Running the gauntlet: challenges to genome integrity in spermiogenesis.

Species' continuity depends on gametogenesis to produce the only cell types that can transmit genetic information across generations. Spermiogenesis, which encompasses post-meiotic, haploid stages of male gametogenesis, is a process that leads to the formation of sperm cells well-known for their motility. Spermiogenesis faces three major challenges. First, after two rounds of meiotic divisions, the genome lacks repair templates (no sister chromatids, no homologous chromosomes), making it incredibly vulnerable to any genomic insults over an extended time (typically days-weeks). Second, the sperm genome becomes transcriptionally silent, making it difficult to respond to new perturbations as spermiogenesis progresses. Third, the histone-to-protamine transition, which is essential to package the sperm genome, counterintuitively involves DNA break formation. How spermiogenesis handles these challenges remains poorly understood. In this review, we discuss each challenge and their intersection with the biology of protamines. Finally, we discuss the implication of protamines in the process of evolution.

Ursonic acid attenuates spermatogenesis in oligozoospermia mice through inhibiting ferroptosis.

Ursonic acid (UNA) is a natural pentacyclic triterpene found in some medicinal plants and foods. The reproductive protective effect of UNA was evaluated in a mouse model of oligozoospermia induced by busulfan (BUS) at 30 mg/kg b.w.. The mice were initially divided into groups with UNA concentrations of 10, 30, 50, 100 mg/kg. Subsequently, based on sperm parameters, the optimal concentration of 50 mg/kg was identified. As a control, an additional group was supplemented with ursolic acid at a concentration of 50 mg/kg. The results indicated that BUS caused the loss of spermatogenic cells in testis, the decrease of sperm in epididymis, the disorder of testicular cytoskeleton, the decrease of serum sex hormones such as testosterone which induced an increase in feedback of androgen receptor and other testosterone-related proteins, the increase of malondialdehyde and reactive oxygen species levels and the increase of ferroptosis in testis while UNA successfully reversed these injuries. High-throughput sequencing revealed that UNA administration significantly upregulated the expression of genes associated with spermatogenesis, such as Tnp1, Tnp2, Prm1, among others. These proteins are crucial in the histone to protamine transition during sperm chromatin remodeling. Network pharmacology analysis reveals a close association between UNA and proteins related to the transformation of histones to protamine. Molecular docking studies reveal that UNA can interact with the ferroptosis-inhibiting gene SLC7A11, thereby modulating ferroptosis. Taken together, UNA alleviated BUS-induced oligozoospermia by regulating hormone secretion, mitigating oxidative stress and promoting recovery of spermatogenesis by inhibiting the ferroptosis.

Molecular and Functional Characterization of Human Sex-Determining Region on the Y Chromosome Variants Using Protamine 1 Promoter.

The male sex-determining gene, sex-determining region on the Y chromosome (SRY), is expressed in adult testicular germ cells; however, its role in regulating spermatogenesis remains unclear. The role of SRY in the postmeiotic gene expression was investigated by determining the effect of SRY on the promoter of the haploid-specific Protamine 1 (PRM1) gene, which harbors five distinct SRY-binding motifs. In a luciferase reporter assay system, SRY upregulates PRM1 promoter activity in vitro in a dose-dependent manner. Through a gel-shift assay involving a 31-bp DNA fragment encompassing the SRY element within the PRM1 promoter, the third SRY-binding site on the sense strand (-373/-367) was identified as crucial for PRM1 promoter activation. This assay was extended to analyze 9 SRY variants found in the testicular DNA of 44 azoospermia patients. The findings suggest that SRY regulates PRM1 promoter activity by directly binding to its specific motif within the PRM1 promoter.

Phase-separated CCER1 coordinates the histone-to-protamine transition and male fertility.

Idiopathic fertility disorders are associated with mutations in various genes. Here, we report that coiled-coil glutamate-rich protein 1 (CCER1), a germline-specific and intrinsically disordered protein (IDP), mediates postmeiotic spermatid differentiation. In contrast, CCER1 deficiency results in defective sperm chromatin compaction and infertility in mice. CCER1 increases transition protein (Tnp1/2) and protamine (Prm1/2) transcription and mediates multiple histone epigenetic modifications during the histone-to-protamine (HTP) transition. Immiscible with heterochromatin in the nucleus, CCER1 self-assembles into a polymer droplet and forms a liquid-liquid phase-separated condensate in the nucleus. Notably, we identified loss-of-function (LoF) variants of human CCER1 (hCCER1) in five patients with nonobstructive azoospermia (NOA) that were absent in 2713 fertile controls. The mutants led to premature termination or frameshift in CCER1 translation, and disrupted condensates in vitro. In conclusion, we propose that nuclear CCER1 is a phase-separated condensate that links histone epigenetic modifications, HTP transitions, chromatin condensation, and male fertility.

Epigenetic aging of mammalian gametes.

The process of aging refers to physiological changes that occur to an organism as time progresses and involves changes to DNA, proteins, metabolism, cells, and organs. Like the rest of the cells in the body, gametes age, and it is well established that there is a decline in reproductive capabilities in females and males with aging. One of the major pathways known to be involved in aging is epigenetic changes. The epigenome is the multitude of chemical modifications performed on DNA and chromatin that affect the ability of chromatin to be transcribed. In this review, we explore the effects of aging on female and male gametes with a focus on the epigenetic changes that occur in gametes throughout aging. Quality decline in oocytes occurs at a relatively early age. Epigenetic changes constitute an important part of oocyte aging. DNA methylation is reduced with age, along with reduced expression of DNA methyltransferases (DNMTs). Histone deacetylases (HDAC) expression is also reduced, and a loss of heterochromatin marks occurs with age. As a consequence of heterochromatin loss, retrotransposon expression is elevated, and aged oocytes suffer from DNA damage. In sperm, aging affects sperm number, motility and fecundity, and epigenetic changes may constitute a part of this process. 5 methyl-cytosine (5mC) methylation is elevated in sperm from aged men, but methylation on Long interspersed nuclear elements (LINE) elements is reduced. Di and trimethylation of histone 3 lysine 9 (H3K9me2/3) is reduced in sperm from aged men and trimethylation of histone 3 lysine 27 (H3K27me3) is elevated. The protamine makeup of sperm from aged men is also changed, with reduced protamine expression and a misbalanced ratio between protamine proteins protamine P1 and protamine P2. The study of epigenetic reproductive aging is recently gaining interest. The current status of the field suggests that many aspects of gamete epigenetic aging are still open for investigation. The clinical applications of these investigations have far-reaching consequences for fertility and sociological human behavior.

DNA groove preference shift upon phosphorylation of a protamine-like cationic peptide.

Protamines, arginine-rich DNA-binding proteins, are responsible for chromatin compaction in sperm cells, but their DNA groove preference, major or minor, is not clearly identified. We herein study the DNA groove preference of a short protamine-like cationic peptide before and after phosphorylation, using all-atom molecular dynamics and umbrella sampling simulations. According to various thermodynamic and structural analyses, a peptide in its non-phosphorylated native state prefers the minor groove over the major groove, but phosphorylation of the peptide bound to the minor groove not only reduces its binding affinity but also brings a serious deformation of the minor groove, eliminating the minor-groove preference. As protamines are heavily phosphorylated before binding to DNA, we expect that the structurally disordered phosphorylated protamines would prefer major grooves to enter into DNA during spermatogenesis.

Protamine folds DNA into flowers and loop stacks.

DNA in sperm undergoes an extreme compaction to almost crystalline packing levels. To produce this dense packing, DNA is dramatically reorganized in minutes by protamine proteins. Protamines are positively charged proteins that coat negatively charged DNA and fold it into a series of toroids. The exact mechanism for forming these ∼50-kbp toroids is unknown. Our goal is to study toroid formation by starting at the "bottom" with folding of short lengths of DNA that form loops and working "up" to more folded structures that occur on longer length scales. We previously measured folding of 200-300 bp of DNA into a loop. Here, we look at folding of intermediate DNA lengths (L = 639-3003 bp) that are 2-10 loops long. We observe two folded structures besides loops that we hypothesize are early intermediates in the toroid formation pathway. At low protamine concentrations (∼0.2 µM), we see that the DNA folds into flowers (structures with multiple loops that are positioned so they look like the petals of a flower). Folding at these concentrations condenses the DNA to 25% of its original length, takes seconds, and is made up of many small bending steps. At higher protamine concentrations (≥2 µM), we observe a second folded structure-the loop stack-where loops are stacked vertically one on top of another. These results lead us to propose a two-step process for folding at this length scale: 1) protamine binds to DNA, bending it into loops and flowers, and 2) flowers collapse into loop stacks. These results highlight how protamine uses a bind-and-bend mechanism to rapidly fold DNA, which may be why protamine can fold the entire sperm genome in minutes.

Sperm chromatin structure and reproductive fitness are altered by substitution of a single amino acid in mouse protamine 1.

Conventional dogma presumes that protamine-mediated DNA compaction in sperm is achieved by electrostatic interactions between DNA and the arginine-rich core of protamines. Phylogenetic analysis reveals several non-arginine residues conserved within, but not across species. The significance of these residues and their post-translational modifications are poorly understood. Here, we investigated the role of K49, a rodent-specific lysine residue in protamine 1 (P1) that is acetylated early in spermiogenesis and retained in sperm. In sperm, alanine substitution (P1(K49A)) decreases sperm motility and male fertility-defects that are not rescued by arginine substitution (P1(K49R)). In zygotes, P1(K49A) leads to premature male pronuclear decompaction, altered DNA replication, and embryonic arrest. In vitro, P1(K49A) decreases protamine-DNA binding and alters DNA compaction and decompaction kinetics. Hence, a single amino acid substitution outside the P1 arginine core is sufficient to profoundly alter protein function and developmental outcomes, suggesting that protamine non-arginine residues are essential for reproductive fitness.

Ability of a selfish B chromosome to evade genome elimination in the jewel wasp, Nasonia vitripennis.

B chromosomes are non-essential, extra chromosomes that can exhibit transmission-enhancing behaviors, including meiotic drive, mitotic drive, and induction of genome elimination, in plants and animals. A fundamental but poorly understood question is what characteristics allow B chromosomes to exhibit these extraordinary behaviors. The jewel wasp, Nasonia vitripennis, harbors a heterochromatic, paternally transmitted B chromosome known as paternal sex ratio (PSR), which causes complete elimination of the sperm-contributed half of the genome during the first mitotic division of fertilized embryos. This genome elimination event may result from specific, previously observed alterations of the paternal chromatin. Due to the haplo-diploid reproduction of the wasp, genome elimination by PSR causes female-destined embryos to develop as haploid males that transmit PSR. PSR does not undergo self-elimination despite its presence with the paternal chromatin until the elimination event. Here we performed fluorescence microscopic analyses aimed at understanding this unexplained property. Our results show that PSR, like the rest of the genome, participates in the histone-to-protamine transition, arguing that PSR does not avoid this transition to escape self-elimination. In addition, PSR partially escapes the chromatin-altering activity of the intracellular bacterium, Wolbachia, demonstrating that this ability to evade chromatin alteration is not limited to PSR's own activity. Finally, we observed that the rDNA locus and other unidentified heterochromatic regions of the wasp's genome also seem to evade chromatin disruption by PSR, suggesting that PSR's genome-eliminating activity does not affect heterochromatin. Thus, PSR may target an aspect of euchromatin to cause genome elimination.

Arginine-rich peptides as crystallization inhibitors for sodium urate.

Inhibiting the formation of urate crystals is the key to prevent hyperuricemia from developing into gout. Although many studies have focused on the influence of biomacromolecules in the crystallization behavior of sodium urate, the role of peptides with specific structures may contribute to unprecedented regulatory effects. Here, for the first time, we studied the effects of cationic peptides on the phase behavior, crystallization kinetics, and size/morphology of urate crystals. The addition of protamine (PRTM, a typical natural arginine-rich peptide) prolongs the nucleation induction time of sodium urate and inhibits crystal nucleation effectively. PRTM binds to the surface of amorphous sodium urate (ASU) through the hydrogen bond and electrostatic attraction between guanidine groups and urate anions, which is conducive to maintaining the state of ASU and inhibiting crystal nucleation. Moreover, PRTM preferentially binds to the MSUM plane and leads to a significant reduction in the aspect ratio of MSUM filamentous crystals. Further studies showed that there are significant differences in the inhibiting effects of arginine-rich peptides with different chain lengths on the crystallization behavior of sodium urate. Both guanidine functional groups and peptide chain length determine the crystallization inhibiting effect of peptides simultaneously. The present work highlights the potential role of arginine peptides in inhibiting the crystallization of urate and provides new insights into the inhibition mechanism in the pathological biomineralization of sodium urate, demonstrating the possibility of using cationic peptides to treat gout.

Possible Molecular Mechanisms Underlying the Decrease in the Antibacterial Activity of Protamine-like Proteins after Exposure of Mytilus galloprovincialis to Chromium and Mercury.

Natural bioactive compounds represent a new frontier of antimicrobial molecules, and the marine ecosystem represents a new challenge in this regard. In the present work, we evaluated the possibility of changes in the antibacterial activity of protamine-like (PL) proteins, the major nuclear basic protein components of Mytilus galloprovincialis sperm chromatin, after the exposure of mussels to subtoxic doses of chromium (VI) (1, 10, and 100 nM) and mercury (1, 10, and 100 pM) HgCl2, since these metals affect some properties of PL. After exposure, we analyzed the electrophoretic pattern of PLs by both acetic acid-urea polyacrylamide gel electrophoresis (AU-PAGE) and SDS-PAGE and determined the MIC and MBC of these proteins on different gram+ and gram- bacteria. PLs, particularly after mussels were exposed to the highest doses of chromium and mercury, showed significantly reduced antibacterial activity. Just at the highest doses of exposure to the two metals, changes were found in the electrophoretic pattern of PLs, suggesting that there were conformational changes in these proteins, which were confirmed by the fluorescence measurements of PLs. These results provide the first evidence of a reduction in the antibacterial activity of these proteins following the exposure of mussels to these metals. Based on the results, hypothetical molecular mechanisms that could explain the decrease in the antibacterial activity of PLs are discussed.

Derepression of Y-linked multicopy protamine-like genes interferes with sperm nuclear compaction in D. melanogaster.

Across species, sperm maturation involves the dramatic reconfiguration of chromatin into highly compact nuclei that enhance hydrodynamic ability and ensure paternal genomic integrity. This process is mediated by the replacement of histones by sperm nuclear basic proteins, also referred to as protamines. In humans, a carefully balanced dosage between two known protamine genes is required for optimal fertility. However, it remains unknown how their proper balance is regulated and how defects in balance may lead to compromised fertility. Here, we show that a nucleolar protein, modulo, a homolog of nucleolin, mediates the histone-to-protamine transition during Drosophila spermatogenesis. We find that modulo mutants display nuclear compaction defects during late spermatogenesis due to decreased expression of autosomal protamine genes (including Mst77F) and derepression of Y-linked multicopy Mst77F homologs (Mst77Y), leading to the mutant's known sterility. Overexpression of Mst77Y in a wild-type background is sufficient to cause nuclear compaction defects, similar to modulo mutant, indicating that Mst77Y is a dominant-negative variant interfering with the process of histone-to-protamine transition. Interestingly, ectopic overexpression of Mst77Y caused decompaction of X-bearing spermatids nuclei more frequently than Y-bearing spermatid nuclei, although this did not greatly affect the sex ratio of offspring. We further show that modulo regulates these protamine genes at the step of transcript polyadenylation. We conclude that the regulation of protamines mediated by modulo, ensuring the expression of functional ones while repressing dominant-negative ones, is critical for male fertility.

DOT1L promotes spermatid differentiation by regulating expression of genes required for histone-to-protamine replacement.

Unique chromatin remodeling factors orchestrate dramatic changes in nuclear morphology during differentiation of the mature sperm head. A crucial step in this process is histone-to-protamine exchange, which must be executed correctly to avoid sperm DNA damage, embryonic lethality and male sterility. Here, we define an essential role for the histone methyltransferase DOT1L in the histone-to-protamine transition. We show that DOT1L is abundantly expressed in mouse meiotic and postmeiotic germ cells, and that methylation of histone H3 lysine 79 (H3K79), the modification catalyzed by DOT1L, is enriched in developing spermatids in the initial stages of histone replacement. Elongating spermatids lacking DOT1L fail to fully replace histones and exhibit aberrant protamine recruitment, resulting in deformed sperm heads and male sterility. Loss of DOT1L results in transcriptional dysregulation coinciding with the onset of histone replacement and affecting genes required for histone-to-protamine exchange. DOT1L also deposits H3K79me2 and promotes accumulation of elongating RNA Polymerase II at the testis-specific bromodomain gene Brdt. Together, our results indicate that DOT1L is an important mediator of transcription during spermatid differentiation and an indispensable regulator of male fertility.

A Molecular Mechanism to Explain the Nickel-Induced Changes in Protamine-like Proteins and Their DNA Binding Affecting Sperm Chromatin in Mytilus galloprovincialis: An In Vitro Study.

Nickel is associated with reproductive toxicity, but little is known about the molecular mechanisms of nickel-induced effects on sperm chromatin and protamine-like proteins (PLs). In the present work, we analyzed PLs from Mytilus galloprovincialis by urea-acetic acid polyacrylamide gel electrophoresis (AU-PAGE) and SDS-PAGE and assessed their binding to DNA by Electrophoretic Mobility Shift Assay (EMSA) after exposing mussels to 5, 15, and 35 µM NiCl2 for 24 h. In addition, a time course of digestion with MNase and release of PLs from sperm nuclei by the NaCl gradient was performed. For all exposure doses, in AU-PAGE, there was an additional migrating band between PL-III and PL-IV, corresponding to a fraction of PLs in the form of peptides detected by SDS-PAGE. Alterations in DNA binding of PLs were observed by EMSA after exposure to 5 and 15 µM NiCl2, while, at all NiCl2 doses, increased accessibility of MNase to sperm chromatin was found. The latter was particularly relevant at 15 µM NiCl2, a dose at which increased release of PLII and PLIII from sperm nuclei and the highest value of nickel accumulated in the gonads were also found. Finally, at all exposure doses, there was also an increase in PARP expression, but especially at 5 µM NiCl2. A possible molecular mechanism for the toxic reproductive effects of nickel in Mytilus galloprovincialis is discussed.

Gradient Nanoconfinement Facilitates Binding of Transcriptional Factor NF-κB to Histone- and Protamine-DNA Complexes.

Mechanically induced chromosome reorganization plays important roles in transcriptional regulation. However, the interplay between chromosome reorganization and transcription activities is complicated, such that it is difficult to decipher the regulatory effects of intranuclear geometrical cues. Here, we simplify the system by introducing DNA, packaging proteins (i.e., histone and protamine), and transcription factor NF-κB into a well-defined fluidic chip with changing spatical confinement ranging from 100 to 500 nm. It is uncovered that strong nanoconfinement suppresses higher-order folding of histone- and protamine-DNA complexes, the fracture of which exposes buried DNA segments and causes increased quantities of NF-κB binding to the DNA chain. Overall, these results reveal a pathway of how intranuclear geometrical cues alter the open/closed state of a DNA-protein complex and therefore affect transcription activities: i.e., NF-κB binding.

Nucleosomes in mammalian sperm: conveying paternal epigenetic inheritance or subject to reprogramming between generations?

The genome of mammalian sperm is largely packaged by sperm-specific proteins termed protamines. The presence of some residual nucleosomes has, however, emerged as a potential source of paternal epigenetic inheritance between generations. Sperm nucleosomes bear important regulatory histone marks and locate at gene-regulatory regions, functional elements, and intergenic regions. It is unclear whether sperm nucleosomes are retained at specific genomic locations in a deterministic manner or are randomly preserved due to inefficient exchange of histones by protamines. Recent studies indicate heterogeneity in chromatin packaging within sperm populations and an extensive reprogramming of paternal histone marks post fertilization. Obtaining single-sperm nucleosome distributions is fundamental to estimating the potential of sperm-borne nucleosomes in instructing mammalian embryonic development and in the transmission of acquired phenotypes.

Effect of hubble-bubble smoking on global DNA methylation and transcription levels of protamine and histone genes in human spermatozoa.

This study was conducted to assess the impact of hubble-bubble smoking on global DNA methylation, DNA fragmentation; protamine deficiency of spermatozoa, and to determine whether the transcription levels of the protamine and histone genes are different in hubble-bubble smokers compared to nonsmokers. Five hundred semen samples were collected from males with an average age of 32.2 ± 6.1 years (300 hubble-bubble smokers "60%" and 200 nonsmokers "40%"). The nucleic acid was isolated from purified sperm, then ELISA and qPCR were used to evaluate the global DNA methylation and transcription level of protamine and histone, respectively. A significant elevation in global DNA methylation, protamine deficiency, and DNA fragmentation was found in hubble-bubble smokers compared to nonsmokers (P < 0.0001). A significant decline was shown in transcription levels of protamine and histone genes in hubble-bubble compared to nonsmokers (P < 0.0001). Additionally, a down-regulation in the transcription levels of protamine and histone was revealed in hubble-bubble compared to nonsmokers with fold change (0.0001 and 0.007, respectively). In conclusion, this study provided proof that hubble-bubble smoking has a negative impact on global DNA methylation, DNA fragmentation, protamine deficiency, and the transcription of protamine and histone genes in spermatozoa, and these findings influence negatively males' fecundity.

Hydrozoan sperm-specific SPKK motif-containing histone H2B variants stabilise chromatin with limited compaction.

Many animals achieve sperm chromatin compaction and stabilisation by replacing canonical histones with sperm nuclear basic proteins (SNBPs) such as protamines during spermatogenesis. Hydrozoan cnidarians and echinoid sea urchins lack protamines and have evolved a distinctive family of sperm-specific histone H2Bs (spH2Bs) with extended N termini rich in SPK(K/R) motifs. Echinoid sperm packaging is regulated by spH2Bs. Their sperm is negatively buoyant and fertilises on the sea floor. Hydroid cnidarians undertake broadcast spawning but their sperm properties are poorly characterised. We show that Hydractinia echinata and H. symbiolongicarpus sperm chromatin possesses higher stability than somatic chromatin, with reduced accessibility to transposase Tn5 integration and to endonucleases in vitro. In contrast, nuclear dimensions are only moderately reduced in mature Hydractinia sperm. Ectopic expression of spH2B in the background of H2B.1 knockdown results in downregulation of global transcription and cell cycle arrest in embryos, without altering their nuclear density. Taken together, SPKK-containing spH2B variants act to stabilise chromatin and silence transcription in Hydractinia sperm with only limited chromatin compaction. We suggest that spH2Bs could contribute to sperm buoyancy as a reproductive adaptation.

Protamine 1 as a secreted colorectal cancer-specific antigen facilitating G1/S phase transition under nutrient stress conditions.

PURPOSE: Cancer testis antigens (CTAs) are optimal tumor diagnostic markers and involved in carcinogenesis. However, colorectal cancer (CRC) related CTAs are less reported with impressive diagnostic capability or relevance with tumor metabolism rewiring. Herein, we demonstrated CRC-related CTA, Protamine 1 (PRM1), as a promising diagnostic marker and involved in regulation of cellular growth under nutrient deficiency. METHODS: Transcriptomics of five paired CRC tissues was used to screen CRC-related CTAs. Capability of PRM1 to distinguish CRC was studied by detection of clinical samples through enzyme linked immunosorbent assay (ELISA). Cellular functions were investigated in CRC cell lines through in vivo and in vitro assays. RESULTS: By RNA-seq and detection in 824 clinical samples from two centers, PRM1 expression were upregulated in CRC tissues and patients` serum. Serum PRM1 showed impressive accuracy to diagnose CRC from healthy controls and benign gastrointestinal disease patients, particularly more sensitive for early-staged CRC. Furthermore, we reported that when cells were cultured in serum-reduced medium, PRM1 secretion was upregulated, and secreted PRM1 promoted CRC growth in culture and in mice. Additionally, G1/S phase transition of CRC cells was facilitated by PRM1 protein supplementation and overexpression via activation of PI3K/AKT/mTOR pathway in serum deficient medium. CONCLUSIONS: In general, our research presented PRM1 as a specific CRC antigen and illustrated the importance of PRM1 in CRC metabolism rewiring. The new vulnerability of CRC cells was also provided with the potential to be targeted in future. Diagnostic value and grow factor-like biofunction of PRM1 A represents the secretion process of PRM1 regulated by nutrient deficiency. B represents activation of PI3K/AKT/mTOR pathway of secreted PRM1.

Carob extract induces spermatogenesis in an infertile mouse model via upregulation of Prm1, Plzf, Bcl-6b, Dazl, Ngn3, Stra8, and Smc1b.

ETHNOPHARMACOLOGICAL RELEVANCE: Ethnopharmacological studies for drug discovery from natural compounds play an important role for developing current therapeutical platforms. Plants are a group of natural sources which have been served as the basis in the treatment of many diseases for centuries. In this regard, Ceratonia siliqua (carob) is one of the herbal medicine which is traditionally used for male infertility treatments. But so far the main mechanisms for effects of carob are unknown. Here, we intend to investigate the ability of carob extract to induce spermatogenesis in an azoospermia mouse model and determine the mechanisms that underlie its function. AIM OF THE STUDY: This is a pre-clinical animal model study to evaluate the effect of carob extract in spermatogenesis recovery. METHODS: We established an infertile mouse model with the intent to examine the ability of carob extract as a potential herbal medicine for restoration of male fertility. Sperm parameters, as well as gene expression dynamics and levels of spermatogenesis hormones, were evaluated 35 days after carob administration. RESULTS: Significant enhanced sperm parameters (P < 0.05) showed that the carob extract could induce spermatogenesis in the infertile mouse model. Our data suggested an anti-apototic and inducer role in the expressions of cell cycle regulating genes. Carob extract improved the spermatogenesis niche by considerable affecting Sertoli and Leydig cells (P < 0.05). The carob-treated mice were fertile and contributed to healthy offspring that matured. Our data confirmed that this extract triggered the hormonal system, the spermatogenesis-related gene expression network, and signaling pathways to induce and promote sperm production with notable level (P < 0.05). We found that the aqueous extract consisted of a polar and mainly well water-soluble substance. Carob extract might upregulate spermatogenesis hormones via its amino acid components, which were detected in the extract by liquid chromatography-mass spectrometry (LC-MS). CONCLUSION: Our results strongly suggest that carob extract might be a promising future treatment option for male infertility. This finding could pave the way for clinical trials in infertile men. This is the first study that has provided reliable, strong pre-clinical evidence for carob extract as an effective candidate for fertility recovery in cancer-related azoospermia.