NC1-Peptide From Collagen α3 (IV) Chains in the Basement Membrane of Testes Regulates Spermatogenesis via p-FAK-Y407

Abstract

The blood–testis barrier (BTB) in the testis is an important ultrastructure to support spermatogenesis. This blood-tissue barrier undergoes remodeling at late stage VII to early stage IX of the epithelial cycle to support the transport of preleptotene spermatocytes across the BTB to prepare for meiosis I/II at the apical compartment through a mechanism that remains to be delineated. Studies have shown that NC1-peptide-derived collagen α3 (IV) chain in the basement membrane is a bioactive peptide that induces BTB remodeling. It also promotes the release of fully developed spermatids into the tubule lumen. Thus, this endogenously produced peptide coordinates these 2 cellular events across the seminiferous epithelium. Using an NC1-peptide complementary deoxyribonucleic acid (cDNA) construct to transfect adult rat testes for overexpression, NC1-peptide was found to effectively induce germ cell exfoliation and BTB remodeling, which was associated with a surge and activation of p-rpS6, the downstream signaling protein of mTORC1 and the concomitant downregulation of p-FAK-Y407 in the testis. In order to define the functional relationship between p-rpS6 and p-FAK-Y407 signaling to confer the ability of NC1-peptide to regulate testis function, a phosphomimetic (and thus constitutively active) mutant of p-FAK-Y407 (p-FAK-Y407E-MT) was used for its co-transfection, utilizing Sertoli cells cultured in vitro with a functional tight junction (TJ) barrier that mimicked the BTB in vivo. Overexpression of p-FAK-Y407E-MT blocked the effects of NC1-peptide to perturb Sertoli cell BTB function by promoting F-actin and microtubule cytoskeleton function, and downregulated the NC1-peptide-mediated induction of p-rpS6 activation. In brief, NC1-peptide is an important endogenously produced biomolecule that regulates BTB dynamics.

In the rodent and human testes, extensive remodeling of cell junctions at the Sertoli cell–cell interface at the site of the Sertoli cell blood–testis barrier (BTB), most notably basal ectoplasmic specialization (ES) and tight junction (TJ), near the basement membrane takes place to facilitate the transport of preleptotene spermatocytes across the BTB at late stage VII to early stage IX of the epithelial cycle (1–4). On the other hand, rapid remodeling also takes place at the Sertoli–spermatid interface called apical ES to support the transport of developing haploid spermatid across the seminiferous epithelium during spermiogenesis and the release of sperm at spermiation (4–8). Interestingly, the biology of these cellular events have remained largely unexplored until recent years. Studies have shown that NC1 (noncollagenous 1 domain) at the C-terminal region of the collagen α3 (IV) chain, the structural and scaffolding component of the basement membrane in the rat testis (9, 10), can be released via the action of MMP-9 (matrix metalloproteinase 9) involving TIMP1 (tissue inhibitor of metalloproteinase 1) near the basal ES/BTB (11). This NC1-peptide also serves as a biologically active peptide that is capable of inducing remodeling of the Sertoli cell TJ permeability barrier either through the use of its purified recombinant protein or overexpression of its complementary deoxyribonucleic acid (cDNA; using mammalian expression vector pCI-neo) based on in vitro (12–14) and in vivo (13) studies, but also on spermatogenesis modulation in vivo (13). These findings are important because they illustrate that the testis is capable of producing NC1-peptide endogenously to support spermatogenesis, most notably through its effects on both actin- and microtubule (MT)-based cytoskeletons (13). However, the biology underlying the NC1-peptide-mediated effects on spermatogenesis remains unknown.

A recent report using primary Sertoli cells cultured in vitro have shown that NC1-peptide (via its overexpression in Sertoli cells) exerts its effects downstream, involving the activation of mTORC1/rpS6 signaling complex, to be followed by an activation of Cdc42 GTPase, which in turn perturbs F-actin cytoskeleton in the Sertoli cell epithelium (14). Other studies have shown that FAK, in particular phosphorylated and activated p-FAK-Tyr397 (15, 16) and p-FAK-Tyr407 (17, 18), exert their effects to promote spermatid adhesion and basal ES/BTB integrity through their corresponding effects at the apical ES and basal ES/BTB, respectively. Here, we sought to delineate the signaling proteins likely involved in NC1-peptide-mediated function in the testis. Specifically, we sought to investigate if these signaling proteins also exert their effects to modify actin cytoskeletons and also MT cytoskeletons in Sertoli cells in the testis. This information, if known, would provide important insights to modify spermatogenesis, but also regarding the transport function at the BTB, unraveling a new window of research to probe the biology of spermatogenesis. This is the subject of this report.

Materials and Methods

Animals

Adult male Sprague Dawley rats, ~275–300 g (body weight), and male pups at 16–18 days of age were purchased from Charles River Laboratories (Kingston, New York). Adult rats were housed in pairs of 2 per cage, while each of the 10 male pups were housed with a foster mother rat at the Rockefeller University (RU) Comparative Bioscience Center with a 12-hour light/dark cycle, and with free access to standard rat chow and water at room temperature of 20 ± 1 °C. For isolation of Sertoli cells for in vitro experiments, male pups were euthanized at 20 days of age. Adult rats were also euthanized at specific time points according to the regimen outlined in Fig. 1A. Rats were sacrificed in a euthanasia chamber by CO₂ asphyxiation, using slow displacement of chamber air (20–30% per minute) from a CO₂ tank via a gas regulator. The use of recombinant DNA materials, such as plasmid DNA from pCI-neo containing cDNA constructs of NC1-peptide or p-FAK-Y407E mutant (and all pertinent experimental procedures reported here) were approved by the RU Institutional Animal Care and Use Committee, with approved Protocol Number 18-043H.

Figure 1.

Overexpression of NC1-peptide in the testis in vivo induces defects in spermatogenesis through mTORC1/rpS6 signaling complex involving p-FAK-Y407. A: Regimen used to obtain rat testes for analysis in this study. Each experiment had 4 rats per time point, and a total of 3 experiments were done over a 2-year period to obtain enough testes for multiple experimental analyses (ie, n = 12 rats/time point). NC1-peptide cloned into the mammalian expression vector pCI-neo was used for transfection on time 0 (and terminated on day 14 for analysis), as detailed in the “Materials and Methods” section. B: Results of qPCR that confirmed overexpression of NC1-peptide at various time points and compared to control testes at time 0, with n = 3 rat testes. * P < 0.05, *** P < 0.001 by ANOVA. C: Defects of spermatogenesis were noted following overexpression of NC1-peptide in the testis and compared to control testes (transfected with pCI-neo empty vector and terminated on day 14; or testes at 0 hours, see [B]). Images in selected areas in the images on the second row were magnified in yellow or red rectangles, and selected areas were further magnified in insets in blue or green to highlight the defects. Defects in spermatogenesis were noted as early as 3 hours, wherein some Sertoli cells (black arrowhead) were no longer localized to the base of the seminiferous epithelium, groups of spermatids (sometimes spermatocytes by 6 hours) were detected in tubule lumen (orange arrowheads), and elongated spermatids (green arrowheads) remained trapped inside the epithelium when spermiation had occurred. These defects were also noted in other time points. By 12 hours, notable defects in spermatid polarity were detected whereby elongated spermatids (red arrowheads) no longer had their heads pointed to the basement membrane but deviated by at least 90^o from the intended orientation noted in control testes (white arrowheads). By day 3 and thereafter, numerous multinucleated round spermatids were detected (blue arrowheads). Also, numerous seminiferous epithelial vacuoles were noted (yellow arrowheads) on days 3, 7, and 14, illustrating Sertoli cell damage. By day 14, more than 90% of the tubules were devoid of advanced germ cells of any type, and only spermatogonia and Sertoli cells remained. Scale bars: 350 µm, 200 µm, 80 µm, 80 µm, 30 µm, and 30 µm apply to corresponding micrographs and insets. Tubules marked with asterisks illustrate tubules with defects in spermatogenesis. See details of criteria to define defects in spermatogenesis in the “Materials and Methods” section. Data shown here are representative findings from an experiment from n = 3 independent experiments, which yielded similar results. D: Composite data (mean ± SD) showing tubules with defects in spermatogenesis by randomly scoring at least 200 tubules per rat testis from n = 4 rats at each time point, with a total of 800 tubules per time point. E: Representative IB data from n = 4 experiments (see composite data in Fig. S1 (19), illustrating a decline in the expression of mTOR and Raptor), with a considerable upregulation on the expression of p-rpS6-S235/S236 and p-rpS6-S240/S244 at time points between 3 hours and 1 to 3 days (but not total rpS6). A considerable downregulation on the expression of c-Src (but not c-Yes), as well as MT regulatory proteins MARK2 and CAMSAP2, were also noted, which is also consistent with a recent report that downregulation on the MT regulatory proteins (eg, EB1) was detected (20). Importantly, it was shown that the expression of p-FAK-Y397 and p-FAK-Y407 was also considerably downregulated, which have been recently shown to be the crucial regulators of spermatogenesis through their promoting effects to maintain cytoskeletons across the seminiferous epithelium (16, 17, 21, 22). α-tubulin, ß-actin, and GAPDH served as protein loading control.

Antibodies

All antibodies used for the experiments reported here were obtained commercially and are summarized in Table S1 (19). All supplementary material and figures are located in a digital research materials repository (19). These antibodies were previously characterized in our laboratory, as noted in corresponding sections in this report, including their specificity, working dilutions, and RRID information (see Table S1) (19). Other detailed information, such as host animal species, including their working dilution used for specific experiments, such as immunoblotting (IB) and immunofluorescence (IF) analysis and working dilutions are shown in Table S1 (19). Antibodies from Abcam (Cambridge, Massachusetts) included α-tubulin (23), GAPDH (24), detyrosinated α-tubulin (25), ß-tubulin (26), acetylated α-tubulin (27), and MAP1a (28). Antibodies from Cell Signaling Technology (Danvers, Massachusetts) included rpS6 (29), p-rpS6-S235/S236 (30), p-rpS6-S240/S244 (31), mTOR (32), Rictor (33), Raptor (34), Akts (35), p-Akt1-T308 (36), p-Akt1-S473 (37), and p-Akt2-S474 (38). Antibodies from Invitrogen (Carlsbad, California) included Occludin (39), ZO-1 (40), N-cadherin (41), ß-catenin (42), Eps8 (43), p-FAK-Y407 (44), and p-FAK-Y397 (45). Antibodies from BD Biosciences (Woburn, Massachusetts) included EB1 (46) and c-Yes (47). Antibodies from Santa Cruz Biotechnology (Dallas, Texas) include actin (48), N-cadherin (49), EB1 (50), FAK (51), c-Src (52), rabbit IgG-HRP (53), and mouse IgG-HRP (54). Antibodies from Proteintech Group (Rosemont, Illinois) included MARK4 (55), CAMSAP2 (56), and MARK2 (57). Antibodies from Thermo Fisher Scientific (Fair Lawn, New Jersey) included goat IgG Alexa Fluor 488 (58), goat IgG Alexa Fluor 555 (59), mouse IgG Alexa Fluor 488 (60), mouse IgG Alexa Fluor 555 (54), rabbit IgG Alexa Fluor 488 (61), and rabbit IgG Alexa Fluor 555 (62). Antibody from Millipore Sigma (Burllington, MA) was Tyrosinated α-tubulin (63). Antibody from Sigma-Aldrich (Allentown, Pennsylvania) was Arp3 (64).

Preparation of cDNA constructs containing NC1-peptide, FAK, and FAK mutants

Plasmid DNA of the mammalian expression vector pCI-neo (Promega, Madison, WI) containing cDNA constructs corresponding to NC1-peptide, FAK (wild type [WT], ie, full-length FAK clone), phosphomimetic (ie, constitutive active) p-FAK-Y407E mutant (prepared by site-directed mutagenesis), or nonphosphorylatable (ie, constitutive inactive) p-FAK-Y407F mutant (prepared by site-directed mutagenesis) were prepared in our laboratory, as previously described (12, 13, 17). Plasmid DNA was obtained and purified by using Plasmid Plus Midi kits (Qiagen, Germantown, MD) by removing residual endotoxin contamination using the protocol provided by the manufacturer.

Overexpression of NC1-peptide and/or FAK (WT) and different FAK mutants in testes of adult rats

Overexpression of either NC1-peptide, FAK-WT, p-FAK-Y407E mutant (ie, constitutively active), p-FAK-Y407F mutant (ie, constitutively inactive and nonphosphorylatable), or NC1-peptide+p-FAK-Y407E mutant (ie, dual overexpression) cloned into pCI-eno was performed in adult rat testes by using 50 µl transfection solution per testis, containing either 10 µg (for singular overexpression of either NC1-peptide or FAK vs its corresponding mutant) or 20 µg (for dual overexpression of both NC1-peptide and p-FAK-Y407E mutant at 10 µg each) plasmid DNA, respectively (for both treatment groups and control group), together with 1.8 µl Polyplus in vivo-jetPEI^® transfection reagent (PolyPlus transfection S.A., Illkirch-Graffenstaden, France) according to the manufacturer’s Protocol, via intratesticular administration (65). This approach was used to examine the effects of corresponding overexpression in altering spermatogenesis by assessing changes of phenotypes across the seminiferous epithleium of rat testes when compared to control testes. Control testes were transfected with the same amount of plasmid DNA (10 or 20 µg) containing empty pCI-neo vector without cDNA insert of either NC1-peptide or FAK (or its mutant), which yielded no detectable changes in the phenotypes in the seminiferous epithelium based on pilot experiments, as this treatment affected <8% of the tubules examined (see Fig. 1). Pilot experiments also illustrated that a single transfection of NC1-peptide was sufficient to induce remarkable changes in phenotypes, with a ~75% to 80% transfection efficiency, as previously reported (13, 65). This transfection solution in 50 µl, inside a 28-gauge 0.5-ml syringe with a 12.7-mm long needle, was loaded into each testis by first inserting it from the apical site of each testicle and gently moving it towards the basal end. As the syringe was slowly withdrawn from the testis, plasmid DNA in the transfection medium inside the syringe was released gradually into the testis, thus avoiding a rapid build-up of hydrostatic pressure that could induce mechanical damage to the seminiferous tubules. Rats at specified time points noted in the regimen in Fig. 1A were euthanized, as described above. Testes were fixed in either modified Davidson’s fixative or frozen in liquid nitrogen until ready for use for different experiments.

Histological analysis

Histological analysis was performed using paraffin-embedded cross-sections (about 5-µm thick and obtained using a microtome) of testes fixed in modified Davidson’s fixative (66) and stained in hematoxylin and eosin. Images of testes were obtained using an Olympus BX61 microscope equipped with an Olympus DP-71 digital camera, and the Olympus MicroSuite Five software package (version 1244; Olympus, Tokyo, Japan). Images were saved in TIFF format and aligned in Photoshop in Adobe Creative Suite (version 6.0) without further manipulations. All data shown here are representative images from independent experiments of n = 4 rats, including both treatment and control groups, which yielded similar results. All samples, including experimental and control groups, were processed in a single experimental session to avoid interexperimental variations. For statistical analysis, at least 80 cross-sections of tubules from an adult rat testis were randomly selected and examined from n = 4 rats, to a total of 320 cross-sections of tubules.

Immunofluorescence analysis by fluorescence microscopy

Immunofluorescence analysis by fluorescence microscopy, including dual-labeled IF, was performed, as detailed elsewhere using the corresponding specific antibodies (Table S1) (19) and as characterized in our earlier reports for CAMSAP2 and MTs (67), EB1 and MTs (68), F-actin and p-rpS6-S240/S244 (69), and with cross-sections of testes from the treatment versus control groups. For studies using primary Sertoli cells cultured in vitro for the visualization of different marker proteins pertinent to the regulation of Sertoli cell and BTB function, including p-FAK-Y407, F-actin, Eps8, Arp3, MTs and their different forms (including detryosinated α-tubulin, acetylated α-tubulin, and tyrosinated α-tubulin), EB1, and MAP1a, were performed using the corresponding antibodies (Table S1) (19) characterized earlier in our laboratory (17, 21, 67, 68, 70, 71). Cell nuclei were visualized by 4’,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). Slides were mounted on ProLong Gold antifade reagent (Invitrogen/Life Technologies/ThermoFisher (Waltham, MA). Fluorescence microscopy was performed using a Nikon Eclipse 90i microscope, and images were acquired using the built-in Nikon DS-Qi1Mc or DS-Fi1 digital camera and Nikon NIS Elements AR 3.2 software package. All samples within an experimental set, including both treatment and control groups, were processed and examined in a single experimental session to avoid interexperimental variations.

Criteria to assess defects in spermatogenesis

Based on histological analysis using cross-sections of testes from n = 4 rats, a seminiferous tubule was scored as a tubule with defects in spermatogenesis when its seminiferous epithelium displayed one or more of the following criteria, as previously reported (72–74). At least 80 tubules were randomly examined and scored per rat testis, to a total of 320 tubules for each treatment group versus control group. These included (1) premature release of germ cells, with the presence of >10 elongated spermatids or spermatocytes in the tubule lumen; (2) defects in spermatid polarity such that spermatid heads no longer pointed toward the basement membrane but deviated by 90^o–180^o from the intended orientation, and with at least >5 spermatids displaying defects in polarity per cross section of testes; (3) defects in Sertoli cell polarity such that Sertoli cell nuclei were no longer confined near the basement membrane of the tunica propria, as noted in control testes, but located at least 1/3 across the epithelium from its intended location; (4) Sertoli cell injury where large vacuoles 10–80 µm in size were detected across the seminiferous epithelium, with at least 2 of these vacuoles per cross section of a tubule; (5) the presence of multinucleated round spermatids, which are the result of defects in spermiogenesis, which would eventually be phagocytosed by Sertoli cells for degradation; (6) thinning of the seminiferous epithelium where due to germ cell exfoliation, a considerable reduction in the width of the epithelial cell layer across the epithelium was detected, >25% reduction when compared to control tubules which were ~70–90 µm depending on stages of the epithelial cycle in adult rat testes (75) was considered defective; and (7) defects in spermatid transport: elongated step 19 spermatids remained trapped inside the seminiferous epithelium when spermiation had taken place in nonstage VIII tubules, intermingling with step 9–14 spermatids in stage IX-XIV tubules.

Overexpression of NC1-peptide and/or FAK (WT) versus different FAK mutants in primary Sertoli cells cultured in vitro

Sertoli cells were isolated from 20-day-old rat testes, as detailed elsewhere (76), with negligible contamination of peritubular myoid cells, Leydig cells, and germ cells based on polymerase chain reaction (PCR) analysis using corresponding markers (77). Also, these cells were functionally similar to Sertoli cells isolated from adult rat testes, as earlier reported (78, 79). Freshly isolated cells were seeded on Matrigel-coated (1:5–1:7, diluted in F12/DMEM; Thermo Fisher Scientific): (1) 6-well dishes (containing 5 ml F12/DMEM/well for lysate preparation for IB or RNA extraction for qPCR); (2) coverslips (for IF microscopy, to be placed in 12-well dishes containing 2-ml F12/DMEM/well); and (3) bicameral units (Millicell-HA mixed cellulose esters-based cell culture inserts, 0.45-µm pore size, 12-mm diameter, effective surface area at 0.6 cm², with units placed in 24-well dishes, with 0.5 ml of F12/DMEM each in the apical and basal compartment for transepithelial electrical resistance measurement to monitor the TJ permeability barrier function) using a Sertoli cell density of 0.5 ×, 0.03 ×, and 1.0 × 10⁶ cells/cm², respectively. Sertoli cells were cultured in serum-free F12/DMEM (Sigma-Aldrich, St. Louis, Missouri) supplemented with growth factors and gentamicin in a humidified atmosphere of 95% air and 5% CO₂ (v/v) at 35 °C, as described (76). The in vitro experiments were performed using the regimen noted in different experiments in corresponding figures. For the overexpression of FAK-WT, p-FAK-Y407E-MT (ie, constitutively active), p-FAK-Y407F-MT (ie, constitutively inactive), and alone (or co-overexpression with NC1-peptide cDNA, also cloned into pCI-neo [see above]) versus empty vector (pCI-neo), Sertoli cells were transfected with the corresponding plasmid DNA: NC1-peptide cDNA at 0.8 µg of plasmid DNA per 10⁶ Sertoli cells; FAK-WT, p-FAK-Y407E-MT, or p-FAK-Y407F-MT cDNA at 0.5 µg of plasmid DNA per 10⁶ Sertoli cells; or 0.5 µg DNA of each target gene/10⁶ cell in double transfection/overexpression experiment for 14 hours with Lipojet In Vitro Transfection Reagent (SignaGen Laboratories, Rockville, Maryland) using a 3 µl transfection medium (1 µg plasmid DNA ratio, according to the manufacturer protocol as detailed elsewhere) (80). Thereafter, transfection reagent was removed by washing Sertoli cells twice with F12/DMEM. Plasmid DNA was labeled with Cy3 using a LabelIT® Tracker Intracellular Nucleic Acid Localization Kit (Mirus, Madison, Wisconsin) to confirm successful transfection by IF microscopy.

Real-Time qPCR

Overexpression of NC1-peptide in lysates of testes and Sertoli cells was quantified by QuantStudio^TM 12K Flex Real-Time PCR System (ThermoFisher, Waltham, Massachusetts) with Power SYBR Green Master Mix (Applied Biosystems, Foster City, California), following the protocols from the manufacturer with n = 5 samples in different experiments (each sample in triplicate) with Gapdh serving as the internal control for normalization. Specificity of the fluorescent signal was confirmed by both melting curve analysis and gel electrophoresis. Expression of the NC1 gene was determined using the 2^-ΔΔC_T method (65).

Lysate preparation, protein estimation, and IB

Lysates from testes or Sertoli cells were obtained for IB using corresponding antibodies (Table S1) (19), as described (81). Protein estimation was performed using the BioRad DC kits using BSA (bovine serum albumin) to calibrate the standard curve. About 40 µg protein per testis sample, or 30 µg protein per Sertoli cell lysate sample, was used for SDS-PAGE and IB analysis.

F-actin and MT spin-down assays

The assays used to assess bundled/polymerized actin filaments and polymerized MTs were performed using lysates of Sertoli cells and kits from Cytoskeleton (Denver, Colorado) for F-actin (Cat Number: BK037) and MTs (Cat Number: BK038) according to the manufacturer’s protocols, as described (14, 82).

Statistical analysis

Statistical analysis was performed using GraphPad Prism 6 software package (GraphPad Software, version 6). Each data point was a mean ± SD of n = 3 to 5 independent experiments (or n = 4 to 6 rats), using either a Student t-test (for 2-group comparisons) or 2-way analysis of variance (ANOVA; for multigroup comparisons), and using the repeated-measures model followed by Dunnett’s test to compare changes between treatment and control groups by assessing within-experimental effects, which were the focus of the analysis. P < 0.05 was considered statistically significantly different.

Results

NC1-peptide-induced defects in spermatogenesis is mediated by mTORC1/p-rpS6, p-FAK-Y397, and p-FAK-Y407

The regimen used for this study is shown in Fig. 1A, from n = 3 independent experiments with n = 4 rats per time point for each experiment, with 7 termination time points so that a sufficient number of rat testes (ie, 12 rats per time point) were obtained for multiple experimental analyses. Rats in the animal group of pCI-neo/Ctrl were terminated on day 14 following transfection of the testis with plasmid DNA from pCI-neo empty vector and designated pCI-neo/Ctrl, as noted in Figs. 1–4. However, rats from the pCI-neo/Ctrl group terminated on day 0 (ie, rats terminated within 30 minutes), day 3, and day 7 (n = 3 rats per group based on pilot experiments) displayed similar results to rats terminated on day 14 and similar to normal control testes without any transfection. Overexpression of NC1-peptide was confirmed by q-PCR, as noted in Fig. 1B from n = 3 rats. Testes from n = 3 rats were used for histological analysis following H&E (hematoxylin and eosin) staining (Fig. 1C). Criteria used to determine defects of spermatogenesis are described in the “Materials and Methods” section, with normal testes (transfected with empty vector, pCI-neo/Ctrl) noted on the top panel in Fig. 1C. In as early as 3 to 6 hours, defects of spermatogenesis were detected in some nonstage VIII tubules, with spermatid loss (orange arrowheads) and spermatocyte loss (orange arrowheads) found in tubule lumen, but some elongated spermatids (step 19) spermatids remained embedded deep inside the seminiferous epithelium (green arrowheads) (Fig. 1C). Some Sertoli cells were moved away from the basement membrane (black arrowhead) due to loss of Sertoli cell polarity (Fig. 1C). Many elongated spermatids had defects in polarity (red arrowheads) when their heads oriented by at least >90^o from the intended orientation of pointing toward the basement membrane when compared to control testes. Also, numerous vacuoles (>16 µm) were noted (yellow arrowheads) in the seminiferous epithelium on days 3, 7, and 14, which represented the early stage of Sertoli cell injury due to defective enlargement of smooth endoplasmic reticulum (73, 83, 84). By day 3 and thereafter, numerous multinucleated round spermatids were also detected (blue arrowheads) in many tubules, which were the defective germ cells to be phagocytosed by Sertoli cells for subsequent degradation and disposal (72, 85). By day 14, extensive thinning of the epithelium in >90% of the tubules was noted due to germ cell exfoliation, and the tubule diameter was considerably reduced (Fig. 1C). A summary of the data based on randomly scored defective tubules in n = 4 rats (200 tubules per rat testis to a total of at least 800 tubules) was shown in Fig. 1D. We next used IB with the corresponding specific antibodies (Table S1) (19), which had been characterized earlier from our laboratory for corresponding marker proteins to assess changes in the expression of a number of signaling proteins shown to be involved in modulating spermatogenesis and BTB function, some of which have also been shown to be putative signaling proteins that work downstream of the NC1-peptide-mediated defects in spermatogenesis, such as mTORC1/p-rpS6 (14). Our findings showed that p-rpS6-S235/S236 and p-rpS6-S240/S244 were considerably upregulated by hour 3, through days 1 to 3, following overexpression of NC1-peptide, which was consistent with a recent report based on a study using Sertoli cells cultured in vitro (14). These findings are also consistent with earlier reports that an activation of mTORC1/p-rpS6 was associated with defects in spermatogenic function both in vitro (69, 86, 87) and in vivo (88, 89). Additionally, the expression of c-Src (but not c-Yes—both of which are members of the Src family kinases) is known to be involved in regulating spermatogenesis (90), and several MT regulatory proteins, including MARK2 (a Ser/Thr kinase that phosphorylates MAPs, causing their detachment from MTs to facilitate MT catastrophe (91)) and CAMSAP2 (an MT minus [-] end targeting protein, -TIP, that binds to the slow-growing minus [-] end of MT to induce destabilization (67)) were downregulated (Fig. 1E). Importantly, p-FAK-Y397 and p-FAK-Y407 were also considerably downregulated, which are also 2 prominent regulators of spermatogenesis in the testis (16, 17). Collectively, these findings support the notion that NC1-peptide is utilizing mTORC1/p-rpS6, p-FAK-Y397, and p-FAK-Y407 downstream to exert its regulatory effects to modulate spermatogenesis.

Figure 4.

NC1-peptide perturbs F-actin organization through a surge in spatial expression of p-rpS6-S240/S244 at the apical and basal ES. On the left panel, the organization F-actin was visualized by FITC-phalloidin (green fluorescence), and the right panel shows F-actin (green fluorescence) co-localized with p-rpS6-S240/S244 (red fluorescence), which is the downstream signaling of mTORC1. In control testes, F-actin appeared as bulb-like structures at the concave side, surrounding the entire tip of spermatid heads (see enlarged boxed areas in red and yellow insets) at the site of the apical ES. F-actin was also notably expressed near the basement membrane (annotated by the dashed white line) at the site of the basal ES/BTB (annotated by yellow arrowheads). However, following overexpression of NC1-peptide, F-actin at the apical ES became notably disorganized by day 1, as F-actin no longer restrictively expressed at the apical and basal ES/BTB sites, but randomly localized across the seminiferous epithelium (see both left and right panels). Interestingly, the expression of p-rpS6-S240/S244 was notably enhanced at the apical and basal ES, which was previously shown to be associated with a degenerating apical and basal ES based on studies in vitro (69, 87) and in vivo (88, 89). This surge of p-rpS6-S240/S244 expression across the seminiferous epithelium is also consistent with the IB data shown in Fig. 1E. See also the boxed yellow and red areas (enlarged in corresponding insets); insets in blue are the merged images. Scale bars: 150 µm, 80 µm, and 40 µm in the left panel; and 80 µm and 30 µm in the right panel; which apply to corresponding micrographs in the same panel. Results shown here are representative findings of an experiment from n = 3 experiments using different testes for examination which yielded similar results. BTB, blood-testis barrier; ES-ectoplasmic specialization; FITC, fluorescein isothiocyanate.

NC1-peptide overexpression in the testis perturbs MT organization

Since studies have shown that p-FAK-Y397 and p-FAK-Y407 exert their regulatory effects on spermatogenesis, such as BTB dynamics, via changes in cytoskeletal organization based on studies in vitro and in vivo (16, 17, 21), including primary human Sertoli cells in vitro (22), we examined any changes in MT organization in the testis following NC1-peptide overexpression in the testis. Indeed, overexpression of NC1-peptide induced considerable defects on MT-based cytoskeleton across the seminiferous epithelium time-dependently (Fig. 2). These defects of MT organization appeared to be associated with disruptive changes in spatial expression of 3 MT regulatory proteins. First, CAMSAP2, a -TIP (MT minus [-] end targeting protein) that binds to the MT minus end known to maintain MT homeostasis by determining the lengths of MT protofilaments (92, 96), which also plays a similar role in the testis (67) (Fig. 2). Second, EB1, a +TIP (MT plus [+] end-tracking protein) that binds to the MT plus end, is known to promote MT stabilization (93, 97), which also plays a similar role in the testis (68) (Fig. 3). Third, MAP1a (an MT-associated protein 1a), an MAP known to bind to the tubulins along the MTs to maintain MT stability (95) (Fig. 3). Based on the representative data from n = 3 rats, all 3 MT regulatory proteins became truncated and disorganized, which were detected as early as hour 6; thereafter, their expression were also considerably downregulated, following overexpression of NC1-peptide, which is consistent with the IB data shown in Fig. 1. By day 14, these MT regulatory proteins that bound to MTs appeared as collapsed structures alongside MTs (Figs. 2 and 3).

Figure 2.

NC1-peptide perturbs spermatogenesis through its disruptive effects on MT organization. Using the regimen outlined in Fig. 1A, the organization of MTs across the seminiferous epithelium was examined by staining rat testes with α-tubulin (which together with ß-tubulin [that create the α-/ß-tubulin dimers] are the building blocks of MTs) (green fluorescence) obtained from rats at different time points following overexpression of NC1-peptide versus the control (pCI-neo/Ctrl) testes (left panel). In the control testes, MTs stretched across the entire epithelium as distinctive track-like structures that aligned perpendicular to the basement membrane (annotated by a dashed white line), which served as tracks to support transports of spermatids and other cell organelles (eg, residual bodies, phagosomes, endocytic vesicles). By hour 6, defects in MTs were noted, since these MTs were truncated (annotated by red arrowheads) and broken into shorter fragments (white arrowheads). More extensive defects in MT organization were noted in subsequent time points, and by days 3 to 7, many MTs were misaligned, and some MT-based tracks laid parallel to the basement membrane (yellow arrowheads). By day 14, MTs almost collapsed (blue arrowheads) that laid parallel to (and near) the basement membrane. As noted in the panel on the right, CAMSAP2 (red fluorescence), a -TIP that binds to the minus (-) end of MTs and is known to destabilize MTs to regulate MT lengths (92), co-localized with MTs, as previously shown in the testis (67), also displayed disruptive spatial expression, with notable truncation and misalignment similar to MTs. Scale bar: 80 µm and 40 µm, which apply to corresponding micrographs. d, day; h, hour; MT, microtubule.

Figure 3.

NC1-peptide perturbs MT. On the panel on the left, the organization of MTs (visualized by ß-tubulin staining, green fluorescence) was supported by EB1 (red fluorescence, a MT plus (+) end-tracking protein that binds to the plus (+) end of MTs, known to stabilize MTs (93, 94)) and also to MAP1a (right panel; red fluorescence, an MAP known to bind to the tubulins along the MTs to maintain MT stability (95)). In control testes, EB1 (left panel) and MAP1a (right panel) co-localized with ß-tubulin and aligned perpendicular to the basement membrane (annotated by dashed white line), which also stretched across the entire length of the seminiferous epithelium. However, by 6 hours after overexpression of the testis with NC1-peptide, MTs were truncated and broken (red arrowheads) into smaller fragments (yellow arrowheads), some of which were misaligned that laid parallel (yellow arrowheads) to the basement membrane. By day 14, many MTs were collapsed (blue arrowheads) and laid close to the basement membrane. Scale bar: 80 µm, which applies to the corresponding micrographs. Results shown here are representative findings of an experiment from n = 3 experiments using different testes for examination which yielded similar results. d, day; h, hour; MAP, microtubule-associated protein; MT, microtubule; qPCP, read-time qPCR.

NC1-peptide overexpression in the testis perturbs F-actin organization

Studies have shown that mTORC1/p-rpS6 signaling complex exerts its effects to modulate F-actin organization based on studies in vitro (69, 86, 87) and in vivo (88, 89). Also, IB data noted in Fig. 1E confirmed the involvement of mTOR/p-rpS6 complex in NC1-peptide-mediated effects on BTB function and spermatogenesis, consistent with a recently reported in vitro study (14). Thus, we examined the spatial expression of p-rpS6-S240/S244 in the seminiferous epithelium in the testis following overexpression of NC1-peptide using the regimen shown in Fig. 1A. As noted in the left panel in Fig. 4, F-actin was notably expressed at the apical ES (appearing as bulb-like structures predominantly at the concave side of spermatid heads) and basal ES/BTB (annotated by yellow arrowheads) near the basement membrane (annotated by dashed white line) in control testes (pCI-neo/Ctrl, empty vector). However, overexpression of NC1-peptide induced a rapid but time-dependent disruptive organization of F-actin across the seminiferous epithelium whereby F-actin was notably disorganized by 1 day until all the tubules were devoid of germ cells (except for some spermatogonia and Sertoli cells) by days 7 and 14 (Fig. 4). Consistent with the IB data shown in Fig. 1E, there was a considerable surge in the expression of p-rpS6-S240/S244 at the sites of both apical ES and basal ES/BTB by hours 3, 6 and 12, but the expression of p-rpS6 gradually diminished by days 7 and 14, also consistent with IB data noted in Fig. 1E (vs Fig. 4, right panel).

Overexpression of pFAK-Y407E-MT (mutant) blocks NC1-peptide-mediated Sertoli cell TJ permeability barrier disruption

Studies have shown that p-FAK-Y397 (16) and p-FAK-Y407 (17) are ES regulators in the testis. p-FAK-Y397 promotes apical ES integrity but perturbs basal ES/BTB function, whereas p-FAK-Y407 promotes both apical and basal ES function (16, 17, 98). As noted in Fig. 1E, NC1-peptide-mediated defects in spermatogenesis were found to associate with a considerable downregulation of p-FAK-Y407 and p-FAK-Y397. To expand these findings, we used a phosphomimetic (ie, constitutively active) mutant of p-FAK versus WT (ie, the full-length FAK) and a nonphosphorylatable (ie, constitutively inactive) mutant of p-FAK called p-FAK-Y407F-MT (see the “Materials and Methods” section for the preparation of these cDNA constructs) for co-transfection studies, as noted in Fig. 5, using the regimen in Fig. 5A. Overexpression of p-FAK-Y407 was confirmed by IB (Fig. 5B, left panel) using an antibody specific to p-FAK-Y407 (Table S1) (19) and overexpression of the NC1-peptide was confirmed by qPCR (Fig. 5B, right panel) using a primer pair specific to NC1-peptide (Table 1). As shown in Fig. 5C, overexpression of p-FAK-Y407E-MT or p-FAK-Y407F-MT indeed promoted and perturbed the TJ barrier function, respectively, consistent with an earlier report (17). Also, NC1-peptide overexpression perturbed Sertoli cell TJ barrier function (Fig. 5C). However, overexpression of p-FAK-Y407E-MT was capable of blocking NC1-peptide-induced TJ barrier disruption (Fig. 5D), illustrating that the signaling protein p-FAK-Y407 noted in Fig. 1E was involved in NC1-peptide-mediated efforts on spermatogenesis. In order to expand this observation, distribution of BTB-associated proteins at the Sertoli cell–cell interface was examined by fluorescence microscopy in the treatment group versus control group (Fig. 5E). NC1-peptide overexpression indeed perturbed the distribution of TJ proteins (eg, occludin, ZO-1) and basal ES proteins (eg, N-cadherin, ß-catenin) at the Sertoli cell cortical zone (Fig. 5E) without altering the steady-state levels of these proteins (Fig. 5F), which was consistent with results of an earlier report (13). However, overexpression of p-FAK-Y407E-MT was able to block the NC1-peptide-mediated disruptive distribution of TJ and basal ES proteins at the Sertoli cell cortical zone (Fig. 5E), also without perturbing the steady-state levels of these proteins (Fig. 5F). These findings thus support the notion that NC1-peptide exerts its regulatory effects on BTB function through p-FAK-Y407.

Figure 5.

Overexpression of p-FAK-Y407E mutant in Sertoli cells cultured in vitro blocks NC1-peptide mediated Sertoli cell TJ-barrier disruption. A: Regimen used to examine whether overexpression of p-FAK-Y407 would alleviate the disruptive effects of NC1-peptide on Sertoli cell BTB function by using the phosphomimetic (ie, constitutively active) mutant-designated p-FAK-Y407E-MT versus the nonphosphorylatable (ie, constitutively inactive) mutant-designated p-FAK-Y407F-MT and the WT-designated FAK-WT on the Sertoli cell TJ permeability barrier function, as well as for IB and IF. B: IB (left panel) and qPCR (right panel) were used to illustrate changes (and overexpression) in the steady-state protein and mRNA levels of p-FAK-Y407 by using a specific anti-p-FAK-Y407 antibody (for IB) (Table S1) (19) and by qPCR using the primer pair specific for p-FAK-Y407 (Table 1). C: Effects of overexpression of NC1-peptide (pCI-neo/NC1) and different phosphomimetic (ie, constitutively active, pCI-neo/p-FAK-Y407E) and nonphosphorylatable (ie, constitutively inactive, pCI-neo/p-FAK-Y407F) mutants versus WT (pCI-neo/WT) of FAK on the Sertoli cell TJ permeability barrier function. Each data point is a mean ± SD of n = 4 bicameral units from an experiment, whereby n = 3 independent experiments yielded similar results. * P < 0.05 when compared to pCI-neo/Ctrl by ANOVA. D: Overexpression of p-FAK-Y407E was effective (and more effective than FAK-WT) to block the NC1-peptide-mediated disruption of the Sertoli cell TJ permeability barrier function. Each data point is a mean ± SD of n = 4 bicameral units from an experiment, whereby n = 3 independent experiments yielded similar results. * P < 0.05, ** P < 0.01 by ANOVA. E: Overexpression of the p-FAK-Y407E mutant was able to block the disruptive effects of NC1-peptide (pCI-neo/NC1) on the distribution of TJ (eg, occludin, ZO-1) and basal ES (eg, N-cadherin and ß-catenin) proteins at the Sertoli cell–cell interface of the Sertoli cell epithelium in vitro. Results shown herein are representative data from an experiment, and a total of n = 3 experiments yielded similar results. F: Results of a study by IB using corresponding specific antibodies (Table S1) (19), which illustrated that the overexpression of either NC1-peptide or p-FAK-Y407E had no apparent effects on the steady-state protein levels of the TJ (eg, occludin, ZO-1) and basal ES (eg, N-cadherin, ß-catenin) proteins in lysates of Sertoli cells from different treatment versus control groups. Results are representative data of an experiment from n = 3 IB experiments, which yielded similar results. ANOVA, analysis of variance; IB, immunoblot analysis; IF, immunofluorescence analysis; qPCR, real-time qPCR; WT, wild-type.

Table 1.

Primer pairs used for real-time qPCR

Gene	Primer Sequence	Position	Length, bp	Temp (°C)	GenBank Accession No.
S16	Sense: 5’-TCCGCTGCAGTCCGTTCAAGTCTT-3’	15–38	385	54	XM_341815
	Antisense: 5’-GCCAAACTTCTTGGTTTCGCAGCG-3’	376–399
NC1	Sense: 5’-CAGCTGCCTACAGCGATTCA-3’	4473–4492	177	60	NM_001135759.1
	Antisense: 5’-ACGGTGCATCTGCTAACGTA-3’	4630–4649

Abbreviations: bp, base pair; temp, annealing temperature.

Overexpression of p-FAK-Y407E-MT protects Sertoli cell actin cytoskeleton organization from NC1-peptide-mediated disruptive changes. Since TJ and basal ES proteins at the BTB examined in Fig. 5E utilized F-actin for their attachments, and p-FAK-Y407E-MT was able to correct the NC1-peptide-mediated disruptive changes in the distribution of these cell adhesion proteins at the Sertoli cell–cell interface, we next examined F-actin organization in Sertoli cells (Fig. 6). Using the regimen shown in Fig. 5A, the efficacy of overexpression of p-FAK-Y407 was confirmed in Fig. 6A (top panel) and also in Fig. 6B by IB. More importantly, the NC1-peptide induced disruptive organization of F-actin, wherein actin filaments no longer stretched across the Sertoli cell cytosol, as noted in the control group, and was blocked when the Sertoli cell epithelium was co-transfected with NC1-peptide and p-FAK-Y407E-MT plasmid cDNA for their overexpression (Fig. 6A). p-FAK-Y407E-MT likely exerted its effects through changes in the spatial expression of the 2 actin regulatory proteins Eps8 (an actin barbed end capping and bundling protein in the testis (71)) and Arp3 (which, together with Arp2—to create the Arp2/3 complex—is a branched actin polymerization protein in the testis (99)) (Fig. 6A). Consistent with findings noted in studies in vivo (Fig. 1E), NC1-peptide exerted its effects through a surge in the expression of p-rpS6 and a concomitant downregulation of p-Akt2, which were also blocked by overexpression of p-FAK-Y407E-MT (Fig. 6B). NC1-peptide also downregulated the expression of Eps8, but not Arp3, which was also blocked by overexpression of p-FAK-Y407E-MT (Fig. 6B). More importantly, using a biochemical assay to monitor actin polymerization activity in the treatment group versus control group, it was noted that NC1-peptide indeed perturbed actin polymerization activity of generating F-actin in cell lysates, but this defect was corrected by p-FAK-Y407E-MT (Fig. 6C).

Figure 6.

Overexpression of p-FAK-Y407E-MT in Sertoli cells blocks NC1-peptide-mediated disruption of actin cytoskeleton through changes in actin organization and polymerization. The regimen used for the experiments reported herein is shown in Fig. 5A. A: Overexpression of p-FAK-Y407-MT was confirmed by immunostaining of Sertoli cells using a specific anti-p-FAK-Y407 antibody (Table S1) (19) in the treatment versus control groups, wherein NC1-peptide was found to downregulate p-FAK-Y407 expression (top panel). NC1-peptide induced F-actin disorganization and downregulation and disruptive spatial expression in actin barbed-end capping, and bundling protein Eps8 and branched actin polymerization protein Arp3, respectively, were noted. However, these disruptive changes were blocked by p-FAK-Y407E-MT. Scale bar: 40 µm. Images shown are representative findings of experiments from n = 3 experiments, which yielded similar results. B: IB data confirmed overexpression of p-FAK-Y407. NC1-peptide also induced downregulation of p-FAK-Y407 and Eps8, but these changes were blocked by p-FAK-Y407E-MT and had no effect on total FAK. On the other hand, NC1-peptide induced an upregulation of p-rpS6-S240/S244 (but not total rpS6), which was downregulated following overexpression of p-FAK-Y407E-MT. NC1-peptide also induced a downregulation of p-Akt2-S474 (but not total Akts), which was also recused by p-FAK-Y407E-MT overexpression. C: The disruptive effect of NC1-peptide on F-actin organization across the Sertoli cell cytosol as noted in (A) was confirmed by biochemical assay that assessed actin polymerization activity, with the corresponding positive and negative controls shown here. However, overexpression of p-FAK-Y407E-MT was able to block the NC1-peptide-induced defects in actin polymerization. Overexpression of p-FAK-Y407 in this experiment was confirmed by IB (see the last lane in [C]). Results shown here are representative findings of an experiment from n = 3 experiments, which yielded similar results. Composite data of the biochemical analysis were shown on the right panel, with each bar representing a mean ± SD of n = 3 experiments.** P < 0.01 by ANOVA, *** P < 0.001 by ANOVA. ANOVA, analysis of variance; IB, immunoblot analysis; MT, mutant.

Overexpression of p-FAK-Y407E-MT protects Sertoli cell MT organization from NC1-peptide-mediated disruptive changes

Microtubules, visualized by α-tubulin staining (since the α-/ß-tubulin oligomers are the building blocks of MTs), stretched across the Sertoli cell cytosol to provide structural support, as noted in control cells (Fig. 7A). NC1-peptide overexpression perturbed MT cytoskeleton, causing extensive truncations so that MTs just wrapped around the cell nuclei instead of extending across the entire cell cytosol; however, these disruptive changes were blocked by overexpression with p-FAK-Y407E-MT (Fig. 7A). Similar changes were noted for detyrosinated (where C-terminal Tyr [Y] was hydrolyzed by exposing Glu [E] at its newly formed C-terminus, rendering MTs to become more stable (100, 101)) and for acetylated (also rendering MTs less dynamic and more stable (101)) α-tubulin versus tyrosinated α-tubulin (which is more dynamic and less stable (101)) (Fig. 7A). Also, overexpression of NC1-peptide induced downregulation and/or disruptive distribution of EB1, a +TIP (MT plus [+] end-tracking protein, known to stabilize MTs (93, 102)) and MT-associated protein 1A (MAP1a, known to stabilize MTs (103)), wherein these regulatory proteins no longer stretched across the cell cytosol with MTs, but, as disruptive spatial distributions, were corrected following overexpression with p-FAK-Y407E-MT (Fig. 7A). Furthermore, using a biochemical assay to monitor MT polymerization activity in the treatment group versus the control group, it was noted that NC1-peptide perturbed MT polymerization activity, and this defect was blocked by p-FAK-Y407E-MT overexpression (Fig. 7B).

Figure 7.

Overexpression of p-FAK-Y407E-MT in Sertoli cells blocks NC1-peptide-mediated disruption on MT cytoskeleton through changes in MT organization and polymerization. The regimen used for the experiments reported herein is shown in Fig. 5A. A: NC1-peptide was found to perturb MT organization, which was visualized by α-tubulin staining (α-/ß-tubulin oligomers are the building blocks of MTs), since MTs no longer stretched across the cell cytosol but became truncated and disorderly localized across the cell cytosol. However, overexpression of p-FAK-Y407E-MT was capable of blocking the NC1-peptide-mediated MT disorganization. Furthermore, NC1-peptide overexpression also affected the organization of both detyrosinated and acetylated α-tubulin (which promoted MT stabilization (101)) and tyrosinated α-tubulin (which promoted MT dynamics, rendering MT less stable (101)), since these 3 forms of MTs no longer stretched across the cell cytosol but retracted across the cell by staying closer to the cell nuclei, thereby impeding MT dynamics across the cell cytosol to maintain MT homeostasis. However, p-FAK-Y407E-MT overexpression blocked the NC1-peptide-mediated changes in their distribution, possibly through corrective spatial expression of EB1 (a +TIP known to stabilize MTs (93, 102)) and MAP1a (an MT-binding protein also known to stabilize MTs (103)) by maintaining MT homeostasis. Scale bar: 40 µm, which applies to all other micrographs. B: The findings noted in (A) are also supported by findings based on a biochemical assay that indicated that NC1-peptide overexpression induced a considerable loss of MT polymerization in Sertoli cells, which was also blocked by overexpression of p-FAK-Y407E-MT. Overexpression of p-FAK-Y407 in the corresponding samples and the effects of NC1-peptide to downregulate p-FAK-Y407 expression was noted in the last lane. All data shown here are representative findings from an experiment of n = 3 experiments, which yielded similar results. * P < 0.05 by ANOVA; ** P < 0.01 by ANOVA, *** P < 0.001 by ANOVA.

Discussion

Studies have shown that biologically active fragments can be released from extracellular matrix components, such as collagens and laminins, via the action of matrix metalloproteinases (104), which are intimately related to health and diseases, since these bioactive fragments designated matrikines are capable of inducing cellular activation and chemotaxis of a variety of cells (105, 106). For instance, endostatin, a 20 kDa fragment derived from NC1 domain of collagen type XVIII, is a potent inhibitor of endothelial cell proliferation, cell migration, angiogenesis, and also tumor growth (107). In fact, matrikines derived from basement membrane collagens are actively being investigated as novel anticancer reagents (108). On the other hand, collagens also serve as an extracellular storage site for bioactive peptides, such as IL-2 (109, 110). Thus, it is not entirely unexpected that NC1-peptide released from the C-terminal region of collagen α3 (IV) chain, a major constituent component of the basement membrane (10), is a novel bioactive peptide capable of regulating BTB and spermatogenesis (13).

A recent report has demonstrated that NC1-peptide exerts its effects via mTORC1/rpS6 signaling complex, involving an activation of Cdc42, but not RhoA GTPase, which in turn modifies actin and also MT cytoskeletal organization (14). These findings are physiologically important, because they illustrate that a manipulation of mTORC1, rpS6, and Cdc42 can considerably impact the action of NC1-peptide in modulating spermatogenesis through its action on actin cytoskeleton, which is consistent with earlier reports (111, 112). Other studies have shown that FAK, specifically p-FAK-Y407, is a crucial regulator of cytoskeletal function in the testis (17). Specifically, using a phosphomimetic (ie, constitutive active) mutant of p-FAK-Y407 called p-FAK-Y407E (by converting Y407 to E407 by site-directed mutagenesis (17)) is capable of promoting Sertoli cell BTB barrier function using an in vitro rat Sertoli cell BTB model (17). In fact, when a human p-FAK-Y407E mutant was prepared, it was also capable of promoting BTB function in human primary Sertoli cell epithelium by blocking perfluorooctanesulfonate (PFOS; an environmental toxicant known to perturb BTB function in rat Sertoli cells (21)) mediated BTB dysfunction (22). As overexpression of NC1-peptide in the testis in vivo was associated with a considerable decline in the expression of p-FAK-Y407, we thus sought to examine if co-overexpression of p-FAK-Y407E mutant would also block NC1-peptide-mediated changes in cytoskeletal organization. Indeed, it was demonstrated for the first time that, besides mTORC1/rpS6 and Cdc42 activation, p-FAK-Y407 is a putative signaling protein utilized by NC1-peptide to exert is regulatory effects on spermatogenesis. Interestingly, this observation is in agreement with an earlier study, which has shown that mTORC1-FAK signaling is crucial to support mesenchymal stem cell proliferation (113), thus supporting the concept that the mTORC1 signaling pathway can merge with the versatile FAK-based signaling pathway, which is crucial to induce integrin receptors clustering to interact with the extracellular matrix on the outside of cells and with the actin cytoskeleton on the inside at the focal adhesion site of cell epithelia (114, 115).

While most of the studies that probe the role of FAK in cytoskeletal regulation focused on F-actin cytoskeleton, emerging evidence has shown that FAK also regulates MTs; aside from F-actin, network function (116), such as through its effects on dynein localization and cell polarity by recruiting dynein/dynactin complex at focal adhesion sites at the leading edge of migrating fibroblasts (117). In this context, it is of interest to note that dynein is a MT-dependent minus (-) end-directed motor protein, recently shown to support cargo transport, including spermatids, across the seminiferous epithelium in the testis (118).

In summary, NC1-peptide released from the collagen α3 (IV) chain in the basement membrane that regulates Sertoli cell function and spermatogenesis is mediated through the mTORC1/p-rpS6/p-Akt1/2 and p-FAK-Y407 signaling cascade. These signaling proteins are also being used by another signaling peptide produced endogenously in the testis called LG3/4/5-peptide, and they are released from the laminin-α2 chain in the basement membrane (119, 120). However, NC1- and LG3/4/5-peptides have contrasting effects on Sertoli cell BTB function and spermatogenesis wherein NC1-peptide promotes junction remodeling at the Sertoli-spermatid interface (ie, apical ES) and Sertoli cell–cell interface (ie, basal ES/BTB) (12–14) versus the LG3/4/5-peptide, which promotes apical and basal ES/BTB integrity (119, 120), respectively. Interestingly, NC1-peptide upregulates p-rpS6 and downregulates p-FAK-Y407 expression, as reported herein, whereas LG3/4/5-peptide downregulates p-rpS6 expression (119, 120). Taken collectively, these findings illustrate the presence of an efficient mechanism in the testis to turn cellular events “on” and “off” across the seminiferous epithelium during the epithelial cycle of spermatogenesis through upregulation or downregulation of the mTORC1/p-rpS6/p-Akts and p-FAK-Y407 signaling cascade.

Additional Information

Disclosure Summary: The authors declare that there is no conflict of interest.

Data Availability: All data generated or analyzed during this study are included in this published article or in the data repositories listed in References.