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Publications from USDA-ARS / UNL Faculty U.S. Department of Agriculture: Agricultural Research Service, Lincoln, Nebraska

1998

Structure of the Avian Oviduct With Emphasis on Sperm Storage Structure of the Avian Oviduct With Emphasis on Sperm Storage

in Poultry in Poultry

Murray R. Bakst United States Department of Agriculture

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Bakst, Murray R., "Structure of the Avian Oviduct With Emphasis on Sperm Storage in Poultry" (1998). Publications from USDA-ARS / UNL Faculty. 623. https://digitalcommons.unl.edu/usdaarsfacpub/623

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618 M.R. BAKSTTHE JOURNAL OF EXPERIMENTAL ZOOLOGY 282:618–626 (1998)

© 1998 WILEY-LISS, INC. †This article is a US Governmentwork and, as such, is in the public domain of the United States ofAmerica.

JEZ 0513

Structure of the Avian Oviduct With Emphasis onSperm Storage in Poultry

MURRAY R. BAKST*U.S. Department of Agriculture, Agricultural Research Service, Germplasmand Gamete Physiology Laboratory, Livestock and Poultry Sciences Institute,Beltsville, Maryland 20705

ABSTRACT The macroanatomy, histology, and fine structure of the avian oviduct is reviewedand related to its role in fertile egg production. The avian oviduct functions as a biological assem-bly line, beginning sequentially with the deposition of the albumen around the fertilized or unfer-tilized ovum, then the shell membrane, and lastly, the shell, all within 25 hr of ovulation. While intransit through the oviduct, the fertilized ovum progresses to the pre-gastrulation stage of devel-opment. J. Exp. Zool. 282:618�626, 1998. © 1998 Wiley-Liss, Inc.†

Each structural component of the laid egg, ex-cept for the yolk (the hen’s ovum) is derived fromthe oviduct. When released from the ovary, theovulated ovum is gathered into the ostium by theaction of the fimbriated region of the infundibu-lum. If sperm are present, the ovum may be fer-tilized. Regardless, the ovum traverses the oviductand accrues the albumen in the magnum, the shellmembrane in the isthmus, and the hard shell inthe uterus. The most caudal segment, the vagina,contributes the cuticle to the egg prior to oviposi-tion and also serves as a conduit between theuterus and cloaca. The time elapsed from ovula-tion to oviposition is about 25 hr. If the laid eggwas fertilized, the blastoderm, which consists ofabout 60,000 cells in the chicken and 30,000 cellsin the turkey, is in the pre-gastrulation stage.

In order to fertilize a nearly daily succession ofova, which amounts to 2 to 7 eggs per week,sperm are slowly but continuously released fromthe oviductal sperm storage sites in the caudalend of the oviduct and are transported to the siteof fertilization at the cranial end of the oviduct.The precise mechanisms which regulate thesecomplex processes remain to be elucidated. How-ever, since the 1980s we have begun to better un-derstand the mechanisms which regulate spermstorage and selection in the avian oviduct (seeBakst et al., ’94).

This paper will examine the structure and func-tion of the avian oviduct relative to its role in pro-ducing a normal fertilized egg. In addition toproviding a brief review of the pertinent litera-ture, new observations and accompanying specu-lation on the significance of these observations will

be presented. Unless otherwise stated, descrip-tions will be limited to Galliformes, specifically,commercial chickens and turkeys.

OVIDUCT STRUCTUREGeneral anatomy

The oviduct in the mature hen in egg produc-tion (Fig. 1) consists of five morphologically andfunctionally distinct segments with a total lengthof 80–85 cm in the chicken and 90–95 cm in theturkey. In each segment but the uterus (sometimesreferred to as the shell gland), the luminal mu-cosa of the oviduct consists of distinct longitudi-nal folds (plicae) which vary in height, coloration,and volume both before and after the passage ofan egg mass (Figs. 2, 3). In the uterus, this orien-tation is difficult to discern as the mucosa formspade-shaped leaflets which collectively appear tohave a pleated, transverse orientation.

The luminal mucosa of the oviduct consists ofnonciliated secretory cells (often referred to asgoblet cells) and ciliated cells. Ciliated cells mayor may not possess secretory granules in theirapical cytoplasm (Makita et al., ’73). The his-tochemical and immunocytochemical stainingproperties (including that of lectins) of the sur-face epithelium suggest that the secretory cellsin each segment elaborate different products(Aitken, ’71; Kami and Yasuda, ’84; Yamamoto etal., ’85; Bakst, ’87).

*Correspondence to: M.R. Bakst, Bldg. 262, BARC-East, Beltsville,MD 20705.

AVIAN OVIDUCT 619

Figures 1–5. (Caption on next page.)

620 M.R. BAKST

The fimbriated region of the infundibulum (alsoreferred to as the funnel portion of the infundibu-lum) (Fig. 2) and the vagina are densely popu-lated with ciliated cells. The abovarian direction(away from the ovary) of the cilia beat may bepartially responsible for the ovum entering theostium of the infundibulum (Fujii et al., ’81). Theratio of ciliated to nonciliated secretory cells isgreater toward the luminal surface of the folds.Nonciliated cells predominate in the basal aspectsof the folds and are found exclusively in the sub-epithelial tubular glands throughout the oviduct.

The structures forming the egg mass envelop-ing the ovum are derived primarily, but not ex-clusively, from the subepithelial tubular glandslocated from the mid-section of the infundibulumthrough the uterus. These exocrine glands fullydifferentiate shortly after the onset of egg produc-tion and appear to vary in complexity and vol-ume. All have a well-defined transition betweenthe surface (luminal) epithelial cells and the epi-thelium forming the tubular gland.

Located in the uterovaginal junction (UVJ)(Figs. 3, 4), which is a narrow band at the ante-rior end of the vagina, are the primary sperm stor-age sites in the oviduct, the sperm-storage tubules(SST). Based on histological (light and electronmicroscopy) and histochemical observations, thesespecialized subepithelial tubular glands are ca-pable of only limited secretory activity (Bakst, ’87).This characteristic may be one of the factors thatcontribute to the successful storage of sperm inthe SST over a succession of daily ovulatory cycles.

The loose connective tissue in the lamina pro-pria is populated with lymphocytes, plasma cells,macrophages, fibroblasts, and mast cells. Nerve,collagen, and elastin fibers are also discernible

and easily visualized in the segments in whichsubepithelial tubular invaginations are absent orless densely distributed. Intraepithelial lympho-cytes, particularly in females in egg productionfor several months, are prevalent in the mucosalsurface and SST epithelia. These, as well asplasma cells in the lamina propria, are IgG posi-tive (Bakst, ’87).

OVIDUCTAL SPERM STORAGE SITESVagina

A more detailed overview of the vagina is war-ranted considering its multiple roles in sperm se-lection, storage, and transport (see Bakst et al.,’94). The primary folds of the vaginal mucosa,which lack exocrine-type tubular glands, are di-vided into smaller parallel secondary and tertiaryfolds (Fig. 3). Scanning electron microscopy (SEM)of the vagina’s luminal surface reveal longitudi-nally oriented, parallel tracts densely covered bya ciliated epithelium.

In hens in egg production, the UVJ is fully dis-cernible after dissecting away the connectivetissue binding the vagina in a tightly coiled con-figuration (compare Fig. 1 with Fig. 4). When thestripped-down vagina is slit longitudinally, its lu-minal mucosa is exposed and the contrastingmorphology and coloration of the uterine leaflets tothe longitudinal folds of the vagina are evident. TheSST generally are localized within the anterior 1 to3 cm of the vagina (Fig. 3) and are easily visual-ized at this point by stereomicroscopy (Fig. 5).

The SST are derived from invaginations of theoviduct’s surface epithelium (Bakst, ’92). The SSTepithelium consists of nonciliated columnar cells,

Figures appear on p. 619.Fig. 1. The turkey oviduct with a hard-shelled egg in the

uterus (U). The fimbriated region (arrowhead) of the in-fundibulum (I) is evident. The vagina (V) is coiled and envel-oped in a connective tissue casing.

Fig. 2. A scanning electron micrograph of the mucosal sur-face of the rim of the fimbria. The complex array of mucosalfolds is lined with a ciliated epithelium. (Bar = 500 µm)

Fig. 3. The vaginal mucosa (V) is composed of numerouslongitudinally oriented plicae which merge with the more com-plex folds of the uterine mucosa (U). A 1.5-cm band (betweenarrowheads) at the anterior end of the vagina contains theSST, which are barely discernible here.

Fig. 4. The uterus (U) and vagina (V) are observed afterthe connective tissue surrounding the vagina is removed. Theuterovaginal junction is evident as a slightly bulbous area.

Fig. 5. Stereomicroscopy of the anterior end of the va-gina reveals the coiled SST, most exceeding 250 µm in length.(Bar = 1 mm)

Fig. 6. A similar preparation to Fig. 5 showing the sub-epithelial tubular glands of the distal infundibulum.

Fig. 7. A similar preparation to Figs. 5 and 6 showingthe subepithelial tubular glands of the distal infundibulumand the magnal subepithelial tubular glands.

Fig. 8. A bright field micrograph of the distal infundibu-lum highlighting the subepithelial tubular glands. The tran-sition from a ciliated epithelium to a nonciliated secretoryepithelium is observed (arrowhead). (Bar = 80 µm)

Fig. 9. A bright field micrograph of the subepithelial tu-bular glands of the distal infundibulum. The denser appear-ing glands are magnal subepithelial tubular glands.

Fig. 10. A DIC micrograph of an unfixed squash prepara-tion of distal infundibulum revealing a single capillary (ar-rowhead) adjacent to a subepithelial tubular gland. (Bar =40 µm)

Fig. 11. Adjusting the focal plane through the epithelialmucosa surface of the distal infundibulum reveals openingsto the subepithelial tubular glands surrounded by epithelialcells.

AVIAN OVIDUCT 621

Figures 6–11.

622 M.R. BAKST

which in the turkey are about 20 µm in height,and in the chicken about 15 µm in height (Meroand Ogasawara, ’70). The intracellular lipid dis-tribution and content may vary between andwithin species. Mitochondria are most prevalentin the basal area of the cell. While lacking anabundance of secretory granules, the tips of sur-face microvilli, which approach about 1 µm inheight, are occasionally observed to bleb, possiblyan indication of limited exocytotic activity. Whilesuch blebs may be an artifact of preparing thespecimen for transmission electron microscopy,their consistency suggests exocytotic activity, pos-sibly representing a source of lipid or protein foruse by resident sperm (Bakst, ’93).

Recent work performed in our laboratory (V.Akuffo, S. Freedman, and M. Bakst, unpublished)has provided some additional insight into the epi-thelial cell structure of the SST. Using an F-ac-tin-specific immunofluorescence probe, we wereable to visualize by confocal microscopy a discrete,dense microfilament network in the immediate vi-cinity of the microvillous border. This network,which is clearly evident in transmission electronmicrographs, may be involved in the release ofresident sperm from the SST.

The lateral faces of SST epithelial cells formcomplex interdigitations, which at the apical sur-face are further characterized by tight and inter-mediate junctions. It is possible to visualize these“terminal bars” in the lumen of individual SST inunfixed squash preparations of UVJ mucosaviewed by differential interference contrast (DIC)microscopy. Desmosomes are distributed along theremaining length of the lateral walls. Myoepithe-lial cells are not observed around any aspect ofthe SST. Contrary to previous authors (Gilbert etal., ’68), we (Freedman, Akuffo, and Bakst, un-published) have demonstrated the immunocy-tochemical localization of nerve fibers in closeassociation with the outer connective tissue sur-rounding the individual SST. In addition, neuronshave also been observed within 100 µm of SST.Whether there are also specialized endocrine orpeptide secreting cells in the oviduct epitheliumhas yet to be determined.

InfundibulumThe most anterior segment of the oviduct is the

infundibulum (Fig. 1). It has been subdivided intothree functionally and morphologically distinctregions: the fimbriated region, which guides theovulated ovum into the ostium of the infundibu-

lum; the funnel region, which lacks subepithelialtubular glands, but is presumably the site wheresperm first contact the recently ovulated ovum;and the chalaziferous region, which will be re-ferred to here as the distal infundibulum (Aitkenand Johnston, ’63; Aitken, ’71; Gilbert, ’79; Fujiiet al., ’81). The distal infundibulum is the “sec-ondary” sperm-storage site in the oviduct (VanDrimmelen, ’51; Fujii and Tamura, ’63; Bakst, ’81)and is characterized by subepithelial tubularglands (Figs. 6–14) which secrete an albumen-likematerial around the ovum (Bain and Hall, ’69),referred to as the outer perivitelline layer. Theouter perivitelline layer provides an additional fi-brous protein investment around the ovum and,relative to the process of fertilization, preventsexcessive sperm penetration of the ovum at theanterior end of the oviduct. It should be noted thatwhile polyspermy is normal in birds, excessivesperm penetration of the germinal disc regionleads to pathological polyspermy (Bakst andHowarth, ’77), which results in early embryonicmortality. In the transition zone between theproximal magnum and the distal infundibulum,the tubular glands from each segment can beclearly viewed by phase contrast (Fig. 7) andbright field microscopy (Fig. 9).

Little is known of the mechanisms of spermtransport to and storage in the infundibulum.

Fig. 12. A dual DIC-fluorescence micrograph of a distalinfundibular subepithelial tubular gland with a single fluo-rescing bisbenzimide stained sperm (arrowhead) in the lu-men. Visualization of this sperm was possible only by reducingthe DIC illumination. (Bar = 40 µm)

Fig. 13. If a hen is inseminated with sperm stained withbisbenzimide immediately after oviposition, some of the dis-tal infundibular subepithelial tubular glands become engorgedwith sperm. In this fluorescence micrograph, three of the fourdistal ends of the tubular glands are fluorescing brightly dueto sperm accumulations. The fourth tubular, which is barelydiscernible, is evident in Fig. 14. Also note that the bis-benzimide has stained the nuclei (arrow) of the tubular glandepithelium with resident sperm. (Bar = 40 µm)

Fig. 14. A DIC micrograph of the same subepithelial tubu-lar glands observed in Fig. 13. While the epithelial nuclei arevisible, only one sperm is observed (arrowhead). Secretory gran-ules are also clearly evident in the apical portion of the epithe-lial cells forming the tubular glands. The arrow highlights thesame nucleus highlighted in Fig. 13. (Bar = 40 µm)

Fig. 15. A dual DIC-fluorescence micrograph of a singleSST with numerous bisbenzimide stained sperm in its lu-men. Connective tissue fibers are densely distributed aroundthe SST. The arrow highlights a nucleus in a SST epithelialcell. (Bar = 50 µm)

Fig. 16. In this fluorescence micrograph, the profile of an-other SST is observed with numerous bisbenzimide stainedsperm in its lumen.

AVIAN OVIDUCT 623

Figures 12–16.

624 M.R. BAKST

During the course of the daily ovulatory cyclesperm are slowly released from the SST andascend to the distal infundibulum. Here theyaccrue in the tubular glands and are probablyreleased with the ovum-induced distention ofthe mucosa combined with the active secretionof the albumen-like proteins.

Squash preparations viewed by phase contrastand DIC microscopy have been useful in develop-ing a better understanding of the role of the in-fundibulum in sperm storage in turkeys (Bakst,’94) and other Galliformes. Using DIC on freshmucosa, the beating pattern of the densely cili-ated surface of the fimbria can be clearly observed.As the number of nonciliated cells increases inthe funnel region, shallow bud-like invaginationsof the surface epithelium begin to appear. Theseinvaginations are composed of nonciliated cellswhich do not contain any appreciable amountof secretory material in their apical cytoplasm.Sperm were not observed in these invagina-tions. With the focal plane at the level of thesurface epithelium, cross sections of polygonalcells with secretory granules (Fig. 11) or nucleican be observed surrounding the openings tothe tubular glands.

The surface topography changes little as oneprogresses toward the distal infundibulum. Bud-like invaginations originating from the surfaceepithelium lining the distal infundibulum appearmore voluminous and begin to branch (Fig. 8).The epithelium is composed of nonciliated cellswith and without secretory granules in their api-cal cytoplasm. More caudally, in the transitionregion between the distal infundibulum and proxi-mal magnum, the tubular glands increase in com-plexity and volume until individual tubularglands are no longer discernible (Figs. 7, 9). Theepithelium forming these glands is clearly secre-tory, possessing abundant secretory granules intheir apical cytoplasm.

Rarely were more than one sperm observed inthe distal infundibular (Fig. 12) or magnal tubu-lar glands (Fujii and Tamura, ’63; Schindler etal., ’67; Bakst, ’94). However, if a hen is subjectto artificial insemination (200 to 300 millionsperm introduced into the distal vagina) immedi-ately after oviposition, large numbers of sperm in-filtrate the infundibular tubular glands (Figs. 13,14) (Burke and Ogasawara, ’69; Brillard, ’90a,b;Bakst et al., ’94) and the proximal 1 cm of themagnum (Fujii and Tamura, ’63; Bobr et al.,’64a,b; Schindler et al., ’67; Bakst, ’81, ’83). A simi-lar infiltration of the infundibulum with sperm

follows intrauterine and/or intramagnal insemi-nations (Ogasawara et al., ’66; Brillard, ’90a,b).In such instances, the abnormally high popula-tion of sperm at the site of fertilization increasesthe possibility of pathological polyspermy and thehigh incidence of embryonic mortality.

Recommended approaches to studyof the hen’s oviduct

I have used a variety of approaches to character-ize various aspects of oviductal anatomy and ovi-duct–sperm interactions in the hen. Routine lightand electron microscopy provided fundamentalhistological information. However, there were limi-tations to these techniques, particularly when at-tempting to evaluate sperm distribution in thesperm storage sites in the UVJ and infundibulum.

I found the most useful and versatile proceduresimply involved using a squash preparation ofunfixed oviductal mucosa. Isolated pieces of pli-cae (no larger than 3 × 3 mm) without the mus-cularis can be viewed by stereomicroscopy, phasecontrast, or DIC microscopy. Compared to rou-tinely stained histological sections, these proce-dures provide a much improved appreciation ofthe spacial distribution of the subepithelial tubu-lar glands and SST with and without sperm inpoultry (Bakst, ’87, ’92, ’94; Bakst et al., ’94) aswell as in feral birds (Bakst and Bird, ’87; Birk-head and Moller, ’92).

Our ability to resolve resident sperm in theSST, infundibulum, and other areas of the ovi-duct is significantly improved if the hen is in-seminated with sperm previously stained withnuclear fluorescent dye bisbenzimide (Hoechst33342, Molecular Probes, Eugene, OR) (Bakst,’94). The sperm staining procedure is applicableto other avian species but preliminary workshould be performed to optimize the staining pro-cedure and evaluate the impact of the stain andstaining procedure on sperm viability and sub-sequent fertility. With fresh turkey semen, thepooled sample is diluted to about 5 billion spermper ml with poultry semen extender and thor-oughly mixed. An equal volume of extender con-taining 120 µg bisbenzimide per ml is added tothe diluted semen and the mixture placed on arotary shaker for 2 hr at room temperature be-fore insemination. This procedure minimizes theinimical effects of the bisbenzimide on hen fer-tility and egg hatchability while maximizingsperm fluorescence.

Dual DIC and fluorescence microscopy of squashpreparations of oviductal mucosae revealed single

AVIAN OVIDUCT 625

sperm as well as dense accumulations of sperm re-siding in SST (Fig. 15) and infundibular tubularglands (Figs. 12, 14). Fluorescing sperm becomemore evident as the DIC light source is reduced oreliminated (Figs. 13, 16). Here again the investi-gator must be prepared to modify some establishedpractices in order to optimize the viewing of ovi-ductal sperm. For example, to view fluorescingsperm residing in the infundibular region, the DICoptics may have to be slightly modified. By slidingthe DIC analyzer out of its proper position, an im-age is created which allows visualization of resi-dent sperm in the voluminous infundibular tubularglands (Bakst, ’94). This is particularly useful whenlarge surface areas need to be evaluated to deter-mine the presence or absence of sperm.

Other techniques have been developed and re-viewed elsewhere which provide means to esti-mate the efficacy of oviductal sperm storage(Bakst et al., ’94; Bakst and Cecil, ’97). These pro-cedures are based on estimating the number ofsperm embedded in the outer perivitelline layerof the fresh laid egg or by estimating the numberof holes (sites where penetrating sperm have hy-drolyzed a path through the ovum’s investment)present in the perivitelline layer overlying the ger-minal disc of a fresh laid egg.

To conclude, a better understanding of the physi-ological and molecular mechanisms of oviductalsperm selection and storage will provide the ba-sis for improvements in the efficiency of poultrybreeding. Furthermore, the same knowledge willaid in the development of improved technologiesfor the propagation of endangered birds and thepreservation of their germplasm.

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