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

Abstract

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.

Keywords: corpus luteum; lipid droplets; perilipin 2; progesterone; prostaglandin F2α; steroidogenesis.

FIGURE 1

PLIN2 is highly enriched in bovine luteal cells. Western blotting was used to determine validated changes in expression of PLIN2 and PLIN3 in freshly isolated bovine granulosa (GC) and theca cells (TC) from large follicles and purified preparations of bovine small and large luteal cells from mature corpora lutea. (A) Representative Western blot of PLIN2, PLIN3, HSD3B, and HSL expression (n = 3). (B) Quantitative analysis of PLIN2 expression. (C) Quantitative analysis of PLIN3 expression. (D) Quantitative analysis of HSD3B expression. (E) Quantitative analysis of HSL expression. Statistics were performed by one-way ANOVA, followed by Tukey’s multiple comparison tests. Data are means ± standard error, n = 3. Bars represent means ± SEM, n = 3. Significant difference between treatment, *p < .05; **p < .01; ***p < .001; ****p < .0001. Perilipin 2 (PLIN2); Perilipin 3 (PLIN3); 3beta-Hydroxysteroid dehydrogenase (HSD3B); Hormone Sensitive Lipase (HSL); Beta-Actin (ACTB; loading control).

FIGURE 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 (10 μM), and phorbol myristate acetate (PMA, 20 nM). (A) Medium progesterone obtained from TC following incubation with or without differentiation media. (B) Representative Western blot of PLIN2, HSL, CYP11A1, and HSD3B expression in TC and differentiated TCs (dTC; n = 4–6). (C) Quantitative analysis of PLIN2 expression. (D) Quantitative analysis of HSL expression. (E) Quantitative analysis of CYP11A1 expression. (F) Quantitative analysis of HSD3B expression. (G) Medium progesterone obtained from GCs following incubation with or without differentiation media. (H) Representative Western blot of PLIN2, HSL, CYP11A1, and HSD3B expression in GC and differentiated TCs (dGC; n = 6). (I) Quantitative analysis of PLIN2 expression. (J) Quantitative analysis of HSL expression. (K) Quantitative analysis of CYP11A1 expression. (L) Quantitative analysis of HSD3B expression. (M) Representative micrographs of lipid droplets obtained from TC (panels a and b) and dTC (panels c and d). (N) Representative micrographs of lipid droplets obtained from GC (panels a and b) and dGC (panels c and d). (O) Representative micrographs of lipid droplets obtained from small luteal (panel a) and large luteal cells (panel b). The micron bar represents 20 μm. Statistics were performed using t-tests to evaluate paired responses. Bars represent means ± SEM. Significant difference between treatments, *p < .05; **p < .01. Perilipin 2 (PLIN2); hormone sensitive lipase (HSL); cholesterol side-chain cleavage enzyme (CYP11A1); 3beta-hydroxysteroid dehydrogenase (HSD3B); and beta actin (ACTB; loading control).

FIGURE 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); beta-actin (ACTB; loading control).

FIGURE 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 (2х10⁸ VP/mL). (C) Quantification of lipid droplet number in small luteal cells infected with Ad.βGal or Ad.PLIN2. (D) Quantification of lipid droplet volume (nm³) in small luteal cells infected with Ad.βGal or Ad.PLIN2. Statistics were performed by t-tests to evaluate paired responses. Data are means ± standard error, n = 3. (E) Medium progesterone obtained from small luteal cells treated with Ad.βGal or increasing concentrations Ad.PLIN2 following stimulation with LH. Statistics were performed by two-way ANOVA 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; ****p < .0001. Micron bar represents 20 μm. Beta Actin (ACTB; loading control); Beta Tubulin (TUBB; loading control).

FIGURE 5

Effects of Prostaglandin (PG) F2α on PLIN2 expression in vivo. Midcycle cows (n = 3–8/time-point) were administered I.M. Prostaglandin F2α (PGF2α; 25 mg) for 4, 12, and 24 h or control saline injections. (A) Serum progesterone concentrations were obtained from animals 0 (n = 8), 4 (n = 3), 12 (n = 6), and 24 h (n = 3) following I.M PGF2α administration. Statistics were performed by one-way ANOVA followed by Tukey’s multiple comparison tests. Mid-luteal phase cows were injected with saline (Control) or PGF2α, (25 mg, i.m.) and ovariectomized after 4 and 12 h to collect corpora lutea. RNA sequencing of whole luteal tissue was performed. (B) mRNA levels of PLIN2 in the bovine corpus luteum at midcycle and 4- and 12-h post-PGF2α injection. (C) mRNA levels of PLIN3 in the bovine corpus luteum at midcycle and 4- and 12-h post-PGF2α injection. Data are presented as mean number of transcripts per million (TPM) ± SEM. n = 4; *p < .05, **p < .01, compared to 0 h by DESeq2 analysis, Benjamini Hochberg correction. P-values shown are adjusted p-values for multiple comparisons. (D) Representative Western blot of PLIN2 expression in bovine corpus luteum at midcycle and 12- and 24 h post-PGF2α injection. (E) Quantitative analysis of PLIN2 expression following 0 (n = 6), 12 (n = 5) and 24 h (n = 3) post-PGF2α injection. Statistics were performed by one-w ay ANOVA followed by Tukey’s multiple comparison tests. (F) Representative immunohistochemistry micrograph of the PLIN2 in luteal tissue 12 h following I.M administration of PGF2α treatment. Micron bar = 5 mm (Insert) and 1 mm (Enlarged). Bars represent means ± SEM. Significant difference between treatments, *p < .05; **p < .01; ***p < .001; ****p < .0001.