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: revised 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 ( I 00 mglrat/day for 24 days). Transmission electron microscopic analysis revealed that in the cauda epididymal epithelium the nuclei of principal cell s were enlarged and the number of coated micropinocytotic vesicles of the apical cytop lasm decreased. Microvilli were mi ssing and mitochondrial cristae and Golgi complex were 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 vacuoli 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 because 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 anatomical, histological and functional differentiation along the epididymis recorded in several mammalian species formed the basis for further studies on this subjed-4

.

India is gifted with abundant natural remedies in the form of herbs, shrubs and mineral e le ments. In an attempt to prevent conception, tribal people are known to use decoctions prepared from plant materials, both orally and locally. Azadirachta indica (syn: Melia azadirachta), commonly known as neem, is an important medicina l plant that grows throughout India and Burma5

·9

_ 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 withdrawal of the treatmene 0

-12

. The morpho logical 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: drnazeerahmcd2003 @yahoo.co. in

fructose content of vas deferens and ultra structural changes in prostate gland and vas deferens in albino

h I b d iS 16 I . d . . rats ave a so een reporte · . n vttro an m v1vo studies have shown that the praneem polyhe rbal pessary (formulated from purified ingredients from A. indica, Sapindus mukerossi fruits and Mentha citrate o il ) has potent spermic idal activity on human sperm when applied on the vagina before coitus, it prevents rabbits from becoming pregnant 17

.

The present study has been aimed to eluc idate the effect of A.indica 18

.19 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 dried in shade. The dried leaves were finely powdered and suspended in distilled wate r for oral administration to a lbino rats. Three to four months old male albino rats (Wistar Strain) weighing 170-200 g, were obtained from the rat colony maintained in the department. They were housed in cages and were fed on pe llet feed ("Gold Mohur" , Hindustan Lever Limited , Bombay) and water ad libitum.

The animals were divided into two groups each consisting of five animals:

1092 IN !DIAN J EXP BIOL. NOVEMBER 2004

Group I : Each rat received lml of distilled water, po, each day for 24 days and served as controls .

Group 11: Each rat was admini stered , po, 100 mg of leaf powder in l ml of disti lied water, each day for 24 days.

The effecti ve dose of lOOmg and the period of trea tment viz., 24 days, have been arrived at afte r pre liminary studies on dose and duration response studies in our laboratory and reported elsewhere 10

.12

.

Afte r fi xation by vascular perfusion , with 3% glutaraldehyde the epididymi s was removed rapidly and again fi xed in 3% g lutaralde hyde for 2-4 hr. The tissue was stored in sodium cacodylate buffer at 4°C (pH 7. 2, 0 .1 M), washed in bu ffe r and pos t fixed in l % osmium tetraox ide for l-2 hr. The ti ssue was washed aga in in the buffer, dehydrated in graded series of alcohol , stained enbloc in 2% uranyl acetate fo r 6 hr, infiltrated w ith araldite : propylene oxide ( I : 1) mixture for over-ni ght, aga in infiltrated with fresh ara ldite and e mbedded in a raldite beam capsule. The bloc ks were cut in a Leica LKB Broma ultramicroto me. U ltra thin secti ons ( 100-300A) were cut , collected on copper g rids and stained in 1% uranyl acetate and lead citrate20

. The sections were observed 111 a Joel-TEM lOOc x II e lectron rmcroscope.

Results

In the control rats the principal ce lls w~~ re present a long the entire length of the cauda epididymi s. T hese ce ll s have a sing le round or e lliptica l nucleus containing granula r chro matin . They are charac te ri zed by we ll developed micro villi on the lumi na l surface . The multi ves ic ul a r bodi es conta in amo rpho us materi a l. Golgi complex is composed o f fe nes trated ci stern ae. T he endopl as mi c re ti culum and mitochondri a are well developed in the cytopl as m. ln higher magni f icati on the ce ll s showed coated ves icles in the vici nity of Golg i compl ex. S upra nuc lear region of the ce ll showed we ll developed mitochondria l c ri stae and endoplasmic reti culum arranged in the form of whorl s (Figs 1 and 2).

The mos t obvious changes in the p rinc ipa l cell of cauda epididymidi s o f A. indica trea ted rats were dec reased in number of coated micro pinocytotic ves ic les , in vaginati on o n the lumina l surface, loss of apical microv illi and di strupti on of mi tocho ndri a l c ri stae and Golg i apparatus. In the nuc leus, the chro matin materi a l was pushed to one s ide, the

nuclear membrane was bulged a nd there was karyokines is. Apical end of the ce ll s was vacuolated and contained particulate materi a l. The multivesi c iular bodies were increased and contained a

ho mogenous or hete rogenous materi al. In the apica l region the mitochondri a atrophied and contributed to vacuolization (Figs 3 and 4).

In the control rats, the clear cells were fo und in between the principal ce lls. They contained ovoid nucle i placed slightly above the basal pos ition and contained granular chromatin material. The cytoplasm was abounded with lipid droplets. Mi cropinocytoti c ves ic les were prominent. The basa l ce ll s were e llipti cal and nucle i were e longa ted and fl attened aga inst the basement me mbrane (Fig. 5).

In the c lear ce ll s of trea ted rat, microv illi were mi ss ing. The size of the ce ll , nucleus and the cytoplasmic granules were increased. The chro matin material was less, nuclear enve lope was bul ged , karyolysis and karyohexis were noticed . M itochondria, Golg i apparatus and endopl asmic re ticulum were di srupted . Micropinocytotic ves ic les were ra re ly seen. Basa l ce ll s appeared decreased (Fig . 6).

Discussion

The epididymi s in general and the princ ipa l ce ll in particul ar, a re androgen-dependent and androgen withdrawal is known to cause exte nsive changes in the principa l cell. 2 1

-23 Thus, the changes in the

princ ipal cell of A. indica treated rats may refl ect a mani fes tati on of hypoandrogeni c status, brought about by A. indica leaf trea teme nt . However, d irec t acti on of A. indica leaf o n the princ ipa l ce ll cannot be excluded. Mic rotubul es constitute a principa l component of the ti ss ue matri x syste m of the epithe lia l ce ll s24

. Vin cristine is a microtu bul e di srupting agent23

-25 and microtubu les of the princ ip;:tl

cell may be the target for A. indica act ion. It is poss ible , therefore, that A. indica may have ca used pa tho logical changes Ill the cauda epi did ymal epithe li a l princ ipa l cell.

Jn the present study, principal ce ll and c lea r ce ll underwent ul trastructural changes fo ll owing A. indim treatmenl. A mong the s igni ficant cha nges observed in the princ ipal cell were dec rease in the number of micropinocytotic ves icl es and red ucti on in the size of mitoc hondria and Golg i appa ratus . Asha prakash r: t a /. 26 have suggested that absorpti ve functi on of p rinc ipal ce ll is impaired fo llow ing admin istrati on of

GHODESA WAR eta/. : ULTRASTRUCTURAL CHANGES IN EPITHELIAL CELL IN A. INDICA TREATED RATS I 093

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), dense microvilli (Mv) are present. The demarcation between 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 ident 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 reticu lum (ER) 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) heterchromatization is decreased.The mitochondrial (M ) are vacuolated. The endocytic vesicles (open arrow) are few x 5200.

1094 IN!DIA K .l EXP BIOL. NOVEMBER 2004

Mv·,. '·

;~ .\ \. ,.·.

'-y

-.. , -·

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 present showing its normal features. The nucleus (N) is normal. lying at the basal region. The Mitochondria(M) and endocytic vesicles are also normal. The cell abounds with 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 long showi ng its normal structure. The nucleus (N) of basal ce ll lies parallel to the basal lamina x 3600. (Fig. 6)- In the clear cell Micropinocytotic vesicles are decreased. Nuc;eus (N) is enlarged and the gap between o uter and inner nuclear membranes has increased (small arrows). The number o f lipid droplets (*) are increased. Mitochondria arc di srupted x 9600

cyproTerone 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 villcristine27

'28

•

In the clear cell of the A. indica treated rats , organelles viz., cytoplasmic vacuoles, electron dense secretory granules and mitochondria, 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 IS affected following A. indica treatment.

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

References

Yeung C H, Nashaw D, Sorg G, Oborpen nin F, Schulze H. Nieschlag E & Cooper T G, Bioi Rep rod. SO (1994 ) 917.

2 Hermo L, Barin K & Oko R, Anat Rec, 240 ( 1994) 86.

3 Gopal Dutt N H, Structure of mammalia epididymis, in The Comp Endocrinol Reprod, edited by K P Joy & C Halder. (Narosa Publishing House, New Delhi) 1999, 201.

4 Akbarsha M A, Avera! H I, Girija R .. Anandhi S & Faridha Banu A, Male reproductive toxici ty of vincristine: Ultrastructural changes in the epididymal epithelial apical cell, Biomed Let!, 102 (2000) 85.

5 Nadkarni A K, Nadkarni's Indian Materica Medica Vol. l, II and III (Popular Book Depot, Bombay) ( 1954).

GHODESAWAR et al.: ULTRASTRUCTURAL CHANGES IN EPITHELIAL CELL IN A. INDICA TREATED RATS 1095

6 Iyangar M A, Bibliography of Indian Medicinal Plants (1950 -1975) Manipal , College of Pharmacy, Kasturba Medical College, Indi a (1976) .

'7 Si nha K C, Ri ar S S, Tiwary R S & Dhawan A K, Neem oi l as a vaginal contraceptives, Indian J Med Res.79 (1984)131.

8 Sinha K C, Ri ar S S, Bardhan J, Thomas P, Kain A K & Jain R K, Antiimplantation effect of neem oil , Indian J Med Res, 80 (1 984) 708.

9 Chaudhary C N, Singh J N, Verma S K & Singh B P, Antifertili ty effects of leaf extracts of some plants in male rats, Indian J Exp Bioi, 28 ( 1990) 714.

10 Shaikh P D, Mani vannan B, Pathan K M, Kasturi M & Nazeer Ahamed R, Antispermatic activi ty of Azadirachta indica leaves in albino rats, Curr Sci, 64 (1993) 688.

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 albino rats, Indian J Physiol Pharmacal, 4 1 (1997) 234.

12 Anjali R J, Nazeer Ahamed R, Pathan K M & Manivannan B, Effect of Azadirachra indica leaves on testi s and its recovery in albino rats, Indian J Exp Bioi, 34 ( 1996) 1091.

13 Aladakatti R H & Nazeer Ahamed R, Effect of Azadirachta indica leaves on rats spermatozoa, Indian J Exp Bioi, 37 (1999) 1251.

14 Aladakatti R H, Nazeer Ahamed R, Mukhtar Ahmed & Ghodesawar M G, Sperm parameters changes induced by Azadirachta indica in albino rats, J Basic & Clin Physiol Phannacol, 12 ( 2001) 69.

15 Ghodesawar M G, Nazeer Ahamed R, Mukhtar Ahmed A W & Aladakatti R H, Azadiraclua indica adversely affects sperm parameters and fructose levels in vas deferens t1uid of albino rats, J Basic & Clin Physiol Pharmacal, 14 (2003) 387.

16 Ghodesawar M G, Nazeer Ahamed R & Aladakatti R H, Ultrastuctural changes in prostate gland and vas deferens induced by Azadirachta indica leaves in albino rats, J Nat Remed, 26 (2004) in press.

17 Poonam R, Bagga R, Malhotra D, Gopalan S & Talwar G P, Spermicidal and contraceptive properties of praneem polyherbal pessary, Indian J Med Res, 13 (2001) 135.

18 Kirtikar K R & Basu B D, Indian medicinal plants Vol. I-IV 2"d edition (Lali tmohan Basu, Allahabad, India) 1933.

19 Dastur J F, Medicinal plants of India & Pakistan (D B Taraporevala Sons & Co Ltd Bombay) 1962.

20 Reynolds E S, The use of lead citrate at hi gh pH as an electron-opeque stain in electron microscopy, J Cell Bioi, 17 (1963) 208 .

2 1 Hermo L & Robaire B, in The Epididymis: From Molecules to Clinical Practice, edited by B Robaire & B T Hinton , Kluwar Academic/Plenum Publi shers, New York (2002) 8 1.

22 Toney T W & Danzo B J, Developmental changes in and hormonal regulation of estrogen & androgen receptors present in the rabbit epididymis, Bioi Reprod, 39 (1998) 818.

23 Akbarsha M A, & Avera! HI, Male reproductive toxicity of vincristine: Ultrastructural changes in the epididymal principal cells, Biomed Lett, 57 (1998) 159.

24 Getzenberg R H, Pienta K J & Coffey D S, The tissue matri x: Cell dynamics and hom1one act ion , Endocr Rev, 11 (1990) 399.

25 Dustin P, Microtubules (Springer- Verlag Berlin) 1984.

26 Asha Prakash, Rajalakshmi M & Prasad M R N, Ultrastructural changes in the principal and clear cells in the rat epididymis fo llowing the admini stration of cyproterone acetate, Indian J Exp Bioi, 17 ( 1979) 1159.

27 Akbarsha M A & A vera] H I, Epididymis as a target organ for the toxic effect of vincristine: Light microscopic changes in the epididymal epithelial cell types, Biomed Lett, 54 (1996) 133.

28 Akbarsha M A & A vera! H I, Epididymis as a target for the toxic manifestation of vincristine: Ultrastructural changes in the clear cell, Biomed Lett, 59 (1999) 149.

29 Raj alakshmi M & Prasad M R N, Steroid, 28 (1976) 143.

30 Akbarsha M A & A vera] H I, Spermatotoxic effect of vincristine: An ultrastructural study in the rat, Biomed Lett, 54 (1996) 239.

31 Pereira M A S, Orsi A M, Molinan S L & Garcia P J, Alcohol effects on the principal cells of the caput epididymi s of albino rats, A nat Hist Embr, 32 (2003) 17.