Immune Netw.  2017 Aug;17(4):261-268. 10.4110/in.2017.17.4.261.

Localization of Serum Amyloid A3 in the Mouse Ovary

• Choi H
• Ignacio RM
• Lee ES
• Roby KF
• Terranova
• Son DS

• KMID: 2388080
• DOI: http://doi.org/10.4110/in.2017.17.4.261

Abstract

Tumor necrosis factor-α (TNF-α) induces serum amyloid A (SAA) 3 among acute-phase proteins in mouse granulosa cells by activating NF-κB signaling via p55 TNF-α receptor type 1. However, the localization of SAA3 within the ovary is unknown. Here we investigated ovarian localization of SAA3 in a mouse ovulation model and in response to IL-1β, a proinflammatory mediator. For the ovulation model, equine chorionic gonadotropin (eCG; 2.5 IU) was administered to mice subcutaneously (sc) to stimulate follicular development on day 25 of age and then 50 h after eCG, human chorionic gonadotropin (hCG; 2.5 IU) was administered sc to induce ovulation. The mouse ovulation model was characterized by the localization of CYP19 mRNA expression to granulosa layers of larger follicles. SAA3 mRNA, determined by in situ hybridization, was broadly expressed throughout the whole ovary. Granulosa layers and small follicles expressed higher SAA3 mRNA compared to thecal-interstitial layers and large follicles, respectively. Interestingly, atretic follicles contained cells expressing intense SAA3 mRNA. After ovulation, SAA3 mRNA expression was intensely evident in ruptured follicles and corpora lutea (CL). The intraperitoneal administration of IL-1β revealed the intense and extensive appearance of specific cells expressing SAA3 mRNA around follicles and in CL. In addition, Gene Expression Omnibus (GEO) database analysis supported expression pattern of SAA3 mRNA observed in mouse ovulation model. Taken together, SAA3 was broadly distributed through the whole ovary, but intensely expressed in atretic follicles and CL. Furthermore, proinflammatory mediators could trigger the intense appearance of SAA3 around follicles and in CL.

Keyword

Serum amyloid A; Ovulation; Ovary

MeSH Terms

Acute-Phase Proteins
Amyloid*
Animals
Aromatase
Chorionic Gonadotropin
Corpus Luteum
Electrocardiography
Female
Gene Expression
Granulosa Cells
In Situ Hybridization
Mice*
Necrosis
Ovarian Follicle
Ovary*
Ovulation
RNA, Messenger
Serum Amyloid A Protein
Acute-Phase Proteins
Amyloid
Aromatase
Chorionic Gonadotropin
RNA, Messenger
Serum Amyloid A Protein

Figure

• 

  Figure 1 Schematic of mouse ovulation model and ovarian localization of CYP19 mRNA. Ovaries were collected at 0, 24, 48 h after eCG (2.5 IU) to initiate follicular development, and 2, 24 h after hCG (2.5 IU) to induce ovulation and luteinization. The mouse ovulation model is characterized by the expected time- and cell-specific expression of CYP19 mRNA using in situ hybridization with digoxigenin CYP19 RNA probe.

• 

  Figure 2 Localization of SAA3 mRNA in the mouse ovary during gonadotropin-induced follicle development and ovulation. Hybridization of antisense SAA3-labeled riboprobes was visualized as purple-blue precipitate in the whole ovary at respective time points (0, 24, 48 h post-eCG injection, and 2, 24 h post-hCG injection). Incubation of tissue sections with labeled sense probe revealed no hybridization.

• 

  Figure 3 IL-1β treatment in vivo induced SAA3 mRNA in the mouse ovary. Adult cycle mice (2 months old) were treated intraperitoneally with IL-1β (1 μg/mouse). Mice were euthanized at 2 h post-injection and ovaries were collected for 10 μm frozen sectioning and in situ hybridization.

• 

  Figure 4 Expression of SAA3 mRNA in mouse granulosa cells. (A) Effect of proinflammatory cytokines on SAA3 mRNA in mouse granulosa cells. Mouse granulosa cells were incubated with vehicle, TNF-α (10 ng/mL), IL-1α (10 ng/mL), and IL-1β (10 ng/mL) for 2 h. RT-PCR was performed using SAA3 primers. L19 was used as a loading control. (B) Expression of SAA3 mRNA in mouse granulosa cells collected from ovaries of mice during the ovulation model and after 2 h post-LPS (100 μg/mouse) challenge. (C) Expression levels of SAA isoforms in mouse granulosa cells based on the NCBI GEO dataset (GSE23084). (D) Expression levels of SAA isoforms in cumulus oocyte complex based on the NCBI GEO dataset (GDS1677). NCBI, National Center for Biotechnology Information; M, molecular marker in base pairs; Con, control.

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