Role of PAFAH1B1 in human spermatogenesis, fertilization and early embryonic development Gui-Dong Yao, Sen-Lin Shi, Wen-Yan Song, Hai-Xia Jin, Zhao-Feng.

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Role of PAFAH1B1 in human spermatogenesis, fertilization and early embryonic development Gui-Dong Yao, Sen-Lin Shi, Wen-Yan Song, Hai-Xia Jin, Zhao-Feng Peng, Hong-Yi Yang, En-Yin Wang, Ying-Pu Sun Reproductive BioMedicine Online Volume 31, Issue 5, Pages 613-624 (November 2015) DOI: 10.1016/j.rbmo.2015.07.010 Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 PAFAH1B1 mRNA and protein expression in testicular tissue and spermatozoa. (A,B) Bar chart and Gaussian nonlinear regression analysis graph for PAFAH1B1 mRNA expression against Johnsen scores 2–10 in testicular tissue. (C) Comparison of PAFAH1B1 mRNA expression among testicular tissue samples with Johnsen score of 8–9 and 10, and sperm purified by swim-up from ejaculated semen. JS = Johnsen score. (D) Representative sections of immunohistochemical staining for PAFAH1B1 protein in testicular tissue sections from two patients with normal spermatogenic ability (Johnsen score 10). The images in panels b and d show higher magnification of views from the boxes marked in panels a and c, respectively. Red arrowheads = spermatogonia; red arrows = spermatocytes; green arrowheads = spermatids; green arrows = spermatozoa. Scale bars (a,c) = 10 µm, (b,d) = 5 µm. Reproductive BioMedicine Online 2015 31, 613-624DOI: (10.1016/j.rbmo.2015.07.010) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Immunofluorescence for PAFAH1B1 protein expression in spermatozoa. The two panels are taken under the same confocal laser scanning microscopy, with the one is under the bright field without laser, and the other is activated with the corresponding wavelength laser. (A) PESA-derived (a–d) and ejaculated (e–h) spermatozoa. (B) Ejaculated spermatozoa, from proven fertile men, following direct centrifugation of semen (a,b), purification by gradient centrifugation (c,d), following a 6-h incubation with oocytes and either unbound (e,f), or bound (g,i,j) to oocytes, and spermatozoa incubated for 6 h with and bound to cleavage staged embryos (the spermatozoa still bound to the abnormally fertilized day 3 embryos from the same couple) (h). Scale bar = 5 µm. Reproductive BioMedicine Online 2015 31, 613-624DOI: (10.1016/j.rbmo.2015.07.010) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 PAFAH1B1 mRNA and protein expression in human oocytes, zygotes, cleaving embryos and blastocysts. (A) Relative PAFAH1B1 mRNA expression in unfertilized MII oocytes, 1-cell zygotes, cleavage stage embryos and expanding blastocysts. NS = no significant difference; P-value < 0.05 indicates a significant difference. (B) Representative immunofluorescence staining for PAFAH1B1 in a MII oocyte (a), 1-cell zygote (b), 4-cell stage embryo (c), 8-cell stage embryo (d), 10-cell stage embryo (e), 16-cell stage embryo (f), morula (g), expanding blastocyst (h) and hatched blastocyst (i). Scale bar = 50 µm. Reproductive BioMedicine Online 2015 31, 613-624DOI: (10.1016/j.rbmo.2015.07.010) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Relationships between PAFAH1B1 mRNA expression in testicular tissue and oocyte fertilization (A), embryo cleavage (B) or high quality embryo (C) rates, or pregnancy outcome (D). PAFAH1B1 mRNA expression was normalized to that of the housekeeping gene GAPDH. Reproductive BioMedicine Online 2015 31, 613-624DOI: (10.1016/j.rbmo.2015.07.010) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions