The story begins with a simple observation. When the researchers profiled rat testicular tissue at 2, 6, 12, and 18 months of age, they found that both overall DNA methyltransferase activity and circulating testosterone levels followed strikingly parallel trajectories, with a pronounced concurrent decline beginning at 12 months. The enzymes that actually build testosterone—CYP11A1, CYP17A1, and the cholesterol transporter StAR—mirrored this drop precisely. Intriguingly, when the team mined existing human multi-tissue transcriptomic datasets, they discovered that DNMT1, the principal maintenance methyltransferase responsible for preserving genomic methylation patterns during cell division, is preferentially and abundantly expressed in the testis compared with 31 other human tissues. This positioned DNMT1 as a prime candidate regulator of testicular function, and indeed, complementary RT-qPCR, Western blotting, and immunohistochemical staining all confirmed a progressive age-related decline in DNMT1 expression in rat testes.

To isolate the cell-intrinsic relationship between DNMT1 and testosterone production without the confounding influences of the living organism, the researchers established an in vitro aging model using primary Leydig cells—the specialized interstitial cells that serve as the testis’s sole source of testosterone. Isolated from juvenile rats and serially passaged to induce replicative senescence, these cells developed a robust aged phenotype, with more than 70 percent staining positive for senescence-associated β-galactosidase. They also upregulated the senescence marker p16 and secreted elevated levels of inflammatory SASP factors including TNF-α, IL-1β, and IL-6. Crucially, these senescent Leydig cells produced markedly less testosterone and expressed significantly less DNMT1, recapitulating the age-related declines observed in living animals and supporting DNMT1 as a cell-intrinsic correlate of Leydig cell functional aging.

Gain- and loss-of-function experiments then established DNMT1 as a genuine positive regulator of steroidogenesis rather than a mere bystander. When the researchers knocked down DNMT1 using lentiviral shRNA, testosterone secretion into the culture medium plummeted, and the protein expression of CYP11A1, CYP17A1, and StAR all fell in concert. Conversely, overexpressing DNMT1 produced the reciprocal phenotype, elevating testosterone output and upregulating the same rate-limiting enzymes. DNMT1 knockdown also drove Leydig cells toward senescence, increasing the proportion of β-galactosidase-positive cells and upregulating p16 and SASP factors. Overexpression of DNMT1 in already-senescent cells reversed this program, reducing senescence markers and restoring testosterone secretion. Together, these findings position DNMT1 as an epigenetic safeguard against the functional decay of the aging testis.

The real surprise came when the team performed RNA sequencing on DNMT1-deficient Leydig cells to identify what DNMT1 actually controls. Among the altered transcripts, the mRNA encoding FTO—the fat mass and obesity-associated protein, better known as a demethylase that erases N6-methyladenosine (m6A) marks from messenger RNA—was significantly upregulated when DNMT1 was silenced. Methylation-specific PCR revealed that the Fto promoter became significantly hypomethylated following DNMT1 knockdown, and chromatin immunoprecipitation coupled with qPCR confirmed that DNMT1 physically binds to the CpG island within the Fto promoter, enriching approximately 398-fold over a control antibody. In other words, DNMT1 normally keeps FTO switched off by methylating its promoter, and when DNMT1 levels fall with age, FTO is derepressed and begins to accumulate.

FTO, it turns out, is not a benign consequence of DNMT1 loss but an active agent of decline. When the researchers overexpressed FTO in Leydig cells, testosterone production dropped and the steroidogenic enzymes CYP11A1, CYP17A1, and StAR were all downregulated—phenocopying the effect of DNMT1 knockdown. Dual knockdown experiments then established the direction of causality: silencing FTO in DNMT1-deficient cells rescued testosterone production and restored enzyme expression, whereas silencing DNMT1 in FTO-deficient cells produced the opposite outcome. Notably, FTO overexpression did not feed back to alter DNMT1 levels, confirming that the regulatory axis is unidirectional, with DNMT1 upstream and FTO downstream.

To find FTO’s actual target, the team turned to computational prediction and RNA immunoprecipitation. Using the catRAPID protein-RNA interaction database, they identified Hsp90aa1—the gene encoding heat shock protein 90α, a molecular chaperone that stabilizes and activates the pro-survival kinase AKT—as the transcript with the highest binding propensity for FTO. RIP-qPCR using an FTO-specific antibody confirmed that Hsp90aa1 mRNA is a direct binding substrate of FTO. MeRIP-qPCR then showed that FTO overexpression reduced the m6A modification level of Hsp90aa1 transcripts, and this demethylation was accompanied by downregulation of Hsp90aa1 mRNA and HSP90AA1 protein. Conversely, FTO knockdown elevated both m6A marks and HSP90AA1 abundance. Importantly, DNMT1 knockdown did not alter the DNA methylation status of the Hsp90aa1 promoter, yet it still reduced HSP90AA1 expression—and this reduction was rescued by concurrent FTO knockdown. The message is clear: DNMT1 regulates HSP90AA1 indirectly, through the FTO-mediated m6A axis, rather than through direct promoter methylation.

The downstream consequences of losing HSP90AA1 are what ultimately kill testosterone production. HSP90AA1 overexpression increased testosterone output and upregulated the steroidogenic enzymes, while simultaneously raising the levels of phosphorylated AKT and the anti-apoptotic protein BCL-2 and lowering the pro-apoptotic protein BAX. Pharmacological inhibition of HSP90AA1 with 17-AAG produced the opposite effects, reducing testosterone secretion, suppressing AKT phosphorylation, and tipping the balance toward cell death. Rescue experiments confirmed the hierarchical architecture of the pathway: FTO knockdown reversed the inhibitory effects of DNMT1 deficiency on HSP90AA1 expression, AKT phosphorylation, and testosterone production, whereas HSP90AA1 overexpression rescued the functional impairments induced by FTO overexpression. TUNEL staining corroborated these molecular findings, confirming that the DNMT1–FTO–HSP90AA1–AKT axis exerts hierarchical control over Leydig cell apoptosis.

When the researchers examined how this axis behaves during natural aging, the picture became even more coherent. In testicular tissue from 18-month-old rats, the Fto promoter was significantly hypomethylated compared with 2-month-old controls, FTO binding to Hsp90aa1 mRNA was markedly enhanced, and the m6A modification level of Hsp90aa1 transcripts was significantly reduced. FTO expression rose progressively with age while HSP90AA1 fell, and immunohistochemical staining revealed elevated FTO, reduced HSP90AA1, and increased pro-apoptotic Caspase-3 in aged testicular sections. TUNEL staining verified a significant age-dependent increase in apoptotic cells, and Western blot analysis showed that phosphorylated AKT and BCL-2 were significantly decreased while BAX was markedly upregulated. Senescent Leydig cells in vitro recapitulated every one of these molecular alterations, and primary Leydig cells isolated from aged rats displayed the same reduced Fto promoter methylation, reduced m6A modification of Hsp90aa1, and impaired AKT signaling.

To validate the pathway in a living organism, the team delivered adeno-associated virus serotype 9 carrying a Dnmt1-targeting construct directly into rat testes via local intratesticular injection. Three weeks later, serum testosterone levels were significantly reduced, the steroidogenic enzymes CYP11A1, CYP17A1, and StAR were downregulated, Fto promoter methylation was decreased, FTO was upregulated, HSP90AA1 was downregulated, and apoptotic cells within the testicular interstitium were markedly increased. Phosphorylated AKT and BCL-2 fell while BAX rose, and senescence markers including p16 and SASP factors were elevated. These in vivo observations mirror the in vitro findings almost exactly, confirming that the DNMT1–FTO–HSP90AA1–AKT cascade operates as a genuine regulatory circuit in the aging testis rather than an artifact of cell culture.

The implications of this work extend well beyond basic reproductive biology. Testosterone replacement therapy, currently the most widely applied intervention for age-related hypogonadism, carries notable contraindications and adverse effects including polycythemia, cardiovascular events, and prostate-related morbidity, and its efficacy remains suboptimal. The DNMT1–FTO–HSP90AA1 axis offers a mechanistic alternative: rather than supplying testosterone from outside the body, it suggests that pharmacological strategies aimed at preserving DNMT1 activity, restraining FTO expression, or sustaining HSP90AA1-dependent AKT signaling could enhance endogenous testosterone synthesis from within. Selective small-molecule inhibitors targeting DNMT1 and m6A modifiers provide feasible therapeutic entry points, and the identification of Leydig cells as among the earliest testicular cell types to undergo senescence makes them a particularly attractive target for interventions designed to postpone reproductive aging. The authors acknowledge several limitations, including the fact that the precise m6A residues on Hsp90aa1 transcripts have not yet been mapped, that the MSP assay lacked certain positive controls, and that the findings have not yet been validated in human testicular samples. Nevertheless, the study establishes a comprehensive molecular model in which the slow erosion of a single epigenetic enzyme—DNMT1—triggers a cascade of RNA-level changes that ultimately silence the testis’s capacity to produce its own testosterone, and it identifies the DNMT1–FTO–HSP90AA1–AKT axis as a promising therapeutic target for enhancing endogenous steroidogenesis during aging.

Subject of Research: Epigenetics and Reproductive Aging

Article Title: DNMT1 Regulates Testosterone Synthesis in Aged Rat Leydig Cells via FTO‐Mediated HSP90AA1 m6A Levels

Article References: Li, Y., Duan, Y., Tian, Z., Gao, Y., Qiao, Y., Zhao, S., Li, W., Gao, Z., & Niu, S. (2026). DNMT1 Regulates Testosterone Synthesis in Aged Rat Leydig Cells via FTO ‐Mediated HSP90AA1 m 6 A Levels. Aging Cell, 25(10), Article e70760. https://doi.org/10.1111/acel.70760

Image Credits: AI Generated

DOI: 10.1111/acel.70760

Keywords: DNMT1, FTO, HSP90AA1, m6A, testosterone, Leydig cells, epigenetics, aging, AKT signaling, reproductive biology, Regulates, Synthesis

Busulfan is a bifunctional alkylating agent widely used in clinical medicine, at low doses in prolonged regimens for chronic myeloid leukemia and ovarian cancer, and at high doses as a conditioning drug before bone marrow transplantation. Its therapeutic power comes with a notorious cost: the drug preferentially destroys rapidly dividing cells, and spermatogonia, the stem cells of sperm production, sit squarely in the blast zone. Previous work has shown that busulfan triggers oxidative apoptosis in spermatogonial stem cells, depletes the germinal epithelium of seminiferous tubules, and can leave cancer survivors with drastically reduced sperm counts. As cancer diagnoses rise globally and survival rates improve, preserving fertility in young male patients has become an increasingly urgent research priority.

Telfairia occidentalis, known locally as ugu, ewuroko, or ikong-ubong depending on the Nigerian region, is an edible leafy cucurbit whose seeds are consumed as snacks, soup condiments, and fermented seasonings. The plant is rich in essential oils, vitamins, minerals, amino acids, flavonoids such as quercetin and kaempferol, alkaloids, carotenes, and cucurbitacines, which together underpin its documented antioxidant, anti-inflammatory, antidiabetic, and antimicrobial properties. Earlier experiments had already hinted at reproductive benefits: fluted pumpkin seed oil reversed alcohol-induced germ cell loss in Sprague-Dawley rats, co-administration of the seeds with caffeine protected the spermatogenesis score index in Wistar rats, and seed fractions attenuated doxorubicin-induced testicular toxicity. The Port Harcourt team set out to dissect which chemical fractions of the seed drive which effects, using two parallel experimental designs over a 54-day treatment period.

The researchers harvested mature fluted pumpkin pods from a local market in Ogoni, Rivers State, verified the plant identity botanically, and macerated the dried, ground seeds in 60 percent aqueous ethanol. After concentrating the crude extract, they split it with a separation funnel into a hexane fraction enriched in non-polar lipids and a freeze-dried aqueous fraction rich in water-soluble compounds. In the injury study, adult male Wistar rats received busulfan at 15 milligrams per kilogram body weight, injected intraperitoneally once weekly for two weeks, alongside oral doses of the hexane fraction at 50, 100, or 200 milligrams per kilogram given twice weekly for the full 54 days. In the companion study, healthy rats received the aqueous fraction three days weekly at the same dose range, with corn oil serving as the vehicle control in both experiments. The 54-day window was chosen to span a full cycle of spermatogenesis, ensuring that any effect on sperm production could manifest.

The biochemical readouts painted a vivid picture of busulfan’s assault and the hexane fraction’s counteroffensive. Busulfan alone significantly shrank testis weight and the gonado-somatic index, spiked lipid peroxidation as measured by malondialdehyde levels, and raised the activities of catalase, superoxide dismutase, glutathione reductase, and glutathione S-transferase, a constellation of changes signaling severe oxidative stress and a disrupted glutathione redox balance. Testicular marker enzymes including acid and alkaline phosphatase and gamma-glutamyl transpeptidase, all of which climb when the seminiferous epithelium degenerates, were also elevated, while lactate dehydrogenase, essential for germ cell energy metabolism, fell. Co-treatment with the hexane fraction reversed virtually every one of these abnormalities in a dose-dependent manner, restoring glutathione status, quenching lipid peroxidation, normalizing enzyme activities, and recovering testis weight toward control levels, with the 200 milligram per kilogram dose showing the greatest efficacy.

Under the microscope, the contrast was equally striking. Control rats displayed intact seminiferous tubules brimming with the full cast of spermatogenic cells and tuffs of mature spermatozoa in the lumen. Busulfan left the tubules vacuolated, distorted, and stripped of spermatozoa and spermatids, a classic signature of maturation arrest. Animals given the hexane fraction showed markedly better tubular architecture, with intact basement membranes lined by spermatogonia and substantially less degeneration, although the authors note that some tubules still contained reduced germ cell layers, indicating partial rather than complete rescue. Stereological analysis with ImageJ software confirmed that the extract prevented the busulfan-induced collapse of tubular diameter and seminiferous epithelial height at all doses tested.

The aqueous fraction told a different, quieter story in healthy rats. After 54 days of treatment, seminiferous tubular diameter, luminal diameter, tubular cross-sectional area, tubular length per gram of tissue, epithelial thickness, and, most intriguingly, the number of testosterone-producing Leydig cells all increased in proportion to dose. Yet the Johnsen spermatogenesis score index and the counts of spermatogonia, spermatocytes, round spermatids, and Sertoli cells remained unchanged, as did body weight, absolute testis weight, and the gonado-somatic index. The researchers interpret this as an enhancement of the testis’s structural and steroidogenic infrastructure, potentially improving sperm passage and androgen synthesis, without an outright acceleration of sperm cell production.

Gas chromatography-mass spectrometry helped explain the divergent behaviors of the two fractions. The hexane fraction yielded 270 identified metabolites, 17 of them abundant, dominated by long-chain fatty acids and related lipids including squalene, n-hexadecanoic acid, octadecanoic acid, conjugated linoleic acid, alpha-linolenic acid, and an ascorbic acid derivative. Many of these lipids are known antioxidants, consistent with the fraction’s ability to blunt busulfan-driven oxidative injury. The aqueous fraction contained 277 metabolites, 14 of them abundant, including glycerin, a compound previously shown to modulate testicular androgen production and testicular morphology. The authors propose that the lipid repertoire underwrites protection against injury, while water-soluble constituents such as glycerin sculpt testicular stereology.

The team is candid about the limits of the work: the two fractions were tested in different animal models rather than head-to-head, and future studies should isolate individual bioactive compounds, test them in additional models of gonadal injury, and evaluate liver and kidney safety markers before any clinical translation. Still, the convergence of biochemical, histological, stereological, and metabolomic evidence makes a compelling case that a common Nigerian food seed contains separable chemical programs, one that shields the testis from chemotherapy’s collateral damage and another that may bolster its hormonal machinery. For a world in which more than a million new cancer cases are diagnosed annually and male infertility is a feared consequence of cure, that duality is precisely the kind of leads translational reproductive medicine has been searching for.

The decision to study seed fractions separately reflects a broader principle in pharmacognosy research. Crude plant extracts contain hundreds of compounds with varying solubilities, and pooling them can obscure which molecules are actually responsible for a given biological effect. By partitioning the aqueous ethanol extract between hexane and water, the Port Harcourt team effectively separated the seed’s lipid-soluble cargo from its water-soluble constituents, allowing each chemical repertoire to be evaluated on its own terms. This approach mirrors earlier work on other edible plants, where non-polar fractions rich in tocopherols, phytosterols, and unsaturated fatty acids often carry antioxidant activity, while polar fractions contribute different bioactivities.

The choice of busulfan as the injury model deserves note. Because the drug reliably depletes spermatogonial stem cells while sparing the somatic framework of the testis, it has become a standard tool for generating reproducible testicular damage in rodents. It is also used experimentally to condition recipients for spermatogonial stem cell transplantation, a technique being explored as a fertility-preservation strategy for prepubertal boys facing gonadotoxic therapy. Any compound that protects the germinal epithelium in a busulfan model is therefore of interest not only as a potential co-adjuvant during chemotherapy but also as a candidate for improving the efficiency of stem cell-based fertility restoration.

The stereological findings in healthy rats also carry implications for how such plant products should be interpreted. An increase in Leydig cell number without a corresponding rise in spermatogenic cell counts suggests that the aqueous fraction may be acting on the interstitial compartment, the site of testosterone synthesis, rather than on the seminiferous tubules directly. Since adequate intratesticular testosterone is required to maintain the blood-testis barrier and support later stages of sperm development, expanding the Leydig cell population could, in principle, strengthen the hormonal support system on which spermatogenesis depends, even if the immediate effect on germ cell numbers is neutral.

It remains important to temper enthusiasm appropriately. Rodent doses of 50 to 200 milligrams per kilogram are far higher, on a body-weight basis, than what a person would obtain from eating fluted pumpkin seeds as food, and the extract was administered as a concentrated fraction rather than a whole seed matrix. Absorption, metabolism, and distribution of the identified lipids and water-soluble compounds in humans may differ substantially from rats. Moreover, the absence of reported toxicity data in this study means that long-term safety, particularly at high doses, has not been established. These caveats notwithstanding, the study illustrates how a detailed fraction-by-fraction dissection of a traditional food plant can yield mechanistically grounded leads for protecting male reproductive health.

Subject of Research: Protective and enhancing effects of Telfairia occidentalis seed extract fractions on rat testicular function and busulfan-induced testicular injury

Article Title: Hexane fraction of Telfairia occidentalis (Cucurbitaceae) ethanol seed extract ameliorates busulfan-induced testicular damage while aqueous fraction enhances testis function in normal rats

Article References: Abarikwu, S. O., Erekeere, C. K., Timi-Johnson, E., Ogbonnaya, A. O., & Ezim, O. E. (2026). Hexane fraction of Telfairia occidentalis (Cucurbitaceae) ethanol seed extract ameliorates busulfan-induced testicular damage while aqueous fraction enhances testis function in normal rats. Discover Toxicology, 3(1), Article 16. https://doi.org/10.1007/s44339-026-00061-1

Image Credits: AI Generated

DOI: 10.1007/s44339-026-00061-1

Keywords: Telfairia occidentalis, fluted pumpkin seeds, busulfan, testicular damage, spermatogenesis, oxidative stress, Leydig cells, seminiferous tubules, male fertility, phytochemicals, testicular stereology, Discover Toxicology