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. 2013 Jul 23;305(6):E687–E699. doi: 10.1152/ajpendo.00254.2013

Fig. 2.

Fig. 2.

FAK Y397E phosphomimetic mutant-induced defects in spermiation in the rat testis are mediated by disrupting the stage-specific and spatiotemporal expression of actin-related protein 3 that induces branched actin polymerization (Arp3) and F-actin at the apical ES. A: in testes overexpressed with pCI-neo empty vector (pCI-neo/Ctrl), Arp3 (red) was high at the apical ES in stage VII tubules, but it was restricted to the concave side of the spermatid head, colocalized with F-actin (green), where the endocytic vesicle mediated protein trafficking known to occur to facilitate protein endocytosis, transcytosis, and recycling, so that “old” apical ES proteins can be used to assemble “new” apical ES for newly formed step 8 spermatids in stage VIII tubules. Thus, the upregulation of Arp3 at this site facilitated endocytic trafficking events. However, Arp3 expression was virtually nondetectable at the basal ectoplasmic specialization (ES) of the blood-testis barrier (BTB) when the BTB was intact. However, in stage VIII tubules, Arp3 expression was high at the basal ES of the BTB, colocalizing with F-actin, to facilitate BTB restructuring to allow the transport of preleptotene spermatocytes across the BTB. Arp3 expression at the apical ES was considerably diminished in stage VIII tubules to an almost nondetectable level, and F-actin was also barely visible at the apical ES, which thus facilitated spermiation. While overexpression of pCI-neo/FAK Y397E did not perturb the expression nor distribution of Arp3 and F-actin considerably at the apical ES in stage VII tubules, its overexpression upregulated Arp3 expression and retained a considerable amount of F-actin at the apical ES, rendering stage VIII tubules to mimic stage VII tubules regarding the expression and/or localization of Arp3 and F-actin at the apical ES. Cell nuclei visualized by DAPI. Scale bar = 60 and 15 μm in inset, which apply to corresponding micrographs and insets. B: image analysis of Arp3 (red) at the apical ES surrounding the head of step 19 spermatids (n = 300 spermatids from 4 rat testes) was performed (see materials and methods) in which the level of Arp3 in pCI-neo/Ctrl in stage VII tubules was arbitrarily set at 1. pCi-neo/FAK Y397E was compared with the control group in stage VII or VIII tubules. *P < 0.05; nd, nondetectable.