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Peroxiredoxin 6 Plays Essential Role in Mediating Fertilization and Early Embryonic Development in Rabbit Oviduct

  • Reproductive Biology: Original Article
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Abstract

The oviduct is a site for early reproductive events including gamete maturation, fertilization, and early embryo development. Secretory cells lining the oviduct lumen synthesize and secrete proteins that interact with gametes and developing embryos. Although previous studies have identified some of the secretory proteins in the oviduct, however, knowledge and their precise specific functions in the oviduct are poorly understood. In this study, by using proteomic approach, we identified a secretory protein, Peroxiredoxin 6 (PRDX6), and evaluated its role in mediating early pregnancy events, fertilization, and embryo development in rabbit oviduct. The expression of PRDX6 was significantly higher in ampulla and isthmus sections of the oviduct in mated animal groups compared to non-mated controls. Furthermore, significant reduction in number of embryos recovered from PRDX6 siRNA-transfected oviductal horn was observed compared to the control contralateral horn. Moreover, in animals receiving PRDX6 siRNA in their oviductal horn, the number of implanted blastocysts was significantly less in the uterus as observed on day 9 post-coital (p.c.). Further, during embryo-rabbit oviduct epithelial cell (ROEC) co-culture, siRNA-mediated PRDX6 silencing attenuated the early embryonic development. Mechanistically, increased levels of ROS and expression of oxidative stress- and inflammation-related proteins were found in PRDX6 siRNA-treated ROEC cells as compared to control cells, implicating that ablation of PRDX6 in the oviduct creates a stress-induced micro-environment detrimental to early embryonic development in oviduct. Taken together, our data suggest that PRDX6 maintains an optimal micro-environment conducive to successful embryo development and can be considered as a candidate to evaluate its therapeutic potential in IVF strategies.

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Abbreviations

ROS:

Reactive oxygen species

p.c.:

Post-coitum

ART:

Assisted reproductive technology

IVF:

In vitro fertilization

ROECs:

Rabbit oviductal epithelial cells

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Acknowledgements

The authors wish to thank Dr. M.P. Singh, Principal Scientist, CSIR-IITR, Lucknow, for his help and valuable suggestions during the study. The authors wish to thank Dr. T.J. Reddy SAIF-facility, CSIR-CDRI, for help in MS/MS analysis. P.P. is recipient of Senior Research Fellowship from the Indian Council of Medical Research, New Delhi. We thank Skyler Hendrix and Anushree Seth for assisting with English language use in association with InPrint, Washington University, St. Louis, MO. The CSIR-CDRI communication number is 10263.

Funding

This work was supported by grants from the CSIR network project “PROGRAM” on “Factors Governing Competent Gamete Production and Reproductive Dysfunction” (BSC0101).

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Authors and Affiliations

  1. Division of Endocrinology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, 226031, India

    Pooja Popli, Vinay Shukla, Jyoti B. Kaushal, Rohit Kumar, Kanchan Gupta & Anila Dwivedi

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  1. Pooja Popli
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  2. Vinay Shukla
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  3. Jyoti B. Kaushal
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Contributions

A.D. conceptualized the study. P.P., V.S., J.B.K., R.K., and K.G. performed the experiments. P.P. and A.D. analyzed the data. P.P. and A.D. wrote the manuscript. All authors have approved the final version of the manuscript.

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Correspondence to Anila Dwivedi.

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Ethics Approval

All experimental procedures were performed with the guidelines and protocols approved by the Institutional Animal Ethics Committee for the Use of Laboratory Animals of CSIR-CDRI, Lucknow, India (Approval No.: IAEC/2012/24).

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Supplementary Information

Supplementary Figure S1:

2-D gel electrophoresis (2-D PAGE) between ampullary region of non-mated (control) versus mated rabbit (10.5 h p.c.) (n=3). (A) Representative gel image of 2-D PAGE is shown here. A total of 15 differentially altered protein spots (upregulated or downregulated) were identified by image master 2-D platinum software. (B) The spot volumes of the differentially expressed proteins (up or down) in ampulla of control (non-mated) and mated rabbit is shown as bar diagram in the lower panel. The data are expressed as mean±SE, n=3. P values are ‘b’ (P<0.01) and ‘c’ (P<0.05) versus control (non-mated). (JPG 5637 kb)

Supplementary Figure S2:

Localization of PDI, PP1, TPM1, PRDX6, PAF in ampulla of control (non-mated) and mated rabbits (10.5 h p.c.). Lower panel shows image analysis of PDI, PP1, TPM1, PRDX6, and PAF in ampulla of both groups. The immunostaining intensity of all proteins in the epithelium, stroma, lamina propria and smooth muscle cells were assessed semi-quantitatively on a four-point scale: 0 for no staining, 1 for mild staining, 2 for moderate staining and 3 for intense staining. The histologic score (HSCORE) was calculated as follows: HSCORE =Σ Pi (i + 1), where i=1, 2 or 3, and Pi is the percentage of each intensity, from 0 to 100%. Str (Stroma), GE (glandular epithelium), and LE (luminal epithelium). Magnification 10X, n=6 in all the groups. (JPG 7493 kb)

Supplementary Figure S3:

Rabbit oviductal epithelial cells (ROECs) were characterized by the expression of cytokeratin, an epithelial marker, by immunofluorescence. Primary ROECs were fixed, permeabilized, incubated with cytokeratin antibody for overnight, and incubated with FITC-conjugated anti-mouse antibody for 1 h. The preparations were washed and counterstained with DAPI and cell images were grasped using a Leica fluorescence microscope at 20X. (JPG 1892 kb)

Supplementary Figure S4:

Unfertilized oocytes recovered from scram or PRDX6 siRNA- transfected oviductal horns. (JPG 618 kb)

Supplementary Table S1:

Differentially expressed proteins identified by MS/MS analysis between ampullary regions of non-mated (control) versus mated (10.5 h p.c.) rabbit oviduct. (DOCX 14 kb)

Supplementary Table S2:

Summary of results obtained from proposed study. (DOCX 13 kb)

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Popli, P., Shukla, V., Kaushal, J.B. et al. Peroxiredoxin 6 Plays Essential Role in Mediating Fertilization and Early Embryonic Development in Rabbit Oviduct. Reprod. Sci. 29, 1560–1576 (2022). https://doi.org/10.1007/s43032-021-00689-x

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