THE ANATOMICAL RECORD 254:508–520 (1999) Postnatal Differentiation of the Ductus Deferens, Tail of the Epididymis, and Distal Body of the Epididymis in Goats Occurs Independently of Rete Testis Fluid HARI O. GOYAL,1* CAROL S. WILLIAMS,1 MOHAMMED K. KHALIL,1 MADAN M. VIG,2 AND M.A. MALONEY3 1Department of Biomedical Sciences, Tuskegee University, Tuskegee, Alabama 36088 2Department of Clinical Sciences, Tuskegee University, Tuskegee, Alabama 36088 3Department of Agricultural Sciences, Tuskegee University, Tuskegee, Alabama 36088 ABSTRACT Observations from extratesticular rete-ligated, mature goats indicated that epithelial morphology in the tail of the epididymis can be maintained without any input from testicular fluid (Goyal et al., Acta Anat., 1994;150: 127–135). Hence, the objective of this study was to determine whether the tail of the epididymis and/or other regions of the male excurrent ducts can differentiate prior to the appearance of lumen in the seminiferous tubules, which is an indicator for the onset of seminiferous tubular fluid secretion. Based on age and scrotal circumference (SC), 20 male goats were divided into four groups of five animals each: 1–4 weeks (SC, 6.5–7.5 cm), 7–10 weeks (SC, 8.5–11.0 cm), 12–15 weeks (SC, 11.0–14.0 cm), and 15–25 weeks (SC, 16.0–19.0 cm). Tissues were collected from the testis, six regions of the epididymis (proximal, middle and distal head; proximal and distal body; and tail), and the ductus deferens, and were processed for light and electron microscopic examination. Changes in epithelial height and cytological features associated with absorption (microvilli, pinocytotic and coated vesicles) and protein secretion (RER, Golgi body) were used as markers for differentiation. Differentiation of all of these features was comparable to that observed in the 15–25-week-old animals in the ductus deferens by ⱖ1 week, in the tail of the epididymis by ⱖ7 weeks, in the distal body of the epididymis by ⱖ12 weeks, and in the proximal body of the epididymis and all three regions of the head of the epididymis by ⱖ15 weeks. Seminiferous tubules developed lumens between 12 and 15 weeks. In conclusion, epithelial differentiation in the ductus deferens, tail of the epididymis, and distal body of the epididymis follows a time-dependent, spatial, ascending order and is achieved before lumen formation in the seminiferous tubules. Conversely, epithelial differentiation in all three regions of the head and the proximal body of the epididymis occurs simultaneously and after lumen formation in the seminiferous tubules. Anat Rec 254:508–520, 1999. r 1999 Wiley-Liss, Inc. Key words: postnatal; epididymis; ductus deferens; differentiation The mammalian epididymis is a long tube where sperm acquire the potential to move in a forward direction and fertilize ova (Bedford, 1975; Orgebin-Crist et al., 1975). Grossly, the epididymis in most species can be divided into three regions—head, body and tail—although further subdivisions have been described in virtually all species (Glover and Nicander, 1971; Hamilton, 1975). While the r 1999 WILEY-LISS, INC. Grant sponsor: NIH; Grant number: MBRS-5-S06-GM08091; Grant sponsor: USDA; Grant number: CSR-EES-ALX-TU-CTIF; Grant sponsor: NIH; Grant number: RCMI-5-G12RRO3059–08. *Correspondence to: H.O. Goyal, Department of Biomedical Sciences, School of Veterinary Medicine, Tuskegee University, Tuskegee, AL 36088. E-mail: email@example.com Received 19 September 1998; Accepted 18 November 1998 POSTNATAL DIFFERENTIATION OF THE EPIDIDYMIS TABLE 1. Age, scrotal circumference (SC), and status of the seminiferous epithelium of animals used in the study Age (weeks) SC (cm) Status of the seminiferous epithelium I 1 6.5 I I I I II II II II II 1 1 3 4 7 8 8 9 10 7.0 7.5 7.0 7.5 9.0 9.0 8.5 10.0 11.0 III 12 11.0 III 12 11.5 III 13 12.5 III III 14 15 12.0 14.0 IV 15 16.0 IV 19 16.5 IV IV IV 21 23 25 18.5 17.5 19.0 Sertoli cells and gonocytes, lumen absent Same as above Same as above Same as above Same as above Same as above Same as above Same as above Same as above Occasional spermatocytes, lumen absent Spermatocytes more frequent, lumen absent Same as above, but lumen present in some tubules Spermatocytes and lumens present in most tubules Same as above Round spermatids, few elongated spermatids Spermatogenesis established in most tubules Spermatogenesis established in all tubules Same as above Same as above Same as above Group head and body are involved in sperm maturation, the tail is associated with sperm storage (Glover and Nicander, 1971; Cooper, 1986). Androgens play many important roles in the epididymis, including prenatal and postnatal differentiation, absorption of testicular fluid, maturation of sperm, and secretion of proteins (Brooks, 1987; Robaire and Hermo, 1988; Hinton and Palladino, 1995). Experiments involving orchidectomy, testosterone substitution, and ligation of the efferent ductules or extratesticular rete indicate regionspecific, differential epididymal responses to androgen deprivation in adult animals (Fawcett and Hoffer, 1979; Abe and Takano, 1989a,b; Robaire and Viger, 1995). Whereas the structure of the tail of the epididymis can be Fig. 1. Approximate boundaries of different regions of the epididymis in adult goats. Regions I, II, and III constitute the proximal, middle, and distal head, respectively; regions IVa and IVb, the proximal and distal body, respectively; and region V, the tail. ED, efferent ductules; DD, ductus deferens. Fig. 2. Epididymides from a 4-week-old goat. The head (h), body (b), and tail (t) regions are identified. Arrowheads demarcate the boundary between the descending and ascending limbs of the head. Note that the tail of the epididymis is as large as or larger than the head of the epididymis at this stage. 3⫻. Fig. 3. A longitudinal section of the descending limb of the head of the epididymis at 1 week. Arrows demarcate the boundary between the efferent ductules (ED) and the epididymis (EP). Note that except for a few epididymal tubules located at its distal end, the entire descending limb is occupied by the efferent ductules. Epididymal tubules, compared to the efferent ductules, have a thicker muscle coat and a larger luminal diameter. 31⫻. 509 maintained with circulating androgens alone, that of the head of the epididymis is dependent upon luminal androgens and/or other factors present in the rete testis fluid (Fawcett and Hoffer, 1979; Goyal et al., 1994). These results raise a hypothesis that the tail of the epididymis and/or other regions of the male reproductive tract may postnatally mature in the absence of rete testis fluid. To test the hypothesis, we propose to study postnatal light and electron microscopic differentiation of different regions of the male reproductive tract in goats and determine which regions complete their differentiation prior to 510 GOYAL ET AL. and the rat ductus deferens (Francavilla et. al., 1987). Similar studies in large animals are few and limited to light microscopic observations of the epididymis in pigs (Wrobel and Fallenbacher, 1974), buffaloes (Goyal and Dhingra, 1975), and rams (Nilnophakoon, 1978). In the present study, goats are used as an experimental animal model for large species because, morphologically, the reproductive tract of male goats, rams, and bulls is essentially similar (Amann, 1987; Goyal, 1985; Goyal and Williams, 1991). Among these three species, male goats are preferred because, they, unlike rams, are non-seasonal (Skalet et al., 1988) and, unlike bulls, are inexpensive and easier to handle for surgical maneuvers. MATERIALS AND METHODS Animals Twenty Nubian bucks, 1 to 25 weeks of age and with 6.5 to 19.0 cm of SC, were divided into four groups of five animals each, depending upon the age and SC of animals (Table 1). All animals were kept in a covered shelter, allowed to walk freely, and given hay and water (except 1–8-week-old kids, which were bottle-fed milk) ad libitum. A general management schedule for deworming and disease prevention was followed. Tissues and Fixation Fig. 4. A comparison of regional differences in mean measurements for the epithelial height (EH), luminal diameter (LD), and tubular diameter (TD) among different age groups. Notable observations are significant (P ⱕ 0.05) increases in EH, LD, and TD in the tail (V) of the epididymis between groups I and II; significant (P ⱕ 0.05) increases in EH and LD in the distal body (IVb) of the epididymis between groups II and III; and significant (P ⱕ 0.05) increases in EH, LD, and TD in the proximal head (I), middle head (II), distal head (III), and proximal body (IVa) of the epididymis between groups III and IV. lumen formation in the seminiferous tubules, an indicator for the onset of seminiferous tubular fluid secretion. Studies involving temporal differentiation of the male reproductive tract at the light and/or electron microscopic levels have concentrated mainly in laboratory animals, including the rat epididymis (Reid, 1959; Leeson and Leeson, 1964; Flickinger, 1969; Sun and Flickinger, 1979; Francavilla et. al., 1987), the mouse epididymis (Takano, 1980; Abou-Haila and Fain-Maurel, 1985; Taggart et al., 1993), the rabbit epididymis (Leeson and Leeson, 1970), Tissues were collected from the testis, six regions of the epididymis (proximal, middle and distal head; proximal and distal body; and tail), and the ductus deferens under general anesthesia (Fig. 1). Tissues from one side of the animal were perfusion-fixed with Bouin’s fluid, and those from the other side were either immersion-fixed or perfusion-fixed (two animals in each group) with 5% glutaraldehyde in 0.2 M s-collidine buffer at pH 7.4. Since it was difficult at the gross level to distinguish between the proximal head of the epididymis and the efferent ductules in young animals, the entire descending limb of the head of the epididymis was collected (note: efferent ductules are located in the descending limb). Tissues were further fixed in Bouin’s fluid for 24 hr using slow agitation. They were cleared of Bouin’s fluid overnight in 70% alcohol, dehydrated in alcohol, cleared in xylene, and embedded in paraffin. Sections at 5–6 µm thickness were collected and stained with hematoxylin and eosin or periodic acid-Schiff (PAS). Fig. 5. A–F: Light micrographs of paraffin sections from the proximal head (A), middle head (B), distal head (C), proximal body (D), distal body (E), and tail (F) of the epididymis in group I animals. Epithelium is undifferentiated and contains only principal cells (p) in all regions, except in the tail of the epididymis where lymphocytes were also occasionally observed (not shown here). A–F, 360⫻. Fig. 6. A: A low power electron micrograph of the epithelium from the distal head of the epididymis at one week. Principal cells (p) are the only cell type present in the epithelium. Most cell organelles, except for some ribosomes and mitochondria, are poorly developed. Lucent areas (stars) containing myelin-like figures may have resulted from a fixation artifact. B: A high power electron micrograph from the apical area of principal cells. Note the presence of a few, short microvilli, but endocytotic features, such as, pinocytotic vesicles and canaliculi are absent. C: Light micrograph of a semithin section of the epithelium from an area similar to that shown in A. D: An electron micrograph showing a junctional complex, zonula occludens (ZO), zonula adherens (ZA), and desmosome (DE), between two principal cells in the epithelium of the middle head of the epididymis at 1 week. A, 7,400⫻; B, 17,800⫻; C, 720⫻; D, 15,500⫻. POSTNATAL DIFFERENTIATION OF THE EPIDIDYMIS Figures 5 and 6. 511 512 GOYAL ET AL. Figures 7 and 8. POSTNATAL DIFFERENTIATION OF THE EPIDIDYMIS In the case of glutaraldehyde fixation, 1-mm thick tissue slices from six regions of the epididymis and the ductus deferens were fixed (or further fixed in the case of perfusion fixation) with 5% glutaraldehyde in 0.2 M s-collidine buffer for 3–6 hr, postfixed with 2% osmium tetroxide in 0.2 M s-collidine buffer, dehydrated in ethanol, cleared in propylene oxide, and embedded in Epon. Sections 1 µm thick were stained with 1% toluidine blue in 1% borax and examined with a light microscope. Ultrathin sections exhibiting silver to gold colors were stained with uranyl acetate and lead citrate and examined with a Philips 201 electron microscope at 60 kV. Histoquantitative Observations Using a computer-assisted image analysis system, histoquantitative data were collected from six regions of the epididymis. Five intermittent sections from each region of each animal were examined. Five tubules with a round profile from each section were randomly analyzed for epithelial height, luminal diameter, and tubular diameter (basal lamina to basal lamina, smooth muscle cells were not included). The data were analyzed by two-way analysis of variance for evidence of significant differences among age groups (P ⱕ 0.05). RESULTS Testis Developmental changes in the seminiferous epithelium of the goat testis are briefly described here for the purpose of comparison with those of the epididymis and the ductus deferens. Seminiferous epithelium contained only Sertoli cells and gonocytes from 1 to 9 weeks (SC, ⱕ10.0 cm). Primary spermatocytes were occasionally found at 10 weeks (SC, 11.0 cm), but were frequently encountered at ⱖ13 weeks (SC, ⱖ12.5 cm). Seminiferous epithelium containing all germ cell lineages was present at ⱖ19 weeks (SC, ⱖ16.5 cm). The lumen was seen in some seminiferous tubules at 12 weeks (SC, 11.5 cm) and in almost all tubules at 15 weeks (SC, 14.0 cm). Epididymis Group 1 (1–4 weeks; SC, 6.5–7.5 cm). The epididymis at week 1 was distinguished into head, body, and tail regions. The head consisted of a descending limb and an ascending limb, and lay over the dorso-caudal border of the testis. The body was a thin thread-like structure attached Fig. 7. A–C: Light micrographs of paraffin sections from the proximal head (A), proximal body (B), and tail (C) regions of the epididymis in group II animals. Whereas the epithelium is undifferentiated in the head and body, it is differentiated in the tail of the epididymis and contains both principal (p) and basal (b) cells. Note significantly taller epithelium in the tail than in other regions. A–C, 360⫻ Fig. 8. A–C: Electron micrographs of epithelial cells from the tail of the epididymis at 8 weeks. A: Apical portions of principal cells containing absorptive features including stereocilia (S), pinocytotic and coated vesicles (arrows), and subapical vacuoles (v). Supranuclear (B) and infranuclear (C) portions of principal cells (p) containing protein synthetic organelles, such as, the Golgi cisternae (G), rough endoplasmic reticulum (arrowhead), and small, dense granules (arrow). The latter are especially abundant near the basal lamina. Basal cell, (b); blood capillary, (bc). Inset: Light micrograph of a semithin section of epithelium from an area similar to that shown in these electron micrographs. A,B, 10,500⫻; C, 8,000⫻; inset, 330⫻. 513 on the caudo-medial border of the testis. The tail was a large promontory-like structure attached on the ventromedial border of the testis (Fig. 2). Except for a few epididymal tubular cross-sections located at the distal end, the entire descending limb of the head was occupied by the efferent ductules (Fig. 3). The epididymis in 1–4-week-old goats showed distinct morphometric differences along the length. The epithelial height was significantly taller in the body and tail than in the head (19–22 µm vs. 12–14 µm). The luminal and tubular diameters were significantly greater in the tail than in the body or the head (65 µm vs. 31–44 µm, 102 µm vs. 64–74 µm, respectively). However, neither parameter differed among different regions of the head or the body (Fig. 4). Except for morphometric differences noted above, epithelial morphology was similar throughout the length of the epididymis (Fig. 5). Simple columnar epithelium contained principal cells in all regions except in the tail of the epididymis where intraepithelial lymphocytes/macrophages were also occasionally encountered. Basal and apical epithelial cells were absent in all regions. Principal cells contained an oval, heterochromatic nucleus that occupied most of the cell cytoplasm. Except for free ribosomes/polysomes, organelles associated with protein synthesis such as RER and Golgi cisternae were poorly developed. Similarly, except for a few short, irregular apical microvilli, organelles associated with fluid absorption such as canaliculi, pinocytotic and coated vesicles, and subapical vacuoles were scarce (Fig. 6A,B). The junctional complex between two adjacent principal cells was differentiated (Fig. 6C). Group II (7–10 weeks; SC, 8.5–11.0 cm). Mean measurements for epithelial height and luminal and tubular diameter did not differ from group I in any region of the head or the body of the epididymis. However, all of these parameters more than doubled in the tail of the epididymis (51 µm vs. 22 µm, 115 µm vs. 52 µm, 212 µm vs. 94 µm, respectively) (Fig. 4). Epithelial morphology at the light or electron microscopic level did not differ from group I in any region of the head or the body (Fig. 7). Principal cells were the only cell type encountered in the epithelium, and cell organelles associated with protein synthesis and/or fluid absorption were poorly developed. Conversely, epithelium in the tail of the epididymis was differentiated. It was pseudostratified and contained principal cells, basal cells and intraepithelial lymphocytes/macrophages. Principal cells were the predominant cell type and developed cytological features suggestive of fluid absorption and protein synthesis (Fig. 8). Basal cells were characterized by a relatively large, slightly indented nucleus and a few vacuoles in the cytoplasm. Intraepithelial lymphocytes were characterized by a heterochromatic nucleus and a pale cytoplasm with few organelles. Group III (12–15 weeks; SC, 11–14 cm). Compared to the previous group, epithelial height and luminal and tubular diameter did not change within any region of the head of the epididymis or in the proximal body of the epididymis, but epithelial height and tubular diameter almost doubled in the distal segment of the body (55 µm vs. 22 µm, 173 µm vs. 94 µm, respectively). None of the parameters differed from group II in the tail of the epididymis (Fig. 4). 514 GOYAL ET AL. Figures 9 and 10. POSTNATAL DIFFERENTIATION OF THE EPIDIDYMIS Epithelial morphology in different regions of the head as well as in the proximal body was still undifferentiated, but that of the distal body was differentiated (Fig. 9). Morphological features associated with differentiation were similar to those observed for the tail of the epididymis in the previous group. Epithelium changed from simple columnar to pseudostratified and contained principal cells, basal cells, and intraepithelial lymphocytes or macrophages. Principal cells acquired cytological features reflective of absorptive and protein synthetic activities. Cisternae of RER containing pale proteinaceous material were especially large in the Golgi area and in the infranuclear cytoplasm (Fig.10). Basal cells and intraepithelial lymphocytes presented a morphology which was similar to that described above in the tail of the epididymis. Group IV (15–25 weeks; SC, 16.0–19.0 cm). Between group III and group IV, all three regions of the head as well as the proximal body of the epididymis underwent a tremendous increase in epithelial height (160–230%), luminal diameter (49–155%), and tubular diameter (130– 190%). However, all of these parameters, except statistically insignificant reduction in epithelial height in the tail of the epididymis, showed modest, but statistically significant, increases in the distal body and the tail (Fig. 4). Epithelium in all three regions of the head of the epididymis presented a differentiated morphology (Fig. 11), although the degree of differentiation in the proximal head somewhat varied among individual animals within the group. For example, epithelial height was lower and more uniform in younger animals (15 and 19 weeks) than in older animals (21, 23, and 25 weeks). Regardless of the region of the head, the epithelium was pseudostratified and contained principal cells, basal cells, apical cells, and intraepithelial lymphocytes. At the light microscopic level, the proximal head was characterized by a stellate lumen and by a pale Golgi area in the supranuclear region of principal cells. The middle head and distal head were morphologically similar and possessed an even lumen lined with principal cells which contained vacuoles in the cytoplasm. At the ultrastructural level, principal cells of all three regions of the head of the epididymis contained a well developed endocytotic apparatus in the apical cytoplasm; a large Golgi complex consisting of concentrically arranged cisternae in the supranuclear cytoplasm; small, flat cisternae of SER distributed throughout the cytoplasm; and long Fig. 9. A–C: Light micrographs of paraffin sections from the proximal head (A), proximal body (B), and distal body (C) of the epididymis in group III animals. Whereas epithelium is still undifferentiated in all three regions of the head and in the proximal body, it is differentiated in the distal body. Note in the distal body the presence of both principal (p) and basal (b) epithelial cells, and significantly taller epithelium than in other regions. A–C, 360⫻. Fig. 10. A–C: Electron micrographs of epithelial cells from the distal body of the epididymis at twelve weeks. Principal cells show an abundance of organelles associated with absorption in the apical portions (A) and those associated with protein synthesis, such as, the Golgi cisternae (G) and rough endoplasmic reticulum in the supranuclear (B) and infranuclear portions (C). Note the distension of RER with a proteinaceous material (arrows). A basal cell (b) with few cell organelles lies next to the basal lamina. Inset: Light micrograph of a semithin section of the epithelium showing principal (p) and basal (b) cells from an area similar to that shown in these electron micrographs. A,B, 10,500⫻; C, 8,000⫻; inset, 330⫻. 515 flat or slightly distended RER cisternae in the infranuclear cytoplasm and on the lateral aspect of the Golgi body (Fig. 12). Apical cells were few in number, interspersed among principal cells, and extended from the lumen of the tubule to the basal lamina. They were characterized by an apical position of the nucleus, a convex luminal border that was free of microvilli, and mitochondrial aggregation in the apical cytoplasm (Fig. 12D). Basal cells and intraepithelial lymphocytes were morphologically similar to those present in the distal body or the tail of the epididymis. The proximal body, like the head of the epididymis, acquired differentiation in this age group and was morphologically similar to the distal body. Both regions of the body were characterized by a tall, irregular epithelium that accounted for the stellate shape of the lumen (Fig. 11C). The pseudostratified epithelium contained principal cells, basal cells and intraepithelial lymphocytes, but apical cells were not encountered. Principal cells in the proximal body acquired absorptive and protein synthetic organelles, similar to those acquired by principal cells of the distal body in group III animals. Basal cells formed a continuous row near the basal lamina and possessed ultrastructural features similar to those described previously in other regions of the epididymis. The epithelium of the tail of the epididymis was as differentiated as noted in earlier groups except that the epithelial height was slightly lower. Ductus Deferens Groups I–IV (1–25 weeks; SC, 6.5–19.0 cm). Unlike the epididymis, the epithelium in the ductus deferens was differentiated at week 1. The pseudostratified epithelium was very tall, actually taller than at week 25 (65 ⫾14 µm vs. 32 ⫾ 8 µm), and contained principal cells, basal cells, and intraepithelial lymphocytes/macrophages (Fig. 13). Basal cells formed a row of nuclei near the basal lamina, and were characterized at the ultrastructural level by empty-appearing vacuoles in the cytoplasm. Principal cells exhibited absorptive features including microvilli, canaliculi, and pinocytotic/coated vesicles, and protein synthesizing features including a stack of Golgi cisternae, RER cisternae, and small, dense granules. The latter were mainly seen near the basal lamina (Fig. 14). DISCUSSION This is the first study in large animals that describes postnatal spatial and temporal differentiation at the light and electron microscopic levels in different regions of the epididymis and in the ductus deferens. Based on morphological differences, the epididymis in adult, 2–3-year-old goats was divided into five regions: proximal head, middle head, distal head, body, and tail (Goyal and Williams, 1991). Regardless of the region, the epithelium contained principal cells and basal cells, and principal cells of all regions contained cytological features reflective of an absorptive function (microvilli, canaliculi, pinocytotic and coated vesicles, and subapical vacuoles) and a protein synthetic function (RER and Golgi bodies) (Goyal and Williams, 1991). Using the presence of both principal and basal cells in the epithelium and the presence of both absorptive and protein synthetic features in principal cells as criteria for differentiation, epithelium can be classified as differentiated in the ductus deferens by ⱖ1 week, in the 516 GOYAL ET AL. Figures 11 and 12. POSTNATAL DIFFERENTIATION OF THE EPIDIDYMIS tail of the epididymis by ⱖ7 weeks, in the distal body of the epididymis by ⱖ12 weeks, and in the proximal body and all three regions of the head of the epididymis by ⱖ15 weeks. From the above data, one can conclude that postnatal epithelial differentiation in the male reproductive tract of goats begins in the ductus deferens and then ascends in a time-dependent manner toward the tail, the distal body, and finally the proximal body and all three regions of the head of the epididymis. An ascending pattern of differentiation is also reported in the male reproductive tract of rats (Francavilla et al., 1987), rabbits (Gaddum, 1964), bulls (Abdel-Raouf, 1960), pigs (Wrobel and Fallenbacher, 1974), and rams (Nilnophakoon, 1978). According to the latter study, epithelium in the epididymis of lambs acquired mature-like characteristics in the distal cauda at 6 weeks, proximal cauda at 10 weeks, distal body at 12 weeks, proximal body at 15 weeks, and head at 18 weeks. In contrast to the above reports, a descending mode of differentiation is reported in the male tract of brown marsupial mice (Taggart et al., 1993), rats (Reid, 1959; Sun and Flickinger, 1979), rabbits (Leeson and Leeson, 1970), and rhesus monkeys (Alexander, 1972). Reasons for differences in the mode of differentiation could be speciesrelated, but are hard to explain between studies within the same species. The significance of androgens in prenatal and postnatal differentiation of the male reproductive tract is well established in all species studied to date (Robaire and Hermo, 1988; Robaire and Viger, 1995). However, regional differences observed in the timing of postnatal differentiation of the male reproductive tract in goats suggest a differential androgen regulation. Observations that the ductus deferens, tail of the epididymis and distal body of the epididymis differentiated prior to the onset of lumen formation in the seminiferous tubules (12–15 weeks, an indicator for the beginning of seminiferous tubular fluid production) suggest that differentiation of all these regions does not require luminal androgens or other components of the rete testis fluid, but may require circulating androgens. The latter possibility is supported by many previous reports in the literature. For example, 1) epithelium in the tail of the epididymis was taller in infantile lambs than in adult lambs (Carreau et al., 1984); 2) testosterone replacement restored epithelial height and volume density of epithelium to the control level in the tail of the epididymis of orchidectomized goats (Goyal et al., 1994); 3) the tail region of the epididymis maintained its normal structure in rete fluid-deprived epididymides of bulls (Gustaffson, Fig. 11. A–C: Light micrographs of paraffin sections from the proximal head (A), distal head (B), and proximal body (C) of the epididymis in group IV animals. Epithelium in all regions presents a differentiated morphology and contains both principal (p)and basal (b) cells. A–C, 360⫻. Fig. 12. A–C: Electron micrographs of epithelial cells from the proximal head of the epididymis at 19 weeks. Notable observations in principal cells (p) include the presence of endocytotic components in the apical portions (A) and rough endoplasmic reticulum (arrows) and Golgi cisternae (G) in the supranuclear (B) and infranuclear (C) portions. Inset: Light micrograph of a semithin section of the epithelium from an area similar to that shown in these electron micrographs. Principal cells in other regions of the epididymis possessed similar cytological features and, therefore, are not shown. D: An electron micrograph of the apical cell. Note the absence of microvilli at the luminal border and an aggregation of mitochondria in the apical cytoplasm. A, 10,500⫻; B, 13,300⫻; C, 8,000⫻; D, 5,500⫻; inset, 330⫻ 517 1966) and rats (Fawcett and Hoffer, 1979); and 4) castration and androgen replacement did not affect protein synthesis ability of the body and tail regions of the rabbit epididymis (Jones et al., 1981). It is also possible that the ductus deferens, tail of the epididymis, and distal body of the epididymis require a lower concentration of androgens (or have a lower threshold) for maintaining their structure since differentiation in these organs is completed at an early age when blood testosterone concentration is low and rete testis fluid is lacking in the epididymal lumen. In this context, it should be noted that testosterone level in blood is low at infancy and/or prepuberty and rises with the onset of puberty in domestic animals including bulls (Rawlings et al., 1972; Lacroix et al., 1977; Amann, 1983), rams (Lee et al., 1976; Olster and Foster, 1986), boars (FlorCruz and Lapwood, 1978), and goats (Chakarborty et al., 1989; Ahmed et al., 1996; Coleman et al., 1998). The latter authors conducted a longitudinal study in Nubian kids and reported that mean plasma testosterone concentration increased over time from 0.37 ⫾ 0.18 ng/ml at 1 month to 2.21 ⫾ 0.18 ng/ml at 6 months, with significant increases occurring between 1 and 2 months and 4 and 6 months. In contrast to the differentiation of the ductus deferens, tail of the epididymis, and distal body of the epididymis, that of the proximal body and all three regions of the head of the epididymis occurred soon after the appearance of lumen in the seminiferous tubules, suggesting a role for the rete testis fluid in their differentiation. These observations are in agreement with those of previous reports in that 1) epithelial height in the proximal head of the lamb epididymis increased by threefold between prepubertal and pubertal stage (Carreau et al., 1984); 2) deprivation of the rete fluid caused appreciable degenerative changes in the head of the epididymis of rats (Fawcett and Hoffer, 1979), bulls (Gustaffson, 1966) and goats (Goyal et al., 1994); 3) hypophysectomy caused significant changes in the head of the boar epididymis (Morat et al., 1980); 4) ligation of the efferent ductules caused changes in protein profiles only in the proximal segment of the mouse epididymis (Holland et al., 1992); and 5) castration and testosterone treatment or ligation of the efferent ductules significantly reduced the protein synthesis ability of the initial segment of the rabbit epididymis (Jones et al., 1981). Of different components of the rete testis fluid, androgens and androgen binding protein (ABP) are considered important in regulating the structure and function of the head of the epididymis (Robaire and Hermo, 1988). It is suggested that ABP secreted by Sertoli cells serves to transport androgen to the epididymis and thus helps in maintaining a high concentration of androgen surrounding the epithelial cells, especially in the head of the epididymis (Pelliniemi et al., 1981; Bardin et al., 1994). Principal cells in the epithelium lining the proximal part of the head of the epididymis are shown to endocytose ABP, which can deliver androgen into the cell where it can activate cell-specific gene expressions (Attramadal et al., 1981; Veeramachaneni and Amann, 1991; Hermo et al., 1998). In conclusion, postnatal differentiation of the ductus deferens, tail of the epididymis, and distal body of the epididymis follows a time-dependent, ascending order and is achieved prior to lumen formation in the seminiferous tubules. Conversely, that of the proximal body and all 518 GOYAL ET AL. Figures 13 and 14. POSTNATAL DIFFERENTIATION OF THE EPIDIDYMIS Fig. 13. A–C: Light micrographs of paraffin sections from the ductus deferens at 3 weeks (A) (group I), 13 weeks (B) (group III), and 19 weeks (C) (group IV). Unlike the epididymis, the epithelium of the ductus deferens in group I animals is differentiated and contains both principal (p) and basal (b) cells. Note a significant reduction in epithelial height between group III and group IV. A–C, 360⫻. Fig. 14. A–C: Electron micrographs of epithelial cells from he ductus deferens at three weeks. Note in principal cells (p) the prevalence of endocytotic features in the apical cytoplasm (A), Golgi cisternae (G) in the supranuclear cytoplasm (B), and mitochondria and small, dense granules (arrow) in the infranuclear cytoplasm (C). b, Basal cells. Inset: Light micrograph of a semithin section of the epithelium from an area similar to that shown in these electron micrographs. A,B, 10,500⫻; C, 8,000⫻; inset, 330⫻. three regions of the head of the epididymis occurs simultaneously and after lumen formation. ACKNOWLEDGMENTS We thank V. Ingram and T. Martin for providing technical assistance, and S. M. Bryant for typing the manuscript. LITERATURE CITED Abdel-Raouf M. 1960. The postnatal development of the reproductive organs in bulls with special reference to puberty. Acta Endocrinol (Copenh) [Suppl] 49:1–119. Abe K, Takano H. 1989a. Early degenerative changes of the epithelial cells in the initial segment of the epididymal duct in mice after efferent duct cutting. 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