Original Research Article PRENATAL DEVELOPMENT OF … · PRENATAL DEVELOPMENT OF EXOCRINE PANCREAS...

Int J Anat Res 2016, 4(2):2365-71. ISSN 2321-4287 2365

Original Research Article

PRENATAL DEVELOPMENT OF EXOCRINE PANCREAS INCROSSBRED GOATS: A HISTOLOGICAL STUDY

ABSTRACT

Address for Correspondence: Sreeranjini. A.R., Assistant Professor, Department of VeterinaryAnatomy, College of Veterinary and Animal Sciences, Mannuthy, Thrissur, Kerala -680 651, India,Tel: +91 9447820178, E-Mail: [email protected]

Back ground: Studies about the normal development of pancreas are beneficial in understanding its normalmode of functioning and thereby help to trace the causes of its dysfunction as in diabetes, pancreatitis or thehighly fatal pancreatic cancer. The mechanisms involved in normal prenatal development of exocrine pancreasin goats are poorly understood.Purpose: The present study was conducted with an aim of contributing some useful information in the field ofdevelopmental and pancreatic biology.Methods and Results: For the present study, the goat embryos from second to fifth month of prenatal life,collected from clinics and farms of Kerala Veterinary and Animal Sciences University and those available in themuseum of Department of Veterinary Anatomy, College of Veterinary and Animal Sciences, Mannuthy, Kerala,India were utilized. Samples were fixed in 10% neutral buffered formalin and Bouin’s solution and were processedfor light microscopic histological studies. Sections of 5-7µ thickness were cut on a rotary microtome, stainedusing various routine and special techniques and were examined under Leica DM 2000 LED microscope. Duringsecond month of prenatal development, the exocrine pancreas comprised ductules of varying size dispersedwithin the mesenchyme of both pancreatic primordia. As age advanced and the pancreatic primordia fused, thenumber and size of ductules increased and developed acini at their end. Later the pancreatic parenchyma wasdivided into lobules containing groups of acini, blood vessels and intralobular ducts. The present study revealedthat the duct system of exocrine pancreas developed before the formation of acini and the number of ductsincreased mainly by branching. Acini developed by the third month of development while distinct lobulationcould be noticed in the fourth month of development. The arrangement of lobules, duct system and acini in thefifth month of foetal life indicated the development of a well-organized exocrine pancreas in goats in the prenatallife itself.Potential implications: The results can also be compared to human data for furthering the knowledge of humanbiology and medicine.KEY WORDS: Exocrine pancreas, Goats, Histology, Prenatal development.

INTRODUCTION

International Journal of Anatomy and Research,Int J Anat Res 2016, Vol 4(2):2365-71. ISSN 2321-4287

DOI: http://dx.doi.org/10.16965/ijar.2016.221

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Received: 26 Apr 2016 Accepted: 16 May 2016Peer Review: 27 Apr 2016 Published (O): 31 May 2016Revised: None Published (P): 31 May 2016

International Journal of Anatomy and ResearchISSN 2321-4287

www.ijmhr.org/ijar.htm

DOI: 10.16965/ijar.2016.221

to the presence of both exocrine and endocrinecomponents. The exocrine pancreas constitutesThe pancreas of mammals is a unique gland due

A.R. Sreeranjini *1, N. Ashok 2.*1 Assistant Professor, Department of Veterinary Anatomy, College of Veterinary and Animal Sciences,Mannuthy, Thrissur, Kerala, India.2 Professor & University Head, Department of Veterinary Anatomy, College of Veterinary and AnimalSciences, Mannuthy, Thrissur, Kerala, India.

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http://dx.doi.org/10.16965/ijar.2016.221

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A.R. Sreeranjini, N. Ashok. PRENATAL DEVELOPMENT OF EXOCRINE PANCREAS IN CROSSBRED GOATS: A HISTOLOGICAL STUDY.

the larger part of the pancreatic parenchyma andfunctions as one among the major digestiveglands in the body. It is composed oftubuloacinar glands that secrete various diges-tive enzymes such as amylase, lipase and pro-tease [1]. The secretory units or acini are ar-ranged as groups of sac like structures at theend of ducts. These ducts of varying diameters,originating as the intercalated duct carry thesecretory products towards duodenum and draininto it through major or minor duodenal papilladepending upon the species. The lining cells ofthese ducts are also secretory in nature and pro-duce mucus and bicarbonate rich fluid that helpsto neutralize the acidic chyme entering theduodenum [2]. Pancreas develops from dorsaland ventral pancreatic primordia that areoutgrowths of endodermal lining at the caudalpart of foregut [3]. Cells of the pancreaticprimordia multiply and differentiate into ductsand secretory acini. Some cells get detachedfrom the duct system and develop into islets ofLangerhans.Studies about the normal development ofpancreas are beneficial in understanding itsnormal mode of functioning and thereby help totrace the causes of its dysfunction as indiabetes, pancreatitis or the highly fatalpancreatic cancer. It has been reported that anydefect in the endocrine part of pancreas as indiabetes adversely affects the function ofexocrine part and pathological conditions affect-ing the exocrine component of pancreas suchas chronic pancreatitis, results in impairment inthe function of endocrine part [4]. In thepharmaceutical industry, for commercialproduction of insulin and several digestiveenzymes, the pancreas of domestic animals isused. The mechanisms involved in normalprenatal development of exocrine pancreas ingoats are poorly understood. Knowledge aboutthese events in goats will add on information tothe existing scientific data and therebycontribute to advances in developmental andpancreatic biology and digestive physiology. Theresults can also be compared to human data forfurthering the knowledge of human biology andmedicine.

clinics and farms of Kerala Veterinary and Ani-mal Sciences University and those available inthe museum of Department of VeterinaryAnatomy, College of Veterinary and AnimalSciences, Mannuthy, Kerala, India were utilized.Immediately after collection, the embryos weregrossly examined and only those without anygross abnormalities were selected. Body weightand crown rump length (CRL) of the embryoswere recorded and their age was calculated [5].The present study was conducted on four groupsof embryos/foetuses from second month (31-60days) to fifth month (121-150 days) of prenatallife. The embryos up to 59 days of age were fixedin toto and were serially sectioned to locate theprimordia and for studying the early stages ofdevelopment of the pancreas. From the foetusesof later age groups, the pancreas was harvestedby careful dissection.After recording the morphological parameters,tissue samples were fixed in various fixativessuch as 10% neutral buffered formalin andBouin’ s solution. Fixed tissues were processedfor histological studies using paraffin embed-ding method. Sections of 5-7µ thickness werecut on a rotary microtome and were stained us-ing Ehrlich’s Haematoxylin and Eosin (H&E)staining technique [6] and special staining tech-niques for light microscopical histological stud-ies such as Lison’s alcian blue method [7].Stained sections were examined under Leica DM2000 LED microscope and digital images werecaptured and recorded.

MATERIALS AND METHODS

RESULTSIn embryos during the second month (31 to 60days) of development, the parenchyma of bothpancreatic primordia contained ductules ofvarying size dispersed within the looselyarranged mesenchymal connective tissue(Fig. 1). A few ductules showed branching andwere lined by cuboidal cells containing darkstained nucleus with high nuclear to cytoplas-mic ratio. Ductular lumen was clear and did notshow any secretory material. Projecting from theperiphery of some ductules, masses of darkstained cells enclosed in a space, indicating thedevelopment of endocrine cells were also seen.The loosely arranged mesenchyme containedfine connective tissue fibres and scatteredmesenchymal cells.For the present study, the embryos collected from



Fig. 1: Pancreatic primordia containing several ductulesin 58 days-old goat embryo. Haematoxylin and Eosinstaining. Magnification: 40 x: 1- dorsal pancreaticprimordium, 2- ventral pancreatic primordium,3- developing stomach, 4- developing intestine.

By third month (61 to 90 days), the pancreaticprimordia fused to form a single mass which wasenveloped by a distinct connective tissuecapsule. The mesenchyme was composed ofloosely arranged connective tissue fibres;numerous stellate mesenchymal cells and largenumber of ductules (Fig. 2). Small ductules weremore in number compared to larger ones andwere lined by simple cuboidal epithelial cellswith well developed dark stained nucleus andhigh nuclear to cytoplasmic ratio. Theseductules showed extensive branching. Adjacentto ductules, solid clusters of cells prior to thedevelopment of tubular lumen were also noticed.These cells were pyramidal in shape and werearranged in circular fashion with the apex of cellscoming close together at the centre. Commence-ment of lumen formation occurred at the centreof cell clumps to form new ductules. Largeductules showed branching to form new ones

Fig. 2: Section of fused pancreas in 65 days-old goatembryo. Haematoxylin and Eosin staining.Magnification: 40 x: 1- capsule, 2- mesenchyme,3- ductules.

and budding to form new islets (Fig. 3). Liningcells of these larger ductules were lightly stainedand taller with clear intercellular borders,whereas the cell buds contained darkly stainedcells with large vesicular nucleus.

Fig. 3: Section of pancreas showing branching of largeductules in 65 days-old goat embryo. Lison’s alcian bluestaining. Magnification: 400 x: 1- lumen of large ductule,2- branching from large ductule, 3- small ductule.

In 70 days-old embryos, the pancreas wascovered by a well developed capsulecontaining connective tissue fibres and bloodvessels. Within the parenchyma, the number ofductules, cell clusters and developing isletsincreased and they started to group together.Capsule and the mesenchyme containedconnective tissue cells and fibres including afine reticulum. As the number of ductules andislets increased, the amount of mesenchyme gotreduced. Ductules showed extensive branchingand elongation. Branches of ductules showedthe formation of acini at their ends (Fig. 4).Developing acini were seen as small sac likeenlargements at the end of ductular branches.The cells lining these developing acini werepyramidal with large, spherical, heterochromaticbasal nuclei and eosinophilic cytoplasm. From71 days, the lobulation of the gland becameevident with grouping of the ductules, acini andislets. Within each group, ductules showedbranching as well as elongation. Ductular cellswere cuboidal with large spherical or ovalheterochromatic nuclei and scanty cytoplasm.The developing acinar cells were cuboidal orpyramidal with large spherical and heterochro-matic basally located nuclei. The apicalcytoplasm was more eosinophilic.



Fig. 4: Section of pancreas showing branching of ductulesand development of acini in 70 days-old goat embryo.Haematoxylin and Eosin staining. Magnification: 200 x:1- branching of ductule, 2- developing acini, 3- develop-ing islet.

By the beginning of the fourth month (91 to 120days) of development, division of thepancreatic parenchyma into clear lobules ofvarying size was observed (Fig. 5). Within thelobules, groups of acini and islets of varying sizewere present. Cross sections of ducts ofvarying size lined by cuboidal cells and largeblood vessels were also seen in the connectivetissue between acini and in the interlobularsepta. Acini contained cells with basally placed,dark stained nuclei and eosinophilic cytoplasmindicating the presence of zymogen granules.Corresponding to the increase in number ofacini, ductules and islets, amount of mesen-chyme was reduced considerably. Within thelobules, pancreas from 98 days old embryosshowed large ducts of varying diameter. Theseducts were lined by simple columnar epitheliumwith microvilli and their lumen showed somesecretory material (Fig. 6). Small intralobularducts with free cells similar to centroacinar cellsin the lumen were observed. After 105 days, thecapsule became thick and each lobule containedseveral secretory acini. Acini were mostlytubular and were lined by cuboidal cells withspherical, dark, basally located nuclei. Amongacini, ducts of varying size, arterioles, venules,nerve bundles and isolated neurons were alsoobserved. Small intralobular ducts were lined bythick simple squamous cells or cuboidal cellshaving lightly stained spherical or oval nuclei.Some of these ducts showed isolated cells within

the lumen. Larger intralobular ducts were linedby darkly stained cuboidal cells with vesicular,spherical or oval, basal nuclei having distinctnucleoli. Large interlobular ducts were lined bycolumnar epithelial cells with large spherical orbasal nuclei. Luminal border presentednumerous microvilli.Fig. 5: Section of pancreas showing division ofparenchyma into clear lobules in 91 days-old goatembryo. Haematoxylin and Eosin staining.Magnification: 40 x: 1- lobule, 2- interlobular connectivetissue septum, 3- developing islets.

Fig. 6: Section of pancreas showing large duct withsecretion and microvilli in 98 days-old goat embryo.Haematoxylin and Eosin staining. Magnification: 400 x:1- artery, 2- vein, 3- large duct with secretion.

In the fifth month (121 to 150 Days) of prenatallife, most of the pancreatic lobules showed atree like arrangement around a central trunk thatcontained blood vessels and intralobular ductswith cuboidal epithelial lining (Fig. 7). Theinterlobular septa made of loosely arrangedcollagen and reticular fibres contained bloodvessels, ducts, nerve bundles and ganglia.Within the lobules, groups of acini and ducts ofvarying size were further separated by thickintra lobular septa. Acini were supported byconnective tissue frame work composed of


collagen and reticular fibres.Fig. 7: Section of pancreas showing well developedlobules in 148 days-old goat embryo. Haematoxylin andEosin staining. Magnification: 100 x: 1- lobule containingacini, 2- large intralobular duct, 3- interlobular septum,4- large islets with sinus.

DISCUSSION

An understanding about the steps involved inthe normal development of exocrine pancreasis important in the field of developmentalbiology and pancreatic cancer biology. In thisstudy, the parenchyma of both pancreaticprimordia in embryos during second month ofdevelopment contained ductules of varying sizedispersed within the mesenchymal connectivetissue and some ductules showed branching.According to earlier reports, the pancreas in 40to 45 days-old sheep foetuses, containednumerous ductules [8]. The loosely arrangedmesenchyme contained fine connective tissuefibres and scattered mesenchymal cells similarto the findings in foetal goats at 42 days ofgestation [9].By third month of development, fusedpancreatic mass with well developed capsulewas noticed. The loosely arranged mesenchymepresented large number of ductules of varyingsize. Small ductules were numerous comparedto larger ones and showed extensive branching.Adjacent to them, solid clusters of cells prior tothe development of tubular lumen were alsonoticed. The presence of large number of smallductules and solid clusters indicated an increasein the mass of the exocrine component. Thebranching of large ductules and presence of cellclusters adjacent to ductules indicated that anincrease in number of ductules occurred bothby budding of new cell masses in the

mesenchyme as well as by branching of largeductules. The pancreas of human embryos atnine weeks of age also showed a similarstructure [10].The pancreas of 60 days-old buffalo foetusescontained cell clusters and ductules, bothsurrounded by an abundant mesenchymal mass[11]. However, the pancreatic primordium in micebetween 9.5 and 12.5 days embryonic agedeveloped multiple micro lumens whichcoalesced later [2]. It was also found that inmice, duct morphogenesis involved initialepithelial stratification and formation of multiplesmall lumens [12]. These further remodelledthrough changes in shape and position of cells,fused and formed a ramifying duct system withsingle lumen. In 70 days-old embryos, thepancreas was covered by a well developedcapsule containing connective tissue fibres andblood vessels. Within the parenchyma, thenumber of ductules, cell clusters anddeveloping islets was more. Capsule and themesenchyme contained connective tissue cellsand fibres including a fine reticulum.As the number of ductules and islets increased,the amount of mesenchyme got reduced.Extensive branching and elongation of ductulesindicated rapid increase in the mass of exocrinetissue. Branches of ductules showed theformation of small sac like acini at their ends.The cells lining these developing acini werepyramidal with large, spherical, heterochromaticbasal nuclei and eosinophilic cytoplasm. In thepancreas of rat foetuses, secretory acini withzymogen granules developed after thedevelopment of ductules [12]. In mouse embryosat 13.5 days, acinar cells developed from theextending tips of several finger-like protrusionsin the mesenchyme and increased in number byduplication [2]. In the pancreas of humanembryos the acini started to appear by 14 to 16weeks and by 20 weeks, well defined acini withsignificant number of zymogen granules werenoticed [9]. Grouping of the ductules and isletswithin the parenchyma from 71 days ofdevelopment was indicative of the initiation oflobule formation within the pancreas.By 91 days of development, division of thepancreatic parenchyma into clear lobules ofvarying size was observed. Within each lobule,



ductules showed branching as well aselongation. Ductular cells were cuboidal withlarge spherical or oval heterochromatic nucleiand scanty cytoplasm. The developing acinarcells were cuboidal or pyramidal with largespherical and heterochromatic basally locatednuclei. The apical cytoplasm was eosinophilic.Similar observations have been reported inbuffalo foetuses by third month [11]. Contraryto the present study, distinct lobulation of thepancreas was recorded at 125 days in buffalofoetuses [13] and at 14 to 16 weeks of gesta-tional age in human foetuses [9, 14].From fourth month of development onwards, thepancreas showed a distinct capsule composedof loose connective tissue and the parenchymawas organized into a large number of lobules ofvarying size. Large ducts of varying diameterwere also seen within the lobules and theirlumen showed some secretory material.Presence of such a ductular arrangement andthe presence of secretory material in large ductscould be a sign of the beginning of secretoryactivity of the exocrine pancreas.In the embryos of fifth month of gestational age,the capsule became thicker and was made ofcollagen and reticular fibres. This gland showeddistinct lobulation with a large number oflobules of varying size. Most of the lobulesshowed a tree like arrangement around acentral trunk that contained blood vessels andintralobular ducts lined by cuboidal epithelium.Such an arrangement is indicative of theestablishment of a well-developed exocrinesystem within the pancreas. Moreover, at thisage, the amount of secretory acini increased toa considerable extent compared to the initialstages of development and a systematicarrangement of ducts was also noticed in thisage group. In correspondence with the advance-ment of foetal age, there was a progressive in-crease in the proportion of exocrine portion ofthe pancreas and a progressive decrease in theamount of mesenchyme and interlobularconnective tissue. These findings support theobservations in the pancreas of human embryosthat the interstitial tissue became progressivelyless conspicuous with age [9]. In buffalofoetuses maximum growth of exocrine pancreasoccurred during late gestation [13].

CONCLUSION

The present study revealed that the ductsystem of exocrine pancreas developed beforethe formation of acini as early as second monthof foetal life. Formation of acini could benoticed in the third month of development whiledistinct lobulation as seen in postnatal animalscould be noticed in the fourth month ofdevelopment. The arrangement of lobules, ductsystem and acini within the pancreas in the fifthmonth of foetal life indicated the formation of awell organized exocrine system in goats in theprenatal life itself.

ACKNOWLEDGEMENTS

Authors are thankful to Kerala Veterinary andAnimal Sciences University for providing partialfinancial support for the present work.

Conflicts of Interests: None

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How to cite this article:A.R. Sreeranjini, N. Ashok. PRENATAL DEVELOPMENT OF EXO-CRINE PANCREAS IN CROSSBRED GOATS: A HISTOLOGICALSTUDY. Int J Anat Res 2016;4(2):2365-2371. DOI: 10.16965/ijar.2016.221

Original Research Article PRENATAL DEVELOPMENT OF … · PRENATAL DEVELOPMENT OF EXOCRINE PANCREAS...

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