Characterization of ovine follicular fluid and granulosa cell-derived extracellular vesicles and their miRNA cargo following in vitro exposure to bisphenols A and S.

This study characterizes the miRNA cargo of ovine granulosa cells and their derived extracellular vesicles following exposure to bisphenols A and S, revealing that while BPA and BPS have limited effects on cellular miRNA expression, they significantly alter the miRNA profiles of secreted vesicles, including the downregulation of miR-1306 homologs.

The Gist

Imagine a sheep's ovary as a bustling, high-tech construction site where a future baby (the egg) is being built. The workers on this site are called Granulosa Cells. They don't just sit there; they chat constantly with each other and with the egg to make sure everything goes smoothly.

Here is the simple story of what this paper discovered, using some everyday analogies:

1. The "Text Messages" of the Ovary (Extracellular Vesicles)

The workers (cells) don't just talk by shouting; they send out tiny, waterproof bubbles called Extracellular Vesicles (EVs). Think of these bubbles as text messages or envelopes floating through the fluid surrounding the egg.

Inside these envelopes are miRNAs. If the cell is a computer, miRNAs are the software updates or instructions that tell the computer what to do next (like "grow faster," "stop dividing," or "make hormones"). These bubbles travel through the Follicular Fluid (the soup inside the ovary) to deliver these instructions.

2. The Villains: The "Plastic Chemicals" (BPA and BPS)

The study looked at two common chemicals found in plastics: Bisphenol A (BPA) and Bisphenol S (BPS). You might find these in water bottles, food containers, or receipts.

• The Problem: These chemicals are "imposters." They look like the body's natural hormones (estrogen), so they trick the cells into listening to them.
• The Experiment: The scientists took sheep cells, put them in a lab dish, and exposed them to these chemicals for 48 hours to see how the "text messages" changed.

3. What Happened to the Workers? (The Hormone Shift)

Before looking at the messages, the scientists checked the workers' output.

• BPA (The Heavy Hitter): It messed up the factory. The cells stopped making Progesterone (a hormone needed for pregnancy) and started overproducing Estrogen. It was like a factory suddenly switching from making car engines to making only tires.
• BPS (The Sneaky One): It didn't stop Progesterone as much, but it still boosted Estrogen. Both chemicals disrupted the normal rhythm of the ovary.

4. The "Text Messages" Got Glitched (The miRNA Changes)

This is the main discovery. The scientists opened the "envelopes" (EVs) and read the "software updates" (miRNAs) to see if the chemicals changed the instructions.

• Inside the Cells (The Factory Floor):

  • BPA: Surprisingly, the instructions inside the cells didn't change much. The workers were confused about the hormones, but their internal software seemed stable.
  • BPS: This one did change one specific instruction inside the cell, turning down a volume knob on a message called oar-24b.

• In the Envelopes (The Text Messages Sent Out):

  • BPA: This chemical was very active in changing the outgoing messages. It altered four different instructions being sent out in the bubbles. It was like the factory manager suddenly sending out four new, confusing memos to the rest of the building.
  • BPS: It changed two outgoing messages.
  • The Common Glitch: Both chemicals reduced the amount of a specific message called miR-1306. In sheep, this message is crucial for deciding how many babies a sheep can have (litter size). By lowering this message, the chemicals might be accidentally telling the ovary, "Hey, maybe we shouldn't make as many eggs."

5. The Big Picture: Why Should We Care?

Think of the ovary as a delicate orchestra. The cells are musicians, the hormones are the music, and the miRNA bubbles are the sheet music.

• The Study's Conclusion: Even though BPA and BPS look similar (they are both "bisphenols"), they play the orchestra differently. BPA is like a conductor who changes the tempo and volume wildly. BPS is more subtle but still changes the sheet music.
• The Risk: Because these chemicals change the "sheet music" (miRNAs) that cells send to each other, they could disrupt the entire process of making a healthy egg. This might explain why exposure to these plastics is linked to fertility problems in humans and animals.

Summary in One Sentence

This study found that common plastic chemicals (BPA and BPS) confuse sheep egg cells, causing them to send out the wrong "text messages" (miRNAs) to their neighbors, which could disrupt fertility and how many babies are born.

Technical Summary

1. Problem Statement

Endocrine-disrupting chemicals (EDCs), specifically Bisphenol A (BPA) and its substitute Bisphenol S (BPS), are known to impair female reproductive function and ovarian steroidogenesis. While previous studies have established that these compounds disrupt granulosa cell (GC) function, the molecular mechanisms involving extracellular vesicles (EVs) and their microRNA (miRNA) cargo as mediators of cellular response to environmental stress remain poorly understood, particularly in the ovine model. Furthermore, there is a lack of comprehensive annotation of the ovine miRNA repertoire compared to humans or bovines. This study aims to:

• Investigate the impact of acute BPA and BPS exposure on the miRNA expression profiles of ovine granulosa cells and their secreted EVs.
• Characterize the miRNA cargo of follicular fluid (FF) EVs versus conditioned media (CM) EVs.
• Expand the annotation of the ovine miRNA genome.

2. Methodology

Experimental Design:

• Model: Primary ovine granulosa cells (GCs) isolated from follicles (2–6 mm) of adult ewes and lambs.
• Treatment: Cells were cultured for 48 hours under three conditions:
  1. Control (0.01% ethanol vehicle).
  2. 10 µM Bisphenol A (BPA).
  3. 10 µM Bisphenol S (BPS).
• Sample Collection:
  • Cellular: GCs harvested at 0h (seeding) and 48h (post-treatment).
  • Extracellular Vesicles (EVs): Isolated from:
    • Native Follicular Fluid (FF EVs).
    • 48-hour Conditioned Media (CM EVs) from treated GCs.

Characterization Techniques:

• Steroidogenesis: Quantification of Progesterone and Estradiol in conditioned media via ELISA/EIA.
• EV Isolation: Differential ultracentrifugation (3000g, 12,000g, and 100,000g).
• EV Validation:
  • Morphology: Transmission Electron Microscopy (TEM).
  • Size/Concentration: Nanoparticle Tracking Analysis (NTA).
  • Protein Markers: Western Blotting for tetraspanins (CD63, CD81, CD9), Alix, HSPA1A, and negative markers (Calnexin, Tubulin).
• miRNA Sequencing:
  • Small RNA sequencing (Illumina NextSeq 2000, 50bp single read).
  • Bioinformatics: Used the nf-core/smrnaseq pipeline mapped to the Ovis aries genome (ARS-UI_Ramb_v3.0).
  • Annotation Strategy: Combined existing genome annotation with miRMachine (homology search) and miRDeep2 + RumimiR database (novel prediction) to extend the ovine miRNA catalog.
  • Statistical Analysis: DESeq2 for differential expression (FDR ≤ 0.05); PCA for clustering.

3. Key Contributions

1. Expansion of Ovine miRNA Annotation: The study identified 533 miRNA sequences in total, including 129 novel sequences (24.2% of the total). This significantly enriches the reference database for ovine reproductive biology.
2. Comparative EV Cargo Profiling: It is the first study to qualitatively and quantitatively compare miRNA cargo between native Follicular Fluid EVs, cellular GCs, and EVs secreted by GCs in vitro under chemical stress.
3. Differential Impact of BPA vs. BPS: The study elucidates that while both bisphenols disrupt steroidogenesis, they exert distinct and subtle effects on the miRNA regulatory network within cells and their secreted EVs.

4. Key Results

A. Steroidogenesis Disruption:

• BPA (10 µM): Significantly decreased Progesterone secretion (-19%) and drastically increased Estradiol secretion (+511%).
• BPS (10 µM): Did not affect Progesterone but significantly increased Estradiol secretion (+46%).
• Conclusion: Both compounds disrupt the steroidogenic balance, but BPA has a more profound effect on progesterone suppression.

B. EV Characterization:

• Morphology: TEM confirmed spherical/cup-shaped vesicles.
• Size Distribution:
  • FF EVs: Peak size ~135 nm; 67.8% small EVs (<200 nm).
  • CM EVs: Peak size ~125 nm; 77.5% small EVs.
• Bisphenol Effect: Neither BPA nor BPS altered the total particle concentration or size distribution of EVs secreted by GCs.
• Markers: EVs were positive for CD63, CD81, and Alix. Detection of HSPA1A and Calnexin suggested the presence of non-vesicular extracellular particles (NVEPs) or early endosomes.

C. miRNA Expression Profiles:

• Global Detection: 533 miRNAs identified (103 known, 129 novel, 301 homology/predicted).
• Matrix Differences:
  • FF EVs and 0h GCs shared ~68% of sequences but showed significant differential expression (312 DE miRNAs).
  • CM EVs contained significantly fewer miRNA reads (0.3% of total counts) compared to FF EVs (38.5%) and GCs (22%), likely due to lower RNA yield requiring higher amplification.
• Bisphenol Effects on miRNA:
  • In Granulosa Cells (GC):
    • BPA: No significant differential expression of miRNAs compared to control.
    • BPS: Significantly downregulated oar-24b (homologous to miR-24b).
  • In Conditioned Media EVs (CM EVs):
    • BPA: Altered 4 miRNAs (2 upregulated, 2 downregulated). Notably, downregulated oar-03746 (homologous to miR-1306-5p) and a novel sequence.
    • BPS: Altered 2 miRNAs (downregulated oar-03746 and a novel sequence).
    • Common Effect: Both BPA and BPS reduced the expression of sequences homologous to miR-1306.

D. Target Prediction:

• Predicted targets for the differentially expressed miRNAs (e.g., miR-1306) include genes involved in apoptosis (BMPR1B) and TGF-β/SMAD signaling pathways, which are critical for folliculogenesis and litter size in sheep.

5. Significance and Conclusion

• Mechanistic Insight: The study suggests that BPA and BPS modulate intercellular communication via EVs, even when cellular miRNA changes are minimal. The specific downregulation of miR-1306 (a known promoter of GC apoptosis via BMPR1B suppression) in EVs suggests a potential mechanism by which bisphenols could induce follicular atresia or alter oocyte competence.
• Species Specificity: The findings highlight that BPA and BPS, despite structural similarities, act through distinct molecular pathways in ovine GCs, with BPS showing a unique effect on cellular miR-24b and BPA showing a broader impact on EV cargo.
• Resource Generation: The identification of 129 novel miRNAs provides a valuable resource for future studies on sheep reproduction, fertility, and the impact of environmental toxins.
• Limitations: The study notes that quantitative comparison between cellular and EV miRNAs was limited by amplification differences in low-yield CM samples. Future work is needed to link these miRNA changes directly to target gene expression and functional outcomes (e.g., apoptosis rates).

Overall Conclusion: Acute exposure to BPA and BPS alters the miRNA cargo of ovine granulosa cell-derived EVs, particularly affecting miRNAs linked to apoptosis and signaling pathways, potentially compromising follicular development and female fertility.