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. 2026 Feb 26;46(10):3403–3414. doi: 10.1002/jat.70129

Assessment of Gold‐Mediated Male Reproductive Dysfunction in Male WISTAR Rats Through Sperm Analysis and Testosterone Measurement

Samira Maghraoui 1,2,✉, Adrian Florea 3, Leila Tekaya 1
PMCID: PMC13532839  PMID: 41749514

ABSTRACT

Gold has been used for centuries in both ornamental and medicinal contexts. More recently, gold compounds, containing ions or nanoparticles, have attracted attention for their anti‐inflammatory, anticancer, and diagnostic applications. Therefore, concerns about systemic toxicity and biodistribution have prompted investigations into the biological effects of gold in animal models and humans. In the present study, we explored the impacts of a gold solution, used in chrysotherapy, on the reproductive function of male Wistar rats. Seven chronic intraperitoneal injections of sodium 3‐aurothio‐2‐hydroxypropane‐1‐sulfonate at 30% of gold (allochrysine) were administered to adult male Wistar rats over a period of 2 weeks. The control rats received 0.9% NaCl solution. Our results showed that gold has no notable effect on the absolute weight of reproductive organs (testicles, epididymides, seminal vesicles, and the two caudae of the epididymides) and of nonreproductive organs (liver and brain) excepting the kidneys. The assessment of the fertility parameters showed a significant decline in the motility and viability of sperm cells. A slight reduction in the sperm density, and many malformations affecting the spermatozoa were detected, and the testosterone levels in rats' blood were also significantly diminished. Our study showed that administration of gold solution reduced sperm parameters and was a disruptive agent for the endocrine function of testicles. The oxidative stress generation, attributed to this heavy metal, led to a significant hypertrophy of the kidneys probably after inflammation. Thus, gold existing in different medical treatments could be classified as an endocrine disruptor and could be a detrimental factor of male fertility.

Keywords: absolute weight, fertility parameters, gold toxicity, nonreproductive organs, reproductive organs, sperm malformations, testosterone measurement

Short abstract

Gold compounds are widely used in medical applications, but their biological effects remain a concern. This study investigated the impact of chrysotherapy gold solution on male reproductive function in Wistar rats. Repeated injections did not affect most organ weights except the kidneys, but significantly reduced sperm motility, viability, and testosterone levels, with increased sperm abnormalities. These findings suggest that gold induces oxidative stress, disrupts testicular endocrine function, and may impair male fertility.

1. Introduction

Gold (Au), a lustrous and highly valued metal, has captivated human society's attention for millennia. Revered since antiquity for its rarity, aesthetic appeal, and corrosion resistance, gold has been used in ornamental artifacts, currency systems, religious objects, and as a symbol of power and wealth across ancient civilizations. While its cultural and economic significance has remained steadfast, modern science and technology have developed new and diverse applications for the heavy metal that extend far beyond its historical roles (Fricker 1996).

In contemporary times, gold plays a crucial role in various fields, including electronics, catalysis, nanotechnology, and medicine (Lansdown 2018). Its exceptional conductivity and chemical stability make it indispensable in the manufacturing of semiconductors and advanced electronic devices (Ghobashy et al. 2024). At the nanoscale, gold exhibits unique physicochemical properties that have led to its incorporation in diagnostics, drug delivery systems, and photothermal therapies for cancer treatment (Bansal et al. 2020). Furthermore, gold‐based compounds have shown therapeutic potential in the management of certain autoimmune diseases, such as rheumatoid arthritis (RA) (Messori and Marcon 2004; Zhang 2015).

In spite of all these applications, gold raised health considerations depending on its form and exposure context. Elemental gold was considered biocompatible, and edible gold was used as the food additive E175, but thin gold sheets seemed to induce gut microbiota alterations and low‐grade inflammation at high exposures, as observed in animal studies (Evariste et al. 2023). Occupational exposure, especially in artisanal and small‐scale mining, carried risks from gold and from associated toxicants such as mercury, arsenic, cyanide, and silica dust, which contributed to respiratory diseases, heavy‐metal toxicity, and environmental contamination (Basu et al. 2023; Ondayo et al. 2024). The reproductive toxicity was also well documented by reports demonstrating testicular damage, decreased fertility, and histopathological changes in the seminiferous tubules following gold exposure in rats and mice (Wang et al. 2018).

In the present study, we aimed to examine whether allochrysine, a non‐biological treatment for RA and inflammations in humans, had any impact on the reproductive functions of the male Wistar rat. This aim was especially corroborated by evidence that this noble metal was concentrated at the subcellular level in the lysosomes of testicular Leydig cells, in the form of very dense microneedles (Maghraoui et al. 2013).

To meet our goal:

  • We measured the absolute weight of reproductive (testicles, epididymides, epididymides caudae, and seminal vesicles) and nonreproductive organs (kidneys, liver, and brain);

  • We explored the sperm cells functionality by assessing the different parameters of fertility, namely, the motility, the viability, and the density of sperm cells, and by studying the sperm cells malformation using the Scanning Electron Microscopy (SEM);

  • We evaluated the endocrine function of the testicle by measuring the testosterone levels in the blood of gold‐treated rats.

2. Material and Methods

2.1. Characterization of the Allochrysine

In the current study, we used the commercial drug allochrysine (sodium aurothiopropanol sulfonate or sodium [2‐hydroxy‐3‐mercaptopropane‐1‐sulphonato(2)]aurate (1‐) (Sarbak, Laboratoire de Thérapeutique Moderne; France)) whose chemical formula is C3H6AuNaO4S2 (AuSCH2‐CHOH‐CH2SO3Na), with the molecular weight of 390,164 g/mol and the purity of ≥ 99%. The use of this drug is known as chrysotherapy, and it constitutes a non‐biological treatment for RA and inflammations in humans. It represents a slow‐acting symptomatic treatment and is usually considered, along with other drugs, as a first‐line alternative in cases of contraindication or intolerance to methotrexate (4‐amino‐10‐methylfolic acid), the conventional treatment of many inflammatory diseases in humans. Since many years, the allochrysine has many analogues and equivalents, all compounds of gold, used for same purposes to treat inflammation, like Aurothiomale, Auranofin, and Myochrisin.

2.2. Animal Experiment

During this study, 16 adult male Wistar rats were used; they were purchased from the Pasteur Institute of Tunis (Tunisia) and had an average initial weight of around 250 g. Animals were randomly divided into two groups (G1 and G2) and conditioned for 8 days (D) in the Experimental Medicine Unit of the Faculty of Medicine of Tunis (Tunisia) where experiments were conducted. The laboratory temperature was 25°C ± 2°C, and the relative humidity of 70% ± 5%, the nycthemeral cycle of 12 h/12 h. For the two groups, food and water were available ad libitum. Food (pellets for rodents were purchased from Société Industrielle de Concentré, SICO Sfax, Tunisia) and water were available ad libitum. Each rat of the first group (G1; N = 8) received allochrysine (at 30% of gold). The total received dose of gold was 2100 mg/kg of body weight as seven chronic intraperitoneal injections of 1 mL each. This dose was very low compared to the LD50, and it was calculated in a preliminary experimental study conducted in our laboratory. The injections of gold were administered every 2 days (D9, D11, D13, D15, D17, D19, and D21) for a period of 2 weeks.

In the same experimental conditions, control rats (G2; N = 8) were injected with 0.9% NaCl solution. Twenty‐four hours after the last injection (22nd day), treated and control rats were anesthetized with ether diethyl (Sigma Aldrich, Germany). Right and left testicles, epididymides, epididymides caudae, and seminal vesicles as well as kidneys, liver, and brain were removed, freed of fats, and weighed. Blood was collected in dry vacuum tubes, centrifuged, and the collected serum was immediately placed in the refrigerator at −20°C for testosterone measurement. At the end of the experiment, animals were euthanized by cervical dislocation. All procedures were carried out according to the guidelines of the National Research Council's Guide for the Care and Use of Laboratory Animals.

2.3. Sample Processing

The collected epididymides caudae were destined to study the functional parameters of male rats, precisely the motility, the viability, and the density of sperm cells. The same biological material was also used to explore the malformations of sperm cells. The serum served to explore the endocrine function of the rat testicles by measuring the testosterone levels.

2.4. Determination of Fertility Parameters

Epididymides caudae (left and right) of gold‐treated and control rats were excised and minced in 10 mL of saline solution, preincubated at 36°C for 15 min. This allows sperm cells to swim out of the epididymides tissues. In order to assess the sperm motility, epididymides caudae fragments were removed from the solution, leaving mainly sperm suspension in saline solution. This solution was considered a sperm solution and was used to determine fertility indicators. The following parameters were determined at 400 × magnification using a light microscope (Optika microscopes, Italy).

2.4.1. Motility

Two or three drops of the sperm solution were mounted between a microscope slide and coverslips. To determine the percentage of motile sperm cells, 10 microscopic fields were observed, and the following formula was used:

Percentage (%) of motile spermatozoa = (number of motile spermatozoa/total number of counted spermatozoa) × 100.

2.4.2. Viability and Sperm Cells Morphological Abnormalities

Droplets of sperm solution were mixed with the same droplets number of eosin (1%) and nigrosin (1%). This mixture was gently shaken, and smears were made and allowed to air dry. The samples were assessed to count the percentage of alive cells and to study the morphological abnormalities. A spermatozoon completely or partly pink colored was counted as dead. The completely uncolored, bright, or slightly pink cells were considered alive. The sperm cells' morphological abnormalities were conducted in alive and dead spermatozoa. The following formula was used to determine the percentage of alive cells:

Percentage (%) of alive spermatozoa = (number of alive spermatozoa/total number of counted spermatozoa) × 100.

2.4.3. Density

The density of sperm solution was counted after dilution (20 times), fixation with formaldehyde solution (2%), and staining with methylene blue. After cell counting in a Malassez chamber, the density of cells was determined using the following formula:

Concentration of SPERM = number of counted cells ×20 × 10 × 1000 = 2 N × 105.

Where 20: dilution factor, 10: volume of sperm solution, and 1000: conversion factor from mm3 to mL.

Obtained values were averaged and considered as the density of sperm solution extracted from the epididymides caudae.

2.5. The SEM Analysis of Sperm

SEM analysis was applied to study the sperm cells malformations. Sperm cells were washed with 0.1‐M phosphate buffer (pH 7.2) and then pelleted by centrifugation. The obtained sperm pellets were prefixed in 2.5% glutaraldehyde (Sigma Aldrich, Germany) for 90 min, washed three times in the phosphate buffer, and postfixed for 90 min in 1.5% osmium tetroxide (Sigma Aldrich, Germany). The cells were then dehydrated in a series of ethanol solutions of increasing concentrations (30%, 50%, 70%, 90%, 96%, and 3 × 100%), 15 min each. The spermatozoa were dropped and dispersed on clean glass coverslips, air‐dried, mounted on SEM stubs, and then sputtered with gold atoms into a Polaron E5100 sputter coater (Polaron Equipment Ltd., England). Examination of the samples was performed at 30 kV at different magnifications with a Jeol JSM‐25 (Jeol, Tokyo, Japan). Micrographs were recorded with a Pixie 3000 system (Deben, England).

2.6. Testosterone Level Measurement

Blood levels of total unconjugated testosterone (free and protein‐bound) were measured with the Radio Immuno Assay (RIA) kit (Testosterone Double Antibody RIA kit) in rat serum. Steps described in the protocol of the manufacturer MP Biomedicals (formerly ICN Biomedicals Inc., CA, USA) were followed to allow testosterone dosage.

2.7. Statistics

The analysis of our results statistical significance was performed using the software SPSS 13.0 and data were reported as means ± standard deviation (M ± SD). The distribution of the data was not normal and the nonparametric test “t” of Student was used to compare the results. The values of p < 0.05 were considered to be significant.

3. Results

3.1. Impact of Gold on Reproduction Organs Absolute Weight: Testicles; Epididymides, Seminal Vesicles, and Epididymides Caudae

Results presented in Figure 1 showed no significant statistical differences in reproductive organs' absolute weights precisely: testicles, epididymides, epididymides caudae, and seminal vesicles after gold administration (test “t” of Student; p > 0.05).

FIGURE 1.

FIGURE 1

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Unchanged absolute weight of reproduction organs: No changes detected in the weight of testicles; epididymides, seminal vesicles, and epididymides caudae (p > 0.05). 2 Ep. C, two epididymides caudae; L. Ep, left epididymides; L. S. V, left seminal vesicles; L.T, left testicles; R. Ep, right epididymides; R. S. V, right seminal vesicles; R.T, right testicles.

3.2. Impact of Gold on Other Organs Absolute Weight: Kidneys, Liver, and Brain

Figure 2 displayed the recorded values of absolute weight of kidneys, liver, and brain in response to treatment with gold solution. Gold induced a statistically significant increase in the absolute weight of right and left kidneys (test “t” of Student; p < 0.01). No significant differences were observed in the absolute weight of the liver and the brain of treated rats when compared to the control ones.

FIGURE 2.

FIGURE 2

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Changes in absolute weight of nonreproduction organs: Weight of the kidneys (right and left) of gold‐treated rats was significantly higher than that of the control organs (p < 0.01). No significant differences were noticed for the weight of the liver and brain of the two treated groups (p > 0.05). p < 0.01. L. K, left kidneys; R. K, right kidneys. ** Very significantly different from the control group using test “t” of Student at p < 0.01.

3.3. Impact of Gold on the Sperm Cells: Motility, Viability Density, and Sperm Cell Malformations

Figure 3 summarizes the effects of gold administration on the fertility parameters of male rats. Intraperitoneal administration of gold solution induced a very significant diminishing in both motility and viability of the sperm cells (test “t” of Student; p < 0.01) when compared to the control rats.

FIGURE 3.

FIGURE 3

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Incidence of gold administration on fertility parameters of male rats: Motility and viability sperm of gold‐treated rats were significantly decreased (p < 0.01), but nonsignificant modifications were observed for the density of sperm (p > 0.05). ** Very significantly different from the control group using test “t” of Student at p < 0.01.

The Figure 4 showed the difference between motile and nonmotile spermatozoa. While motile cells have a blurred appearance because they are in continuous movement, the nonmotile and fixed ones appear with a clear and translucent look.

FIGURE 4.

FIGURE 4

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(Magnification × 400): The difference between motile and nonmotile spermatozoa: Photomicrograph taken with an optical microscope showing some motile (M: with blurred appearance) and nonmotile (N: with clear appearance) spermatozoa in the sperm of gold‐treated rats.

On eosin and nigrosin stained slides, alive cells with a bright appearance were detected (Figure 5A), and dead spermatozoa appeared partly or totally pink colored (Figure 5B).

FIGURE 5.

FIGURE 5

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(A) Magnification × 600; (B) magnification × 400: The difference between alive and dead spermatozoa: Photomicrograph observed with an optical microscope showing the difference between alive ((A) two bright control cells) and dead cells (B) three totally or partly pink of gold‐treated rats.

The statistical analysis revealed that in the gold‐treated rats, the concentration of sperm cells was insignificantly decreased (test “t” of Student; p > 0.05) when compared to the control group.

Malformations of the studied spermatozoa were detected on eosin and nigrosin stained slides (sperm morphology analysis) and were confirmed by SEM technique observation.

3.4. Study of the Sperm Malformations Using SEM Technique

The observation of spermatozoa shapes and aspect of the gold‐treated rats and control ones was conducted using the SEM technique. This qualitative study showed the presence of many changes in the shape of gold‐treated rat cells when compared to the control cells (Figure 6). However, it looks important to take into account that no standard classification is available for rat sperm malformations, to take as a reference, like the classification of David related to the human sperm malformation.

FIGURE 6.

FIGURE 6

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SEM micrographs showing the differences between normal control cells (A) and (B) and malformations in gold‐treated spermatozoa (C–H): (A) normal spermatozoon of control rat. (B) High magnification of the head rat spermatozoa. In this micrograph, the insertion of the tail to the head called the connecting piece is well observed (arrowhead). (C) A free head of spermatozoon with a hook form. The first level of weakness is observed (arrowhead). (D) A decapitated cell observed in gold‐treated rat sperm. (E) Tail cut into two parts showing the second level of weakness (arrowhead) observed in the spermatozoon of gold‐treated rats. (F) A cut tail where the first (arrowhead) and the second (arrowhead) levels of spermatozoon weakness can be observed. (G) A bent neck spermatozoon observed in gold‐treated rat sperm. (H) Bent tail observed in gold‐treated rat sperm.

According to our results, the spermatozoon of rats seems to have two levels of weakness. The first is just under the head; it is in the connecting piece (Figure 7A). The second, approximately halfway through the tail, is in the annulus (Figure 7B).

FIGURE 7.

FIGURE 7

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SEM micrographs showing the levels of weakness in the rat spermatozoa where they could be bent or broken: (A) The first level of weakness of the rat spermatozoon, located in the connecting piece. (B) the second level of weakness of the rat spermatozoon corresponding to the annulus.

3.5. Incidence of Gold Administration on Testosterone Production

The dosage of the total unconjugated testosterone (free and protein‐bound) in the blood of gold‐treated and control rats showed a very significant decrease (p < 0.01) in this hormone concentration in the treated group when compared to the control group (Figure 8).

FIGURE 8.

FIGURE 8

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Effect of gold administrations to rats on total unconjugated testosterone levels: Gold induced a very significant decrease in total unconjugated testosterone levels measured in the blood of rats.** Very significantly different from the control group using test “t” of Student at p < 0.01.

4. Discussion

4.1. Effect of Gold on Absolute Weight of Reproductive Organs: Testicles; Epididymides, Seminal Vesicles, and Epididymides Caudae

No changes in the absolute weight of reproductive organs (testicles, epididymides, seminal vesicles, and epididymides caudae) were observed after gold administration. These results are concordant with previous research that found no significant changes in absolute organ weights for male and female rats including testicles, seminal vesicles, kidneys, liver, and brain following chronic intravenous injection of gold solution for 28 days (Parveen et al. 2017). On the contrary, the recent study of Abdulhaq et al. showed a significant diminishment in absolute testicle weights when gold chloride was intraperitoneally administered to rats (Adulhaq et al. 2023). Authors explained this divergence by the detrimental effects of gold on germ cell mass leading to a decrease in absolute reproductive organs' weight (Adulhaq et al. 2023; Habas et al. 2021).

4.2. Effect of Gold on the Absolute Weight of Nonreproductive Organs: Kidneys, Liver, and Brain

Gold induced a statistically significant increase in the absolute weight of right and left kidneys (p < 0.01). No significant differences were observed in the absolute weight of the liver and the brain of treated rats when compared to the control ones.

In high‐dose studies, gold‐induced oxidative stress in the liver, kidneys, and brain, but effects remained dose‐dependent, and no reproductive organ hypertrophy was observed even at higher exposure levels (Bekhti Sari et al. 2022).

The kidney is a primary organ of gold excretion via glomerular filtration and tubular reabsorption and secretion, especially for small or soluble gold species. Previous studies found that gold injections resulted in the concentration of its particles under electron‐dense microneedles in lysosomes of proximal convoluted tubular cells (Galle 1974). The biochemical studies showed that the intralysosomal enzyme arylsulfatase was described to concentrate gold with sulfur (Berry et al. 1984; Berry 1996), causing tubular necrosis and swelling, leading to weight gain (Enea et al. 2020; Pereira et al. 2021).

Dose‐dependent histological injuries in kidney, like congested vessels, tubular degeneration, and necrosis, were confirmed after administration of gold, corresponding to renal structural disruption that would raise absolute kidney weight (Bekhti Sari et al. 2022). The hypertrophy induced by the nephrotoxicity of gold is similar to that mentioned for cadmium in rats (El‐Demerdash et al. 2004).

Increase in organ weight typically involves cell proliferation, inflammation, hyperplasia, hypertrophy, or fluid accumulation (Galle 1974; Khan et al. 2013). The latter is the most important explanation for the significant increase in kidney weight in our study. In the same perspective, many reports insist that gold‐induced injuries and oxidative stress lead to toxicity and inflammation, via TNFα and IL6 secretion, and hypertrophy of the renal tissues (Khan et al. 2013; Muller et al. 2017; Enea et al. 2020; Pereira et al. 2021). Despite the confirmed toxicity of gold in kidneys, these organs still activate their defensive scavenging capacity involving the antioxidant system against inflammation, in order to maintain the excretion function (Gottlieb 1979; Jellum et al. 1980; Massarella and Pearlman 1987; Lopez‐Chaves et al. 2018). This confirms the important role of kidneys in the clearance of the blood against exogenous and toxic mineral elements, especially heavy metals like gold (Gottlieb 1979; Jellum et al. 1980; Massarella and Pearlman 1987).

Our results related to the brain and the liver are in line with many others. These reports showed that even when gold particles were intravenously (Parveen et al. 2017) or orally (Jo et al. 2015) administered to rats, there were no changes in brain and liver absolute weight.

Other available data described the gold particles to be safe for humans since they do not induce change or damage in the brain after their administration as a cure for colorectal cancer (Muller et al. 2017). We could explain this outcome by the low capacity of the brain to concentrate heavy metals because of the efficient role of the blood–brain barrier.

The important retention of gold in the liver is expected, but this does not necessarily change the organ mass, especially at low to moderate doses (Yahyaei et al. 2019; Maghraoui et al. 2026). Thus, the capacity of the liver to concentrate heavy metals is guaranteed without incidence on the weight of the organ (Bekhti Sari et al. 2022). To further explain this result, we can evoke the relatively short period of treatment of 2 weeks, the properties of the gold‐containing molecules, and the defensive status of organs. Regarding all these facts, gold accumulation in tissues and the very likely installation of oxidative stress (Bekhti Sari et al. 2022) do not lead to hypertrophy, proliferation, or edema, and the organ weights remain stable (Jo et al. 2015; Parveen et al. 2017).

Further explanation could be advanced, such as the preferential uptake of gold by kidneys, liver, and spleen via phagocytosis or plasma protein adsorption. These organs, known to be organs of blood clearance, activate the elimination of heavy metals in urine and bile solutions. This will minimize the presence of heavy metals, like gold in our case, in reproductive organs as if there is a lack of stimulus for hypertrophy or atrophy of organs, leading to an unchanged absolute organ weight. Meanwhile, oxidative stress or endocrine disruption may alter function without changing organ mass.

4.3. Incidence of Gold on the Exocrine Function of Rat Testicle: Motility, Viability, and Density of Sperm Cells

The motility and viability are important parameters for evaluation of sperm cells' functional capability, whose decrease is a marker of male reproductive alteration and an indicator of infertility (Perreault 1997).

The analysis of gold‐treated rats' sperm quality displayed very significant statistical decreases in sperm motility and viability percentages. The density of sperm solution was insignificantly declined. Figures 4 and 5 showed respectively the differences between motile (M) and nonmotile (N) (Figure 4) and the alive and dead (Figure 5) sperm cells.

These results remind those of Wiwanitkit et al., reporting the action of mixing gold particles with a fresh semen sample taken from a healthy human male (Wiwanitkit et al. 2009); 15 min after exposure to gold solution, they showed that the motility of the sperm declined to 25%, and some human sperm cells were agglutinated and fragmented with an accumulation of gold in the sperm tails and heads. Another study conducted by Taylor et al. (2014) on bovine spermatozoa reported detrimental effects on sperm motility, morphology, and fertilizing capability after mixing with gold particles (Taylor et al. 2014). Nazar et al. (2016) reported an important decrease in the motility of spermatozoa after chronic intraperitoneal injection of gold to mice. These harmful effects of gold particles on sperm parameters were explained by the induction of reactive oxygen species (ROS), resulting in oxidative stress and mitochondrial damage with subsequent metabolic dysfunction (Habas et al. 2021).

A new approach in the development of reproductive biotechnologies is to eliminate germ cells or treat local cancers using gold (Coimbra et al. 2023). That study aimed to evaluate the gold animal sterilizing after intratesticular injections and long‐term adverse reproductive and systemic effects (Coimbra et al. 2023). Testicles sampled from treated rats had histological abnormalities, thinner seminiferous epithelia, and oxidative stress. Although the sperm density was diminished, no alterations in cell morphology were observed in the gold‐treated rats. That study concluded that gold had a long‐term impact on reproduction with very slight alterations in animal health (Coimbra et al. 2023). Available research reported that particles of silver, another noble metal, were generally more toxic than the gold ones after their intraperitoneal administration to male rats. The former metal caused an observable decrease in sperm quantity in the lumen of epididymis tubules with a simultaneous increase in the number of extraepididymis cells in young animals while the latter type of particles had a slight effect on the reproductive system (Kalynovskyi et al. 2016). Kahdem et al. (2020) reported the negative effect of gold metal on male fertility summarized as a significant decrease (p < 0.01) of testosterone level and an extremely significant decrease (p < 0.01) of sperm concentration as compared to the control group.

To further disclose this, many discordances were described by Godatwar et al. (2021). These authors concluded that gold given orally to rats as “Suvarna Bhasma,” a treatment for autoimmune diseases, significantly increased the body and testicular weight, the total sperm count, and the percentage of sperm motility in epididymis fluid. The histological study showed a significant increase in interstitial area of testis, proliferation, and branching of the epithelial layer of seminal vesicle (Godatwar et al. 2021).

The negative effect of gold‐containing molecules administration on spermatogenesis in the present study may be explained by their ability to enhance ROS production, leading to the induction of oxidative stress and disruption of cellular metabolism. This could be associated with the promotion of the related inflammatory response (Yan et al. 2016), as well as with the induction of DNA damage that in turn leads to cell cycle arrest and cytotoxic effects in the germ cells. This may explain why the sperm cell count was slightly decreased.

The presence of gold in cells, especially in the testicular tissues, as it has already been proven in our previous reports, by the Electron Probe Microanalysis (EPMA)—a sensitive analytical technique (Maghraoui et al. 2013), and by the Coupled Plasma–Mass Spectrometry (ICP‐MS)—a quantitative technique (Maghraoui et al. 2026), cannot occur without consequences. This could induce lipid peroxidation of the membranes and organelles of germinal cells. This also destroys the spermatozoa lipid matrix structure. Thus, gold could be described as a disruptive element of the spermiogenic phases in rat testicles, potentially associated with loss or change in the function of the testicles (Chianese and Pierantoni 2021).

4.4. Study of the Sperm Malformations

The purpose of the description of the different malformations presented in our study was to highlight the modifications in spermatozoa shapes after gold administration to rats and to evaluate the incidence of this heavy metal on the quality of rat sperm. This qualitative study allowed the detection of many changes in the shape of the gold‐treated rat spermatozoa when compared to the control cells. These modifications are described here:

  • Normal spermatozoa: Micrographs of normal control rat spermatozoa are presented in Figure 6A,B. Like that of other mammals, the normal rat spermatozoon is composed of three pieces, a head, a midpiece and a tail (Figure 6A). The specificity of the rat sperm cell is the head, which resembles a hook (Figure 6B). In this figure, it is easy to detect the connecting point linking the head to the tail (arrowhead). This region represents one of the fragility levels of the sperm cell.

  • Free head: This malformation is also called a detached head. It is seen as a spermatozoon head separated from the tail. It is presented in Figure 6C where the connection of the head to the tail is well visible (arrowhead).

  • Decapitated cell: It is a tail detached from the head cell. This malformation is presented in Figure 6D.

  • Cut tail: This malformation was observed after the spermatozoa lost the lower part of the tail (Figure 6E) and/or their head (Figure 6F). In these figures, the arrowheads indicate the levels of weakness of the spermatozoon.

  • Bent neck: This malformation has alternative names including kinked neck or coiled neck. It is observed in Figure 6G.

  • Bent tail: This malformation could also be called kinked tail or coiled tail. This malformation is presented in Figure 6H.

Spermatozoa are susceptible cells, reason for which they are suspended in a protective and nourishing medium (Juyena and Stelletta 2012). This fluid is the result of the work of many organs and reproductive glands like seminiferous tubules, epididymides, seminal vesicles, and prostates (Juyena and Stelletta 2012). It provides a unique ionic and hormonal medium with an exact composition that supports reproductive cell development (Gholizadeh et al. 2023), since the controlled ionic environment has a great influence on sperm cells' maturation and function (Hamameh and Gatti 1998). International World Health Organization's guidance on the assessment of seminal plasma includes analysis of some macro‐ and microelements, such as Zinc (Zn) and Selenium (Se), which are associated with sperm quality in humans due to their antioxidant and protective properties (Chia et al. 2000; Massányi et al. 2003; Agarwal and Sekhon 2011).

Thus, exposition of such cells to any exogenous and toxic agents, like gold, or changes in the composition of this fluid could easily disturb their balance, leading to a reduction in their motility, viability, density, as well as to an increase in their breakage and destruction. Many reports described gold as a disturbing agent leading to malformations of spermatozoa including at the level of heads and tails (Nazar et al. 2016; Liu et al. 2020; Adulhaq et al. 2023), but available micrographs showing these structural modifications are very rare.

In the present study, the SEM technique allowed the detection of many malformations of the head and the tail of the rat's spermatozoon that were summarized in Figure 6. The same technique allowed detecting the weakness levels where the spermatozoa could be broken when exposed to toxic metals (Figure 7). These different weaknesses induce the apparition of many malformations, reminding those already observed after indium (In) and aluminum (Maghraoui et al. 2023) administration to male rats. The observed modifications are strongly associated with ROS generation, mitochondrial damage, and DNA or chromatin disruption as well as a decrease in testosterone levels leading to fragility and breakage of spermatozoa at different weakness levels (Figure 7) already detected in the cell.

4.5. Incidence of Gold Administration on the Endocrine Function of the Rat Testicle: Testosterone Production

The measurements of the testosterone levels in the blood of gold‐treated rats showed a very significant decrease as compared to the control ones.

This result is in agreement with previous studies. Adulhaq et al. (2023) demonstrated that administration of gold chloride significantly decreased the testosterone concentration in the blood of rats (Adulhaq et al. 2023). Behnammorshedi et al. reported that daily intraperitoneal administration of gold particles in different doses resulted in a decline of the testosterone level with the increasing gold doses (Behnammorshedi et al. 2015). Similarly, Liu et al. observed that the daily intravenous administration of gold particles solution in mice decreased testosterone production by the Leydig cells (Liu et al. 2020). According to their report, this was due to the downregulation of the expression of the 17α hydroxylase enzyme, playing a crucial role in testosterone production (Liu et al. 2020).

Regarding our results, the decreased testosterone levels may be attributed to alteration of Leydig cells and of their organelles functions, like modification of mitochondrial energetic activity and decline of capacity of lysosomes in storage of lipids. This is correlated to our previous studies showing that gold was concentrated in lysosomes of Leydig cells as electron‐dense microneedles (aurosomes) leading very probably to alterations of these organelles' functions (Maghraoui et al. 2013). The presence of aurosomes at subcellular level enhanced the production of ROS and altered the cell cycle resulting in cytotoxicity and DNA damage, with a significant reduction of testosterone production and thus alteration of the endocrine function of the testicles (Hazout et al. 2008; Liu et al. 2020). Consequently, gold ions have an inhibitory action in mitochondrial membrane depolarization and/or inactivation of mitochondrial enzymes, inducing mitochondrial damage, leading to imbalance of oxidative status and promoting cell necrosis or apoptosis (Umair et al. 2016). All the changes observed in endocrine Leydig cells and their subsequent degeneration explain the decrease in testosterone levels and impaired sperm cell production and maturation of the gold‐treated rats (Liu et al. 2020).

In conclusion, the effects of gold on the organs' weight, except the hypertrophy noticed in the kidneys, were literally insignificant, and this could be explained by the low reactivity of the ions of this metal. However, when functional effects occur, like the decline of fertility (exocrine and endocrine functions), this often happens at the cellular or molecular level, without any impact on the organ weight. All these observed and discussed effects were related to the impact of allochrysine at the administered doses (2100 mg/kg of body weight), which could vary if the doses were changed.

Previous reports show that the decline of fertility could often be reversed when gold exposure stops (Bai et al. 2010; Ren et al. 2016). As a perspective, this hypothesis of reversible effect may be tested a few months after allochrysine administration to rats. This is encouraged especially after the emergence of the facts that gold can be found in breast milk of mothers treated with injectable aurothiomalate (RA treatment) (Ostensen et al. 1986; Ostensen 1992) and that small amounts of gold are detectable in the infant's urine, indicating absorption by the newborn. Also, gold can pass through the placental barrier (Badri et al. 2017; Badri et al. 2020; Myllynen and Vähäkangas 2013), leading to potential teratogenic effects (Ostensen 1992; Muoth et al. 2016); thus, no barrier in the body could stop gold invasion and every defense system could be overcome.

Author Contributions

Samira Maghraoui: conceptualization, methodology, investigation, writing – original draft preparation, data curation. Adrian Florea: software, visualization, investigation, validation, supervision, writing – reviewing and editing. Leila Tekaya: supervision, validation, writing – reviewing and editing.

Funding

The authors have nothing to report.

Disclosure

The work described has not been published previously. The article is not under consideration for publication elsewhere. The article's publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out.

Ethics Statement

Animals used in this study were treated in accordance with the ethic directives in animal experimentation. They were housed in polystyrene cages for acclimation for 8 days prior to initiation of studies. Animals were maintained in an environmentally controlled room with a 12‐h light/12‐h dark cycle, 25°C ± 2°C and humidity of 70% ± 5% and allowed ad libitum access to commercial rat food (SICO society, Sfax, Tunisia) and water (Reference: Committee for the Update of the Guide for the Care and Use of Laboratory Animals; National Research Council. 2010. 243 p). Before starting our experiments, the experimental protocol gets the agreement of two Ethic Committees:

  • 1‐

    In Tunisia: The Ethic Committee of Pasteur Institute of Tunis on behalf with the Medical Experimentation Unit of the Medical School of Tunis; Tunisia. The study was registered under the number: UTM2011/07.01.2011/18.

  • 2‐

    In Romania: The Ethic Committee of the “Iuliu Haţieganu” University of Medicine and Pharmacy, Cluj‐Napoca, Romania registered the protocol under the number 320/20.05.2011.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Data S1: Supporting information.

JAT-46-3403-s001.docx (20.1KB, docx)

Acknowledgements

Open Access funding enabled and organized by CNUDST.

Data Availability Statement

Data are available from the corresponding author on reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Data S1: Supporting information.

JAT-46-3403-s001.docx (20.1KB, docx)

Data Availability Statement

Data are available from the corresponding author on reasonable request.

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