Understanding testicular single cell transcriptional atlas: from developmental complications to male infertility

Spermatogenesis is a multi-step biological process where mitotically active diploid (2n) spermatogonia differentiate into haploid (n) spermatozoa via regulated meiotic programming. The alarming rise in male infertility has become a global concern during the past decade thereby demanding an extensive profiling of testicular gene expression. Advancements in Next-Generation Sequencing (NGS) technologies have revolutionized our empathy towards complex biological events including spermatogenesis. However, despite multiple attempts made in the past to reveal the testicular transcriptional signature(s) either with bulk tissues or at the single-cell, level, comprehensive reviews on testicular transcriptomics and associated disorders are limited. Notably, technologies explicating the genome-wide gene expression patterns during various stages of spermatogenic progression provide the dynamic molecular landscape of testicular transcription. Our review discusses the advantages of single-cell RNA-sequencing (Sc-RNA-seq) over bulk RNA-seq concerning testicular tissues. Additionally, we highlight the cellular heterogeneity, spatial transcriptomics, dynamic gene expression and cell-to-cell interactions with distinct cell populations within the testes including germ cells (Gc), Sertoli cells (Sc), Peritubular cells (PTc), Leydig cells (Lc), etc. Furthermore, we provide a summary of key finding of single-cell transcriptomic studies that have shed light on developmental mechanisms implicated in testicular disorders and male infertility. These insights emphasize the pivotal roles of Sc-RNA-seq in advancing our knowledge regarding testicular transcriptional landscape and may serve as a potential resource to formulate future clinical interventions for male reproductive health.


Introduction
During the past two decades, an alarming rise in infertility particularly in developed/developing countries has become a matter of great concern (1).Around 15% of couples suffering from infertility globally, out of which 50%, is exclusively caused by the male partner (2).Male infertility is a multifaceted pathological condition with diverse manifestations, ranging from total absence of testicular sperm to specific changes in sperm quality (3).Chromosomal aberrations (like Klinefelter syndrome 47XXY) or variance (like 9qh+ heteromorphism) and other genetic mutations contribute upto 15-20% of total male infertility; however around 30-50% of cases remain untreatable due to idiopathic in origin/nature (4)(5)(6).Therefore, the mechanistic details of testicular spermatogenesis need to be reexamined at the cellular and molecular level (7,8).
Spermatogenesis is gonadotropin regulated, highly synchronized, developmental programme involving multi-step events like stem cell renewal/differentiation, epigenomic remodeling, and meiotic divisions for generating millions of spermatozoa inside testes (9)(10)(11).The continuity of this process throughout adult life is supported by an active spermatogonial stem cell (SSC) population that critically maintains the key balance between the capacity of self-renewal (to sustain the stem cell population) and differentiation [to produce spermatogonial progenitor cells (SPCs), which subsequently develop into mature sperm via meiotic divisions] (12-14).Therefore, the cellular and molecular landscape associated with the SSC micro-environment turns out to be critical for regulating male fertility.The rapid intrinsic speed (around 1000-1500 sperm per heartbeat in adult men) of spermatogenic progression significantly highlights the degree of orchestration governing the transcriptional dynamics of testicular cells (15)(16)(17).Figure 1 represents the histological architecture of the testis showing arrays of multiple developing germ cells (Gc) (from SSC to mature motile sperms) alongside with visual representation of the different stages and cellular interactions essential for spermatogenesis.
Functional and/or anatomical defects in testicular somatic and/ or Gc lead to spermatogenic impairment (with a qualitative and quantitative decline in sperm count clinically defined as azoospermia, oligozoospermia, teratozoospermia, and asthenospermia, etc.) causing male sub-infertility/infertility (18).Azoospermia, (the most severe form where semen without sperm) are of two types: obstructive azoospermia (OA) and non-obstructive azoospermia (NOA).OA stems from normal sperm production but obstructed sperm delivery, while NOA, accounting for around 10% of male infertility, results from lack of spermatogenesis in the testis (19).Three specific clinical categories are described in NOA as primary testicular failure like Sertoli cell only syndrome (SCOS)/Gc aplasia, Gc maturational arrest and hypo-spermatogenesis (20,21).Supplementary Table 1 highlights (A) clinical categories of semen quality, (B) etiologies and frequencies of male infertility and (C) the hallmarks of primary testicular failure.
Notably, multiple genetic complexities involving over 2,000 spermatogenic genes observed in different forms of male infertility having substantial variations in semen quality and testicular histology (5,22).Over recent decades assisted reproductive techniques (ART) e.g.in vitro fertilization (IVF) and intra-cytoplasmic sperm injection (ICSI) have successfully facilitated biological fatherhood for men with extremely low sperm counts (3).However, addressing the fundamental basis of poor sperm quality and quantity remains challenging due to a limited understanding of the intricate molecular and cellular interactions driving human sperm production (18,23).An impressive progress in deploying high-throughput sequencing technologies in biomedical research has been observed during the past decade (24).Within this scenario, both bulk and single-cell RNA-sequencing (Sc-RNA-seq) collectively have emerged as revolutionary tools, revealing the molecular complexities governing testicular physiology and elucidating mechanisms underlying testicular dysregulation/disorders (25).However, analyzing bulk RNAs from diverse cell types faces adverse challenges in detecting expression data in rare cell populations, possibly diluting the presence of uncommon transcripts.In contrast, Sc-RNA-seq generates distinct RNA fingerprints for each cell (at single-cell resolution) type, transforming our understanding of differential transcriptomic profiles from complex tissues like the testis and therefore found to be critically informative in the field of clinical endocrinology, especially in deciphering infertility (26,27).Table 1 summarizes the advantages of the Sc-RNA-seq strategy over the conventional bulk transcriptomic approach.
Despite being critically relevant, comprehensive reviews of recent literature revealing the dynamic landscape of single-cell testicular transcriptomics are currently limited (25,32).The current study dissects the spermatogenic landscapes viz., SSC/SPC niche, and cellular transcriptional dynamics during the meiotic onset and progression.Finally, this review attempts to reveal the intricate tapestry of testicular development and disorders through next-generation sequencing technologies.
2 Methodologies adopted for Sc-RNA-seq The Sc-RNA-seq generates high-resolution transcriptomic data that precisely identifies minor transcriptomic variations at a singlecell level from a complex organ (33,34).This tool has significant implications for gonadal development and disorders [viz., infertility, Disorders/Differences of Sex Development (DSDs), and cancer] (26,27).Sc-RNA-seq produces transcriptomic data employing methods like conventional cellular purification via enzyme-based crude mechanical isolation or transgenic reporter gene/antibody-based sorting of target cells by sophisticated magnetic-activated cell sorter (MACS) or fluorescence-activated cell sorter (FACS) columns (35).Figure 2 represents the fundamental working principles of bulk and Sc-RNA-seq.
Single-cell isolation and sequencing can be achieved at two different platforms, first droplet-based 10× Genomics Chromium where individual cells get encapsulated in aqueous droplets along with micro-beads loaded with barcoded primers (36) and second Fluigidm C1, where individual cells are physically captured on microfluidic chips (37).After generating Sc-RNA-seq libraries, 10x Genomics utilizes 3′ end-counting unique molecular identifiers (UMIs), a genetic barcode that ensures a distinct cell population is traced and categorized as a specific lineage within a particular tissue/organ (32).In contrast, Fluidigm C1 libraries are automatically generated having full-length transcriptomes with all possible mRNA isoforms/variants (due to alternative splicing/ transcription start site, etc.) (37).Notably, a new method Smart-seq2 has been developed recently having enhanced yield and length of cDNA libraries with better reverse transcription, template switching, and pre-amplification, improving the accuracy of transcript detection at a cheaper cost (38).Figure 3 schematically illustrates the available Sc-RNA-seq platforms and downstream workflow for data analyses.
Sc-RNA-seq data are plotted in bi-dimensional space and interpreted by methods like t-Distributed Stochastic Neighbor Embedding (t-SNE) and/or Uniform Manifold Approximation and Projection (UMAP) (33,34).Although, these methods clearly depict cellular heterogeneity, Sc-RNA-seq data demonstrate cellular condition in a non-functional state as the tissue architecture gets dissociated/disaggregated (39).Therefore, new technology has been developed that captures the spatial transcriptomic data from the tissues via individual-cell laser-capture micro-dissection or multiplexed in situ hybridization (39).However, this method also shows limited sensitivity and depends on prior information on types of cells and/or genes to be targeted/analyzed.Recent Slide-seq technology overcomes these limitations and is considered to be the most suitable for generating high-throughput spatial transcriptomic information at 10-mm resolution (40).Figure 4 depicts the workflow of four popular methodologies being deployed for spatial transcriptomics.

Fetal testicular morphogenesis
The ontology of testicular germ and somatic cell lineages are developmentally independent (15,41,42).The bipotential fetal gonads are formed at the gonadal ridge (GR) during embryonic life [embryonic days (E) 8.0-9.5 in mice and weeks of gestation (W) 3.5-4 in humans] and appear distinctly (by E9.5-10 and W5) with thickening on the ventral side of the mesonephros (43).However, during E6.5-7.5 and W3 murine and human primordial gem cells (PGCs) emerged from posterior-epiblast and amnion respectively.After getting specified (at E7.5 in mice and W4 in humans), these PGCs start migrating towards the developing GR and finally colonize the nascent gonads by E10.5 in mice and W8 in humans (44).The gonadal PGCs stop proliferation, lose pluripotency to gain competence for meiotic commitment via upregulating RNA binding DAZL protein (45).During E11.5 in mice and W6-7 in humans these nascent gonads undergo sex determination (SD), triggered by Sry expression in pre-Sertoli cells of XY gonads (46).The SD event involves step-wise transcriptional cascades generating male (Sry, Sox9, Fgf9, Amh, Dmrt1 etc.) and female (Wnt4, Rspo1, Foxl2, Runx1, etc.) specific robust genetic programming (47).The hallmark of fetal testicular morphogenesis is completed with the formation of the testicular cord and associated vasculature pattern due to selective (XY specific) migration of the mesonephric

Exploring cellular heterogeneity in the testis
Testis is a highly complex organ showing remarkable cellular heterogeneity constituted by both germline and somatic cells.Multiple somatic cell types [like nourishing Sc within seminiferous tubules (ST), supportive PTc covering the ST, interstitial (in inter-tubular space) steroidogenic Lc, interstitial macrophages, etc.] and diverse arrays of developing Gc [including pre-meiotic SSCs/SPCs (2n), different stages of meiotic Gc like primary spermatocytes (2n), secondary spermatocytes (n), round and elongated spermatids (n) and post-meiotic spermatozoa (n) etc] within ST contribute the tissue-organization of testis (Figure 1).Notably, almost all possible cellular events [like mitosis, meiosis, cellular differentiation, apoptosis, epigenetic remodeling, histone-protamine exchange and meiotic sex chromosome inactivation (MSCI) etc.] are evident in testis (52).Table 2 summarizes distinct/key cell markers of individual testicular cells with respect to the appropriate functional significance regulating male fertility.We here have discussed individual major cell-types contributing to the tissue organization of adult testis.

The germ-line cell types
The Gc residing within ST passes through a series of developmental stages [spermatogonia (2n), primary and secondary spermatocytes (n), round and elongated spermatids (n) etc] culminating in the production of mature sperm.Here we discuss different stages of developing Gc.

SSCs
SSCs are established from gonocytes post-birth (postnatal age 2-3 days in mice) which guarantee the continuity of sperm production throughout adult life (53).In rodents and primates, the duration of spermatogenesis varies among species, (in days; man 64, rhesus monkey 42, mouse 35, and rat 52) typically spanning 35 to 70 days (10,54).In rodents A s (A single spermatogonia) and in primates A pale (proliferative stem/progenitor pool) or A dark (slow diving reserve stem pool) are considered to be the functional SSC populations respectively (13,14).The key balance between selfrenewal and differentiation of these cells remains critical for spermatogenic maintenance throughout adult life (13).The Sc and PTc contribute to establishing the micro-environment of SSC that governs such regulated balance (55).

SPCs
SPCs are constituted with undifferentiated Spermatogonia (Spg), type A paired or A aligned connected with common cytoplasmic bridges.In rodents, these pre-meiotic (mitotic) Gc exhibit clonal fragmentation and get differentiated into a transit amplifying (TA) population (A1 to A4) induced by RA and then further transformed to differentiated Spg type B stage having meiotic fate (56).In primates, no such TA population is observed however, in rhesus monkeys (not human) 4 categories of Spg B (B1, B2,B3,B4) are found.In rodents, RA further drives the meiotic entry of Spg B by inducing Stra8, Rec8 and Meiosin expressions in these cells (50, 57).

Meiotic Gc
Meiotic Gc represent the most critical stage of spermatogenesis (58) with two successive divisions.In the first division, diploid (2n) primary spermatocytes transform into haploid (n) secondary spermatocytes followed by in the second division, these spermatocytes further divide to produce round spermatids.Meiotic Gc are pivotal in reducing the chromosome number by half, ensuring that the resulting sperm cells possess the correct genetic material for fertilization upon union with an egg cell.The juxtacrine testosterone (T) signaling through Sc regulates the meiotic progression (59).

Post-meiotic Gc
Post-meiotic Gc maturation mainly involves the transformation of a round spermatid to an elongated structure and such elongated spermatids get condensed and further mature into spermatozoa.In this step, the histone-protamine transition is observed leading to massive hetero-chromatinization leading to transcriptional silencing.However, the functional maturation of male gamete occurs with spermiogenesis, which involves complex cellular changes, viz., development of acrosome and flagellum, etc. Unraveling this Illustrating fundamental difference between bulk and single-cell RNA sequencing (Sc-RNA-seq).metamorphosis is crucial for addressing male fertility concerns and developing strategies for male reproductive health and contraception (60).Figure 5 elucidates the testicular transcriptional dynamics concerning complex cellular heterogeneity.Figure 5A exhibits the differential degrees of heterogeneous/diverse gene expression profiles observed in multiple developmental stages of human (i) spermatogonia, (ii) spermatocytes, (iii) spermatids, and (iv) the entire adult human testis.Figure 5B is the graphical representation of developing Gc-specific marker expression during the entire spermatogenic progression.Figure 6 illustrates the comparative heat maps showing the expression profiles of critical genes involved/ associated with different developmental/maturational stages of (A) spermatogonia (B) spermatocytes (C) spermatids and (D) whole adult testis.

The somatic cells
Five distinct somatic cell types (that undergo mitosis but not meiosis) provide the critical structural and nutritional support are discussed in the following section.

Sertoli cells
Sertoli cells (Sc) are the major somatic cells that are the target of both FSH and T, known to dictate spermatogenesis by providing direct structural and nutritional support to all stages of developing Gc within the ST.Sc nurture and regulate the meiotic progression and completion of Gc.Sc-Sc tight junctions establish the bloodtestis barrier (BTB) essential for Gc development and their directional movement towards the tubular lumen (61).FSH is critical for Sc proliferation and expansion of the pre-meiotic Gc population, whereas T governs the BTB formation, dynamics and Gc meiosis (4,62).Sc derived glial cell line-derived neurotrophic factor (GDNF) and stem cell factor (SCF) play crucial role in determining the SSC fate decision from self-renewal to differentiation.Fetal Sc originate from the coelomic epithelium (63) and get specified (at E11.5 in mice and W6-7 in humans) by Sry driven Sox9 expression (43).The development of Sc is distinctly divided into three phases, immature (during fetal/neonatal/ perinatal phase), maturing (during juvenile/pre-pubertal life) and mature (at puberty and adulthood) (64)(65)(66).Hormonal (FSH & T) stimulations to fetal/neonatal/infant Sc, show limited responses like self-proliferation and local expansion of pre-meiotic Gc but are found to be insufficient to induce robust onset of spermatogonial differentiation.The functional immaturity in terms of hormonal responsiveness of younger Sc is considered the underlying cause.Restricted plasma membrane localization of FSH-Receptor (Fsh-r), inadequate expression of associated Gas sub-units and poor binding of T with Androgen Receptor (Ar) have been demonstrated in immature Sc during infancy (67-70).Therefore, the functional maturation of pubertal Sc is found to be developmentally critical for inducing Gc differentiation.Such pubertal maturational event in Sc become a prerequisite for spermatogenic onset and male fertility (15, 65).This is further evident by the presence of persistent immature Sc in adult testes that results in severe oligozoospermic conditions with infertility in rodents (71)(72)(73)(74)(75) and in humans (64, 76-79).

Peritubular cells
Peritubular cells (PTc) constitute the outermost covering of the ST and further provides structural support by creating peristaltic waves through their contractile elements, potentially prompting fluid movement within the tubule lumen to facilitate the expulsion of spermatozoa (80).PTc are the targets of T signaling and shown to promote Sc maturation.Furthermore, crosstalk between PTc with adjacent Lc play a vital role in spermatogenic development.Along with Sc, these cells govern the maintenance of the SSC microenvironment (81, 82).

Leydig cells
Leydig cells (Lc) are the key steroidogenic cells located within the inter-tubular interstitium and the sole target of LH for T

Macrophages
Macrophages, (being CSF1R + , CD206 + MHCII -) are present in the testicular interstitium and regulate the steroidogenic activity of ALc (84,89).Another distinct population of adult macrophages (CSF1R -, CD206 -, MHCII + ) has been observed at various patches of the peritubular region which actively contributes to the SSC niche and promotes spermatogonial differentiation (80,90).Intriguingly in fetal life, yolk-sac-derived murine macrophages (at E7.5) play a critical role in directing the selective migration of mesonephric endothelial cells towards developing XY gonads (not XX) and directs testicular cord (at E12.5) formation (49).Recent findings indicate that monocytes derived from hematopoietic stem cells (HSCs) of fetal liver get colonized in embryonic mouse testes in Sc dependent manner and further differentiate into testis resident macrophages during postnatal life (84).During adulthood, CD206 + interstitial macrophages promote ALc proliferation and steroidogenesis and thereby indirectly regulate male fertility.Fu rthermore, these m acro phages also mai ntain the immunosuppressive environment within the testis (91).

Endothelial cells
Endothelial cells (ECs) form the border walls of blood vessels, facilitating nutrient exchange for developing Gc/sperm.Dysfunction in these cells can affect sperm development and overall testicular health, impacting fertility.During murine testicular morphogenesis, a key feature involves sex-specific vascularization where endothelial cells migrate from the neighboring mesonephros into the fetal XY gonads (during E11-12.5),encircling Sc-Gc clusters and prompting the formation of seminiferous cords.These cells are potent sources of vascular endothelial growth factor A (Vegf A) critical for tissue remodeling/homeostasis (92).ECs also contribute to the SSC niche by producing GDNF that sustains SSCs during extended culture periods (93).
Notably, moderate expressions of Dmrt1, Sox9, Wt1, Amh, Dhh and Aldh1a1 were found in murine Sc from E18.5 to P2D upto P7D.However, the transcript levels of the genes essential for induction of Gc differentiation like Scf/Kit-ligand, Gdnf and Csf-1 etc, remained low during this time of testicular development.In consistent with previous reports, Lc specific Insl3, Cyp17a1, Hsd3b1, Lh/cg-receptor and PTc restricted Ar expressions were observed from E18.5 to P7D; whereas meiotic gatekeeper Stra8 expression was enriched at Gc on P7D (Figure 7C).Figures 7A, B show age-dependent dynamic gene expression profiles observed in different cell types during three [(i) E18.5, (ii) P2D (post-natal 2 days of age) & (iii) P7D (post-natal 7 days of age)] distinct developmental phases of murine testicular maturation.Figure 7C depicts the comparative heat maps revealing the expression profiles of key spermatogenic genes in different cell types during (i) fetal (E18.5)testicular developmental and neonatal [(ii) P2D, (iii) P7D] testis maturation.
To reduce the likelihood of overlooking pertinent studies, particularly preprint studies, an additional search is conducted on the GEO dataset (https://www.https://www.ncbi.nlm.nih.gov/geo/)employing combinations of refined above search terms.The searches yielded approximately ~132 records from the last 10 years (2015-2024).Among these, 60 articles contained original Sc-RNA-seq data (from at least one donor) related to human testicular samples, while the remaining 68 articles involved re-analyzed data from prior publications or the datasets from other primates, rodents, livestock animals, or non-mammalian species.Starting in 2015, Guo et al. pioneered Sc-RNA-seq on 233 individual male and female human PGCs from 15 embryos between 4 and 19 weeks of gestation (94), followed by two studies in 2017 that sequenced adult testicular cells (95,96).Interestingly, despite being from different teams, both adult studies focused on spermatogonia.Since 2018, numerous studies have conducted Sc-RNA-seq on human testicular samples, encompassing both prenatal and postnatal stages, normal and abnormal testes.Figure 8 represents the timeline of literature published/available in PubMed.Table 3 is the chronological summary of original research articles published on Sc-RNA-seq data for normal testicular samples collected from (A) primates (human and monkeys) and (B) rodents.Table 4 discusses countrywise Sc-RNA-seq data on clinical human samples with multiple forms of impaired spermatogenesis/infertility reported so far.Notably, Tables 3, 4 will serve as a critical repositories/valuable resource for Sc-RNA-seq datasets specific to distinct testicular cell types generated to date.

New progress
This comprehensive review of literature on testicular Sc-RNAseq highlights the complexity of testicular tissue composition and the uniqueness of cellular transcriptional dynamics.The cuttingedge approach has unveiled a heterogeneous landscape within the testis, providing insights into spermatogenic Gc, somatic PTc and interstitial Lc and supportive Sc associated with SSC microenvironments.Studies employing Sc-RNA-seq have examined the transcriptional profiles of various Gc populations at distinct developmental stages elucidating the molecular regulation of spermatogonial differentiation, meiosis, and sperm maturation.The application of Sc-RNA-seq has not only enriched our understanding of testicular cell heterogeneity but has also identified novel markers and regulatory pathways critical for male infertility and reproductive health (147).Integration of multi-omics data and advanced in-silico analyses offers a comprehensive view of the testicular transcriptomic landscape, fostering discoveries with profound implications for both basic biology and clinical applications in reproductive medicine.

B C A
Single-cell transcriptomic analysis of testicular cells at different developmental age groups.(A) UMAP visualizations representing the cellular composition of testicular cells at (i) embryonic days 18.5 (E18.5),(ii) postnatal days 2 (P2D), and (iii) postnatal days 7 (P7D).Each dot represents a single cell, color-coded by identified cell types: stroma (cyan), Leydig cells (yellow), Sertoli cells (dark blue), endothelial cells (green), innate lymphoid cells (purple), peritubular myoid cells (red), macrophages (light blue), and germ cells (orange).(B) UMAP plots displaying cell cycle phases for testicular cells on E18.5, P2D, and P7D.Cells are color-coded according to their phase in the cell cycle: G1 (blue), S (red), G2/M (green), and cells not in cycle (black).Arrows indicate the direction of the developmental trajectory.(C) Heatmaps illustrating the expression profiles of selected marker genes across different cell types on E18.5, P2D, and P7D.Gene expression levels are represented by a gradient from low (blue) to high (red) expression.Beneath each UMAP plot in B, stacked bar charts show the proportion of cells in each cell cycle phase for two replicates at each developmental stage (EMB1, EMB2, P2D1, P2D2, P7D1, P7D2).
A significant development in the field of testicular transcriptomics requires an inclusive assimilation/integration of data obtained from Sc-RNA-seq analyses with spatial transcriptomic information (32).For example, Chen and colleagues employed the "spatial transcriptomic analysis" technology, creating a spatial atlas that delineates testicular gene expression at near-singlecell resolution in the human testis (111).Similarly, Garcia-Alonso et al. conducted integrated analyses of male gonadal development by fusing Sc-RNA-seq and spatial transcriptomics, offering a more comprehensive and in-depth perspective (27).

Current challenges
The integration of these methodologies is anticipated to augment future investigations on the human testis and male infertility, affording a comprehensive understanding of functional/impaired spermatogenesis.Although Sc-RNA-seq analyses have corroborated the notable presence of immune cells (e.g., macrophages -both M1 and M2, T cells, mast cells, and B cells) in the testicular microenvironment, the functional roles (and also transcriptional patterns) of these cells in regulating male infertility development remain elusive (85).However, despite the current high cost of Sc-RNA-seq, several of the studies sincerely lack a direct clinical orientation Therefore, diverse testicular pathologies, viz., cryptorchidism, AZFc deletions, azoospermia post chemo/radiotherapy, and teratospermia, demand focused exploration of Sc-RNA-seq analyses.The ongoing refinement and cost mitigation of this technology holds future promise for clinical applications, such as diagnostics, classification, prediction of sperm retrieval, and evaluation of hormonal treatment efficacy.Figure 9 illustrates the probable applications of Sc-RNA-Seq data towards framing appropriate preclinical studies (both in vitro and in vivo approach) leading to potential future diagnostic (assays/tools/kits) in male reproductive health care.

Concluding remarks and future directions
Exploring the intricate landscape of testicular function and pathology requires a comprehensive understanding spanning from the fundamental processes of development to the complexities of the disorders.This review has examined the uncharted territory of the testicular single-cell transcriptional atlas, illuminating the diverse cellular populations and their orchestrated molecular dynamics.From the genesis of Gc to the intricate orchestration of somatic cell interactions, this exploration navigates the transcriptional intricacies shaping testicular development.Furthermore, it delves into the disruptive signatures underlying various testicular disorders, shedding light on potential avenues for diagnostic and therapeutic advancements.By unraveling the cellular intricacies from a single-cell perspective, this review aims to provide comprehensive insights bridging the developmental trajectory to the manifestations of disorders within the testicular microenvironment.We also FIGURE 8 Trends in publication on testicular Sc-RNA-seq studies.The bar graph illustrates the number of articles published by year, from 2015 to 2024 available at PubMed.A noticeable peak is observed in 2022, followed by a decline in subsequent years.The x-axis represents the years, while the y-axis quantifies the number of articles published.The data suggests a period of heightened research activity in 2022, with a gradual decrease in the volume of publications thereafter.Human spermatogonia form discrete groups, whereas baboon and rhesus spermatogonia are found to be less heterogeneous.
A cross-species analysis reveals significant differences between primate and mouse SSCs.For example, genes coding for components and regulators of the actin cytoskeleton cortical for cell-adhesion are enriched in primate-specific SSC.In humans, both SSC and progenitor spermatogonia were found to be Adark, while Apale spermatogonia appears biased towards differentiation.
( This study provides a blueprint for understanding the development of the male germline and supporting somatic cells in humans.The germ cell subset markers identified are candidates to be used for clinical applications, including SSC therapy for treating infertility.Their identification of RNA helicase DDX43 as a crucial regulator of chromatin remodeling in spermiogenesis showcases its pivotal role in this process.These findings underscore the significance of DDX43 in spermiogenesis and emphasize the efficacy of the single-cell-based approach in delineating cell-state-specific control in male germline development.The histone demethylase KDM6A, exhibits transient expression just before and during meiotic entry in spermatogenesis, playing a crucial role in maintaining epigenetic states across generations in the male germline.summarize the previous attempts deploying Sc-RNA-Seq on clinical testicular biopsies (Table 4) that have significantly enriched our understanding of testicular physiology e.g.-regulation of SSC micro-environment and testicular somatic cell maturation, etc.Finally, the increasing availability of publicly accessible testicular Sc-RNA-seq datasets provides researchers with greater possibilities for conducting personalized data analyses to address diverse research requirements.We anticipate that this review will not only facilitate the comprehensive understanding of spermatogenic transcription during normal and impaired (sub-fertile/infertile) conditions but also serve as a valuable reference for critical diagnosis and cure for some forms of unexplained/idiopathic male infertility.

1
FIGURE 1 Testicular tissue architecture and Spermatogenic Development.(A) Testis Anatomy: Cross-section illustrating the testis with internal cellular composition and associated structures: the epididymis and vas deferens.(B) Seminiferous Tubule: Histological image showing the transverse section of a seminiferous tubule where spermatogenesis occurs.(C) Spermatogenesis Overview: Schematic representation of spermatogenesis within the seminiferous tubule, from spermatogonial stem cells (SSC) to mature spermatozoa.(D) Sperm Maturation: Detailed progression of sperm development from type A spermatogonium through meiosis to form mature sperm.(E) Human vs. Rodent Spermatogenesis: Comparative stages of spermatogenesis between humans and rodents, highlighting the differentiation of spermatogonial cells.(F) Sperm Cell Morphology: Detailed diagram of a motile sperm cell, showcasing its various structural components from head to tail.
(A) Bulk Processing: The left diagram illustrates the enzymatic and mechanical dissociation of testicular tissue into a heterogeneous mixture of cells including Sertoli cells, Leydig cells, spermatogonia, spermatocytes, spermatids etc.Following dissociation, cells are processed in bulk, as shown by the central test tube graphic, including DNA barcoding and sequencing to yield average gene expression profiles.(B) Single Cell-Suspension and Enrichment: The top middle flow diagram transitions from an unselected testicular cell suspension to cell type enrichment via methods like transgenic reporters (e.g., GFP) and antibody labeling etc.The bottom middle panel depicts the conversion of the unselected cellular mixture into a single-cell suspension, followed by cell sorting based on type-specific markers, with each cell type represented by a different color.The right side shows the cell clustering outcome, visualized by a t-SNE plot, and the subsequent sequencing of individual cells resulting in a detailed gene expression heatmap, with colors indicating expression levels from high (red) to low (green).

3 4 Molecular
FIGURE 3 Comprehensive platforms of Single-Cell Transcriptomics.(A) 10× Genomics Chromium: The workflow initiates with encapsulation of individual cells with barcoded primer gel beads into oil droplets to generate Gel bead-in-emulsions (GEMs), ready for collecting and processing.Subsequent steps include sequencing, which generates data for proportion of cell type bar charts, a volcano plot showing differential gene expression, and a violin plot depicting quantification of average gene expression across cell types.(B) Fluigidm C1: Individual cells are physically captured on microfluidic chips.(C) Data Integration and Analysis: The t-SNE plot illustrates cell cycle analysis of individual cells.UMAP with pseudotime calculation provides insights into the developmental trajectory of the cells.The chord diagram highlights cell-to-cell interaction analysis, elucidating the complex interplay between different cell types.

( 85 )
. Although the extent of LH responsiveness of ALc remain unaltered with testicular aging, overall T production declines with testicular aging and testicular metabolic complications (86-88).

6
FIGURE 6 Gene expression profiles during human spermatogenesis.(A) Gene expression heatmap of spermatogonia: The heatmap illustrates the gene expression levels at different steps of spermatogonial development, from pre-meiotic to SSCs.(B) Gene expression heatmap of spermatocytes: Displays the gene expression patterns during the meiotic stages (spermatocytes, from pre-leptotene to post-meiotic).(C) Gene expression heatmap of spermatids: Depicts variations of gene expression in spermatids, highlighting the transition from early to late spermatid stages.(D) Comparative expression heatmap of adult human testis: Compares gene expression across different cell types and stages of sperm development, including leptotene and zygotene spermatocytes and diplotene to secondary spermatocytes.

TABLE 1
Summarizes the essential aspects of different RNA-Seq strategies, including their aims, key features, popular platforms, merits, limitations, and direct applications.

TABLE 2 Essential
Genes Influencing Spermatogenesis Identified Across Multiple Single-Cell RNA Sequencing Research Studies.Assembly and stability of the synaptonemal complex, as well as for the progression of meiotic recombination and synapsis between homologous chromosomes.

TABLE 2 Continued
Involved in the repair of DNA double-strand breaks during meiotic recombination, ensuring proper chromosome segregation and genetic diversity in sperm cells.

TABLE 2 Continued
Promote Proliferation of Sertoli cells and support pre-meiotic germ cell differentiation.
They utilize single-cell RNA sequencing to create a resource on mouse spermatogenesis which aims to unravel the diverse cell types within the adult testis, pinpoint factors influencing germ cell differentiation, and uncover various types of somatic cells involved in this process.This study reveals the first detailed map of aging in mouse testes at the single-cell level.It identifies changes in gene profiles, disruptions in stem cell balance, and the emergence of specific aging-related cell types, like macrophages.The findings offer a vast resource for studying age-related subfertility and suggest potential paths for diagnostics and targeted treatments against testicular aging by focusing on oxidative stress and inflammation.

TABLE 4
List of Human Participants Exhibiting Spermatogenic Dysfunction or Infertility: A Detailed Examination.earlytesticulardamage, leading to fewer Gc, loss in Sc, and ALc changes.Genetic studies pinpoint specific gene alterations, especially in X-escapee genes, driving these issues.Sc dysfunction is emphasized as pivotal in Gc loss, urging wider research across ages and more KS patients to understand this condition better.This study has compared testicular cells (from 10 normal and 7 NOA) and found prominent maturational defects.Blocking Wnt signaling helps immature cells to support germ cell survival, offering new diagnostic /treatment strategies for NOA.-related genes, such as the upregulation of Sqstm1 and the down-regulation of Lc3a in spermatids, were identified in NOA Gc , while Cst3-mediated autophagy potentially aids in maintaining SSC through Oct4, Id1, and Nanos3; additionally, inhibition of Cst3 disrupted meiosis and spermatid formation.Gc aplasia viz., immaturity of ALc, chronic tissue inflammation, fibrosis, and senescence phenotype of the testicular somatic cells, ratio of Lamin A/C transcripts and an active DNA damage response in Lc and PTc.This study identifies a subpopulation of Sc (within multiple individuals having KS) lacking transcription from the XIST locus resulting elevated X-linked gene expressions.Furthermore, 72 pathways upregulated in KS suggesting changes in interstitial cells due to loss of X inactivation in Sc.