Blood-testis Barrier Remodeling Research Articles

Eugenol alleviates acrylamide-induced rat testicular toxicity by modulating AMPK/p-AKT/mTOR signaling pathway and blood-testis barrier remodeling.

This study aimed to investigate the effects of eugenol treatment on reproductive parameters in acrylamide (ACR)-intoxicated rats. The study evaluated alterations in relative testes and epididymides weights, sperm quality, serum hormonal status, seminal plasma amino acids, testicular cell energy and phospholipids content, oxidative and nitrosative stress parameters, adenosine monophosphate-activated protein kinase/ phosphoinositide 3-kinase/phosphor-protein kinase B/mammalian target of rapamycin (AMPK/PI3K/p-AKT/mTOR) signaling pathway, blood-testis barrier (BTB) remodeling markers, testicular autophagy and apoptotic markers, as well as histopathological alterations in testicular tissues. The results revealed that eugenol treatment demonstrated a significant improvement in sperm quality parameters, with increased sperm cell concentration, progressive motility live sperm, and a reduction in abnormal sperm, compared to the ACR-intoxicated group. Furthermore, eugenol administration increased the levels of seminal plasma amino acids in a dose-dependent manner. In addition, eugenol treatment dose-dependently improved testicular oxidative/nitrosative stress biomarkers by increasing oxidized and reduced glutathione levels and reducing malondialdehyde and nitric oxide contents as compared to ACRgroup. However, eugenol treatment at a high dose restored the expression of AMPK, PI3K, and mTOR genes, to levels comparable to the control group, while significantly increasing p-AKT content compared to the ACRgroup. In conclusion, the obtained findings suggest the potential of eugenol as a therapeutic agent in mitigating ACR-induced detrimental effects on the male reproductive system via amelioration of ROS-mediated autophagy, apoptosis, AMPK/p-AKT/mTOR signaling pathways and BTB remodeling.

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

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.

NC1-peptide regulates spermatogenesis through changes in cytoskeletal organization mediated by EB1.

During the epithelial cycle of spermatogenesis, different sets of cellular events take place across the seminiferous epithelium in the testis. For instance, remodeling of the blood-testis barrier (BTB) that facilitates the transport of preleptotene spermatocytes across the immunological barrier and the release of sperms at spermiation take place at the opposite ends of the epithelium simultaneously at stage VIII of the epithelial cycle. These cellular events are tightly coordinated via locally produced regulatory biomolecules. Studies have shown that collagen α3 (IV) chains, a major constituent component of the basement membrane, release the non-collagenous (NC) 1 domain, a 28-kDa peptide, designated NC1-peptide, from the C-terminal region, via the action of MMP-9 (matrix metalloproteinase 9). NC1-peptide was found to be capable of inducing BTB remodeling and spermatid release across the epithelium. As such, the NC1-peptide is an endogenously produced biologically active peptide which coordinates these cellular events across the epithelium in stage VIII tubules. Herein, we used an animal model, wherein NC1-peptide cloned into the pCI-neo mammalian expression vector was overexpressed in the testis, to better understanding the molecular mechanism by which NC1-peptide regulated spermatogenic function. It was shown that NC1-peptide induced considerable downregulation on a number of cell polarity and planar cell polarity (PCP) proteins, and studies have shown these polarity and PCP proteins modulate spermatid polarity and adhesion via their effects on microtubule (MT) and F-actin cytoskeletal organization across the epithelium. More important, NC1-peptide exerted its effects by downregulating the expression of microtubule (MT) plus-end tracking protein (+TIP) called EB1 (end-binding protein 1). We cloned the full-length EB1 cDNA for its overexpression in the testis, which was found to block the NC1-peptide-mediated disruptive effects on cytoskeletal organization in Sertoli cell epithelium and pertinent Sertoli cell functions. These findings thus illustrate that NC1-peptide is working in concert with EB1 to support spermatogenesis.

MTORC1/rpS6 signaling complex modifies BTB transport function: an in vivo study using the adjudin model.

Studies have shown that the mTORC1/rpS6 signaling cascade regulates Sertoli cell blood-testis barrier (BTB) dynamics. For instance, specific inhibition of mTORC1 by treating Sertoli cells with rapamycin promotes the Sertoli cell barrier, making it "tighter." However, activation of mTORC1 by overexpressing a full-length rpS6 cDNA clone (i.e., rpS6-WT, wild type) in Sertoli cells promotes BTB remodeling, making the barrier "leaky." Also, there is an increase in rpS6 and p-rpS6 (phosphorylated and activated rpS6) expression at the BTB in testes at stages VIII-IX of the epithelial cycle, and it coincides with BTB remodeling to support the transport of preleptotene spermatocytes across the barrier, illustrating that rpS6 is a BTB-modifying signaling protein. Herein, we used a constitutively active, quadruple phosphomimetic mutant of rpS6, namely p-rpS6-MT of p-rpS6-S235E/S236E/S240E/S244E, wherein Ser (S) was converted to Glu (E) at amino acid residues 235, 236, 240, and 244 from the NH2 terminus by site-directed mutagenesis, for its overexpression in rat testes in vivo using the Polyplus in vivo jet-PEI transfection reagent with high transfection efficiency. Overexpression of this p-rpS6-MT was capable of inducing BTB remodeling, making the barrier "leaky." This thus promoted the entry of the nonhormonal male contraceptive adjudin into the adluminal compartment in the seminiferous epithelium to induce germ cell exfoliation. Combined overexpression of p-rpS6-MT with a male contraceptive (e.g., adjudin) potentiated the drug bioavailability by modifying the BTB. This approach thus lowers intrinsic drug toxicity due to a reduced drug dose, further characterizing the biology of BTB transport function.

F5-peptide enhances the efficacy of the non-hormonal male contraceptive adjudin

ObjectiveThe bioavailability of the non-hormonal male contraceptive adjudin is low in rats due to the blood-testis barrier (BTB). This study was designed to examine if F5-peptide, an endogenously produced reversible BTB modifier, could enhance the bioavailability of adjudin to affect spermatogenesis and provide a contraceptive effect in rats while reducing systemic toxicity. Study DesignWe overexpressed F5-peptide in adult male rats (n=10 rats; with 3 or 4 rats for each of the three different experiments noted in the three regimens) by intratesticular injection of a mammalian expression vector pCI-neo (pCI-neo/F5-peptide) vs. empty vector alone (pCI-neo/Ctrl) to be followed by treatment with adjudin by oral gavage at a dose of 10 or 20 mg/kg. The status of spermatogenesis was assessed by histological analysis and dual-labeled immunofluorescence analysis on Day 16. To assess fertility, we allowed treated males (n=3–4 rats) to mate with mature female rats (n=3–4) individually, and assessed the number of pups on Days 23, 36 and 82 to assess fertility and reversibility. ResultsAll 4 treated rats overexpressed with F5-peptide and low-dose adjudin were infertile by Day 36, and half of these rats were fertile by Day 82, illustrating reversibility. However, overexpression of F5-peptide alone (or low-dose adjudin alone) had no effects on fertility in n=3 rats. These findings were consistent with the histology data that illustrated the BTB modifier F5-peptide promoted the action of adjudin to induce germ cell exfoliation, mediated by changes in cytoskeletal organization of F-actin and microtubules across the epithelium, thereby reducing the systemic toxicity of adjudin. ConclusionIn this proof-of-concept study, it was shown that overexpression of the F5-peptide prior to administration of adjudin to rats at a low (and ineffective dose by itself) was found to induce reversible male infertility. ImplicationsOverexpression of F5-peptide, an endogenously produced biomolecule in the testis known to induce BTB remodeling, enhanced the contraceptive effect of adjudin in rats, supporting proof of concept studies of BTB disrupters in men.

Regulation of blood-testis barrier dynamics by the mTORC1/rpS6 signaling complex: An in vitro study.

During spermatogenesis, developing germ cells that lack the cellular ultrastructures of filopodia and lamellipodia generally found in migrating cells, such as macrophages and fibroblasts, rely on Sertoli cells to support their transport across the seminiferous epithelium. These include the transport of preleptotene spermatocytes across the blood-testis barrier (BTB), but also the transport of germ cells, in particular developing haploid spermatids, across the seminiferous epithelium, that is to and away from the tubule lumen, depending on the stages of the epithelial cycle. On the other hand, cell junctions at the Sertoli cell–cell and Sertoli–germ cell interface also undergo rapid remodeling, involving disassembly and reassembly of cell junctions, which, in turn, are supported by actin- and microtubule-based cytoskeletal remodeling. Interestingly, the underlying mechanism(s) and the involving biomolecule(s) that regulate or support cytoskeletal remodeling remain largely unknown. Herein, we used an in vitro model of primary Sertoli cell cultures that mimicked the Sertoli BTB in vivo overexpressed with the ribosomal protein S6 (rpS6, the downstream signaling protein of mammalian target of rapamycin complex 1 [mTORC1]) cloned into the mammalian expression vector pCI-neo, namely, quadruple phosphomimetic and constitutively active mutant of rpS6 (pCI-neo/p-rpS6-MT) versus pCI-neo/rpS6-WT (wild-type) and empty vector (pCI-neo/Ctrl) for studies. These findings provide compelling evidence that the mTORC1/rpS6 signal pathway exerted its effects to promote Sertoli cell BTB remodeling. This was mediated through changes in the organization of actin- and microtubule-based cytoskeletons, involving changes in the distribution and/or spatial expression of actin- and microtubule-regulatory proteins.

Regulation of spermatogenesis by a local functional axis in the testis: role of the basement membrane–derived noncollagenous 1 domain peptide

Spermatogenesis takes place in the epithelium of the seminiferous tubules of the testes, producing millions of spermatozoa per day in an adult male in rodents and humans. Thus, multiple cellular events that are regulated by an array of signaling molecules and pathways are tightly coordinated to support spermatogenesis. Here, we report findings of a local regulatory axis between the basement membrane (BM), the blood-testis barrier (BTB), and the apical ectoplasmic specialization (apical ES; a testis-specific, actin-rich adherens junction at the Sertoli cell-spermatid interface) to coordinate cellular events across the seminiferous epithelium during the epithelial cycle. In short, a biologically active fragment, noncollagenous 1 (NC1) domain that is derived from collagen chains in the BM, was found to modulate cell junction dynamics at the BTB and apical ES. NC1 domain from the collagen α3(IV) chain was cloned into a mammalian expression vector, pCI-neo, with and without a collagen signal peptide. We also prepared a specific Ab against the purified recombinant NC1 domain peptide. These reagents were used to examine whether overexpression of NC1 domain with high transfection efficacy would perturb spermatogenesis, in particular, spermatid adhesion (i.e., inducing apical ES degeneration) and BTB function (i.e., basal ES and tight junction disruption, making the barrier leaky), in the testis in vivo We report our findings that NC1 domain derived from collagen α3(IV) chain-a major structural component of the BM-was capable of inducing BTB remodeling, making the BTB leaky in studies in vivo Furthermore, NC1 domain peptide was transported across the epithelium via a microtubule-dependent mechanism and is capable of inducing apical ES degeneration, which leads to germ cell exfoliation from the seminiferous epithelium. Of more importance, we show that NC1 domain peptide exerted its regulatory effect by disorganizing actin microfilaments and microtubules in Sertoli cells so that they failed to support cell adhesion and transport of germ cells and organelles (e.g., residual bodies, phagosomes) across the seminiferous epithelium. This local regulatory axis between the BM, BTB, and the apical ES thus coordinates cellular events that take place across the seminiferous epithelium during the epithelial cycle of spermatogenesis.-Chen, H., Mruk, D. D., Lee, W. M., Cheng, C. Y. Regulation of spermatogenesis by a local functional axis in the testis: role of the basement membrane-derived noncollagenous 1 domain peptide.

Basement Membrane Laminin α2 Regulation of BTB Dynamics via Its Effects on F-Actin and Microtubule Cytoskeletons Is Mediated Through mTORC1 Signaling.

A local axis connects the apical ectoplasmic specialization (ES) at the Sertoli-spermatid interface, the basal ES at the blood-testis barrier (BTB), and the basement membrane across the seminiferous epithelium functionally in rat testes. As such, cellular events that take place simultaneously across the epithelium such as spermiation and BTB remodeling that occur at the apical ES and the basal ES, respectively, at stage VIII of the cycle are coordinated. Herein, laminin α2, a structural component of the basement membrane, was found to regulate BTB dynamics. Sertoli cells were cultured in vitro to allow the establishment of a tight junction (TJ) barrier that mimicked the BTB in vivo. Knockdown of laminin α2 by transfecting Sertoli cells with laminin α2-specific short hairpin RNA vs the nontargeting negative control was shown to perturb the Sertoli cell TJ barrier, illustrating laminin α2 was involved in regulating BTB dynamics. This regulatory effect was mediated through mammalian target of rapamycin complex 1 (mTORC1) signaling because the two mTORC1 downstream signaling molecules ribosomal protein S6 and Akt1/2 were activated and inactivated, respectively, consistent with earlier findings that mTORC1 is involved in promoting BTB remodeling. Also, laminin α2 knockdown induced F-actin and microtubule (MT) disorganization through changes in the spatial expression of F-actin regulators actin-related protein 3 and epidermal growth factor receptor pathway substrate 8 vs end-binding protein 1 (a MT plus-end tracking protein, +TIP). These laminin α2 knockdown-mediated effects on F-actin and MT organization was blocked by exposing Sertoli cells to rapamycin, an inhibitor of mTORC1 signaling, and also SC79, an activator of Akt. In summary, laminin α2-mediated regulation on Sertoli cell BTB dynamics is through mTORC1 signaling.

Actin binding proteins in blood-testis barrier function.

The present review examines the role of actin binding proteins (ABPs) on blood-testis barrier (BTB), an androgen-dependent ultrastructure in the testis, in particular their involvement on BTB remodeling during spermatogenesis. The BTB divides the seminiferous epithelium into the basal and the adluminal compartments. The BTB is constituted by coexisting actin-based tight junction, basal ectoplasmic specialization, and gap junction, and also intermediate filament-based desmosome between Sertoli cells near the basement membrane. Junctions at the BTB undergo continuous remodeling to facilitate the transport of preleptotene spermatocytes residing in the basal compartment across the immunological barrier during spermatogenesis. Thus, meiosis I/II and postmeiotic spermatid development take place in the adluminal compartment behind the BTB. BTB remodeling also regulates exchanges of biomolecules between the two compartments. As tight junction, basal ectoplasmic specialization, and gap junction use F-actin for attachment, actin microfilaments rapidly convert between their bundled and unbundled/branched configuration to confer BTB plasticity. The events of actin reorganization are regulated by two major classes of ABPs that convert actin microfilaments between their bundled and branched/unbundled configuration. We provide a model on how ABPs regulate BTB remodeling, shedding new light on unexplained male infertility, such as environmental toxicant-induced reproductive dysfunction since the testis, in particular the BTB, is sensitive to environmental toxicants, such as cadmium, bisphenol A, phthalates, and PFOS (perfluorooctanesulfonic acid or perfluorooctane sulfonate).

AKAP9, a Regulator of Microtubule Dynamics, is Essential for Remodeling of the Blood‐Testis Barrier

The blood‐testis barrier (BTB) is formed by tight, adherens and gap junctions between adjacent Sertoli cells of the seminiferous tubules. The BTB undergoes extensive remodeling during the seminiferous epithelial cycle of spermatogenesis to allow the transport of preleptotene spermatocytes from the basal compartment to the adluminal compartment for further development. The actin cytoskeleton serves unique structural and supporting roles in this process, but little is known about the role of microtubules and their regulators in BTB remodeling. The cAMP responsive scaffold protein AKAP9 has been shown to regulate microtubule dynamics. We demonstrate that conditional and inducible deletion of AKAP9 in mice after the initial formation of the BTB leads to infertility despite evidence of germ cell meiosis. AKAP9 deletion results in marked alterations in the integrity of microtubules in Sertoli cells, mislocalization of gap and tight junctional molecules at the BTB and a progressive loss of barrier integrity despite normal localization of F‐actin at the BTB. These changes were accompanied by an arrest of meiosis and spermiogenesis. Unexpectedly, barrier integrity and meiosis were restored in older AKAP9 deficient animals. Yet, significant defects in spermiogenesis persisted suggesting a role for AKAP9 in the transport of developing spermatids across the adluminal compartment during spermiogenesis. These results suggest that AKAP9 is critical for both dynamic restructuring of the BTB during the epithelial cycle and subsequent spermiogenesis.

N-wasp is required for structural integrity of the blood-testis barrier.

During spermatogenesis, the blood-testis barrier (BTB) segregates the adluminal (apical) and basal compartments in the seminiferous epithelium, thereby creating a privileged adluminal environment that allows post-meiotic spermatid development to proceed without interference of the host immune system. A key feature of the BTB is its continuous remodeling within the Sertoli cells, the major somatic component of the seminiferous epithelium. This remodeling is necessary to allow the transport of germ cells towards the seminiferous tubule interior, while maintaining intact barrier properties. Here we demonstrate that the actin nucleation promoting factor Neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) provides an essential function necessary for BTB restructuring, and for maintaining spermatogenesis. Our data suggests that the N-WASP-Arp2/3 actin polymerization machinery generates branched-actin arrays at an advanced stage of BTB remodeling. These arrays are proposed to mediate the restructuring process through endocytic recycling of BTB components. Disruption of N-WASP in Sertoli cells results in major structural abnormalities to the BTB, including mis-localization of critical junctional and cytoskeletal elements, and leads to disruption of barrier function. These impairments result in a complete arrest of spermatogenesis, underscoring the critical involvement of the somatic compartment of the seminiferous tubules in germ cell maturation.