Localization of angiotensin-II type 1(AT1) receptors on buffalospermatozoa: AT1 receptor activation during capacitationtriggers rise in cyclic AMP and calcium

Sivaram Vedantam • Rita Rani • Monica Garg •

Suresh K. Atreja

Received: 4 December 2012 / Accepted: 4 January 2014

� Springer Science+Business Media Dordrecht 2014

Abstract The purpose of this study was to determine the

role of Ang-II in buffalo spermatozoa; localize angiotensin

type 1 (AT1) receptors on the sperm surface and under-

stand the signaling mechanisms involved therein. Immu-

noblotting and immunocytochemistry using polyclonal

Rabbit anti-AT1 (N-10) IgG were performed to confirm the

presence of AT1 receptors. Intracellular levels of cyclic

adenosine monophosphate (cAMP) were determined by

non-radioactive enzyme immunoassay, while that of Cal-

cium [Ca2?] were estimated by fluorimetry using Fura2AM

dye. The results obtained showed that AT1 receptors were

found on the post-acrosomal region, neck and tail regions.

Immunoblotting revealed a single protein band with

molecular weight of 40 kDa. Ang-II treated cells produced

significantly higher level of cAMP compared to untreated

cells (22.66 ± 2.4 vs. 10.8 ± 0.98 pmol/108 cells,

p \ 0.01). The mean levels of Ca2? were also higher in

Ang-II treated cells compared to control (117.4 ± 6.1 vs.

61.15 ± 4.2 nmol/108 cells; p \ 0.01). The stimulatory

effect of Ang-II in both the cases was significantly inhib-

ited in the presence of Losartan (AT1 antagonist; p \ 0.05)

indicating the involvement of AT1 receptors. Further,

presence of neomycin (protein kinase C inhibitor) inhibited

significantly the Ang-II mediated rise in Ca2? indicating

the involvement of PKC pathway. These findings confirm

the presence of AT1 receptors in buffalo spermatozoa and

that Ang-II mediates its actions via the activation of these

receptors. Ang-II stimulates the rise in intracellular levels

of cAMP and Ca2? during capacitation.

Keywords Capacitation � Spermatozoa � Angiotensin �Cyclic AMP � Calcium

Introduction

Mammalian spermatozoa undergo extensive biochemical

and physiological changes under the influence of various

factors present in the seminal plasma and the female

reproductive tract. Angiotensin-II (Ang-II) is one such

factor found in the seminal plasma [1]. Ang-II is the

principal hormone of the Renin-Angiotensin system (RAS)

largely recognized for its role in the electrolyte homeo-

stasis and haemodynamics. Although the impact of RAS on

reproduction is not completely elucidated, there are reports

implicating RAS in various reproductive functions [2–4].

The presence of Ang-II in ovaries [5] and follicular fluid

[6] further substantiates the role of RAS in reproduction.

Studies in the past have demonstrated the ability of Ang-II

to induce hyperactivated motility [7], accelerate capacita-

tion [8] and acrosome reaction [9]. Our own studies

involving buffalo spermatozoa showed that Ang-II induces

capacitation and acrosome reaction in a concentration and

time dependent manner [10]. These actions are mediated

via specific interactions between Ang-II and its specific cell

surface receptors [11].

Till date, four types of angiotensin receptors, viz., AT1,

AT2, AT3, & AT4 have been reported differing in their

tissue distribution and signaling mechanisms [12, 13]. Of

these, mainly type 1 (AT1) and type 2 (AT2) receptors have

been characterized by pharmacological and molecular

biology techniques [12]. Previous reports suggest that in

case of spermatozoa, Ang-II primarily interacts with AT1

receptors [9, 14, 15]. Although AT1 receptors have been

localized on mammalian spermatozoa, there is considerable

S. Vedantam (&) � R. Rani � M. Garg � S. K. Atreja

Division of Animal Biochemistry, National Dairy Research

Institute, Karnal 132001, Haryana, India

e-mail: sivaram3@gmail.com

123

Mol Biol Rep

DOI 10.1007/s11033-014-3043-7

variation across the species in terms of their location. They

have been localized on the sperm tail in rat [14], while the

post-acrosomal region in bovine spermatozoa [9]. Further,

activation of AT1 receptors have been linked to two distinct

signaling pathways, one mediated by cAMP [16] and the

other by Ca2? [13].

In view of these variations, and lack of information on

the role of Ang-II in buffalo sperm functions, the present

study was undertaken to localize Ang-II receptors on buf-

falo sperm surface and to understand the subsequent

intracellular signaling mechanisms.

Materials and methods

Chemicals

Bovine serum albumin (BSA fraction V), heparin, fura-2-

AM, neomycin, 3-isobutyl-1-methyl xanthine (IBMX),

cAMP enzyme immunoassay kit CA-201, and PD123319

were procured from Sigma Chemical Company (St. Louis,

MO, USA). Rabbit anti-AT1 (N-10) IgG and blocking

peptide (sc1173p) were procured from Santa Cruz Biotech.

Inc. California, USA. Goat anti-rabbit IgG (whole mole-

cule)-FITC conjugate, and protein molecular weight

markers were procured from Bangalore Genei Pvt. Ltd.,

India. All other chemicals used in this study were of ana-

lytical grade and purchased from local suppliers.

Culture media

A modified Tyrode’s bicarbonate-buffered medium (sp

TALP) described previously [17, 18] was used for sperm

processing and culture. The media (29-stock) was first

prepared in the absence of Ca2?, BSA, pyruvate and

bicarbonate. Working sp-TALP medium was prepared by

adding Ca2? (2 mM), pyruvate (1 mM) and NaHCO3

(25 mM). For spermatozoa washing, media was supple-

mented with 1 mg/ml BSA (washing media) and for

capacitation 6 mg/ml BSA was used.

Sperm processing

Semen was collected from Murrah buffalo bulls (Bubalus

bubalis) housed at the artificial breeding complex of the

National Dairy Research Institute (NDRI), Karnal, using

artificial vagina (IMV, France) maintained at 40 �C. A total

of 46 ejaculates were obtained from 5 bulls with an average

(SEM) volume of 2.12 (0.8) ml. Only those semen samples

having mass activity of ?3.0 and above on a subjective

scale of 0–5, were used in this study. Semen was diluted in

the ratio of 1:6 with washing media. The sample was then

washed twice by centrifugation (2759g) for 5 min,

replacing the supernatant with washing media. The final

washing was done with sp-TALP containing 6 mg/ml BSA.

Finally, the sperm pellet was re-suspended in the same

medium and final sperm concentration was adjusted to

100 9 106 cells/ml.

Sperm culture

Capacitation of spermatozoa was performed in 1.5 ml

centrifuge tubes. Briefly, requisite quantities of Ang-II or

other test agents were added to 250ll of sp-TALP (with

6 mg/ml BSA) in centrifuge tubes. To this 250ll of sperm

suspension prepared as mentioned above was added mak-

ing the final sperm concentration 50 9 106 cells/ml. The

final concentration of Ang-II was adjusted to 200 nM.

Samples were incubated for 6 h at 38.5 �C with 5 % CO2

and [90 % humidity. At the end sperm samples were

processed for the assessment of extent of capacitation,

intracellular cAMP and Ca2? production.

Protein extraction

Whole cell proteins were extracted from capacitated

spermatozoa as described previously [18]. Briefly, sperm

suspensions were pooled together taking 75 9 106 cells,

and washed thrice with 1 ml PBS (pH 7.4) containing

1 mM sodium vanadate (PBS-V) by centrifugation. The

pellet was re-suspended in sample buffer (150 ll/75

million cells) containing 5 mM dithiothreitol, 1 mM

sodium vanadate and 1 mM phenylmethylsulfonyl fluo-

ride (PMSF), mixed thoroughly by using vortex mixer.

The contents were kept in boiling water bath for 5 min.

and then allowed to cool. All the tubes were then cen-

trifuged at 10,0009g for 20 min. The protein extract in

the supernatant was used either immediately or stored at

-20 �C for further use.

Immunoblotting

Whole cell proteins were resolved by sodium dodecyl

sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)

[19]. The resolved proteins were then electro transferred

onto PVDF membrane as described earlier [20]. The

membrane was probed with rabbit anti AT1 (N-10) poly-

clonal IgG (1:2,500 dilution) followed by goat anti rabbit

IgG HRPO conjugate (1:50,000 dilution). The blot was

then developed by autoradiography using ECL plus kit

from Amersham. The specificity of the primary antibody

was confirmed by control experiments in which the pri-

mary antibody was replaced with rabbit non-immune

serum, or preadsorption of the primary antibody with

excess blocking peptide (SC-1173 P) using the manufac-

turer’s protocol.

Mol Biol Rep

123

Immunocytochemistry

AT1 receptors were localized in both capacitated and non-

capacitated cells by indirect immunofluorescence technique

using rabbit anti-AT1 (N-10) IgG as primary antibody and

goat anti-rabbit IgG (whole molecule)-FITC conjugate as the

secondary antibody. Sperm suspension (100 ll) was incu-

bated with equal volume of primary antibody diluted five

times in TALP containing 3 mg/ml BSA for 2 h at 37 �C

with gentle intermittent agitation. The suspension was then

washed thrice with washing media by centrifugation. The

pellet was resuspended in goat anti-rabbit IgG-FITC conju-

gate diluted 20 times with TALP containing 3 mg/ml BSA

and incubated in dark for 3–4 h at 37 �C. The suspension was

again washed five times with washing media. Finally the

pellet was resuspended in TALP with 3 mg/ml BSA. 2 ll of

this was spread on a cover slip and before drying, the smeared

surface was kept on a 20 ll drop of 1.5 % (w/v) DABCO in

90 % (v/v) glycerol (an anti-fade mounting medium) on a

glass slide. The slide was immediately viewed under a

fluorescence microscope using a blue filter and photographs

were taken by using automatic photomicrographic system

(Olympus, PM10SP). Control experiments were run with

secondary antibody only.

Estimation of intracellular cAMP

The amount of intracellular cAMP produced during the course

of capacitation was determined using a non-radioactive

enzyme immunoassay kit. Assays were performed as descri-

bed in the instructions provided with the kit. To see the effect

of receptor specific inhibitors, 1lM Losartan (AT1 specific);

and 0.1 mM IBMX an inhibitor of phosphodiesterases, on

cAMP production the spermatozoa were pre-incubated for

30 min in the presence or absence of inhibitors before the

Ang-II treatment followed by cAMP measurement.

Estimation of Ca2?

Intracellular calcium was measured by using fluorescent

calcium indicator Fura-2, as described prviously [21, 22].

Briefly spermatozoa were incubated under capacitation

conditions in absence or presence of 200nM Ang-II, and

then 20 lM Fura-2/AM was added for a further 60 min.

The loaded cells were then washed twice with phosphate

buffer saline (pH 7.4) to remove extracellular Fura-2. The

cells were used immediately for fluorescence measure-

ments using a Cary1 Eclipse; Varian (California USA)

fluorescence spectrophotometer with an excitation wave-

length of 340 nm and emission of 500 nm. To see the

effect of various inhibitors (1lM Losartan (AT1 specific);

5lM PD123319 (AT2 specific); and 1 mM Neomycin

(Phospholipase C specific) on Ang-II induced Ca2?

production, spermatozoa were pre-incubated with respec-

tive inhibitor for 30 min before the Ang-II treatment fol-

lowed by Ca2? measurement.

Statistical analysis

All the experiments were repeated at least 3 times and the

data were analyzed by ANOVA (analysis of variance).

Statistical differences between the effects of various

treatments were determined by Tukey’s multiple compar-

ison tests using graph pad PRISM 3.02. A difference with

p \ 0.05 was considered statistically significant. Data are

expressed as mean ± SEM.

Results

Localization of Ang-II type 1 receptors

Immunoblotting

Immunoblotting using anti-rabbit AT1 antibody clone AT1

(N-10) revealed a single protein band with approximate

molecular weight of 40 kDa (Fig. 1). In the control experi-

ment where the primary antibody was omitted, no band

could be visualized. Further, adsorbing the primary antibody

with blocking peptide (sc-1173p) also did not reveal any

band indicating the specificity of the primary antibody.

Immunocytochemistry

The distribution pattern of the type 1 angiotensin receptors as

revealed by immunocytochemistry using anti-AT1 antibody

clone AT1 (N-10) shows that the AT1 receptor is localized

on the post acrosomal region and also on the tail region

Fig. 1 Detection of AT1 receptor from buffalo sperm whole protein

extract by immunoblotting

Mol Biol Rep

123

(Fig. 2). No change could be observed in the immunostain-

ing pattern before and after capacitation (Fig. 3).

Effect of Ang-II on intracellular cAMP levels

Angiotensin-II significantly increased the intracellular cAMP

production during the buffalo sperm capacitation (Fig. 4).

Ang-II treated cells produced significantly higher level

(p \ 0.01) of cAMP as compared to untreated cells

(22.66 ± 2.4 vs.10.8 ± 0.98 pmol/108 cells). These levels

were further increased upon pretreatment with IBMX

(28.46 ± 2.67) before Ang-II treatment, compared to cells

treated with only IBMX (20.9 ± 1.59 pmol/108 cells). This

stimulatory effect of Ang-II was significantly (p \ 0.05)

inhibited in the presence of 1 lM losartan (11.66 ±

2.02 pmol/108 cells).

Fig. 2 Localization of AT1 receptors on the buffalo sperm surface by immunocytochemistry. a Indirect immunofluorescence. b Corresponding

phase-contrast image

Fig. 3 Distribution of AT1 receptors on buffalo sperm surface before

and after Capacitation. a AT1 receptors distribution before capaci-

tation by indirect immunofluorescence, b AT1 receptors distribution

before capacitation phase contrast image, c AT1 receptors distribution

after capacitation by indirect immunofluorescence, d AT1 receptors

distribution after capacitation phase contrast image

Mol Biol Rep

123

Effect of Ang-II on intracellular free calcium Ca2?

The results presented in Fig. 5 clearly show that Ang-II at

200nM concentration significantly increased (p \ 0.01) the

intracellular free calcium (117.4 ± 6.1 nmol/108 cells) dur-

ing the course of capacitation as compared to the control

(61.15 ± 4.2 nmol/108 cells). This stimulatory effect was

significantly inhibited in the presence of 1lM Losartan

(73.7 ± 7.93 nmol/108 cells) but not PD123319 (113.7 ±

5.77 nmol/108 cells). Further, this was also inhibited in the

presence of 1 mM neomycin, (70.76 ± 7.16 nmol/108 cells;

p \ 0.05). These effects were also reflected in the extent of

capacitation (Fig 6). In the presence of neomycin, Ang-II

treated cells capacitated to the extent of 30.66 ± 3.71 %

which is significantly lower (p \ 0.01) than Ang-II treatment

alone (50.7 ± 2.45 %). While losartan inhibited Ang-II

induced capacitation (31.7 ± 4.45 %), no effect could be

seen in the presence of PD123319 (50.46 ± 4.8 %).

Discussion

Angiotensin-II has been shown to induce the capacitation

and acrosome reaction in buffalo spermatozoa in a con-

centration and time dependent manner. Our previous study

showed that the optimum concentration of Ang-II for

capacitation was 200 nM [10] and therefore, in the present

study the same concentration was used. The results

obtained in this study confirmed the presence of AT1

receptors in buffalo spermatozoa. Activation of these

receptors by Ang-II leads to rise in the intra-cellular levels

of cAMP and calcium. In view of the species specific

variations in the processes leading to sperm fertilization,

these findings are relevant and a step further in our

understanding of the buffalo sperm physiology. The pre-

sence of AT1 receptors was detected by using a rabbit

polyclonal anti-AT1 IgG with its epitope mapping within

the N-terminal extracellular domain (amino acids 15–24) of

AT1 of human origin (N-10). The fact that the antibody

raised against the AT1 of human origin could identify AT1

of buffalo origin shows that its structure is highly con-

served across the species as reported previously [12, 13].

Indirect immunofluorescence showed that AT1 receptors

were localized to the post acrosomal region, neck and also

over the tail region. These findings are similar to those

reported in other species such as bovine [9] equine [3] rat

and human [14, 23] except for the fact that in the current

study there was no re-distribution of these receptors before

as reported earlier [23]. Since the epitope was against

the extracellular domain, there was no need to permeabi-

lize the cells which otherwise is a common practice in

immunocytochemistry.

Upon immunoblotting, a single protein band corre-

sponding to molecular weight of 40 kDa was identified in

0

10

20

30

40

ControlAng-IILosartan + Ang-IIIBMXIBMX + Ang-II

cAM

P p

mo

les/

108 C

ells

aa

a

b

Fig. 4 Effect of Ang-II on the intracellular cAMP production by

capacitating buffalo spermatozoa. Data represent mean ± S.E. of four

different samples. ap \ 0.05 vs. control, bp \ 0.05 vs. Ang-II

0

50

100

150

ControlAng-IILosartan + Ang-IIPD123319 + Ang-IINeomycin + Ang-II

Ca2+

nm

ole

s/10

8 Cel

ls

b

a

b

a

Fig. 5 Effect of Ang-II on intracellular free calcium [Ca2 ? i] levels

during capacitation. Data represent mean ± S.E. of four different

samples. ap \ 0.01 vs. control, bp \ 0.05 vs. Ang-II

0

25

50

75

Control

Ang-II

Losartan + Ang-II

PD123319 + Ang-II

Neomycin + Ang-II

% C

apac

itat

ion

b

a

b

a

Fig. 6 Extent of capacitation at 6 h in Angiotensin-II treated cells in

the presence of neomycin (PLC inhibitor). Data represent

mean ± S.E. of four different samples. ap \ 0.01 vs. control,bp \ 0.05 vs. Ang-II

Mol Biol Rep

123

ejaculated buffalo spermatozoa, which is typical of many

mammalian cell types [11]. Similar molecular mass of AT1

was reported in case of bovine [9]. However, it is quite

different from that of 60 kDa reported in equine, rat and

human [3, 14]. Although there is no direct evidence to

explain these differences, species specific variations in the

post translational modifications such as the degree of gly-

cosylation may contribute to some extent.

Although there are several mechanisms involved in the

processes leading to sperm fertilization, the calcium fluxes

are known to be central to these processes [24–26].

Therefore, it was hypothesized that any agent, which

modulates sperm functions has to involve Ca2? either

directly or indirectly.

The use of dual wavelength dyes such as fura-2, is the

fluorescent probe of choice to assay Ca2? [27, 28]. Fura-2-

AM (Acetoxymethyl ester) is a cell permeable analogue of

the dye fura 2 that fluoresces when bound to calcium. Ca2?

alters the wavelengths and not just amplitudes of the

fluorescence excitation or emission peaks [21]. Dual

wavelength probes facilitate a ratiometric approach to

measure Ca2? levels. Any variability caused by different

levels of dye loading because of cellular size or other

factors will not affect the results [21, 24].

The present study showed a significant rise in calcium

level in response to the Ang-II treatment. The mean basal

Ca2? levels observed in this study were similar to that

reported previously [29, 30]. The stimulatory effect was

significantly inhibited in the presence of losartan indicating

the involvement of AT1 receptors in this process. Further, it

was also inhibited in the presence of neomycin, indicating

the involvement of phosphoinositide (PI) pathway. Pre-

sence of neomycin also decreased the extent of capacitation

in the Ang-II treated cells, further substantiating these

findings. AT1 receptors have been shown to be coupled to

‘Gq’ [12] which is known to interact with PLC [31].

Indeed, the major physiological functions of Ang-II are

expressed through Gq-mediated activation of PLC fol-

lowed by phosphoinositide hydrolysis and Ca2? signaling

[13].

The present study also demonstrated the ability of

Ang-II to induce cAMP production during buffalo sperm

capacitation. These results confirm the previous report [4]

that Ang-II stimulates cAMP production in mouse sper-

matozoa. The stimulatory effect of Ang-II was completely

inhibited in the presence of 1 lM Losartan, again sug-

gesting the involvement of AT1 receptors. Pretreatment of

the cells with IBMX, a broad spectrum inhibitor of phos-

phodiesterases [32, 33] resulted in further increasing the

cAMP levels.

Ang-II mediated rise in cAMP appears to be indirect,

since the ubiquitous stimulator of Adenylate Cyclase (AC)

is ‘Ga(s)’ [34], while the AT1 receptors are coupled to Gq/11

[12, 13] which is known to stimulate PLC. However, ACs

are known to be differentially regulated by diverse mech-

anisms including hetero-trimeric GTP regulatory proteins,

protein kinases and particularly by Ca2? and/or calmodulin

[35–37]. Transmembrane AC type III and VIII, from rat

spermatozoa are known to be stimulated by Ca2? [35]. A

soluble AC (sAC), molecularly unrelated to transmem-

brane ACs, responsive to bicarbonate and calcium has been

identified in mammalian spermatozoa [38, 39]. Further

studies are required to elucidate this signaling cross-talk.

Conclusion

This study confirms the presence of AT1 receptors on

buffalo sperm surface. Ang-II exerts its influence on buf-

falo sperm capacitation via the activation of these recep-

tors. It stimulates the rise in intracellular levels of cAMP

and Ca2? the two key secondary messengers central to the

signaling mechanisms leading to sperm fertilization. These

findings are in favor of attributing a substantial role for

Ang-II in buffalo sperm physiology.

Acknowledgments The authors are grateful to the Indian Council

of Agricultural Research (ICAR) for funding this project.

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