Ovarian expression of functional MTTP and apoB for VLDL assembly and secretion in chickens

Abstract

In mammals, tissues other than liver and intestine are known to possess functional MTTP (microsomal triglyceride transfer protein) and apoB (apolipoprotein B) capable of VLDL (very low-density lipoprotein) assembly. Birds are oviparous and possess unique capabilities in lipid biology to accommodate yolk formation through massive deposition of hepatically assembled yolk-targeted VLDLy into ovarian follicles. Following identifications of MTTP and ApoB expression within chicken ovarian stroma, granulosa, theca, and epithelial cells of various classes of follicles, we sought to define the functionality of ovarian MTTP and ApoB in VLDL assembly. In situ hybridization analysis found that ApoB transcripts are most abundant in thecal layers, whereas immunohistochemistry showed that MTTP predominates in the granulosa layers. MTTP lipid transfer activity was greater in small yellow follicles than in hierarchical follicles. Metabolic labeling, electron microscopy, and Western blot studies confirmed the functionality of ovarian apoB and MTTP as newly assembled VLDL around 50-200 nm in diameter and lacking ApoVLDL-II dissimilar to VLDLy, were secreted from cultured follicular cells. Lomitapide and the ApoB-antisense oligonucleotide Mipomersen dose-dependently decreased MTTP activity and VLDL-apoB secretion from cultured follicular cells, while oleate addition or acute heat stress enhanced VLDL-apoB secretion. Ultrastructural images showed VLDL assembly and trafficking toward the secretion route. The findings support the notion that VLDL assembly and secretion within avian ovarian tissues functions as a protective mechanism against fuel and physical stressors to secure follicle development and/or nutritional quality control of yolk for embryo development.

Keywords: Apolipoprotein B; Chickens; Microsomal triglyceride transfer protein; Ovary; VLDL secretion.

Fig. 1

Expression of ApoB and MTTP in chicken ovarian tissues and their secretion of ApoB. Total RNA extracts from ovarian tissues including stroma (OS), hierarchical follicles (F2-F5), small yellow follicles (SYFs), large white follicles (LWFs), freshly isolated granulosa (GC), theca (TH), and epithelial (EP) cells (from F2-F5 follicles), and liver (L) from a laying hen were used to amplify ApoB and the M subunit of MTTP (MTTP-M) transcripts by RT-PCR (panel A). After seeding, EP, TH, and GC cells (from F3 and F4 follicles) and total cells from LWF, SYF, OS, and F2 follicles at 85 % confluence were cultured overnight prior to culture medium harvest for VLDL isolation. The d < 1.006 g/ cm³ fraction of harvested medium and cells were used for ApoB (panel B and D, respectively) and ApoVLDL-II (panel C) analysis by Western blot at equal total protein. Amounts of ApoB and MTTP-M in freshly isolated EP, TH, and GC cells (from F2 and F3 follicles) and ovarian tissues were measured by Western blot (panel E and F, respectively). Laying hens’ plasma VLDL (PV) and liver (L) served as references. M; marker.

Fig. 2

In situation hybridization of ApoB mRNA in chicken ovarian tissues. Ovarian follicles (panel A and B) and stroma (panel C) were used for in situ hybridization of ApoB mRNA using a commercial kit with a specific target probe. In panel C, ovarian cortex and medulla, and various classes of stromal small follicles were noted for ApoB expression. Scale bars; 10 μm, magnification 20 × . EP; epithelial layer; TE; theca externa, TI; theca interna, GC; granulosa layer, PM; plasma membrane (oolemma), BL; basal lamina, YG; yolk granules. .

Fig. 3

Immunohistochemical analysis of MTTP expression in chicken ovarian tissues. Ovarian follicles (panel A and B) and stroma (panel C) were used for immunohistochemistry using an antibody raised against the synthetic epitope of chicken MTTP-M subunit. In panel C, various classes of stromal small follicles were noted for MTTP-M expression. Scale bars; 10 μm, magnification 20 × . EP; epithelial layer, TE; theca externa, TI; theca interna, GC; granulosa layer, PM; plasma membrane (oolemma), BL; basal lamina, YG; yolk granules.

Fig. 4

MTTP activity in chicken ovarian follicles and cells. Freshly isolated epithelial (EP), theca (TH), and granulosa cells (GC) from ovarian F2 and F5 follicles and whole tissues of F3, F4, small yellow follicles (SYFs), large white follicles (LWFs), and stroma (OS) were used for MTTP activity analysis (n = 3 hens). Hens’ breast muscle and mouse 3T3 fibroblasts served as negative references, while hens’ livers and isolated chick hepatocytes were used as a positive control.

Fig. 5

MTTP activity of chicken ovarian follicle cells respond to Lomitapide and Mipomersen inhibition and oleate stimulation. Ovarian granulosa (GC), theca (TH), and epithelial (EP) cells (from F2-F4 follicles) grown to 85 % confluence were treated with Lomitapide (Lom) or Mipomersen (Mip) (a MTTP and ApoB inhibitor, respectively, panel A and B) at indicated concentrations, or their combination (2.5 μM for each, panel C) for 2 hr and replaced with new medium in the presence or absence of oleate (OA, 0.1 mM) for overnight culture. Cells were thereafter harvested for MTTP activity analysis. *; significant difference (P < 0.05, vs. corresponding control, n = 3).

Fig. 6

Metabolic labeling analysis of ApoB secretion by ovarian follicle cells. Ovarian granulosa (GC), theca (TH), and epithelial (EP) cells (from F2-F4 follicles) at 85 % confluence were treated with Lomitapide and Mipomersen (Lom and Mip, a MTTP and ApoB inhibitor, respectively, 2.5 μM) for 2 hr and replaced with new medium containing azidohomoalanine (AHA, 50 μM) in the presence or absence of oleate (OA, 0.1 mM) for overnight culture. Culture medium was collected for VLDL isolation. The newly synthesized proteins identified by AHA incorporation in VLDL extracts were labeled with biotin azide and analyzed by the regular Western blot method using streptavidin-HRP conjugate as a probe (panel A and B). The secretion of ApoB following treatment with Lom and Mip inhibitors or OA stimulation was validated by Western blot analysis using an antibody raised against chicken ApoB (panel C and D). The protein extracts of laying hens’ plasma VLDL (PV) and cell-free culture medium (CM) after ultracentrifugation against the density buffer at 1.006 g/cm³ served as references. M; marker.

Fig. 7

Heat stress induces ApoB secretion from ovarian follicle cells. Ovarian granulosa (GC), theca (TH), and epithelial (EP) cells (from F2-F4 follicles) were grown at 37 °C to 85 % confluence were subjected to heat stress (HS) by incubation at 42 °C for 3 hr and then returned to 37 °C for overnight culture. Control cultures were maintained at constant 37 °C for the same incubation time. In the next day, culture medium was collected for VLDL isolation and collected VLDL was used for ApoB quantification using a commercial ELISA kit (panel A). Western blot analysis with an antibody raised against chicken ApoB was used to validate ApoB presence (panel B). The protein extracts of laying hens’ plasma VLDL (PV) served as a reference. M; marker.

Fig. 8

Ultrastructure analysis of secreted VLDL by follicle cells. Ovarian granulosa, theca, and epithelial cells (from F2-F4 follicles) grown to 85 % confluence were cultured overnight prior to culture medium harvest for VLDL isolation. The d < 1.006 g/ cm³ fraction of harvested medium was imaged using scanning electron microscopy (SEM). VLDL isolated from laying hens’ plasma and medium harvested from chick hepatocyte cultures served as positive controls. Cell-free medium served as a negative reference. Scale bars; 1 μm, magnification;10,000 × .

Fig. 9

Electron micrograph of chicken ovarian follicles. Ovarian follicles were fixed and stained by imidazole-buffered osmium tetroxide procedure and imaged using transmission electron microscopy (TEM). Scattered VLDL (black to dark grey particles indicated by arrows) were observed in epithelial, theca, and granulosa cells of the hierarchical (F3 or F4) and small yellow follicles (SYFs). Scale bars; 1 μm, magnification; 2,550 × .

Fig. 10

Analysis of VLDL assembly and traffic in chicken ovarian follicle cells. Ovarian follicles were fixed and stained by imidazole-buffered osmium tetroxide procedure and imaged using transmission electron microscopy (TEM). VLDL particles with diameters around 50-60 nm were observed in epithelial, theca, and granulosa cells of the hierarchical (F3 or F4) and small yellow follicles (SYFs). Images were noted with VLDL trafficking from the ER to GI (black arrows), GI toward cell surface (black arrow heads), VLDL accumulation within ER or GI (blank stars), and coated pits (black stars). Scale bars; 0.2 μm, magnification; 7,000 × . ER; endoplasmic reticulum. GI; Golgi apparatus.

Fig. 11

Coated pits and VLDL transport vesicles in chicken ovarian follicle cells. Ovarian follicles were fixed and stained by imidazole-buffered osmium tetroxide procedure and imaged using transmission electron microscopy (TEM). Very few VLDL were observed in the interstitial spaces. Coated pits on the cell surfaces were notably devoid of VLDL clustering in the pocket (black stars, panel A). VLDL were trafficked in secretory vesicles; mostly with single particle per vesicle, departing from ER to GI (black arrows) and from GI toward cell membranes (black arrow heads). Scale bars; 0.2 μm and 100 nm and magnification; 7,000 × and 19,500 × for panel A and B, respectively. ER; endoplasmic reticulum. GI; Golgi apparatus.