ultrastructural characterization of the pars distalis …787 int. j. morphol., 28(3):787-801, 2010....

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Int. J. Morphol.,28(3):787-801, 2010.

Ultrastructural Characterization of the Pars Distalis of the IndianFemale Sheath-Tailed Bat, Taphozous longimanus (Hardwicke)

Caracterización Ultraestructural de la Pars Distalis del MurciélagoCola de Vaina Hembra Indio, Taphozous longimanus (Hardwicke)

Archana A. Nerkar & Mohan M. Gadegone

NERKAR, A. A. & GADEGONE, M. M. Ultrastructural characterization of the pars distalis of the Indian female sheath-tailed bat,Taphozous longimanus (Hardwicke). Int. J. Morphol., 28(3):787-801, 2010.

SUMMARY: The present ultrastructural observations demonstrate the presence of six cell types in the pars distalis of non-pregnant and pregnant bats of Taphozous longimanus. In the pars distalis of T. longimanus, STH cells are round to oval with eccentricallyplaced nucleus, numerous secretory granules and well developed Golgi indicate a cell under vigorous synthetic activity while those filledwith secretory granules with reduced Golgi complex suggest reserve or storage state of cells. LTH cell is characterized by the largesecretory granules, dilated endoplasmic reticulum and numerous mitochondria in the cytoplasm which indicate that these cells arehypertrophied and synthetically very active during pregnancy. ACTH cells are found either singly or in groups and are elongated orangular with long cytoplasmic processes. The size and peripheral arrangement of secretory granules are characteristic of ACTH cell.TSH cells are distributed mostly towards the periphery of the pars distalis of T. longimanus. They are elongated, polygonal or triangularin shape. The secretory granules are small, electron dense, 150-200 nm in diameter. The rough endoplasmic reticulum is very welldeveloped. In FSH, the secretory granules are small (200 to 400 nm) and less in number and are distributed towards the periphery of thecell. FSH cells show well developed mitochondria, Golgi and rough endoplasmic reticulum indicating active state of FSH during estrusand pregnancy. The hypertrophy of FSH and LH cells during pregnancy is associated with filigreed cytoplasmic pattern giving a bizarreappearance. At late pregnancy, FSH and LH cells are highly active and synthesize large quantities of hormone as indicated by thedevelopment of cell organelles.

KEY WORDS: Bat; Pars distalis; Ultrastructure; Pregnancy.

INTRODUCTION

One of the most compelling questions addressed bybat Physiologist today is how neuroendocrine mechanismsupports the diverse array of reproductive patterns that haveevolved within the Chiropteran order. The ultrastructuralstudy of the pars distalis has been mainly investigated inlaboratory mammals, with the goal of defining not onlymicroscopic characteristics but also the physiologicalsignificance of different cell categories with respect toreproduction. The influence of pituitary hormones, such as,LH, FSH and prolactin are fundamental to reproductivephysiology. These cells of pars distalis not only play a pivotalrole in reproductive processes of mammals but also showchanging morphology during different phases ofreproductive cycle of bats (Herlant, 1962; Peyre & Herlant,1963; Badwaik, 1988, 1991; Richardson, 1979; O’Brien et.al., 1996; O’Brien, 2003).

Cell Biology Section, Department of Zoology, Institute of Science, India.

Morphological studies at electron-microscopic levelcontinue to extend our knowledge of the pituitary’s functionalorganization. Azzali (1971) studied the cytology andphysiology of the adenohypophysis of Vesperugo savi andVesperugo piccolo and identified six types of secretory cellsand a seventh type without secretory granules. Bhiwgade,et al. (1989) studied the ultrastructural and functionalcharacteristics of the anterior pituitary cells in the Indianfruit bat, Rousettus leschenaulti. Ultrastructural changes ingonadotropic and prolactin cells of Myotis myotis under ex-perimental conditions have been studied by Muniz et al.(1991). In Vespertilionid bat, Scotophilus heathi electronmicroscopic studies revealed six distinct cell types in thepars distalis on basis of specific morphological characters,staining reactions and immunocharacteristics (Singh &Krishna, 1994; 1997).

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Although Chiropterans are the second largest orderof mammals, detailed reproductive patterns and theirassociated endocrine characteristic have been documentedin only a few species. The detailed reproductive patternshave been described in the emballonurid bat, T. longimanusfrom India (Gopalakrishna, 1955, Krishna & Dominic,1982). T. longimanus shows geographic variation in thereproductive patterns. In Varanasi, India, T. longimanusshows a seasonally dioestrus pattern (Krishna & Dominic)while in Nagpur, central India exhibits an aseasonalpolyestrus (continuous) breeding pattern (Gopalakrishna).

There is an alternation of the two sides of genitalia,when one ovary shows corpus luteum during pregnancy,the contralateral ovary is not quiescent but showsfolliculogenesis and at late pregnancy ovary shows fullyformed Graafian follicle. The endocrine characteristic ofthe anomalous reproductive pattern in T. longimanus havenot yet been studied. The present electron microscopicstudy was designed to elucidate the cytology of the parsdistalis of female bat, T. longimanus during the physiologyof reproduction.

MATERIAL AND METHOD

The emballonurid bat, T. longimanus (Hardwicke)was selected for the present study because of its uniquereproductive habits. The specimens were collected fromNagpur, throughout the year representing differentreproductive states. The specimens were brought to thelaboratory alive. Mature females were separated fromimmature females after observing mammary glands andpelvic dugs.

For the electron-microscopic study, the intermediateand posterior lobes were separated from the pituitary glandand the isolated anterior pituitary was fixed in fresh ice-cold 3% glutaraldehyde for three hours and then four hoursin 0.1 M cacodylate buffer. The tissues were washed inbuffer and then post fixed for one to two hours in 1% 0.067M cacodylate-buffered osmium tetroxide. Afterdehydration with graded series of alcohol, the tissues werecleared in propylene oxide solution and embedded inAraldite resin which would be polymerized at 60ºC. Then,ultrathin sections from selected blocks were cut with glassknife and picked up on 400-mesh copper grids. Sectionswere double stained with 10% alcoholic uranyl acetate for20 min and for 10 min in Reynold’s lead citrate. Thesections were examined under a JEM Jeol-100s electronmicroscope (Japan) at 80KU accelerating voltage andphotographed.

RESULTS

The current concept of cell identification anddifferentiation in the mammalian pituitary gland is based onthe activity of the Golgi-apparatus, the elaboration ofsecretory granules, liberation of the granules from the cellmembrane, development of the ergastoplasm andmorphology of the mitochondria. Beside this, the shape andthe size of the cell, nucleus and the distribution andmorphological features of the secretory granules are alsotaken into consideration. All these constitute visible changeswhich allow us to determine the state of the secretory activityof each of the adenohypophysial cell. The fine structure ofthe pars distalis of T. longimanus is studied during thedifferent phases of the reproductive cycle. The presentultrastructural observations demonstrate the presence of sixcell types in the pars distalis of non- pregnant and pregnantbats of T. longimanus.

Somatotroph (STH-cells)

These are the most prevalent cell type in the parsdistalis of bat, T. longimanus. These are situated generallyfar from the capillaries. The pars distalis of estrus femaleshows that these cells are ovoid to polyhedral in shape andare found distributed throughout the pars distalis. The nucleusis irregular in shape with a little indentation and eccentricallyplaced, mostly euchromatic with varying amount ofheterochromatin. A thin rim of heterochromatin is seen atthe periphery of nucleus. Nucleolus is not visible.Mitochondria are spherical with lamellar cristae, distributedthroughout the cytoplasm. Rough endoplasmic reticulum isnot well developed and it is in the form of short tubularcisternae. Free ribosomes are seen scattered throughout thecytoplasm. Golgi complex is indistinct. The electron densecytoplasm of these cells is filled with varying number oflarge, ovoid or markedly pleomorphic electron densesecretory granules. The size of granules ranges from 250-400 nm in diameter (Fig.1).

During pregnancy the fine structure of STH cellschanges significantly. Mitochondria are relatively fewduring pregnancy and mitochondrial cristae are collapsed.In contrast to the inconspicuous Golgi complex in estrus,the Golgi zone of STH cell during pregnancy is welldeveloped. Rough endoplasmic reticulum is not abundantin STH cells of pregnant bat. Secretory granules are ovoidto pleomorphic or irregular in shape with high electrondensity. They are large and small mostly seen towards oneof the poles of cell. They are seen below the plasmamembrane and near the Golgi complexes. Heterophagosomesare observed in the cytoplasm (Fig. 2).

NERKAR, A. A. & GADEGONE, M. M. Ultrastructural characterization of the pars distalis of the Indian female sheath-tailed bat, Taphozous longimanus (Hardwicke).Int. J. Morphol., 28(3):787-801, 2010.

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In pars distalis of bat, T.longimanus two types of STH cells areobserved. One cell type corresponds toa medium sized cell with a large Golgiarea and few granules while the othercell type observed corresponds to a largecell, the cytoplasm of which is filled withlarge secretory granules and contains areduced Golgi area. The first cell typerepresents an active cell engaged inhormone secretion while the second celltype is in a reserve state. This aspectcorresponds to so-called functionalalternation phenomenon.

Lactotroph (PRL-cells)

LTH cells are easilyrecognizable with electron microscopyin this bat. The number of these cellsvaries in female during its reproductiveactivity such as pregnant and non-pregnant stage. During estrus, LTH cellsare large, irregular in outline. Nucleusis eccentrical and indented.Heterochromatin flakes are distributedthroughout cytoplasm and a rim ofheterochromatin is seen attached toinner surface of nuclear membrane.Nuclear pores are visible. Mitochondriaare spherical to rod shaped with lamellarcristae and are distributed throughoutthe cytoplasm. Some of themitochondria are hypertrophied andcristae are collapsed. The roughendoplasmic reticulum is in the form ofshort and long tubular profiles dottedwith ribosomes are seen scattered in thecytoplasm. Free ribosomes aredistributed throughout the cytoplasm.Golgi zone is well developed and presentnear the nucleus. This zone is made upof number of Golgi complexes arrangedin circle. Large number of electron den-se secretory granules is present in closeproximity to Golgi saccules. They aremarkedly pleomorphic or irregular inshape and measure 300-500 nm indiameter. These electron dense granulesare seen mostly towards the apicalcytoplasm, near or attached to the plas-ma membrane. No exocytosis isobserved in the cell (Fig. 3).

Fig. 1. Electron micrograph of STH cells in the pars distalis of female bat during estrus.Note irregular nucleus (N), large number of electron dense round to ovoid secretorygranules (Sg) and globular mitochondria (M) with lamellar cristae. X 10 K.Fig. 2. Electron micrograph of the STH cell during early pregnancy showing lobed nucleus(N), spherical mitochondria (M) with lamellar cristae, well developed Golgi apparatus(G) with dilated cisternae and lysosome (Ly). Note large number of electron dense secretorygranules (Sg) mostly seen at periphery of the cells. X 12000.


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LTH cells in the pars distalis during early andmid- pregnancy are hypertrophied and granulated.Mitochondria are numerous and more or less sameas in estrus bat. A well developed Golgi zone issituated towards one pole of cytoplasm as in estrusbat. Lysosomes and multivesicular bodies are alsopresent in the cytoplasm. The electron dense granulesare pleomorphic. Large number of secretory granulesis seen scattered in the cytoplasm. The structure ofcellular organelles indicates that the LTH cell showshigh secretory activity during early phases ofpregnancy (Figs. 4, 5). The most conspicuousultrastructural features of LTH cells during latepregnancy is a well developed rough endoplasmicreticulum. It is mostly concentrated in the apicalcytoplasm. Mitochondria are round to rod shapedwith lamellar cristae are distributed in the cytoplasm.The electron dense granules are pleomorphic. Thesesecretory granules are large in number and aredistributed throughout the cytoplasm (Fig. 6).

Corticotroph (ACTH-cells)

These cells are found either singly or ingroups. These are not very abundant and correspondto a small number of adenohypophysial cellpopulations. These cells are elongated or angular withlong cytoplasmic processes with eccentric nucleus.Nucleus is large, irregular with marked indentationsoccupying major part of the cytoplasm.Heterochromatin is in the form of granules distributedthroughout the nucleoplasm. Mitochondria are roundwith lamellar cristae, distributed throughout thecytoplasm. Some mitochondria are hypertrophiedwith collapsed cristae. Golgi apparatus is not welldeveloped. Rough endoplasmic reticulum is in theform of elongated tubular profiles arranged parallelto one another and is distributed throughout thecytoplasm. Ribosomes are more and lying free inthe cytoplasm. Secretory granules are spherical orovoid shaped and exhibit variation in electron density.The diameter of granule is 200-250 nm (Fig. 7).

Thyrotroph (TSH cells)

TSH cells in estrus bat are elongated,polygonal or triangular in shape with largecytoplasmic processes. Nucleus is irregular in outlineand shows indentations. It is placed eccentrically,clumps of heterochromatin are seen distributed innucleoplasm. A rim of heterochromatin is adherentto the inner surface of nuclear membrane.Mitochondria are distributed throughout the

Fig. 3. Electron micrograph of the LTH cell during estrus. Note indentednucleus (N), spherical to oval mitochondria (M) with lamellar or collapsedcristae and well developed Golgi apparatus (G) with stacks of dilated Golgisaccules arranged in semicircle contains many immature secretory granules.Cisternae of rough endoplasmic reticulum (RER) are tubular and electrondense secretory granules (Sg) are pleomorphic and are seen below the plas-ma membrane. X 10000.Fig. 4. Electron micrograph of the LTH cell during early pregnancy. Notewell developed Golgi apparatus (G), hypertrophied mitochondria (M) andparallely arranged cisternae of rough endoplasmic reticulum (RER). Notelysosome (Ly) and secretory granules (Sg) are pleomorphic. X 12000.


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cytoplasm. They are hypertrophied with collapsedcristae. In contrast to TSH cells of estrus bat, theTSH cells of pregnant bats contain a well developedendoplasmic reticulum. Golgi apparatus is not welldeveloped in TSH cells of estrus and pregnant bats.Secretory granules are small, somewhat sphericaland vary in electron density. It measures 150-200nm in diameter. In sparsely granulated cells, theyshow a peripheral distribution. In more granulatedcells, they are scattered throughout the cytoplasm(Figs 8, 9).

Gonadotrophs (FSH and LH cells)

In the present study, the identification andcharacterization of the FSH cell is based onmorphological features of the cellular constituentssuch as secretory granules, ergastoplasm, Golgiapparatus and mitochondria. The characteristicchanges occurring in these cells during the variousphysiological states (estrus and different stages ofgestation) also confirm characterization of thesecells. FSH cells in the pars distalis of estrus batsare large ovoid to polyhedral. Euchromatic nucleusis irregular in outline and shows indentations.Heterochromatin in the form of chromatin granulesare seen scattered throughout the nucleoplasm. Athin rim of heterochromatin is seen in the peripheryof nucleus. Mitochondria are hypertrophied and arepresent in the Golgi zone. Elements of Golgicomplex outline a cytoplasmic area nearly as largeas the nucleus. Associated with dilated saccules,large number of small vesicles is present. Secretorygranules are spherical, variable size and electrondensity and are distributed throughout the cytoplasm(Fig. 10).

The fine structure of FSH cells changesduring pregnancy. Mitochondria are spherical or rodshaped with lamellar cristae. Some are withcollapsed cristae. Rough endoplasmic reticulum isin the form of lamellar or tubular cisternae studdedwith ribosome. Secretory granules are electron den-se and variable in size. They are distributed in apicalcytoplasm (Fig. 11). As pregnancy advances,cytoplasm appears vacuolated because of dilationof rough endoplasmic reticulum. Endoplasmicreticulum characteristically occurs in a vesicularform of varied sizes and irregular shapes. Thesedilated cisternae are sparsely dotted with ribosomesand are distributed throughout the cytoplasm. Thelumina of cisternae are filled with less osmiophilicmaterial. The hypertrophy of FSH cells during late

Fig. 5. Electron micrograph of the LTH cell during mid-pregnancy showingnucleus (N), hypertrophied mitochondria (M) with collapsed cristae, tubularcisternae of rough endoplasmic reticulum, lysosome (L) and less electrondense secretory granules (Sg). X 12000.Fig. 6. Electron micrograph of part of the LTH cell during late pregnancyshowing nucleus (N) with rim of chromatin material, parallely arrangedtubular cisternae of rough endoplasmic reticulum (RER), and sphericalmitochondria (M) with lamellar cristae. Note Secretory granules (Sg) arearranged just below the plasma membrane X 12000.


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pregnancy is associated with the filigreedcytoplasmic pattern giving a bizarre appearance(Fig. 12). In contrast to FSH cells of estrus bats,the FSH cells contain inconspicuous Golgicomplex in pregnant bat. Lysosomal bodies andmultivesicular bodies containing 3-4 granules arealso observed in the cytoplasm. A very fewelectron dense granules are present. They arescattered throughout the cytoplasm and moretowards the apical cytoplasm. The size ofsecretory granules ranges from 200 to 400 nm indiameter during different phases of thereproductive cycle. Thus, the FSH cell showswell developed cell organelles such asmitochondria, Golgi and rough endoplasmicreticulum during pregnancy. This indicates thatthe FSH cells are active during pregnancy andelaborate large amounts of follicle stimulatinghormone.

During early part of gestation, LHgonadotroph cells are large polygonal with largebilobed nucleus occupying maximum part of thecytoplasm. Heterochromatin flakes aredistributed throughout the nucleoplasm. A thickrim of heterochromatin and two prominentnucleoli are seen below the nuclear membrane.Structure of mitochondria is more or lessidentical during the course of pregnancy.Mitochondria are round with lamellar cristaedistributed throughout the cytoplasm. Somemitochondria are hypertrophied with loss ofcristae. The cisternae of rough endoplasmicreticulum are dilated and are distributedthroughout the cytoplasm, but more towards thenucleus. These dilated cisternae are studded withribosomes. Because of dilation of roughendoplasmic reticulum, it gives vacuolatedappearance to the cytoplasm. Golgi zone ispresent juxtanuclear in position. Golgi complexis made up of 7-8 Golgi saccules associated withsmall vesicles. In the region of Golgi, smallgranules are observed. Golgi saccules are curvedand dilated at the ends. Heterophagosomes areobserved in the cytoplasm. Large number ofsecretory granules of varying electron density isobserved distributed throughout the cytoplasm.These secretory granules measure 200-300 nmin diameter (Fig. 13).

Hypertrophied LH gonadotroph cells inthe pars distalis of pregnant bat (late pregnancy)show well developed endoplasmic reticulum.

Fig. 7. Electron micrograph of the triangular ACTH cell during early pregnancyshowing indented nucleus (N) with rim of heterochromatin below nuclearmembrane. Spherical mitochondria (M) and electron dense secretory granules(Sg) are distributed throughout the cytoplasm. Cisternae of rough endoplasmicreticulum (RER) are also observed. X 8000.Fig. 8. Electron micrograph of TSH cell during early pregnancy. Note the angu-lar shape of TSH cell with irregular, enlarged nucleus (N), round to oval shapedmitochondria (M) and stack of parallel cisternae of rough endoplasmic reticulum(RER). Cytoplasm is filled with large number of small sized secretory granules(Sg). X 8000.


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The profiles of rough endoplasmic reticulumoccupy a large part of the cytoplasm, displacingother cell constituents. The cisternae of roughendoplasmic reticulum are arranged in parallelarrays. In some cells, cytoplasm is extremelyvacuolated is due to dilation of cisternae of roughendoplasmic reticulum. The hypertrophy of LHcells is associated with the filigreed cytoplasmicpattern giving a highly bizarre appearance.Ribosomes are distributed throughout thecytoplasm. Golgi apparatus is indistinct. Thesecretory granules are less in number and aredistributed towards the periphery of the cell.During pregnancy, LH cells show gradualhypertrophy and show marked development ofmitochondria, Golgi and rough endoplasmicreticulum. Thus, these cells are very active andelaborate large amounts of luteinizing hormoneduring the pregnancy (Fig. 14).

DISCUSSION

Electron microscopic study offers theadvantage of revealing the state of activity ofthe cells and their morphological features in aprecise manner, which allows formulating thecriteria for their functional analysis. Inconjunction with our knowledge of theultrastructure of the mammalian pituitary gland(Herlant, 1964; Harris & Donovan, 1966; Nunezet al., 1980; Bhiwgade et al.; Muniz et al.; Singh& Krishna, 1994, 1997), the presentultrastructural observations demonstrate thepresence of six cell types in the pars distalis ofadult female emballonurid bat, T. longimanus.The increase in nuclear size, enlargement ofnucleoli and hypertrophy of organelles andnature of secretory granules of the cells havetaken as the indicators of stimulation andincreased synthetic activity.

Somatotroph (STH-cells)

Ultrastructural characteristics of STHcells of Indian fruit bat, R. leschenaulti have beenstudied by Bhiwgade et al. These cells are roundto oval in shape with centrally placed roundnucleus. The secretory granules are numerous,uniform, round and very dense about 350-400nm in diameter. The Golgi apparatus isinconspicuous and mitochondria are round and

Fig. 9. Electron micrograph of the angular TSH cell during mid-pregnancy. Notethe presence of indented nucleus (N) with nucleolus, hypertrophied mitochondria(M) with collapsed cristae, rough endoplasmic reticulum (RER) and electrondense secretory granules (Sg). X 8000.Fig. 10. Electron micrograph of the FSH cell during estrus. Note indented nucleus(N), hypertrophied mitochondria (M) with collapsed cristae, tubular cisternaeof rough endoplasmic reticulum (RER), secretory granules (Sg) andhypertrophied Golgi apparatus (G) consists of dilated saccules and small vesiclesforming a circlet near the nucleus. X 12 K


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scattered in the cytoplasm. In S. heathi, Singh& Krishna (1994), reported dense secretorygranules ranging from 240 to 480nm indiameter, round mitochondria and Golgiapparatus in the STH cells.

In Hipposideros lankadiva (Seraphim,2004), STH cells are round to oval witheccentrically placed nucleus. The secretorygranules are numerous, mostly round to ovalwith uniform electron density. The welldeveloped Golgi, endoplasmic reticulum andsmall amount of secretory granules indicate acell under vigorous synthetic activity whilethose filled with secretory granules withreduced Golgi complex suggest reserve orstorage state of cells, supporting the presentobservations. The oval to round STH cells in parsdistalis is reported in Miniopterus schreibersiifuliginosus (Mikami, et al., 1988). In generalsecretory granules in the STH cells of bats aredense, membrane enclosed and from 350-400 nmin diameter (Herlant, 1962, 1963; Azzali). In T.longimanus, the diameter of secretory granulesranges from 250-400 nm. The STH cells thus,identified in the pars distalis of T. longimanusare comparable with STH cells identified andreported in the other species of bats confirmingour observations (Bhiwgade et al., Singh &Krishna, 1994, Seraphim).

Lactotroph (PRL-cells)

The ultrastructure features of LTH cellsin the pars distalis of pregnant bats differ fromthat of the other cells of the estrus bat. Thesecells are hypertrophied and granulated duringpregnancy. Mitochondria are numerous, Golgiapparatus and rough endoplasmic reticulum arewell developed. The electron dense granulesare pleomorphic and are seen scattered incytoplasm. The size of granules ranges from300-500 nm diameter. The most conspicuousultrastructural feature of LTH cells during latepregnancy is well developed endoplasmicreticulum. The structure of cell organellesindicates that the LTH cells show high secretoryactivity during different phases of pregnancy.Lysosomes and multivesicular bodies whichwere not observed during early part of gestationare observed in the LTH cells during latepregnancy, thus indicating digestion of excesssecretory material.

Fig. 11. Electron micrograph of the FSH cell during mid-pregnancy showingnucleus (N), hypertrophied mitochondria (M) with lamellar or collapsed cristae,rough endoplasmic reticulum (RER) is in the form of dilated cisternae and a veryfew secretory granules. X 12000Fig.12. Electron micrograph of the FSH cell during late pregnancy. Note indentednucleus (N) with nucleolus, globular mitochondria (M) with lamellar cristae, dilatedcisternae of rough endoplasmic reticulum (RER) and electron dense secretorygranules (Sg) at the periphery of the cell. X 12000


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Bhiwgade et al. reported that the LTH cellsare characterized by the largest secretory granules,well developed endoplasmic reticulum and numerousmitochondria in the cytoplasm. They further reportedthat an accumulation of secretory material in the PRLcells associated with the retention of milk followingthe removal of litter contributes favorably foridentification of the prolactin in bat. An accumulationof secretory granules in PRL cells after 8 hrs ofnonsuckling and decrease of granules prompted by10 minutes of suckling has been confirmed.

In S. heathi, Singh & Krishna (1994) reportednumerous mitochondria, dilated endoplasmicreticulum and extensive Golgi complex, largenumber of secretory granules in the LTH cells.Ishibashi and Shiino (1989) identified the LTH cellsin the pars distalis of Pipistrellus abramus. Thesecells show morphologically active features inpregnant and lactating females and have a tendencyto contain smaller secretory granules. In H. lankadivathe LTH cells in the pars distalis show hypertrophiedmitochondria with lamellar cristae, dilated roughendoplasmic reticulum and well developed Golgiapparatus. The secretory granules are arranged alongthe plasma membrane.

Muniz et al. reported prolactin granules of va-riable shapes and electron density. The degranulationof LTH cells of pregnant female of M. myotis isobserved under experimental conditions, which displaygreatly increased development of the roughendoplasmic reticulum, Golgi apparatus, a largenumber of lysosomes. The higher concentration of theLTH prolactin cells for mammary growth and milksecretion during gestation and lactation has beenreported (Harris & Donovan, 1966; Austin & Short,1972).

The mammotrophs occupy the pars distalis ofhibernating bat, P. abramus during pregnancy andlactation suggesting the elevated prolactin contentduring this period (Ishibashi & Shiino). Theproliferation activity of mammotrophs in female batwas observed during pregnancy and lactation(Kawamoto, 2003). The morphometrical activity andPRL level were mostly low during folliculardevelopment and the early implantation and thisactivity and PRL level increases after implantation(Anthony, 2000).

The ultrastructural observations on LTH cellsshowed that size of secretory granules vary from

Fig. 13. Electron micrograph of the LH cell during mid-pregnancy showingthe indented nucleus (N), hypertrophied Golgi apparatus (G), tubular anddilated cisternae of granular endoplasmic reticulum (RER), hypertrophiedmitochondria (M) with collapsed cristae and small secretory granules (Sg)distributed towards the periphery of the cell. X 6000.Fig. 14. Electron micrograph of the LH cell during late pregnancy. Notethe irregularly shaped LH cell, with eccentrically placed nucleus (N), ex-treme dilation of endoplasmic reticulum (RER) giving bizarre appearance,hypertrophied mitochondria (M) and very few small secretory granules(Sg) scattered throughout the cell. Golgi is well developed and presentnear the nucleus. X 8000.


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species to species (Richardson). The size of secretorygranules in bat, M. myotis ranges from 170-550 nm diameter(Herlant, 1963); V. savi and V. piccolo from 700-800 nmdiameter (Azzali); R. leschenaulti from 400-600 nmdiameter; S. heathi, from 300-600 nm diameter (Singh &Krishna, 1994) and M. myotis from 180-510 nm diameter(Muniz et al. 1991). In the present study the size of secretorygranules in T. longimanus ranges from 300 to 500 nmdiameter. The LTH cells, thus identified in pars distalis ofnonpregnant and pregnant bat of T. longimanus with electronmicroscopy are comparable with LTH cells of the otherspecies of bats confirming our observations.

Apart of lactogenic function, LTH cells are alsoluteotrophic in function in bats, M. schreibersii (Peyre &Herlant, 1963; Herlant, 1968) Macrotus californicus (Bleier,1975); H. lankadiva (Seraphim) and T. melanopogon(Badwaik, 1988). Singh & Krishna (2000) observed twopeaks of progesterone concentration during the reproductivecycle coincide with two pregnancies in T. longimanus. SinceT. longimanus invariably bears only one conceptus at a time,the variation in progesterone concentration during pregnancyappear to depend on stages of embryonic development.

In the present study, LTH cells in par distalis ofpregnant bat, T. longimanus, which is a continuous breeder,are synthetically active and elaborate large quantity ofprolactin during pregnancy. This hormone may bestimulating luteal cells of corpus luteum to synthesizeprogesterone. The ultrastructural features of luteal cells inTaphozous indicate that these cells are steroidogenicallyactive during pregnancy (Nerkar & Gadegone, 2007),suggesting that the LTH cells are luteotrophic in this speciesof bat as observed in other bat species. Our findings correlatewith the findings of other workers on bats.

Corticotroph (ACTH cells)

ACTH cells are elongated or angular found eithersingly or in groups in pars distalis of T. longimanus duringpregnancy. Mitochondria are round with lamellar cristae.Rough endoplasmic reticulum is in the form of elongated andshort tubular profiles. Golgi complex is inconspicuous. Thespherical or ovoidal secretory granules (200-250 nm indiameter) of variable electron density are present just belowthe plasma membrane. The size and peripheral arrangementof secretory granules are characteristic of ACTH cell of T.longimanus.

Similar ultrastructural characteristics of ACTH cellsare reported in S. heathi (Singh & Krishna, 1994, 1997); R.leschenaulti (Bhiwgade et al.); H. lankadiva (Seraphim). TheACTH cells were hypertrophied and became largest cells in

the pars distalis of R. leschenaulti after the adrenalectomy andmetapyrone treatment. The mitochondria, rough endoplasmicreticulum and Golgi apparatus are poorly developed in nor-mal state. Secretory granules of about 100-150 nm in diameterbut become more extensive after adrenalectomy. In S. heathi,Singh & Krishna (1994, 1997), revealed the presence of smallsecretory granules of about 100-190 nm in diameter. The nucleiwere ovoid to irregular. The mitochondria with spaced cristaeare also observed. In M. schreibersii fuliginosus, the largeround or polygonal ACTH cells are reported in rostroventraland ventrolateral region of the pars distalis (Mikami et al.).Ultrastructural characteristics of the ACTH cells i.e. peripheralarrangement of small sized secretory granules along theplasmalemma correspond to those reported in rat (Nakayamaet al., 1969; Siperstein & Miller, 1970); rabbit (Cameron &Foster, 1972); cow (Mikami, 1970) and mink (Murphy &James, 1976) supporting the present observations. TheMalayan flying bat, Pteropus vampyrus, Reeder et al. (2006)reported that the bats restraint stress for 15-60 minutes resultsin a significant increase in plasma levels of ACTH andglucocorticoid. ACTH levels quickly returns to baselinefollowing restraint, whereas glucocorticoid levels remainselevated for at least 30 minutes after restraint ended. Widmaieret al. (1994) reported that the chiropteran adrenal gland ishypersensitive to ACTH or possess active intracellularsteroidogenic mechanism. They conclude that the bat possessesa stress response and the high constitutive and stress inducedlevel of glucocorticoids in the Megachiroptera are uniqueamong the mammals.

Although there are many different neuroendocrinecomponents involved in regulating reproduction and theresponse to changing environment the hypothalamic-pituitary-adrenal (HPA) axis and the hypothalamic-pituitary gonadal(HPG) axis are particularly important (Reeder & Kramer, 2005;Reeder et al.). The ACTH regulates the synthesis and releaseof glucocorticoids in the adrenal cortex. Glucocorticoidshormones (cortisol and corticosterone) are important regulatorsof energy balance and elevations in glucocorticoid hormonesare considered a hallmark of the vertebrate stress response(Reeder & Kramer, 2005). The HPA and HPG axes interactwith one another in complex ways and in part, serve to regulatebehaviour and fertility (DeVries, 2002; Viau, 2002).Ultrastructural features of ACTH cells and adrenocortical cellsof T. longimanus during pregnancy (Nerkar & Gadegone,2009) suggest that these cells are metabolically active and maybe involved in the reproductive processes and management ofstress, bat is undergoing during pregnancy.

Thyrotroph (TSH cells)

TSH cells during pregnancy show similarultrastructural features that observed in the TSH cells during


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estrus, except well developed rough surfaced endoplasmicreticulum. It is in the form of array of elongated, tubular orlamellar cisternae frequently localized at one pole orperiphery of the cell. The profiles of cisternae are parallelto one another or curved. Free ribosomes are seen scatteredin the cytoplasm. Golgi is inconspicuous. The smallsecretory granules are 150-200 nm in diameter, in varyingelectron density are scattered throughout the cytoplasm orshow peripheral distribution. Thus, ultrastructural featuresof TSH cells during estrus and pregnancy suggest that thesecells elaborate large quantity of thyrotrophic hormones.

In S. heathi, TSH cells showed the presence of afew secretory granules which ranged in diameter from 100-160 nm. The cytoplasm contained rounded to elongatedmitochondria, free ribosomes and poorly developed Golgicomplex (Singh & Krishna, 1994). In H. lankadiva,Seraphim reported angular TSH cells in the pars distalis ofestrus bat. The TSH cells show fine secretory granuleswhich are relatively uniform in size (100 to 200 nm indiameter), small Golgi arranged in circular array and rodshaped mitochondria and several stacks of rough surfacedendoplasmic reticulum. The size of secretory granules isthe characteristic of TSH cell which is of 150-200 nmdiameter is also reported in the TSH cells of T. longimanus.

Bhiwgade et al. presented most convincingevidence for the presence of TSH cells in anterior pituitaryof bat, R. leschenaulti by surgical and chemicalthyroidectomy. The experiments resulted in hypertrophiedTSH cells containing a small saccular dilations of roughendoplasmic reticulum studded with ribosome in thecytoplasm. Golgi complex and mitochondria arehypertrophied. The above findings provide direct evidencefor the existence of TSH cells in the bat pituitary. Ourobservations are supported by many research workers onbats.

In the present study, TSH cells exhibit the presenceof well developed Golgi apparatus, hypertrophiedmitochondria, extreme dilation of cistenae of rough surfacedendoplasmic reliculum and large number of secretorygranules indicates increased synthetic and metabolicactivity of the TSH cells during estrus and pregnancy. Theultrastructural features of thyroid gland of T. longimanusduring estrus and pregnancy indicate that the thyroid issynthetically very active (Nerkar & Gadegone, 2009). Thus,the active TSH cells during estrus and pregnancy may beactiving thyroid gland to produced large quantities ofthyroxines which is necessary for normal transition fromthe reproductively inactive to active condition anddevelopment and growth of embryo of this species of bat,which is a continuous breeder.

Gonadotrophs (FSH and LH cells)

Several researchers have shown by electronmicroscopy (Farquhar & Rinehart, 1954a; Purves, 1961)that morphological differences between FSH and LHproducing cells exist. Herlant (1963, 1964, 1968), Azzaliand Bhiwgade et al. studied cytological variations in

gonadotrophs in different species of bats under variousphysiological conditions. They have differentiated thegonadotrophs into two distinct entities, the FSH and LHsecreting gonadotrophs. According to Bhiwgade et al.(1989) the variations observed in the electron density ofthe secretory granules is sufficient to differentiate twotypes of gonadotrophs. These cells also differ in theircytoplasmic organelles and do not show similar changesunder altered conditions such as estrus, pregnancy andlactation.

The present ultrastructural studies on par distalisfrom non pregnant and pregnant bats, T. longimanus pointto the existence of two types of gonadotrophs in bats. TheFSH-secreting cells described in bats correspond to thepresumptive rat FSH cells of Nakane (1970), bat FSH cellsof Herlant (1964) and mink FSH cells of Murphy & James.The LH gonadotrophs in bats correspond to the LH cellsof Nakane, gonadotrophs of Rennels et al. (1971) and LHcells of Herlant (1964). Electron microscopic studies haveconfirmed the presence of two gonadotroph cell types inthe rat (Farquhar & Rinehart, 1954a, 1954b) and bat(Herlant, 1964; Bhiwgade et al.; Singh & Krishna, 1994).

FSH cells in the pars distalis of estrus female arelarge ovoid to polyhedral. Golgi apparatus ishypertrophied. Mitochondria are hypertrophied withcollapsed cristae. Rough surfaced endoplasmic reticulumis well developed. Secretory granules are spherical, 200-400 nm in diameter and show variable electron density.These ultrastructural features suggest that FSH cells aresecretory and metabolically active during estrus. Duringpregnancy, FSH cells undergo hypertrophy. Nucleus isirregular in outline. Large number of mitochondria withcollapsed cristae is present in the cytoplasm. The dilatedcisternae of rough endoplasmic reticulum are sparselydotted with ribosome and are distributed throughout thecytoplasm. Thus the cytoplasm appears vacuolated. A fewelectron dense granules are seen more towards the apicalcytoplasm. The hypertrophy of FSH cells during latepregnancy is associated with filigreed cytoplasmic patterngiving a bizarre appearance, because of further dilationof cisternae of endoplasmic reticulum. Thus, ultrastructuralfeatures suggest that though the FSH cells appeardegranulated during pregnancy but they are syntheticallyactive and elaborate large amount of hormone.


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Ultrastructural characteristics of FSH cells of T.longimanus are similar to that reported in other bat species(Herlant, 1964; Azzali; Singh & Krishna, 1994; Bhiwgadeet al.; Seraphim). Muniz et al. studied ultrastructuralcharacteristics of gonadotrophs of pregnant bat, M. myotis.The granules of the gonadotrophic cells are columnar orcuboidal and are medium electron density. Thegonadotrophs of pregnant bat under experimentalconditions show degranulation which display greatlyincreased development of the rough endoplasmic reticulumand Golgi apparatus, a large number of lysosomes and alarge amount of degraded material. Azzali observedcytological changes in the LH cells of V. savi and V. piccolowith electron microscopy and suggest that delayedovulation may be the result from insufficient quantities ofLH and that the Graafian follicle is maintained by tonicsecretion of FSH.

T. longimanus at Nagpur is a continuous breeder andpregnancies follow in quick succession. There is analternation of two sides of genitalia. When one ovary showscorpus luteum and development of embryo in uterine hornduring pregnancy, the contralateral ovary is not quiescentbut shows folliculogensis, and at late pregnancy, ovaryshows fully formed Graafian follicle. In the present study,ultrastructural features of FSH cells indicate that they aresynthetically very active during estrus and pregnancy whenovary shows folliculogenesis and a Graafian follicle. Ourobservations suggest that the hypertrophied FSH cells atlate pregnancy are necessary for maintaining Graafianfollicle in the contralateral ovary, which is going to ovulate.The present observations are supported by the hormonalstudies carried out by Singh & Krishna (2000) on T.longimanus at Varanasi, where it exhibits diatocousbreeding habits.

LH cells in the pars distalis of bat during earlypregnancy show inconspicuous Golgi apparatus.Mitochondria are round with lamellar cristae. The cisternaeof rough surfaced endoplasmic reticulum are dilated andare distributed throughout the cytoplasm. Large numbersof secretory granules (250-350 nm) of varying electrondensity are observed throughout the cytoplasm. However,during mid-pregnancy, Golgi zone is well developed. Thecytoplasm is vacuolated because of dilation of cisternae ofthe rough endoplasmic reticulum. Secretory granules arevery few and are distributed throughout the cytoplasm.

During late pregnancy, hypertrophied LH cells showthe profiles of rough surfaced endoplasmic reticulumoccupying a large part of the cytoplasm, displacing othercell constituents. Cytoplasm is extremely vacuolated dueto dilation of cisternae of rough endoplasmic reticulum.

The hypertrophy of LH cells is associated with the filigreedcytoplasmic pattern giving a highly bizarre appearance tothe cell as seen in the FSH cells during late pregnancy. Thesecretory granules are small and less in number and aredistributed towards the periphery of the cell. Theseultrastructural features indicate that though LH cellsappeared degranulated during pregnancy but they are acti-ve and elaborate large quantities of hormone.

In R. leschenaulti, (Bhiwgade et al.) LH cells areangular in shape and have secretory granules 100-150 nmdiameter. These secretory granules are of equal density andare irregularly distributed throughout the cytoplasm.Mitochondria are elongated and have light matrix. Golgiapparatus is prominent. In S. heathi, (Singh & Krishna,1994) LH cells have secretory granules 175-350 nmdiameter in the pars distalis. These granules are irregularlydistributed throughout the cytoplasm. The mitochondria areelongated or round shaped and Golgi complex isconspicuous. H. lankadiva (Seraphim) exhibits thephenomenon of embryonic diapause during pregnancy. TheLH cells during embryonic diapause show well developedGolgi complex and rough surfaced endoplasmic reticulum.Mitochondria are numerous and have lamellar cristae andare present near the rough endoplasmic reticulum and Golgicomplex. The secretory granules are few in number. TheLH cells during embryonic diapause are not inactive butsynthetically active, although activity is slowed down. TheLH gonadotrophin volume in M. lucifugus lucifugusdecreases after ovulation and remain low during pregnancyand lactation (Anthony & Gustafson, 1984) and M.californicus (Richardson).

The ultrastructural characteristics exhibited by LHcells of different species of bats are similar to theultrastructural features exhibited by LH cells of T.longimanus, supporting the present observations.Hypertrophy and hyperplasia of LH cells during latepregnancy and lactation is reported in a number of bats, T.melanopogon (Badwaik, 1988); M. lucifugus lucifugus(Anthony & Gustafson); H. lankadiva (Seraphim) and H.speoris (Patil, 1974). The ultrastructural features of LH cellsindicate that these cells are synthetically very active duringpregnancy and there is a hypertrophy of cells at latepregnancy, supporting observations on LH cells of differentspecies of bats during pregnancy.

Luteotrophic role of LH cells is reported in M. myotis(Herlant, 1956); M. schreibersii (Peyre & Herlant, 1963a,b); T. melanopogon (Badwaik, 1988) and H. lankadiva(Seraphim). In the present study, ultrastructural features ofLH cells indicate that these cells are synthetically very ac-tive during pregnancy and are hypertrophied at the end of


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pregnancy. Similarly the luteal cells of T. longimanus aresteroidogenically very active (Nerkar & Gadegone). Thus,LH cells may be stimulating luteal cells during pregnancyto secrete progesterone which is responsible formaintenance of pregnancy as reported in T. longimanus(Singh & Krishna, 2002).

In the present study, the cell types, FSH, LH andLTH in the pars distalis of T. longimanus during pregnancyare active, elaborating hormones which are needed for thedevelopment of embryo in one uterine horn and

folliculogenesis in contralateral horn as this bat iscontinuous breeder and pregnancies follow in quicksuccession.

ACKNOWLEDGEMENTS

The electron microscopy facilities provided by Dr.Arun Chitale, Department of Histopathology, Jaslok Hos-pital and Research Centre, Mumbai, are gratefullyacknowledged. Our thanks are due to Mr. Dilip Kanaskarand Shivaji Bhosale for their excellent technical assistance.

NERKAR, A. A. & GADEGONE, M. M. Caracterización ultraestructural de la pars distalis del murciélag cola de vaina hembra Indio,Taphozous longimanus (Hardwicke). Int. J. Morphol., 28(3):787-801, 2010.

RESUMEN: Las observaciones ultraestructurales actuales demuestran la presencia de seis tipos de células en la pars distalis demurciélagos Taphozous longimanus preñadas y no preñadas. En la pars distalis del T. longimanus, las células STH son redondas uovaladas con un núcleo excéntrico, numerosos gránulos de secreción y un Golgi bien desarrollado que indican una célula en actividad desíntesis vigorosa, mientras que las llenas de gránulos de secreción con un complejo de Golgi reducido sugieren un estado celular dereserva o almacenamiento. Las células LTH se caracterizan por grandes gránulos de secreción, el retículo endoplásmico dilatado ynumerosas mitocondrias en el citoplasma, indicando que estas células están hipertrofiadas y con una actividad sintética muy activadurante el embarazo. Células de ACTH se encuentran de forma individual o en grupos, son alargadas o angulares, con largos procesoscitoplásmicos. El tamaño y la disposición periférica de los gránulos de secreción de ACTH son característicos de la célula. Células deTSH se distribuyen principalmente hacia la periferia de la pars distalis del T. longimanus. Ellos son alargadas, poligonales o de formatriangular. Los gránulos de secreción son pequeños, electrodensos, de 150-200 nm de diámetro. El retículo endoplasmático rugoso estámuy bien desarrollado. En células FSH, los gránulos de secreción son pequeños (200 a 400 nm), menores en número y se distribuyenhacia la periferia de la célula. Células FSH muestran mitocondrias bien desarrolladas, Golgi y retículo endoplasmático rugoso que indicael estado activo de la FSH durante el estro y la preñez. La hipertrofia de las células de FSH y LH durante la preñez se asocia con un patróncitoplasmático filigrana dando una extraña apariencia. Al final de la preñez, las células de FSH y LH son muy activas y sintetizan grandescantidades de hormonas, como producto del desarrollo de las organelos celulares.

PALABRAS CLAVE: Murciélago; Pars distalis; Ultrastructura; Preñez.

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Correspondence to:Archana A. NerkarCell Biology SectionDepartment of ZoologyInstitute of ScienceR. T. Road, Civil LinesNagpur 440001INDIA E mail: [email protected]

Received: 21-07-2009Accepted: 24-03-2010


ultrastructural characterization of the pars distalis …787 int. j. morphol., 28(3):787-801, 2010....

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