Apoptosis leads to Decrease, Sperm count

The biological plausibility of this KER is strong, based on extensive mechanistic understanding of spermatogenesis and the role of apoptosis in regulating testicular cell populations.

Spermatogenesis is a continuous and highly coordinated process of germ cell proliferation and differentiation, leading to the production of mature spermatozoa. Apoptosis (programmed cell death) is an important mechanism that removes damaged cells and maintains tissue homeostasis (Shaha et al., 2010). During spermatogenesis, physiologic apoptosis occurs in all testicular cells (Oldereid et al., 2001). Meanwhile, stress-induced apoptosis can be triggered by exogenous stressors, such as environmental toxicants, heat, and radiation, which can markedly increase apoptotic cell death in testis (Shaha et al., 2010).

Mechanistically, the primary link between apoptosis and reduced sperm counts is the loss of developing male germ cells, which decreases the number of cells available to complete spermatogenesis and produce mature spermatozoa. However, apoptosis of testicular Sertoli and Leydig cells can also contribute to reduced sperm counts through indirectmechanisms, such as disrupting structural support and hormonal regulation of spermatogenesis.

Apoptosis in male germ cells

In the early stage of spermatogenesis, spermatogonial stem cells (SSCs) maintain the male germline through self-renewal, while a subset of SSCs commits to differentiation to produce mature sperm (Nakagawa et al., 2010). As long-term germ cell reservoirs, SSCs have enhanced DNA repair capacity, which renders them less sensitive to damage or stress-induced apoptosis compared with their differentiating progeny (Meistrich et al., 1982b; Rübe et al., 2011). However, excessive apoptosis of SSCs at higher doses can compromise the ability of the testis to replenish the pool of developing germ cells and cause sustained or irreversible impairment of spermatogenesis (Meistrich et al., 2013).

Differentiating spermatogonia are particularly sensitive to stress-induced apoptosis because they undergo rapid mitotic divisions (Meistrich et al., 1982a, 1982b, 2013). Apoptosis of differentiating spermatogonia results in a progressive loss of the more mature germ cells in a process known as maturation depletion (Meistrich et al., 2013). Although later-stage germ cells continue to develop, minimal replacement occurs from progenitor cells. Consequently, the pool of developing germ cells is depleted over time, leading to reductions in sperm counts that become evident after a delay. This delay reflects the time required for developing germ cells to complete spermatogenesis before the effects of progenitor cell depletion become apparent in mature sperm output. As a result, the timing of the decline in sperm counts is generally consistent with the duration of spermatogenesis in the species of interest (Meistrich et al., 1992).

Primary spermatocytes undergo extensive meiotic reorganization and division to generate haploid spermatids. Disruption of meiotic progression induces checkpoint mediated apoptosis to eliminate defective cells, particularly at the pachytene stage (Roeder and Bailis, 2000; Li et al., 2025). Compared with pre-meiotic germ cells, apoptosis in spermatocytes is often detected during meiotic progression rather than as an immediate response following exposure (Meistrich et al., 1982b, 2013). The subsequent reduction in sperm counts is usually temporary when SSCs survive, as these remaining stem cells rapidly replenish spermatogenic cells and restore sperm counts in ~45 days in mice and ~12 weeks in humans (Meistrich et al., 2013). 

After meiosis, spermatids differentiate into mature spermatozoa during spermiogenesis. At this stage, male germ cells undergo chromatin and morphological remodeling when the sperm genome becomes highly condensed and transcriptionally silenced (Aitken et al., 2010). In parallel, mitochondria are relocalized, either becoming confined to the sperm midpiece or transferred into residual bodies for degradation and elimination (Varuzhanyan and Chan, 2020). These structural changes limit the execution of canonical caspase-dependent apoptotic pathways. Instead, post-meiotic germ cells undergo truncated or abortive apoptosis, which is mediated primarily by reactive oxygen species and lipid peroxidation; the formation of lipid adducts leads to a rapid loss of sperm motility within hours, reflecting immediate functional loss (Aitken and Baker, 2013). Notably, these cells remain viable, and this form of apoptosis does not typically result in immediate DNA fragmentation and will not be detected by the TUNEL assay (Aitken and Baker, 2013). Accordingly, compared with apoptosis in early-stage germ cells, apoptosis occurring at post-meiotic stages is less likely to cause substantial germ cell loss and reduction in sperm counts despite causing functional defects.

Apoptosis in other testicular cells

In addition to germ cells, apoptosis can also happen in other testicular cells and indirectly influence the progression of spermatogenesis. Sertoli cells provide essential structural support in the seminiferous epithelium and play multiple roles in maintaining testicular functions, including supporting germ cell development, facilitating germ cell movement across the epithelium, enabling spermiation (release of mature spermatids), and secreting regulatory factors (Murphy and Richburg, 2015). As one Sertoli cell only has a finite supportive capacity, the size of Sertoli cell population is a key determinant of the total germ cell numbers and, indirectly, Leydig cell numbers in the testis (Rebourcet et al., 2017). Under physiological conditions, spontaneous apoptosis is an important mechanism to maintain the Sertoli cell to germ cell ratio. Toxicant-induced Sertoli cell apoptosis has been reported to cause germ cell loss through loss of structural and metabolic support, ultimately leading to reduced sperm output (Murphy and Richburg, 2015). Among germ cell populations, the developing spermatocytes are the most sensitive to Sertoli cell-selective apoptosis, while spermatogonia are relatively resistant (Murphy and Richburg, 2015). Leydig cells are responsible for testosterone synthesis, which is essential for multiple key processes during spermatogenesis, including germ cell survival and progression (Smith and Walker, 2014). Hormonal deprivation significantly increases the incidence of apoptotic germ cells, particularly late-stage spermatocytes that are androgen-dependent (Hikim et al., 1997). Excessive apoptosis in Leydig cells is strongly linked to lower intratesticular testosterone levels, and therefore can impair germ cell survival, indirectly leading to reduced sperm counts.

Together, these well-established mechanisms provide strong biological support for a causal relationship between increased apoptosis in testicular cells and reduced sperm counts.

The empirical support for this KER is strong. Concordant increases in apoptosis in testicular cells and reductions in sperm counts have been consistently observed across multiple in vivo rodent studies, and numerous intervention studies provide evidence of essentiality by demonstrating preservation or recovery of sperm counts following attenuation of apoptotic signaling. Temporal concordance is strongly supported by the well-established kinetics of spermatogenesis, although it is often inferred rather than directly measured within individual studies. Quantitative dose-response information is limited because many studies employ single exposure or intervention conditions. In addition, apoptosis frequently co-occurs with oxidative stress and inflammatory responses, which may contribute to spermatogenic impairment and complicate attribution to apoptosis alone. Nevertheless, these limitations do not detract from the overall consistency, biological coherence, and weight of evidence supporting this KER.

A large number of studies demonstrated the use of chemicals with anti-apoptotic, anti-inflammatory, and anti-oxidant properties to attenuate toxicant-induced testicular injury. These studies consistently show that attenuation of apoptosis is associated with recovery of sperm counts, providing supportive, albeit indirect, evidence for the essentiality of apoptosis. Since this is a data-rich field and the biological plausibility is strong, select empirical studies are presented as examples.

In a study by Yaman et al (2018), 30 day-old male Wistar albino rats were exposed to a single intraperitoneal dose of 5 mg/kg cisplatin. Half of the rats were euthanized at 31 days of age (pre-pubertal) and observed for the presence of testicular cell apoptosis; the other half was euthanized at 90 days (fertile age) to evaluate epididymal sperm counts. In exposed pre-pubertal rats, 107 ± 5.9 apoptotic (TUNEL-positive) cells were observed in 100 seminiferous tubule cross-sections, compared with only 24.6 ± 6.1 cells in control rats (p<0.001). The TUNEL+ cells were predominantlyspermatogonia and spermatocytes. Consistent with the expected temporal sequent, the average sperm counts measuredlater in adulthood were significantly reduced by ~54% (175 ± 67.1 million/mL in treated rats compared to 379.2 ± 45.9 million/mL observed in the controls) (Figure 1). Moreover, co-treatment with L-carnitine (250 mg/kg, for three consecutive days) significantly decreased the numbers of TUNEL+ cells in the tubules and resulted in a near-complete restoration of sperm counts (Figure 1). This study provides strong evidence of temporal concordance and indirect evidence of essentiality, as modulation of apoptosis was associated with recovery of sperm production.

Udefa et al. (2020) reported preventive effects of a tigernut plant extract on lead acetate-induced testicular damage in rats. Male Wistar rats were injected intraperitoneally with 20 mg/kg lead acetate once daily, with or without oral co-treatment with the extract (500 or 1000 mg/kg) for three weeks. Lead exposure significantly increased apoptosis in the testes, evidenced by pro-apoptotic shift in the Bax/Bcl-2 ratio (~7-fold increase) and elevated cleaved caspase-3 levels (~2.3-fold increase), indicating activation of pro-apoptotic pathways. Concordant decreases in serum and testicular testosterone levels suggested toxicity to Leydig cells. These changes were accompanied by a ~32% reduction in epididymal sperm counts. Co-treatment with the plant extract reversed changes in apoptotic markers and restored sperm counts in a dose-dependent manner. A negative correlation (r= -0.813, p<0.05) between the Bax/Bcl-2 ratio and sperm counts was reported, based on a small number of treatment group-level data. Since the Bax/Bcl-2 ratio is an indirect marker of apoptotic signaling, this correlation provides only limited quantitative support for a direct response-response relationship between the two KEs.

Oyovwi et al. (2023) investigated whether quercetin, a bioflavanol known for anti-oxidant and anti-apoptotic properties, could mitigate testicular dysfunction as a side effect of the antiepileptic drug levetiracetam (LEV). Male Wistar rats (10-12 weeks old) were exposed to saline or LEV (300 mg/kg/day) by oral gavage for 56 days, a period that covers a full cycle of spermatogenesis in rats. A separate group of rats were co-treated with quercetin (20 mg/kg/day) during days 28-56, administered 30-minute after each LEV dose. LEV exposure altered apoptosis-related factors in testicular tissues, including decreased expression of anti-apoptotic protein Bcl-2, and increased pro-apoptotic signaling. Specifically, the cytochrome c level in mitochondrial supernatant was increased by ~60%, while expression of caspase-3 and p53, and sperm DNA fragmentation index (indirectly predicted by aniline blue staining) showed about 2 to 3-fold increases relative to controls (Figure 2). These changes were accompanied by lower testicular weights, a ~30% decrease in epididymal sperm counts, and histological evidence of germ cell loss at all stages, along with decreases in Sertoli cell and Leydig cell numbers. Co-treatment with quercetin reversed all changes in apoptotic markers to control levels and prevented the LEV-induced reductions in testicular weights and sperm counts. This study provides high-quality evidence supporting essentiality of apoptosis in male germ cells.

In a mouse study examining the effects of X-ray radiation on male infertility, selenium nanoparticles (SeNPs) and a probiotic Lactobacillus casei were shown to mitigate testicular damage through anti-apoptotic and anti-oxidant mechanisms (Ehghaghi et al., 2022). Sixty-four Syrian male mice were divided into eight groups (n=8 per group) and orally treated with vehicle (PBS), SeNPs (0.2 mg/kg/day), probiotic (1 × 10⁸ CPU), or their combination. Four groups received whole body X-ray radiation (2 Gray for 5 minutes) after each daily oral dosing for 30 days, and the other four groups were used as non-irradiated controls. Apoptosis of testicular cells was assessed using Annexin V and propidium iodide (PI) double staining and flow cytometry. In the absence of irradiation, none of the groups exhibited significant changes in testicular parameters (Figure 3). X-ray exposure caused a ~20-fold increase in late apoptotic testicular cells, alongside a ~65% decrease in spermatogonia cell numbers and > 50% decrease in testicular sperm counts (Figure 3). Treatment with either SeNPs or the probiotic reduced apoptotic signaling, as indicated by downregulation of caspase-3 and caspase-9 transcripts. These changes were accompanied by partial restoration of spermatogonia and sperm counts. The combined treatment restored both apoptotic markers and downstream endpoints to near-control levels.

In addition to the select studies presented above, multiple rodent studies have reported chemo-preventive effects of anti-apoptotic interventions and corresponding recovery of impaired spermatogenesis caused by a wide range of stressors, including high-fat diet (Abdulwahab et al., 2021), nanomaterials (reviewed by Sun et al., 2025), and toxicants such as aflatoxin B1 (Ijaz et al., 2023), tartrazine (Essawy et al., 2024), atrazine (reviewed by Abarikwu et al., 2023), suggesting a broad applicability of this KER. Mitigation of testicular cells apoptosis is consistently concordant with preservation of sperm counts.

Together, these findings demonstrate concordant changes in apoptosis and sperm counts across treatment groups and provide indirect evidence of essentiality, as attenuation of apoptosis (and other stress responses) was associated with recovery of sperm production. Although evidence for dose-response relationships is limited, the studies generally support dose and temporal alignment and a moderate empirical relationship for this KER.

A key uncertainty arises from the use of whole testis homogenates to measure apoptotic markers, which reflect mixed cell populations and do not distinguish responses of germ cells and somatic cells. Histological evaluation of seminiferous tubules supports germ cell loss as a primary driver of reduced sperm counts (Yaman et al., 2018; Udefa et al, 2020; Ehghaghi et al., 2022; Oyovwi et al., 2023; Ijaz et al., 2023). However, reductions in Sertoli cell and Leydig cell numbers can occur in parallel (Oyovwi et al., 2023). Therefore, the reduced sperm output is likely caused by combined effects on mixed populations of testicular cells and cumulative damages over the course of spermatogenesis, although the relative contribution of each cell type is not clear.

An apparently inconsistent study was noted. In a rat study, exposure to 10 mg/kg of sodium arsenite for 14 days induced a near 100% increase in the mRNA expression of Bax and caspase-3, and a ~40% decrease in Bcl-2 expression in testicular tissues, indicating activation of apoptotic signaling. Although histological analysis showed remarkable loss of spermatogenic cells, particularly spermatogonia, no changes were observed in testicular weights or epididymal sperm counts. Suppression of apoptotic signaling by carvacrol treatment did not alter sperm density across groups (Gur et al., 2023). The inconsistency likely reflects a limitation of the study design rather than a lack of biological linkage between apoptosis and sperm counts. Samples were collected immediately following the 14-day exposure period , which is insufficient to observe downstream changes in sperm output given the duration of spermatogenesis in rats.

Multiple biological pathways can influence sperm output and introduce variability and uncertainty in this KER.Inflammation and oxidative stress frequently co-occur with apoptosis, and therefore reductions in sperm output may reflect overlapping or combined effects of these stress-related responses. In many studies, attenuation of apoptosis is often accompanied by simultaneous suppression of oxidative stress and inflammatory markers (Udefa et al., 2020; Oyovwi et al, 2023). Given the extensive interplay among stress responsive pathways, it is difficult to separate their individual contributions. In addition, disruption of hormonal regulation in the hypothalamic-pituitary-gonadal axis can indirectly affect spermatogenesis and sperm production. Such interacting mechanisms may partly explain inconsistencies observed across studies and should be considered when interpreting this KER.