TOPOGRAPHIC AND ULTRASTRUCTURAL VARIATIONS IN INFUNDIBULAR SEGMENT OF GOAT OVIDUCT

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1 Journal of Cell and Tissue Research Vol. 7 (1) (2007) ISSN: Original Article TOPOGRAPHIC AND ULTRASTRUCTURAL VARIATIONS IN INFUNDIBULAR SEGMENT OF GOAT OVIDUCT SHARMA, R. K.? AND RITA, S. Reproductive Physiology Laboratory, Department of Zoology, Kurukshetra University, Kurukshetra E mail: rkszookuk@yahoo.co.in Received: June 20, 2006; Revised: July 7, 2006 ; Accepted: August 1, 2006 Abstract: Topographic and ultrastructural changes in the infundibular segment of goat (Capra hircus) oviduct has been studied during the follicular and luteal phases of the oestrous cycle. Histologically, the mucosal epithelium of the infundibulum revealed characteristic pattern of changes in the number, height, branching of the mucosal folds and relative frequency of ciliated and secretary cells during both the phases of the oestrous cycle. Topographic analysis of the infundibular epithelium in the follicular phase demonstrated uniform distribution of ciliated cells. The frequency of the non-ciliated cells were higher and restricted zones of ciliated cells was observed during the luteal phase. Secretory cells appeared as collapsed envelopes (ghost cells) at some places. The presence of low electron dense cytoplasm in the ciliated cells was the characteristic of the follicular phase. Densely stained secretory cells possessed small secretory granules with dense homogeneous matrices. The non-ciliated cells of the luteal phase showed large finger like cytoplasmic protrusions. Ciliated cells were interspersed with secretory cells at some restricted zones. The results demonstrate that the oviductal epithelium of goat revealed marked cyclic variations in the topography and fine structure. Keywords: Topography, Ultrastructure, Fallopian Tubes. INTRODUCTION The mammalian oviduct, a tubular conduit is the site of fertilization and early development [1,2]. The epithelial ciliated and non-ciliated cells of the oviduct are involved in the transport of oocytes and secrete stage specific proteins and glycoproteins that provide environment necessary for the maturation of gametes, their interactions and early embryonic development [1,3-5]. Detailed electron microscopic observation of the oviductal segments has been carried out in many mammals [6-10]. Most of these studies were restricted to laboratory animals, bovine or ovine species [11-14]. However in caprine species only, ultrastructural features of goat oviduct has been described [15]. The correlative light, scanning and transmission electron microscopic studies have not been carried out so far. The main objective of this study is to define the structure and functions of infundibulum of goat oviduct in follicular and luteal phases of the oestrous cycle. MATERIALS AND METHODS The genitalia of mature normal cycling female goats (Capra hircus) were collected from the slaughter houses of Delhi (28 o 38 1 N, 77 o 12 1 E). On gross morphology of the ovary and ferning pattern of vaginal smear, the genitalia were divided into two groups. Ovaries possessing (>5.0 mm in diameter) normal developing Graafian follicle and vaginal smear showing clear primary, secondary and tertiary fern pattern were designated in follicular phase. While ovaries possessing corpus luteum (>5.0 mm in diameter) and vaginal smear, showing primary and secondary fern pattern were categorized into luteal phase. After morphometric analysis, tissues were processed for light microscopy [16]. For scanning electron microscopy, the infundibular segment of the oviduct from both phases were fixed in 4% gluteraldehyde for 24 hours at 4 o C and post fixed in 1% osmium tetroxide for 4 h. [17]. After dehydration and critical point drying in liquid CO 2 the samples 889

2 J. Cell Tissue Research were gold plated by sputtering and subjected for topographic analyses under JSM-6100 Jeol Tokyo, Japan installed at sophisticated analytical instrumentation facility, Panjab University, Chandigarh. For transmission electron microscopic analysis, infundibular segment of both phases were fixed in 2.5% gluteraldehyde, post fixed in 1% osmium tetroxide. After fixation, the samples were embedded in Epon 812 and ultrathin sections were cut [18]. The sections were stained with uranyl acetate and lead citrate and studied at All India Institute of Medical Sciences, New Delhi. RESULTS AND DIUSSION Infundibulum, the anterior portion of goat oviduct, is comprised of serosa, musculosa, submucosa and mucosa. These four structural entities undergo variations in their dimensions. Height of the mucosal folds and mucosal epithelium (ciliated cells and secetory cells) showed higher values during the follicular phase as compared to luteal phase (Table 1). The mucosal epithelium revealed characteristic pattern of changes in different phases of oestrous cycle. From the histomorphometric data, it is evident that as goat proceeds from follicular phase to luteal phase, the height of mucosal folds and thickness of mucosa (both ciliated and non-ciliated cells) decreased significantly (P<0.05). Similar reports on cattle, buffalo, sows and wild collared peccary oviductal histomorphology has already been documented [19-23]. Goat infundibulum in its histomorphological variations revealed a generalized bovine pattern. During the estrogen dominant phase, ciliated cell number and size were higher than that of progesterone dominant phase. Hormone dependent oviductal epithelial Table-1 : Histometric data on the mucosal components of infundibulum during follicular and luteal phases of oestrous cycle of goat. *P<0.05, Values are expressed as Mean ± S.E. (range in parenthesis). Histological Components Follicular Phase Luteal Phase Serosa (µm) 29.5± 3.4 ( ) 13.5 ± 1.0* ( ) Musculosa (µm) 28.5 ± ± 2.8* ( ) (22.90 ± 30.80) Submucosa (µm) 11.0 ± 0.6 ( ) 14.0 ± 1.0* ( ) Height of Mucosal ± ± 62.0* folds (µm) ( ) ( ) Width of Mucosal 83.5 ± ± 4.1* folds at base (µm) ( ) ( ) Width of Mucosal 51.5 ± ± 2.3* folds at tip (µm) ( ) ( ) Ciliated Cells (%) * Secretory Cells (%) * Height of the ciliated 31.8 ± ± 0.3* cells (µm) ( ) ( ) Height of the 32.3 ± ± 0.8* secretory cells (µm) ( ) ( ) cyclicity has also been reported in sheep [14,24] and cannine uterine tubes [25]. Scanning electron microscopic studies showed a thick coat of microvilli on the ciliated cells in the mucosal epithelium of the infundibulum during the follicular phase. Ciliogenesis revealed a well organized rosette or flame pattern. A uniformed length cilia were evenly distributed and extended above the apices of secretory cells. The ciliated cells concealed the secretory cells to some extent. The ciliated cells in the infundibulum were larger and the number of cilia projected from each cell was similar (Fig. 1). The apical surfaces of the secretory cells were rounded, elliptical or flat and were covered with cilia. Apical surfaces of the non-ciliated cells also possessed numerous stubby microvilli. Cytoplasmic blebs were also observed (Fig. 2). During the luteal phase, the Fig. 1-2 : Infundibulum from the oviduct of goat in the follicular phase. Fig. 1. Showing uniform distribution of cilia (Ci) and variations in the size and shape of secretory cell processes. Bar = 10 µm. Fig. 2. The apical surfaces of non-ciliated cells (NC) are concealed by the cilia and possessed short prominent microvilli (Mv). Bar = 1 µm Fig. 3-4 : Infundibulum from the oviduct of goat in the luteal phase. Fig. 3. Showing the conspicuous non-ciliated cell processes (P). Many cilia (CI) are concealed by bulbous processes of the nonciliated cells. Bar = 10 µm. Fig.4. Bulbous apical processes (P) of non-ciliated cells. Some of these processes lack microvilli. Bar = 1 µm Fig. 5-6 : Electron micrograph of luminal surface of infundibular oviduct in the follicular phase. Fig. 5. Ciliated cells (CC) with medially located nuclei, rough endoplasmic reticulum and mitochondria. Dome shaped secretory cells () possessed numerous secretory granules in the apical protrusions. Bar = 2 µm. Fig.6. Large vacuole (V) containing fine granular substance in the apical region of secretory cell. Bar = 1 µm. Fig. 7-8 : Electron micrograph of luminal surface of infundibular oviduct in the luteal phase. Fig. 7. The secretory cells () display large finger like partially-collapsed cytoplasmic protrusions. Bar = 2 µm. Fig. 8. Apical protrusion of the secretory cell () showing secretory granules, glycogen granules, few coated vesicles and mitochondria. Bar = 1 µm. 890

3 Sharma and Rita Mv NC 2 P P Ci 3 4 N CC SG M V 5 6 M

4 J. Cell Tissue Research infundibular epithelium was extensively covered by the bulbous processes of the secretory cells. Majority of these processes were elliptical shaped and of varied sizes (Fig. 3). Extensive hypertrophy of the secretory cells were observed. A number of swollen or fully extended peg cells/goblet cells were seen. Stubby microvilli were seen on apical surfaces of some of the secretory cells. At few places, the secretory material of these cells were lying in close vicinity and the cells revealed collapsed envelopes. The ciliated cells were concealed by the secretory cell processes as these processes extended beyond the tips of cilia (Fig.4). The results of the present investigation demonstrate that infundibulum of goat oviduct undergo marked changes in ciliation and secretory activity during the estrous cycle. Heavily ciliated fimbriae in follicular phase changed to largely secretory epithelium during luteal phase. The large number of cilia were hidden during this phase. Similar cyclic changes have been observed in the pig-tailed monkey [7], Chinese Meishan pig [9] and cows [1]. In fimbriae, the cilia are primarily responsible for the pick up and transport of ovulated eggs [3]. It is, therefore, a richly ciliated epithelium is developed at ovulation. It seems that the cyclic changes observed in the present study reflect the functions of cilia in the fimbriae of goat oviduct. The number of cilia on fimbriae tracts declined at the luteal phase. These changes are largely due to dramatic decrease in the height of ciliated cells. The height of non-ciliated cells remain more stable, thus ciliated cells seems to have got reduced in number. An identical cyclic phenomenon has also been reported in cows [1]. The reduction in ciliated cells in luteal phase can be attributed to the loss of the cilia themselves from the ciliated cells. The regular cycle of ciliogenesis and deciliation by epithelial cells of mammalian oviducts is dependent upon the level of circulating estrogen and progesterone [26,27]. It is therefore, suggested that increase in the height of secretory cells and deciliation together contribute to the reduction in number of ciliated cells during progesterone dominant luteal phase. Transmission electron microscopic analysis revealed low electron dense cytoplasm in the ciliated cells of the infundibulum during follicular phase. These cells showed the presence of fibrous granules and basal bodies along the apical border of the cells, which were characterized by medially located nuclei with chromatin material, rough endoplasmic reticulum, ribosomes and mitochondria. The basal bodies possessed electron dense glycogen particles. The dome shaped secretory cells stained more darkly than that of ciliated cells and possessed numerous secretory granules with in the apical protrusions. Secetory granules showed electron-dense homogeneous matrices and electron leucent areas within them (Fig.5). A large vacuole containing a fine granular substance was observed in the apical region of secretory cell of the follicular phase (Fig.6). During luteal phase, the non-ciliated cells were prominent and were interspersed with the ciliated cells at some restricted belts. Non-ciliated secretory cells showed large finger-like partially collapsed cytoplasmic protrusions (Fig. 7). A few coated vesicles and small homogeneous secretory granules were observed in the apical protrusion of the secretory cells. The cytoplasm contained glycogen granules. Partially surrounded mitochondria by the cisternae of endoplasmic reticulum was observed (Fig.8). Lamellated secretory granules were also seen at some places. The non-ciliated secretory cells of the fimbriae were characterized by the presence of prominent secretory granules of different sizes and electron densities. The relative abundance and electron density of these granules was higher during the luteal phase. It, therefore, appears likely that synthesis of secretory granules occurs mainly during the follicular phase and may be under hormonal control of estrogen as reported in sheep [13,28-30] and goats [31]. The apical protrusions of the secretory cells into the oviductal lumen throughout the oestrous cycle, and the location of nuclei within these protrusions during luteal phase were more prominent. These protrusions possessed a number of swollen mitochondria, lysosome like bodies and a number of cell organelles of various shapes and sizes. The secretory granule characteristics represent formation, maturation and release of secretory granules as also reported in other species [2,13,30,32-34]. The presence of apical vesicles in ciliated cells of the infundibulum and their apocrine release into lumen has not been found in goat. However, only single statement on sheep isthmus, a similar secretory activity has been reported [13]. The role of these cells around estrous need to be analysed further for their attributes in reproduction. ACKNOWLEDGEMENTS The authors acknowledge the financial assistance 892

5 Sharma and Rita provided by the Kurukshetra University, Kurukshetra. Authors are also thankful to the officers in-charge, Electron Microscopy, Panjab University, Chandigarh and All India Institute of Medical Sciences, New Delhi. REFERENCES [1] Abe, H. and Oikawa, T.: Anat. Record, 235: (1993). [2] Lauschova, I.: Scripta Medica (BRNO), 76(4): (2003). [3] Odor, D.L. and Blandau, R.J.: Fert. Steril., 24: (1973). [4] Oliphant, G.: In: The Fallopian Tube, Basic Studies and Clinical Contributions (Siegler, A.M. ed.) Futura, Mount Kisco, New York, pp (1986). [5] Abe, H. and Hoshi, H.: Cytotechnology, 23(1-3): (1997). [6] Nilsson, O. and Reinius, S.: In: The Mammalian Oviduct. (Hafez, E.S.E. and Blandau, R.J. eds). The University of Chicago Press, Chicago and London, pp (1969). [7] Rumery, R.E., Gaddum-Rosse, P., Blandau, R.J. and Odor, D.L.: Am. J. Anat., 153: (1978). [8] Odor, D.L., Gaddum-Rosses, P., Rumery, R.E. and Blandau, R.J.: Anat. Rec., 198: (1980). [9] Abe, H. and Oikawa, T.: Anat. Rec., 233: (1992). [10] Hunter, R.H.F. : Reprod. Nutr. Dev., 45: (2005) [11] Hafez, E.S.E. and Kanagawa, H.: Cornell Vet., 63: (1973). [12]Nayak, R.K. and Ellington, E.F.: J. Animal Sci., 35: (1977). [13] Hollis, D.E., Frith, P.A., Vaughan, J.D., Chapman, R.E. and Nancarrow, C.D.: Am. J. Anat., 171: (1984). [14] Murray, M. K.: Biol. Reproduc., 53: (1995). [15] Abe, H., Onodera, M., Sugawara, S., Satoh, T. and Hoshi, H.: J. Anat., 195: (1999). [16] Pearse, A.G.E.: Histochemistry: Theoretical and Applied. Churchill. London (1968). [17] Sharma, R.K. and Guraya, S.S.: Acta Embr. Morph. Exp., 11: (1990). [18] Zamboni, L.: In: Ovulation in Human (Crosignani, P.G. and Mischell, D.R. eds.) London and New York, pp 1-30 (1976). [19] Snedecor, G.W. and Cochran, W.G.: Statistical Method. 6th ed., Oxford and IBH Publishing Company, New Delhi (1968). [20] Natarajan, T., Prasad, R.V., Kakde, K. and Jamuna. K.V.: Ind. J. Animal Sci., 73 (5): (2003). [21] Natarajan, T., Prasad, R.V., Kakde, K. and Jamuna. K.V.: Ind. J. Animal Sci., 73 (5): (2003) [22] Sant Ana, F.J.F., Nascimento, E.F., Nogueira, J.C. and Serakides, R.: Arq. Bras. Med. Vet. Zootech., 55(2): 3-8 (2003) [23] Mayor, P., Jori, F. and Lopez-Bezar, M.: Anot. Histol. Embryol., 33: (2004). [24] Lewis, A.W. and Berardinelli, J.G.: J. Anim. Sci., 79: (2001). [25] Steinhauer, N., Boos, A. and Gunzel-Apel, A.R.: Reprod. Domest. Anim., 39(2): (2004). [26] Brenner, R.M., Resko, J.A. and West, N.B. : Endocrinology, 95 : (1974). [27] Verhage, H.G., Bareither, M.L., Jaffe, R.C. and Akbar, M.: Amer. J. Anat., 156: (1979). [28] Willemse, A.H.: A light microscopical study, Tijdschr. Diergeneeskd, 100: (1975). [29] Willemse, A.H., and Van, Vorstenbosch, C.J.A.H.V.: An electron microscopical study. Tijdschr. Diergeneeskd, 100: (1975). [30] Russe, I., and Liebich, H.G.: Cell Tissue Res., 201: (1979). [31] Krajnicakova, M., Cigankova, V., Lenhardt, L., Kostecky, M. and Maracek, I.: Acta Vet. Brno, 7: (2002). [32] Willemse, A.H. and Van Vorstenbosch, C.J.A.H.V.: Tijdschr. Diergeneeskd, 100: (1975). [33] Nayak, R.K., Albert, E.N. and Kassira, W.N.: Amer. J. Vet. Res., 37: (1976). [34] Odor, D.L., Gaddum-Rosse, P. and Rumery, R.E.: Amer. J. Anat., 166: (1983). 893