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Objective The 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 Design We 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. Results All 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. Conclusion In 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. Implications Overexpression 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.

Spermatogenesis is comprised of a series of cellular events that lead to the generation of haploid sperm. These events include self-renewal of spermatogonial stem cells (SSC), proliferation of spermatogonia by mitosis, differentiation of spermatogonia and spermatocytes, generation of haploid spermatids via meiosis I/II, and spermiogenesis. Spermiogenesis consists of a series of morphological events in which spermatids are being transported across the apical compartment of the seminiferous epithelium while maturing into spermatozoa, which include condensation of the genetic materials, biogenesis of acrosome, packaging of the mitocondria into the mid-piece, and elongation of the sperm tail. However, the biology of spermiation remains poorly understood. In this review, we provide in-depth analysis based on the use of bioinformatics tools and an animal model that mimics spermiation through treatment of adult rats with adjudin, a non-hormonal male contraceptive known to induce extensive germ cell exfoliation across the seminiferous epithelium, but nost notably elongating/elongated spermatids. These analyses have shed insightful information regaridng the biology of spermiation.

Spermatogenesis is comprised of a series of cellular events that lead to the generation of haploid sperm. These events include self-renewal of spermatogonial stem cells (SSC), proliferation of spermatogonia by mitosis, differentiation of spermatogonia and spermatocytes, generation of haploid spermatids via meiosis I/II, and spermiogenesis. Spermiogenesis consists of a series of morphological events in which spermatids are being transported across the apical compartment of the seminiferous epithelium while maturing into spermatozoa, which include condensation of the genetic materials, biogenesis of acrosome, packaging of the mitocondria into the mid-piece, and elongation of the sperm tail. However, the biology of spermiation remains poorly understood. In this review, we provide in-depth analysis based on the use of bioinformatics tools and an animal model that mimics spermiation through treatment of adult rats with adjudin, a non-hormonal male contraceptive known to induce extensive germ cell exfoliation across the seminiferous epithelium, but nost notably elongating/elongated spermatids. These analyses have shed insightful information regaridng the biology of spermiation.

The blood-testis barrier (BTB), conferred by Sertoli cells in the mammalian testis, is an important ultrastructure that supports spermatogenesis. Studies using animal models have shown that a disruption of the BTB leads to meiotic arrest, causing defects in spermatogenesis and male infertility. To better understand the regulation of BTB dynamics, we report findings herein to understand the role of ribosomal protein S6 (rpS6), a downstream signaling protein of mammalian target of rapamycin complex 1 (mTORC1), in promoting BTB disruption in the testis in vivo, making the barrier “leaky.” Overexpression of wild-type rpS6 (rpS6-WT, the full-length cDNA cloned into the mammalian expression vector pCI-neo) and a constitutively active quadruple phosphomimetic mutant cloned into pCI-neo (p-rpS6-MT) vs. control (empty pCI-neo vector) was achieved by transfecting adult rat testes with the corresponding plasmid DNA using a Polyplus in vivo-jetPEI transfection reagent. On the basis of an in vivo functional BTB integrity assay, p-rpS6-MT was found to induce BTB disruption better than rpS6-WT did (and no effects in empty vector control), leading to defects in spermatogenesis, including loss of spermatid polarity and failure in the transport of cells (e.g., spermatids) and organelles (e.g., phagosomes), to be followed by germ exfoliation. More important, rpS6-WT and p-rpS6-MT exert their disruptive effects through changes in the organization of actin- and microtubule (MT)-based cytoskeletons, which are mediated by changes in the spatiotemporal expression of actin- and MT-based binding and regulatory proteins. In short, mTORC1/rpS6 signaling complex is a regulator of spermatogenesis and BTB by modulating the organization of the actin- and MT-based cytoskeletons.

The mechanism that regulates sperm release at spermiation is unknown. Herein, we used an animal model wherein rats were treated with adjudin, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide, via oral gavage to induce premature release of elongating/elongated spermatids, followed by round spermatids and spermatocytes. Spermatid release mimicking spermiation occurred within 6 to 12 hours following adjudin treatment and, by 96 hours, virtually all tubules were devoid of elongating/elongated spermatids. Using this model, we tracked the organization of F-actin and microtubules (MTs) by immunofluorescence microscopy, and the association of actin or MT regulatory proteins that either promote or demolish cytoskeletal integrity through changes in the organization of actin microfilaments or MTs by coimmunoprecipitation. Adjudin treatment induced an increase in the association of (1) epidermal growth factor receptor pathway substrate 8 (an actin barbed-end capping and bundling protein) or formin 1 (an actin nucleator) with actin and (2) end-binding protein 1 (an MT stabilizing protein) with MT shortly after adjudin exposure (at 6 hours), in an attempt to maintain spermatid adhesion to the Sertoli cell at the apical ectoplasmic specialization (ES). However, this was followed by a considerable decline of their steady-state protein levels, replacing with an increase in association of (1) actin-related protein 3 (a branched actin nucleator that converts actin filaments into a branched/unbundled network) with actin and (2) MT affinity-regulating kinase 4 (an MT destabilizing protein kinase) with MTs by 12 hours after adjudin treatment. These latter changes thus promoted actin and MT disorganization, leading to apical ES disruption and the release of elongating/elongated spermatids, mimicking spermiation. In summary, spermiation is a cytoskeletal-dependent event, involving regulatory proteins that modify cytoskeletal organization.

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.

Laminin α2 is one of the constituent components of the basement membrane (BM) in adult rat testes. Earlier studies that used a mouse genetic model have shown that a deletion of laminin α2 impedes male fertility by disrupting ectoplasmic specialization (ES; a testis-specific, actin-rich anchoring junction) function along the length of Sertoli cell in the testis. This includes ES at the Sertoli cell–elongating/elongated spermatid interface, which is known as apical ES and possibly the Sertoli–Sertoli cell interface, known as basal ES, at the blood–testis barrier (BTB). Studies have also illustrated that there is a local regulatory axis that functionally links cellular events of spermiation that occur near the luminal edge of tubule lumen at the apical ES and the basal ES/BTB remodeling near the BM at opposite ends of the seminiferous epithelium during the epithelial cycle, known as the apical ES-BTB-BM axis. However, the precise role of BM in this axis remains unknown. Here, we show that laminin α2 in the BM serves as the crucial regulator in this axis as laminin α2, likely its 80-kDa fragment from the C terminus, was found to be transported across the seminiferous epithelium at stages VIII–IX of the epithelial cycle, from the BM to the luminal edge of the tubule, possibly being used to modulate apical ES restructuring at these stages. Of more importance, a knockdown of laminin α2 in Sertoli cells was shown to induce the Sertoli cell tight junction permeability barrier disruption via changes in localization of adhesion proteins at the tight junction and basal ES at the Sertoli cell BTB. These changes were found to be mediated by a disruption of F-actin organization that was induced by changes in the spatiotemporal expression of actin binding/regulatory proteins. Furthermore, laminin α2 knockdown also perturbed microtubule (MT) organization by considerable down-regulation of MT polymerization via changes in the spatiotemporal expression of EB1 (end-binding protein 1), a +TIP (MT plus-end tracking protein). In short, laminin α2 in the BM seems to play a crucial role in the BTB-BM axis by modulating BTB dynamics during spermatogenesis.—Gao, Y., Mruk, D., Chen, H., Lui, W.-Y., Lee, W. M., Cheng, C. Y. Regulation of the blood–testis barrier by a local axis in the testis: role of laminin α2 in the basement membrane.

SUMMARY Single-cell RNA sequencing has revealed extensive molecular diversity in gene programs governing mammalian spermatogenesis but fails to delineate their dynamics in the native context of seminiferous tubules, the spatially confined functional units of spermatogenesis. Here, we use Slide-seq, a spatial transcriptomics technology, to generate an atlas that captures the spatial gene expression patterns at near-single-cell resolution in the mouse and human testis. Using Slide-seq data, we devise a computational framework that accurately localizes testicular cell types in individual seminiferous tubules. Unbiased analysis systematically identifies spatially patterned genes and gene programs. Combining Slide-seq with targeted in situ RNA sequencing, we demonstrate significant differences in the cellular compositions of spermatogonial microenvironment between mouse and human testes. Finally, a comparison of the spatial atlas generated from the wild-type and diabetic mouse testis reveals a disruption in the spatial cellular organization of seminiferous tubules as a potential mechanism of diabetes-induced male infertility., Graphical Abstract, In brief Chen et al. generate a spatial transcriptome atlas of the mammalian testis at near-single-cell resolution that recapitulates spermatogenesis by accurately localizing testicular cell types and reconstructing tissue structures. The atlas is used to reveal the spatial organization of testicular microenvironment and profile its changes under diabetic conditions.

Planar cell polarity (PCP) has been extensively investigated for decades in Drosophila and also hair cells in cochlea. Recent studies have shown that many of the PCP proteins are well conserved, from flies to rodents and humans. In rodents and humans, the Van Gogh (Vang)/Prickle (Pk) complex, the frizzled (Fz)/dishevelled (Dvl)/inversin (Ivs) (or Diego (Dgo) in Drosophila) complex, and the Celsr (or flamingo (Fmi) in Drosophila) protein are crucial to confer PCP, both during embryogenesis such as gastrulation and also in adult cells/tissues. While there are few reports in the literature that examine the role of PCP proteins in spermatogenesis, emerging evidence has illustrated the importance of PCP during the seminiferous epithelial cycle of spermatogenesis. In this chapter, we carefully evaluate recent published data in light of the significance of PCP in Drosophila and highlight some of the areas of research that deserve attention in future research.

The blood-testis barrier is a unique ultrastructure in the mammalian testis, located near the basement membrane of the seminiferous tubule that segregates the seminiferous epithelium into the basal and the adluminal (apical) compartment. Besides restricting paracellular and transcellular passage of biomolecules (e.g., paracrine factors, hormones), water, electrolytes, and other substances including toxicants and/or drugs to enter the adluminal compartment of the epithelium, the BTB is an important ultrastructure that supports spermatogenesis. As such, a sensitive and reliable assay to monitor its integrity in vivo is helpful for studying testis biology. This assay is based on the ability of an intact BTB to exclude the diffusion of a small molecule such as sulfo-NHS-LC-biotin (C20H29N4NaO9S2, Mr. 556.59, a water-soluble and membrane-impermeable biotinylation reagent) from the basal to the apical compartment of the seminiferous epithelium. Herein, we summarize the detailed procedures on performing the assay and to obtain semiquantitative data to assess the extent of BTB damage when compared to positive controls, such as treatment of rats with cadmium chloride (CdCl2) which is known to compromise the BTB integrity.

Drebrin is a family of actin-binding proteins with two known members called drebrin A and E. Apart from the ability to stabilize F-actin microfilaments via their actin-binding domains near the N-terminus, drebrin also regulates multiple cellular functions due to its unique ability to recruit multiple binding partners to a specific cellular domain, such as the seminiferous epithelium during the epithelial cycle of spermatogenesis. Recent studies have illustrated the role of drebrin E in the testis during spermatogenesis in particular via its ability to recruit branched actin polymerization protein known as actin-related protein 3 (Arp3), illustrating its involvement in modifying the organization of actin microfilaments at the ectoplasmic specialization (ES) which includes the testis-specific anchoring junction at the Sertoli-spermatid (apical ES) interface and at the Sertoli cell-cell (basal ES) interface. These data are carefully evaluated in light of other recent findings herein regarding the role of drebrin in actin filament organization at the ES. We also provide the hypothetical model regarding its involvement in germ cell transport during the epithelial cycle in the seminiferous epithelium to support spermatogenesis.

In the rat testis, studies have shown that cell polarity, in particular spermatid polarity, to support spermatogenesis is conferred by the coordinated efforts of the Par-, Crumbs-, and Scribble-based polarity complexes in the seminiferous epithelium. Furthermore, planar cell polarity (PCP) is conferred by PCP proteins such as Van Gogh-like 2 (Vangl2) in the testis. On the other hand, cell junctions at the Sertoli cell–spermatid (steps 8–19) interface are exclusively supported by adhesion protein complexes (for example, α6β1-integrin-laminin-α3,β3,γ3 and nectin-3-afadin) at the actin-rich apical ectoplasmic specialization (ES) since the apical ES is the only anchoring device in step 8–19 spermatids. For cell junctions at the Sertoli cell–cell interface, they are supported by adhesion complexes at the actin-based basal ES (for example, N-cadherin-β-catenin and nectin-2-afadin), tight junction (occludin-ZO-1 and claudin 11-ZO-1), and gap junction (connexin 43-plakophilin-2) and also intermediate filament-based desmosome (for example, desmoglein-2-desmocollin-2). In short, the testis-specific actin-rich anchoring device known as ES is crucial to support spermatid and Sertoli cell adhesion. Accumulating evidence has shown that the Par-, Crumbs-, and Scribble-based polarity complexes and the PCP Vangl2 are working in concert with actin- or microtubule-based cytoskeletons (or both) and these polarity (or PCP) protein complexes exert their effects through changes in the organization of the cytoskeletal elements across the seminiferous epithelium of adult rat testes. As such, there is an intimate relationship between cell polarity, cell adhesion, and cytoskeletal function in the testis. Herein, we critically evaluate these recent findings based on studies on different animal models. We also suggest some crucial future studies to be performed.

In adult mammalian testes, spermatids, most notably step 17-19 spermatids in stage IV-VIII tubules, are aligned with their heads pointing toward the basement membrane and their tails toward the tubule lumen. On the other hand, these polarized spermatids also align across the plane of seminiferous epithelium, mimicking planar cell polarity (PCP) found in other hair cells in cochlea (inner ear). This orderly alignment of developing spermatids during spermiogenesis is important to support spermatogenesis, such that the maximal number of developing spermatids can be packed and supported by a fixed population of differentiated Sertoli cells in the limited space of the seminiferous epithelium in adult testes. In this review, we provide emerging evidence to demonstrate spermatid PCP in the seminiferous epithelium to support spermatogenesis. We also review findings in the field regarding the biology of spermatid cellular polarity (e.g., head-tail polarity and apico-basal polarity) and its inter-relationship to spermatid PCP. Furthermore, we also provide a hypothetical concept on the importance of PCP proteins in endocytic vesicle-mediated protein trafficking events to support spermatogenesis through protein endocytosis and recycling.

Spermatogenesis in humans is comprised of a series of highly complicated cellular events, necessary to support the production of an upward of 200 million sperm daily from puberty through the entire adulthood of a healthy man. Recent advances in the field using the techniques of cell and molecular biology, genetics, and biochemistry have unraveled many of the mysteries in spermatogenesis. In this Chapter, we highlight some recent advances in the field regarding the biology of human spermatogenesis. We also summarize and discuss recent advances regarding the regulation of spermatogenesis in humans. Due to rapid advances in our understanding of spermatogenesis and the large number of published reports in the literature in the last 2–3 decades, we focus on rapidly developing areas to stimulate the interest of our readers, in particular in areas that offer advances for the treatment of infertility in men.

In adult mammalian testes, spermatogenesis is comprised of several discrete cellular events that work in tandem to support the transformation and differentiation of diploid spermatogonia to haploid spermatids in the seminiferous epithelium during the seminiferous epithelial cycle. These include: self-renewal of spermatogonial stem cells via mitosis and their transformation into differentiated spermatogonia, meiosis I/II, spermiogenesis and the release of sperms at spermiation. Studies have shown that these cellular events are under precise and coordinated controls of multiple proteins and signaling pathways. These events are also regulated by polarity proteins that are known to confer classical apico-basal (A/B) polarity in other epithelia. Furthermore, spermatid development is likely supported by planar cell polarity (PCP) proteins since polarized spermatids are aligned across the plane of seminiferous epithelium in an orderly fashion, analogous to hair cells in the cochlea of the inner ear. Thus, the maximal number of spermatids can be packed and supported by a fixed population of differentiated Sertoli cells in the limited space of the seminiferous epithelium in adult testes. In this review, we briefly summarize recent findings regarding the role of PCP proteins in the testis. This information should be helpful in future studies to better understand the role of PCP proteins in spermatogenesis.

Planar cell polarity (PCP) proteins confer polarization of a field of cells (eg, elongating/elongated spermatids) within the plane of an epithelium such as the seminiferous epithelium of the tubule during spermatogenesis. In adult rat testes, Sertoli and germ cells were found to express PCP core proteins (eg, Van Gogh-like 2 [Vangl2]), effectors, ligands, and signaling proteins. Vangl2 expressed predominantly by Sertoli cells was localized at the testis-specific, actin-rich ectoplasmic specialization (ES) at the Sertoli-spermatid interface in the adluminal compartment and also Sertoli-Sertoli interface at the blood-testis barrier (BTB) and structurally interacted with actin, N-cadherin, and another PCP/polarity protein Scribble. Vangl2 knockdown (KD) by RNA interference in Sertoli cells cultured in vitro with an established tight junction-permeability barrier led to BTB tightening, whereas its overexpression using a full-length cDNA construct perturbed the barrier function. These changes were mediated through an alteration on the organization actin microfilaments at the ES in Sertoli cells, involving actin-regulatory proteins, epidermal growth factor receptor pathway substrate 8, actin-related protein 3, and Scribble, which in turn affected the function of adhesion protein complexes at the ES during the epithelial cycle of spermatogenesis. Using Polyplus in vivo-jetPEI reagent as a transfection medium to silence Vangl2 in the testis in vivo by RNA interference with high efficacy, Vangl2 KD led to changes in F-actin organization at the ES in the epithelium, impeding spermatid and phagosome transport and spermatid polarity, meiosis, and BTB dynamics. For instance, step 19 spermatids remained embedded in the epithelium alongside with step 9 and 10 spermatids in stages IX-X tubules. In summary, the PCP protein Vangl2 is an ES regulator through its effects on actin microfilaments in the testis.

The blood-testis barrier (BTB) is one of the tightest blood-tissue barriers in the mammalian body. It divides the seminiferous epithelium of the seminiferous tubule, the functional unit of the testis, where spermatogenesis takes place, into the basal and the adluminal (apical) compartments. Functionally, the BTB provides a unique microenvironment for meiosis I/II and post-meiotic spermatid development which take place exclusively in the apical compartment, away from the host immune system, and it contributes to the immune privilege status of testis. However, the BTB also poses major obstacles in developing male contraceptives (e.g., adjudin) that exert their effects on germ cells in the apical compartment, such as by disrupting spermatid adhesion to the Sertoli cell, causing germ cell exfoliation from the testis. Besides the tight junction (TJ) between adjacent Sertoli cells at the BTB that restricts the entry of contraceptives from the microvessels in the interstitium to the adluminal compartment, drug transporters, such as P-glycoprotein and multidrug resistance-associated protein 1 (MRP1), are also present that actively pump drugs out of the testis, limiting drug bioavailability. Recent advances in drug formulations, such as drug particle micronization (