Lead as a Reproductive Endocrine Disruptor in Aquatic Species and Agricultural Livestock

Abstract

Lead (Pb) is a naturally occurring metal that is environmentally ubiquitous due to industrial activities, such as mining, smelting, and fossil fuel combustion. Exposure to Pb adversely affects the central nervous system, gastrointestinal tract, lungs, liver, bones, and cardiovascular system, leading to a multitude of negative health impacts, such as anemia and neurological disorders. While significant research has focused on the effects of Pb on the nervous and immune systems, Pb’s impact as a reproductive endocrine disruptor remains largely understudied. The first objective of this review was to collate the current literature regarding the effects of Pb on the reproductive system of aquatic species (primarily fish) and agricultural livestock to highlight the ecological significance and impacts on animal health. Literature supports the hypothesis that exposure to Pb can impede reproductive processes by affecting hormone levels, reproductive organ development, and fertility. A second objective of this review was to elucidate putative mechanisms underlying Pb as a reproductive endocrine disruptor using molecular data and computational approaches. Based on transcriptomics data, Pb is hypothesized to perturb key pathways important for hypothalamic–pituitary–gonadal axis functions, such as circadian regulation and estrogen receptor signaling. Given the widespread environmental presence of Pb, understanding these mechanisms is essential for improving risk assessments and protecting animal reproductive health.

1. Introduction

Lead (Pb) is a naturally occurring metal found in many minerals of the Earth’s crust. Following the Industrial Revolution, when human activities such as smelting, mining, and burning fossil fuels intensified, the presence of Pb in the environment began to increase. This metal is now ubiquitously detected in the environment and is present in oceans, rivers, plants, and many food items. Pb is also used in the manufacturing of pipes, drains, and soldering materials [1]. Both the World Health Organization (WHO) and the U.S. Environmental Protection Agency (EPA) recommend a Pb limit between 10 and 15 µg/L in drinking water, but Pb can exceed 100 µg/L in contaminated areas like Flint, Michigan [2,3]. Generally, Pb levels in surface and groundwater are below 100 µg/L, but may be higher at industrial or mining sites, as well as in unregulated regions [4]. Urban soils may have elevated Pb levels (e.g., up to 500 mg/kg) due to past use of leaded gasoline and paint [5]. At other contaminated sites, such as near smelters or battery recycling operations, Pb can exceed 1000 mg/kg [4].

Pb is an impactful environmental pollutant that exerts toxic effects on many body organs (i.e., central nervous system (CNS), gastrointestinal (GI) system, cardiovascular system, lungs, liver, and bones) [6]. It is currently listed as the second most toxic chemical pollutant on the list of the Agency for Toxic Substances and Disease Registry (ATSDR) for public health concerns. The major route of Pb absorption in the body is through inhalation and ingestion, and its distribution within the body following absorption varies in different compartments. For instance, when Pb interacts with red blood cells, it has a half-life of 25 days and can cross the placenta to affect the CNS of a developing fetus [7]. Pb is also stored in muscles and bones and replaces calcium (Ca²⁺) in the body, causing osteoporosis under conditions of estrogen deficiency [8]. Additionally, modes of action of Pb toxicity include oxidation balance disruption [9], inflammatory response [10,11], and disruption of cellular processes requiring Ca²⁺ influx [12]. Biochemically, Pb exposure increases serum endothelin and erythropoietin levels, causing hematological-related changes (i.e., anemia) and reduced antioxidant levels (i.e., glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)) [6]. Pb is also a neurotoxicant as it has been linked to behavioral and intellectual deficits [13,14], as well as nerve damage, which may contribute to the development of neurodegenerative diseases (i.e., Alzheimer’s and Parkinson’s) [15].

While most studies have focused on the effects of Pb on bone tissue, the central nervous system, and the immune system, there are additional sub-lethal effects on various physiological systems in both aquatic and terrestrial organisms, including the reproductive system [1]. Pb can cross the blood–brain barrier, causing neuroendocrine disruption along the reproductive axes [16,17]. The major reproductive endocrine axes that are negatively affected by Pb toxicity are the hypothalamic–pituitary–fetal–gonadal (HPF) axis, hypothalamic–pituitary–gonadal (HPG) axis, and the hypothalamic–pituitary–adrenal (HPA) axis, a pathway important for steroid hormone biosynthesis. Clinical and biochemical endpoints that are related to the reproductive system, which are negatively impacted by Pb, include impaired organ development in fetuses, cognitive function, reproductive behavior, still births, abortion, male sterility, semen volume, semen density and motility, oxidative stress enzymes, luteinizing hormone (LH), follicle-stimulating hormone (FSH), 17β-estradiol (E2) hormone levels, and miscarriages. Such alterations in reproductive processes can have significant detrimental effects on species that are important for aquaculture and agriculture. Pb has a similar ionic radius and charge to Ca²⁺, allowing it to enter cells via Ca²⁺ channels for incorporation into Ca²⁺-binding signaling pathways [18], which can disrupt reproductive processes. In females, Ca²⁺ contributes to GnRH release, placental development, oocyte maturation, and fertilization [19,20]. When replaced by Pb, menstrual irregularities, infertility, miscarriage, low birth weight, and egg development/viability are all established consequences [21]. In males, Ca²⁺ signaling in mammalian spermatozoa plays many essential roles, including role in fertilization, and sperm motility, capacitation, and the acrosome reaction [22,23].

Pb, as a relevant reproductive endocrine disruptor, is an important aspect of toxicological profile that has been given less attention in recent years. The mechanisms and pathophysiologies involved in Pb-induced toxicity of reproductive axes have significant implications in aquaculture and agriculture, where exposure can occur through contaminated water and animal feed. Therefore, this review aims to assimilate the existing body of research in both aquatic and livestock species to better understand the mechanisms by which reproductive toxicity may occur. Although this review focuses on animals in unique ecological niches, such data are highly relevant to human and public health. The National Institute of Health (NIH) has emphasized several naturally occurring toxic metals due to their potency, systemic effects, and exposure risks in humans. Herein, we describe endpoints related to both ecological and public health while identifying knowledge gaps to inform future risk assessments. Toxic metals like Pb are ubiquitous in the environment and are commonly associated with adverse health outcomes. The presence of Pb in water and animal feed spans critical sectors of concern, including aquatics, agriculture, and public health impacts through multiple exposure mechanisms.

2. Literature Search Criteria

A comprehensive literature search was conducted using the NCBI PubMed database to identify relevant studies published over the past 20 years, up to 10 April 2025. The search strategy employed a combination of the following keywords: “reproduction”, “endocrine”, “lead”, in association with “fish”, “cow”, “sheep”, or “livestock”. These search terms were selected to capture studies investigating the effects of Pb exposure on the endocrine system, specifically targeting species of ecological or agricultural importance.

All retrieved articles were subjected to a systematic screening process based on predefined inclusion and exclusion criteria. Studies were included in this review if they: (1) involved one or more of the target species (aquatic or livestock) and (2) reported direct effects of Pb exposure on endocrine-related endpoints of reproduction, specifically alterations in pituitary or gonadal structure, function, or hormone levels. The initial search yielded 292 articles. After applying the inclusion criteria, 31 studies were deemed eligible for full-text review and were included in the manuscript. Of these included manuscripts, those that were experimental studies related to Pb exposure in fish and livestock were further summarized in

Table 1. Summaries of studies related to Pb exposure in fish.

Species	Exposure	Results	Reference
Zebrafish (Danio rerio)	2.5 or 5 µg/L Pb during embryogenesis until 8 dpf	Delayed growth rates, spine deformities, yolk sac edema, and swim bladder inflation.	[24]
Freshwater teleost (Astyanax bimaculatus)	15, 50, or 100 μg/L Pb during gametogenic stages for 28 days	Females had higher follicular atresia and lower vitellogenic follicle ratios. Reduction in seminiferous tubule diameter and spermatozoa production, and an increase in apoptotic germ cells in males. Increased nuclear ERα and ERβ expression. Hormone communication was disturbed in both sexes.	[25]
Common carp (Cyprinus carpio L.)	13 or 68 mg Pb/kg d.w. in adults for 6 months	Reduced ovulation, fecundity, and ovulation rates. Immature oocyte production. Impaired vitellogenesis. Pb accumulated in brain tissue.	[26]
Zebrafish (Danio rerio)	10 µM Pb in embryos for 24 h	Significant changes in brain transcriptome, including unexposed F2 offspring. Noted epigenetic modifications were linked to neurogenesis and synaptic function, thus compromising brain health. F0 and F2 generation displayed differential expression of genes involved in the endocrine system.	[27]
Zebrafish (Danio rerio)	10 µg/L Pb for 3 months in parental fish	Enhanced developmental neurotoxicity in offspring and altered reproductive and thyroid endocrine systems.	[28]
Catfish (Heteropneustes fossilis)	0.1, 1.0, 10, or 50 ng/mL Pb for up to 24 h in oocytes	At 4 and 8 h, Pb caused an increase in germinal vesicle breakdown and ovulation across all tested concentrations. Dose-dependent reduction in oocyte response was observed at 16 and 24 h. Pb promoted ovulation more effectively than the control group at 16 and 24 h.	[29]
Common carp (Cyprinus carpio L.) and Capoeta sp.	Environmental Pb contamination in adults	Average gonad concentration of 0.52 ± 0.09 mg/kg. E2 concentrations were lower in females collected from a heavily polluted sample area.	[30]
Curimata-pacu (Prochilodus argenteus)	Environmental Pb contamination in adults	Average concentrations were 10-fold greater in liver or muscle tissue compared to safe limits. Males had disorganized and degenerating spermatocysts in the testes. Females had a higher incidence of atretic follicles, disturbed yolk deposition, and overripe oocytes.	[31]

and

Table 2. Summaries of studies related to Pb exposure in livestock.

Species	Exposure	Results	Reference
Cow (Bos taurus)	Environmental Pb contamination in adults	E2 blood plasma levels are higher in contaminated smelter areas, causing impaired fertility and sterility.	[32]
Cow (Bos taurus)	Environmental Pb contamination in adults	Higher levels of Pb in follicular fluids and higher MDA levels.	[33]
Cow (Bos taurus)	Environmental Pb contamination in adults and offspring	Increased morbidity and mortality. Higher rates of stillbirths and calf deaths. Prolonged elevated blood Pb levels.	[34]
Porcine (Sus scrofa domesticus)	0.46, 0.63, 0.83, 2.5, or 5.0 mg/10 mL Pb acetate trihydrate for 18 h in oocytes	Release of Mg2+ and K+ in GCs. Reduced total antioxidant and glucose levels. Ca2+ release is lowest in cells cultured with the highest Pb concentration.	[35]
Porcine (Sus scrofa domesticus)	0.046–0.250 mg/mL Pb for 18 h in GCs	Inhibition of IGF-1 release from GCs at low doses. Cyclin B1 and CASP3 were impacted.	[36]
Bovine (Bos taurus)	1, 2, 3, 5, or 10 μg/mL Pb acetate for 2 h in GCs	ROS accumulation and carbonylation of proteins in GCs. Decreased cell viability and expression markers of cell proliferation genes and cell cycle arrest in GCs. Downregulated Nrf2 and NF-κB TF and increased expression of ER stress marker genes and proapoptotic genes in GCs.	[37]
Buffalo (Bubalus bubalis)	0.005–10 μg/mL Pb for 24 h in oocytes	Decreased oocyte viability, hatching, morula/blastocyst yield, ICM, and TCN of blastocysts. Increased morphological abnormalities in oocytes.	[38]
Bovine (Bos taurus)	1, 2, 3, 5, or 10 µg/mL Pb acetate for 2 h in GCs	Aberrant blastocyst phenotype. ROS generation and reduced blastocyst cell numbers.	[39]
Porcine (Sus scrofa domesticus)	10, 20, or 40 μM Pb acetate for 48 h in SCs	Decreased AMH level expression. Loss of FSH-r integrity and E2 production. Increased AKT and mTOR expression in SCs.	[40]
Deer (Dama dama)	15, 30, 60, 125, or 250 μM PbCl2 for 24 h in gametes	Inhibition of antioxidant enzymes and glutathione peroxidase in SCs.	[41]
Goat (Capra hircus)	0.5, 5, 10, or 20 ppm Pb acetate for 3 h in mature spermatoza	Decrease in sperm kinematic parameters. Induced spontaneous or premature acrosome reaction. Increased DNA damage and apoptosis. Plasma membrane and acrosomal damage. Collapsed mitochondrial cristae.	[42]
Ram (Ovis aries)	Up to 5000 ng/mL Pb for 3 h in mature spermatoza	Sperm motility, acrosome integrity, sperm viability, and membrane functionality were negatively correlated with Pb level.	[43]
Goat (Capra hircus)	4.5 and 6.0 mg Pb/kg b.w. during gestation until abortion	Increase in GGT and aminotransferase activity in blood. Decrease progesterone level in a dose-dependent manner in the blood.	[44]
Pig (Sus scrofa domesticus)	10 mg/kg Pb for 120 days during the postnatal juvenile stage	Decreased body weight, feed efficiency, LH, and E2 levels in serum.	[45]

, respectively. Google Scholar was also used as a secondary search to ensure all relevant manuscripts were captured, using the same criteria.

3. Lead as an Endocrine Disruptor of Reproductive Processes

3.1. Impacts on Reproduction: Aquatic Species

Pb exposure impacts gonadal development through HPG and HPA axes interference, as well as through reactive oxygen species (ROS) production. These mechanisms affect bioavailability, metabolism, and excretion of various biomolecules, including hormones related to reproduction [46]. The instances of such indirect impacts on reproduction are numerous, including their endocrine-disrupting potential. Curcio et al. [24] exposed larval zebrafish (Danio rerio) to environmentally low (2.5 μg/L) or high (5 μg/L) Pb doses during one of two windows: early (1–7 days post-fertilization (dpf)) or late (2–8 dpf). Phenotypic abnormalities such as delayed growth rates, spine deformities, yolk sac edema, and swim bladder inflation were identified with both treatments. Such parameters have the potential to impact overall fitness and lessen the number of fish that reach reproductive maturity. In a separate study conducted by Ferreira et al. [25], freshwater teleost (Astyanax bimaculatus) were exposed to 15, 50, or 100 μg/L Pb for 28 days. In males, a reduction in seminiferous tubule diameter and spermatozoa production, as well as an increase in apoptotic germ cells, were noted in exposed teleosts. Vitellogenin (VTG), the egg yolk precursor protein, and metallothionein (MT) were measured in the liver, along with E2 and 11-ketotestosterone in blood plasma. Hormone communication was disturbed in both sexes, resulting in reduced reproductive efficiency. Specifically, MT was unaltered; however, VTG, E2, and 11-ketotestosterone were altered with 100 µg/L Pb. Females showed skewed reproductive cycles, in which higher follicular atresia and lower vitellogenic follicle ratios were observed despite increased plasma E2 levels. Consequently, females were unable to appropriately produce oocytes or undergo vitellogenesis. Some evidence of compensation for the disruption of estrogen signaling by increasing nuclear ERα and ERβ expression was observed. In another study, Sionkowski et al. [26] fed female common carp (Cyprinus carpio L.) 13 or 68 mg Pb/kg d.w. (dry weight) Pb for 6 months. Adverse outcomes related to Pb exposure included decreased ovulation and reduced fecundity (egg number and weight). Interestingly, Pb accumulated in brain tissue and affected other chemical communication networks. These fish also exhibited impaired vitellogenesis and produced immature oocytes, which was likely mechanistically due to attenuated LH and FSH release. Other studies report decreased or inhibited LH secretion in fish species exposed to Pb, including prussian carp (Carassius gibelio), fathead minnow (Pimephales promelas), African catfish (Clarias gariepinus), and zebrafish [26,47]. Changes to LH levels suggest HPG axis disruption, an effect that Chakraborty [48] discusses in addition to inhibited steroidogenic enzymes and significantly altered expression of hormone receptors.

Pb exposure can alter gene expression or epigenetic marks, like DNA methylation, which can be passed down to offspring. Consequently, these alterations can disrupt vital biological systems and may elevate the transgenerational genetic risk for toxicity-related phenotypes, including decreased fecundity and survival of offspring. DNA methylation influences neurodevelopment, and various studies on the human genome elaborate on methylation modifications following Pb exposure [49,50]. Overall, most studies to date that assess the transgenerational effects following Pb exposure evaluate neurotoxicity endpoints, while data regarding endocrine-related transgenerational effects are not as abundant. In fish, Meyer et al. [27] found that zebrafish exposed to 10 µM Pb during early development showed significant changes in their brain transcriptome, as well as their unexposed offspring. Noted epigenetic modifications were linked to neurogenesis and synaptic function, indicating compromised brain health. Additionally, zebrafish of the F0 generation displayed differential expression of genes involved in the endocrine system, and these effects persisted through the F2 generation. Another study exposing 10 µg/L Pb to zebrafish adults also observed enhanced developmental neurotoxicity in offspring and altered reproductive and thyroid endocrine systems in zebrafish adults, along with an abnormal transfer of reproductive and thyroid hormones to their offspring [28].

In addition to Pb, other heavy metals induce reproductive effects on fish. Gautam and Chaube [29] investigated the impact of cadmium (Cd), cobalt (Co), mercury (Hg), and Pb on oocyte maturation and ovulation in catfish (Heteropneustes fossilis) using an in vitro incubation model. Post-vitellogenic oocytes were exposed to these metals at concentrations of 0.1, 1.0, 10, and 50 ng/mL for up to 24 h. The metals exhibited differential effects, mostly attributed to their influence on oocyte maturation and ovulation following the order: Pb > Hg > Cd > Co. In the early stages of incubation, specifically at 4 and 8 h, Pb induced an increase in germinal vesicle breakdown and decreased ovulation rates across all tested concentrations, indicating a strong stimulatory effect; however, a dose-dependent reduction in oocyte response was observed at 16 and 24 h, suggesting a temporal limit to the stimulatory capacity of these metals. Exposure to other metals, including Cd, chromium (Cr), copper (Cu), and zinc (Zn), also causes reproductive effects in pejerrey fish (Odontesthes bonariensis), including altered expression of reproductive genes (e.g., upregulation of gnrh genes, downregulation of cyp19a1b, and reduced fshr mRNA) and structural testicular damage such as fibrosis and shrinkage of spermatic lobules [51].

While most direct evidence for reproductive impairment by Pb comes from laboratory studies, a small number of studies have captured fish from polluted environments to evaluate the relationship between reproductive health and the concentration of heavy metals within tissues. For example, Ebrahimi and Taherianfard [30] evaluated the concentrations of arsenic (As), Cd, Hg, and Pb in the organs of common carp and Capoeta sp. from different sampling sites at Iran’s Kor River. Regarding Pb concentration in the gonads (both testes and ovaries), levels were higher compared to other organs, and the average concentration was 0.52 ± 0.09 mg/kg. Additionally, E2 concentrations in female fish collected from the heavily polluted sample area were significantly lower compared to the other sampling sites, suggesting a direct effect of metal contamination on steroidogenesis. In another study conducted by Paschoalini et al. [31], curimata-pacu (Prochilodus argenteus) collected from the Paraopeba River, Brazil, contained average concentrations of Pb that were 10-fold greater in liver or muscle tissue compared to safe limits. Additionally, when compared to a reference site containing fish with unaltered reproductive parameters, male fish from the Paraopeba River had disorganized and degenerating spermatocysts in the testes, while females exhibited a higher incidence of atretic follicles, disturbed yolk deposition, and overripe oocytes, indicating negative gonadal development.

Findings of Pb in aquatic environments are particularly concerning due to potential human consumption, driving the need for regulatory limitations in food products. Currently, within European Union member states, the maximum permissible concentration of Pb in all types of fish products (except for beluga, swordfish, and tuna) is 1 mg/kg. Regarding the exceptions, the maximum permissible concentration in tuna, swordfish, and beluga is 2 mg/kg [52]. Due to high variability in recommended safe limits for contaminants, more universally applied concentration guidelines are needed to ensure safe human consumption.

3.2. Impacts on Reproduction: Livestock Species

Livestock are an integral part of a sustainable food chain while concomitantly serving as large animal and biomedical human research models for reproduction and infertility, including genetic, epigenetic, toxicity, and stem cell applications. Livestock have been widely exposed to many toxic heavy metals, including Pb. Due to previously described Pb actions as an endocrine disruptor, there is a growing concern for effects of Pb on gametogenesis and consequences on livestock production, reproduction, and products for human consumption [29]. Livestock exposures to Pb may arise from environmental contamination associated with coal mining and smelting operations, improper disposal of Pb-acid batteries, ingestion of Pb-contaminated feed or water, proximity to areas with elevated Pb levels, and use of Pb-based materials within agricultural settings. Pb concentrations in edible tissues of livestock vary based on species, geography, and local pollution levels. According to the European Commission [52], when consuming muscle meat of cattle, pigs, and sheep, the relative maximum Pb concentration for consumption is 0.10 mg/kg. Regarding offal (i.e., liver and kidney), concentrations are set to 0.20 mg/kg in bovine and sheep and 0.15 mg/kg in pig.

Pb can alter sex steroid levels in livestock, impacting reproductive cyclicity. In cows, E2 blood plasma levels were reportedly higher in contaminated Zn/Pb smelter areas, which resulted in impaired fertility and sterility [32]. In a separate study, coal-fired power plants were shown to have high environmental levels of Pb, and cows near these plants presented with higher levels of Pb in their follicular fluids and higher malondialdehyde (MDA) levels, an indicator of oxidative stress [33]. There are other examples of adverse associations between Pb and fertility in livestock. For example, heifers associated with accidental exposure to Pb-containing batteries experienced increased morbidity and mortality, including higher rates of stillbirths and calf deaths. Affected animals also showed prolonged elevated blood Pb levels, with little reduction even after months of exposure [34]. The authors concluded that environmental exposure to Pb resulted in disturbed reproductive endocrine function and altered levels of various reproductive hormones, which are critical for reproductive cyclicity and fertility.

Environmental toxicant exposure has been a major concern for declining fertility and reduced sperm count not only in humans but also in ruminants. There has been potential damage to the fertility of livestock species as heavy metals cross the blood–testes barriers, and bioaccumulate in the testes, where metals are not easily degraded [53]. Often, sperm quality, quantity of semen, and androgen level are negatively affected by heavy metal exposure [53]. For one study conducted in bulls, Pb-induced oxidative stress markers and semen quality, including additional fertility indices, were evaluated. The results suggested that higher Pb in blood and semen were associated with an increase in oxidative stress markers (catalase, MDA, SOD), a reduction in semen quality, and poor fertility of bulls [54]. Similar studies were conducted in rams to determine the effect of natural Pb exposure and levels of Pb in plasma on various male reproductive function parameters (i.e., spermatozoa and epididymal characteristics, oxidative stress markers, histology of testes and epididymis tissues, and apoptosis level in testes) [55,56]. The level of serum testosterone and major seminal characteristics was negatively correlated with higher Pb exposure. In agreement with the previous findings, a study utilizing red deer (Cervus elaphus) as a representative wildlife species, concluded that high Pb environmental exposure resulted in a higher Pb level in the parenchyma of the testes compared to unexposed bucks [57]. Pb exposure was related to the highest testis mass, reduced spermatozoa membrane integrity, and decreased SOD and GPx activity in the tissue. The same exposure also resulted in compromised acrosome integrity and higher DNA fragmentation, resulting in chromatin damage and compromised spermatozoa membrane integrity, which is critical for sperm function [57]. This study concluded that males may be sensitive to environmental Pb exposure, which can (1) cause alterations in the structure, physiology, genomic, and epigenomic landscape of spermatozoa and (2) cause disturbance in oxidative stress biomarker levels in semen, resulting in reduced fecundity.

Regarding in vitro data, granulosa cells (GCs) of livestock species are a good model to study the pervasive effects of Pb as a reproductive toxicant and endocrine disruptor. For example, the antioxidant status and biochemical parameters of porcine ovarian GC exposed to Pb acetate in vitro have been utilized to determine the negative consequences on ovarian function [35,36]. Total antioxidant and glucose levels were higher in the control groups than the treated groups, and SOD levels were lowest in the control group. Ca²⁺ release was noted to be lowest in cells cultured with the highest concentration of Pb (5 mg). Pb also caused the release of magnesium (Mg²⁺) and potassium (K⁺) from GCs [35]. Regarding secretory activity of GCs, Pb inhibited IGF-1 release at lower doses while P4 secretion was not influenced by Pb addition, whereas the expression of Cyclin B1 and caspase-3 (CASP3) was significantly increased by Pb [36]. As such, Pb appears to affect the intracellular path of proliferation and apoptosis of GCs through intracellular mechanisms involving, but not limited to, cyclins and caspase-3 signaling. In another study, Aglan et al. [37] studied the effect of Pb on the regulatory pathways of two transcriptional factors (Nrf2 and NF-κB) in bovine GCs. Pb caused ROS accumulation and carbonylation of proteins when exposed to as low level as 1 µg/mL. Additionally, GCs showed decreased cell viability and decreased expression markers of cell proliferation genes and cell cycle arrest. Pb also downregulated both Nrf2 and NF-κB TF as low level as 1 µg/mL and increased expression of ER stress marker genes and proapoptotic genes (CASP3) at 3 µg/mL. Like GC, oocyte culture systems are used to study the reproductive toxicity of Pb on oocytes, viability, and in vitro development as key parameters. Nandi et al. [38] studied the effect of Pb exposure on in vitro-derived oocytes from buffalo. Exposure to Pb resulted in decreased oocyte viability, higher morphological abnormalities, decreased hatching rates, and morula/blastocyst yield from as low level as 0.5 µg/mL. In addition, the total cell counts (TCN) of blastocysts and inner cell mass (ICM) were significantly lower in PB-treated groups as compared to controls. In a similar in vitro study conducted by Aglan [39] on bovine preimplantation embryos, Pb exposure resulted in an aberrant blastocyst phenotype in the Pb-exposed group. Pb exposure increased the production of ROS and reduced blastocyst cell numbers. In complement to oocyte and embryo studies, effects of Pb on Sertoli cells (SCs) provide an additional model to explore the effects of environmental toxicants on male infertility, as changes in the SC niche disturb germ cells and spermatogenesis. The effects of sub-toxic levels of Pb on porcine SCs resulted in decreased expression of anti-Mullerian hormone (AMH) as low as 10 µM [40]. Decreased expression of AMH subsequently resulted in loss of FSH-r integrity and loss of E2 production if stimulated by FSH. Additionally, protein kinase B (AKT) and mammalian target of rapamycin (mTOR) expression increased, indicating that Pb is toxic to SCs [40]. Similarly, SCs derived from wild deer yielded similar results after Pb exposure compared to porcine [41]. Consequences included cell perturbation in Pb-treated groups, including observations of antioxidant enzyme and glutathione peroxidase inhibition.

Swine testis (STs) cells have also been used as a model to determine the toxic effects of Pb on reproductive parameters. These cells are a collection of immature Sertoli cells. Pb exposure to STs in culture increased ROS, disrupted the antioxidant system, upregulated the pro-apoptotic gene PTEN, and inhibited the anti-apoptotic PI3K/AKT pathway, thereby promoting apoptosis [58]. In another study exposing environmentally relevant levels (0.5, 5, 10, and 20 ppm) of Pb to goat sperm in vitro, a decrease in sperm kinematic parameters at concentrations as low as 5 ppm was reported [42]. Pb also caused changes in Ca²⁺ levels and induced spontaneous or premature acrosome reaction, increased DNA damage and apoptosis, and resulted in acrosomal and plasma membrane damage, as well as collapsed mitochondrial cristae [42]. Similarly, Castellanos et al. [43] described that sperm motility, acrosome integrity, sperm viability, and membrane functionality of ram spermatozoa were negatively correlated with Pb exposure.

In vivo experimental studies of Pb exposure have been difficult to conduct because of ethical concerns, but some studies are still reported in smaller livestock species. Two groups of pregnant goats were fed with Pb acetate at 4.5 and 6.0 mg Pb/kg body weight (b.w.) until abortion (~14th week of pregnancy). Results indicated an increase in gamma-glutamyl transferase (GGT) with Pb exposure. Furthermore, aminotransferase activity in blood and progesterone levels were significantly lower in the treated groups than in control males, further revealing dose-dependent observations [44]. Comparable studies in pigs have assessed the effects of dietary Pb exposure by evaluating parameters such as body weight, growth, and feed efficiency. Pb exposure (10 mg/kg) resulted in decreased body weight and feed efficiency. While there was no difference in uterus and ovary weight, decreased serum LH and E2 levels were noticed, confirming that Pb may have disturbed the HPG axis [45].

4. Molecular Biomarkers of Lead and Reproductive Dysfunction

The Comparative Toxicogenomics Database (CTD) was queried to retrieve molecular responses related to Pb [59]. We extracted gene information and conducted an enrichment analysis to reveal cellular processes regulated by Pb exposure. Most of the top hits for hormone systems were related to the reproductive axis.

Table 3. Pathways related to reproduction affected by Pb exposure.

Pathway Name	Reproductive Relevance
Estrogen signaling pathway	Female reproductive system development, regulation, and puberty
GnRH secretion	Controls GnRH release, which regulates reproductive hormone secretion and gonadal response
GnRH signaling pathway	Regulates LH and FSH pituitary release
Oocyte meiosis	Key process in the maturation of female gametes
Ovarian steroidogenesis	Steroid hormone production in ovaries
Progesterone-mediated oocyte maturation	Involved in the maturation of the oocyte prior to ovulation
Prolactin signaling pathway	Involved in reproductive health, milk production, and metabolism
Steroid hormone biosynthesis	Biosynthesis of hormones (i.e., estrogen, progesterone, testosterone)

lists all reproductive-related pathways discovered. Pathway enrichment analyses were conducted using iPathway software (Advaita Corporation, Ann Arbor, MI, USA) to identify biological processes and hallmark signatures. Analyses utilized pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG, Release 100.0+/11–12 November 2021) [60] and gene ontologies from the Gene Ontology Consortium (4 November 2021) [61,62]. Enrichment analyses incorporated miRNA data from miRBase (v22.1, October 2018) and TargetScan (Mouse v8.0, Human v8.0) [63,64], regulatory networks from BioGRID (v4.4.203, October 2021) [65], and toxicant and disease associations from the Comparative Toxicogenomics Database and KEGG databases [59].

There were 2237 differentially expressed (DE) genes used in the analysis that are known to be altered by Pb based on CTD, and enrichment was performed against all the genes in the Advaita Knowledge Base (AKB). The analysis revealed a significant number of pathways (Figure 1) perturbed by Pb exposure depicted in the chord diagram. According to pathway enrichment analysis, Pb impacts processes such as response to estradiol, positive regulation of transcription by RNA polymerase II, and cellular response to amyloid-beta, all of which are important to reproductive processes. Regarding the enrichment of the response to estradiol, brain-derived neurotrophic factor (BDNF) was upregulated and, though well-known for its neuronal association, BDNF is expressed in ovarian tissue and contributes to follicle and embryonic development, as well as oocyte maturation [66]. One previous study completed on embryonic Pb-exposed zebrafish noted noticeable behavioral impairments in larvae, which were associated with BDNF disruption; however, gene expression analysis revealed reduced levels of bdnf [67]. To date, no studies report on BDNF-associated disruption in livestock; however, various studies report its alterations following Pb exposure in rats [68,69] and humans [70]. Regarding the enrichment of the positive regulation of transcription by RNA polymerase II and cellular response to amyloid-beta, along with the upregulation of BCL2 and CASP3, respectively, these results imply impacted roles in reproductive homeostasis [71]. The enrichment of estrogen early response pathways and upregulation of nfkbil1 (NFKB inhibitor-like 1) suggest potential estrogen receptor (ER)-mediated effects.

Figure 2 depicts an estrogen signaling pathway perturbed by Pb exposure based on molecular data. Red color indicates that the transcript has been identified in transcriptome or proteome studies to be altered by Pb based on the Comparative Toxicogenomics Database. BCL2, AKT, ERK1/2, CREB, and eNOS, which were all upregulated in our analysis, play important roles in follicular development, oocyte maturation, and uterine receptivity. Similar dysregulation has not been reported in much literature, specifically in livestock; however, experimental fish studies do elaborate on similar transcript alteration due to Pb toxicity [72,73,74]. Pb may dysregulate BCL2, which may inhibit follicular atresia [75] and Pb can inhibit AKT signaling and disturb ERK1/2 activation, leading to impaired GC function and disrupted steroidogenesis [76]. Dysregulation of CREB and eNOS further compromises uterine gene expression and vascular function, affecting implantation, and placental development [77]. Collectively, these molecular disruptions highlight Pb’s potential to impair fertility through estrogen-mediated signaling.

Figure 3 depicts an upregulated estrogen-responsive gene network following Pb exposure. Within the center are key signaling molecules (i.e., EGFR, GH1, STAT3) involved in signal transduction pathways. Various genes within the network are both estrogen- and reproductive-related. For example, MMP2 (Matrix Metallopeptidase 2) is involved in the breakdown of the extracellular matrix, contributing to endometrial remodeling, implantation, and spermatogenesis [78]. ESR1 (Estrogen Receptor 1) mediates physiological responses to estrogen (i.e., follicle development, ovulation, and sperm maturation), in which its deficiency has been shown to contribute to infertility in animal models [79]. Additionally, TGFB1 (Transforming Growth Factor Beta 1), which is shown to interact with ESR1, is involved in tissue remodeling required for implantation, fertilization, spermatogenesis, angiogenesis, and inflammatory modulation [80,81].

5. Conclusions

According to the U.S. EPA [82], Pb enters drinking water through plumbing materials, and if concentrations exceed 15 µg/L, action must be taken to control levels. In contrast, the European Union and India have a stricter limit of 10 µg/L Pb in drinking water [83,84]. Regarding soil, the EPA sets a residential soil screening level at 200 mg/kg [85], while some European countries hold a more conservative limit of 100 mg/kg (i.e., Germany) [86]. Additionally, India maintains an upper limit of about 500 mg/kg for agricultural soils [87]. However, many experimental studies investigating reproductive effects use Pb concentrations far exceeding these environmental levels, such as those elaborated on in this study. While these elevated doses help elucidate mechanisms of Pb-induced reproductive dysfunction, they do not accurately reflect real-world ecological exposures. Therefore, additional studies using lower, environmentally relevant Pb levels are warranted to better assess toxicity risks.

Beyond its molecular and physiological impacts on reproduction, Pb represents a significant public health concern due to bioaccumulation in aquatic and terrestrial animals consumed by humans. Given Pb’s pervasive environmental presence and its ability to disrupt critical reproductive signaling pathways (i.e., BCL2, AKT, ERK1/2, CREB, eNOS), there is a pressing need for more rigorous monitoring and protective regulatory measures. Even low-level Pb exposure can compromise fertility, hormone regulation, placental development, and embryonic viability, affecting both animal and human populations. As Pb’s biochemical mimicry of calcium enables its integration into cellular pathways vital for reproduction, multigenerational consequences are likely to be impacted. Future research on the endocrine-disrupting effects following Pb exposure, inducing transgenerational endocrine disruptions, is warranted. Such studies can investigate the role of epigenetic modifications, including DNA methylation, histone modifications, and non-coding RNA regulation in fish, livestock, and large animal models for human biomedical sciences. Determining the impact of Pb exposure in combination with other environmental toxins, including endocrine-disrupting chemicals, that may produce synergistic effects to exacerbate endocrine dysfunction may also be beneficial. A comprehensive understanding of Pb’s reproductive toxicity will be key to safeguarding both ecological and public health in the face of continued environmental contamination.