Studies of the deferent ducts from the testis of the African elephant

J. Aiat. (1981), 132 3, pp. 371--386 371Witlh 23 figures

Priilted in GCleat Britaii,

Studies of the deferent ducts from the testis of the Africanelephant, Loxodonta africana. I. Structural differentiation

R. C. JONES AND M. F. BROSNAN

Department of Biological Scienices, University of New'castle,N.S. W. 2308, Alistralia

(Accepted II August 1980)

INTRODUCTION

Although there is considerable variation amongst mammals in the structure of themale genital ducts (Benoit, 1926; Nicander, 1957, 1958; Reid & Cleland, 1957;Hoffer & Greenberg, 1978; Flickinger, Howards & English, 1978; Bedford, 1977;Bedford & Rifkin, 1979) detailed structural studies have not been carried out on asufficiently wide variety of species to establish the degree of variation which mayoccur, and too little is known about the function (Hamilton, 1975) of the genitalducts to satisfactorily assess the value of any attempt (Glover & Nicander, 1971;Nicander & Glover, 1973) to establish homologous parts of the system. Conse-quently, a series of studies on the male genital ducts of the African elephant (andother vertebrates) is being carried out to provide information for the interpretationof homologous regions. This report describes a detailed light microscope study of thestructure of the ductuli efferentes testis, ductus epididymidis and ductus deferensof the elephant and it provides a basis for the interpretation of other work on theultrastructure, cytochemistry and function (e.g. Jones, Rowlands & Skinner, 1974;Jones, 1977, 1978, 1980; Holt, Jones & Skinner, 1979).The elephant is of particular interest to comparative reproductive biologists

because its testes and genital ducts reside deep within tlhe abdominal cavity andbecause of the animal's immense size and unspecialized habitat. Earlier reports on theelephant are not extensive and not in full agreement about the degrees of structuraldifferentiation of the epididymis. Short, Mann & Hays (1967) found little histologicaldifferentiation and concluded that the organ was not anatomically differentiated.On the other hand, Glover (1968, 1973) indicated that it was anatomically differen-tiated into a head, body and tail, and Hanks (1977) showed some histological differen-tiation in the proximal end.

MATERIALS AND METHODS

Strluctuire of dluctsThe material was collected during October 1972, July 1974, and October 1977, in

Kruger National Park, South Africa, from elephants which were culled to avoidoverstocking (Jones, 1973a). The testes and genital tract were dissected fromanimals immediately after death and in all studies a sample of a testis from each bullwas fixed for subsequent histological examination to assess spermatogenic activity.The gross anatomy of the reproductive tract was examined in five freshly collected

and three fixed specimens (10°' v/v formol-saline) after dissection free of the sup-porting mesentery. The ductuli efferentes from two of the fresh and one of the fixed

R.C. JONES AND M.F. BROSNAN

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Fig. 1. A scale diagram showing a lateral view of a testis and genital ducts as they appear in situin a 26 years old African elephant. The ductuli efferentes are indicated by the shaded area abovethe testis. Numerals above the diagram show the location of sampling sites along the epididymisand ductus deferens for the 1974 study summarized biometrically in Figs. 2-7: the asterisksshow the location of other sites sampled in 1977 (see Fig. 9 for sampling sites 1-3 along theductuli efferentes). The heading above the diagram indicates how the epididymis may beclassified into three anatomically distinct regions. The dotted area shows the middle segment.

specimens were separated from one another as shown in Figure 8; several of thelongest ductuli efferentes were then carefully dissected free of their connective tissuestroma in order to determine their course and (for one duct from each animal) length.For the histological studies samples of the genital tract were fixed in Bouin's fluid,

embedded in paraffin wax, sectioned at 5,m and stained with haematoxylin andcosin. Due to the immense size of the genital tract it was most practical to sampletissue from different sites for histological examination rather than to attempt toprepare serial sections of the whole duct. Consequently, in 1972, three sites weresampled from 3 bulls for histological examination and the nine sites examined in1974 (4 elephants studied) were chosen using the information gained from studyingthe 1972 samples. Sites 1-3 were in the ductuli efferentes (Fig. 9) and sites 4-8 (Fig. 1)were from the ductus epididymidis. Sample site 4 was taken between the pointswhere the seventh and eighth (counted from anterior end) ductuli efferentes joinedthe ductus epididymidis, site 5 was located just distal to the point where the mostdistal ductulus efferens joined the ductus epididymidis, site 6 was opposite the originof the posterior gonadal ligament, site 7 was at the distal end of the neck of theepididymal isthmus and site 8 was at the centre of the distal region of the epididymis(Fig. 1). Site 9 was in the ductus deferens. Samples from sites 1-9 and intermediatesites (see asterisks in Fig. 1) were also taken from 2 elephants in 1977 to confirm thegeneral trends shown in Figures 2-7. For the cytological studies (4 bulls were exa-mined; 2 in 1974 and 2 in 1977) tissue was taken from site 2, the site shown by theasterisk proximal to site 4, sites 4 to 8 and mid-way between sites 5 and 6. They werefixed in PFG-cacodylate (Jones, 1 973b) for 2 hours at 5 °C, stored in sucrose-cacody-late at 5 °C for 2 weeks, then osmicated and embedded in Araldite. Thick sections (1,am) were cut on an ultramicrotome and stained with Azur II (Richardson, Jarett &Finke, 1960), or 1% toluidine blue in 0 5% borax, for light microscopy.

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Figs. 2-7. Summary of the biometry of the genital ducts of 4 elephants. The abscissa indicate thesample sites shown in Figs. 1 and 9. The ordinates are: Fig. 2, duct diameter; Fig. 3, length offolds; Fig. 4, mean scores of sperm concentration in the duct lumen; Fig. 5, epithelial height;Fig. 6, nuclear shape expressed as a ratio of height: width; Fig. 7, length of epithelial stereocilia.Horizontal bars indicate the magnitude of standard errors.

Biometry of ductsMeasurements of the main characteristics of the ducts were made using the

histological preparations collected from four bulls in 1974 (a 9 x 4 randomisedblock design with 9 sample sites; see Figs. 2-7). The histological preparations were

cross sections of the genital tract in which there were about 30 profiles of the ductsite under consideration (except for the ductus deferens for which 6 cross sectionswere prepared, each containing one profile of the dluct per section). A sufficient

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number of sections were prepared to ensure that for each sample site, for each animal,one section was chosen in which there was at least one profile of the genital duct cutin cross section (i.e. the boundary was circular and the lining epithelium was cut in aplane perpendicular to the basement membrane) and the basic structure of the profilewas typical of the other profiles in the section. A transmission light microscope fittedwith a calibrated eyepiece micrometer was used to make the following measurementsof the profile which was considered the best cross section of the part of the genitalduct under consideration (i.e. one profile was measured per sample site per animal:in practice a number of other profiles were measured in order to choose the mostsuitable one): duct diameter (between the opposite basement membranes of thelining epithelium), thickness of smooth muscle surrounding the duct (periductalmuscle), height of the epithelium, length of the epithelial stereocilia, length andwidth of the nuclei of principal cells (the ratio of height: width was calculated), andthe average length of all invaginations of the epithelium into the lumen of the duct(villi). In practice, for each profile two measurements of the dimensions of eachcharacteristic were made corresponding to the longest and shortest dimension (e.g.the two duct diameters were oriented at 90° to one another), and the mean of the twomeasurements (which were identical or almost identical) was used as unit observation.In order to estimate the effect of the folds on the surface area of epithelium facingthe duct lumen, photomicrographs (magnification = x 100) of the duct underexamination were prepared and the lumen circumference measured with a plani-meter. The index calculated was the ratio of measured lumen circumference tocalculated circumference of a circle with the same diameter as the duct's lumen. Thewhole extent of the histological section used for the previous measurements wasexamined and scored (on a scale from 0 to 4; 0 = absent and 4 = numerous)according to the frequency of folds within the ducts and the number of spermatozoain the lumen of the ducts.

Analyses of variance were carried out on untransformed data (except for measure-ments of periductal muscle), and the standard errors given in Figures 2-7 werecalculated from the residual error in each analysis. This corresponds to the first orderinteraction between sampling sites and replicates (elephants). Due to heterogeneityof the error variance for measurements of thickness of periductal muscle, the datawere transformed to logarithms for statistical analyses. However, the standard errorsstated in the Results for this data were estimated from the variance between animals,within sampling site (values correspond to untransformed data). In the analyses ofvariance, differences between sampling sites were statistically significant for all theparameters measured (P<001 for all parameters except length of stereocilia, forwhich P < 0 05). Consequently, for the sake of brevity the levels of probability are notstated in the Results and it may be assumed that only differences which are statisti-cally significant are described.

Maturation of spermatozoaSpermatozoa were collected (Jones, 1971) along the epididymis of six elephants

and used for the preparation of eosin-nigrosin smears for determining the location ofthe cytoplasmic droplet on the middle piece.

374

Structure of the elephant genital ducts

RESULTS

Testes from all of the animals studied were actively producing spermatozoa.However, specimens from some animals had a higher proportion of seminiferoustubules containing later stages of spermatogenesis than others and this was correlatedwith the number of spermatozoa in the epididymis (see below).The testes hung from the dorsal wall of the abdominal cavity medial to the kidneys.

The mesorchium folded around the ductuli efferentes and anterior part ofthe epididy-mis which was medial to the dorsal pole of the testis, and the epididymis coursedposteriorly within the mesenteric fold. Figures 1 and 8 respectively show the overalldimensions of these structures before and after dissection from the mesenteric fold.Further, Figures 2-7 summarise, for comparative purposes, six of the main histologi-cal features of the genital ducts. However, in order to keep the following descriptionsbrief these figures are not repeatedly referenced.

Ductuli efJerentes (Figs. 8, 9, 11, 12, 22)The ducts were highly convoluted and within their connective tissue stroma each

formed a cord (Fig. 9) 5 to 8 cm long. The cords radiated dorsally from the extra-gonadal rete testis (Fig. 8) which was located on the superior surface of the testisabout one third caudad from the anterior end. Most of the ducts were pigmentedreddish-brown (Fig. 9). In the most anterior ducts, all except the most distal 0 5 cmof each duct was pigmented. The more caudal ducts were shorter and a smallerproportion of them (proximal end) was pigmented; the pigmentation was absent inthe most caudal 3-6 ducts which were less convoluted and white like the connectivetissue (and epididymis), and were difficult to distinguish from the connective tissue.In three animals in which the ducts were dissected in detail, 14 to 16 ductuli efferenteswere identified in two fresh specimens whilst 19 distinct and 3 ill-defined (caudal)ducts were counted in a fixed specimen.For three separate elephants, one of the longer ductuli efferentes was dissected

free of the enmeshing connective tissue. In two specimens, which were completelyunravelled, their lengths were 63 and 70 cm. It was also found that there was someanastomosing between different parts of the same duct (Fig. 11). However, it was notvery extensive (in two specimens 12 and 14 anastomoses were counted) and waslimited to the first 2 cm.Anatomical studies indicated that all the ductuli efferentes appeared to join the

ductus epididymidis at right angles (Fig. 8). However, histological examinationindicated that the most anterior ducts (about 6) joined together ultimately to form asingle duct, which continued caudally as the ductus epididymidis, and received eachof the remaining ductuli efferentes at right angles. The anterior ducts successivelyjoined together in pairs so that the two most anterior ducts merged to form a singleciliated duct which was then joined by the next duct, and so on.

In histological sections the ductuli efferentes were narrow, lined by a low pseudo-stratified columnar epithelium, contained very few spermatozoa and were surroundedby a thin layer of circularly arranged smooth muscle (Fig. 12) which was thicker atthe distal than at the two proximal sites (means ± S.E. for sites I to 3 respectivelywere 42 + 7, 41 + 4 and 66 + 1O pm). The stroma around the ductuli efferenteswas not as well vascularized as the epididymal stroma. The duct epithelium increasedin height from the proximal to the distal end. It was composed mainly ofprincipal andciliated cells and a few basal and 'halo' cells (Reid & Cleland, 1957; Fig. 22). The

375

376 R. C. JONES AND M. F. BROSNAN

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Structure of' the elephant genital ducts 377principal cells were stereociliated (i.e. microvilli), but, otherwise, structurally similarto the ciliated cells. Both contained oval, slightly basal nuclei which became roundertowards the distal end of the duct. They contained small and large densely stainedbodies in the supra- and infranuclear cytoplasm (Fig. 22). The bodies were brown inparaffin sections and occurred most frequently in the pigmented parts of the ducts.

Ductus epididymidisAnatomical differentiationEach epididymis was a long cord (150-200 cm long, depending on the age of the

bull) composed of a highly convoluted duct in a connective tissue stroma. Threeanatomically distinct regions could be recognised when the cord was carefullydissected from the supporting mesenteric fold (Figs. 1, 8), a proximal (head), isthmicand distal (tail) region. The proximal region partly enclosed the ductuli efferentesand extended distally to an isthmus beginning beyond the posterior gonadal ligament(i.e. beyond site 6, Fig. 1). In freshly collected material the external diameter of theduct was less than 1 mm, proximal to the posterior end of the isthmus. As the cordwidened beyond the isthmus to form the distal region, the duct also graduallywidened to a diameter of about 2 mm. Further along the duct its diameter increasedmore abruptly to form the ductus deferens (10 mm) which was loosely coiled andwas more solid than the ductus epididymidis.

It is noteworthy that the isthmus of the epididymis was more distinct in somespecimens than others. This morphological variation was partly a consequence ofvariation in duct patency, but mainly due to variation in the length of the epididymis.The latter varied with the age of the bull; consequently, larger bulls had a long, moredistinct isthmus. In about 80% of elephants studied (the genital ducts of about 40male elephants have been examined over the past 8 years; see Introduction) theductus epididymidis in the distal region was markedly distended with spermatozoa.In these elephants there was a high proportion of seminiferous tubules showing latestages of spermatogenesis (i.e. spermatozoa protruding into the tubule lumen)whereas in the animals without distended ducts there was a low proportion oftubules showing late stages of spermatogenesis.

Histological differentiationThe ductus epididymidis was lined by a stereociliated, pseudostratified, columnar

epithelium composed mainly of principal and basal cells (Figs. 13-15, 17-20); a

Fig. 8. The genital ducts dissected free of their supporting mesentery. T, part of the testis,showing its relationship to the ductuli efferentes (one shown by arrow) and epididymis; L,posterior gonadal ligament; AE, anterior end of ductus epididymidis; PE, posterior end of theproximal region of epididymis; IE, isthmus of epididymis; DE, distal region of epididymis;DD, ductus deferens.Fig. 9. A ductulus efferens enmeshed in connective tissue to form a cord connecting the ductusepididymidis on the left side and the rete testis on the right side. Note that the ductulus efferensis dark due to reddish pigmentation. Sampling sites 1, 2 and 3 were in the proximal, middle anddistal thirds of the pigmented part of the cord. x 0-9.Fig. 10. High magnification photomicrograph of one of the folds shown in Fig. 16. Note thevery low principal cells and the basal cell (arrow). Paraffin, H & E. x 180.Fig. 11. A section of a ductulus efferens dissected from its connective tissue stroma showing ananastomosis (arrow) between separate parts of the duct. x 3 5.Fig. 12. Transverse section of a ductulus efferens. Note the sparsity of sperm in the lumen.Paraflin, H- &E. x 100.


small proportion of 'apical' (Fig. 19) and 'halo' cells was also present but ciliatedcells were rare. The duct was surrounded by a layer of circularly arranged, smoothmuscle (Figs. 13-15) which was about the same thickness in the proximal and isthmicregions (means + s.E. for sites 4 to 7 were respectively 63 + 7, 72 + 8, 61 + 4 and68 + 4 /im), but of increased thickness along the distal region (mean + S.E. for site 8was 191 + 84). Invaginations of the duct epithelium into the lumen formed circularlyarranged folds (Figs. 13, 14, 21) in which only a thin layer of well vascularized looseconnective tissue (mainly collagen fibres) lay between the opposing basementmembranes of the epithelium. Indeed, it was concluded that the degree of vasculari-zation of the ductus epididymidis was approximately proportional to the frequency offolds. Where the epithelium was stretched over the crest of a fold it usually appearedlower and carried fewer stereocilia than adjacent epithelium, and in the depths of thegrooves between the folds the height was greater than in adjacent areas. There wasa close relationship between the number of folds seen in a region and their length soonly the latter is shown in Figure 3.

Figures 2-7 show how the ductus epididymidis is divided into two parts showingcharacteristic proximodistal gradients. Proximal to site 6 there was a gradualincrease in epithelial height, length of stereocilia and concentration of spermatozoa,and decreases in length of folds and of lumen diameter. Distal from site 6 there weremarked increases in lumen diameter and fold length, a decrease in epithelial heightand length of stereocilia and the lumen was packed with spermatozoa. The proximaland distal parts of the ducts have been referred to as the initial and terminal segmentsrespectively, and the region around site 6 as the middle segment.

I,iitial segmiienit (Figs. 13, 17, 21)Overall the ducts contained a low to moderate number of spermatozoa which

showed structural signs of immaturity, i.e. most had a proximally located cyto-plasmic droplet. The duct was wide but the lumen was almost obliterated by foldswhich increased the surface area of epithelium facing the lumen by a factor of about3-5-fold. The epithelium was tall and the principal cells contained relatively roundnuclei and long stereocilia. Moderate numbers of basal cells were present but apicaland 'halo' cells were quite rare. In paraffin sections the cytoplasm was quite homo-geneous, but in Araldite sections a few or moderate (Figs. 17, 21) numbers of denselystained bodies were present in the cytoplasm, and some very small vacuoles were alsopresent in the supranuclear cytoplasm. fn the latter context the supranuclear cyto-plasm was more homogeneous at site 4 than site 5 (Fig. 17 vs. Fig. 21).

Middle segmnenit (Figs. 15, 18, 19)The region around site 6 (i.e. where the ductus epididymidis was narrowest) could

be distinguished from the segments on either side. It was a short transitional zone inwhich spermatozoa were more concentrated than elsewhere in the duct and thecytoplasmic droplet of spermatozoa was proximal, distal or intermediately locatedalong the middle piece. In general the lumen of the duct was narrower and its epithe-lium higher than elsewhere. The stereocilia of the principal cells were long, the nucleioval and the cytoplasm was dominated by lipid droplets. The droplets were presentin all sections examined although there was considerable variation in lipid contentbetween sections and throughout the epithelium in the same section. The dropletswere much more frequent at site 6 (Fig. 19) than on either side of this site (Fig. 18).However, even at site 6 some sections showed a greater degree of aggregation of the

Structure of tde elephant genital ducts 379'4

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16Fig. 13. Transverse section of the initial segment of the ductus epididymidis. Note the homo-geneous supranuclear cytoplasm of the epithelium, the folds of the epithelium and the sparsityof spermatozoa in the lumen. Paraffin, H & E. x 80.Fig. 14. Longitudinal section of the terminal segment of the ductus epididymidis. Note the lowepithelium, long epithelial folds and dense concentration of spermatozoa in the lumen. Paraffini,H&E. x 70.Fig. 15. Transverse section of the middle segment of the ductus epididymidis. Note the highepithelium with inhomogeneous supranuclear cytoplasm, the absence of epithelial folds and thehigh concentration of spermatozoa in the lumen. Paraffin, H & E. x 110.Fig. 16. Transverse section of the ductus deferens showing the lumen almost obliterated by theinvaginations of the duct wall. Paraffin, H & E. x 40.

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Fig. 17. Longitudinal section of epithelium from site 5 in the initial segment of the ductusepididymidis. Note the round nuclei and long, thick stereocilia. The dense bodies occur morefrequently than in site 4 (Fig. 21). Araldite, Azur II. x 1000.Fig. 18. Longitudinal section of epithelium from site 7 (about the distal end of the middle seg-ment of the ductus epididymidis). Note the dominant Golgi apparatus and the presence of largelipid droplets. Araldite, Azur 1H. x 1000.Fig. 19. Longitudinal section of epithelium from site 6 (middle segment of ductus epididymidis).Note the numerous lipid droplets, the oval nuclei and the apical cell. Araldite, Azur II. x 1000.Fig. 20. Longitudinal section of epithelium from site 8 (terminal segmentofductusepididymidis).Note the large lipid droplets; other areas are dominated by regularly shaped dense bodies.Aralkite,Azurll. x 1000.

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Strutcture of the elephant genital ductsdroplets (mainly in the supranuclear cytoplasm) than is shown in Figure 19. Smallervacuoles were also present in the apical cytoplasm and some small densely stainedbodies were present (mainly) throughout the supranuclear cytoplasm. There were nodistinct folds present in the sections examined, but slight undulations of the ductepithelium were often present (Fig. 15). Basal, apical and 'halo' cells occurred morefrequently in this than in the other segments and the basal cells contained morecytoplasm than they did elsewhere in the duct.

Terl,iinal segmenit (Figs. 14, 20)The duct lumen was usually so packed with spermatozoa that, on macroscopic

examination, it appeared distended. In general, the spermatozoa were mature beforethey entered the terminal segment.

Histologically the duct was quite different to the more proximal epididymis; it waswider, the epithelium and stereocilia lower and the nuclei of the principal cells weremore elongated. The lumen was interrupted by folds which increased in length andfrequency in a proximodistal gradient along the duct.

There was some variation in structure between the anatomical isthmus and distal(tail) regions of the terminal segment. In the proximal part the duct was narrowerand was lined by a higher epithelium with longer stereocilia and fewer folds than inthe distal region. In the distal region spermatozoa appeared to be packed moredensely than in the proximal region of the terminal segment, and the epithelium wasvery low and formed numerous folds; the effect of this at site 8 was to increase thesurface area of epithelium facing the lumen by a factor of 1¢5-fold. The epithelialcytoplasm in the distal region contained several large lipid particles and a variablenumber of densely stained bodies. The latter were either small and round or largeelongate bodies half to two thirds the length of the cells. One of the most noticeablefeatures of the distal region was the gradual increase in thickness of the smoothmuscle that surrounded the duct (see above).

Ductus deferens (Figs. 10, 16, 23)The ductus deferens was anatomically distinct from the ductus epididymidis due

to its thicker and more solid wall and its looser convolutions. It was lined by avery low epithelium with short stereocilia. There were numerous spermatozoa in theduct lumen of animals in which large numbers of spermatozoa were found in thedistal segment of the epidkdymis, but there were few spermatozoa in the ductusdeferens of other animals. The folds were longer and more frequent than elsewherein the duct and obliterated much of the lumen; in appropriate thin sections therewas no more connective tissue between opposing epithelial basement membranesthan in the epididymis (Fig. 21). The folds increased the surface area of epitheliumfacing the lumen by a factor of about 5-fold. The cytoplasm of the principal cellscontained large lipid particles and dense bodies. The smooth muscle surroundingthe duct was arranged in circular, oblique and longitudinal layers and increased inthickness along the duct. The mean ± S.E. thickness at site 9 was 2-6 ± 0 3 mm.

DISCUSSION

Early workers referred to those parts of the genital ducts which are adjacent to thetestis as the epididymis and those posterior to the testis as the ductus deferens(Oudemans, 1892 and Weber, 1898: quoted by Glover, 1968). However, this classifi-

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Fig. 21. Part of a section of the initial segment of the ductus epididymidis at site 4 showing thevasculature of an epithelial fold and the relatively homogeneous supranuclear cytoplasm ofthe epithelium. Araldite, Azur II. x 400.Fig. 22. Longitudinal section of the epithelium lining the ductuli efferentes. Note the densebodies throughout the epithelium, the ciliated and stereociliated cells and halo cell (arrow).Araldite, Toluidine blue. x 1000.Fig. 23. Longitudinal section of the epithelium lining the ductus deferens showing the presenceof lipid and dense bodies in the cytoplasm. Araldite, Azur II. x 1000.

cation is misleading and uninformative for comparative purposes, particularly as theterm epididymis may include derivatives of the mesonephric tubules (ductuli efferen-tes) and mesonephric duct (ductus epididymidis). Consequently, we have distinguishedbetween the ductuli efferentes, ductus epididymidis and ductus deferens and haveadopted Glover's (1968) proposal that the cord formed by the ductus epididymidisand its stroma (i.e. 'epididymis') be recognized as an organ which extends beyondthe testis.The ductuli efferentes of the elephant are similar to those described in other

eutherian mammals in that they are numerous, relatively long, narrow ducts whichare arranged in parallel, are lined by a low columnar epithelium composed mainly ofciliated and stereociliated cells and contain very few spermatozoa (Benoit, 1926;

382

Structure of the elephant genital ductsLadman & Young, 1958; Ladman, 1967; Yokoyama & Chang, 1971; Wrobel, 1972;Ramos & Dym, 1977; Flickinger et al. 1978). The doubling in mean score for concen-tration of spermatozoa between sites 1 and 4 (Fig. 4) indicates that the ductuliefferentes are reabsorbing fluid as in the boar and bull (Crabo, 1965). However, thereseem to be no reports of pigmentation of the ductuli efferentes of mammals otherthan the elephant. The distribution of the pigmentation in the ducts indicates thatthere may be some variation in function along the length of a duct and betweenindividual ducts. Another unusual feature of the elephant ductuli efferentes is thepresence of an anastomosis between separate parts of a duct. However, we did notfind that the anastomoses were as extensive as described by Hanks (1977). Thedifference between these findings may be because we were particularly critical inaccepting evidence of an anastomosis. Our initial work with fresh material gavemisleading results. They were particularly misleading when we used Hank's methodof injecting India ink into the rete testis to demonstrate the anastomoses, since,during dissection of this material, the ink tended to break through the duct walls,falsely indicating that an anastomosis was present.These studies resolve the disagreement (see Introduction) about the extent to which

the elephant ductus epididymidis is structurally differentiated. The findings are inagreement with Hanks (1977) who recognized some structural differentiation at thetissue level of organization and it is suggested that Short et al. (1967) did not recog-nize this differentiation because their sampling sites were in the vicinity of site 6 anddistal to this location, where sperm concentration remains much the same, wherethere is a continuous decrease in epithelial height, and where there is a continualincrease in epithelial folding and duct and lumen diameter (see Figs. 2-7). The presentreport also shows that some structural differentiation of the duct may be recognizedat the cytological and anatomical levels of organization. The anatomical differentia-tion into thick cephalad and caudal ends of the epididymis and the distinctionbetween the ductus epididymidis and ductus deferens is in agreement with a reporton the Indian elephant (Schulte, 1937). We are also in agreement with the suggestionby Glover (1968) that the three anatomically distinct regions of the elephant epididy-mis are homologous with the head, body and tail of the epididymis of scrotalmammals. However, the distinction between the regions is more marked in mostscrotal mammals (e.g. mouse, rat, rabbit, guinea-pig, ram and bull) than in theelephant because in the former the thick ends of the epididymis are folded on thesuperior and inferior poles of the testis, and relative to their body size, they store alarger number of spermatozoa in the tail of the epididymis than does the elephant(Jones & Djakiew, 1978). It is noteworthy that there appears to be considerablevariation between individual elephants in the fluid output of the testes (Jones et al.1974) and in the number of spermatozoa present in the tail of the epididymis.The objective of the biometric approach to the histological study was to better

characterize the ducts in order to facilitate comparisons between different parts of thegenital ducts of the elephant (see below) and future comparisons between the elephantand other species; and to facilitate the interpretation of functional studies (Jones,1980). However, for the sake of clarity in presentation as well as facilitating com-parisons with some other reports, the ductus epididymidis has been classified into thethree major segments proposed by Glover & Nicander (1971). The initial segment inthe elephant is in accord with Benoit's (1926) original description, as a region wherethe duct is lined by a tall epithelium with long stereocilia and has a narrow lumencontaining few spermatozoa. The finding that the mean score for concentration of

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spermatozoa nearly doubled between sites 4 & 6 (Fig. 4) indicates that the initialsegment of the elephant epididymis is probably involved with considerable fluidreabsorption like the initial segment in the bull, boar and rat (Crabo, 1965; compareTuck, Setchell, Waites & Young, 1970, and Levine & Marsh, 197 1). The classificationof the terminal segment of the elephant ductus epididymidis is in accord with Glover& Nicander's (1971) structural and functional description, i.e. the duct is very wideand packed with spermatozoa and it is lined by an epithelium which is lower thanelsewhere and which has short slender stereocilia. The greater number of periductalmuscle layers in this segment, compared to more proximal parts of the duct, is alsoconsistent with the proposal (Glover & Nicander, 1971) that it is a region for storingspermatozoa available for ejaculation (Baumgarten, Holstein & Rosengren, 1971).The region designated the middle segment of the elephant ductus epididymidis is alsoin accord with Glover & Nicander's (1971) proposal that this is a region wherespermatozoa show signs of maturation and the lining epithelium contains supra-nuclear vacuoles in paraffin sections. However, until more information is available onthe structure and function of the epididymis of the elephant and other vertebrates,the possibility should be entertained that the elephant ductus epididymidis containsonly the two distinct segments (initial and terminal) which are histologically charac-terized by the proximodistal gradients on either side of site 6, Figure 1 (see Resultsand Figs. 2-7). This suggestion recognizes that the initial segment may not behomogeneous (Fawcett & Hoffer, 1979).The main difference between the histological structure of the ductus epididymidis

of the elephant and that of other mammals studied (see review by Hamilton, 1975) isthe extensive folding of the epithelium which occurs in the proximal and distal partsof the duct. If the folds are an adaptation serving to increase the surface area, orvolume, of epithelium facing the duct lumen, it may be assumed that their develop-ment probably has a selective advantage over increasing the magnitude of thesefactors by increasing the length of the duct. This, in turn, may indicate that theoverall length of the elephant ductus epididymidis is relatively shorter than inother mammals studied and, that, consequently, spermatozoa spend a shorter time intheir passage through the duct. This suggestion is supported by Bedford's (1978)recent demonstration that the duration of sperm transit is reduced in the experi-mentally cryptepididymal rabbit.

SUMMARY

The testes of the elephant are located medial to the kidneys and their excurrentducts are also located deep within the abdominal cavity. The ductuli efferentes testis(about 20 ducts) radiate from the superior pole of the testis; the most anterior ofthese ducts (about 6) successively join together, two at a time, to form a single ductwhich continues distally as the ductus epididymidis and receives the more caudalductuli efferentes at right angles. Each ductulus efferens is highly convoluted andanastomoses along the proximal one third of its length. The lining epithelium is alow pseudostratified columnar type composed of mainly ciliated and stereociliatedcells and some basal and 'halo' cells.The ductus epididymidis is lined by a pseudostratified, stereociliated, columnar

epithelium which is considerably folded towards each end of the duct. The epitheliumis composed mainly of stereociliated principal cells and some basal, apical and'halo' cells. The epididymis is structurally differentiated along its length. It is anato-

Structure of the elephant genital ductsmically differentiated into a thick, proximal head, which courses over the superiorpole of the testis, a thinner, distal tail (where the duct is swollen with spermatozoa),located towards the posterior end of the abdominal cavity, and an even thinneristhmus which courses under the dorsal wall of the abdominal cavity and joins thetwo thicker regions. It is suggested that these three regions correspond respectivelyto the head, tail and body of the epididymis of scrotal mammals. The duct has twohistologically distinct segments with characteristic proximodistal gradients. Theinitial segment has a narrow lumen containing few spermatozoa and it is lined byhigh epithelium with long stereocilia. The duct narrows distally and there is anincrease in epithelial height, length of stereocilia and concentration of spermatozoa.The terminal segment of the duct has a wide lumen and is packed with spermatozoa.It is lined by a low epithelium and is surrounded by a thick layer of smooth muscle.The duct widens and the p.riductal muscle thickens distally along its length. Thetransitional region between the two segments, the middle segment, is characterizedby very tall epithelium (with long stereocilia) containing numerous lipid droplets anda narrow lumen containing a dense concentration of spermatozoa; the cytoplasmicdroplet was located midway along the middle piece in a relatively high proportion ofthe spermatozoa.The ductus deferens is loosely coiled and has a thick muscular wall. The lining

epithelium is made up of principal (cuboidal) and basal cells and is extensively folded.

We are indebted to Professor J. D. Skinner, Mammal Research Institute, Univer-sity of Pretoria, for encouragement and generous support and hospitality, to thestaff of Kruger National Park for their generous help and hospitality, Mrs D. M.Rhodes and Ms K. Jurd for technical assistance, and to the Council for Scientific andIndustrial Research, South Africa, and the Australian Research Grants Com-mittee for financial support.

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Studies of the deferent ducts from the testis of the African elephant