Morphological Aspects by Light and Scanning Electron ...€¦ · estrous cycle. Materials and...

Thai J Vet Med. 2013. 43(1): 23-32.

Morphological Aspects by Light and Scanning Electron

Microscopic Studies of Swamp Buffalo Endometrium at

Follicular and Mid-luteal Phases

Paisan Tienthai* Kriengyot Sajjarengpong

Abstract

This study was carried out to observe morphological changes of swamp buffalo endometrium at follicular

and mid-luteal phases. Uterine horns were collected from female buffaloes at a local abattoir and the selected estrous stages were categorized into the follicular (n = 10) and mid-luteal (n = 10) phases. General histology and histomorphometry were examined under light microscope (LM) whereas a scanning electron microscope (SEM) was used to study surface epithelial changes. The results showed that the height of the endometrial epithelium, the number of superficial endometrial glands and the number of capillaries were significantly greater (p < 0.05) at the follicular phase. By SEM examination, the ciliated and secretory cells with different patterns, i.e. abundant microvilli on the apical part or secretory protrusion in various degrees, distinctly increased at the follicular phase. In the meantime, numerous secretory cells with stubby microvilli were covered throughout the endometrial surface with secretory vesicles on endometrial glandular orifices at the mid-luteal phase in which the ciliated cells were sparsely seen. It was concluded that the swamp buffalo endometrium obviously revealed modifications during the estrous cycle for physiological events, i.e. sperm transport, early embryonic development and implantation.

Keywords: estrous cycle, morphology, scanning electron microscopy, swamp buffalo, uterus Department of Anatomy, Faculty of Veterinary Science, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand *Corresponding author: E-mail: [email protected]

Original Article

24 Tienthai P. and Sajjarengpong K. / Thai J Vet Med. 2013. 43(1): 23-32.

บทคดยอ

ลกษณะทางสณฐานวทยาโดยการศกษาดวยกลองจลทรรศนแสงสวางและอเลกตรอนแบบสองกราดของเนอเยอโพรงมดลกกระบอปลกในระยะฟอลลคลารและลเทยลชวงกลาง ไพศาล เทยนไทย* เกรยงยศ สจจเจรญพงษ

งานวจยนตองการศกษาการเปลยนแปลงทางสณฐานวทยาของเนอเยอโพรงมดลกกระบอปลกในระยะฟอลลคลารและลเทยลชวงกลาง เกบปกมดลกกระบอปลกเพศเมยจากโรงฆาสตวทองถนจานวน 20 ตว แบงวงรอบการเปนสดออกเปนระยะฟอลลคลารจานวน 10 ตว และระยะลเทยลชวงกลางจานวน 10 ตว ศกษาลกษณะทวไปและตรวจวดตวแปรตาง ๆ ของเนอเยอโพรงมดลกดวยกลองจลทรรศนแสงสวาง ขณะทนาเยอบผวมดลกนามาศกษาดวยกลองจลทรรศนอเลกตรอนแบบสองกราด ผลการศกษาพบวาความสงเยอบผวมดลก จานวนตอมมดลกชนผว และจานวนของหลอดเลอดฝอยบรเวณชนใตเยอบผวเพมขนอยางมนยสาคญ (p < 0.05) ในระยะฟอลลคลาร จากลกษณะเยอบผวมดลกภายใตกลองจลทรรศนอเลกตรอนแบบสองกราดพบวา เยอบผวปกคลมไปดวยเซลลทมซเลย และเซลลคดหลงทมลกษณะแตกตางกน ตงแตการพบเซลลคดหลงทมไมโครวลไลจานวนมากและทมลกษณะเปน secretory protrusion ในระดบตาง ๆ กน กระจายอยเปนจานวนมากในระยะฟอลลคลาร ขณะทในระยะลเทยลชวงกลาง พบเซลลคดหลงทมไมโครวลไลสนปกคลมอยางหนาแนน โดยพบ secretory vesicles ทบรเวณรเปดของตอมมดลกกระจายอยเปนจานวนมาก การศกษานสรปไดวา ลกษณะของเนอเยอโพรงมดลกกระบอปลก เปลยนแปลงตามวงรอบการเปนสดเพอรองรบกลไกตาง ๆ ทางสรรวทยาทเกดขนตามลาดบ ตงแตการขนสงเซลลอสจ การเจรญของตวออนในระยะแรก และการฝงตวของตวออน

คาสาคญ: วงรอบการเปนสด สณฐานวทยา จลทรรศนอเลกตรอนแบบสองกราด กระบอปลก มดลก ภาควชากายวภาคศาสตร คณะสตวแพทยศาสตร จฬาลงกรณมหาวทยาลย ปทมวน กรงเทพฯ 10330 *ผรบผดชอบบทความ E-mail: [email protected]

Introduction

Buffaloes (Bubalus bubalis) in South-East Asia are principally of the swamp type and are raised for their draught power and meat in incorporated crop-livestock agricultural systems (Perera, 2008). In Thailand, the swamp buffalo population in 1980 was 5.65 million head which had declined to 1.62 million head by 2010 (FAO, 2012). In addition to genetic selection programs and quality of feed resources, important factors such as delayed puberty, long calving interval and poor detection of estrus phase including reproductive disturbances, e.g. early embryonic death, repeat breeding, and reduced conception rates, have been the main constraints on enhanced productivity in this species (Nanda et al., 2003; Pasha and Hayat, 2012). Importantly, most of the risk factors have occurred in relation to vital female reproductive organs which are composed of ovary, oviduct and uterus. To increase swamp buffalo population and production, a fundamental knowledge with reference to the endocrinology, physiology and morphology influencing the female reproductive organs should be further studied in

parallel with improvement of semen quality (Koonjaenak et al., 2007a; 2007b) and advanced reproductive bio-techniques as it has already started in river buffaloes (Singh et al., 2009; Perera, 2011). Recently, morphological features by light microscopy (LM) and scanning electron microscopy (SEM) of the swamp buffalo oviduct, where diverse critical events of fertilization occurred, have been thoroughly reported at both follicular and luteal phases (Tienthai et al., 2008; 2009). In contrast, fundamental studies of endometrium in swamp buffalo uterine horns have rarely been observed. Generally, the most important function of the uterine horns in ruminant is involved in sperm transport, implantation, pregnancy and parturition in associated with adjustment of cellular structures and micro-environments (Barnes, 2000). In cows, uterine fluid produced by transudation of blood serum, uterine epithelial cells and endometrial glands is compulsory for sperm maturation and capacitation including early embryonic development, in which blastocysts take a prolonged period before their attachment in endometrium (Kings et al., 1981). In bovines, most cells and associated structures in the endometrial compartments undergo proliferation, differentiation and activity in correlation to fluctuation of steroid

Tienthai P. and Sajjarengpong K. / Thai J Vet Med. 2013. 43(1): 23-32. 25

hormonal levels, i.e. estrogen and progesterone, throughout the estrous cycle (Boos et al., 1996; Wang et al., 2007). Therefore, supplementary information concerning the morphology of the swamp buffalo endometrium is definitely required to distinguish normal physiological status or events during cycling phases from abnormalities. The purpose of this study was to investigate the morphological modifications by general histological, histomorphometric and SEM approaches in the swamp buffalo endometrium at the follicular phase in comparison to the mid-luteal phases of the estrous cycle.

Materials and Methods Sample collection and tissue preparation: Female reproductive organs of swamp buffaloes at various ages (3-6 years) were brought to the laboratory of the Anatomy Department in a cool container from an abattoir immediately after slaughter for macroscopic examination. The reproductive tracts were classified into the follicular phase (days 18-20, n= 10) and the mid-luteal phase (days 5-10, n= 10) based on the characteristics of the corpus luteum and the dominant follicle as described by Roy et al. (2006). The uterine horns were dissected out of the mesometrium and the middle segment was cut about 2 cm in length. One piece of uterine sample was immersed in 10% buffered formalin for studying by LM while the other portion was longitudinally exposed and immersed in 2.5% glutaraldehyde in 0.1 M phosphate buffer saline (PBS) for routine exploration by SEM.

General histology and histomorphometry: After fixing in 10% buffered formalin, the samples were embedded in paraffin. Tissue blocks were cut into 4 µm-thick sections and stained with hematoxylin and eosin (H&E). Evaluation was accomplished under LM (BX50, Olympus, Tokyo, Japan) equipped with a digital camera Micropublisher 5.0 (Qimage, Surrey, BC, Canada) at a magnification of 200x and 400x by movement of the ocular micrometer across the entire tissue slide as described by Tienthai et al. (2008). The heights were measured by use of the selected programs of Image Pro-Plus version 6 (Media Cybernatics Inc., MD, USA). In each microscopic field, the following parameters were evaluated. A) the types of uterine epithelium and endometrial glandular epithelium (superficial and deep glands) at 400x; B) the height (μm) of the uterine epithelium from the basement membrane to the tip of epithelial cell (not including the cilia or secretory droplets) at 400x (40 different positions); C) the height (μm) of the endometrial glandular epithelium (superficial and deep glands) at 400x (40 glands); D) the number of superficial and deep endometrial glands (gland/ocular field area) at 200x (40 fields); and E) the number of capillary (number/ocular field area) in the subepithelial layer close to the uterine epithelium at 400x (40 fields).

SEM procedures: The uterine tissues preserved in 2.5% glutaraldehyde were stored at 40C for 24 hours and cut into small pieces. The samples were rinsed in distilled water, post-fixed for 1 hour in 1% osmium

tetroxide (Merk, Darmstadt, Germany) in 0.1 M PBS, dehydrated in graded ethanol (30-100%) and substituted with acetone. The tissues were then subsequently subjected to critical point drying using liquid CO2 substitution. Dehydrated samples were adhered to the stubs with clear nail polish, coated with gold-palladium, and analyzed under JEOL 5800 LV SEM (JEOL, Tokyo, Japan) at an accelerating voltage of 15 kV.

Statistical analyses: Data were statistically analyzed using the SAS statistical package (version 9.0, SAS Institute Inc, Cary, NC, USA). Least squares means were analyzed by analysis of variance (ANOVA) via the General linear model (GLM) procedure and compared using the least significant difference test. A value of p < 0.05 was considered statistically significant.

Results General histological remarks

In swamp buffalo, there were different regions of endometrium by general histology, i.e. the intercaruncular area and caruncular area (Fig 1a-b). Noticeably, the caruncular area had, no gland within its own subepithelial connective tissue (CNT) layer (Fig 1b) whereas the intercacuncular area had, like domestic animal uterine horns, superficial and deep endometrial glands throughout the subepithelial CNT layer were found (Fig 1a). The types of uterine e p i t h e l i u m e x h i b i t e d h i g h t o m e d i u m

Figure 1 Light microscopic photographs of the

intercaruncular area (a) and the caruncular area (b) in the swamp buffalo uterine horn by LM. The intercaruncular area exhibits uterine lumen (a), the uterine epithelium (b), the subepithelial connective layer (c), the muscular layer (d), the superficial endometrial glands (arrows) and the deep endometrial gland (arrowheads). H&E staining, Bar = 200 µm.


Figure 2 Light microscopic photographs of the uterine epithelium (a, b) the superficial endometrial glands (c, d) and the deep

endometrial glands (e, f) of the swamp buffalo endometrium at the follicular phase (a, c, e) and the mid-luteal phase (b, d, f): Uterine epithelium (EP); Subepithelial layer (SE); Uterine fluid (UF); Capillary (arrows). H&E staining, Bar = 30 µm.

pseudostratified columnar epithelium during the follicular phase and small secretory droplets were easily noticed on the luminal epithelium (Fig 2a). Medium to low pseudostratified columnar or simple columnar epithelia were observed at the mid-luteal phase (Fig 2b). The superficial glandular epithelium was pseudostratified or had simple columnar types which were almost the same at both phases (Fig 2c-d). However, secretory droplets clearly protruded from the superficial glandular epithelium at the mid-luteal phase and the uterine fluid accumulated within the lumen of these glands (Fig 2d). The low simple columnar epithelium was of the type of deep endometrial glandular epithelium at both phases (Fig 2e-f).

Evaluation of histomorphometry

In this study, we focused on the intercaruncular area due to the structural compartments being completed as they appeared in the endometrium of other domestic animals. The height of uterine epithelium was significantly greater (p< 0.05) at the follicular phase than at the mid-luteal

Table 1 Mean±SD of the uterine epithelial (UE), the superficial endometrial glandular epithelial (SEGE) and the deep endometrial glandular epithelial (DEGE) height (µm) of the swamp buffalo endometrium at selected stages of the estrous cycle

Estrous cycle stages UE SEGE DEGE Follicular 34.1 ± 5.7a 25.4 ± 3.7a 13.2 ± 2.9a Mid-luteal 25.7 ± 3.3b 26.8 ± 1.4a 12.2 ± 1.3a

a,b Different superscript within column differed significantly (p < 0.05)

Table 2 Mean±SD of the capillary underneath the uterine epithelium as number/62,500 μm2, superficial endometrial gland (SEG) and deep endometrial gland (DEG) as number/15,625 μm2 of the swamp buffalo endometrium at different stages of the estrous cycle

Estrous cycle stages Capillary SEG DEG Follicular 7.4 ± 1.4a 1.9 ± 0.5a 14.6 ± 3.0a Mid-luteal 3.5 ± 1.1b 2.8 ± 1.4b 15.8 ± 2.8a

a,b Different superscript within column differed significantly (p < 0.05)


phase (Table 1). The number of capillaries within the subepithelial CNT layer near the uterine epithelium was also greater (p < 0.05) at the follicular phase and

only the number of superficial endometrial glands was significantly higher (p < 0.05) during the mid-luteal phase (Table 2).

Figure 3 SEM photomicrographs of the uterine epithelial surface in the swamp buffalo endometrium at proestrus of the follicular

phase (a-d) showing sparsely ciliated cells (Ci) surrounded by abundant flat secretory cells (Sc) that are covered with short microvilli (a, b) or swelling secretory cells with numerous microvilli on the surface epithelium (c, d).

Figure 4 SEM photomicrographs of the uterine epithelial surface in the swamp buffalo endometrium at estrus of the follicular phase

(a-d) show the increase in ciliated cells (Ci) with very long cilia and the distension of secretory cells (Sc) with or without microvilli (a, b). In some areas, the secretory cells protruded in varying degrees and patterns from the apical part and the ciliated cells were usually located around the endometrial gland opening (c, d).


Figure 5 SEM photomicrographs of the uterine epithelial surface in the swamp buffalo endometrium at the mid-luteal phase (a-d)

exhibit abundant flat secretory cells covered with short microvilli without ciliated cells. The spherical structures (white arrows) which presumably were the secretory vesicles from the endometrial gland appear at the opening of the endometrial glands (a, c, d) and are also distributed on the surface epithelium (b). Notice the numerous craters on the surface epithelium (a, c) which might occur when the vesicles are released from the epithelium.

Uterine epithelium by SEM

The luminal epithelium of swamp buffalo endometrium was clearly composed of ciliated cells and various patterns of protruding secretory cells during the follicular phase (Fig 3-4). In some specimens that were expected to be proestrus (Fig 3a-b), ciliated cells with medium length of cilia were rarely found and were surrounded by abundant flat secretory cells. In some areas of the same samples, we observed a few scattered ciliated cells encircled by a plentiful number of swelling secretory cells with numerous stubby microvilli on the apical part which were sketched out by continuous cell border depression (Fig. 3c-d). In other samples that were supposed to be estrus, the ciliated cells were easy to observe and were evenly distributed on the surface when compared to the secretory cells (Fig 4a-b). The ciliated cells in some regions were usually located around the endometrial gland openings and the secretory cells were distinguished by several characteristics, i.e. smooth surface cells or slightly convex cells with or without microvilli, and rounded protruding cells in various degrees without microvilli (Fig 4c-d). During the mid-luteal phase, the ciliated cells declined in number or disappeared whereas the flat secretory cells with short microvilli and borders of a mosaic-like shape were covered throughout the luminal epithelium (Fig 5a-d). On the luminal surface, the round and smooth secretory vesicles were noticeably protruding from the opening of endometrial glands (Fig. 5a, c-d) and were scattered on the epithelium covered by microvilli (Fig 5b). Conspicuously, these secretory vesicles were sometimes released or washed away during the routine SEM process and a lot of crater-like invaginations on the surface epithelium were revealed instead (Figs 5a, c).

Discussion

In the present report, the swamp buffalo endometrium was investigated by general histological, histomorphometric and scanning electron microscopic studies to obtain fundamental knowledge for further biotechnological and clinical application. This data indicated that the various structures and cells combined within the endometrium of swamp buffalo uterine horn could be modified depending on the stages of the estrous cycle for appropriate functions.

It has been known that the placental shape of ruminants is classified as cotyledonary placenta (Dantzer and Leiser, 2006), therefore, the caruncular and intercaruncular regions undoubtedly appeared in the swamp buffalo endometrium. Importantly, Dhaliwal et al. (2002) indicated that in cows this area demonstrate the physiological activity more than the caruncular area. In this study, we tried to carry out morphological changes that mainly occur in the intercaruncular area where the structural compartment is related to the endometrium of other farm animals. The results showed that the uterine epithelium of the swamp buffalo endometrium was covered by pseudostratified columnar at the follicular phase or simple columnar types at the mid-luteal phase similar to a previous report by Priedkalns and Leiser (2006). The height of the uterine epithelium and number of capillaries that are located close to uterine epithelium obviously increased during the follicular phase in our report which is accordance with studies in porcine (Kaeoket et al., 2002). This data pointed out the influence of estrogen of which a high level was found at proestrus and estrus, i.e. the follicular phase, in swamp buffalo (Kanai and Shimizu, 1984). Furthermore, the most important function of a high estrogen level was that it caused an increase in


cellular proliferation, uterine epithelial cell secretion, uterine blood flow and endometrial vascular permeability (Johnson et al., 1997; Barnes, 2000). By histological study, we also confirmed this data through the appearance of the secretory droplets protruding from the uterine epithelial cells and uterine edema within the subepithelial CNT layer at the follicular phase (Fig 2a, c). With regard to progesterone, Kanai and Shimizu (1984) suggested that the plasma progesterone concentrations in swamp buffaloes increased after day 5 (day 0 = day of estrus) and climbed to a high level by about day 10 of the estrous cycle. Therefore, the uterine samples examined in the present study were precisely under the control of progesterone. It is also known that the main function of progesterone is associated with the growth and secretory activity of endometrial glands (Barnes, 2000; Priedkalns and Leiser, 2006) which corresponds to the present results, which exhibit the secretory vesicles bulged from the glandular epithelial cells or building up within the glandular lumens. With regard to the endometrial glands, the superficial glands increased at the mid-luteal phase in this study. On the contrary, Wang et al. (1997) found that a total number of superficial and deep endometrial glands in cows were unchanged between cycle phases but the area of the subepithelial CNT layer enlarged at the follicular phase. For this reason, superficial endometrial glands that increased during the mid-luteal phase in our study might occur as a result of the reduction in uterine edema of subepithelial CNT layer. However, further details in the quantity of both endometrial glands could be circumspectly estimated throughout this area by the advanced efficiency methods.

The SEM micrographs emphasized the estrous cyclic modifications of the swamp buffalo uterine surface epithelium and disclosed the characterization of ciliated and secretory cells that corresponded with earlier studies in pigs (Wu et al., 1976), rabbits (Barberini et al., 1978), cows (Almeida et al., 1986), dogs (Van Cruchten et al., 2003) and horses (Al-Bagdadi et al., 2004). In our preliminary study, the uterine epithelial surface of prepubertal swamp buffalo depicted a smooth surface of presumably secretory cells with rarely scant microvilli or without microvilli and prominent mosaic-shape boundaries whereas the ciliated cells were only sparsely observed and importantly they were only situated at the rims of the endometrial glandular openings (P. Tienthai, unpublished observations). This finding confirmed the morphological changes of uterine epithelial surface when the swamp buffaloes reach puberty. Furthermore, in ovariectomized rats in which the uterine epithelial characteristics were similar to prepubertal swamp buffalo, the microvilli lengths and numbers greatly increased after injection with estradiol-17β within a short time whereas the progesterone decreased these incidents (Anderson et al., 1975; Rambo and Szego, 1983). These investigations support the theory that the epithelial cell modification is induced by ovarian steroid hormones and could be concluded that when the swamp buffaloes reach puberty, the uterine epithelial

surface will change rapidly. Unquestionably, the SEM micrographs in the present study can be shown in different patterns of cell and structural modifications from proestrus to estrus due to the reasons above. In several animals (Wu et al., 1976; Wick and Kress, 2002; Van Cruchten et al., 2003), some areas of the uterine epithelial were covered by a flat mosaic-shape with scant microvilli of cells to a convex appearance with abundant long microvilli of cells and very sparse number of ciliated cells at proestrus whereas the ciliated cells obviously increased at estrus, which is in accordance with our observations. In this study, the ciliated cells were largely distributed on the endometrial surface at the follicular phase but they almost vanished by the mid-luteal phase. In the ampulla and infundibulum, the increase in the ciliogenesis of ciliated cells was influenced by estrogen in which the greatest density of ciliated cells was reached at the estrus phase (Brenner, 1969; Hagiwara et al., 1992) and deciliogenesis was induced by the progesterone (Nayak and Ellington, 1977). Additionally, ovariectomized rabbits which were treated with estrogen demonstrated the appearance of ciliogenesis on the endometrial epithelium (Tsutsumi et al., 1981). Therefore, the increase in ciliated cells on the swamp buffalo endometrial surface might be associated with the effect of estrogen as it also occurred in swamp buffalo oviducts at the follicular phase and these cells facilitate spermatozoa and oocyte transport at the time of fertilization (Tienthai et al., 2009). It is known that the microvilli in the ruminant uterine epithelium are important to the implantation process because the interdigitation of cytoplasmic projections of trophectoderm cells and uterine epithelial microvilli has occurred during apposition (Bazer et al., 2012). In addition, it is possible that the microvilli might be associated with absorption and electrolyte transport during uterine metabolism (Anderson et al., 1975). In comparison to cilia, as many investigators suggest, the ciliated cells are only normal cellular components of the uterine surface epithelium and there is no clear evidence of their function in endometrium (Masterson et al., 1975; Almeida et al., 1986; Van Cruchten et al., 2002; 2003). To our knowledge, the uterine horns play a major role in sperm transport after insemination, sperm maturation and perhaps sperm capacitation before they travel toward the utero-tubal junction (UTJ) to be stored and await fertilization (Hawk, 1983; Suarez, 2007). After natural or artificial insemination, sperm motility is not important for the rapid transport of sperms through the uterus, but actually the myometrial contractions convey sperms to the UTJ and oviduct (Katila, 2001). Seemingly, the direction of ciliary movement could function to support a subpopulation of spermatozoa during uterine contraction because the optimal amount of spermatozoa was observed in UTJ or the caudal part of the oviduct within 30 min after breeding (Rodriguez-Martinez et al., 2005). However, the precise function of ciliated cells observed in the uterine epithelium of swamp buffalo is controversial and requires further study for clarification.


Considering the activity of secretory epithelial cells and of endometrial glands, in the present study, the secretory cells and the endometrial gland had different morphological changes between the follicular and the mid-luteal phases. The cytoplasmic protrusions of secretory cells in varying degrees were noticed at the follicular phase (Figs. 4c-d) but the secretory vesicles from the endometrial glands were clearly observed on the glandular openings during the mid-luteal phase (Fig 5a). The cytoplasmic blebs in the rabbit endometrial epithelium detached from secretory cells at secretory phase occurred by the apocrine mechanism (Motta and Andrews, 1976) whereas Guillomot et al. (1986) suggested that these protrusions in cow uterine epithelium were principally associated with secretory activity and involved in merocrine. For further study in mare endometrium at estrus, Tunon et al. (1995) reported the appearance of concentrate cellular vacuolation which indicates the high synthesis of proteins in secretory cells and by transmission electron microscopy (TEM) the abundance of Golgi complex, mitochondria and rough endoplasmic reticulum is revealed in these secretory cells verifying the merocrine mode. Nevertheless, the understanding of swamp buffalo uterine epithelium, especially the mechanisms of secretory cells, requires to be resolved in future studies by TEM. On the other hand, the secretory vesicles from endometrial glands were observed during the mid-luteal phase. In general, the uterine secretory fluid in domestic animals mainly originates from blood serum, endometrial gland secretions and in part by the synthesizing of droplets of uterine epithelial cells (McRae, 1988). The uterine fluid is composed of enzymes, energy substrates, hormones, amino acids, peptides, serum proteins and uterine proteins which are essential for sperm capacitation and early embryonic development (Ing et al., 1989; Barnes, 2000). Particularly, many studies have suggested that there are different molecular weights of proteins and macromolecules in the uterine fluid which varies during the estrous cycle in bovines (McRae, 1988; Alavi-Shoushtari et al., 2008). These observations may imply that the components in the secretory droplets produced by uterine epithelial cells are essential for the appropriate events related to sperm activity, e.g. sperm transport, maturation or perhaps capacitation, at the follicular phase. Meanwhile, the appearance of uterine vesicles close to the endometrial glandular orifices in this study was also reported in cows at days 6 to 7 after estrus (Almeida et al., 1986) that implied to the mid-luteal phase and confirmed the impact of progesterone on the uterine milk protein synthesized from endometrial glands (Ing et al., 1989). Additionally, the occurrence of these vesicles might involve in the accumulation of uterine fluid within the superficial endometrial glandular lumen during mid-luteal phase (Fig. 2d). All information above absolutely signifies the uterine micro-environment fluid forming for the subsequent situations that consist of early embryonic growth and implantation during the mid-luteal phase.

In conclusion, the swamp buffalo endometrium is also like the other vital female tubular reproductive organ, e.g. the oviduct, which

showed the modification of cellular and structural compartments depending on the phases of the estrous cycle to adjust appropriate conditions for diverse physiologic events associated with the appearance of spermatozoa or initial embryos.

Acknowledgements

The authors are grateful to Mr. Silpchai

Pienchop and Mr. Witoon Mabutr, Department of Veterinary Anatomy, Faculty of Veterinary Science, Chulalongkorn University, for their excellent technical assistance. The SEM technical expertise of Miss Bung-orn Wattana-umpai, Scientific and Technology Research Equipment Centre (STREC), Chulalongkorn University, is also gratefully acknowledged. The present research was financially supported by Faculty of Veterinary Science Research Fund, Chulalongkorn University.

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Morphological Aspects by Light and Scanning Electron ...€¦ · estrous cycle. Materials and...

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