Maternal short and medium chain fatty acids supply during early pregnancy improves embryo survival through enhancing progesterone synthesis in rats
Manipulating maternal lipid metabolism to maintain sufficient progesterone level is an effective way
- Maternal SB, SH, or SC supply significantly improved live litter size and embryo implantation in rats
- The expression of key genes involved in ovarian steroidogenesis and granulosa cell luteinization were elevated in ovaries and primary cultured granulus cells, including cluster of differentiation 36 (CD36), steroidogenic acute regulatory protein (StAR), and cholesterol side-chain cleavage enzyme
- Our results have important implications that short or medium chain fatty acids have the potential to prevent miscarriage in women or early pregnancy loss in mammals
Early embryo loss has a direct impact on pregnancy outcome in humans and animals
- In women, about 15% of miscarriages occur during early pregnancy
- Although great advances have been accomplished with in-vitro fertilization (IVF) technology, the pregnancy rate of IVF is still low (~25%)
- The quality of implantation determines the quality of ongoing pregnancy, and strategies to alleviate early pregnancy loss and improve implantation are of great theoretical interest, as well as practical urgency for reproduction improvement in human and mammals.
Animals and diets
- To investigate the effect of maternal short and medium chain fatty acids on pregnancy outcome and embryo implantation, pregnancy was induced by overnight caging of two females with one male of proven fertility.
- The presence of spermatozoa in the vaginal smear in the next morning was deemed as day 1 of pregnancy (1 dpc).
- Both the male rats and non-pregnant female rats were provided the basal diet while the pregnant rats were given the pregnancy diets
Animal study design
- Pregnancy rate, implantation rate, and the number of rats used in each experimental group are described in Table 2.
- Each experimental group contained 9 rats on each day. Each group was randomly assigned to one of three treatment groups fed the pregnancy diets respectively supplemented with 0.1%, SB, SH, or SC during the whole pregnancy, and kept for 21-23 d.
Uterus tissues isolation and in vitro culture
- Uterine tissues were obtained within 1 h from rats at 5 dpc for protein analysis, and female rats under regularly physiological cycle for mRNA analysis.
Granulosa cells isolation and in vitro culture
- Female SD rats aged 22~26 days old were injected with 0.5 IU/g PMSG to induce superovulation.
- After 48 h, ovaries were obtained, and granulosa cell were harvested from punctured ovarian follicles. The oocytes were removed by filtering cell suspension through a 40 μm nylon cell strainer (BD Falcon).
Fatty acids profile and lipid metabolites
- In diet, short-chain fatty acids were determined by ion chromatography; medium and long chain fatty acids by gas chromatography
- In serum, fatty acids profile was measured by GC-MS; lipid metabolites by high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS).
Biochemistry analysis in serum
- Serum progesterone (P4), estrogen (E2), and cholesterol were measured by ELISA using a commercially available kit (Cusabio Biotech Company, Wuhan, China).
mRNA analysis
- Total RNA extraction, and qPCR analysis of tissues and granulosa cells were performed as previously described [23]
- The quality and quantity of total RNA were determined by gel electrophoresis and a NanoDrop Spectrophotometer (P330, Implen, Germany). The extracted RNA (1 μg) was reverse-transcribed to complementary DNA (cDNA) using Kit (Takara, Ostu, Japan).
- Primers were designed using Primer3.0 input online and shown in Table 3.
- All reactions were run in triplicate and normalized with the housekeeping gene β-actin. Relative gene expression data was determined by the 2-ΔΔCt method
Immunoblotting staining
- Total protein extraction and Western blot analysis of tissues were conducted as previously described
- The frozen ovarian/uterine samples were homogenized in RIPA lysis buffer containing protease inhibitors (Applygen, Beijing, China) and total protein content was measured by the BCA assay
- Protein was electrophoresed on SDS polyacrylamine gels and electrotransferred to PVDF membranes (Millipore)
- Blots were stripped and reprobed with anti-β-actin antibody (Cell Signaling Technology, 13E5) to demonstrate equal loading
- After washing of membranes with 1× TBST, membranes were incubated with the HRP-conjugated goat anti-rabbit IgG
Immunofluorescence staining
- Frozen ovaries were cryosectioned using a microtome within a cryostat (Bright Instrument Company Limited, Huntingdon, UK) to a thickness of 5 μm onto uncoated glass microscope slides, then stained within 1 h of sectioning, and followed with three times washing for 5 min in phosphate-buffered saline (PBS).
- Monoclonal rabbit antiserum to recombinant rat StAR and CYP11A1 were applied respectively to the section for 2 h at room temperature.
- Goat anti-rabbit IgG and DAPI was added to the secondary antibody.
Statistical Analysis
- Statistical analysis and graphic construction were undertaken using SPSS and Graph Pad Prism, respectively.
- Each rat was considered an experimental unit, and the outliers were removed according to the Grubbs test. Data are shown as means ± SEM unless stated otherwise. Statistical tests were two-tailed, and used unpaired Student’s t-test. The different superscript between bars indicates the significance at P<.05.
Maternal short and medium chain fatty acids supply improves pregnancy outcome and embryo implantation
- Increased litter sizes and live litter size, respectively compared to Con group.
- Lower parturition rate and implantation rate of SH and SC groups than that of SB and Con groups compared to the control group (Table 2).
Short and medium chain fatty acids increase progesterone level and decrease TC, HDL and LDL levels
- Progesterone levels in rats of SB, SH, and SC groups were significantly increased compared to control group at 5 dpc, whereas no significant difference was observed at 6 dpc.
- Serum high-density lipoprotein cholesterol (HDL) level in both SB and SH groups, but not in SC group, was significantly reduced, whereas serum TC, LDL, and HDL levels did not differ.
Short and medium chain fatty acids strengthen ovarian steroidogenesis process via enhancing FAT/CD36, StAR and CYP11A1 expression
- We determined the expressions of key molecules involved in steroidogenesis, membrane transport of lipoprotein (CD36), and G-protein-coupled-receptors
- Relative mRNA abundance of CD36, steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme, and GPR43 in ovarian tissues at 5 dpc increased in the SB, SH, and SC groups compared to the Con group (Fig. 2B).
- However, no significant difference was observed in the relative abundance of cytochrome P450 17α-hydroxylase (CYP17A1), Cytochrome 19α-Hydroxyase, GPR40
- Notably, SB increased the relative mRNA abundance in granulosa cells in a dose-dependent manner suggesting an induced effect of luteinization.
Short and medium chain fatty acids modulate fatty acids profile and lipid metabolism
- The SB group at 5 and 6 dpc had a lower serum palmitic acid (C16:0) level than the control group (Figs. 3 and S3).
- Interestingly, maternal SB, SH, or SC supply consistently resulted in a higher serum arachidonic Acid (C20:4n6) level.
- Levels of serum butyrate, hexanoate, and caprylate in the SB group and dietary SC treatment group were lower than the detection limit, while in dietary SB and SH treatments, only a small amount of serum Butyric and Hexanoate were detected
- A total of 273 lipid metabolites were detected, with 23 metabolites were significantly different in rats fed the FA diet compared to the Con diet
Short and medium chain fatty acids promote uterine LPA3 and COX2 expression
- The expression of key genes associated with phospholipid metabolism and embryo implantation were detected in both the SH and SC groups compared to the Con group at 5 dpc (Fig. 5A).
- Protein abundance of uterine LC/LPA3 in SB group was significantly increased compared to Con group.
- Similarly, the protein abundance of *** COOX2 in SB increased significantly, and mRNA abundance increased.
Discussion
- Modulating a balanced metabolism of lipids and fatty acids can be a potential strategy to integrate nutrition and reproduction
- Our results indicated that maternal short and medium chain fatty acids supply improve pregnancy outcome in rats
- The lower parturition rate and implantation rate in SH and SC groups may be the dose of SH/SC used in this study decreased the fertilization rate or terminated the pregnancy during gestation.
- We speculated that SCFA increased the ability of luteal secretion of progesterone before embryo implantation
- Whether SCFA exert impact on placental growth or not remains unknown
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