Effect of Manganese Exposure on the Reproductive Organs in Immature Female Rats

Abstract

Manganese (Mn²⁺) is a trace element that is essential for normal physiology, and is predominantly obtained from food. Several lines of evidence, however, demonstrated that overexposure to MnCl₂ exerts serious neurotoxicity, immunotoxicity and developmental toxicity, particularly in male. The present study aimed to evaluate the effect of 0, 1.0, 3.3, and 10 mg/kg/day doses of MnCl₂ on the reproductive organs in the immature female rats. Rats (PND 22; S.D. strain) were exposed to MnCl₂ (MnCl₂ ∙ 4H₂O) dissolved in drinking water for 2 weeks. The animals were sacrificed on PND 35, then the tissues were immediately removed and weighed. Histological studies were performed using the uteri tissue samples. Serum LH and FSH levels were measured with the specific ELISA kits. Body weights of the experimental group animals were not significantly different from those of control group animals. However, ovarian tissue weights in 1 mg and 3.3 mg MnCl₂ dose groups were significantly lower than those of control animals (p<0.05 and p<0.01, respectively). Uterine tissue weights of 3.3 mg dose MnCl₂ groups were significantly lower than those of control animals (p<0.01), while the 1 mg MnCl₂ dose and 10 mg MnCl₂ dose failed to induce any change in uterine weight. Similarly, only 3.3 mg MnCl₂ dose could induce the significant decrease in the oviduct weight compared to the control group (p<0.05). Non-reproductive tissues such as adrenal and kidney failed to respond to all doses of MnCl₂ exposure. The uterine histology revealed that the MnCl₂ exposure could affect the myometrial cell proliferation particularly in 3.3 mg dose and 10mg dose group. Serum FSH levels were significantly decreased in 1mg MnCl₂ dose and 10 MnCl₂ mg groups (p<0.05 and p<0.01, respectively). In contrast, treatment with 1 mg MnCl₂ dose induced a significant increment of serum LH level (p<0.05). The present study demonstrated that MnCl₂ exposure is capable of inducing abnormal development of reproductive tissues, at least to some extent, and altered gonadotropin secretions in immature female rats. Combined with the well-defined actions of this metal on GnRH and prolactin secretion, one can suggest the Mn²⁺ might be a potential environmental mediator which is involved in the female pubertal process.

INTRODUCTION

Manganese (Mn²⁺) is a trace metal and is essential element that is required for normal mammalian physiology. Several enzyme systems have been reported to interact with or depend on Mn²⁺ for their optimal catalytic or regulatory function (Gunter et al., 2006). On the other hand, excessive exposure to Mn²⁺ seems to cause serious neurotoxicity, immunotoxicity and developmental toxicity, particularly in male (Michalke et al., 2007).

Concerning the reproductive toxicity of Mn²⁺, studies have been focused on the mammalian testicular dysfunction. Chronic exposure to Mn₃O₄ (1,050 ppm) retarded the sexual development shown significantly smaller testis, seminal vesicle, and preputial gland weights in mice (Gray & Laskey, 1980; Webster & Valois, 1987). In human, occupational exposure to Mn²⁺ decreased libido and impotency (Emara et al., 1971; Mena et al., 1967), and may result in lowered sperm count and semen quality (Hjollund et al., 1998).

So far, however, research on the effect of Mn²⁺ exposure on female reproductive physiology has been conducted poorly. The present study aimed to evaluate the effect of 0, 1.0, 3.3, and 10 mg/kg/day doses of MnCl₂ on the reproductive organs in the immature female rats.

MATERIALS & METHODS

1. Animals

Timely pregnant Sprague-Dawley rats were obtained from DBL (Chungcheongbuk-do, Korea) and reared in sangmyung university animal facility under conditions of 12-h light/dark cycle (lights on at 07:00 h) and constant temperature of 22±1 °C. During pregnancy and lactation, the mothers had free access to normal chow and tap water. All procedures used were approved by the Animal Care and Use Committee at Sangmyung University.

2. Experimental design

The day after weaning (postnatal day 22, PND 22), female dams were randomly assigned to the following exposure groups (n=8 dams/group) from PND 22 until PND 35: (1) oral administration of 0.9% Saline (300 μℓ /100 g BW/day, Control group), (2) oral administration of 1.0 mg/kg BW/day MnCl₂ ∙ 4H₂O (Sigma-Aldrich), (3) oral administration of 3.3 mg/kg BW/day MnCl₂ ∙ 4H₂O, and (4) oral administration of 10 mg/kg BW/day MnCl₂ ∙ 4H₂O. Both saline and Mn²⁺ solution were supplied daily for 2 weeks. At PND 35, animals were sacrificed and the tissues(ovary, uterus, oviduct, adrenal, and kidney) were immediately removed and weighed at 1,800 hour.

3. Histology

Uterine tissue specimens were fixed 4% paraformaldehyde then were serially dehydrated in graded ethanol and xylene. Specimens were paraffin embedded and sectioned at 5 μm thickness. Sections were stained with Hematoxylin-Eosin (H & E) stain and examined under light microscope.

4. Measurement of serum gonadotropin levels

The trunk blood samples were collected and centrifuged at 3,000×g for 15 min. The measurements of the serum FSH and LH were carried out according to the commercial instructions for the specific ELISA kits (USCN, China). The sensitivities of the FSH and LH assay were 0.55 ng/ml and 122.5 pg/ml, respectively. The intra-assay and inter-assay coefficients of variation were <10% and <12% for both hormones, respectively.

5. Statistical analysis

All values are expressed as the means (±S.E.). Differences between control and treatment groups were analysed by Student’s t-test. P values less than 0.05 were considered significant. The IBM PC programs INSTAT and PRISM 3.0 (GraphPad, San Diego, CA, USA) were used to calculate and plot the results.

RESULTS

1. Tissue weights

In order to evaluate a potential effect of Mn²⁺ on female reproductive organs, we measured the tissues weights of ovary, uterus and oviduct. Kidney and adrenal weight served as non-reproductive tissues. After 2 weeks of administration, body weights of the all Mn²⁺ exposure group animals were not significantly different from those of control group animals (Fig. 1, A). However, ovarian tissue weights (Fig. 1, B). in 1 mg MnCl₂ dose group (19.06±0.98 mg, p<0.05) and 3.3 mg MnCl₂ dose group (17.47±1.16 mg, p<0.01) were significantly lower than those of control animals (22.39±0.97 mg). Uterine tissue weights (Fig. 1, C). of 3.3 mg dose MnCl₂ groups were significantly lower than those of control animals (Control : 3.3 mg dose MnCl₂ = 67.52±5.15 : 50.04±2.41 mg, p<0.01), while the 1 mg MnCl₂ dose (Control : 1 mg dose MnCl₂=67.52±5.15 : 67.11±3.28 mg) and 10 mg MnCl₂ dose (Control : 10 mg dose MnCl₂ = 67.52±5.15 : 66.70±4.03 mg) failed to induce any change in uterine weight. Similarly, only 3.3 mg MnCl₂ dose could induce the significant decrease in the oviduct weight (Fig. 1, D). compared to the control group (Control : 3.3 mg dose MnCl₂ = 7.52±0.40 : 6.35±0.29 mg, p<0.05). Non-reproductive tissues such as adrenal (Control : 1 mg : 3.3 mg : 10 mg dose MnCl₂ = 12.13±0.28 : 11.93±0.38 : 11.75±0.42 : 12.59±0.43 mg) and kidney (Control : 1 mg : 3.3 mg : 10 mg dose MnCl₂ = 574.74±8.99 : 584.56±12.38 : 592.42±7.82 : 603.67±14.06 mg) failed to respond to all doses of MnCl₂ exposure (Fig. 1, E & F, respectively).

Fig. 1.

Effect of MnCl₂ exposure on changes in body weights and tissues weights. Animals were daily supplied 0.9% Saline (300 μℓ/100 g BW/day, Control) or MnCl₂ ∙ 4H₂O (1.0 mg/kg BW, 3.3 mg/kg BW, 10 mg/kg BW, respectively) for 2 weeks. A, body weight; B, ovary; C, uterus, D, oviduct, E, adrenal, and F, kidney. Values are expressed as mean±S.E. (n=8 per group). *, Significantly different from control group, p<0.05. **, Significantly different from control group, p<0.01.

2. Uterine histology

To access the histological changes in MnCl₂ exposured uteri, standard paraffin section and hematoxylin-eosin staining method were employed. 3.3 mg and 10 mg MnCl₂ dose groups shown the thickend myometrial layer when compared to control (Fig. 2, Mn3.3 & Mn10). In contrast, 1 mg MnCl₂ exposure reduced the thickness of myometrium (Fig. 2, Mn1).

Fig. 2.

Effect of MnCl₂ exposure on histological changes in uteri from immature rats. Standard paraffin section and hematoxylin-eosin staining method were employed. Mn 1, 1.0 mg/kg BW/ day of MnCl₂ ∙ 4H₂O exposure; Mn 3.3, 3.3 mg/kg BW/day of MnCl₂ ∙ 4H₂O exposure; Mn 10, 10 mg/kg BW/day of MnCl₂ ∙ 4H₂O exposure, respectively.

3. ELISA

Serum LH and FSH levels were measured using specific ELISA kits. Fig. 3 (A) shows that the secretion of FSH was significantly decreased by treatment with 1 mg or 3.3 mg MnCl₂ (Control:1 mg dose MnCl₂ = 3.54±0.18 : 3.07±0.14 ng/ml, p<0.05; Control : 10 mg dose MnCl₂ = 3.54±0.18:2.75±0.10 ng/ml, p<0.01). Serum LH level was significantly elevated by 1 mg MnCl₂ exposure (Control : 1 mg dose MnCl₂ = 39.85±8.93:76.71±12.36 ng/ml, p<0.05, Fig. 3, B). Higher doses of MnCl₂ exposure failed to change the serum LH levels.

Fig. 3.

Measurement of serum FSH and LH levels in response to Mn²⁺ exposure. The trunk blood samples were collected and centrifuged at 3,000×g for 15 min. The measurements of the serum FSH and LH were carried out according to the commercial instructions for the specific ELISA kits (USCN, China). Values are expressed as mean±S.E. (n=6 per group). *, Significantly different from control group, p<0.05. **, Significantly different from control group, p<0.01.

DISCUSSION

In the present study we demonstrated that the Mn²⁺ exposure could change the weights of reproductive tissues in immature female rats. Although we failed to find the advance or delay of puberty onset in the Mn²⁺ exposured animals (data not shown), the potential reproductive toxicity of this metal in immature female rats cannot be ruled out. Mn²⁺ exposure, Indeed, not only affected the proliferative activity in myometrial layer, but induced significant changes in the serum FSH and LH levels.

More defined doses and exposure periods will be helpful to verify the physiological relevance of Mn²⁺ exposure in female reproduction.

Occupational exposure to Mn²⁺ could be occurred often at the workplaces such are mines and dried battery factories (Emara et al., 1971; Mena et al., 1967). In this respect, most studies on the toxicological effects of Mn²⁺ have been focused on the men. Furthermore, a relatively small portion of the studies dealt with reproductive toxicity of Mn²⁺ exposure using male animal models. Mn²⁺ exposure for 2 and 4 h inhibited rat primary Leydig cell steroidogenesis by decreasing StAR protein expression while 24 and 48 h exposure of MnCl₂ caused adverse effects on both StAR protein and P450scc and 3b-HSD enzyme activity to reduce steroidogenesis (Cheng et al., 2003).

Pine et al. (2005) reported that Mn²⁺ administered acutely into the third ventricle shown dose-dependently to stimulate LH release in prepubertal female rat, and this effect was due to a Mn²⁺-induced stimulation of GnRH. The authors demonstrated that Mn²⁺ can stimulate specific puberty-related hormones and suggested that it may facilitate the normal onset of puberty. According to them, Mn²⁺ may contribute to precocious puberty if an individual is exposed to elevated levels of Mn²⁺ too early in developmental process. This hypothesis was verified by same research group; Lee et al. (2006) demonstrated that Mn²⁺ is a direct stimulator of prepubertal GnRH/LH secretion and may facilitate the normal onset of male puberty. Their data that Mn²⁺ can cause GnRH release in adult males, suggested this action should be considered in relation to age, gender, as well as mechanistic and functional differences between adult and immature animals.

In the present study we failed to confirm the advanced puberty onset in Mn²⁺ exposed female rats (up to 10 mg/kg BW, data not shown). Pine et al. (2005) shown that same dose of Mn²⁺ exposure initiated a moderate but significant advancement in age at vaginal opening (VO) in terms of days (1.5 days). The only difference between the two studies was timing and duration of Mn²⁺ exposure; Our treatment regimen was PND 21-35 (14 days), and theirs was PND 12-29 (18 days). Though Mn²⁺ can acutely induce GnRH secretion in adult males, one should consider that additional action of Mn²⁺ to release GABA, a GnRH inhibitor, may ultimately contribute to suppressed reproductive function observed in adult animals following exposure to high chromic levels of Mn²⁺ (Prestifilippo et al., 2008). Because neurotransmitter secretory circuits in female during peripubertal period are remarkably unstable, the extent of GABA GnRH regulation, and probably more neuronal regulation, may vary responding to the minor difference. Studies indicate the more specific action mechanism of Mn²⁺ within the hypothalamus; Mn²⁺ activates soluble guanylate cyclase (sGC) directly and/or as a cofactor with available nitric oxide (NO), hence generating cGMP and resulting in prepubertal GnRH release (Lee et al., 2007). More recently, Mn²⁺, through the upregulation of IGF-1 and COX-2, may promote maturational events and glialneuronal communications facilitating the increased neurosecretory activity, including that of GnRH, resulting in precocious pubertal development (Hiney et al., 2011).

Special emphasis should be made on the action of Mn²⁺ in brain. Inhalation of the mixture of MnCl₂ and Mn(OAc)3 for 5 months developed movement abnormalities, significant loss of substantia nigra compacta (SNc) dopaminergic neurons; these symptoms similar to those observed in Parkinson disease (PD) (Ordoñez-Librado et al., 2011). Dopamine (DA) depletion is closely related to pituitary prolactin biosynthesis. Male rats exposed to Mn²⁺ for 4 or 13 weeks showed a progressive and significant decrease in hypothalamic DA, whereas prolactin and Pit-1 mRNA levels increased in response to Mn²⁺ exposure (Kim et al., 2009). These results suggest that exposure to Mn²⁺ decreases hypothalamic DA and promotes the production of prolactin in the pituitary and that Pit-1 might be a regulator of DA and prolactin. Furthermore, such Mn²⁺ exposure induced significant increase in serum prolactin levels seemed to be highly correlated with the testis toxicity (Lee, 2009). In this context, relationship between Mn²⁺ exposure and DA/prolactin secretions in immature female rats will be helpful to understand the function of the metal during pubertal development.