Interleukin 1alpha-induced disruption of the Sertoli cell cytoskeleton affects gap junctional communication

Cellular Signalling 28 (2016) 469–480

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Interleukin 1alpha-induced disruption of the Sertoli cell cytoskeleton affects gap junctional communication Katarzyna Chojnacka a, Barbara Bilinska b, Dolores D. Mruk a,⁎ a b

Center for Biomedical Research, Population Council, 1230 York Avenue, New York, NY, USA Department of Endocrinology, Institute of Zoology, Jagiellonian University, Gronostajowa 9, 30-387 Krakow, Poland

a r t i c l e

i n f o

Article history: Received 22 December 2015 Accepted 8 February 2016 Available online 13 February 2016 Keywords: Sertoli cell Testis Interleukin 1alpha Gap junction Cytoskeleton

a b s t r a c t Gap junctions (GJ) are transmembrane channels that connect the cytoplasms of adjacent cells, thereby facilitating the rapid exchange of ions, second messengers, and metabolites smaller than 1 kDa. Connexin 43, the beststudied GJ protein, is a component of the Sertoli cell barrier/blood–testis barrier (BTB). To gain insight into the biology of the BTB, we investigated the effects of interleukin 1alpha (IL1A), a pro-inflammatory cytokine that disrupts BTB function, on gap junctional communication (GJC) in Sertoli cells. Compared with controls, the levels of connexin 43 and connexin 43 (Ser 368) increased ~30- and 20-fold, respectively, at 24 h after IL1A treatment. To assess GJ function, we investigated fluorescence recovery in photobleached Sertoli cells after vehicle or IL1A treatment. Compared with the control, IL1A affected the ability of calcein to return to photobleached cells, indicating that GJC was compromised. To explain the effects of IL1A on GJ function, the involvement of the Sertoli cell cytoskeleton was investigated. Stress fibers aggregated at the periphery of Sertoli cells treated with IL1A. These results were substantiated by a biochemical assay that showed IL1A to disrupt the bundling of exogenous Factin by Sertoli cells. In summary, IL1A regulates GJC in Sertoli cells, which is critical for BTB restructuring. © 2016 Elsevier Inc. All rights reserved.

1. Introduction Spermatogenesis relies on critical interactions between Sertoli cells, as well as between Sertoli and germ cells, in the seminiferous epithelium of the mammalian testis. In general, cell–cell interactions can be transient such as those occurring between immune cells and a pathogen, or stable such as those occurring between cells connected by cell junctions. Gap junctions are transmembrane channels that connect the cytoplasms of two adjacent cells, thereby allowing for the rapid exchange of ions, second messengers, and metabolites smaller than 1 kDa (for reviews, [1–3]). They are critical for many biological processes that include the contraction of cardiomyocytes, neurotransmission across electrical synapses in neurons, and germ cell development throughout spermatogenesis (for reviews, [4–7]). Structurally, the gap junction is composed of two apposed homotypic or heterotypic hemichannels or connexons, with each connexon consisting of six identical (defined as a homomeric connexon) or different (defined as a heteromeric connexon) connexins arranged around a 1.2-nm diameter central pore. Connexins, the building blocks of gap junctions in vertebrates, constitute a large family of evolutionarily conserved proteins with 21 and 20 members in the human and rat genomes, respectively, originally named according to their predicted molecular weights (e.g., connexin ⁎ Corresponding author at: Center for Biomedical Research, Population Council, 1230 York Avenue, New York, NY 10021, USA. E-mail address: [email protected] (D.D. Mruk).

http://dx.doi.org/10.1016/j.cellsig.2016.02.003 0898-6568/© 2016 Elsevier Inc. All rights reserved.

43, a 43-kDa protein in the human, corresponds to CX43) (for reviews, [3,8–10]). However, this naming system can be confusing when connexins from different species are discussed within the same context. Thus, connexins have been renamed according to evolutionary characteristics (e.g., connexin 43 corresponds to GJA1). Connexin is a fourpass transmembrane protein having one cytoplasmic loop, two extracellular loops, and cytoplasmic amino acid and carboxy termini. Although the four transmembrane domains and two extracellular loops are well conserved, the cytoplasmic loop and the carboxy terminus are variable across the different connexins. Furthermore, the carboxy terminus serves as the site for protein–protein interactions (for reviews, [11,12]). In the testis, 14 connexins have been found to be expressed: connexins 26, 30.2, 31, 31.1, 31.9, 32, 33, 37, 40, 43, 45, 46, 50, and 57 (for reviews, [6,13]). Of these, connexin 43 is the best-studied gap junction protein, and it is highly expressed by Sertoli, germ, Leydig, and peritubular myoid cells in the adult mammalian testis (for a review, [6]). In the seminiferous epithelium of the adult mouse/rat testis, connexin 43 is present between Sertoli cells at the Sertoli cell barrier/ blood–testis barrier (BTB), as well as between Sertoli cells and spermatogonia, spermatocytes, and possibly spermatids [14–17]. Not surprisingly, Cx43-null mice die shortly after birth from a failure in pulmonary gas exchange caused by the swelling and blockage of the right ventricular outflow tract that connects to the pulmonary artery [18]. Although Cx43-knockin mice, produced by replacing the Cx43 coding region with the coding region of Cx32 or Cx40, rescue the postnatal

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lethality of the Cx43-null mice, homozygotes display enormous defects in the testis [19]. Specifically, spermatogonia and secondary spermatocytes are absent, and only Sertoli cells occupy seminiferous tubules, revealing that connexin 43 is indispensable for fertility. By contrast, connexin 43 knockdown in cultured rat Sertoli cells by RNA interference has no apparent effect on cell function [15], suggesting that there is functional compensation by other members of the connexin family of proteins in this model. Other in vitro and in vivo studies report that environmental toxicants (e.g., cadmium, bisphenol A, perfluorooctane sulfonate) affect gap junction function by inhibiting cell–cell communication, which may explain some cases of unexplained infertility/ subfertility in humans ([20–22]; for reviews, [23–25]). Thus, connexin 43 is critical for spermatogenesis. To gain new insight into the function and regulation of gap junctions at the Sertoli cell barrier/BTB, we investigated the effects of the potent pro-inflammatory cytokine interleukin 1alpha (IL1A) on gap junctional communication in cultured rat Sertoli cells. Except for a study performed on the 42GPA9 Sertoli cell line [26], the effects of IL1A on gap junctional communication in Sertoli cells have not been previously investigated. IL1 is a multifunctional cytokine that is comprised of two isoforms: IL1A and interleukin 1beta (IL1B); both proteins are encoded by separate genes. Although many tissues constitutively produce IL1A in the absence of inflammation, IL1B is generally produced by activated macrophages (for reviews, [27–32]). In the rat testis, IL1A is developmentally [i.e., detectable from postnatal day 20–25] and stagespecifically [i.e., undetectable at stage VII of the seminiferous epithelial cycle but high at all other stages] expressed by Sertoli cells as a 31kDa precursor protein that is cleaved by calpain to produce a 17-kDa mature protein. Both precursor and mature proteins also exert bioactivity in a murine proliferation assay. Indeed, recombinant IL1A stimulates DNA synthesis in intermediate and type B spermatogonia [33], indicating that it is a mitogenic factor. In terms of its function in the testis, IL1A participates in the paracrine regulation of spermatogenesis [34–38]. Although germ cells appear to express IL1A [39], they do not secrete it into the culture medium [40]. Furthermore, IL1A disrupts the function of the Sertoli cell barrier/BTB ([41,42]; for a review [31]), suggesting that it participates in the restructuring of the BTB, which allows meiotic germ cells to enter the adluminal compartment of the seminiferous epithelium. In this study, we show that IL1A affects both actin and microtubule cytoskeletons, resulting in the disruption of gap junctional communication in Sertoli cells. 2. Materials and methods 2.1. Animals Twenty-day old (body weight, ~ 50 g) male Sprague Dawley® rats (Crl:SD) were purchased (Charles River Laboratories, Kingston, NY) and housed at the Comparative Bioscience Center of the Rockefeller University for 24–72 h under conditions of 12 h light:12 h dark with access to standard rat chow and fresh water ad libitum. Rats were terminated by CO2 asphyxiation using a slow displacement (20–30%/min) of chamber air. All other guidelines issued by the Institutional Care and Use Committee of the Rockefeller University were followed under protocol numbers 12-506 and 15780-H. 2.2. Isolation of Sertoli cells and treatment of cells with interleukin 1α Sertoli cells were isolated from the testes of 20-day-old Sprague Dawley® rats and cultured in serum-free Dulbecco's Modified Eagle's Medium/Nutrient F-12 Ham (DMEM/F-12; Sigma-Aldrich, St. Louis, MO) containing gentamicin, penicillin–streptomycin, transferrin, insulin, epidermal growth factor (receptor-grade), and phenol red (sodium salt, containing negligible estrogenic activity [43]) as previously described [44–46]. Sertoli cells were isolated from the testes of 20-dayold rats due to the difficulty of obtaining highly pure adult Sertoli cells

[47,48]. Sertoli cells were plated at a density of 1.0 × 106 cells/cm2 on Millicell® culture plate inserts (HA membrane, 0.45 μm pore size, 12 mm insert size; EMD Millipore, Billerica, MA) for transepithelial electrical resistance readings, 0.5 × 106 cells/cm2 on 6-well polystyrene plates for lysate preparation, 0.04–0.075 × 106 cells/cm2 on 18-mm diameter round no. 1 cover glasses (Thomas Scientific, Swedesboro, NJ) for fluorescent immunostaining/F-actin labeling or 0.2 × 106 cells/cm2 on 35-mm diameter no. 1.5 glass-bottom culture dishes for fluorescence recovery after photobleaching (FRAP). Under these experimental conditions, Sertoli cells were confluent at all densities, with cell junctions (i.e., tight junctions, ectoplasmic specializations, gap junctions, and desmosomes) assembling by day 3 in vitro [49–55]. This conclusion is based on several studies in which junction assembly and stability were evaluated by different approaches that included the use of mono-specific antibodies against component proteins (e.g., claudin 11, N-cadherin, connexin 43) for immunoblotting and fluorescent staining, as well as functional assays (e.g., transepithelial electrical resistance readings, fluorescence recovery after photobleaching) that assess junctional integrity. Culture plate inserts, plates, and cover glasses were coated with Matrigel™ (BD Biosciences, San Jose, CA), which was diluted 1:7 with medium. Cultures were treated with a hypotonic buffer (20 mM Tris, pH 7.4) for 2.5 min at 48 h after plating to lyse contaminating germ cells [56,57]. The contamination of cultures with peritubular myoid and Leydig cells was periodically assessed by cell-specific markers (α-smooth muscle actin (ACTA2) for peritubular myoid cells [58]; hydroxy-δ-5-steroid dehydrogenase, 3β- and steroid δ-isomerase 1 (HSD3B1) for Leydig cells [59]) and immunoblotting as previously described [60]. The contamination of Sertoli cell cultures by other cells was negligible, and the purity of Sertoli cells was 95–98%. Sertoli cells were incubated on day 3 after plating with Escherichia coli-derived recombinant rat IL1A (molar mass, 17,5000 g/mol; R&D Systems, Minneapolis, MN) at a concentration of 100 pg/ml (5.71 pM) as previously described unless stated otherwise [42]. (The concentration of IL1A in the adult rat testis is 75 ± 8 pg/mg protein by rat IL-A-specific ELISA; ~200 pM [34]). IL1A was dissolved in phosphate-buffered saline (PBS; 10 mM NaH2PO4, 0.15 M NaCl, pH 7.4) containing 0.1% BSA (w/v) to yield a stock concentration of 5 μg/ml. DMEM/F-12 containing BSA served as the vehiclecontrol. Medium containing vehicle or IL1A was replaced daily until the end of the experiment. Sertoli cells were terminated at the indicated time points beginning on day 3 and ending on day 6 after plating. 2.3. Treatment of Sertoli cells with protein kinase C-α inhibitor Gö6976 and interleukin 1α This experiment determined whether IL1A-induced connexin 43 phosphorylation at Ser368 is mediated by protein kinase C-α (PKC-α). Gö6976 (5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3a]pyrrolo[3,4-c]carbazole-12-propanenitrile; molar mass, 378.43 g/ mol; IC50 = 7.9 nM) is a potent second-generation inhibitor of PKC-α and -β, but not PKC-δ, − ε, and ζ, indicating that it differentiates between Ca2+-dependent and -independent isoforms [61]. In brief, Sertoli cells were isolated from the testes of 20-day-old Sprague Dawley® rats and plated at a density of 0.5 × 106 cells/cm2 on 12-well polystyrene plates for lysate preparation. Cultures were treated with a hypotonic buffer, and Sertoli cells were allowed to recover for 24 h before treatment. On day 3 after plating, Sertoli cells were pretreated with Gö6976 at a concentration of 0.38 ng/ml or 1.89 ng/ml (1 nM or 5 nM, respectively) in growth factor-supplemented DMEM/F-12 for 3 h. Gö6976 was dissolved in dimethyl sulfoxide (DMSO) to yield a stock concentration of 10 mM. After pretreatment, the medium was aspirated, and cells were incubated with Gö6976 or Gö6976 and IL1A at the indicated concentrations. DMEM/F-12 containing only IL1A served as the positive control, and DMEM/F-12 containing DMSO served as the vehicle-control. At 24 h after treatment, Sertoli cells were lysed in lysis buffer [10 mM Tris, 0.15 M NaCl, 2 mM EDTA, 1% Nonidet™ P40 (v/v), 10% glycerol (v/v), pH 7.4] freshly supplemented with a broad-

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spectrum protease inhibitor cocktail (Sigma-Aldrich), 20 mM NaF, and 20 mM Na3VO4. Medium containing vehicle, IL1A, Gö6976 or Gö6976 and IL1A was replaced in the remaining wells. Sertoli cells were cultured for an additional day, and cultures were terminated thereafter. Sertoli cell lysates were used for immunoblotting. 2.4. Transepithelial electrical resistance The integrity of the permeability barrier in Sertoli cells treated with vehicle or IL1A was assessed by transepithelial electrical resistance as previously described [62,63]. In brief, an ~ 2-s pulse of current was passed through the Sertoli cell epithelium with a Millicell®-ERS volt– ohm meter (EMD Millipore). The first measurement was taken 1 d after plating cells, and four readings were obtained for each cell culture insert, which were then averaged. An averaged reading obtained from Matrigel™-coated blank inserts, which did not contain Sertoli cells, was subtracted from an averaged reading from inserts that did contain cells. Each value was multiplied by the surface area of the insert (i.e., 0.6 cm), and data were presented as Ω·cm2. Sertoli cells were treated with vehicle or IL1A from day 3 onwards. 2.5. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting Sertoli cells were lysed in lysis buffer. Protein concentrations were determined by the Pierce™ BCA protein assay (Thermo Fisher Scientific Inc., Waltham, MA) with bovine serum albumin as a standard. Equal amounts of protein were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and then transferred to nitrocellulose membranes. Membranes were blocked at room temperature with 5% nonfat milk (w/v) in PBS– Tris (10 mM NaH2PO4, 0.15 M NaCl, 10 mM Tris, pH 7.4) containing 0.1% Tween® 20 (v/v) and incubated with primary antibodies (Table 1), followed by species-compatible horseradish peroxidaseconjugated secondary antibodies diluted 1:1000 (Santa Cruz Biotechnology Inc., Santa Cruz, CA). Immunoreactive proteins were visualized by enhanced chemiluminescence as previously described [64]. Images were captured with an ImageQuant™ LAS 4000 mini imaging system (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and analyzed with Multi Gauge software (version 2.0), followed by densitometric analysis with Scion Image software (version 4.03, Scion Corp., Frederick, MD). Protein bands that were marked as saturated by the imaging system were not included in the final analysis. Actin served as the protein loading control. The minimum amount of protein resolved was 15 μg; the maximum amount was 30 μg; the average amount was 25 μg. 2.6. Fluorescent immunostaining of Sertoli cells and morphological analysis At 24 h after the treatment of Sertoli cells with vehicle or IL1A, cells were fixed at room temperature with 4% paraformaldehyde (w/v) in PBS or methanol at −20 °C for 10 min, permeabilized with 0.1% Triton™ X-100 (v/v) in PBS for 4 min, and blocked with 10% normal goat serum (v/v) in PBS for 30 min. Thereafter, the cells were incubated with primary antibodies overnight at 4 °C (Table 1), followed by speciescompatible Alexa Fluor®-conjugated secondary antibodies at 1:200 (Invitrogen/Life Technologies) in PBS for 30 min at room temperature. Sertoli cells were extensively washed with PBS on an orbital shaker after incubation with primary and secondary antibodies. Cells were mounted with ProLong® Gold antifade reagent containing 4′,6diamidino-2-phenylindole (DAPI). Images were acquired with a Nikon DS-Qi1 cooled digital camera attached to a Nikon Eclipse 90i microscope and Nikon Imaging Software-Elements Advanced Research software (version 3.2; Nikon, Melville, NY), and they were saved as .nd2 images. Raw .nd2 images were analyzed, saved as .tiff images, and merged with Photoshop CS5 Extended software (version 10.0.1; Adobe Systems Inc., San Jose, CA). No other image adjustments were performed.

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The relative distance in which α-tubulin and end-binding protein 1 (EB-1) retracted from the Sertoli cell plasma membrane after IL1A treatment was quantified. These results were compared with controls and then cross-compared with F-actin. Four measurements were taken from the center of the nucleus to the edge of cytoplasmic F-actin, αtubulin or EB-1 and then averaged; and four measurements were taken from the center of the nucleus to membrane-bound γ-catenin or claudin 11 and then averaged. The center of the nucleus was determined with Nikon Imaging Software-Elements Advanced Research software. Measurements were taken at 90° increments on raw .nd2 images within the same software. The relative distance in which the actin and microtubule cytoskeletons pulled away from the Sertoli cell plasma membrane after treatment was calculated as follows: [distance from the center of the nucleus (a) to the edge of cytoplasmic F-actin, αtubulin or EB-1 (b)] / [distance from the center of the nucleus (a) to γ-catenin or claudin 11 (c)] × 100%. 2.7. Fluorescent labeling of F-actin Oregon Green® 488 phalloidin (Molecular Probes, Eugene, OR) was used to label F-actin in Sertoli cells as previously described [42]. In brief, Sertoli cells were incubated on day 3 after plating with vehicle or IL1A for 24 h. Thereafter, the cells were used for F-actin labeling. In selected experiments, Sertoli cells were concurrently stained for γcatenin and F-actin. 2.8. F-actin bundling assay The F-actin bundling assay was performed using the actin binding protein spin-down assay (Cytoskeleton, Inc., Denver, CO) as previously described [65,66]. In brief, this method assesses the intrinsic ability of actin-bundling proteins present within Sertoli cell lysates to bundle exogenous F-actin, which is provided in the assay kit. It then separates by differential sedimentation the F-actin pool (polymerized actin, actinbundling and other actin-binding proteins; pellet) from the G-actin pool (non-polymerized actin; supernatant), and their relative ratios are then determined and compared between treatment groups by immunoblotting. G-actin binding proteins, F-actin severing proteins, and non-actin-binding proteins are also present in the supernatant. At 24 h after the treatment of Sertoli cells with vehicle or IL1A, cells were lysed in assay buffer (20 mM Tris, 20 mM NaCl, 0.5% Triton™ X-100 (v/v), pH 7.5) freshly supplemented with a broad-spectrum protease inhibitor cocktail, 20 mM NaF, and 20 mM Na3VO4, followed by centrifugation at 20,800 × g at 4 °C for 1 h. Thereafter, G-actin was polymerized into F-actin (i.e., exogenous F-actin), and lysates (~25 μg of protein) from Sertoli cells treated with vehicle or IL1A were combined with an equal amount of F-actin. Samples were incubated at room temperature for 1 h to facilitate bundling of F-actin, followed by centrifugation at 14,000 × g at 24 °C for 5 min to pellet bundled Factin microfilaments. The pellet and an aliquot from the supernatant were analyzed by immunoblotting and an anti-actin antibody (Table 1). F-actin served as the negative control. 2.9. Fluorescence recovery after photobleaching Fluorescence recovery after photobleaching (FRAP) was performed on calcein-stained Sertoli cells treated with vehicle or IL1A as previously described [22,53]. Calcein AM (molar mass, 994.87 g/mol) is a viablecell membrane-permeable non-fluorescent dye. Upon entering the cell, intracellular esterases cleave its acetoxymethyl ester group, which produces calcein (molar mass, 622.55 g/mol), a membraneimpermeable negatively charged fluorescent dye with excitation and emission wavelengths of 495 and 515 nm, respectively. Thus, the recovery of fluorescence at a specific velocity indicates that calcein passed from adjacent non-photobleached cells to the single photobleached cell via gap junctions. In brief, Sertoli cells were plated at a density of

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Table 1 Antibodies used in this study. Experimental conditions Approved symbol

Apparent Mr (kDa)

Vendor

Catalog no.

Lot no.

Host species

Connexin 43

GJA1

39-43

Sigma-Aldrich

C6219

113m4756

Rabbit

Connexin 43 (Ser 368)

–

39-43

Cell Signaling Technology

3511

3

Rabbit

Connexin 26

GJB2

26

Santa Cruz Biotechnology Inc.

sc-7261-R

C-1008

Rabbit

ZO-1

TJP1

210

Invitrogen/Life Technologies

61-7300

QA213101

Rabbit

Claudin 11

CLDN11

22

Invitrogen/Life Technologies

36-4500

387613 A

Rabbit

N-cadherin

CDH2

127

Invitrogen/Life Technologies

33-3900

PK211030

Mouse

alpha-Catenin

CTNNA1

102

Santa Cruz Biotechnology Inc.

sc-7894

C0318

Rabbit

beta-Catenin

CTNNB1

92

Invitrogen/Life Technologies

71-2700

564500A

Rabbit

gamma-Catenin

JUP

82

BD Biosciences

610254

28548

Mouse

c-SRC

SRC

60

EMD Millipore

05-184

2193143

Mouse

FAK

PTK2

125

BD Transduction Laboratories

F15020-150

20

Mouse

Cortactin

CTTN

80

Millipore

05-180

2013166

Rabbit

PKC-alpha

PRKCA

80

Santa Cruz Biotechnology Inc.

sc-208

H052

Mouse

PKC-epsilon

PRKCE

80

Santa Cruz Biotechnology Inc.

sc-214

E0605

Rabbit

Palladin Epidermal growth factor receptor kinase substrate 8

PALLD

95

Proteintech

10853-I-AP

18344

Rabbit

EPS8

97

BD Biosciences

610143

8603

Mouse

Drebrin E

DBN1

110

Abcam

ab11068

GR2304-2

Mouse

Santa Cruz Biotechnology Inc.

sc-15347

K1709

Rabbit

EB-1

MAPRE1

38

BD Biosciences

610535

3025569

Mouse

alpha-Tubulin Vimentin Actin

TUBA VIM ACTB

55 57 42

Abcam Santa Cruz Biotechnology Inc. Santa Cruz Biotechnology Inc.

ab7291 sc-6260 sc-1616

GR12217-1 I2413 E0714

Mouse Mouse Goat

1 2 3 4

Abbreviations: IB, immunoblotting; IF, immunofluorescence. Host species-compatible secondary antibodies conjugated to either Alexa Fluor® 488- or 555 were used at 1:250. Sertoli cells were fixed with 4% paraformaldehyde. Sertoli cells were fixed with methanol.

Immunogen Synthetic peptide corresponding to amino acids 363-382 of human/rat connexin 43 Synthetic phosphopeptide corresponding to amino acids near Ser 368 of human connexin 43 Epitope mapping at the N-terminus of human connexin 26 Fusion protein corresponding to amino acids 463-1109 of human ZO-1 Synthetic peptide corresponding to part of the C-terminus of human/mouse/rat claudin 11 Recombinant protein corresponding to the intracellular domain of chicken N-cadherin Epitope mapping at the C-terminus of human alpha-catenin Synthetic peptide corresponding to part of the C-terminus of human/mouse beta-catenin Recombinant protein corresponding to amino acids 553-738 of human gamma-catenin Recombinant protein corresponding to amino acids 82-169 of chicken c-SRC Recombinant protein corresponding to amino acids 354-533 of chicken FAK Affinity-purified tyrosine phosphoproteins from chicken fibroblasts expressing activated c-SRC Epitope mapping at the C-terminus of human PKC-alpha Epitope mapping at the C-terminus of human protein kinase C-epsilon Fusion protein corresponding to human palladin Recombinant protein corresponding to amino acids 628-821 of mouse epidermal growth factor receptor kinase substrate 8 Synthetic peptide corresponding to amino acids 22-42 of human drebrin E Epitope mapping at the C-terminus of human EB-1 Recombinant protein corresponding to amino acids 107-268 of human EB-1 Chicken alpha-tubulin, native protein Vimentin from porcine eye lens, native protein Epitope mapping at the C-terminus of human actin

IB1

IF2

1:8000

1:3004

1:1000 1:100 1:100 1:1004 1:100 1:200 1:400 1:100 1:500 1:200 1:100 1:200 1:200 1:100 1:100 1:500

1:1003

1:200 1:3004 1:10,000 1:200 1:200

1:4004 1:2503

K. Chojnacka et al. / Cellular Signalling 28 (2016) 469–480

Protein

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Fig. 1. Interleukin 1α disrupts the Sertoli cell barrier. (A) Representative images in which Sertoli cells treated with vehicle (DMEM/F-12 containing BSA, V-Ctrl) or IL1A at a concentration of 100 pg/ml for 24 h were fluorescently immunostained with antibodies against claudin-11 (green), zona occludens 1 (ZO-1, green), N-cadherin (red), or β-catenin (red) (Table 1). Small rectangles delineate regions that are magnified; these high magnification views are shown to the left and at the bottom of low magnification images. Sertoli cell nuclei were stained with DAPI (blue). Bar in (A), 10 μm; bar in (A, inset), 10 μm. (B) The integrity of the permeability barrier in Sertoli cells treated with vehicle or IL1A from day 3 onwards was assessed by transepithelial electrical resistance readings. Each data point represents the mean of 3–5 averaged readings from one representative experiment. This experiment was performed three independent times. **p b 0.01 (one-way ANOVA, followed by Newman–Keuls post hoc comparison test). These results agree with a previous study [42].

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Fig. 2. Interleukin 1α significantly upregulates the connexin 43 level in Sertoli cells. (A, C) Representative immunoblots in which lysates from Sertoli cells treated with vehicle (DMEM/F-12 containing BSA, V-Ctrl) or IL1A at concentrations from 0 to 100 pg/ml (A, B) or at a concentration of 100 pg/ml (C, D) were resolved by SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes for immunoblotting. Membranes were probed with antibodies against different component proteins of the gap junction, ectoplasmic specialization, and cytoskeleton, as well as against several kinases (Table 1). Actin, vimentin, and α-tubulin served as the protein loading controls. SRC, src proto-oncogene, non-receptor tyrosine kinase; FAK/(PTK2), focal adhesion kinase/(protein tyrosine kinase 2); PKC-α, protein kinase C-alpha; EB-1, end-binding protein 1. (B, D) Histograms summarizing protein changes. Each IL1A treatment point was normalized against the 0 pg/ml treatment point (A, B) or its time-matched vehicle treatment point (C, D). Each bar represents the mean of 3–6 normalized values ± S.D. from five independent Sertoli cell cultures. Within each Sertoli cell culture, each treatment was performed at least in duplicate wells. *p b 0.05, **p b 0.01, ***p b 0.001 (one-way ANOVA, followed by Newman–Keuls post hoc comparison test). (E) Representative images in which Sertoli cells treated with vehicle or IL1A for 24 h were fluorescently immunostained with an antibody against connexin 43 or drebrin E (both red). Sertoli cell nuclei were stained with DAPI (blue). Arrowheads point to connexin 43 at cell–cell contacts. Bar in (E), 15 μm.

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0.2 × 106 cells/cm2 on Matrigel™-coated no. 1 glass bottom culture dishes (MatTek Corp., Ashland, MA) and then incubated on day 3 with vehicle or IL1A at a concentration of 100 pg/ml. At 36 h after treatment, cells were labeled with 5 μM calcein AM (Molecular Probes) in DMEM/ F-12 for 30 min at 35 °C, followed by washing twice with DMEM/F-12 and then FRAP analysis. Calcein AM was dissolved in dimethyl sulfoxide (DMSO) to yield a stock concentration of 0.5 mM, and the final concentration of DMSO during labeling was 0.1% (v/v). A Zeiss/Perkin–Elmer confocal laser scanning microscope (Waltham, MA) equipped with the Photonics Instrument Digital Mosaic system (St. Charles, IL) and a Solent Scientific chamber (Segensworth, UK) for temperature and CO2 control was used for FRAP. A single Sertoli cell was irreversibly photobleached for 5 s with a high-powered focused laser beam at 488 nm in each treatment group. Images were recorded at low laser power and acquired with an Andor Technology iXon 512 × 512 electron multiplying charge-coupled device camera (South Windsor, CT) for high sensitivity/speed imaging and MetaMorph® Microscopy Automation and Image Analysis software (version 7.7; Molecular Devices, Sunnyvale, CA). Two prebleach and 60 postbleach images were acquired at 2-s intervals for each photobleached Sertoli cell. The stacks of images were aligned with the StackReg plugin in ImageJ, and fluorescence intensity was quantified with ImageJ software (version 1.45; http://www.rsb.info.nih.gov/ij/). The FRAP curves were normalized by taking the prebleach and bleach fluorescence intensities as 1 and 0, respectively [67]. During calcein AM labeling and FRAP analysis, Sertoli cells were maintained in DMEM/F-12 containing vehicle or IL1A. FRAP was performed on ~ 50 Sertoli cells in each treatment group in three independent experiments. The optimal Sertoli cell density was determined in preliminary experiments. These experiments also established that functional gap junctions were present between Sertoli cells. This experiment was performed at the Bio-Imaging Resource Center of the Rockefeller University. 2.10. Statistics Each variable was tested using the Shapiro–Wilk W-test for normality. The homogeneity of variance was assessed with Levene's test. Data from the analysis of the effects of IL1A on connexin 43 and connexin 43 (Ser 368) levels were log transformed to obtain normal distributions. Statistical differences in protein levels were analyzed by one-way ANOVA, followed by Newman–Keuls post hoc comparison test. Statistical analyses were performed on raw data using Statistica 10 software (StatSoft Inc., Tulsa, OK). Data were expressed in arbitrary units as means ± standard deviation (S.D.). Data were considered statistically significant at *p b 0.05, **p b 0.01, and ***p b 0.001. 3. Results 3.1. Interleukin 1α disrupts the Sertoli cell barrier In previous in vitro and in vivo studies, we have shown that IL1A affects the F-actin network, resulting in changes in the levels, as well as in the localization and stability, of several tight junction and ectoplasmic specialization component proteins [41,42]. Not surprisingly, the function of the Sertoli cell barrier/BTB is disrupted, suggesting that IL1A participates in the restructuring of the BTB. As a starting point for this study, Sertoli cells treated with vehicle or IL1A were used in fluorescent immunostaining experiments that employed antibodies against tight junction (i.e., claudin 11, zona occludens 1 [ZO-1]) and ectoplasmic specialization (i.e., N-cadherin, β-catenin) component proteins. In agreement with results from our previous study [42], there were changes in the levels, as well as in the localization, of ZO-1, N-cadherin, and β-catenin at the plasma membrane at 24 h after the treatment of Sertoli cells with IL1A (Fig. 1A). However, there was no change in the localization of claudin-11 after IL1A treatment compared with the control. In this study, we also confirm that IL1A disrupts the Sertoli cell barrier

Fig. 3. Protein kinase C-α phosphorylates connexin 43 at Ser368 in Sertoli cells in response to interleukin 1α. (A, C) Representative immunoblots in which lysates from Sertoli cells treated with vehicle (DMEM/F-12 containing DMSO, V-Ctrl), IL1A at a concentration of 100 pg/ml, Gö6976 at 1 or 5 nM, or Gö6976 and IL1A at the indicated concentrations for 24 h were resolved by SDS-PAGE under reducing conditions and transferred onto nitrocellulose membranes for immunoblotting. Membranes were probed with antibodies against protein kinase C-alpha (PKC-α), protein kinase C-epsilon (PKC-ε) and connexin 43 (Ser368) (Table 1). Actin served as the protein loading control. (B, D) Histograms summarizing protein changes. Each treatment point was normalized against the vehicle (B) or IL1A treatment point (D). Each bar represents the mean of 5–6 normalized values ± S.D. from three independent Sertoli cell cultures. Within each Sertoli cell culture, each treatment was performed at least in triplicate wells. *p b 0.05, **p b 0.01, ***p b 0.001 (one-way ANOVA, followed by Newman–Keuls post hoc comparison test).

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Fig. 4. Interleukin 1α affects gap junctional communication in Sertoli cells. (A) Representative images from a calcein dye-transfer experiment that determined the velocity of fluorescence recovery in photobleached Sertoli cells treated with vehicle (DMEM/F-12 containing BSA, V-Ctrl) or IL1A at a concentration of 100 pg/ml for 36 h. Prebleach images show the initial fluorescence intensity. Representative images at 0, 60, and 120 s after photobleaching are shown. (B) FRAP curve and analysis corresponding to the images. (C) Table summarizing FRAP results. Each treatment point represents the mean of 10–12 Mf determinations ± S.D. from one representative FRAP experiment. In total, ~50 Sertoli cells in each treatment group from four independent FRAP experiments were analyzed. Results were similar across all FRAP experiments. **p b 0.01 (one-way ANOVA, followed by Newman–Keuls post hoc comparison test). Mf, mobile fraction: the fraction of fluorescent molecules that can exchange between photobleached and non-photobleached regions; IMf, immobile fraction: the fraction of fluorescent molecules that cannot exchange between photobleached and non-photobleached regions; Ii, initial intensity; I∞, infinite intensity.

(Fig. 1B). At 24 h after the treatment of Sertoli cells with IL1A, transepithelial electrical readings significantly decreased by ~ 20% (p b 0.01). 3.2. Interleukin 1α significantly upregulates the connexin 43 level in Sertoli cells In this experiment, the levels of several junction component and regulatory proteins in Sertoli cells after IL1A treatment were examined by

immunoblotting. Compared with the control, the levels of connexin 43 (Fig. 2A–D) and connexin 43 (Ser 368) (Fig. 2C, D) significantly increased ~ 30- and 20-fold (p b 0.001), respectively, at 24 h after the treatment of Sertoli cells with IL1A. The levels of both proteins decreased at 72 h after IL1A treatment. The reason for this is not known. Furthermore, the levels of β-catenin (a component protein of the cadherin complex, p b 0.01) and protein kinase C-α (a Ser-/Thr-specific protein kinase, p b 0.05) significantly increased after treatment (Fig. 2C, D). The increase in the level of β-catenin after IL1A treatment is in

Fig. 5. Interleukin 1α disrupts the Sertoli cell cytoskeleton. (A) Representative images in which Sertoli cells treated with vehicle (DMEM/F-12 containing BSA, V-Ctrl) or IL1A at a concentration of 100 pg/ml for 24 h were fluorescently immunostained with antibodies against cortactin (red), palladin (green) or epidermal growth factor receptor kinase substrate 8 (EPS8, red) (Table 1). Oregon Green® 488 phalloidin was used to label F-actin (green). Small boxes delineate regions that are magnified; these high magnification views, as well as corresponding monochrome images, are shown below low magnification images. Arrows point to the aggregation of F-actin and palladin (promotes actin crosslinking) at the periphery of Sertoli cells after IL1A treatment, whereas asterisks point to the mislocalization of F-actin and cortactin (promotes actin nucleation). Sertoli cell nuclei were stained with DAPI (blue). Bar in (A), 10 μm; bar in (A, high magnification view), 10 μm. Interleukin 1α disrupts F-actin bundling in Sertoli cells. (B) Representative immunoblots in which lysates from Sertoli cells treated with vehicle (DMEM/F-12 containing BSA, V-Ctrl) or IL1A for 24 h were used for an F-actin bundling assay. The resulting samples [pellet and supernatant (S/ N)] were resolved by SDS-PAGE under reducing conditions and transferred to a nitrocellulose membrane. The membrane was probed with an antibody against actin (Table 1). F-actin served as the negative control (i.e., Sertoli cell lysate was omitted; thus, F-actin was non-bundled and not pelleted). Histogram summarizing protein changes (right panel). The IL1A treatment point was normalized against the V-Ctrl treatment point. Each bar represents the mean of 3 normalized values ± S.D. from three independent Sertoli cell cultures. ***p b 0.001 (oneway ANOVA, followed by Newman–Keuls post hoc comparison test). (C) Representative images in which Sertoli cells treated with vehicle or IL1A for 24 h were fluorescently immunostained with antibodies against α-tubulin or EB-1 (both green). Bar in (C), 10 μm. (D) Representative images in which Sertoli cells treated with vehicle or IL1A for 24 h were fluorescently immunostained with antibodies against α-tubulin or EB-1 (both green) (Table 1). Oregon Green® 488 phalloidin was used to label F-actin. Small boxes delineate regions that are magnified; these high magnification views are shown to the right of low magnification images. Brackets show the relative distance in which F-actin, α-tubulin, and EB-1 pulled away from the Sertoli cell plasma membrane after IL1A treatment. The Sertoli cell plasma membrane was marked by γ-catenin or claudin-11 immunostaining (Table 1). Bar in (A), 10 μm; bar in (A, high magnification view), 10 μm. Histograms summarizing morphological analyses (right panel). The illustration shows how morphological analyses were performed. The IL1A treatment point was normalized against the V-Ctrl treatment point. Each bar represents the mean of 50 distance determinations ± S.D. from three independent Sertoli cell cultures. *p b 0.05 (one-way ANOVA, followed by Newman–Keuls post hoc comparison test).

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agreement with results shown in Fig. 1A and a previous study [42]. By contrast, there was no change in the level of connexin 43 in Sertoli cells treated with 20-fold-less IL1A (Fig. 2A, B), as well as in the levels of other proteins (e.g., drebrin E, EB-1) in Sertoli cells treated with IL1A at a concentration of 100 pg/ml (Fig. 2C, D). The effect of IL1A on

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the connexin family of proteins was specific to connexin 43 because there was no change in the connexin 26 level (Fig. 2C, D). In agreement with these results, there was an increase in connexin 43 at cell–cell contacts at 24 h after the treatment of Sertoli cells with IL1A (Fig. 2E). However, there was no change in the localization of the actin-binding

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protein drebrin E, which binds connexin 43 and links gap junctions to the cytoskeleton [68], after treatment compared to the control (Fig. 2E). It is not known whether there are changes in the levels of other connexin 43-binding proteins after IL1A treatment. 3.3. Protein kinase C-α phosphorylates connexin 43 at Ser368 in Sertoli cells in response to interleukin 1α This experiment determined whether IL1A-induced phosphorylation of connexin 43 at Ser368 is mediated by protein kinase C-α (PKCα). For this purpose, Gö6976, a potent inhibitor of PKC-α and -β, but not PKC-δ, -ε, and ζ [61], was used together with IL1A. The ability of Gö6976 to inhibit the basal level of PKC-α in Sertoli cells was first examined. Compared with the control, Gö6976 at 5 nM significantly decreased (p b 0.05) the basal level of PKC-α in Sertoli cells (Fig. 3A, B). There was no change in the basal level of PKC-α after treatment with Gö6976 at 1 nM, as well as no change in the basal level of PKC-ε at both concentrations, revealing that Gö6976 is a specific inhibitor of PKC-α. The ability of Gö6976 to inhibit the phosphorylation of connexin 43 at Ser368 after IL1A treatment was then examined. Gö6976 at both concentrations significantly inhibited (p b 0.001) the IL1A-induced phosphorylation of connexin 43 at Ser 368 (Fig. 3C, D), indicating that PKC-α phosphorylates connexin 43 at Ser368 under these experimental conditions. The increase in the levels of PKC-α and connexin 43 (Ser368) after IL1A treatment are in agreement with results shown in Fig. 2C, D. 3.4. Interleukin 1α affects gap junctional communication in Sertoli cells To assess gap junction function, a calcein dye-transfer assay was performed, which essentially quantified the fluorescence recovery in photobleached Sertoli cells treated with vehicle or IL1A. FRAP curve analysis showed that there was a significant decrease (IL1A, 0.315 ± 0.184 s versus vehicle, 0.640 ± 0.126 s; p b 0.01) in the mobile fraction (i.e., the fraction of fluorescent molecules that can exchange between photobleached and non-photobleached regions) in Sertoli cells treated with IL1A compared with that in Sertoli cells treated with vehicle at 120 s after photobleaching (i.e., plateau) (Fig. 4A–C). In other words, IL1A affected the recovery of fluorescence (Fig. 4B, C), indicating that calcein could not diffuse from adjacent non-photobleached cells to the photobleached cell via gap junctions. 3.5. Interleukin 1α disrupts the Sertoli cell cytoskeleton In this experiment, the most pronounced change was in the localization of F-actin. Compared with the control, F-actin aggregated at the periphery of Sertoli cells after IL1A treatment, with few stress fibers running across IL1A-treated Sertoli cells (Fig. 5A). The disruption in gap junctional communication is likely the result of a disrupted Factin network. In addition, there were striking changes in the localization of actin binding proteins cortactin and palladin after IL1A treatment; these changes resembled those of F-actin. Cortactin, which promotes actin nucleation, and palladin, which promotes actin bundling, both concentrated at the periphery of Sertoli cells (Fig. 5A). However, there was no change in the localization of epidermal growth factor receptor kinase substrate 8 (EPS8), which promotes actin bundling and barbed-end actin capping (depending on the cellular context), after IL1A treatment compared with the control. To understand the effects of this pro-inflammatory cytokine on the F-actin network, an F-actin bundling assay was performed using lysates from Sertoli cells treated with vehicle or IL1A at a concentration of 100 pg/ml for 24 h (Fig. 5B). Compared with the control, IL1A affected the ability of Sertoli cells to bundle exogenous F-actin as shown by the significant decrease (p b 0.001) in the level of actin in the Sertoli cell pellet. There was no change in the actin level in the supernatant, which consisted of Gactin and its binding proteins, F-actin severing proteins, and non-actin

binding proteins, between treatment groups (Fig. 5B). The decrease in the ability of Sertoli cells to bundle F-actin agrees with the change in the localization of palladin. In addition, there was a change in the localization of α-tubulin, as well as in the tubulin-binding protein EB-1, which regulates microtubule dynamics ([69,70]; for a review, [71]). Compared with the control, α-tubulin and EB-1 pulled away from the Sertoli cell plasma membrane, and both proteins concentrated at the nucleus (Fig. 5C). To substantiate this conclusion, the relative distance in which α-tubulin and EB-1 retracted from the Sertoli cell plasma membrane after IL1A treatment was quantified. These results were compared with controls and then cross-compared with F-actin. In agreement with the results shown in Fig. 5A, IL1A did not disrupt the ability of F-actin to interact with the Sertoli cell plasma membrane (Fig. 5D). However, the ability of α-tubulin and EB-1 to interact with the plasma membrane was significantly affected (p b 0.05) in IL1Atreated Sertoli cells (Fig. 5D). 4. Discussion The objective of this study was to gain new insight into the function and regulation of gap junctions at the Sertoli cell barrier/BTB by determining whether IL1A affects gap junctional communication and identifying the mechanism of action to partly explain its effects (Fig. 6). Previous studies show that gap junctions are a key component of the BTB, where they facilitate the rapid exchange of ions, second messengers, and metabolites smaller than 1 kDa across Sertoli cells (for reviews, [1–3,6,13]). At stages VIII–XI of the seminiferous epithelial cycle, preleptotene/leptotene spermatocytes traverse the BTB while enclosed within a dynamic compartment that is primarily sealed by Sertoli cell tight junctions and ectoplasmic specializations ([72,73]; for a review, [74]). Gap junctions and desmosomes are also found at the BTB, co-existing and co-functioning with tight junctions and ectoplasmic specializations (for reviews, [75,76]). Thus, it is befitting to assume that BTB restructuring involves precise coordination across different cell junctions. As spermatocytes move across the BTB, structural proteins are thought to be internalized and then recycled or degraded, which results in the simultaneous disassembly of old junctions ahead of moving germ cells and in the assembly of new junctions behind germ cells. Furthermore, the cytoskeleton reshapes and remodels as cell junctions restructure, which is supported by our results from this study (Fig. 6). Connexin 43, the best-studied gap junction protein, is present at the BTB [14–17], where it presumably contributes to the function of the barrier. However, connexin 43 knockdown in cultured Sertoli cells by RNA interference does not affect barrier function [15]. The reason for this is not known. Instead, concurrent knockdown of two genes, connexin 43 and plakophilin-2 (a desmosomal plaque protein), affects the integrity of the permeability barrier in Sertoli cells. Although the relationship between gap junctions and the other cell junctions at the BTB is not completely known, a better understanding is needed as it may help in the treatment of testicular dysfunction in the future. Throughout the seminiferous epithelial cycle, IL1A bioactivity is high at all stages except stage VII [34,77], which precedes the movement of preleptotene/leptotene spermatocytes across the BTB (for a review, [76]). Previous studies show that IL1A disrupts the function of the Sertoli cell barrier/BTB ([41,42]; for a review, [31]), suggesting that it participates in the restructuring of the BTB. In this study, the levels of connexin 43 and connexin 43 (Ser 368) increased ~30- and 20-fold, respectively, after IL1A treatment (Fig. 6). Connexin 43 in Sertoli cells is very susceptible to the effects of IL1A because there was no comparable increase in the level of any other protein examined in this and a previous study [42]. The increase in the connexin 43 (Ser 368) level, which was prompted by PKC-α, is particularly important because connexin 43 phosphorylation induces restructuring of gap junctions ([78]; for reviews, [79–81]), suggesting that IL1A exerts its effects ahead of moving germ cells. However, our results disagree with those of Fiorini et al. who report a time- and dose-dependent decrease in the connexin 43 level in

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Fig. 6. Effects of IL1A on cytoskeletal dynamics and gap junctional communication in Sertoli cells under physiological and pathophysiological conditions. In the left panel, the lower region of two polarized Sertoli cells is emphasized. The plasma membrane of these cells is denoted in red. Here, functional gap junctions at the Sertoli cell barrier/BTB are supported by intact actin and microtubule networks, as well as by interacting proteins that include end-binding protein 1 (EB-1), drebrin E (DBN1), and palladin (PALLD). Occludin, a tight junction component protein, is included based on results from a previous study [42]. In the right panel, the lower region of two polarized Sertoli cells is depicted. It shows what may happen when the level of IL1A is aberrantly elevated in Sertoli cells, which may lead to testicular dysfunction (for reviews, [32,85,86]). The plasma membrane of these cells is denoted in blue. Here, actin and microtubule networks are disrupted, with mostly unbundled actin microfilaments aggregating at the periphery of Sertoli cells and microtubules concentrating at the nucleus. Although the levels of connexin 43 and connexin 43 (Ser368) are upregulated in Sertoli cells with a high level of IL1A, gap junctions are closed. In a previous in vitro study, occludin was internalized, resulting in the disruption of the Sertoli cell barrier/BTB. At this point, it is not completely known whether the effects of IL1A on gap junctional communication are direct or indirect.

the 42GPA9 Sertoli cell line [26]. Although the reason for this discrepancy between studies is not known, there are several differences in experimental design (e.g., the use of a cell line, the inclusion of fetal calf serum and testosterone into cultures, the source of recombinant IL1A, the method used to detect and quantify changes in connexin 43) that may have contributed to these conflicting results. To validate the effects of IL1A on gap junction function and to place changes in protein levels into the correct perspective, FRAP was performed on calcein-stained Sertoli cells in the absence and presence of IL1A. After photobleaching, the recovery of fluorescence was affected in Sertoli cells treated with IL1A, revealing that gap junctional communication was disrupted (Fig. 6). Although there was no change in the connexin 26 level after IL1A treatment by immunoblotting, it is likely that other gap junction proteins were also affected, thereby contributing to the overall loss in gap junction function. IL1A also affected the Sertoli cell cytoskeleton (Fig. 6), suggesting that the effects of IL1A on cell junctions may be secondary to those on the actin and microtubule networks. The most pronounced change was in stress fibers, which aggregated at the periphery of IL1A-treated Sertoli cells as unbundled F-actin microfilaments. Furthermore, the α-tubulin network failed to extend from the nuclear region to the Sertoli cell plasma membrane, possibly due to this region being largely occupied by F-actin. An alternate interpretation may be that microtubules cannot interact with unbundled F-actin microfilaments. Microtubules are dynamic cytoskeletal elements that exhibit remarkable polarity (i.e., a plus-end and a minus-end). The failure of microtubules to extend to the plasma membrane suggests that IL1A may have also disrupted their polarity. These results support studies from other systems, which illustrate that cell junction function cannot be sustained in the absence of a robust cytoskeleton (for reviews, [82,83]), and this also agrees with our results from this study. Prospective studies should incorporate germ cells, especially primary spermatocytes, into in vitro models to determine whether IL1A induces Sertoli cell barrier/BTB restructuring. Germ cells contribute to the function of the Sertoli cell barrier [84]. Thus, the response of Sertoli cells to IL1A in the presence of germ cells may be different. Other studies should investigate the mechanism of gap junction disassembly, specifically whether gap junction proteins are internalized and then recycled or degraded. In summary, our results show that IL1A is a regulator of gap junction function at the Sertoli cell barrier/BTB, which is critical for the movement of preleptotene/leptotene spermatocytes across the barrier.

Declaration of conflicts of interests The authors declare no conflicts of interest. Funding Dr. Chojnacka was supported by a HARMONIA 3 grant (2012/06/M/ NZ4/00,146) from the Polish National Science Centre awarded to Professor Barbara Bilinska. Author contributions KC performed the experiments and analyzed the data; BB developed analytical tools; and DDM designed and performed the experiments, interpreted the data, and wrote the paper. Acknowledgments We thank Drs. Alison North and Pablo Ariel (Rockefeller University) for their expertise in FRAP experiments. We also thank Drs. Elizabeth Tang and Nan Li (Population Council) for their assistance throughout this study. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15]

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