Map-1a regulates Sertoli cell BTB dynamics through the cytoskeletal organization of microtubule and F-actin.

Microtubule-associated protein 1a (Map1a) is a microtubule (MT) regulatory protein that binds to the MT protofilaments in mammalian cells to promote MT stabilization. Maps work with MT cleavage proteins and other MT catastrophe-inducing proteins to confer MT dynamics to support changes in the Sertoli cell shape to sustain spermatogenesis. However, no functional studies are found in the literature to probe its role in spermatogenesis. Using an RNAi approach, coupled with the use of toxicant-induced testis (in vivo)- and Sertoli cell (in vitro)-injury models, RNA-Seq analysis, transcriptome profiling, and relevant bioinformatics analysis, immunofluorescence analysis, and pertinent biochemical assays for cytoskeletal organization, we have delineated the functional role of Map1a in Sertoli cells and testes. Map1a was shown to support MT structural organization, and its knockdown (KD) also perturbed the structural organization of actin, vimentin, and septin cytoskeletons as these cytoskeletons are intimately related, working in concert to support spermatogenesis. More importantly, cadmium-induced Sertoli cell injury that perturbed the MT structural organization across the cell cytoplasm was associated with disruptive changes in the distribution of Map1a and a surge in p-p38-MAPK (phosphorylated p38-mitogen-activated protein kinase) expression but not total p38-MAPK. These findings thus support the notion that p-p38-MAPK activation is involved in cadmium-induced Sertoli cell injury. This conclusion was supported by studies using doramapimod, a specific p38-MAPK phosphorylation (activation) inhibitor, which was capable of restoring the cadmium-induced disruptive structural organization of MTs across the Sertoli cell cytoplasm. In summary: this study provides mechanistic insights regarding restoration of toxicant-induced Sertoli cell and testis injury and male infertility.

The planar cell polarity (PCP) protein Fat 1 in Sertoli cell function.

Fat (FAT atypical cadherin) and Dchs (Dachsous cadherin-related protein) in adjacent Sertoli:Sertoli, Sertoli:spermatid, and spermatid:spermatid create an important intercellular bridge, whose adhesive function is in turn supported by Fjx1, a non-receptor Ser/Thr protein kinase. This concept is derived from earlier studies of Drosophila, which has been confirmed in this and earlier reports as well. Herein, we use the approach of knockdown of Fat1 by RNAi using primary cultures of Sertoli cells that mimicked the blood-testis barrier (BTB) in vivo, and a series of coherent experiments including functional assays to monitor Sertoli cell tight junction (TJ)-permeability barrier and a functional in vitro TJ integrity assay to assess the role of Fat1 in the testis. It was shown that planar cell polarity (PCP) protein Fat1 affected Sertoli cell function through its modulation of actin and microtubule cytoskeletal function, altering their polymerization activity through the Fat1/Fjx1 complex. Furthermore, Fat1 is intimately associated with ß-catenin and α-N-catenin, as well as with Prickle 1 of the Vangl1/Prickle 1 complex, another PCP Core Protein to support intercellular interactions to confer PCP. In summary, these findings support the notion that the Fat:Dchs and the Vangl2:Fzd PCP intercellular bridges are tightly associated with basal ES/TJ structural proteins to stabilize PCP function at the Sertoli:Sertoli, Sertoli:spermatid, and spermatid:spermatid interface to sustain spermatogenesis.

Discovering a Reversible Male Contraceptive Agent Derived from Lonidamine.

There is a huge demand for safe and effective non-hormonal male contraceptives to prevent unintended pregnancy, but research on male contraceptive drugs lacks far behind the pills for women. Lonidamine and its analog adjudin are two of the best studied potential male contraceptives. However, the acute toxicity of lonidamine and the subchronic toxicity of adjudin had impeded their development for male contraception. Here, we designed and synthesized a whole new series of molecules derived from lonidamine according to a structure ligand-based design strategy and obtained a new effective and reversible contraceptive agent (BHD), and their efficacy was demonstrated in male mice and rats. Results showed that BHD had a 100% contraceptive effect on male mice after 2 weeks following a single oral dose of BHD at 100 mg/kg body weight (b.w.) or 500 mg/kg b.w. treatments. The fertility of mice was reduced to 90 and 50% after 6 weeks with a single oral dose of BHD-100 and BHD-500 mg/kg b.w. treatments, respectively. We also revealed that BHD induced the apoptosis of spermatogenic cells rapidly and disrupted the blood-testis barrier effectively. It appears to be a new potential male contraceptive candidate for future development.

Regulation of Sertoli cell function by planar cell polarity (PCP) protein Fjx1.

Four-jointed box kinase 1 (Fjx1) is a planar cell polarity (PCP) protein and a member of the Fat (FAT atypical cadherin 1)/Dchs (Dachsous cadherin-related protein)/Fjx1 PCP complex. Fjx1 is also a non-receptor Ser/Thr protein kinase capable of phosphorylating Fat1 at is extracellular cadherin domains when it is being transported across the Golgi system. As such, Fjx1 is a Golgi-based regulator of Fat1 function by determining its extracellular deposition. Herein, Fjx1 was found to localize across the Sertoli cell cytoplasm, partially co-localized with the microtubules (MTs) across the seminiferous epithelium. It was most notable at the apical ES (ectoplasmic specialization) and basal ES, displaying distinctive stage-specific expression. The apical ES and basal ES are the corresponding testis-specific cell adhesion ultrastructures at the Sertoli-elongated spermatid interface and the Sertoli cell-cell interface, respectively, consistent with the role of Fjx1 as a Golgi-associated Ser/Thr kinase that modulates the Fat (and/or Dchs) integral membrane proteins. Its knockdown (KD) by RNAi using specific Fjx1 siRNA duplexes versus non-targeting negative control siRNA duplexes was found to perturb the Sertoli cell tight junction function, as well as perturbing the function and organization of MT and actin. While Fjx1 KD did not affect the steady-state levels of almost two dozens of BTB-associated Sertoli cell proteins, including structural and regulatory proteins, its KD was found to down-regulate Fat1 (but not Fat2, 3, and 4) and to up-regulate Dchs1 (but not Dchs2) expression. Based on results of biochemical analysis, Fjx1 KD was found to be capable of abolishing phosphorylation of its putative substrate Fat1 at its Ser/Thr sites, but not at its Tyr site, illustrating an intimate functional relationship of Fjx1 and Fat1 in Sertoli cells.

Cadmium-induced Sertoli Cell Injury Through p38-MAPK and Related Signaling Proteins-A Study by RNA Sequencing.

Environmental toxicants, such as cadmium, found in foods, water, and consumer products are known to induce male reproductive dysfunction. However, the underlying molecular mechanism(s) by which cadmium-induced Sertoli cell injury as manifested by a disruption of the blood-testis barrier (BTB) remains unknown. Interestingly, one of the primary targets of cadmium toxicity in the testis is the cytoskeletons of the Sertoli cells, which, in turn, impedes cell junctions in the seminiferous epithelium. In order to expand these earlier observations and to provide a roadmap for future studies, we embarked a study using RNA sequencing to identify the pertinent genes involved in cadmium-induced Sertoli cell injury. Using bioinformatics analyses, multiple gene sets that regulated actin and microtubule (MT) cytoskeletons were identified along with components of the mitogen-activated protein kinase (MAPK) signaling protein and several signaling pathways. More important, we have also discovered that while the gene expression of p38-MAPK (also JNK or c-Jun) was considerably up- or downregulated during cadmium-induced Sertoli cell injury, the activated (phosphorylated) form was upregulated. Importantly, doramapimod (also known as BIRB 796), a specific p38-MARK inhibitor, that was shown to selectively block cadmium-induced p-p38 MAPK activation via phosphorylation in Sertoli cells, was indeed capable of blocking cadmium-induced Sertoli cell injury including disruption of the Sertoli cell-permeability barrier function, disruptive distribution of BTB-associated proteins, and disruptive organization of the actin and MT cytoskeletons. These data provide a helpful source of information for investigators to probe the role of signaling proteins and/or their signaling cascades, besides MAPKs, that likely utilized by cadmium to induce reproductive dysfunction.

Exploring the evolving function of soluble intercellular adhesion molecule-1 in junction dynamics during spermatogenesis.

Intercellular adhesion molecule-1 (ICAM-1) is a transmembrane glycoprotein expressed on immune, endothelial, and epithelial cells. Its ectodomain can be proteolytically cleaved to release a circulating soluble form called sICAM-1. Clinical studies demonstrate sICAM-1 is upregulated in various diseases and associated with disease severity. Research has identified sICAM-1 as a regulator of the blood-testis barrier (BTB) and spermatogenesis. Overexpression of sICAM-1 weakened the BTB in vitro and in vivo, downregulated junction proteins including N-cadherin, γ-catenin, and connexin 43, and caused germ cell loss. This contrasts with barrier-strengthening effects of membrane-bound ICAM-1. sICAM-1 may act as a molecular switch enabling germ cells to open BTB and Sertoli-germ cell adhesion for transport across the seminiferous epithelium. While the mechanism remains unclear, reduced SRC family kinase (SFK) signaling was observed following sICAM-1 overexpression. SRC promotes BTB protein endocytosis and degradation, influences cytoskeletal dynamics, and affects cell polarity. As sICAM-1 overexpression phenocopies SRC inhibition, SRC may operate downstream of sICAM-1 in regulating BTB dynamics and spermatogenesis. Investigating sICAM-1's structure-function regions and downstream targets will elucidate the molecular mechanisms of junction disruption. This knowledge could enable strategies targeting sICAM-1/SRC to modulate BTB permeability and treat male infertility or diseases involving endothelial/epithelial barrier dysfunction.

Defects of microtubule cytoskeletal organization in NOA human testes.

The importance of actin and microtubule (MT) cytoskeletons in testis function in rodents is known to some extent, but its role in the etiology of azoospermia in humans remains unexplored. Here, we examined if MT cytoskeleton was defective in NOA (non-obstructive azoospermia) testes versus normal human testes based on histopathological, immunofluorescence (IF), and scRNA-Seq transcriptome profiling. Testis biopsy samples from n = 6 normal men versus n = 3 Sertoli cell only (SCO) and n = 3 MA (meiotic arrest) of NOA patients were used for histopathological analysis. IF analysis was also used to examine MT organization across the seminiferous epithelium, investigating the likely involvement of microtubule-associated proteins (MAPs). scRNA-Seq transcriptome profiling datasets from testes of 3 SCO patients versus 3 normal men in public domain in Gene Expression Omnibus (GEO) Sample (GSM) with identifiers were analyzed to examine relevant genes that regulate MT dynamics. NOA testes of MA and SCO patients displayed notable defects in MT organization across the epithelium with extensive truncation, mis-alignments and appeared as collapsed structures near the base of the tubules. These changes are in contrast to MTs in testes of normal men. scRNA-Seq analyses revealed considerable loss of spermatogenesis capacity in SCO testes of NOA patients versus normal men. An array of genes that support MT dynamics displayed considerable changes in expression and in spatial distribution. In summary, defects in MT cytoskeleton were noted in testes of NOA (SCO) patients, possibly mediated by defective spatial expression and/or distribution of MAPs. These changes, in turn, may impede spermatogenesis in SCO testes of NOA patients.

MARK2 and MARK4 Regulate Sertoli Cell BTB Dynamics Through Microtubule and Actin Cytoskeletons.

Microtubule affinity-regulating kinases (MARKs) are nonreceptor Ser/Thr protein kinases known to regulate cell polarity and microtubule dynamics in Caenorhabditis elegans, Drosophila, invertebrates, vertebrates, and mammals. An earlier study has shown that MARK4 is present at the ectoplasmic specialization and blood-testis barrier (BTB) in the seminiferous epithelium of adult rat testes. Here, we report the function of MARK4 and another isoform MARK2 in Sertoli cells at the BTB. Knockdown of MARK2, MARK4, or MARK2 and MARK4 by RNAi using the corresponding siRNA duplexes without apparent off-target effects was shown to impair tight junction (TJ)-permeability barrier at the Sertoli cell BTB. It also disrupted microtubule (MT)- and actin-based cytoskeletal organization within Sertoli cells. Although MARK2 and MARK4 were shown to share sequence homology, they likely regulated the Sertoli cell BTB and MT cytoskeleton differently. Disruption of the TJ-permeability barrier following knockdown of MARK4 was considerably more severe than loss of MARK2, though both perturbed the barrier. Similarly, loss of MARK2 affected MT organization in a different manner than the loss of MARK4. Knockdown of MARK2 caused MT bundles to be arranged around the cell periphery, whereas knockdown of MARK4 caused MTs to retract from the cell edge. These differences in effects on the TJ-permeability barrier are likely from the unique roles of MARK2 and MARK4 in regulating the MT cytoskeleton of the Sertoli cell.

Human obstructive (postvasectomy) and nonobstructive azoospermia - Insights from scRNA-Seq and transcriptome analysis.

A substantial number of male infertility is caused by azoospermia. However, the underlying etiology and the molecular basis remain largely unknown. Through single-cell (sc)RNA sequencing, we had analyzed testis biopsy samples from two patients with obstructive azoospermia (OA) and nonobstructive azoospermia (NOA). We found only somatic cells in the NOA samples and explored the transcriptional changes in Sertoli cells in response to a loss of interactions with germ cells. Moreover, we observed a germ cell population discrepancy between an OA (postvasectomy) patient and a healthy individual. We confirmed this observation in a secondary study with two datasets at GSM3526588 and GSE124263 for detailed analysis wherein the regulatory mechanisms at the transcriptional level were identified. These findings thus provide valuable information on human spermatogenesis, and we also identified insightful information for further research on reproduction-related diseases.

Exposed and Sequestered Antigens in Testes and Their Protection by Regulatory T Cell-Dependent Systemic Tolerance.

Continuous exposure of tissue antigen (Ag) to the autoantigen-specific regulatory T cells (Treg) is required to maintain Treg-dependent systemic tolerance. Thus, testis autoantigens, previously considered as sequestered, may not be protected by systemic tolerance. We now document that the complete testis antigen sequestration is not valid. The haploid sperm Ag lactate dehydrogenase 3 (LDH3) is continuously exposed and not sequestered. It enters the residual body (RB) to egress from the seminiferous tubules and interact with circulating antibody (Ab). Some LDH3 also remains inside the sperm cytoplasmic droplets (CD). Treg-depletion in the DEREG mice that express diphtheria toxin receptor on the Foxp3 promoter results in spontaneous experimental autoimmune orchitis (EAO) and Ab to LDH3. Unlike the wild-type male mice, mice deficient in LDH3 (wild-type female or LDH3 NULL males) respond vigorously to LDH3 immunization. However, partial Treg depletion elevated the wild-type male LDH3 responses to the level of normal females. In contrast to LDH3, zonadhesin (ZAN) in the sperm acrosome displays properties of a sequestered Ag. However, when ZAN and other sperm Ag are exposed by vasectomy, they rapidly induce testis Ag-specific tolerance, which is terminated by partial Treg-depletion, leading to bilateral EAO and ZAN Ab response. We conclude that some testis/sperm Ag are normally exposed because of the unique testicular anatomy and physiology. The exposed Ag: 1) maintain normal Treg-dependent systemic tolerance, and 2) are pathogenic and serve as target Ag to initiate EAO. Unexpectedly, the sequestered Ags, normally non-tolerogenic, can orchestrate de novo Treg-dependent, systemic tolerance when exposed in vasectomy.

Kinesins in Mammalian Spermatogenesis and Germ Cell Transport.

In mammalian testes, the apical cytoplasm of each Sertoli cell holds up to several dozens of germ cells, especially spermatids that are transported up and down the seminiferous epithelium. The blood-testis barrier (BTB) established by neighboring Sertoli cells in the basal compartment restructures on a regular basis to allow preleptotene/leptotene spermatocytes to pass through. The timely transfer of germ cells and other cellular organelles such as residual bodies, phagosomes, and lysosomes across the epithelium to facilitate spermatogenesis is important and requires the microtubule-based cytoskeleton in Sertoli cells. Kinesins, a superfamily of the microtubule-dependent motor proteins, are abundantly and preferentially expressed in the testis, but their functions are poorly understood. This review summarizes recent findings on kinesins in mammalian spermatogenesis, highlighting their potential role in germ cell traversing through the BTB and the remodeling of Sertoli cell-spermatid junctions to advance spermatid transport. The possibility of kinesins acting as a mediator and/or synchronizer for cell cycle progression, germ cell transit, and junctional rearrangement and turnover is also discussed. We mostly cover findings in rodents, but we also make special remarks regarding humans. We anticipate that this information will provide a framework for future research in the field.

Multiomics analysis of male infertility†.

Infertility affects 8-12% of couples globally, and the male factor is a primary cause in ~50% of couples. Male infertility is a multifactorial reproductive disorder, which can be caused by paracrine and autocrine factors, hormones, genes, and epigenetic changes. Recent studies in rodents and most notably in humans using multiomics approach have yielded important insights into understanding the biology of spermatogenesis. Nonetheless, the etiology and pathogenesis of male infertility are still largely unknown. In this review, we summarized and critically evaluated findings based on the use of advanced technologies to compare normal and obstructive azoospermic versus nonobstructive azoospermic men, including whole-genome bisulfite sequencing, single-cell RNA-seq, whole-exome sequencing, and transposase-accessible chromatin using sequencing. It is obvious that the multiomics approach is the method of choice for basic research and clinical studies including clinical diagnosis of male infertility.

PCP Protein Inversin Regulates Testis Function Through Changes in Cytoskeletal Organization of Actin and Microtubules.

Inversin is an integrated component of the Frizzled (Fzd)/Dishevelled (Dvl)/Diversin planar cell polarity (PCP) complex that is known to work in concert with the Van Gogh-like protein (eg, Vangl2)/Prickle PCP complex to support tissue and organ development including the brain, kidney, pancreas, and others. These PCP protein complexes are also recently shown to confer developing haploid spermatid PCP to support spermatogenesis in adult rat testes. However, with the exception of Dvl3 and Vangl2, other PCP proteins have not been investigated in the testis. Herein, we used the technique of RNA interference (RNAi) to examine the role of inversin (Invs) in Sertoli cell (SC) and testis function by corresponding studies in vitro and in vivo. When inversin was silenced by RNAi using specific small interfering RNA duplexes by transfecting primary cultures of SCs in vitro or testes in vivo, it was shown that inversin knockdown (KD) perturbed the SC tight junction-barrier function in vitro and in vivo using corresponding physiological and integrity assays. More important, inversin exerted its regulatory effects through changes in the organization of the actin and microtubule cytoskeletons, including reducing the ability of their polymerization. These changes, in turn, induced defects in spermatogenesis by loss of spermatid polarity, disruptive distribution of blood-testis barrier-associated proteins at the SC-cell interface, appearance of multinucleated round spermatids, and defects in the release of sperm at spermiation.

Cell-Cell Interaction-Mediated Signaling in the Testis Induces Reproductive Dysfunction-Lesson from the Toxicant/Pharmaceutical Models.

Emerging evidence has shown that cell-cell interactions between testicular cells, in particular at the Sertoli cell-cell and Sertoli-germ cell interface, are crucial to support spermatogenesis. The unique ultrastructures that support cell-cell interactions in the testis are the basal ES (ectoplasmic specialization) and the apical ES. The basal ES is found between adjacent Sertoli cells near the basement membrane that also constitute the blood-testis barrier (BTB). The apical ES is restrictively expressed at the Sertoli-spermatid contact site in the apical (adluminal) compartment of the seminiferous epithelium. These ultrastructures are present in both rodent and human testes, but the majority of studies found in the literature were done in rodent testes. As such, our discussion herein, unless otherwise specified, is focused on studies in testes of adult rats. Studies have shown that the testicular cell-cell interactions crucial to support spermatogenesis are mediated through distinctive signaling proteins and pathways, most notably involving FAK, Akt1/2 and Cdc42 GTPase. Thus, manipulation of some of these signaling proteins, such as FAK, through the use of phosphomimetic mutants for overexpression in Sertoli cell epithelium in vitro or in the testis in vivo, making FAK either constitutively active or inactive, we can modify the outcome of spermatogenesis. For instance, using the toxicant-induced Sertoli cell or testis injury in rats as study models, we can either block or rescue toxicant-induced infertility through overexpression of p-FAK-Y397 or p-FAK-Y407 (and their mutants), including the use of specific activator(s) of the involved signaling proteins against pAkt1/2. These findings thus illustrate that a potential therapeutic approach can be developed to manage toxicant-induced male reproductive dysfunction. In this review, we critically evaluate these recent findings, highlighting the direction for future investigations by bringing the laboratory-based research through a translation path to clinical investigations.

Planar cell polarity (PCP) proteins support spermatogenesis through cytoskeletal organization in the testis.

Few reports are found in the literature regarding the role of planar cell polarity (PCP) in supporting spermatogenesis in the testis. Yet morphological studies reported decades earlier have illustrated the directional alignment of polarized developing spermatids, most notably step 17-19 spermatids in stage V-early VIII tubules in the testis, across the plane of the epithelium in seminiferous tubules of adult rats. Such morphological features have unequivocally demonstrated the presence of PCP in developing spermatids, analogous to the PCP noted in hair cells of the cochlea in mammals. Emerging evidence in recent years has shown that Sertoli and germ cells express numerous PCP proteins, mostly notably, the core PCP proteins, PCP effectors and PCP signaling proteins. In this review, we discuss recent findings in the field regarding the two core PCP protein complexes, namely the Van Gogh-like 2 (Vangl2)/Prickle (Pk) complex and the Frizzled (Fzd)/Dishevelled (Dvl) complex. These findings have illustrated that these PCP proteins exert their regulatory role to support spermatogenesis through changes in the organization of actin and microtubule (MT) cytoskeletons in Sertoli cells. For instance, these PCP proteins confer PCP to developing spermatids. As such, developing haploid spermatids can be aligned and orderly packed within the limited space of the seminiferous tubules in the testes for the production of sperm via spermatogenesis. Thus, each adult male in the mouse, rat or human can produce an upward of 30, 50 or 300 million spermatozoa on a daily basis, respectively, throughout the adulthood. We also highlight critical areas of research that deserve attention in future studies. We also provide a hypothetical model by which PCP proteins support spermatogenesis based on recent studies in the testis. It is conceivable that the hypothetical model shown here will be updated as more data become available in future years, but this information can serve as the framework by investigators to unravel the role of PCP in spermatogenesis.

Single-cell ATAC-Seq reveals cell type-specific transcriptional regulation and unique chromatin accessibility in human spermatogenesis.

During human spermatogenesis, germ cells undergo dynamic changes in chromatin organization/re-packaging and in transcriptomes. In order to better understand the underlying mechanism(s), scATAC-Seq of 5376 testicular cells from 3 normal men were performed. Data were analyzed in parallel with the scRNA-Seq data of human testicular cells. In all, 10 germ cell types associated with spermatogenesis and 6 testicular somatic cell types were identified, along with 142 024 peaks located in promoter, genebody and CpG Island. We had examined chromatin accessibility of all chromosomes, with chromosomes 19 and 17 emerged as the leading chromosomes that displayed high chromatin accessibility. In accessible chromatin regions, transcription factor-binding sites were identified and specific motifs with high frequencies at different spermatogenesis stages were detected, including CTCF, BORIS, NFY, DMRT6, EN1, ISL1 and GLI3. Two most remarkable observations were noted. First, TLE3 was specifically expressed in differentiating spermatogonia. Second, PFN4 was found to be involved in actin cytoskeletal organization during meiosis. More important, unique regions upstream of PFN4 and TLE3 were shown to display high accessibility, illustrating their significance in supporting human spermatogenesis.

mTORC1/rpS6 and p-FAK-Y407 signaling regulate spermatogenesis: Insights from studies of the adjudin pharmaceutical/toxicant model.

In rodents and humans, the major cellular events at spermatogenesis include self-renewal of spermatogonial stem cells and undifferentiated spermatogonia via mitosis, commitment of spermatogonia to differentiation and transformation to spermatocytes, meiosis, spermiogenesis, and the release of spermatozoa at spermiation. While details of the morphological changes during these cellular events have been delineated, knowledge gap exists between the morphological changes in the seminiferous epithelium and the underlying molecular mechanism(s) that regulate these cellular events. Even though many of the regulatory proteins and biomolecules that modulate spermatogenesis are known based on studies using genetic models, the underlying regulatory mechanism(s), in particular signaling pathways/proteins, remain unexplored since much of the information regarding the signaling regulation is unknown. Studies in the past decade, however, have unequivocally demonstrated that the testis is using several signaling proteins and/or pathways to regulate multiple cellular events to modulate spermatogenesis. These include mTORC1/rpS6/Akt1/2 and p-FAK-Y407. While selective inhibitors and/or agonists and antagonists are available to examine some of these signaling proteins, their use have limitations due to their specificities and also potential systemic cytotoxicity. On the other hand, the use of genetic models has had profound implications for our understanding of the molecular regulation of spermatogenesis, and these knockout (null) models have also revealed the factors that are critical for spermatogenesis. Nonetheless, additional studies using in vitro and in vivo models are necessary to unravel the signaling pathways involved in regulating seminiferous epithelial cycle. Emerging data from studies, such as the use of the adjudin pharmaceutical/toxicant model, have illustrated that this non-hormonal male contraceptive drug is utilizing specific signaling pathways/proteins to induce specific defects in spermatogenesis, yielding mechanistic insights on the regulation of spermatogenesis. We sought to review these recent data in this article, highlighting an interesting approach that can be considered for future studies.

A laminin-based local regulatory network in the testis that supports spermatogenesis.

In adult rat testes, the basement membrane is structurally constituted by laminin and collagen chains that lay adjacent to the blood-testis barrier (BTB). It plays a crucial scaffolding role to support spermatogenesis. On the other hand, laminin-333 comprised of laminin-α3/ß3/γ3 at the apical ES (ectoplasmic specialization, a testis-specific cell-cell adherens junction at the Sertoli cell-step 8-19 spermatid interface) expressed by spermatids serves as a unique cell adhesion protein that forms an adhesion complex with α6ß1-integrin expressed by Sertoli cells to support spermiogenesis. Emerging evidence has shown that biologically active fragments are derived from basement membrane and apical ES laminin chains through proteolytic cleavage mediated by matrix metalloproteinase 9 (MMP9) and MMP2, respectively. Two of these laminin bioactive fragments: one from the basement membrane laminin-α2 chain called LG3/4/5-peptide, and one from the apical ES laminin-γ3 chain known as F5-peptide, are potent regulators that modify cell adhesion function at the Sertoli-spermatid interface (i.e., apical ES) but also at the Sertoli cell-cell interface designated basal ES at the blood-testis barrier (BTB) with contrasting effects. These findings not only highlight the physiological significance of these bioactive peptides that create a local regulatory network to support spermatogenesis, they also open a unique area of research. For instance, it is likely that several other bioactive peptides remain to be identified. These bioactive peptides including their downstream signaling proteins and cascades should be studied collectively in future investigations to elucidate the underlying mechanism(s) by which they coordinate with each other to maintain spermatogenesis. This is the goal of this review.

Role of laminin and collagen chains in human spermatogenesis - Insights from studies in rodents and scRNA-Seq transcriptome profiling.

Studies have demonstrated that biologically active fragments are generated from the basement membrane and the Sertoli cell-spermatid adhesion site known as apical ectoplasmic specialization (apical ES, a testis-specific actin-based anchoring junction) in the rat testis. These bioactive fragments or peptides are produced locally across the seminiferous epithelium through proteolytic cleavage of constituent proteins at the basement membrane and the apical ES. Studies have shown that they are being used to modulate and coordinate cellular functions across the seminiferous epithelium during different stages of the epithelial cycle of spermatogenesis. In this review, we briefly summarize recent findings based on studies using rat testes as a study model regarding the role of these bioactive peptides that serve as a local regulatory network to support spermatogenesis. We also used scRNA-Seq transcriptome datasets in the public domain for OA (obstructive azoospermia) and NAO (non-obstructive azoospermia) human testes versus testes from normal men for analysis in this review. It was shown that there are differential expression of different collagen chains and laminin chains in these testes, suggesting the possibility of a similar local regulatory network in the human testis to support spermatogenesis, and the possible disruption of such network in men is associated with OA and/or NOA.