Excessive dietary calcium in the disruption of structuraland functional status of adult male reproductive system in ratwith possible mechanism

Amar K Chandra • Pallav Sengupta •

Haimanti Goswami • Mahitosh Sarkar

Received: 21 August 2011 / Accepted: 21 December 2011 / Published online: 20 January 2012

� Springer Science+Business Media, LLC. 2012

Abstract Calcium is essential for functioning of different

systems including male reproduction. However, it has also

been reported as chemo-castrative agent. The study has

been undertaken to elucidate the effect of excessive dietary

calcium on male reproductive system in animals with

possible action. Adult male healthy rats fed CaCl2 at dif-

ferent doses (0.5, 1.0 and 1.5 g%) in diet for 13 and

26 days to investigate reproductive parameters as well as

the markers of oxidative stress. Significant alteration was

found (P \ 0.05) in testicular and accessory sex organs

weight, epididymal sperm count, testicular steroido-

genic enzyme (D5 3b-HSD and 17b-HSD) activities, serum

testosterone, LH, FSH, LPO, activities of antioxidant

enzymes, testicular histoarchitecture along with adrenal D5

3b-HSD activity with corticosterone level in dose- and

time-dependent manner. Overall observations suggest that

excessive dietary calcium enhances the generation of free-

radicals resulting in structural and functional disruption of

male reproduction.

Keywords Calcium chloride � Steroidogenic enzyme �Oxidative stress � Antioxidant enzyme �Lipid peroxidation � Superoxide dismutase

Abbreviations

ROS Reactive oxygen species

ASg Type A spermatogonia

pLSc Preleptotene spermatocytes

mPSc Mid-pachytene spermatocytes

7Sd Step 7 spermatids

LH Luteinizing hormone

FSH Follicle stimulating hormone

HSD Hydroxysteroid dehydrogenase

NAD Nicotinamide adenine dinucleotide phosphate

LPO Lipid peroxidation

TBARS Thiobarbituric acid reactive substances

MDA Malondialdehyde

SOD Superoxide dismutase

CAT Catalase

EDTA Ethylenediamine tetraacetic acid

BSA Bovine serum albumin

TMB Tetramethylbenzidine

ANOVA Analysis of variance

HPG Hypothalamo–pituitary–gonadal axis

HPA Hypothalamo–pituitary–adrenal axis

PVN Paraventricular nucleus

CRH Corticotrophin releasing hormone

ACTH Adreno-corticotropic hormone

PUFA Poly unsaturated fatty acid

IP3 Inositol tri-phosphate

Introduction

Calcium, being one of the most important cation in physio-

logical system, plays a pivotal role in the physiology

and biochemistry of physiological system, and its critical

functions in human health have been recognized for many

years, as reflected by a long history of calcium intake

A. K Chandra (&) � P. Sengupta � H. Goswami

Endocrinology & Reproductive Physiology Laboratory,

Department of Physiology, University College of Science &

Technology, University of Calcutta, 92, Acharya Prafulla

Chandra Road, Kolkata 700 009, West Bengal, India

e-mail: amark_chandra@yahoo.co.in

M. Sarkar

Department of Physiology, Gurunanak Institute of Dental

Science & Research, Kolkata, India

123

Mol Cell Biochem (2012) 364:181–191

DOI 10.1007/s11010-011-1217-3

recommendations [1]. Its deficiency leads to osteoporosis,

arthritis, hypertension and other diseases [2], whereas its

excess have detrimental effects on health. The most exten-

sively studied adverse effects of excess calcium include

nephrolithiasis, hypercalcemia and renal insufficiency

(milk-alkali syndrome) [3, 4], and the adverse effects of

calcium on the metabolism of other minerals, such as iron

and zinc, have also been reported [5, 6]. The effect of excess

of calcium in the body may be because of excessive intake of

the increasing number of calcium-fortified food products [7]

or may be because of over calcium supplementation [8] or

drinking of hard water containing excessive calcium salt [9].

Excess calcium salt has been reported as castrative agent

[10], whereas some other experiments contradict its chem-

ocastrative role [11]. There are very few references on the

effect of excess calcium on testicular spermatogenesis or

androgenesis, showing excess calcium may alter steroido-

genesis in the Leydig cells [12]. Therefore, information

about the effects of excess dietary calcium on male repro-

ductive physiology is not inclusive. Thus, this investigation

has been undertaken to explore the effect of excessive dietary

calcium on adult male gonadal system, as consumed by the

people living in hard water areas, and also to find out the

chemosterilizing potentiality of dietary calcium in adult

male rats which is non-invasive and does not require any

special care of administration, as in the earlier studies of

chemocastrative action of calcium chloride has only been

investigated after intratesticular injection [10, 11]. To sub-

stantiate the activity of excessive calcium, the effects of

supplemented calcium chloride at different concentra-

tions for different durations in male rats on testicular hist-

oarchitecture, serum levels of testicular and adrenal steroid

hormones, namely, testosterone and corticosterone, steroi-

dogenic enzymes activities, namely, testicular and adrenal

D5 3b-hydroxy steroid dehydrogenase and testicular 17b-

hydroxy steroid dehydrogenase and serum LH and FSH

levels were investigated. As calcium has also been reported

to have oxidative and nitrosative stress effects [13], thus the

development of oxidative stress, if any, was evaluated by

assaying antioxidant enzymes (namely, superoxide dismu-

tase [SOD] and catalase [CAT]) profile and lipid peroxida-

tion (LPO) in testis of the calcium-treated animals to explore

the possibility, if any, of the development of oxidative stress

in testis and its role in male reproductive impairment.

Materials and methods

Reagents

Calcium chloride (fused) (CaCl2), thiobarbituric acid

(TBA), NAD, NADPH, chloramines-T and -estradiol

(98%) were procured from Sigma Chemical Company,

St. Louis, M.O., USA. Potassium dichromate (K2Cr2O7),

triton X, triethanol amine, diethanolamine, ethylenedia-

mine tetraacetic acid (EDTA) and MnCl2 from E-Merck,

Mumbai, India. ELISA kit obtained from Equipar Diag-

nostic, SRL, Italy (code no. 74010). All other reagents

were procured from Sisco Research Laboratories (SRL),

Mumbai, India, and all were of analytical grade.

Animal maintenance and dietary calcium treatment

A total of 64 healthy adult (90 ± 10 days) male albino rats

(Rattus norvegicus) of Wistar strain weighing 140 ± 10 g

were used in this study. The animals were collected and

maintained as per national guidelines and protocols and the

proposal was approved by the Institutional Animal Ethics

Committee (PHY/CU/IAEC/16/09 dated 15.05.2008). The

animals were housed in clean polypropylene cages and

maintained in an air-conditioned animal house (temperature:

22 ± 2�C; relative humidity: 40–60%) with constant 12:12

light: dark schedule. The animals were fed on standardized

normal diet (20% protein) prepared in laboratory that con-

sisted of 70% wheat, 20% Bengal gram, 5% fish meal

powder, 4% dry yeast powder, 0.75% refined til oil, 0.25%

shark liver oil, 4% salt and water ad libitum [14].

In the 13 and 26 days treatment, respectively, the

experimental animals were divided into eight groups of eight

animals each, as follows (four groups for each duration):

First group was kept as control and fed with normal diet.

Second group received 0.5 g CaCl2/100 g diet/day. Third

group was treated with 1.0 g CaCl2/100 g diet/day. Fourth

group was subjected to 1.5 g CaCl2/100 g diet/day treatment

[15]. All animals were caged individually. Feed consump-

tion, corrected for feed waste, was measured. Treatment

schedule was selected to determine the effect of calcium on

one and two seminiferous cycles as duration of one semi-

niferous cycle is 13.2 days in albino rats [16]. All animals

were sacrificed 24 h after the last treatment following pro-

tocols and ethical procedures by cervical dislocation. Blood

samples for hormone assay were collected from the hepatic

vein. Plasma samples were separated by centrifugation,

frozen and stored at -50�C until assayed. The testis and

accessory sex organs were dissected out, trimmed off the

attached tissues and weighed. The left testis of each rat was

fixed immediately for histological study and the right for

other biochemical estimations.

Food consumption pattern

Food consumption per rat per day was recorded daily

during the period of treatment. Food consumption (g/rat/

day) was calculated, for each rat as,

Food given gð Þ � Food wasted gð Þ¼ Food Consumption gð Þ:

182 Mol Cell Biochem (2012) 364:181–191

123

Body weight and organ weights

The body weights were recorded on the first day before

treatment (initial) and on the day of sacrifice (final). The

testicles and accessory sex organs, namely, seminal vesi-

cles, ventral prostate, cauda epididymis and coagulating

gland were dissected out, trimmed off the attached tissues

and weighed. The relative weight of organs was expressed

per 100 g body weight.

Histopathological study

Immediately after removal, the testis, accessory sex organs

and adrenal gland were fixed in formol fixative and testis

was fixed in Bouin’s fluid and embedded in paraffin. Sec-

tions of 5 lm thickness were taken from the mid portion of

each organ and stained with hematoxylin–eosin Fig. 1.

Each slide was examined under a light microscope. Sem-

iniferous tubular diameter was measured by an ocular

micrometer under light microscope. Quantitative analysis

of spermatogenesis was carried out by counting the relative

number of each variety of germ cells at stage VII of the

seminiferous epithelium cycle, i.e., type A spermatogonia

(ASg), preleptotene spermatocytes (pLSc), mid-pachytene

spermatocytes (mPSc) and step 7 spermatids (7Sd), fol-

lowing the method of Leblond and Clermont [17]. Theo-

retically, the mPSc to 7Sd ratio should be 1:4 [18]. The

percentage of 7Sd degeneration was calculated from this

ratio. Subtraction of the percentage of 7Sd degeneration in

vehicle-treated rats showed the effective percentage of

spermatid degeneration.

Sperm count

Sperm samples were collected from the cauda epididymis

and counted by a haemocytometer chamber under light

microscope [19]. To minimize error, count was repeated at

least five times for each rat.

Determination of LPO

It was measured following the method of Ohkawa [20] using

TBA–TCA–HCl reagent. A mass of 200 mg of tissue was

taken with 2 ml of phosphate buffer (50 mM, pH 7.4) solu-

tion, and was homogenized at 4�C. Following homogeni-

zation, 2 ml of homogenate was mixed thoroughly with 2 ml

of TBA–TCA–HCl mixture and heated in a boiling water

bath for about 15–20 min. After cooling, the precipitate was

removed by centrifugation at 10,000 rpm for 10 min.

Finally, reading of the sample was measured against the

blank at 532 nm.

Fig. 1 Photomicrographs of paraffin-embedded H&E-stained rat

testicular sections (9400) showing the effect of calcium in exposed

animals. (A) Testicular section from control rats and testicular

sections from calcium-treated animals with 0.5 g% of CaCl2(B) 1.0 g% CaCl2 (C) and 1.5 g% CaCl2 (D), respectively, for

13 days; testicular sections from calcium-treated animals with 0.5 g%

of CaCl2 (E) 1.0 g% CaCl2 (F) and 1.5 g% CaCl2 (G), respectively,

for 26 days. ASg spermatogonia A, pLSc preleptotene spermatocytes,

mPSc mid-pachytene spermatocytes, 7Sd step 7 spermatid

Mol Cell Biochem (2012) 364:181–191 183

123

Determination of CAT activity

Catalase activity was determined following the method of

Aebi [21].The tissue was homogenized with sucrose

(0.25 M) solution at 4�C. It was centrifuged at 10,000 rpm

for 20 min. The pellet was removed and the remaining

supernatant was again centrifuged at 2,500 rpm for 10 min

and post mitochondrial supernatant (PMS) was prepared.

100 ll of tissue sample containing PMS and 2.8 ml of

phosphate buffer (50 mM, pH 7.8) are mixed in a cuvette

and decrease in the absorbance was recorded at 240 nm for

5 min during a 60 s interval before the addition of 60 mM

H2O2.

Determination of SOD activity

Superoxide dismutase was determined by the method of

Marklund et al. [22]. A mass of 100 mg of tissue was

mixed with phosphate buffer (50 mM, pH 7.8) solution and

tissue homogenate was prepared at 4�C. Tissue homoge-

nate was centrifuged at 10,000 rpm for about 25 min. A

volume of 100 ll of supernatant was taken in a cuvette and

the assay volume contain 100 mM triethanolamine–dieth-

anolamine–HCl buffer (pH 7.4) along with 7.5 mM

NADPH, 100 mM EDTA and 50 mM MnCl2 (pH 7.0). The

solution was kept at room temperature for about 5 min to

stabilize. The decrease in absorbance was noted at 340 nm

for 20 min during a 5 min interval at 25�C after the addi-

tion of 10 mM mercaptoethanol.

Assay of steroidogenic enzymes

D5 3b - Hydroxysteroid dehydrogenase (HSD)

To study testicular and adrenal D5 3b - HSD enzyme

activity [23], tissues were homogenized, maintaining

chilling conditions (4�C) in 20% spectroscopic-grade

glycerol containing 5 mM of potassium phosphate and

1 mM of EDTA at a tissue concentration of 100 mg/ml

homogenizing mixture in a Potter–Elvehjem glass

homogenizer. This mixture was centrifuged at 10,000 g for

30 min at 4�C in a cold centrifuge (REMI, C40). A volume

of 200 ll supernatant was mixed with 1 ml of 100 lM

sodium pyrophosphate buffer (pH 8.9) and 20 ll of 30 lg

17-estradiol. 53-HSD activity was measured after the

addition of 1 ml of 0.5 lM nicotinamide adenine dinu-

cleotide phosphate (NAD) to the cuvette in a UV spec-

trophotometer (UV-1240 Shimadzu, Japan) at 340 nm

against a blank without NAD. One unit of enzyme activity

is equivalent to a change in absorbance of 0.001/min at

340 nm.

17b - HSD

For testicular 17-HSD enzyme assay [24], the same

supernatant fluid (200 ll) (prepared as described earlier)

was added with 1.5 ml of 440 lM sodium pyrophosphate

buffer (pH 8.9), 0.5 ml of bovine serum albumin (BSA)

(25 mg crystalline BSA) and 40 ll of 0.3 lM 17-estradiol.

17-HSD activities were measured after the addition of 1 ml

of 1.35 lM NAD to the cuvette in a UV spectrophotometer

(UV-1240 Shimadzu, Japan) at 340 nm against a blank

without NAD. One unit of enzyme activity is equivalent to

a change in absorbance of 0.001/min at 340 nm.

Protein estimation

Proteins were estimated by the method of Lowry et al. [25]

using BSA as the standard protein.

Estimation of tissue and serum calcium

Tissue and serum calcium was estimated by the method of

Baginski et al. [26] and values are expressed in lg/g of

tissue wet weight and lg/g of tissue, respectively.

ELISA of serum testosterone

Serum testosterone was assayed using ELISA kit obtained

from Equipar Diagnostici, srl, Italy (code no. 74010). In this

method, serum sample (25 ll) was taken in a micro-plate

well and enzyme–testosterone conjugate was added, then the

reactant was mixed. After the completion of the required

incubation period (60 min at 37�C), the antibody-bound

enzyme testosterone conjugate was separated from the

unbound enzyme testosterone conjugate by decantation. The

activity of the enzyme present on the surface of the well is

quantitated by the reaction with tetramethylbenzidine

(TMB) substrate solution with 15 min incubation and finally

by adding 0.3 M H2SO4 as stop solution. The absorbance

was read against blanking well at 450 nm within 30 min in

ELISA Reader (Merck). The sensitivity of the testosterone

assay was 5 pg/ml and inter- and intra-run precision had a

coefficient of variation of 3.9 and 6.2%, respectively.

Radioimmunoassay (RIA) of follicle stimulating

hormone (FSH) and luteinizing hormone (LH)

Serum levels of FSH and LH were assayed by RIA [27] using

reagents supplied by Rat Pituitary Distribution and NIDDK

(Bathesda, MD, USA). Carrier-free 125I for hormone iodin-

ation was obtained from Bhabha Atomic Research Center

(BARC), Mumbai, India. Pure rat FSH (NIDDK-r FSH-I-11)

184 Mol Cell Biochem (2012) 364:181–191

123

and LH (NIDDK-r LH-I-11) were iodinated using chloram-

ines-T as the oxidizing agent following the standard proce-

dure [28]. NIDDK anti-rat FSH-S-11 and anti-rat LH-S-11 in

rabbit were used as antiserum at a final dilution of 1:35000

and 1:70000, respectively. The second antibody was

goat anti-rabbit-globulin purchased from Indo-Medicine

(Friendswood, TX, USA). The intra-assay variation for FSH

and LH was 5.0 and 4.5%, respectively. All samples were run

in one assay to avoid inter-assay variation.

Spectrofluorometric determination of serum

corticosterone

Serum corticosterone level was determined by spectrofluo-

rometry according to the method of Glick et al. [29] modified

by Silber [30] and Biswas et al. [31, 32]. The fluorescence

was measured at 463 nm (excitation) and 518 nm (emission)

by setting the instrument at a spectrofluorometric reading 80

with standard corticosterone (Sigma Chemical Company,

St. Louis, MO, USA) solution having concentration 1.6 lg/

ml. A minimum 1.6 lg of corticosterone per 100 ml serum

can be measured by this method.

Statistical analysis

Results were expressed as mean ± standard deviation (SD).

One-way analysis of variance (ANOVA) test was first car-

ried out to test for any differences between the mean values

of all groups. If differences between groups were estab-

lished, the values of the treated groups were compared with

those of the control group by a modified t test. A value of

P \ 0.05 was interpreted as statistically significant [33].

Results

Body weight and accessory sex organ weights

Excessive dietary calcium resulted in a significant reduc-

tion of net body weight gain and all reproductive organ

weights over the control values in dose and time depen-

dently (Tables 1, 2), though all the control and treated

animals taken food and water properly.

Food consumption pattern

No significant alteration was found in food consumption

pattern (g/rat/day) in any of the cases of calcium treatment

(Table 3).

Tissue and serum calcium levels

A significant increase in both testicular (lg/g) and serum

calcium (mg/dl) was observed (Table 4).

Testicular and adrenocortical steroidogenic enzymes

activities

On calcium treatment, there was a significant reduction in the

activities of testicular D53b-HSD and 17b- HSD over control

values (Fig. 2A, B). On the contrary, adrenal D53b-HSD

activity has been found to be significantly increased (Fig. 3A).

Epididymal sperm count

Epididymal sperm count in calcium-treated rats was sig-

nificantly decreased (Fig. 4).

Hormone levels

In rats, calcium treatment lowered serum testosterone levels

over the control values (Fig. 5A). In all calcium-treated rats,

LH levels were significantly reduced than the corresponding

value for the respective control rats (Fig. 5C). But serum

FSH was elevated following treatment (Fig. 5B). Serum

corticosterone level was also found to be increased than

respective controls in dose- and time-dependant manner

(Fig. 3B).

Histopathological changes

Histological section of testis from control rats revealed the

presence of normal seminiferous tubules undergoing sper-

matogenic cycle composed of spermatogonia, spermato-

cytes, round spermatids and elongated spermatids consistent

with intact spermatogenesis. In contrast, testicular sections

Table 1 Alterations in the body weight (g) of experimental animals subjected to different doses of calcium treatment for different durations

Parameters 13 Days treatment 26 Days treatment

Control Calcium

0.5%

Calcium

1.0%

Calcium

1.5%

Control Calcium

0.5%

Calcium

1.0%

Calcium

1.5%

1. Body weight (initial) 148 ± 5.5 149.2 ± 6.1 144.5 ± 4.7 143.5 ± 7.8 148.2 ± 4.94 147.2 ± 3.5 145.5 ± 5.8 147.2 ± 6.1

2. Body weight (final) 173.5 ± 5.7 175 ± 6.3 164.2 ± 8.3 160.5 ± 11.3 191.7 ± 6.88 190.2 ± 5.0 182.7 ± 9.2 172.2 ± 8.9

3. Body weight

gain (%)

17.2% 17.1% 14.1% 11.7% 29.3% 29.1% 25.5% 16.9%

Data are presented as the mean ± SD, n = 8

Mol Cell Biochem (2012) 364:181–191 185

123

from treated rats showed testicular lesions ranging from

absence of elongated spermatids to reduction of postsper-

matogonial germ cells. Testicular sections from rats treated

with calcium showed significant reduction in the number of

ASg, pLSc, mPSc and step 7 spermatid (7Sd) over the

control values. The percentage spermatid degeneration was

highly significant after calcium treatment when compared

with those of the control (Table 5).

Testicular LPO level and antioxidant enzyme profile

An increase in the status of testicular antioxidant enzymes,

namely, SOD and CAT, in 13 days treated rats and significant

decrease in 26 days treated rats was observed (Fig. 6B, C)

with a concomitant rise in the rate of LPO in all the calcium-

treated groups was observed in time and dose dependently

(Fig. 6A) when compared with the respective control group.

Discussion

Diet containing excess calcium found to decrease body

weight, weights of testis and other accessory sex organs in

dose- and time-dependent approach (Tables 1, 2). The net

body weight gain of the animals treated with CaCl2 was

found to be markedly less at doses of 1.0 and 1.5 g% as

compared with their respective controls in time-dependent

manner, though the average daily food intake did not differ

between the groups during study period (Table 3) which is

consistent with reports that suggest calcium has negative

association with body weight [34] signifying the mecha-

nism either by increasing core body temperature [35] or

reduced body fat accumulation [36, 37].

Ingestion of calcium regularly showed a decrease in

testicular weight in dose-dependent manner. The weight of

testis is largely dependent on the mass of the differentiated

spermatogenic cells and hence a reduction in the weight of

the testis is because of the decreased number of germ cells

and elongated spermatids [38]. In histopathological

examination of testicular sections, it is clear that higher

doses of calcium affect growth and development/degener-

ation of germ cells at their different levels of maturation.

The mechanism of testicular germ cell degeneration

may be down to transient Ca2? influx that altered redox

potential of the cells leading to generation of reactive

oxygen species (ROS) that facilitates LPO of germ cells

resulting in cell death [39]. It is evident from the study that

regular ingestion of calcium significantly increases both

testicular and serum calcium level (Table 4). When ROS-

induced damage occurs in testicular cells, its antioxidant

enzymes come into play, SOD, the first enzyme involved in

the antioxidant defense, and hemoprotein CAT are the

major marker of cellular oxidative stress [40]. This defenseTa

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186 Mol Cell Biochem (2012) 364:181–191

123

mechanism has been reflected in elevated SOD and CAT

activities in 13 days calcium-treated animals reflecting that

the antioxidant system of testis tries to compensate the

altered cellular environment; however, after chronic

exposure of calcium, activities of these enzymes were

decreased possibly for the break down of cellular antioxi-

dant defense mechanism. The results of this study show

that CaCl2 treatment at doses of 1.0 and 1.5 g% caused a

marked increase in testicular LPO. Thus, it is plausible to

speculate that CaCl2 treatment enhances peroxidation of

PUFAs on the membrane of germ cells, resulting in the

degeneration of phospholipids as mammalian spermatozoa

are rich in PUFAs [41] that ultimately reflects in cellular

deterioration of testis. Various studies also suggested

strong correlation between calcium-induced toxicity and

the induction of LPO [42, 43] that is considered as the key

mechanism of the ROS-induced damage in both testis and

sperm leading to infertility [41].

The testicular germ cell degeneration is reflected by a

marked reduction in the number of ASg, pLSc, mPSc and

step 7 spermatid (7Sd) as compared with those of control.

Theoretically, the ratio of mPSc:7Sd is 1:4 [18]. This ratio

was 1:3.12 and 1:2.66 after CaCl2 exposure at dose of

1.5 g% after 13 and 26 days treatment, respectively, when

compared with control (1:3.36). According to Bansal and

Davies [44], any alteration or disturbances in this ratio

Table 4 Testicular and serum calcium levels obtained from different groups of rats

Parameters 13 Days treatment 26 Days treatment

Control Calcium

0.5%

Calcium

1.0%

Calcium

1.5%

Control Calcium

0.5%

Calcium

1.0%

Calcium

1.5%

1. Serum calcium (mg/dl) 131.0 ± 5.5 133.2 ± 6.3 138.5 ± 4.8a 141.5 ± 7.5a,b 135.2 ± 4.94 141.2 ± 3.6a 145.5 ± 5.7a 147.6 ± 6.6a,b

2. Testicular

calcium (lg/g)

303.6 ± 34.7 303.0 ± 42.3 312.2 ± 38.3a 315.6 ± 21.3a,b 305.1 ± 36.8 307.2 ± 15.0 321.7 ± 29.2a 327.3 ± 18.9ab

Data are presented as the mean ± SD, n = 8. Values bearing superscripts are significantly different by ANOVA at P \ 0.05a When compared with controlb When compared with calcium

Table 3 Comparison of food consumption pattern of different treated groups with respective controls

Parameters 13 Days treatment 26 Days treatment

Control Calcium

0.5%

Calcium

1.0%

Calcium

1.5%

Control Calcium

0.5%

Calcium

1.0%

Calcium

1.5%

Food intake (g/rat/day) 24.5 ± 0.92 26.5 ± 2.07 25 ± 2.13 25 ± 2.61 25.2 ± 1.48 26 ± 1.51 26.2 ± 1.66 25.7 ± 1.58

Data are presented as mean ± SD

Table 5 Effect of calcium on the relative number of germ cells per tubular cross section at stage VII of the seminiferous epithelial cycle

Parameters 13 Days treatment 26 Days treatment

Control Calcium

0.5%

Calcium

1.0%

Calcium

1.5%

Control Calcium

0.5%

Calcium

1.0%

Calcium

1.5%

Sp

erm

ato

gen

esis

pat

tern

atst

age

VII ASg 0.67 ± 0.04 0.66 ± 0.05 0.63 ± 0.05 0.57 ± 0.05a 0.66 ± 0.04 0.63 ± 0.03 0.54 ± 0.05a 0.48 ± 0.04a

pLSc 18.42 ± 1.17 18.3 ± 1.05 17.1 ± 0.9 15.3 ± 1.2a 18.3 ± 0.81 17.9 ± 1.44 15.0 ± 0.72a 14.4 ± 1.1b

mPSc 19.2 ± 1.44 19.1 ± 1.5 18.7 ± 1.2 15.4 ± 1.14a 19.1 ± 0.9 18.9 ± 1.08 15.3 ± 1.23a 14.7 ± 0.87b

7Sd 64.62 ± 1.2 63.6 ± 1.2 61.1 ± 1.2 48.0 ± 1.4a 64.0 ± 1.14 62.7 ± 1.14 48.1 ± 1.41a 39.2 ± 1.11b

mPSc:7Sd 1:3.36 1:3.33 1:3.27 1:3.12 1:3.35 1:3.32 1:3.15 1:2.66

7Sd Degeneration (%) 16.0 16.75 18.25 22.0 16.25 17.0 21.25 33.5

Effective 7 Sd degeneration – ?0.75 ?2.25 ?6.0 – ?0.75 ?5.0 ?17.25

Data are presented as the mean ± S.D., n = 8. Values bearing superscripts are significantly different by ANOVA at P \ 0.05

ASg spermatogonia A, pLSc preleptotene spermatocytes, mPSc mid-pachytene spermatocytes, 7Sd step 7 spermatida When compared with controlb When compared with calcium

Mol Cell Biochem (2012) 364:181–191 187

123

indicate impairment of spermatogenesis process. The per-

centage of spermatid degeneration as calculated from

above ratio was highly significant after CaCl2 treatment at

dose of 1.0 g% for 26 days and 1.5 g% for both 13 and

26 days. Thus, a graded decrease in epididymal sperm

count was observed that has been reflected in testicular

histoarchitechture as evidenced by progressive degenera-

tive changes with graded doses of CaCl2.

On the other hand, the activities of testicular P450 ste-

roidogenic enzymes (namely, D5 3b HSD and 17b HSD)

were decreased. Several lines of evidence indicate that

during spermatogenesis, ROS and LPO occur for electron

leakage outside the electron transfer chain [45] and these

oxygen radicals can initiate LPO to inactivate P450

enzymes [46, 47]. As calcium treatment increases, LPO of

testicular germ cells, as found in this study, it may cause

inhibition of these testicular steroidogenic enzymes. This

has been ultimately resulted in decreased serum testoster-

one level. Thus, it may be the another cause of germ cell

degeneration, as testosterone plays a key role in growth and

maturation of testicular germ cells [48] and the reduced

testosterone fails to maintain proper spermatogenesis, that

is evident by decreased germ cells count in seminiferous

epithelial cycle. In culture study, different amount of cal-

cium was also found to cause decreased testosterone pro-

duction in Leydig-cell suspension [12].

Testicular Δ 5 3β - hydroxysteroid dehydrogenase activity

0

0.05

0.1

0.15

0.2

0.25

ΔO

D /

min

/ m

g p

rote

in

Control calcium 0.5% calcium 1.0% calcium 1.5%

13 days treatment 26 days treatment

c

db

a

a

Testicular 17β - hydroxysteroid dehydrogenase activity

0

0.005

0.01

0.015

0.02

0.025

0.03

ΔO

D /

min

/ m

g p

rote

in

Control calcium 0.5% calcium 1.0% calcium 1.5%

13 days treatment 26 days treatment

a

b

bc

d

A

B

Fig. 2 Effect of calcium of different doses (0.5, 1.0 and 1.5 g%) for

different durations (13 and 26 days treatment) on testicular D5-3b-

HSD (A) and 17b- HSD (B) activities of adult rats. Each bar denotes

mean ± SD of eight animals per group. Mean values are significantly

different by ANOVA at P \ 0.05. A a Control versus other groups in

26 days treatment, b 1.0 g% 13 days group versus 1.0 g% 26 days

group, c 1.5 g% 13 days group versus 1.5 g% 26 days group and d0.5 g% 26 days group versus 1.5 g% 26 days group. B a Control

versus other groups in 13 days treatment, b control versus other

groups in 26 days treatment, c 1.5 g% 13 days group versus 1.5 g%

26 days group and d 0.5 g% 26 days group versus 1.5 g% 26 days

group

Adrenal Δ 5 3β - hydroxysteroid dehydrogenase activity

0

0.05

0.1

0.15

0.2

0.25

ΔO

D /

min

/ m

g p

rote

in

Control calcium 0.5% calcium 1.0% calcium 1.5%

13 days treatment 26 days treatment

ab

b

c

Serum Corticosterone level

0

10

20

30

40

50

µg

/dl

Control calcium 0.5% calcium 1.0% calcium 1.5%

13 days treatment 26 days treatment

c

bb

a

d

A

B

Fig. 3 Effect of calcium different doses (0.5, 1.0 and 1.5 g%) for

different durations (13 and 26 days treatment) on testicular D5-3b-

HSD activity (A) serum corticosterone level (B) of adult rats. Each

bar denotes mean ± SD of eight animals per group. Mean values are

significantly different by ANOVA at P \ 0.05. A a Control versus

other groups in 13 days treatment, b control versus other groups in

26 days treatment and c 0.5 g% 26 days group versus 1.5 g% 26 days

group. B a Control versus other groups in 13 days treatment, b control

versus other groups in 26 days treatment, c 1.5 g% 13 days group

versus 1.5 g% 26 days group and d 0.5 g% 26 days group versus

1.5 g% 26 days group

Epididymal Sperm Count

0

20

40

60

80

100

120

140

mill

ion

cells

/ cau

da e

pidi

dym

is

Control calcium 0.5% calcium 1.0% calcium 1.5%

13 days treatment 26 days treatment

b

ab

b

b

cd

e

f

Fig. 4 Effect of different doses of calcium (0.5, 1.0 and 1.5 g%) for

different durations (13 and 26 days treatment) on epididymal sperm

count of adult rats. Each bar denotes mean ± SD of eight animals per

group. Mean values are significantly different by ANOVA at

P \ 0.05. a Control versus other groups in 13 days treatment,

b control versus other groups in 26 days treatment, c 0.5% 13 days

group versus 0.5 g% 26 days group, d1.0 g% 13 days group versus

1.0 g% 26 days group, e 1.5 g% 13 days group versus 1.5 g%

26 days group and f 0.5 g% 26 days group versus 1.5 g% 26 days

group

188 Mol Cell Biochem (2012) 364:181–191

123

In addition to testicular tissues, the ROS that generated

also act through HPA-axis. Acute or chronic stress causes

hypothalamus to release certain important peptides,

including CRH from paraventricular nucleus (PVN)

causing increased secretion of adreno-corticotropic hor-

mone (ACTH) from pituitary that facilitates increased

release of glucocorticoids including corticosterone that acts

as a defense against stressful situations [49]. Attributable to

excess secretion of glucocorticoids including corticoste-

rone from adrenal cortex, hypertrophy of adrenal cortical

cells resulting in increased adrenal gland weight was also

observed that is in line with the report that during stress,

Serum Testosterone level

0

0.5

1

1.5

2

2.5

3

3.5

4

ng/m

l

Control calcium 0.5% calcium 1.0% calcium 1.5%

13 days treatment 26 days treatment

a

b

b

c

d

e

f

g

Serum FSH level

0

5

10

15

20

25

ng

/ml

Control calcium 0.5% calcium 1.0% calcium 1.5%

13 days treatment 26 days treatment

ab

bb

cd

e

f

Serum LH level

0

1

2

3

4

5

6

ng

/ml

Control calcium 0.5% calcium 1.0% calcium 1.5%

13 days treatment 26 days treatment

a

b

b

c

d

eg

f

A

B

C

Fig. 5 Effect of different doses of calcium (0.5, 1.0 and 1.5 g%) for

different durations (13 and 26 days treatment) on serum testosterone

(a) FSH (b) and LH (c) level of adult male rats. Each bar denotes

mean ± SD of eight animals per group. Mean values are significantly

different by ANOVA at P \ 0.05. A a Control versus other groups in

13 days treatment, b Control versus other groups in 26 days

treatment, c 1.0 g% 13 days group versus 1.0 g% 26 days group,

d 1.5 g% 13 days group versus 1.5 g% 26 days group, e 0.5 g%

13 days group versus 1.5 g% 13 days group, f 0.5 g% 26 days group

versus 1.0 g% 26 days group and g 0.5 g% 26 days group versus

1.5 g% 26 days group. B a Control versus other groups in 13 days

treatment, b Control versus other groups in 26 days treatment,

c 1.0 g% 13 days group versus 1.0 g% 26 days group, d 1.5 g%

13 days group versus 1.5 g% 26 days group, e 0.5 g% 13 days group

versus 1.5 g% 13 days group, f 0.5 g% 26 days group versus 1.5 g%

26 days group. C a Control versus other groups in 13 days treatment,

b Control versus other groups in 26 days treatment, c 1.0 g% 13 days

group versus 1.0 g% 26 days group, d 1.5 g% 13 days group versus

1.5 g% 26 days group, e 0.5 g% 13 days group versus 1.5 g%

13 days group, f 0.5 g% 26 days group versus 1.0 g% 26 days group

and g 0.5 g% 26 days group versus 1.5 g% 26 days group

Testicular Lipid Peroxidation level

0

2

4

6

8

10

12

nm

ol T

BA

RS

/g o

f ti

ssu

e

Control calcium 0.5% calcium 1.0% calcium 1.5%

13 days treatment 26 days treatment

a

c

b

b

e

d

Testicular Superoxide Dismutase activity

02468

10121416

un

it /

mg

pro

tein

/min

Control calcium 0.5% calcium 1.0% calcium 1.5%

13 days treatment 26 days treatment

a

b

c

d

Testicular Catalase activity

0123456789

ΔO

D /

min

/ m

g p

rote

in

Control calcium 0.5% calcium 1.0% calcium 1.5%

13 days treatment 26 days treatment

a

c

b

b

f

e

d

A

B

C

Fig. 6 Effect of different doses of calcium (0.5, 1.0 and 1.5 g%) for

different durations (13 and 26 days treatment) on testicular LPO level

(A), SOD (B) and CAT (C) activities of adult rats. Each bar denotes

mean ± SD of eight animals per group. Mean values are significantly

different by ANOVA at P \ 0.05. A a Control versus other groups in

13 days treatment, b control versus other groups in 26 days treatment,

c 1.0 g% 13 days group versus 1.0 g% 26 days group, d 1.5 g%

13 days group versus 1.5 g% 26 days group and e 0.5 g% 26 days

group versus 1.5 g% 26 days group. B a Control versus other groups

in 13 days treatment, b control versus other groups in 26 days

treatment, c 1.5 g% 13 days group versus 1.5 g% 26 days group and

d 0.5 g% 26 days group versus 1.5 g% 26 days group. C a Control

versus other groups in 13 days treatment, b control versus other

groups in 26 days treatment, c 1.0 g% 13 days group versus 1.0 g%

26 days group, d 1.5 g% 13 days group versus 1.5 g% 26 days group,

e 0.5 g% 26 days group versus 1.0 g% 26 days group and f 0.5 g%

26 days group versus 1.5 g% 26 days group

Mol Cell Biochem (2012) 364:181–191 189

123

the weight of the adrenal gland is increased [50]. Adrenal

D5 3b HSD activity and serum corticosterone level were

significantly elevated after 26 days of treatment that is an

indicator of increased serum ACTH level. Decreased tes-

tosterone level also elevates serum corticosterone level that

in turn inhibits LH secretion [48].

Thus, the serum LH levels as observed in this study are

decreased in dose- and time-dependent manner. In contrast,

serum FSH levels are found to be elevated in calcium-

treated animals possibly to compensate the testosterone

level as low testosterone stimulates FSH secretion acting

on hypothalamo–pituitary–gonadal-axis (HPG). Therefore,

the altered serum LH and FSH levels may be the other

contributor for the inhibition in the activities of testicular

steroidogenic enzymes as their activities are dependent on

the concentration of respective FSH and LH [51].

On the other hand, a decrease in the mean ventral

prostrate, epididymis, seminal vesicle and coagulating

gland weight correspond with the decrease of serum tes-

tosterone concentration. As testosterone plays a major role

in the maintenance and structural integrity and functional

activities of accessory sex organs [52] and therefore a

reduction in accessory sex organ weight is a reflection of

decreased testosterone level in blood.

Thus, this study elucidates that chronic consumption of

excessive dietary calcium generates ROS that interferes

with both the HPG and HPA axis which in turn responsible

for testicular germ cell degeneration and eventually causes

testicular impairment and male infertility.

Conclusion

In view of the findings of our investigation on male repro-

ductive system, it can be speculated that calcium in its dif-

ferent concentrations as used for longer durations induces

oxidative stress that causes testicular cell damage resulting in

deterioration of testicular morphology and function.

Acknowledgments Acknowledgment is due to the Project of Uni-

versity Potential for Excellence (UPE), University Grants Commis-

sion (UGC), New Delhi, India for providing scholarship to second

author for conducting this work. The authors are thankful to Dr. Syed

N. Kabir, Scientist, Cell Biology & Physiology Division, Indian

Institute of Chemical Biology (IICB), Kolkata, India for his help in

conducting RIA of FSH and LH.

Conflict of interest None.

References

1. Mertz W (2000) Three decades of dietary recommendations. Nutr

Rev 58:324–331

2. Nordin BEC (1960) Osteomalacia, osteoporosis and calcium

deficiency. Clin Ortho Relat Res 17:235–258

3. Andon MB, Ilich JZ, Tzagournio MA, Matkovic V (1996)

Magnesium balance in adolescent females consuming a low- or

high-calcium diet. Am J Clin Nutr 63:950–953

4. Curhan GC, Willett WC, Rimm EB, Stampfer MJ (1993) A

prospective study of dietary calcium and other nutrients and the

risk of symptomatic kidney stones. The N Engl J Med 328:

833–838

5. Ilich-Ernst JZ, McKenna AA, Badenhop NE (1998) Iron status,

menarche and calcium supplementation in adolescent girls. Am J

Clin Nutr 68:880–887

6. Wood RJ, Zheng JJ (1997) High dietary calcium intake reduces

zinc absorption and balance in humans. Am J Clin Nutr 65:

1803–1809

7. Park YK, Yetley EA, Calvo MS (1997) Calcium intake levels in

the United States: issues and considerations. Food Nutr Agric

20:34–43

8. Weaver CM, Heaney RP (2006) Calcium and Human Health, 1st

edn. Humana Press, Totowa, NJ, pp 313–318

9. Chandra AK, Tripathy S, Mukhopadhyay S, Lahari D (2003)

Studies on endemic goiter and associated iodine deficiency dis-

orders (IDD) in the rural area of gangetic West Bengal. Ind J Nutr

Diet 40:53–58

10. Jana K, Samanta PK (2006) Evaluation of single intratesticular

injection of calcium chloride for nonsurgical sterilization in adult

albino rats. Contraception 73:289–300

11. Canpolat I, Gur S, Gunay C, Bulut S, Eroksuz H (2006) An

evaluation of the outcome of bull castration by itra-testicular

injection of ethanol and calcium chloride. Rev Med Vet 157:

420–425

12. Murono EP, Payne AH (1979) Testicular maturation in rats:

In vivo effect of gonadotrophins on steroidogenic enzymes in

hypophysectomized immature rats. Biol Reprod 20:911–916

13. Annunziato L, Cataldi M, Pignataro G, Secondo A, Molinaro P

(2007) Glutamate-independent calcium toxicity: introduction.

Stroke 38:661–664

14. Chandra AK, Tripathy S, Debnath A, Ghosh D (2007) Bio-

availability of iodine and hardness (magnesium and calcium salt)

in drinking water in the etiology of endemic goitre in Sundarban

Delta of West Bengal (India). J Environ Sci Eng 49:139–142

15. Etling N, Fouque F, Garabedian M (1986) Effects of dietary

supply of calcium on thyroid function in rats. Reprod Nutr Dev

26:841–847

16. Sarkar M, Roy Chaudhury G, Chattopadhyay A, Biswas NM

(2003) Effect of sodium arsenate on spermatogenesis, plasma

gonadotrophins and testosterone in rats. Asian J Androl 5:27–31

17. Leblond PC, Clermont Y (1952) Definition of the stages of the

seminiferous epithelium in the rat. Ann N Y Acad Sci 55:

548–573

18. Clermont Y, Morgentaler H (1955) Quantitative study of sper-

matogenesis in hypophysectomized rats. Endocrinology 57:

369–382

19. Majumder GC, Biswas R (1979) Evidence for the occurrence of

ecto (adenosine triphosphatase) in rat epididymal spermatozoa.

Biochem J 183:737–743

20. Ohkawa H, Ohishi N, Yagi K (1989) Assay for lipid peroxidases

in animal tissues by thiobarbituric acid reaction. Anal Biochem

95:351–358

21. Aebi H (1983) Catalase. In: Bergmeyer H (ed) Methods of

enzymatic analysis. Academic press, New York, pp 276–286

22. Marklund S, Marklund G (1974) Involvement of superoxide

anion radical in the autoxidation of pyrogallol and a convenient

assay for superoxide dismutase. Eur J Biochem 47: 469–474

23. Talalay P (1962) Hydroxysteroid dehydrogenases. Methods

Enzymol 5:512–532

24. Jarabak J, Adams JA, Williams-Ashman HG, Talalay P (1962)

Purification of 17 beta-hydroxysteroid dehydrogenase of human

190 Mol Cell Biochem (2012) 364:181–191

123

placenta and studies on its transhydrogenase function. J Biol

Chem 237:345–357

25. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein

measurement from phenol reagent. J Biol Chem 193:265–270

26. Baginski ES et al (1973) Clin Chim Acta 46:49

27. Moudgal NR, Madhawa Raj HG (1974) Pituitary gonadotropin.

In: Jafe BM, Berham HR (eds) Methods of hormone radioim-

munoassay. Academic Press, New York, pp 57–85

28. Greenwood FC, Hunter WM, Glover JS (1963) The preparation

of 131I-labelled human growth hormone of high specific activity.

Biochem J 89:114–123

29. Glick D, Recllich DV, Seyniour L (1964) Fluorometric deter-

mination of corticosterone and cortisol in 0.02–0.05 ml of plasma

or submilligram samples of adrenal tissue. Endocrinology 74:653

30. Silber RH (1966) In: Glick D (ed) Methods in Biochemical

Analysis. Interscience Publishers, New York, p 63

31. Biswas NM, Chattopadhdyay A, Sengupta R, Roychowdhury G,

Sarkar M (1999) Protection of adrenocortical activity by dietary

casein in rats treated with estrogen. Med Sci Res 27:415

32. Biswas NM, Roychowdhury G, Sarkar M, Sengupta R (2000)

Influence of adrenal cortex in testicular activity in the toad during

the breeding season. Life Sci 66:1253

33. Fisher RA, Yates R (1974) Statistical Tables for Biological,

Agricultural and Medical Research. Longman Group, London

34. Davies KM, Heaney RP, Recker RR, Lappe JM, Barger-Lux MJ,

Rafferty K, Hinders S (2000) Calcium intake and body weight.

J Clin Encrinol Metab 85:4635–4638

35. Welberg JW, Monkelbaan JG, de Vries EG, Muskiet FA, Cats A

(1994) Effect of supplimental dietary calcium on quantitative

fecal fat excretion in man. Ann Nutr Metab 38:185–191

36. Zemel MB, Morgan K (2002) Interaction between calcium,

dairyband dietary macronutrients in modulating body composi-

tion in obese rats. FASEB J 16:A369

37. Zemel MB, Shi H, Greer B, Dirienzo D, Zemel PC (2000) Regu-

lation of adiposity by dietary calcium. FASEB J 14:1132–1138

38. Chapin RE, Harris MW, Davis BJ, Ward SM, Wilson RE, Ma-

uncy MA, Lockhart AC, Smialowicz RJ, Moser VC, Burka LT,

Collins BJ (1997) Fundam Appl Toxicol 40:138–157

39. Hansson MJ, Mansson R, Morota S et al (2008) Calcium induced

generation of reactive oxygen species in brain mitochondria is

mediated by permeability transition. Free Radic Biol Med 45:

284–294

40. Bandyopadhyay U, Das D, Banerjee RK (1999) Reactive oxygen

species:oxidative damage and pathogenesis. Curr Sci 77:658–666

41. Maneesh M, Jayalekhshmi H, Dutta S, Singh TA, Chakrabarti A

(2006) Impaired Hypothalamo-pituitary-gonadal function in men

with diabetes mellitus. Ind J Clin Biochem 21:165–168

42. Ermak G, Davies KJA (2002) Calcium and oxidative stress: from

cell signaling to cell death. Mol Immunol 38:713–721

43. Bruckbauer A, Zemel MB (2009) Dietary Calcium and Dairy

Modulation of Oxidative Stress and Mortality in aP2-Agouti and

wild-type Mice. Nutrients 1:50–70

44. Bansal MR, Davies AG (1986) Effects of testosterone oenanth-

ante on spermatogenesis and serum testosterone concentration in

adult mice. J Reprod Fertil 78(1):219–224

45. Hanukoglu I, Rapoport R, Weiner L, Sklan D (1993) Electron

leakage from the mitochondrial NADPH-adrenodoxin reductase-

adrenodoxin-P450scc (cholesterol side chain cleavage system).

Arch Biochem Biophys 305:489–498

46. Hornsby PJ (1980) Regulation of cytochrome P-450 supported

11-hydroxylation of deoxycortisol by steroids, oxidants and anti-

oxidants in adrenocortical cell culture. J Biol Chem 255:4020–4027

47. Chandra AK, Chatterjee A, Ghosh R, Sarkar M, Chaube SK

(2007) Chromium induced testicular impairment in relation to

adrenocortical activities in adult albino rats. Reprod Toxicol

24:388–396

48. Chandra AK, Ghosh R, Chatterjee A, Sarkar M (2007) Effects of

vanadate on male rat reproduction tract histology, oxidative stress

markers and androgenic enzyme activities. J Inorg Biochem

101:944–956

49. Viau V (2002) Functional cross talk between hypothalamic-

pituitary-gonadal and adrenal axis. J Neuroendocrinol 14:

506–513

50. Pellegrini A, Grieco M, Materazzi G, Gesi M, Ricciardi MP

(1998) Stress-induced Morphohistochemical and Functional

Changes in Rat Adrenal Cortex, Testis and Major Salivary

Glands. The Histochem J 30:695–701

51. Janszen FH, Cooke BA, Van driel JA, Vander molen HJ (1976) J

Biochem 160:433–437

52. Steinberger E, Steinberger A (1975) Hormonal control of testic-

ular function in mammals. In: Hamiltol DW, Greep RO (eds)

Handbook of Physiology, Endocrinology, vol. IV, Part 2. Wil-

liams and Wilkins, Baltimore, MD, pp 325–345

Mol Cell Biochem (2012) 364:181–191 191

123