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II Kasturi M, Nazeer Ahamed R, Pathan K M, Shaikh P D &. Manivannan B, Effect of Azadirachta indica leaves on the seminal vesicles and ventral prostate in ...
Indian Journal of Experimental Biology Voi.42.November2004.pp.I091-1095

Ultrastructural changes in cauda epididymidal epithelial cell types of Azadirachta indica leaf treated rats M G Ghodesawar, R Nazeer Ahamed*, A W Mukhtar Ahmed & R H Aladakatti Department of Post-Graduate Studies & Research in Zoology, Karnatak University, Dharwad 580003, India Received 21 Mav 2003: re vised 12 Aug us! 2004

To assess if cauda epididymis is a target for the effect of A. indica leaves, Wistar strain male albino rats were administered (po) A. indica leaves ( I00 mglrat/day for 24 days). Transmission electron microscopic analysis revealed that in the cauda epididymal epithelium the nuclei of principal cell s we re enlarged and the number of coated micropinocytotic vesicles of the apical cytop lasm decreased. Microvilli were mi ssing and mitochondrial cristae and Golgi complex we re highly disrupted. The cytoplasm was abounding wi th lysosomal bodies . The clear cell s increased in perimeter and their nuclei increased in size and contained lesser chromatin . The nuclear membrane bulged out. The cytoplasm was vac uoli zed. Further, there was decrease in size of the lipid droplets, mitochondria, Golgi complex, endoplasmic reticulum and there was accumulation of lysosomal bodies. The changes in the principal and clear cells appear to be due to the effect of the hypoandrogen status caused by treatment with A. indica leaves and a direct action on the epididymal epithelium. Key words: Ultrastructure. Cauda epididymis, Epithelial cells, Azadirachla indica, Albino rat.

The mammalian epididymis has attracted the attention of investigators beca use of its important role in sperm maturation. It could be the extragonadal site to inte rfere within the control of fertility without impairing libido and potency. The anatomica l, histological and functional differentiation along the epididymis recorded in several mammalian species 4 formed the basis for further studies on this subjed- . India is gifted with abundant natural remedies in the fo rm of herbs, shrubs and mine ra l e le me nts. In an attempt to prevent conception, tribal people are known to use decoction s prepared from plant materials, both orally and locally. Azadirachta indica (sy n: Melia azadirachta), commonly known as neem, is an important medicin a l plant that grows throughout 9 India and Burma 5· _ Jt is known to cause the decrease in the weights of accessory sex glands like epididymis, ventral prostate and semina l vesic les and the effects are reversible after w ithdrawal of the 0 12 treatme ne - . The morph o logica l changes in the head of rat spermatozoa and sperm parameters induced by A. indica leaves have been reported 13" 14 . Recently, significant reduction tn sperm parameters and *Con·espondent author Phone 0836-2747121 (0); 2777674(R) E-mail: drnazee rahmcd2003 @yahoo .co. in

fructose content of vas deferens and ultra structural changes in prostate gland and vas deferens in albino . . v1vo . rats have a Iso been reporte d iS·16 . I n vttro an d m studies have shown that the praneem polyhe rbal pessary (formul ated from purified in gredients from A. indica, Sapindus mukerossi fruits and Mentha citrate o il ) has potent spermic ida l activity on human sperm when applied on the vagina before coitus, it prevents 17 rabbits from becoming pregnant . The present study has been aimed to eluc id ate the 18 19 effect of A.indica . leaves on the ultrastructural organ izat ion of the epitheli al cells of rat cauda epididymidis.

Materials and Methods The leaves of A. indica were collected and dri ed in shade. The dried leaves were finely powdered and suspended in distilled wate r for oral administration to a lbin o rats. Three to four month s old mal e albino rats (Wistar Strain) weighing 170-200 g, were obtained from the rat colony maintained in the departme nt. They were housed in cages and were fed on pe ll et feed ("Gold Mohur" , Hindustan Leve r Limited , Bombay) and water ad libitum. The animals were divided into two groups each consisting of five anima ls:

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Group I : Each rat received lml of distilled water, po, each day for 24 days and served as controls . Gro up 11: Each rat was admini stered , po, 100 mg of leaf po wder in l ml of disti lied wate r, eac h day fo r 24 days. The effecti ve dose of lOOmg a nd the peri od of trea tment viz., 24 days, have been arrived at afte r pre limina ry studi es on dose and duration response 10 12 st udi es in our laboratory and reported elsewhere . . Afte r fi xation by vascular perfu sion , with 3% glutaraldehyde the epididymi s was re moved rapidl y and again fi xed in 3% g luta ra lde hyde for 2-4 hr. The tiss ue was stored in sodium cacody late buffe r at 4°C (pH 7. 2, 0 .1 M ), was hed in bu ffe r a nd pos t fix ed in l % os mium tetraox ide for l-2 hr. The ti ss ue was was hed aga in in the buffe r, de hydrated in graded seri es of alcohol , stain ed enbloc in 2 % uranyl acetate fo r 6 hr, infiltrated w ith araldite : propyle ne oxide ( I : 1) mixture for ove r-ni ght, aga in infiltrated with fres h ara ldite and e mbedded in a ra ldite beam capsul e. The bl oc ks we re cut in a Le ic a LKB Broma ultrami cro to me. U ltra thin secti o ns ( 100-300A) were c ut , collected on copper g rids and stained in 1% 20 uranyl acetate and lead citrate . The section s we re o bserved 111 a Joe l-TEM lOOc x II e lectro n rm c rosco pe.

Results In the control rats the principal ce lls w~~ re prese nt a long the e ntire length of the cauda e pididy mi s. T hese ce ll s ha ve a sing le round o r e llipti ca l nuc le us conta inin g g ranula r c hro matin . Th ey a re cha rac te ri zed by we ll develo ped mi c ro villi o n the lumi na l surface . The multi ves ic ul a r bodi es co nta in a mo rpho us materi a l. Golgi co mpl ex is co mposed o f fe nes trat ed ci stern ae. T he e ndopl as mi c re ti c ulum a nd mitocho ndri a are we ll deve loped in the cytopl as m. ln higher magni f icati o n the ce ll s showed coa ted ves icl es in the vici nity of Go lg i compl ex. S upra nuc lea r reg ion of the ce ll showed we ll deve loped mitoch o ndri a l c ri stae and e nd o plasmic reti c ulum a rranged in the form of who rl s (Figs 1 and 2). The mos t obvious c han ges in the p rinc ipa l cell of ca ud a epidid ymidi s o f A. indica trea ted rats were dec reased in numbe r of coated mi c ro pin ocytotic ves ic les , in vagin ati o n o n the lumin a l s urface, loss of api cal mi c rov illi a nd di strupti o n of mi toc ho ndri a l c ri stae and Go lg i apparatu s. In the nuc le us, the c hro matin mate ri a l was pushed to one s id e, the

nuclear membrane was bulged a nd there was karyo kines is. Apica l end of the ce ll s was vac uolated and co nta ined particul ate materi a l. The multivesi c iular bodies we re increased and co nta ined a ho mogeno us or hete rogenou s materi al. In the api ca l region the mitochondri a atrophied and contributed to vacuolization (Fi gs 3 and 4). In the control rats, the cl ear ce lls were fo und in between the principal ce ll s. They contained ovoid nucl e i pl aced slig htly above the basal pos iti on and contained g ra nular chromatin material. The cytopl as m was abo unded with lipid drop lets. Mi cropin ocytoti c ves ic les were pro mine nt. The basa l ce ll s we re e llipti cal and nucl e i we re e lo nga ted and fl atte ned aga in st the base me nt me mbra ne (F ig. 5). In the c lea r ce ll s of trea ted rat, microv illi were mi ss in g. The size of the ce ll , nucle us and the cytoplasmic granul es were increased. The chro matin mate ri al was less, nu c lear e nve lope was bul ged , karyolysis and ka ryohex is were noticed . M itochondri a, Gol g i apparatu s and endopl as mi c re ticulum were di srupted . Mi c rop inocytoti c ves ic les were ra re ly seen. Basa l ce ll s appea red decreased (Fig . 6).

Discussion The epididymi s in gene ra l a nd the princ ipa l ce ll in particul ar, a re androgen-depe nde nt and androge n withdra wa l is known to ca use exte nsive c hanges in 2 1 23 the princ ipa l cell. Thu s, the c ha nges in the princ ipal ce ll of A. indica treated rats may refl ect a ma ni fes tati o n of hypoandrogeni c status, bro ug ht abo ut by A. indica leaf trea te me nt . H owever, d irec t acti o n of A. indica leaf o n the prin c ipa l ce ll cannot be e xclud ed . Mic ro tubul es constitute a principa l co mponent of the ti ss ue matri x syste m of the 24 epithe lia l ce ll s . Vin cristin e is a mi crotu bul e 23 25 di srupting agent - a nd mi crotubu les of the princ ip;:tl cell may be the target for A. in dica act io n. It is poss ible , the refore, that A. indica may have ca used pa tho log ical c ha nges Ill the ca ud a e pi did yma l e pithe li a l prin c ipa l ce ll. Jn th e present stud y, prin c ipa l ce ll a nd c lea r ce ll und e rwent ul trastru ctura l c ha nges fo ll ow in g A. indim treatmenl. A mo ng the s ig ni ficant cha nges observed in the prin c ipa l ce ll were dec rease in th e numbe r of mi c ro pinocyto ti c ves icl es and red ucti o n in the size of mitoc hond ria and Go lg i appa ratus . As ha prakas h 26 r: t a /. have suggested th at a bsorpti ve fun cti on of p rin c ipa l ce ll is impa ired fo ll ow in g admin istrati o n of

GHODESA WAR eta/. : ULTRASTRUCTURAL CHANGES IN EPITHELIAL CELL IN A. INDICA TREATED RATS

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Figs 1-4---(Figs I, 2)- Electron micrographs of control rat cauda epiclidymidis. (Fig. I) - Principal ce ll (PC) and basal cell (BC) wi th normal Golgi complex (G). The nucleus (N) and endoplasmic reti culum (ER) are normal. Mitochondria (M) are abundant and intact. At the apical region of principal cell (PC), den se mi crovilli (Mv) are prese nt. The demarcation betwee n the two princi pal cells is clear x 4500. (Fig. 2)- The nucleus (N) of Principal cell (PC) is normal. The Golgi complex (G) is ev ide nt showing its normal features. Few lysosomal bodies (Ly) are clearly visible. The mitochondria (M) are intact. Supranuclear region of the principal cell shows features typical of the cell. Endoplasmic reti cu lum (E R) is visible and appears normal x 2000. Figs 3 and 4 Electron micrographs of cauda epididymidis of A. indica treated rat. (Fig. 3)- The nucleus (N) of principal cell is enlarged. There is bulging of nuclear envelope (small arrow). The chromatin material is reduced. Microvilli (Mv) are di srupted and coated micropinocytotic vesicles (CV) are decreased. The lysosomal bodies (large arrow) show complete disruption. Mitochondrial cri s!ue (M) are disrupted x 9000. (Fig. 4) - The lipid droplets in the principal cells (small arrow) are increased. Multi vesicular bodies(*) are also more electron dense. In the nucleus (N) heterchromatizati on is decreased.The mitochondrial (M ) are vacuolated. The endocy tic vesicles (open arrow) are few x 5200.

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Fi gs 5,6 - (Fig. 5)- TEM picture show ing c lear (CC) and basal cells (BC). In between two principal cells (PC) clear cell is prese nt showin g its normal features. The nucleus (N) is normal. lying at the basal region. The Mitocho ndria(M) and endocytic vesicles are also norm al. The cell abounds w ith lysosomal bodies (Ly). At the apical region of principal cell microvil li (Mv) appear normal. The basal lamina (BL) is normal. The basal cell (BC) is lo ng showi ng its normal structure. The nucleus (N) of basal cell lies parallel to the basal lamina x 3600. (Fig. 6)- In the clear cell Micropinocytotic vesicles are decreased. Nuc;eus (N) is enlarged and th e gap between o uter and inner nucl ea r membranes has increased (small arrows). The number o f lipid droplets (*) are increased. Mitochondria arc di srupted x 9600

cyproTeron e acetate an antiandrogen. Thus, the findings in the present study lead us to infer hypoandrogenic status caused due to treatment of A. indica leaves. The result of the present study further indicated that in response to A. indica treatment, the clear cells undergo hypertrophy, hyperplasia and hyperactivity in an attempt to remove the cel l debris reach ing the ductus epididymal lumen from the testis in the form of res idual bodies, Sertoli cell fragments and dead and deformed sperms. Similar observations have been made in the cauda epididymid is of rats treated with villcristine 27 '28 • In the clear cell of the A. indica treated rats , organell es viz., cytoplasmic vacuoles, electron dense sec retory granules and mitochondri a, exhibited a reduction in abundance. Also the lipid droplets in the cells of the treated rats were smaller as compared with those in control rats. Further, there was a reduction in the number of micropinocytotic vesicles and multi vesicular bodies. These findings indicate that the

endocytic functions of the clear cells following A. indica treatment.

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affected

The above ultrastructural chan ges indicate that the principal cell and clear cell arc affected, thu s altering the composition of epididymal fluid which in turn may affect the sperm maturation. This contention ' 4· 23?6 . supported b y t"1n d'mgs o f ear11' er stu d 1es IS ·- ·-'8-3 1 also.

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