Changes in the Male Reproductive Organs of the …dugong.id.au/publications/JournalPapers/1984/Marsh...

Aust. J. Zool., 1984, 32, 721-42

Changes in the Male Reproductive Organs of the Dugong, Dugong dugon (Sirenia : Dugondidae) with Age and Reproductive Activity

H. Marsh,* G. E. Heinsohn* and T. D. GloverB

A Zoology Department, James Cook University, Townsville, Qld. 481 1. * Department of Veterinary Anatomy, University of Queensland, St Lucia, Qld 4067.

Abstract

The anatomy and histology of the male reproductive tract of the dugong (Dugong dugon) is described. Each testis and its adjacent epididymis lie immediately caudal to the corresponding kidney. The seminal vesicles are large but there is no discrete prostate gland and the bulbo-urethral glands are also diffuse. Both qualitative and quantitative examination of the testes and epididymides of 59 males whose ages have been estimated from tusk dentinal growth layer counts indicate that the male dugong does not produce spermatozoa continuously, despite the absence of a distinct breeding season. Individual dugongs were observed with testes at all stages between complete quiescence and full spermatogenesis, and only 10 of the 40 mature males had fully spermatogenic testes and epididymides packed with spermatozoa. Androgenic and spermatogenic activity of the testes appeared to be in phase, but the testicular histology of some old males suggested that they may have been sterile for long periods.

Introduction

The dugong, Dugong dugon (Miiller), one of the four extant species of Sirenia or seacows, is of particular interest as the only existing herbivorous mammal which is strictly marine. Although the dugong is listed as vulnerable to extinction (Thornback and Jenkins 1982), aerial surveys over the last six years have confirmed that sizeable populations still occur in the shallow seas around northern Australia (Heinsohn et al. 1976, 1978; Ligon 1976; Marsh et al. 1981).

Since 1969, specimens which ideally included skulls and reproductive tracts have been collected from 160 north Queensland dugongs, in order to obtain information about the animal's life history that is basic to the development of effective conservation and management procedures. Marsh (1 980) counted tusk dentinal growth layers to determine the ages of these animals and presented strong evidence for annual layer deposition, indicating that dugongs have a life span of 50-60 years. Concomitant preliminary examination of the reproductive tracts suggested a minimum pre-reproductive period of 9-10 years for both sexes, and a cycle of male sexual activity in which not all animals are in rut simultaneously (Marsh 1980).

Subsequent qualitative and quantitative histological studies of the testes and epididymides of 59 dugongs of a wide range of ages have been performed with the aim of defining precisely the terminal reproductive state of each male. Results of a parallel study of 49 female reproductive tracts, and on breeding cycle, life history and population dynamics are published separately (Marsh et al. 1984a, 1984b).

Although Home (1820), Riha (1911), Hill (1945), Gohar (1957), Petit (1925), and Harrison (1969) have described various aspects of the gross reproductive anatomy of the male dugong, even the exact location of the testes (Harrison 1969) seems unclear and no previous histological records are known to us. Accordingly, this paper also includes details of the gross anatomy and histology of the male reproductive organs.

H. Marsh, G. E. Heinsohn and T. D. Glover

Materials and Methods

Material

Source. Male reproductive tracts were obtained between May 1969 and October 1981 from the following dugongs in northern Queensland: 3 1 animals that were accidentally drowned in nets (usually shark nets set for swimmer protection) near Townsville (1 9" SS., 146" 1 5'E.) had material collected from them within 2 h-2.5 days of death (Heinsohn 1972; Heinsohn and Spain 1974); 25 that were speared and drowned for food by Aborigines near Mornington I. (16"301S., 13Y30'E.) had material collected from them within 0.5-12 h of death (Marsh et al. 1981); and three that were similarly killed for food by Islanders in Torres Strait (10"30fS., 142'3'E.) were sampled within 0.5-12 h of death.

Description. Body-length frequencies of the dugongs from Townsville and Mornington I. are illustrated (Fig. 1) arranged in 0.2-m body-length classes. The ages were estimated from dentinal growth layers in the tusks (Marsh 1980), which erupt and wear in adult males, so that minimum age estimates only are available for these animals. The age-frequency distribution and tusk-eruption status of males from Townsville and Mornington I. are summarized in Fig. 2. Data on all males including the three from Torres Strait are shown in Tables 1, 3 and 4.

Fig. 1 12 r

Fig. 2

Body length im) Age (YI

Fig. 1. Body-length frequency diagrams (by 0.2-m classes) of male dugongs collected in 1969-81 from: (a) Mornington I.; (b) near Townsville. Open bars, tusks not erupted; stippled bars, tusks erupting; solid bars, tusks erupted and worn. Fig. 2. Frequency diagrams of 5-year age classes of male dugongs collected in 1969-81 from: (a) Mornington I.; (b) near Townsville. Open bars, tusks not erupted; stippled bars, tusks erupting; solid bars, tusks erupted and worn, age estimates a minimum only. Animals have been assigned to the age class which corresponds to the number of growth layer groups in their tusks, irrespective ofwhether the tusks are worn.

Field collection procedures. Each carcass was measured and dissected, attention being focussed on the collection of the stomach contents, skull and reproductive tract. One-centimetre cubes were removed from the centre ofthe right testis and from the right epididymis opposite the caudal pole of the testis. These samples were fixed in Cleland's solution (Rowley and Heller 1966) for 24 h. After incision of each testis, the entire reproductive tract was preserved by injection with, and immersion in, 10% seawater formalin.

The reproductive tracts of six freshly killed adults were examined in more detail by excising samples from the efferent ductules, epididymis and ductus deferens at several levels. Other accessory organs were also sampled. The samples were fixed in Bouin's fluid for 24 h. Before the epididymis was fixed, spermatozoa were collected from an arbitrarily chosen level of the mesonephric duct (opposite the caudal pole ofthe testis) by smearing the cut surface across several slides and allowing them to dry in the air. One testis and the corresponding testicular artery from an adult were removed and frozen.

Dugong Male Reproductive Organs

Measurement, Histological Preparation and Examination of Reproductive Specimens

Each fixed testis was trimmed of epididymis and fat and weighed to 0.1 g. The specimen of testis that was fixed in Cleland's solution was included in the weighing. Testis length, width at midlength, and dorso-ventral thickness at midlength were measured with vernier calipers. In order to check that all regions ofboth testes from each dugong were at the same stage of reproductive activity, three extra tissue blocks were taken from each testis along the longitudinal midline and two from along the transverse midline. These specimens were post-fixed in Bouin's fixative (Lillie and Fullmer 1976, p. 61) for 24 h before subsequent processing.

Tissues for histology were dehydrated in ethanol, cleared in xylene, embedded in Paraplast and sectioned at 5 ym or 6 ym. Sections were stained with Mayer's haemalum and Young's eosin-erythrosin (Hand E) (Marsh et al. 1977), Masson's trichrome, or the PAS procedure and Mayer's haemalum (Culling 1974). Some sperm smears were stained with Mayer's haemalum; others were coated with gold-palladium and examined with the scanning electron microscope.

After being thawed, the testicular artery of the frozen testis was injected with latex. The testis was subsequently cleared with salicylate for examination of the arterial architecture. The blood vessels were then examined.

Region of efferent ductules Testis Middle level of epididymis

Lower level of epididymis

(sperm store)

Ductus deferens

Seminal vesicle

Disseminated bulbo-urethral glands Pen~s (cut o f f )

Fig. 3. Schematic diagram of the male reproductive tract of the dugong.

Each slide was scanned qualitatively at xlOO and x400 in order to assess overall spermatogenic activity. The diameters of 100 round seminiferous tubules, randomly selected from all samples from each testis, were measured with a calibrated ocular micrometer at x400 magnification. In retrospect, this sample was unnecessarily big, because the standard deviation per testis was not very large (see Tables 1, 3,4). Subsequent quantitative histological studies were limited to well preserved material. The cell types present were identified under oil immersion at x1000. The sections that were used for cell identification included those stained with PAS, trichrome, and H and E. At least 10 cells of each type were measured by an ocular micrometer at x1000.

Quantitative information on testicular activity was obtained by use of phase analysis. This analysis was based on a series of phases, each of which shows a certain cellular association of the seminiferous epithelium. A series of 12 phases was recognized with this procedure, which differed slightly from standard methods developed for mammals which undergo continuous spermatogenesis (Courot et a!. 1970).

Since no qualitative differences were observed in the histological appearance of the seminiferous tubules in different regions of one testis or between contralateral testes, the phase analysis was performed on one H-and-E-stained section from the Cleland's fixed sample from each dugong. (The fixation of these samples was superior to the others.) The phase in each of 200 transverse sections of tubules was identified at a magnification of x400. This sample size was comparable to those used for


Fig. 4. (A) Ventral view of the left kidney and testis of an adult dugong in situ. The testis is 9 cm long. t , Testis; k, kidney. (B) Ventral view of a dissection of part of the left side of the reproductive tract of a 10-year-old dugong with fully spermatogenic testes. The testis is 11 cm long. The lower epididymis has been displaced to the right. e, epididymis; s, seminal vesicle. (C) Photomicrograph of seminal vesicle from a dugong aged at more than 28 years, with fully spermatogenic testes. The pseudostratified epithelium consists of numerous cuboidal principal cells and a few round basal cells. H and E. (D) Photomicrograph of cross-section of bulbo-urethral gland of the same dugong as Fig. 4C. Note the columnar cells with basal nuclei. PAS and Mayer's haemalum. (E) Scanning electron micrograph of spermatozoa from the lower level of the epididymis of a dugong aged at least 36 years.


similar analyses of the testes of the African elephant (Johnson and Buss 1967) and the albino rat (Roosen-Runge and Giesel 1950).

Within the lobules of each testis, the volume fractions of the tubular and intertubular components (Leydig cells, other cells, space) were estimated by use of a Weibel graticule (Wild Heerbrugg Ltd) and the principles of stereology (Schaefer 1970). This technique involved recording the elements appearing at 42 points (the ends of each of 21 lines of equal length arranged in 7 equally spaced rows) in each of a series of random fields at x400. Slides from three dugongs (one immature and two mature animals) were sampled at 10, 15 and 20 fields at x400 in order to determine an appropriate sample size. The testis of one of the two mature dugongs was producing spermatozoa but the seminiferous tubules of the other contained spermatogonia and Sertoli cells only. On the basis of results calculated at each of the above levels, a sample of 10 fields from one section of Cleland's fixed tissue per testis was selected. Counts beyond this level produced only slight variations.

Observations

General Anatomy Fig. 3 summarizes diagrammatically the principal anatomical features of the male

reproductive tract.

, Testis

Large epididymal arteries

The testes are dorso-ventrally flattened and ovoid. They are abdominal, each lying caudal and slightly lateral to the corresponding kidney (Fig. 4A). Inguinal canals and inguinal ligaments are absent. Each testis is encased in a cream-coloured fibrous tunica albuginea from which connective tissue septa converge on the mediastinum, dividing the testis internally into distinct lobules which are easily recognizable macroscopically. The dugong has a straight testicular artery (Fig. 5) which divides into three or four branches before entering the substance of the testis. The absence of a pampiniform plexus, as we11 as the lack of a helical testicular artery, characterize the testis as that of a typical testicond mammal.

At least 14 ductuli efferentes emerge from the cranial pole of each testis to join the proximal region of the mesonephric (Wolffian) duct. Adjacent to the dorsolateral side of each testis, the mesonephric duct is highly convoluted and forms a distinct epididymis (Fig. 4B) which extends several centimetres caudally and medially beyond the testis before gradually straightening out into the ductus deferens.

Large, thick-walled, ovoid, seminal vesicles (Figs 3,4B, 4C) lie dorsal to the bladder. The tissue surrounding the prostatic urethra consists mainly of bundles of smooth muscle and erectile tissue with limited, poorly developed, urethral mucous glands. We have found no discrete structure corresponding to a pars compactum of a prostate gland, so prostatic tissue in dugongs is likely to be disseminated along the wall of the pelvic urethra. Bulbo-urethral glands are reported to be absent in sirenians (Riha 191 1; Harrison 1969) and we have not been able to identify distinct glands macroscopically. Histologically, however, bulbo-urethral type glandular tissue (Fig. 40) was identified dispersed among the muscle


surrounding the urethra close to the root of the penis, in a dugong whose testes were producing spermatozoa. Because of the limited amount of adequately fixed tissue available, we were not able to determine how this bulbo-urethral type tissue changed with the level of testicular activity.

The penis (Fig. 6) is not visible externally unless erected, when it is extruded through the preputial orifice about halfway between the anus and umbilicus. On morphological grounds the penis should be classified as vascular, meaning that erection is probably achieved entirely by vascular engorgement. The penile blood vessels are extensive and the corpus cavernosum penis substantial. At the root of the penis the corpus cavernosum is undivided but in most of the shaft it is divided by a septum. Near to the apex of the penis, the corpus cavernosum penis extends upward and laterally to fill two lateral and ventral bulges that form a glans penis or corona glandis. Beyond this expansion, the urethra, surrounded at first by both corpus cavernosum erectile tissue and corpus spongiosum, extends beyond this corona to form an erectile urethral process. Similar bulbs of erectile tissue occur in other eutherian species and have been seen in the penis of the nine-banded armadillo Dasypus novemcinctus (personal observation). The anatomy of the penis and its muscles has been described in detail by Riha (191 1) and Petit (1925). Harrison (1969) includes photographs of the glans.

Fig. 6. Ventral view of the 20-cm-long penis of the same dugong as in Fig. 4B.

Immature Dugongs

(i) The seminiferous tubules The seminiferous tubules of immature dugongs are narrow and range in mean diameter

from about 36 to 66 pm (Table 1; Fig. 7). The basement membrane of each tubule is lined with numerous spermatogonia and Sertoli cell nuclei (Fig. 8A). Variable numbers ofprimary spermatocytes (Table 1) are sometimes present in the centre of the tubules but spermatids and spermatozoa are absent. Lumina are only clearly apparent in the seminiferous tubules of some adult animals.

The Sertoli cell nuclei (Fig. 8A) are oval, up to 10.5 pm long, and usually aligned with their long axes perpendicular to the basement membrane. Each nucleus has a typical and conspicuous nucleolus, evenly distributed chromatin, and a distinct nuclear membrane of irregular outline. The Sertoli cell cytoplasm is characteristically indistiinct.

Two types of spermatogonia are present. Type A spermatogonia (sensu Bloom and Fawcett 1975) (Fig. 8A) each have a round to oval nucleus up to 10.5 pm in diameter with fine, evenly distributed chromatin granules, one or two distinct nucleoli and a distinct membrane. The cytoplasm stains faintly so that the cell borders may be difficult to see. Type B spermatogonia tend to be slightly smaller than type A, the nucleus of these cells is about 7.5 pm in diameter and has coarser and more abundant chromatin than those of type A.


Table 1. Age, body length, mean formalin-fixed testis weight and details of the seminiferous tubules of dugongs with immature testes

Values for tubule diameters are means t standard deviations. NA, not available

Accession Body Age Mean testis Tubule Tubules with number length (m) (y) weight (g) diameter (pm) spermatocytes (Yo)

Townsville 2 5.1 N A

6.5 12.5 14.4 N A

N A

11.6 N A

15.5 t l l

Mornington I 7.8

15.8 16.5 K A

16.2

Torres Strait 9.1

Mean tubule diameter (gm)

Fig. 7. Relationship between the seminiferous tubule diameter, testis weight and stage of testicular development or activity. .Immature. approaching puberty. @Mature: resting (phase - 3 predominant). A Mature: intermediate (phase -2 predominant). o Mature: recrudescent (phases - 1,0, or 1-5 predominant, few or no spermatozoa in epididymis). o Mature: fully spermatogenic (phases 1-5, many spermatozoa in epididymis). rn Mature: spermiogenesis finishing (phase 6 predominant).


Fig. 8. (A) Photomicrograph of immature testicular tissue from a 5.5-year-old dugong showing narrow closed seminiferous tubules, the peripheries of which are lined with spermatogonia (arrowed) and numerous Sertoli cell nuclei (arrowhead). Sertoli cell nuclei, spermatogonia, 'and occasional spermatocytes may also occur in the centre of the tubules. Leydig cells (L) are prominent and vacuolated. (B)-(F) The five cellular associations observed in the seminiferous tubules of mature dugongs with testes in a state of arrested or incomplete spermatogenesis. Phase -4 is the least active phase; phase 0 the most. All material obtained within 0.5 h of death from dugongs with erupted tusks and estimated to be older than 20 years. (B) Phase -4. Sertoli cell nuclei (arrowhead) and types A and B spermatogonia (arrowed) line the periphery of solid tubules which have connective tissue fibres; occcasional Sertoli cell nuclei and vacuolated Sertoli cell cytoplasm in the central area. The lamina propria surrounding the basement membrane is about 5 pm thick. Note the interstitial fibrosis and the vacuolated Leydig cells (L). (Q Phase - 3. Like phase - 4 but with low numbers of spermatocytes and more Sertoli cell nuclei in the central area of the tubules. Note the intratubular pigment deposits (arrowed) and type A spermatogonium (arrowhead). This phase was also observed in the testes of No. 112, which was considered to be approaching puberty. (D) Phase - 2. Like phases -4 and - 3 but with numerous primary spermatocytes (arrowed) in tubules which may be open or closed. This phase was also observed in the testes of No. 1 12, which was considered to be approaching puberty. (E) Phase - 1. Primary (arrowed) and secondary (arrowheads) spermatocytes present. Cellular associations organised. Lumen usually present. Leydig cells (L) hypertrophied in this animal. (F) Phase 0. Like phase - 1 but with a few spermatids (arrowed) or spermatozoa present. All sections stained with H and E.


The primary spermatocytes each have a round nucleus about 8.5-10.5 pm in diameter, in which the chromatin configurations typical of pachytene spermatocytes are clearly visible. The nuclear membrane is poorly defined, but the cytoplasm is conspicuous.

(ii) The intertubular tissue Intertubular tissue (Fig. 8A) is prominent and abundant. The Leydig cells are polyhedral

and each has an eccentric round or oval nucleus with one or two centrally placed nucleoli, granular chromatin, and a prominent nuclear membrane. The cell boundary is often indistinct. Most cells contain a large cytoplasmic vacuole (Fig. 8A); however, delayed fixation must be taken into account in interpreting this feature.

Leydig cells, about 5 pm in nuclear diameter and with cytoplasmic vacuoles about 6.5 pm in diameter, were visible in sections from the youngest dugong from which well fixed testicular tissue was available (No. 150, estimated age 1.5 years), even though much of its other intertubular tissue was undifferentiated. The Leydig cells of the other two immature animals examined in detail (No. 103, 5.5 years; No. 117, 15.5 years) were slightly larger, with nuclei averaging 6.8 pm and 6.3 pm in diameter and cytoplasmic vacuoles averaging 9.5 pm and 7 Fm in diameter, respectively. Of the lobular tissue, 27% in No. 103 and about 19% in No. 117 consisted of Leydig cells (Table 2).

Table 2. Details of the relative volumes of intertubular and tubular tissue in the interlobular area of the testes of dugongs

Dugongs are ranked in ascending order of reproductive maturity and activity as determined by testicular histology. The phase of spermiogenesis follows the reproducrive category; for interpretation, see text

Reproductive Acc. Reproductive category and Tubule Inter-tubule volume (Yo) activity No. predominant phase volume Space Leydig Other ranking (yo) cells

Immature Immature Approaching puberty (-2, - Regressed (- 4) Resting (-3) Resting (- 3) Intermediate (- 2) Intermediate (-2) Intermediate (-2) Recrudescent (- 1) Recrudescent (- 1, 0, 1) Recrudescent (1) Recrudescent (1-4) Fully spermatogenic (1-5) Fully spermatogenic (1-5) Spermiogenesis finishing (6)

(iii) Testis size If males from Townsville and Mornington I. are considered separately, our results

indicate that the size of the testes of immature dugongs tends to increase with increasing age (Fig. 9) and body length (Table 1). Single testis weights of the 18 immature dugongs examined ranged from 2 g in an animal of body length 1.28 m, to 16.5 g in one of unknown body length; testis lengths ranged from 2.5 to 4.8 cm in the same animals. The body lengths of these immature dugongs ranged from 1.28 to 2.49 m and their ages from 0 to 6 years (Townsville population, from which specimens aged between 6.5 and 8.5 years inclusive were unavailable) and up to 15.5 years (Mornington I. population) (Table 1).

(iv) Tusk eruption None of the immature dugongs examined had erupted tusks.


(v) Criteria for classifying a male dugong as immature A dugong was classified as immature if it had a single testis weight of less than 20 g and its

testicular histology was similar to that shown in Fig. 8A.

Adult Dugongs and those Approaching Puberty

Forty-one dugongs from 9 to more than 36 years old had testes which were larger than, and histologically distinct from, those of the immature animals, as can be seen by comparing Figs 8B-8F and Fig. 10 with Fig. 8A. Spermiogenesis was occurring in the testes of only 16 dugongs. These animals spanned the entire adult age range and included four whose tusks had not yet erupted (Nos 4, 38, 146, 156; Table 3). This suggests that the tusks erupt after puberty (first spermiogenesis). It was not possible to use testicular histology alone to distinguish males approaching puberty from those whose testes were recrudescing after a period of quiescience. This distinction was made more difficult by the variation in the age of sexual maturity and the absence of a sharply defined breeding season (see Marsh et al. 1984b).

(i) Cell types present in the seminiferous tubules Spermatogonia. Types A and B spermatogonia, similar to those in immature testes, lie in

typical fashion adjacent to the basement membrane (Figs 8B, 8C).

Fig. 9. Relationship between testis weight and age for immature dugongs from Townsville (0) and Mornington I . (m).

Spermatocytes. Mature primary spermatocytes (Fig. 8 0 ) are similar to those in immature testes, pachytene forms being especially conspicuous. Secondary spermatocytes (Fig. 8E) are relatively difficult to find, as is usual in mammalian testes where equational divisions are completed rapidly. The occasional secondary spermatocyte that is seen has a round nucleus about 7 in diameter (intermediate in size between a primary spermatocyte and a round spermatid) with a distinct membrane. The cytoplasm is conspicuous.

Spermatids. Early spermatids (Fig. 10A) each have a spherical nucleus about 5 ym in diameter surrounded by an easily discernible membrane and distinct cytoplasm, but as spermateliosis commences the nucleus elongates and its affinity for haemotoxylin increases (Fig. 10B). The cytoplasm is progressively reduced (Fig. 10C) and a distinct tail becomes visible (Fig. 10E). Remnants of spermatid cytoplasm are to be seen as residual bodies (Fig. 1OD).

Spermatozoa. The spermatozoon of the dugong (Fig. 4-5) has a flattened head about 5 ym long and approximately 3 ym wide, which stains lightly with haematoxylin. The tail is about 60 pm long. Testicular spermatozoa retain the cytoplasmic droplet proximally in the region

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of the neck of the cell but, as yet, the level of the epididymis at which the droplet migrates to the end of the midpiece has not been determined.

Fig. 10. Photomicrographs of cross-sections of several seminiferous tubules from a dugong (minimum age 36 years) with fully spermatogenic testes. The diameter ofindividual tubules is larger and the tubule lamina propria thinner (c. 2 l m thick) than in Fig. 7. The intertubular connective tissue is reduced; the Leydig cells are large and vacuolated. (A) Phase 1. Sertoli cell nuclei and spermatogonia line basement membrane. Primary and secondary spermatocytes organised into layers. Lumen surrounded by round spermatids. (B) Phase 2. Spermatids elongating and becoming associated with Sertoli cells. (C) Phase 3. Spermatid bundles well organized. Maturation divisions occurring among primary and secondary spermatocytes. This stage does not usually occupy whole tubule cross-section. (D) Phase 4. Two generations of spermatids present. Elongate spermatids have tails and are more central to lumen. (E ) Phase 5. Sperm line tubule lumen. Round spermatids also present. (F) Phase 6. Spent tubule at the end of period of sexual activity. Lumen of tubule very wide and surrounded by an incomplete assemblage of cell layers which may contain a subset of any of the cell types. All sections stained with H and E. Material fixed within 0.5 h of death.

Sertoli cells. Sertoli cells (Figs 8B, 8C) are morphologically similar to those in immature dugongs but they occur less frequently.

Conspicuous deposits of light brown pigment (possibly lipofuscin) (Fig. 8C) occurred in the seminiferous tubules of all dugongs more than 20 years old. Small amounts were similarly present in No. 154 which was 15 years old.


(ii) The phase analysis Eleven phases of activity were recognized in the seminiferous epithelia of adult dugongs.

These phases are illustrated in Figs 8B-8F and 10A-1 OF, and are described in the legends to the plates. Phases 1-5 (Fig. 10) are characteristic of active spermiogenesis and represent the orderly sequence ofevents that occurs during the transformation of round spermatids (phase 1) into mature spermatozoa (phase 5). They correspond to phases 9 and 5-8 used by Johnson and Buss (1967) for African elephant testes, and represent a simplified version of the more elaborate phase series developed for animals which undergo continuous spermatogenesis (Courot et al. 1970).

Some dugongs with testes in which spermatogenesis was active had epididymides which were relatively devoid of spermatozoa. These animals, i.e. Nos 1 16, 140 and 2 1 1 (Table 4), had a low proportion of tubules at phases 4 and 5 combined, in contrast to dugongs at the height of reproductive activity with large numbers of spermatozoa in their epididymides, e.g. Nos 38 and 146 (Table 3), and Nos 109, 210, 213 and 214 (Table 4). This observation indicates that spermiogenesis was just starting in Nos 1 16, 140 and 21 1, a conclusion supported by the observation that a majority of the tubules ofNo. 1 16 were still at phases - 3 to 0 inclusive (Table 4). These phases are characteristic of dugongs with testes in which spermatogenesis is arrested or incomplete (see below and Table 2). However, even though No. 104 (Table 4) had few spermatozoa in its epididymis, it had a high proportion (45%) of tubules at phases 4 and 5.

We consider tubules at phase 6 (Fig. 10F) which were only observed in Nos 1 14 and 214 (Table 4) to be characteristic of spent testes towards the end of a period of production of spermatozoa. No. 2 14, which still had epididymides packed with spermatozoa, had only 4% of tubules at phase 6. In contrast, No. 114, which had 68.5% of tubules at phase 6, had few tubules at any of the other phases and only moderate numbers of spermatozoa in its epididymides.

Adult dugongs in which spermatogenesis is arrested or incomplete have tubules in the phase range from - 4 to 0. Phase - 4 (Fig. 8B) represents extreme inactivity and only three animals (No. 68, minimum age 31.5 years; No. 80, minimum age 24.5 years; No. 127, minimum age 33 years) had at least 50% of tubules at this stage. The shapes of the pulp cavities of the tusks of these animals suggest that they were all considerably older than the minimum estimated age. They may have been aspermatogenic for long periods or even permanently. The testes of 13 dugongs had 50% or more of their tubules at phase - 3 (Fig. 8C), which suggests that this is the usual resting phase. Phase -2 (Fig. 80) is an intermediate phase and was the predominant (>50°/o of tubules) phase in the testes of eight dugongs. We consider that phases - 1 and 0 (Figs 8E and 8 F respectively) are part of the sequence of increased activity that precedes spermiogenesis, and that animals with a large proportion (>50%) of tubules at one or more of these phases, e.g. Nos 39,83, 179 (Table 3) and No. 116 (Table 4), were recrudescing, i.e. in the process of becoming fully spermatogenic.

No. 112, aged 14.5 years from Mornington I., had half its seminiferous tubules at phase -3, the remainder at phase -2, and a mean single testis weight of 24.6 g, about halfway between that of the heaviest immature testis (16.5 g) and the lightest of the other post-immature testes which were not producing spermatids or spermatozoa (the weights of which ranged from 35 to 96 g; see below). We considered that No. 112 was probably approaching puberty.

(iii) The intertubular tissue As in other mammals, dugong testes showing active spermatogenesis contain more fluid

than resting, intermediate or recrudescent testes and are thus more difficult to fix. The relative volume of testicular intertubular tissue also tends to decrease with increasing spermatogenic activity, as a result of the concomitant increase in tubule diameter (Table 2). In comparison with that in dugongs with resting, intermediate or recrudescent testes (Fig. 8),


the intertubular connective tissue of fully spermatogenic dugongs appears conspicuously reduced (Fig. 10). However, when the relative volume of Leydig cells in testes at various stages of activity (Table 2) was multiplied by the corresponding testis weight to give an estimate of total Leydig cell weight per dugong, it showed that the total Leydig cell weight actually tends to increase with increasing testicular activity (Fig. 11). This has also been regularly observed in the testes of other eutherians.

This result (Fig. 11) suggests that there is an increase in androgenic activity in animals approaching puberty, and that androgenic activity in adult dugongs is synchronous with the activity of the seminiferous epithelium. The relationship between the spermatogenic cycle in the testes and blood androgen levels clearly needs to be analysed.

(iv) Testis size The mean testis weight ofa dugong which is approaching puberty or is mature, reflects the

mean testis tubule diameter (Fig. 7) and depends on the animal's age and reproductive stage; testis weight usually increasing with increased activity. We observed little size difference between contralateral testes. In general, the size of the testes of dugongs older than 20 years was greater than that of younger dugongs at the same phase (Table 5). Among dugongs less than 20 years old, the testes of animals with unerupted tusks were usually lighter than those, at the same phase, from animals with erupted tusks (Table 5). The single testis weights of dugongs with testes which were producing spermatozoa ranged from 82.7 to 332 g; testis

Reproductive activity ranking

- 30 - - - 8 m

X m - 9 - E

I 10

lengths from 8.7 to 12.5 cm. Three young animals-Nos 36, 38 and 156 (Table 3)-all of which had epididymides packed with spermatozoa, had single testis weights ranging from 82.7 to 105 g, appreciably less than those ofthe six other dugongs at the same phase, whose testis weights ranged from 153 to 332 g. The tusks ofNo. 38 (9 years) and 156 (10.5 years) had not erupted; those of No. 36 (12 years) were erupting. In contrast, the testis of another young dugong with unerupted tusks (No. 146, 10.5 years) weighed 225 g at the same phase of activity, a weight comparable with that of some of the older dugongs (Nos 2, 109,2 13) at the same phase.

r . . . . . Fig. 11. Relationship between reproductive activity ranking of male dugongs (see Table 2) . a and an estimate of the weight of Leydig cells in -

a . their testes calculated from the following . a formula: volumetric fraction of Leydig cells , (O/o) X mean testis weight X 2.

(v) Epididymis and ductus deferens

0 2 4 6 8 10

We did not attempt a detailed anatomical study of these organs. However, examination of the sections of tissue taken at several standardized positions from three mature animals, each of which were sampled within '/2 h of death, indicates, not unexpectedly, that the histology of the epididymis changes with the level of testicular activity and generally seems to conform to descriptions of that in other species (see Nicander 1957a, 19573; Glover and Nicander 197 1 ; Nicander and Glover 1973).

Nos 2 10 and 2 13 (Figs 12A,128,120), both of which were at the height of spermatogenic activity, had similar epididymal histology. The density and distribution of spermatozoa were also similar. Near the cranial pole of the testis, the efferent ductules (Fig. 12A) and the initial segment of the epididymis contained a low concentration of spermatozoa. In this


region, the lumen of the epididymis was lined with a pseudostratified epithelium composed of principal and basal cells. The principal cells were columnar and 25-65 ym high, with round basal nuclei about 6 ym in diameter, and basophilic cytoplasm. These cells bore stereocilia up to 16 ym long. The distribution and occurrence of basal cells was typically irregular and occasional; the nuclei of these cells were about 8 ym long. The epithelium and adjacent highly vascular lamina propria were extensively folded into finger-like projections up to 0.5 mm long, causing the outline of the lumen to be irregular.

These projections became more numerous and elaborate in more distal parts of the epididymis, reaching their maximum development in the region just above the caudal pole of the testis (Fig. 12B), where the height of the principal cells ranged up to 80 ym and their oval nuclei were up to 16 ym long.

Table 5. Summary of details of testes weights of adult dugongs according to stage of testicular activity

Values of testes weights are means, with ranges in italics; sample sizes in square brackets. No. 68, which had 50% of tubules each at phase -3 and -4, is classed as -4; No. 112, with 50% each at phase -2 and -3, is classed

as -3

Reproductive Predominant Formalin-fixed testis weight (g) category phase in Dugongs t 2 0 y old Dugongs >20 y,

testes Unerupted tusks Erupting or erupted tusks erupted tusks

Regressed -4

Resting - 3

Intermediate -2

Recrudescent - I -1, 0, 1

Fully spermatogenic 1-5c

Spermiogenesis 6 finishing

AClassified as approaching puberty. BFew or no sperm in epididymis. CMany sperm in epididymis.

The epididymis gradually became less convoluted near the caudal pole of the testis. In this region the epididymal lumen was about 1.6 mm in diameter. This appeared to be the main sperm store (Fig. 120). We believe that this level of the duct is both homologous and analogous to the terminal segment of the epididymis (Glover and Nicander 1971). The ductus deferens, which was also fairly well filled with spermatozoa, was entirely straight. The mucosal folding persisted in the terminal segment and ductus deferens but was less elaborate. The principal cells were lower, only 16-25 pm high, and, they had shorter nuclei (6-12 pm high). Basal cells in the ductus deferens were still abundant.

The surrounding muscle layers exhibited a proximodistal increase in thickness throughout the epididymis. Initially, the contractile cells were very slender and the bundles they formed were mostly oriented circumferentially. Near the sperm store the layer of circular fibres was surrounded by a layer of longitudinal and oblique fibres. The ductus deferens had very thick muscular walls.

The epididymides of No. 145 (Fig. 12C), a mature dugong with resting testes (majority of tubules at phase - 3), were devoid of spermatozoa and differed histologically from those of the others in the following aspects: (I) the principal cells were much shorter and lacked most


of their supranuclear cytoplasm, and were also devoid of stereocilia, which is not typical for eutherians; (2) the epididymal lumen was narrower; (3) the surrounding muscle layer(s) were thicker.

Fig. 12. (A) Photomicrograph of a transverse section of one of the efferent ductules near the cranial pole of the testis of a dugong (minimum age 28 years) with fully spermatogenic testes. H and E. (B) An epithelial fold extending into the lumen of the epididymis of the same animal as Fig. 12 A just above the caudal pole of the testis. Note the stratified epithelium with elongate ciliated principal cells, the round basal cell nuclei and the lumen packed with spermatozoa. H and E. (C) Cross-section of the epididymis just above the caudal pole of the testis of an adult dugong (minimum age 27.5 years) with resting testes (most of the seminiferous tubules at phase - 3). Compare with B and note the complete absence of spermatozoa and the reduced supranuclear cytoplasm of the principal cells. H and E. (D) Part of the main sperm store near the caudal pole of the testis. This specimen is from the same animal as Figs 12A and 12B.

No spermatozoa were observed in the epididymides of any of the 24 adult dugongs whose testes were not producing spermatids or spermatozoa. Few or no spermatozoa were observed in the epididymides of five dugongs with active testes, at least three of which showed only the early stages of spermateliosis, i.e. spermatozoa were not observed but various stages of spermatid development were evident. Moderate amounts of spermatozoa were observed in the epididymides of one dugong whose testes were at the end of a period of spermiogenesis, and large quantities of spermatozoa were observed in the epididymides of only 10 dugongs, all of which were at the height of spermiogenesis.

(vi) Criteria for classifying a male dugong as approaching puberty One dugong was classified as approaching puberty. Its single testis weight was less than

30 g, its testis was similar histologically to Figs 8C and 80 , and spermatozoa were absent from its epididymides.


(vii) Criteria for classifying a male dugong as mature A dugong was classified as mature if its single testis weight was 30 g or more and its testis

was similar histologically to Figs 8B-8Fandlor Fig. 10, irrespective ofwhether spermatozoa were present in its epididymides.

Discussion

Activity of the Testis and Epididymis

Gross and histological studies of the testes and epididymides of 59 dugongs from north Queensland indicate that adult male dugongs are not always fertile. However, there is no definite correlation between testicular activity and season (Marsh et al. 19843) and both spermatogenically active and inactive animals may be found simultaneously in the same population.

The testes of a large proportion (approximately 60%) of the 41 dugongs that were either approaching puberty or fully adult showed arrested or incomplete spermatogenesis. These animals spanned the entire range of the ages and sizes of adult dugongs and included 18 individuals with erupted and worn tusks. (The tusks of male dugongs begin to erupt after puberty (Table 3; Marsh 1980; Marsh et al. 1984b). We therefore consider it highly unlikely that all these dugongs were merely approaching puberty, although No. 112 was classified as being at that stage on the basis of its testis weight. At least some mature male dugongs are thus not in continuous breeding condition in the wild. Some old males have unusually regressed testes (most tubules at phase -4). These animals are probably sterile for long periods or even permanently.

The reproductive condition of many of the pubertal and adult dugongs is intermediate between complete testicular quiescence (phases -4 and -3) with no epididymal spermatozoa, and full spermatogenesis (phases 1-5) with large numbers of epididymal spermatozoa (see Tables 3,4; Figs 8, 10). The androgenic and spermatogenic activities of the testis appear to be in phase (Fig. 11) and only animals with fully spermatogenic testes have large numbers of spermatozoa in the epididymis.

We are unable to consider the relationship between male reproductive activity and social status, as no pertinent information on dugong social behaviour is available except for reports by Australian Aborigines of the presence of 'whistler' dugongs (e.g. Roughsey 1972) which are considered to be dominant males. If male reproductive activity is dependent on social factors, the presence of a significant proportion (about 20%) of mature males with testes entering or leaving the fully spermatogenic state suggests that social status is very fluid.

The breeding pattern of the male dugong in relation to that of the female is discussed in Marsh et al. (1984b).

Comparison of the Breeding Pattern of the Male Dugong with that of Other Paenungulates

Eye-lens protein analyses have confirmed the monophyletic origin of the paenungulate orders Hyracoidea, Sirenia and Proboscidea, and place the paenungulates as the second offshoot from the main eutherian line after the endentates (de Jong and Zweers 1980). Male African elephants show evidence of a seasonal cycle in the mean diameter of their seminiferous tubules (Laws 1969) but, in contrast to the dugong, adults with arrested or incomplete spermatogenesis have not been reported. However, the male breeding patterns of the different genera of hyrax (e.g. Procavia and Heterohyrax) have many similarities to that of the dugong, especially in the histological changes in the testis and epididymis (Glover and Sale 1968; Millar and Glover 1970). Although away from the equator hyrax reproduction is markedly seasonal, in the tropics many species do not have a well defined breeding season, and observations on behaviour of rock species indicate that, as in the north Queensland dugong (Marsh et al. 1984b), the periods of activity of individual males are not


coordinated (Glover and Sale 1968). Sometimes there are seasons of widespread sexual activity in a population; conversely, there may be periods of very little sexual activity. There is also evidence that some male rock hyraxes remain reproductively quiescent for indefinitely long periods or even permanently (Glover and Sale 1968).

The Dugong as a Testicond Mammal

The dugong's testes are permanently situated in the abdominal cavity immediately caudal to the kidneys. In this respect they are similar to the testes ofthe other paenungulates, the elephant (Short et al. 1967) and the hyrax (Glover and Sale 1968). Also like the other testicond mammals the dugong has a straight testicular artery. However, the structure of the dugong epididymis suggests the beginnings of some caudal migration. The terminal segment of the epididymis or conventional sperm store, appears, for example, to be situated just caudal to the distal pole of the testis and is thus unlike the corresponding structures in the hyrax and the elephant which (although they exhibit important anatomical differences) are both remotely situated from the testes in, or near to, the pelvic cavity.

The presumed advantages of testicular migration, which occurs in most eutherian mammals, have been the subject of much discussion (e.g. Bedford 1977; Carrickand Setchell 1977) but they still remain obscure. Although the detrimental effects of abdominal temperatures on spermatogenesis are well known, and the susceptibility of spermatozoa in the epididymis of scrotal mammals to increased temperature has been demonstrated (Glover 1960; Cummins and Glover 1970), both spermatogenesis and sperm storage apparently take place unimpeded at abdominal temperature in testicondids.

Could this be, perhaps, because abdominal temperatures in testicond mammals are lower than those of scrotal mammals? Available evidence does not suggest this although results for sirenians suggest some thermolability. Gallivan et al. (1983) concluded that Amazonian manatees Trichechus inunguis have a limited capacity for thermiogenesis. The core temperature of an animal weighing 210 kg (about the weight of a 2.25-m dugong) ranged from 34 to 35.6'C at water temperatures between 20 and 34°C. Our unpublished observations on a dugong showed an abdominal temperature of 37°C after we opened its body cavity within 25 min of its death by drowning. In contrast, low rectal temperatures have been recorded for sirenians (Dekeyser 1952; unpublished observation). However, other large testicondids such as the African elephant (Short et al. 1967), the Indian elephant Elephas elephas (Waites and Setchell1969, quoted by Carrick and Setchell1977) and various cetaceans (for references see Irving 1969; Ridgway 1972) have body temperatures which are as high as those of many scrotal mammals.

The anatomical barriers to testicular descent in cetaceans, and the advantages of their having abdominal testes, have been discussed by de Smet (1977). His arguments apply equally to sirenians. Weber (1893) claimed (without firm evidence) that the abdominal position of the testes in cetaceans was a secondary condition and that their terrestrial forebears may have had scrotal testes. As the testes remain just caudal to the kidneys in all paenungulates, their position in sirenians is obviously not a secondary adaptation to the marine environment.

The gross anatomy of the testes and excurrent ducts of the dugong resembles that of cetaceans (personal observation) much more closely than it does that of the African elephant (Short et al. 1967) or the rock hyrax (Glover and Sale 1968). The similarities (relatively elongate testes each with an adjacent distinct epididymis) reflect the similarities in the general morphology of the abdominal cavity in these sirenians and cetaceans. This is dictated by the morphological adaptations ofboth groups to their aquatic environments, e.g. absence of hind limbs, and associated reduction of the pelvic girdle and generally streamlined shape.

The microscopic anatomy of the dugong epididymis (Fig. 12) is unusual, with a number of features similar to that in the other large testicondids, including the African elephant


(Short et al. 1967) and various odontocete cetaceans (Simpson and Gardner 1972; Kasuya 1978, personal communication 1979; Kasuya and Brownell 1979). For instance, the epididymal epithelium and adjacent highly vascular lamina propria are elaborately folded (Fig. 12), resulting in a very large epithelial surface area with an excellent blood supply. This arrangement may be functionally significant in relation to the maturation and storage of spermatozoa at relatively high temperatures, since in most eutherian mammals these events occur at lower temperatures in a scrotum. Yet, maturation of spermatozoa can occur at body temperature where no such specialized epididymal structure exists (Glover 1973; Bedford 1977). However, the elaborately folded epithelium could be associated with sperm storage since, in the dugong, the sperm store is more deeply situated within the abdomen than it is in, for example, the rock hyrax. Alternatively, the folding may be necessitated by the diameter of the epididymis in large testicondids, which is at least twice that in large scrota1 mammmals (personal observation).

Our examinations of the epididymis of the dugong have so far been relatively superficial, largely because of problems of fixation, and the maturation and storage of spermatozoa in this species warrant closer scrutiny.

Concluding Remarks

In the absence of a sharply defined breeding season, the variation in testicular and epididymal activity between individual dugongs documented in this study raises many interesting questions. For example, does the dugong's apparent lack of a pineal gland (Dexler 1912) render it unable to show seasonal changes in reproductive activity induced by photoperiod? Although some of these questions could be answered by a carcass study on a larger sample, field and laboratory studies of known individuals over several years will be necessary to understand the reproductive biology of the male dugong. Monitoring the hormonal status of animals whose reproductive behaviour is being studied would be particularly valuable.

Acknowledgments

Dugong research at James Cook University has been financed by the Australian National Parks and Wildlife Service, the Australian Research Grants Commission and the Marine Sciences and Technologies Grants Scheme. We acknowledge the assistance of the many people, particularly Dr A. V. Spain and Messrs B. R. Gardner and P. W. Channells, who have helped in collecting dugong specimen material since 1969. We also thank Messrs L. Winsor and P. Osmond for technical assistance with histology, Mr R. Hall, Ms P. Moreton, Ms S. Francis, Mr Z. Florian and the Photography Department at James Cook University for assistance with the figures, and Professors J. M. Taylor, P. K. Anderson and R. P. Kenny, Drs D. Domning and G. Rathbun, and two anonymous referees for their critical comments on the manuscript, which was largely completed while the senior author was a visiting investigator at the University of British Columbia.

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Manuscript received 6 May 1983; accepted 26 April 1984

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