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.

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.

PFOS-induced Sertoli cell injury through c-Jun N-terminal kinase (JNK) - a study by RNA-Seq.

Per- and polyfluoroalkyl substances (PFAS) are a family of "forever chemicals" including PFOS (perfluorooctane sulfonate). These toxic chemicals do not break down in the environment nor in our bodies. In the human body, PFOS and PFOA (perfluoroctanoic acid) have a half-life (T1/2) of about 4-5 years so low daily consumption of these chemicals can accumulate in the human body to a harmful level over a long period. Although the use of PFOS in consumer products was banned in the U.S. in 2022/2023, this forever chemical remains detectable in our tap water and food products. Every American tested has a high level of PFAS in their blood (https://cleanwater.org/pfas-forever-chemicals). In this report, we used a Sertoli cell blood-testis barrier (BTB) model with primary Sertoli cells cultured in vitro with an established functional tight junction (TJ)-permeability barrier that mimicked the BTB in vivo. Treatment of Sertoli cells with PFOS was found to perturb the TJ-barrier, which was the result of cytoskeletal disruption across the cell cytoplasm, disrupting actin and microtubule polymerization. These changes thus affected the proper localization of BTB-associated proteins at the BTB. Using RNA-Seq transcriptome profiling, bioinformatics analysis, and pertinent biochemical and cell biology techniques, it was discovered that PFOS-induced Sertoli cell toxicity through the c-Jun N-terminal kinase (JNK; also known as stress-activated protein kinase, SAPK) and its phosphorylated/active form p-JNK signaling pathway. More importantly, KB-R7943 mesylate (KB), a JNK/p-JNK activator, was capable of blocking PFOS-induced Sertoli cell injury, supporting the notion that PFOS-induced cell injury can possibly be therapeutically managed.

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.

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.

A local regulatory network in the testis mediated by laminin and collagen fragments that supports spermatogenesis.

It is almost five decades since the discovery of the hypothalamic-pituitary-testicular axis. This refers to the hormonal axis that connects the hypothalamus, pituitary gland and testes, which in turn, regulates the production of spermatozoa through spermatogenesis in the seminiferous tubules, and testosterone through steroidogenesis by Leydig cells in the interstitium, of the testes. Emerging evidence has demonstrated the presence of a regulatory network across the seminiferous epithelium utilizing bioactive molecules produced locally at specific domains of the epithelium. Studies have shown that biologically active fragments are produced from structural laminin and collagen chains in the basement membrane. Additionally, bioactive peptides are also produced locally in non-basement membrane laminin chains at the Sertoli-spermatid interface known as apical ectoplasmic specialization (apical ES, a testis-specific actin-based anchoring junction type). These bioactive peptides are derived from structural laminins and/or collagens at the corresponding sites through proteolytic cleavage by matrix metalloproteinases (MMPs). They in turn serve as autocrine and/or paracrine factors to modulate and coordinate cellular events across the epithelium by linking the apical and basal compartments, the apical and basal ES, the blood-testis barrier (BTB), and the basement membrane of the tunica propria. The cellular events supported by these bioactive peptides/fragments include the release of spermatozoa at spermiation, remodeling of the immunological barrier to facilitate the transport of preleptotene spermatocytes across the BTB, and the transport of haploid spermatids across the epithelium to support spermiogenesis. In this review, we critically evaluate these findings. Our goal is to identify research areas that deserve attentions in future years. The proposed research also provides the much needed understanding on the biology of spermatogenesis supported by a local network of regulatory biomolecules.

Role of cell polarity and planar cell polarity (PCP) proteins in spermatogenesis.

Studies on cell polarity proteins and planar cell polarity (PCP) proteins date back to almost 40 years ago in Drosophila and C. elegans when these proteins were shown to be crucial to support apico-basal polarity and also directional alignment of polarity cells across the plane of an epithelium during morphogenesis. In adult mammals, cell polarity and PCP are most notable in cochlear hair cells. However, the role of these two groups of proteins to support spermatogenesis was not explored until a decade earlier when several proteins that confer cell polarity and PCP proteins were identified in the rat testis. Since then, there are several reports appearing in the literature to examine the role of both cell polarity and PCP in supporting spermatogenesis. Herein, we provide an overview regarding the role of cell polarity and PCP proteins in the testis, evaluating these findings in light of studies in other mammalian epithelial cells/tissues. Our goal is to provide a timely evaluation of these findings, and provide some thought provoking remarks to guide future studies based on an evolving concept in the field.

Whole-exome sequencing of a large Chinese azoospermia and severe oligospermia cohort identifies novel infertility causative variants and genes.

Rare coding variants have been proven to be one of the significant factors contributing to spermatogenic failure in patients with non-obstructive azoospermia (NOA) and severe oligospermia (SO). To delineate the molecular characteristics of idiopathic NOA and SO, we performed whole-exome sequencing of 314 unrelated patients of Chinese Han origin and verified our findings by comparing to 400 fertile controls. We detected six pathogenic/likely pathogenic variants and four variants of unknown significance, in genes known to cause NOA/SO, and 9 of which had not been earlier reported. Additionally, we identified 20 novel NOA candidate genes affecting 25 patients. Among them, five (BRDT, CHD5, MCM9, MLH3 and ZFX) were considered as strong candidates based on the evidence obtained from murine functional studies and human single-cell (sc)RNA-sequencing data. These genetic findings provide insight into the aetiology of human NOA/SO and pave the way for further functional analysis and molecular diagnosis of male infertility.

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.

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.

AKAP9 supports spermatogenesis through its effects on microtubule and actin cytoskeletons in the rat testis.

In mammalian testes, extensive remodeling of the microtubule (MT) and actin cytoskeletons takes place in Sertoli cells across the seminiferous epithelium to support spermatogenesis. However, the mechanism(s) involving regulatory and signaling proteins remains poorly understood. Herein, A-kinase anchoring protein 9 (AKAP9, a member of the AKAP multivalent scaffold protein family) was shown to be one of these crucial regulatory proteins in the rat testis. Earlier studies have shown that AKAP9 serves as a signaling platform by recruiting multiple signaling and regulatory proteins to create a large protein complex that binds to the Golgi and centrosome to facilitate the assembly of the MT-nucleating γ-tubulin ring complex to initiate MT polymerization. We further expanded our earlier studies based on a Sertoli cell-specific AKAP9 knockout mouse model to probe the function of AKAP9 by using the techniques of immunofluorescence analysis, RNA interference (RNAi), and biochemical assays on an in vitro primary Sertoli cell culture model, and an adjudin-based animal model. AKAP9 robustly expressed across the seminiferous epithelium in adult rat testes, colocalizing with MT-based tracks, and laid perpendicular across the seminiferous epithelium, and prominently expressed at the Sertoli-spermatid cell-cell anchoring junction (called apical ectoplasmic specialization [ES]) and at the Sertoli cell-cell interface (called basal ES, which together with tight junction [TJ] created the blood-testis barrier [BTB]) stage specifically. AKAP9 knockdown in Sertoli cells by RNAi was found to perturb the TJ-permeability barrier through disruptive changes in the distribution of BTB-associated proteins at the Sertoli cell cortical zone, mediated by a considerable loss of ability to induce both MT polymerization and actin filament bundling. A considerable decline in AKAP9 expression and a disruptive distribution of AKAP9 across the seminiferous tubules was also noted during adjudin-induced germ cell (GC) exfoliation in this animal model, illustrating AKAP9 is essential to maintain the homeostasis of cytoskeletons to maintain Sertoli and GC adhesion in the testis.

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.

Testis Toxicants: Lesson from Traditional Chinese Medicine (TCM).

The testis is one of the organs in the mammalian body that is sensitive to toxicants. Accumulating evidence has shown that human exposure to toxic ingredients in Traditional Chinese Medicine (TCM), such as triptolide, gossypol, cannabidol, piperine, α-solanine, matrine, aristolochic acid, and emodin, lead to testis injury and reproductive dysfunction. The most obvious phenotype is reduced sperm counts due to defects in spermatogenesis. Studies have also shown that Sertoli cells in the seminiferous tubule, the functional unit of the testis that supports spermatogenesis, are the cell type that is most sensitive to the disruptive effects of toxicants. Since Sertoli cells are the "mother cells" that nurture germ cell development, Sertoli cell injury thus leads to failure in germ cell development in the seminiferous epithelium. Mounting evidence has shown that the Sertoli cell cytoskeletons, mitochondria function, Leydig cells steroidogenesis pathways and sperm ion channels are some of the prime targets of toxicants from TCM. We carefully evaluate recent findings in this area of research herein, and to provide a summary of these findings, including some insightful information regarding the underlying molecular basis of toxicant-induced testis injury that impede spermatogenesis.

Unraveling the Regulation of Cancer/Testis Antigens in Tumorigenesis Through an Analysis of Normal Germ Cell Development in Rodents.

Cancer/testis (CT) antigens are proteins aberrantly overexpressed in various tumorigenic cells, but they can also be normally expressed in the mammalian germline. Most CT antigens are highly immunogenic and known to be involved in cancer cell proliferation and tumor metastasis. A recent genome-wide analysis systematically identified CT antigen expression in 19 cancer types, significantly expanding the repertoire of CT antigens by 5-fold, from over 200 to approximately 1000. However, their function and regulation in tumorigenesis remain poorly understood. The shared functional characteristics between germ cells and cancer cells, if methodically defined, offer a unique gateway to understanding the regulation of CT antigens in cancers by studying gametogenesis. Nonetheless, such studies also provide insightful information on the role of CT antigens in spermatogenesis. Herein, we analyzed publicly available next generation sequencing datasets generated from normal adult testes in rodents, primordial germ cells and cancer samples across a series of published studies and databases. Based on these analyses, we report that a subset of CT antigens belonged to the core fitness gene family. Furthermore, super-enhancers both in normal testes and various cancers controlled specific CT antigens. We found that DNA methylation of CT antigens, such as TEX101 and TAF7L, was inversely correlated with their expression in both normal primordial germ cells and various cancers, which was mediated at least partly by DNA methyltransferase1 (DNMT1). By analyzing data from a testis knockout model, we showed that TAF7L could further influence the expression of additional CT antigens, which also held true in tumors. These findings not only confirmed the previous notion that CT antigens regulate cancer dynamics, but also showed that understanding the regulation of CT antigens during gametogenesis can offer new insights for cancer research.

Male Infertility in Humans: An Update on Non-obstructive Azoospermia (NOA) and Obstructive Azoospermia (OA).

Non-obstructive azoospermia (NOA) and obstructive azoospermia (OA) are two common causes of infertility that affect a considerable number of men. However, few studies were performed to understand the molecular etiology of these disorders. Studies based on bioinformatics and genetic analyses in recent years, however, have yielded insightful information and have identified a number of genes that are involved in these disorders. In this review, we briefly summarize and evaluate these findings. We also discuss findings based on epigenetic modifications of sperm DNAs that affect a number of genes pertinent to NOA and OA. The information summarized in this Chapter should be helpful to investigators in future functional studies of NOA and OA.

Spermiation: Insights from Studies on the Adjudin Model.

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.

Motor Proteins and Spermatogenesis.

Unlike the intermediate filament- and septin-based cytoskeletons which are apolar structures, the microtubule (MT) and actin cytoskeletons are polarized structures in mammalian cells and tissues including the testis, most notable in Sertoli cells. In the testis, these cytoskeletons that stretch across the epithelium of seminiferous tubules and lay perpendicular to the basement membrane of tunica propria serve as tracks for corresponding motor proteins to support cellular cargo transport. These cargoes include residual bodies, phagosomes, endocytic vesicles and most notably developing spermatocytes and haploid spermatids which lack the ultrastructures of motile cells (e.g., lamellipodia, filopodia). As such, these developing germ cells require the corresponding motor proteins to facilitate their transport across the seminiferous epithelium during the epithelial cycle of spermatogenesis. Due to the polarized natures of these cytoskeletons with distinctive plus (+) and minus (-) end, directional cargo transport can take place based on the use of corresponding actin- or MT-based motor proteins. These include the MT-based minus (-) end directed motor proteins: dyneins, and the plus (+) end directed motor proteins: kinesins, as well as the actin-based motor proteins: myosins, many of which are plus (+) end directed but a few are also minus (-) end directed motor proteins. Recent studies have shown that these motor proteins are essential to support spermatogenesis. In this review, we briefly summarize and evaluate these recent findings so that this information will serve as a helpful guide for future studies and for planning functional experiments to better understand their role mechanistically in supporting spermatogenesis.

Regulation of spermatid polarity by the actin- and microtubule (MT)-based cytoskeletons.

It is conceivable that spermatid apico-basal polarity and spermatid planar cell polarity (PCP) are utmost important to support spermatogenesis. The orderly arrangement of developing germ cells in particular spermatids during spermiogenesis are essential to obtain structural and nutrient supports from the fixed number of Sertoli cells across the limited space of seminiferous epithelium in the tubules following Sertoli cell differentiation by ∼17 day postpartum (dpp) in rodents and ∼12 years of age at puberty in humans. Yet few studies are found in the literature to investigate the role of these proteins to support spermatogenesis. Herein, we briefly summarize recent findings in the field, in particular emerging evidence that supports the concept that apico-basal polarity and PCP are conferred by the corresponding polarity proteins through their effects on the actin- and microtubule (MT)-based cytoskeletons. While much research is needed to bridge our gaps of understanding cell polarity, cytoskeletal function, and signaling proteins, a critical evaluation of some latest findings as summarized herein provides some important and also thought-provoking concepts to design better functional experiments to address this important, yet largely expored, research topic.