Indian Journal of Experimental Biology Vol. 42, May 2004, pp. 481-485

Influence of histamine and HI-receptor antagonists on ejaculated human spermatozoa: Role of intrasperm Ca2

+

A Gupta, R Khosla, S Gupta & A K Tiwary*

Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala 147002, India

Received 25 July 2003; revised 20 Ja/l.uary 2004

Hi stamine red uced sperm viability in a dose- and time-dependent manner, accompanied by ri se in intrasperm Ca2+. Further, 2', 4'-dichlorobenzamil hydrochloride (DBZ), a Na+-Ca2+ exchange inhibitor, known to elevate intrasperm Ca2

+,

potentiated both , elevatio n of intrasperm Ca2+ and spermi cidal action of histamine. Pretreatment of sperm with very low doses of HI-receptor antagonists (chlorpheniramine, promethazine or diphenhydramine) prevented the hi stamine-induced elevation of intrasperm Ca2

+ as well as its spermicidal action . However, pretreatment with famotidine, a Hr receptor antagoni st did not produce suc h a protective action. The results strongly suggest that hi stamine elicits its spermic idal action via HI -receptors present on sperm cells.

Keywords: Di chlorobenzamil, H I-receptor antagonists, Human sperm motility Hi stamine, Intrasperm calcium IPC Code: Int. C17

: A61K

Intrasperm ci+ is known to playa vital role in sperm function . An increase in intrasperm Ca2+ by propranolol produced irreversible loss of sperm viability' . In addition, 2', 4'-dichlorobenzamil hydrochloride (DBZ), a Na+-Ca2+ exchange inhibitor, produced spermicidal action accompanied with elevation of intrasperm calcium 2.3.

Intracellular Ca2+ has also been reported to be increased in human ciliary muscles4

, gingival fibroblasts5 and capillary endothelial cell s6 after the addition of histamine. Histamine is known to act on H,­receptors and induce the formation of inositol-I , 4, 5-triphosphate (IP3) from phospholipids in the cell membrane. Since, IP3 receptors have been reported to be present on neck and acrosome of human spermatozoa7

-9, hi stamine can be envisaged to

modulate the intrasperm Ca2+ and thereby influence sperm viability. Furthermore, H,-receptor antagonists can be expected to antagonize histamine-induced modulation of intrasperm Ca2+.

Therefore, the present investigation has been designed to evaluate the modulation of sperm viabi lity and intrasperm Ca2+ by histamine and H ,-receptor antagonists in ejaculated human semen samples.

Materials and Methods Chlorpheniramine maleate, diphenhydramine

hydrochloride and promethazine hydrochloride were

*Correspondent author- Phone: +91-175-228246 1 Extn. 6184 Fax: +91-175-2283073; E. mail : aktiwary2@rediffmail.co lll

obtained as gift samples from Saviour Enterprises (Patiala, India). 2', 4'-dichlorobenzamil hydrochloride (DBZ) was obtained as gift sample from SRI, USA as a part of Chemical Synthesis Program of the National Institute of Mental Health (Contract NOIMH30003). Histamine dihydrochloride was purchased from HiMedia Lab. Ltd. (Mumbai, India). Quin2-AM was purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals were of AR grade and were purchased from S.D. Fine Chemicals Ltd. , India.

Semen collection-The semen samples wfl'e collected by masturbation from five non-drinkers and non-smoker male volunteers with a J11 ~'<l:1

spermatozoal count of > 20 x 106 spermatozoa/ml Jnd > 70% normal sperm morphology '0. A minim uI-:-! abstinence of not less than 48 hr and not more than 5 days between two collections was adhered to fo r collecting the semen samples" . Samples \h:r:_

collected into a warm glass beaker to avoid co le shock to the sperm. The fresh samples were allowed to liquefy and further investigations were calTied out at 35°C.

Influence on sperm viability-Drug solutions were prepared in Biggers Whitten Whittingham (BWW) medium l 2 and mixed with an equal volume of liquefied semen sample. The mixture was incubated at 37°C. Sample (0.1 ml) of this admixture was taken out at different time intervals and mixed with 0.5 % w/v eosin dye solution (0.05 ml). This was observed

482 INDIAN J EXP BIOL, MA Y 2004

microscopically and the number of viable (unstained) and dead (red stained) spermatozoa were counted 10.

Same process was employed for control sample. The result was expressed as fractional motility (% motile sperm in drug treated sample + % motile sperm in control sample) at each time interval.

In studies involving combination of histamine and DBZ, the drugs were separately dissolved in BWW medium and mixed with semen sample (0.5:0.5: 1.0). The mixture was incubated at 37°C and fractional motility was calculated at various time intervals.

Protective action of Hrreceptor antagonists - Different <;oncentrations of HI-receptor antagoni sts belonging to different classes (chlorphen-iranune, promethazine and diphenhydramine) were prepared in BWW medium and mixed with an equal volume of liquefied semen sample. The mixtures were incubated at 37°C for 15 min followed by washing with fresh BWW medium (1 :5). This was centrifuged at 1000 rpm for 5 min to remc'.·e any extra drug present in solution. The resulting pellet was resuspended in BWW medium (1 ml) and treated with equal volume of histamine (125 mM). The results were expressed as time required to produce complete loss of sperm vi ability . Each set of expel1ments was terminated after 60 min since histamine per se (125 mM) required 50 min to produce the same effect.

Sperm revival test-Glucose solution was added to the sample of immotile sperm to obtain a final glucose concentration of 250 mg/ml. The mixture was incubated at 35°-37°C for 60 min and then observed for revival of sperm motilit/ 3

.

Measurement of i11lraspenn Ca2+-Intrasperm Ca2+ in spermatozoa (separated from human semen and treated with histamine as well as its combination with DBZ) was determined by using fluorescent dye, Quin 2-AM at an excitation wavelength of 339 nm and an emission wavelength of 492 nm. Intrasperm Ca2+ was calculated according to the method described by White et al. 1 and Tsien et a/ 14

•

Results and Discussion Histamine is reported not to produce any change in

sperm motility up to a concentration of 10 mM 15. At

higher concentrations (>50 mM) histamine produced a dose- and time-dependent spermicidal activity requIring a concentration of 165 mM to produce complete loss of sperm viability immediately upon addition to semen samples (Fig. 1). This action of histamine was accompanied with an elevation of intrasperm Ca2+. Histamine (125 mM) per se elevated

the intrasperm Ca2+ (Fig. 2) . Also, DBZ (2 mM) per se elevated the intrasperm Ca2+ in a time-dependent manner. Complete loss of sperm viability by histamine (125 mM) and DBZ (2 mM) was observed upon reaching an intrasperm Ca2+ threshold of 1150-1200 nM, at 50 min and 120 min, respectively . A combination of histamine and DBZ rapidly increased the intrasperm Ca2+, thus achieving the critical Ca2+ threshold level in about 40 min, indicating that elevation of intrasperm Ca2+ plays vital role in producing complimentary spermicidal action of histamine and DBZ. Histamine potenti ated the spermicidal action of DBZ by three-folds (Table 1).

Pretreatment with r.: Jati vely low concentrations (0.01-1.0 mM) 01 HI-receptor antagoni sts (chlorpheniramine, diphenhydramine and prometha­zine) antagonized the spermicidal action of hi stamine (Table 2) . Chlorpheniramine (0.1 mM) was found to prevent the histamine-induced elevation of intrasperm Ca2+ (Fig. 3). All these HI-receptor antagonists

0.9

0.8

0.7

0.6

j?;o

~ 0.5 E co c o

"n ~ 0.4

LL

0.3

0.2

0. 1

·_ 50 mM ... <> . . 75 mM _ 100 mM

· · ·0· · 125 mM ---.-1 50 mM ___ 165 mM

w ~ ~ 00 100 1W 1~ 1~ 1~

Time(min}

Fig .I-Influence of hi stamine on motility of sperm in human semen samples.

GUPTA et al.: SPERMICIDAL ACTION OF HISTAMINE AND HI-RECEPTOR ANTAGONISTS 483

~ .s ~ u en :2 Qj ()

~ E

1200

1000

800

600

400

200

I

~

I

~ HTM(125 mM) perse

_ DBZ(2 mM) per se

.. ... .. DBZ(2 mM) + HTM (125 mM)

o L-__ ~ ____ -L ____ ~ ____ L-___ ~ ____ ~

o 20 40 60

Time (min)

80 100 120

Fig.2- Influence of combined treatment of 2', 4'-dichlorobenzamil hydrochloride (OBZ) and histamine (HTM) on intracell ular Ca2

+ in human spermatozoa.

1200

1000

--*- HTM per se 800

~ . . • .. CPM + HTM

.s . . . • .. FMT + HTM N",

U :0 600 :2 -a; u ~ ~

400

oJ;. .. .. ...... .. . .. ... .. ..... -200

o o 10 20 30 40 50 60 70

Time (min)

Fig. 3- Influence of 125 mM hi stamine (HTM) per se and afl er pretreatment with 0.1 mM chlorpheniramine (CPM ) or 0.5 111M famotidine (FMT) on intracellular Ca2

+ in human spermatozoa.

Table I-Spermicidal potentiation studies following combined treatment of different concentrations of 2' , 4'-dichol orbenzamil hydrochloride (OBZ) and histamine

l Values expressed as fracti onal motility of sperm are mean ± SO]

Time (min)

Fractional motility

2.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0

OBZ (2 mM) + Histamine* mM

* 100 *125 *150

0.48 ± 0.Q2 0.42 ± 0.Q2 0.38 ± 0.05 0.44 ± 0.01 0.30 ± 0.02 0.28 ± 0.02 0.39 ± 0.03 0.22 ± 0.03 0.15 ± 0.01 0.32 ± 0.02 0.08 ± 0.01 0.06 ± 0.03 0.26 ± 0.04 0 0 0.17 ± 0.0 1 0.03 ± 0.03

0

Histamine (125 mM) + OBZ* mM

*0.5 *1 *2

0.50 ± 0.02 0.41 ± 0.01 0.42 ± 0.u2 0.43 ± 0.02 0.35 t ; '.03 0.30 ± 0.02 0.33 ± 0.02 0.24 ± 0.Q2 0.22 ± 0.03 0.25 ± 0.01 0.12 ± 0.01 0.08 ± 0.0 1 0.20 ± 0.01 0.06 ± 0.03 0 0. 14 ± 0.04 0 0.08 ± 0.01 0.Q2 ± 0.02

0

Blank rows indicate termination o f experiment after observing 100% immotility

protected the sperm from losing their viability till 50 min (time required by 125 rnM concentration of histamine, per se) when employed at 0.1,0.5 and 1.0 mM concentrations, respectively. However, only promethazine was effective at a lower concentration (0 .01 mM) in eliciting its protective action . Contrary

to this observation, pretreatment with famotidine, a H2-receptor antagoni st was not able to prevent the histamine-induced elevation of intrasperm Ca2

+ (Fig. 3), indicating th&t the spermicidal activity of histamine is due to its action on HI-receptors and not due to Hz-receptors .

484 INDIAN J EXP BIOL, MAY 2004

0. 9 ®

0.8

0.7

--+- I mM ·· ·0 · · 2 m M ___ 4 mM

--- 0 -- 801 M

- .-16mM

--- 6 " 20m M __ 24mM

0 7 ®

06

0.5 i.

-+-1mM

···0 ·· 2 mM ___ S mM

···0 ·· 10 mM

-'-12.SmM

-*- 1SmM

0 .9

0 .8

0 .7

0.6

--+- 1 mM

·" 0 ·· 201M

---+---4 mM

···0·· 8 m M

-*-10mM

J I ,\

~ T '"

0.4 0 .5

0.3

20 40 60 80 100 120 20 40

'"

60

Time (min)

80

0.4

100 o 20 40 60 80 100 120

Fig.4 - lnfluence of (a) d iphenhydramine, (b) chlorpheniramine and (c) promethazine on motility of sperm in human se men sa mples.

Table 2 - Influence of pre treatme nt ( IS min ) of humun semen sumples with various doses o f H ,·· receptor antagonis ts on spermicidal aClivily of hi stamine ( 125 mM)

Time (m in) afte r H ,-Rccptor ,1I1tagonist prelreatment with

CPM (mM) PMZ (mM) DPH (mM ) H ,-antagoni sl

O.OJ 0.1 0.5 1.0 O.OJ 0.1 0.5 1.0 O.OJ 0 . 1 0.5 1.0

10 + + + + + + + + + + + + 20 + + + + + + + + + + + 30 + + + + + + + + + + 40 + + + + + + + + + + SO + + + + + + + + + + 60 + + + + + + + + + +

CPM = Chlorpheniramine, PMZ = Prometkzine, DPH = Diphenhydramine

Note: + represents >70% viable sperm ; - represents comple te loss of sperm viability. The observations were di scontinued at 60 min, the time taken by hi stamine ( 125 mM) fo r produc ing complete loss o f sperm viability

It is interesting to note that all these HI-receptor antagonists, when employed at higher concentrations ranging from 1-24 mM, reduced the sperm viability in a dose- and time-dependent manner (Figs 4a-4c) . Promethazine required the lowest concentration of 10 mM whereas, chlorpheniramine and diphenhydramine required 15 mM and 24 mM concentration, respectively for producing complete loss of sperm viability immediately upon addition to semen samples. This spermicidal activity of HI-receptor antagonists was found not to be associated with an increase in

intraspenn Ca2+. Although, the present data are not

sufficient to suggest the exact mechanism for spermicidal activity of these HI-receptor antagonists, the role of their membrane stabilizing property '5 cannot be ruled out.

Histamine has been reported to induce elevation of intrasperm ci+ in human ciliary muscle cells4

, gingival fibroblasts5 and capillary endothelial cells6 due to release of ci+ from intracellular IP3-sensitive stores and H,-receptor operated ci+ influx from extracellular sites 16. IP3 receptors are also reported to be present in

GUPTA et al.: SPERMICIDAL ACTION OF HISTAMINE AND HI-RECEPTOR ANTAGONISTS 485

human spermatozoa7-9

. Therefore, the histamine­induced elevation of intrasperm Ca2

+ may be ascribed to the formation of IP3 .

The present investigation has revealed that histamine produces irreversible loss of sperm viability due to elevation of intrasperm Ca2

+. This action was antagonized by relatively low concentrations of HI-receptor antagonists. This strongly suggests the presence of HI-receptors on sperm cells. In addition, the finding that these HI-receptor antagonists themselves produce irreversible loss of sperm viability indicates their novel use as spermicidal agents. These drugs are already approved as anti-allergic drugs in clinical practice. In addition, the results of this study show that their concentrations required for spermicidal action are lower than those administered orally. Therefore, HI -receptor antagonists seem to possess a great potential to be developed as spermicidal agents for local action on ejaculated human spermatozoa.

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3 Reddy P R, Sharma A, Gupta S & Tiwary A K, Effect of2' , 4'­dichlorobenzamil hydrochloride, a Na+Ca2+ exchange inhibitor on human spermatozoa, EliI' J Plwl'lnacol, 418 (2001) J 53.

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7 Walen sky L D & Snyder S H, Inositol I , 4, 5-triphosphate (IP3) receptors selectively locali zed to the acrosome of mammalian sperm, J Cell Bioi, 130 (1995) 857.

8 Kuroda Y, Kaneko S, Yoshimura Y, Nozawa S & Mikoshiba K. Are there inositol I, 4, 5-triphosphate (IP3) receptors in human sperm? Life Sci, 65 (1999) 135.

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J J Reynolds T R & Narang B S, in Medical laboratOlY manllal jor tropical countries, Vol II, edited by M Cheeseborough (B utterworth and Co. Ltd., Kent) 1984, 186.

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14 Tsien R Y, Pozzan T & Rink T J, Calcium homeostasis in intact lymphocytes: Cytoplasmic free calcium monitored with a new intracellularly trapped fluorescent indicator, J Cell Bioi, 94 (1982) 325.

15 Thomas M & Tumer P, Effect of chlorpheniramine, promethal.ine and cimetidine on human sperm motility in­vitro, J Pharm Phal'lnacol, 35 (1983) 761.

16 Niisato N, Ogata Y, Furuyama S & Sugiya H, Hi stamine HI receptor-induced Ca2+ mobi lization and prostaglandin E2 release in human gingival fibroblasts, Biochem Pharmacol. 52 (1996) 1015.