional 48 h at 37°C in an atmosphere of 95% humidified air and 5% CO 2 .The medium was changed, and cells were equilibrated for 2 h before treatment with the control medium or LH (10 ng/ mL; 4 h).Following treatment, the protein was extracted, quantified, and subjected to Western blotting, or cell cultures were prepared for confocal microscopy.

| Western blotting analysis
Following incubation, cells were immediately placed on ice and rinsed three times with 1 mL of ice-cold PBS.Cells were lysed with 100 μL cell lysis buffer and removed from the culture dish using a cell scraper for sonication at 40% power setting (VibraCell, Model CV188) as previously described. 41Protein concentrations were determined using a Bradford protein assay (Bio-Rad Protein Assay).Samples were adjusted with water to ensure equal protein concentrations and then suspended in 6× Laemmli buffer before being placed on a dry heat bath at 100°C for 6 min.
Proteins (20 μg/sample) were resolved using 10% SDS-PAGE or 4%-20% Mini-PROTEAN® TGX™ precast protein gel and then transferred to nitrocellulose membranes.Membranes were blocked with Tris-buffered saline + 0.1% Tween-20 (TBS-T) containing a 5% non-fat milk solution at room temperature for 1 h.Membranes were incubated with primary antibodies (Table 1) for 24 h at 4°C for the detection of total and phosphorylated proteins.Membranes were rinsed three times with TBS-T for 5 min.Membranes were then incubated with an appropriate horseradish peroxidase-linked secondary antibody (Table 1) for 1 h at room temperature.Blots were then rinsed with TBS-T, and chemiluminescent substrate was applied per the manufacturer's instructions.Blots were visualized using a UVP Biospectrum 500 Multi-Spectral Imaging System (UVP, Upland, CA, USA), and the percent abundance of immunoreactive protein was determined using densitometry analysis in VisionWorks (UVP).Total proteins were normalized to a beta-actin or beta-tubulin prior to the calculation of fold induction.Fold increases due to treatment were then calculated.

| Immunohistochemistry
Bovine ovaries were sliced, and portions were fixed in 10% formalin for 24 h and then changed into 70% ethanol until embedded in paraffin.Tissues were cut into 4 μm sections and mounted onto polylysinecoated slides.Slides were deparaffinized through three changes of xylene and through graded alcohols to water and microwaved in an unmasking solution (Vector H-3300) for antigen retrieval.Endogenous peroxidase was quenched with 0.3% hydrogen peroxide in methanol for 30 min.Sections were incubated with anti-PLIN2 overnight at 4°C, as indicated in Table 1, and subsequently with anti-guinea pig HRP for 1 h at room temperature.Slides were counterstained with Mayer's hematoxylin, dehydrated through graded alcohols, and mounted with Fluoromount-G.Non-immune IgG from the host species was used as a control.

| Progesterone analysis
Progesterone concentrations from conditioned culture media were determined using a commercially available ELISA kit per the manufacturer's protocol (intra-assay CV = 4.83%; inter-assay = 12.02%).The analytical sensitivity of the kit is 0.045 ng/mL.

| Confocal microscopy
For all confocal microscopy experiments, sterile No. 1.5 glass coverslips (22 × 22 mm) were individually placed in each well of a 6-well culture dish.Enriched small luteal cell cultures were seeded at 5 × 10 5 cells/well.
To determine the effects of exogenous, PLIN2 or PLIN3 proteins on lipid droplet number and volume, the adenoviruses Ad.βGal (control virus), Ad.PLIN2, or Ad.PLIN3 were added to cell cultures in a serum-free culture medium.After 2 h, the media was replaced with M199 enriched with 5% FBS and maintained for an additional 48 h at 37°C in an atmosphere of 95% humidified air and 5% CO 2 .
Cells were fixed with 200 μL of 4% paraformaldehyde and incubated at 4°C for 30 min.Cells were rinsed 3× with 1 mL 1× PBS following fixation and then incubated with 200 μL of 0.1% Triton-X in 1× PBS-T (0.1% Tween-20) at room temperature for 10 min to permeabilize the membranes.The permeabilized cells were rinsed 3× with PBS and then blocked in 5% BSA for 24 h at 4°C.Cells were then rinsed, and appropriate antibodies (Table 1) were added to each coverslip and incubated at room temperature for 60 min.Following incubation, cells were rinsed 3× with PBS to remove the unbound antibody.Cells were then incubated with appropriate secondary antibodies (Table 1) at room temperature for 60 min.Cells were rinsed 3× with 1 mL 1× PBS to remove unbound antibodies.Following labeling with antibodies, coverslips containing labeled cells were mounted to glass microscope slides using 10 μL Fluoromount-G (Electron Microscopy Sciences).Coverslips were sealed to glass microscope slides using clear nail polish and stored at −22°C until imaging.
To determine the effects of exogenous PLIN2 or PLIN3 proteins on lipid droplet number and volume, images were collected using a Zeiss 800 confocal microscope equipped with a 63× oil immersion objective (1.4 N.A) and an acquisition image size of 1024 × 1024 pixels (101.31μm × 101.31 μm).The appropriate filters were used to excite each fluorophore, and the emission of light was collected between 450 and 1000 nm.Cells were randomly selected from each slide, and z-stacked (0.33 μm) images were generated from bottom to top of each cell.Z-stacked images were converted to maximum intensity projections and processed utilizing ImageJ (RRID:SCR_003070; National Institutes of Health) analysis software.To determine the size and number of lipid droplets in cells infected with Ad.PLINs, images were quantified with ImageJ using the AnalyzeParticles function in threshold images, with size (square pixel) settings from 0.1 to 100 and circularity from 0 to 1. Outputs were then converted into microns.Red Angus] beef cows from the beef physiology herd at the Eastern Nebraska Research and Extension Center (ENREC) were used in this study.Cows were synchronized using two intramuscular injections of PGF2α (25 mg; Lutalyse®) 11 days apart, as described in Part I.At mid-cycle (days 9-10), cows were treated with an intramuscular injection of saline or PGF2α (25 mg).At each of the four time-points post injection (0, 4, 12, and 24 h), cows were subjected to a bilateral ovariectomy through a right flank approach under local anesthesia as previously described. 31,42,43The corpus luteum was removed from each ovary, weighed, and <5 mm 3 sections were snap frozen in liquid N 2 for subsequent protein analysis or fixed in 10% formalin for immunohistochemistry.The University of Nebraska-Lincoln Institutional Animal Care and Use Committee approved all procedures and facilities used in this animal experiment, and animal procedures were performed at the University of Nebraska-Lincoln, Animal Science Department.

| Progesterone analysis
Plasma progesterone concentrations were determined using a radioimmunoassay (RIA) to detect progesterone concentrations as previously described. 44Progesterone concentrations were determined using the ImmuChemTM Coated Tube Progesterone 125I RIA kit (intra-assay CV = 2.0%, inter-assay CV = 4.46%).The sensitivity of the kit is 0.02 ng/mL.

| RNA sequencing
We mined bovine RNA sequencing data from the NCBI GEO repository (GSE217053) to analyze the expression of the lipid droplet coat proteins PLINs 1-5 s in corpora lutea obtained from mid-luteal phase cows (Day 10; n = 6) and corpora lutea obtained from animals treated with 4 (n = 6) and 12 h (n = 6) i.m administration of PGF2α.Details of the RNA isolation and analysis were previously published. 45

| Western blotting
Approximately 100 mg of tissue was homogenized in RIPA Buffer supplemented with 1× Halt Protease and Phosphatase Inhibitor Cocktail and sonicated at 40% power setting (VibraCell, Model CV188) as previously described. 41Following sonification, tissue homogenates were centrifuged at 13000 × g at 4°C for 15 min.Protein was collected, and concentrations were determined using the BCA protein assay.Samples were suspended in 6× Laemmli buffer and placed on a dry heat bath at 100°C for 6 min.Proteins (30 μg/sample) were resolved and visualized as described in Part 1.Total PLIN2 was normalized to ACTB.

| Statistics
Each experiment was performed at least three times, each using cell preparations from separate cows and dates of collection.Specifics of statistical testing are described in the relevant figure legends.The differences in means were analyzed by one-way ANOVA followed by Tukey's multiple comparison tests to evaluate multiple responses, one-way ANOVA followed by Dunnett's posttests to compare means, or by t-tests to evaluate paired responses.A two-way ANOVA was used to evaluate repeated measures with Dunnett's posttests to compare means.All statistical analysis was performed using GraphPad Prism software (GraphPad Prism, RRID:SCR_002798).All data are presented as the means ± SEM.

| Perilipin 2 (PLIN2) expression in the bovine ovary
Following ovulation, during the follicular to luteal transition, the granulosa and theca cells of the ovarian follicle differentiate to form the large and small luteal cells, respectively, of the bovine corpus luteum. 46We mined bovine gene expression arrays from the NCBI GEO repository (GSE83524) to analyze the expression of the lipid droplet coat proteins PLINs 1-5 s in freshly isolated bovine granulosa (GC, n = 4) and theca (TC, n = 3) cells from large follicles and from purified preparations of small (SLC, n = 3) and large (LLC, n = 3) bovine luteal cells from mature corpora lutea.Figure S1 shows the relative mRNA expression of PLIN1, PLIN2, PLIN3, PLIN4, and PLIN5 in follicular cells and their luteal cell counterparts.Levels of mRNA expression for PLIN1, PLIN4, and PLIN5 were low and not different among cell types.Transcripts for PLIN2 and PLIN3 were similar in granulosa and theca cells.Levels of PLIN2 were increased 3.0-fold in large luteal cells compared to granulosa cells (p < .001)and increased 4.4-fold in small luteal cells when compared to theca cells (p < .001).Levels of PLIN3 were not influenced in large luteal cells compared to granulosa cells (p > .05)but were 2-fold greater in small luteal cells when compared to theca cells (p < .001).Levels of ACTB mRNA were not significantly different among cell types.When all cell types were combined, the relative expression of the ACTB mRNA count was 7845 ± 164, mean ± SEM.
Western blot was used to validate expression of PLIN2, PLIN3, and 3beta-Hydroxysteroid dehydrogenase (HSD3B) in granulosa, theca, and their respective luteal cell counterparts (Figure 1A).Protein expression for PLIN2, PLIN3, HSD3B, and HSL was similar in granulosa and theca cells (p > .05).Levels of PLIN2 were increased 4.2-fold in large luteal cells compared to granulosa cells (p < .001)and increased 2.2-fold in small luteal cells when compared to theca cells (p < .001; Figure 1A,B).Levels of PLIN3 were increased 34.8-fold in large luteal cells compared to granulosa cells (p < .001)and 9.7-fold in small luteal cells when compared to theca cells (p < .001; Figure 1A,C).Levels of HSD3B were increased 5.7-fold in large luteal cells compared to granulosa cells (p < .0001)and increased 4.9-fold in small luteal cells when compared to theca cells (p < .0001; Figure 1A,D).Levels of HSL were increased 11.1-fold in large luteal cells compared to granulosa cells (p < .05)and 23.9-fold in small luteal cells when compared to theca cells (p < .01; Figure 1A,E).

| Perilipin 2 (PLIN2) expression following follicular cell differentiation
The differentiation of ovarian cells into their respective luteal cells is driven by mimicking the surge of LH, which activates PKA and PKC signaling. 47To determine the effects of cellular differentiation on PLIN2 expression, theca and granulosa cells were stimulated with FSK (10 μM), an activator of PKA signaling, PMA (20 nM), an activator of PKC signaling, and 1× ITS for 96 h, and protein lysis and conditioned media were collected.Differentiation of theca cells for 96 h stimulated a 9.8-fold increase in progesterone production (p < .05; Figure 2A).Western blotting was used to determine changes in protein expression following theca cell differentiation (Figure 2B).Western blotting revealed that differentiation of theca cells increased the expression of PLIN2 1.8-fold compared to untreated theca cells (p < .05; Figure 2B,C).Although we observed an increase in the expression of HSL in small luteal cells compared to theca cells (Figure 1A,E), we did not observe a significant difference in HSL expression following incubation with differentiation media (p > .05; Figure 2B,D).Western blotting further revealed a tendency for cholesterol side-chain cleavage enzyme (CYP11A1) expression (p = .0855;Figure 2B,E) and an increase in HSD3B expression (p < .05; Figure 2B,F) in differentiated theca cells when compared to untreated theca cells.
Next, we examined the effects of differentiation in granulosa cells.Differentiation of granulosa cells for 96 h stimulated an 8.4-fold increase in progesterone production (p < .01; Figure 2G).Western blotting revealed differentiation of granulosa cells increased the expression of PLIN2 2.1-fold compared to untreated granulosa cells (p < .05; Figure 2H,I).Moreover, we observe a 4.3-fold increase in HSL expression following incubation with differentiation media (p < .05; Figure 2H,J), but difference in the expression of steroidogenic enzymes CYP11A1 (p > .05; Figure 2H,K) and HSD3B (p > .05; Figure 2H,L).
Confocal microscopy was used to visualize the lipid droplet content between the differentiated and untreated theca (Figure 2M) and granulosa cells (Figure 2N).Untreated theca (Figure 2M panels a and b) and granulosa cells (Figure 2N panels a and b) had fewer lipid droplets than the differentiated follicular cells (p > .05; Figure 2M,N panels c and d; Figure S2), as assessed by BODIPY staining of lipid droplets.Lipid droplet content was also visualized in small and large luteal cells as a comparison (Figure 2O).

| PLIN2 knockdown increases acute progesterone production in small luteal cells
We employed specific siRNA targeting of PLIN2 to evaluate the role of PLIN2 on progesterone production in small luteal cells (Figure 3A).Western blotting revealed a 75 ± 5.6% decrease in expression of PLIN2 in siPLIN2treated luteal cells compared to control cells (p < .05; Figure 3B).Treatment of small luteal cells with siPLIN2 did not significantly influence the expression of steroidogenic enzymes (p > .05;data not shown).Treatment of small luteal cells with siPLIN2 did not significantly influence basal progesterone secretion when compared to siControl cells (p > .05; Figure 3C).However, siRNAmediated knockdown of PLIN2 increased LH-induced progesterone secretion compared to siControl treated cells (p < .05; Figure 3C).

| Overexpression of Perilipin 2 (PLIN2) and Perilipin 3 (PLIN3) attenuates progesterone production in small luteal cells
The bovine steroidogenic luteal cells express mRNA for both PLIN2 and PLIN3 (Figure S1).Therefore, we set out to determine the role of overexpression of PLIN2 and PLIN3 on lipid droplet abundance and progesterone production in small luteal cells.To determine the role of overexpression of PLIN2 on acute progesterone production, luteal cells were infected with increasing concentrations of adenovirus (Ad) expressing PLIN2, and Ad.β-Gal is used as a control.(Figure 4).Western blotting revealed that Ad.PLIN2 increased the expression of PLIN2 in small luteal cells (Figure 4A).We set out to determine the effects of exogenous PLIN2 expression on lipid droplet number and volume in small luteal cells (Figure 4B).
Overexpression of PLIN2 induced a 1.6-fold increase in the number of lipid droplets (p < .05; Figure 4C) and a 1.4fold increase in average lipid droplet volume (p < .0001; Figure 4D) when compared to cells treated with Ad.β-Gal control.To examine the effects of exogenous PLIN2 expression on progesterone production, enriched populations of small luteal cells were treated with increasing concentrations of Ad.PLIN2 and then stimulated with or without LH (10 ng/mL) for 4 h.We observed no difference in basal progesterone production in cells treated with Ad.PLIN2 compared to Ad.β-Gal control (Figure 4E).There was a concentration-dependent decrease in acute progesterone production in cells treated with increasing titers of Ad.PLIN2, when compared to the Ad.β-Gal control cells following stimulation with LH (p < .05; Figure 4E).
Increasing concentrations of adenovirus expressing PLIN3 lead to a concentration-dependent increase in the expression of PLIN3 in small luteal cells (Figure S3A).We determined the effects of exogenous PLIN3 expression on lipid droplet number and volume in small luteal cells (Figure S3B).Overexpression of PLIN3 increased lipid droplet volume (p < .05; Figure S3C) and induced a tendency to reduce the number of lipid droplets (p = .09;Figure S3D) when compared to cells infected with Ad.β-Gal control.We observed no difference in basal progesterone production in cells treated with Ad.PLIN3 compared to Ad.β-Gal control (p > .05; Figure 3E).However, a decrease in LH-stimulated progesterone production was observed in cells infected with Ad.PLIN3 when compared to Ad.β-Gal control cells (p < .05; Figure S3E).

| Effects of Prostaglandin F2 alpha on Perilipin 2 (PLIN2) expression in vivo
To evaluate the temporal effects of PGF2α on progesterone production, cows were administered a single dose of saline or PGF2α (i.m.) and corpora lutea were collected at zero time, 4, 12, and 24 h post i.m administration of PGF2α.Serum progesterone decreased 33.6% 4 h post-injection of PGF2α (p < .05)and further decreased 73.7 and 82.3%, respectively, 12 and 24 h post-injection of PGF2α (p < .0001; Figure 5A).
Western blotting was used to confirm the observed increase in PLIN2 expression following i.m administration of PGF2α (Figure 5D).Western blot revealed an acute 1.9-fold increase (p-value < .01) in PLIN2 expression 12 h post-treatment with PGF2α and a 2.8-fold increase 24 h post-PGF2α, respectively (p-value < .001; Figure 5D,E).Immunohistochemistry of luteal tissue revealed an observed increase in PLIN2 (Figure 5F, panel a and b) 12 h post i.m administration of PGF2α.Moreover, there was a notable presence of PLIN2 localized to the steroidogenic large and small luteal cell populations (Figure 5F panels a and b), supporting our hypothesis that PGF2α regulates PLIN2 expression in bovine luteal tissue.

| DISCUSSION
Perilipins 1-5 are major coat proteins associated with lipid droplets that target and regulate intracellular lipid storage and hydrolysis. 48Specifically, the highly conserved 11-mer repeat regions of PLINs 1-3 form amphipathic helices on the lipid droplet surface and coordinate lipid release from these droplets. 49These organelles store cholesterol esters and are enriched with proteins that regulate both lipid homeostasis and steroid production in steroidogenic tissues, such as the corpus luteum. 2,5,50The small and large steroidogenic cells of the corpus luteum exhibit a unique abundance of cholesteryl ester-storing lipid droplets, which have been proposed to contribute to sex steroid synthesis. 5Our study focuses on PLIN2, a coat protein associated with lipid droplets, and examines its role in luteal cells and its impact on progesterone production.We report that PLIN2 is highly expressed in ovarian steroidogenic cells and correlates with follicular cell differentiation.Importantly, genetic manipulation of PLIN2 alters acute LH-stimulated progesterone production in small luteal cells.Additionally, our findings indicate that administering the luteolytic hormone, PGF2α, increases PLIN2 expression, a change that is accompanied by reduced serum progesterone levels.
The transformation of ovarian follicular granulosa and theca cells into steroidogenic luteal cells is a key aspect of corpus luteum development.By comparing follicular cells before and after differentiation, we gain insights into the cellular changes that occur during luteinization.The role of lipid droplets in energy storage and as precursors for steroid hormones is crucial, and the observed increase in PLIN2 expression, along with the accumulation of lipid droplets in differentiated cells, underscores their significance in the metabolic adaptation of luteal cells.This study enhances our understanding of the intricate processes of cellular differentiation, hormonal regulation, and metabolic shifts that are essential in luteal formation.
Steroidogenic cells synthesize steroids upon stimulation, requiring a steady cholesterol supply.Hormone stimulation, particularly LH via cAMP/PKA signaling, prompts lipid droplets to release cholesterol for steroid hormone synthesis, affecting processes like HSL phosphorylation and localization on lipid droplets. 10This mechanism aids in transporting cholesterol to mitochondria for progesterone biosynthesis in bovine luteal cells.Our findings indicate that PLIN2 knockdown enhances hormone-stimulated progesterone production, likely by enhancing HSL access and promoting cholesteryl ester hydrolysis, akin to the role of PLIN2 in adrenal tissue cholesterol flux. 51Conversely, overexpression of PLIN2 inhibits acute LH-stimulated progesterone production in small luteal cells, accompanied by an increase in the number and volume of lipid droplets per cell.A similar effect is seen in cardiac tissue 52 and cultured fibroblasts, 53 whereby overexpression of PLIN2 leads to an accumulation of intracellular lipid droplets and triacylglycerol content.Such overexpression of F I G U R E 2 PLIN2 expression following follicular cell differentiation.Bovine granulosa (GC) and theca cells (TC) were cultured for up to four days in medium containing 1% fetal calf serum with or without insulin/transferrin/selenium, the adenylyl cyclase activator forskolin in adipocytes also impedes the association of adipose triglyceride lipase, a critical lipase responsible for the hydrolysis of triacylglycerol, with the lipid droplet. 54 similar mechanism involving HSL may be occurring in luteal cells, leading to a decrease in intracellular cholesterol available for steroidogenesis.
Lipid droplets and the lipid droplet-associated protein, PLIN2, are a predominant feature of the steroidogenic cells of the bovine corpus luteum. 5It is hypothesized that PLIN2 may function as a regulatory 'brake' regarding progesterone synthesis.Notably, under basal conditions, large luteal cells exhibit a greater capacity for progesterone synthesis compared to small luteal cells.Supporting this theory, microarray analysis of luteal cells shows higher expression of PLIN2 in small luteal cells compared to large luteal cells, hinting at a potential regulatory role.Knockdown of PLIN2 in small luteal cells enhances acute progesterone production, whereas overexpressing PLIN2 in small luteal cells suppresses synthesis, further indicating its inhibitory impact on steroidogenesis.This concept gains additional support following treatment with PGF2α.After such treatment, a significant increase in PLIN2 expression is observed, especially in large luteal cells, coinciding with a marked decrease in serum progesterone levels.These observations support the idea that PLIN2 may play a vital role in modulating progesterone synthesis, potentially serving as a key regulatory factor in hormonal control within the corpus luteum.
Bovine luteal cells express PLIN3, which translocates between the lipid droplet and cytoplasm. 55As the second most common perilipin in these cells, 5 the exact role of PLIN3 in the corpus luteum is unclear.In neutrophils, PLIN3 is crucial for lipid droplet formation and prostaglandin E2 production, 56 a luteotropic hormone also synthesized by the corpus luteum.Overexpressing PLIN3 in our study led to larger lipid droplets and reduced hormonestimulated progesterone synthesis.In muscle cells, PLIN3 overexpression increases triacylglycerol accumulation, 57 suggesting that PLIN3 may act as a regulator of intracellular triacylglycerol content by limiting lipase activity.Given that luteal lipid droplets primarily contain triacylglycerol, 5 F I G U R E 3 Knockdown of lipid droplet-associated proteins, PLIN2, promotes acute progesterone production in bovine small luteal cells.PLIN2 mRNA was silenced using siPLIN2 in small bovine luteal cells.Following knockdown, cells were treated without (control; CTL) or with luteinizing hormone (LH; 10 ng/ mL) for 4 h.(A) Representative Western blot analysis showing expression of PLIN2 in siPLIN2 knockdown small luteal cells.(B) Quantitative analysis of the expression of PLIN2 in siPLIN2 knockdown small luteal cells.(C) medium progesterone.Statistics were performed by a two-way ANOVA, which was used to evaluate repeated measures with Tukey's multiple comparison tests.Bars represent means ± SEM, n = 3. Significant difference between treatments, *p < .05;**p < .01;***p < .001.Steroidogenic acute regulatory protein (STAR); cholesterol side-chain cleavage enzyme (CYP11A1); 3beta-hydroxysteroid dehydrogenase (HSD3B); betaactin (ACTB; loading control).similar effect in luteal could lead to reduced progesterone synthesis and increased lipid droplet size.PLIN3 facilitates the transport of lysosomal hydrolases from the Golgi to lysosomes via mannose 6-phosphate receptors, 58 which hydrolyze lysosomal cholesteryl esters from lipoproteins. 59This mechanism might explain the decreased progesterone synthesis with PLIN3 overexpression, possibly due to altered lysosomal lipid transfer to droplets.Further studies are needed to understand the function of PLIN3 in bovine luteal cells and steroidogenesis.
The luteolytic hormone, PGF2α, induces luteal regression in the bovine. 60Loss of progesterone and accumulation of lipid droplets in the cytoplasm of luteal cells is a characteristic of luteal regression reported across various species, including primates, 61 domestic animals, 28,62 and rodents. 63Perilipins, particularly PLIN2, play a crucial role in lipid droplet formation and metabolism regulation. 64,65Our study found that administering PGF2α led to an increase in PLIN2 mRNA expression while simultaneously decreasing the levels of PLIN3, PLIN4, and PLIN5, indicating a role in lipid droplet formation through the upregulation of PLIN2.In 3 T3-L1 cells, PGF2α augments the formation of lipid droplets and triacylglycerol synthesis via DGAT, 66 whereas in hepatocytes, it reduces lipid droplet accumulation and promotes autophagy and lysosomal aggregation. 67This suggests that the impact of PGF2α on lipid droplets varies across cell types and likely involves modulation of various signaling pathways.
Our study investigates the impact of luteolytic and luteotropic hormones, specifically LH and PGF2α, on the expression of PLIN2.The cellular interactions and signaling pathways associated with these hormones in relation to luteal function are complex.While we have shed light on the role of perilipins in steroidogenesis, the exact molecular mechanisms underlying their influence have not been fully elucidated.This gap in understanding limits our ability to make definitive assertions about their specific roles regarding luteal function.Furthermore, the effects of genetic F I G U R E 4 Overexpression of lipid droplet-associated proteins, PLIN2, in bovine luteal cells.Replication-deficient adenoviruses (Ad) containing beta-galactose (Ad.βGal; control) or PLIN2 (Ad.PLIN2) were utilized to overexpress PLIN2 in bovine small luteal cells.(A) Representative Western blot of dose-dependent overexpression of Ad.PLIN2 [VP/mL] in small luteal cells.Small luteal cells were infected with Ad.βGal or Ad.PLIN2 as described above.After 48 h, luteal cells were equilibrated for 2 h and stimulated with luteinizing hormone (LH; 10 ng/mL) for 4 h.Small luteal cells were treated with Ad.βGal or Ad.PLIN2 and lipid droplets were labeled (Lipi-blue 1 μM) and visualized by confocal microscopy.(B) Representative micrographs of lipid droplets obtained from small luteal cells infected with Ad.βGal or Ad.PLIN2 (2х10 8  of PLIN2 on the droplet proteome have yet to be determined.Conducting a comparative analysis of other proteins associated with lipid droplets under conditions of knockdown/overexpression would provide a more comprehensive understanding of the unique roles of perilipins in bovine luteal cells.Another aspect yet to be explored is the long-term impact of altering PLIN2 on follicular cell differentiation, luteal function, and reproductive outcomes.While our findings indicate changes in progesterone synthesis, further studies are needed to determine the precise molecular mechanisms and their implications for ovarian health and fertility.

CONCLUSION
Our findings offer into the regulation, function, and significance of the lipid droplet-associated protein, PLIN2, in bovine ovarian cells.We confirm the presence of PLIN2 in both ovarian follicular and steroidogenic luteal cells, particularly noting elevated levels during the transition from the follicular to luteal phase.Additionally, our research strongly supports the pivotal role of PLIN2 in regulating progesterone synthesis within the bovine corpus luteum.Moreover, our in vivo experiments establish a correlation between luteolytic hormone administration and PLIN2 expression levels, highlighting its regulatory significance in luteal function.Overall, our findings highlight PLIN2 as a promising therapeutic target for modulating luteal function in bovine species.
Characteristics of antibodies used for Western blotting and microscopy.
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