Postnatal differentiation and development of the rat epididymisA stereological study.код для вставкиСкачать
THE ANATOMICAL RECORD 238~191-198 (1994) Postnatal Differentiation and Development of the Rat Epididymis: A Stereological Study F.X. JIANG, P. TEMPLE-SMITH, AND N.G. WREFORD Department of Anatomy, Monush University, Clayton, Vic 31 68, Australia ABSTRACT Postnatal development and differentiation of the rat epididymis was studied in the rat from 15 to 120 days of life using stereological techniques. Both the relative volume (volume density) and absolute volume of the epithelial, interstitial, and luminal compartments in the initial segment, caput, corpus, and cauda epididymides were determined. In all segments the volume density of the epithelial compartment increased between days 15 and 30 before falling to adult values at 45 days in the initial segment (0.476 & 0.0311, at 60 days in the caput (0.258 2 0.028) and at 90 days in the corpus (0.245 -t 0.007) and cauda (0.140 & 0.004). The relative volume of the interstitiurn decreased, whilst that of the lumen increased over the same period with adult values being achieved earlier in the proximal segments than in the distal segments. In contrast to volume fraction the absolute volume of all compartments in all segments increased from day 15 to day 90. Between 90 and 120 days the absolute volumes of compartments in the initial segment and caput showed little volume change. All compartments in the corpus and cauda showed significant increases in volume over the same period. A similar pattern of development was observed with respect to the surface area of both the luminal and basement membrane aspects of the epithelium; surface area per unit volume (surface density) in all segments reached adult values at approximately 60 days, whilst the increase in absolute area of the surfaces ceased at 90 days in the initial segment and caput and continued to 120 days in the corpus and cauda. The total length of the epididymal tubule showed the same pattern with no increase in length apparent in the initial segment and caput after 90 days. Length however continued to increase in the corpus and cauda between 90 and 120 days. Tubule and luminal diameter reached their definitive values in the initial segment on day 60 but continued to increase until day 90 in the distal segments. Epithelial height increased between 15 and 30 days in all segments; in the caput, epithelial height was greatest on day 30 before decreasing slightly; and in the corpus and cauda maximal epithelial height was observed on day 45 decreasing to stable values on day 60 and 90,respectively. All the data is consistent with a proximal to distal differentiation and development of the epididymis with growth continuing in all segments after the establishment of the definitive architecture. Growth of the distal s e g ments continued after its cessation in the proximal segments. 0 1994 Wiley-Liss, Inc. There have been few stereological studies of the rat the spatial distribution of the surfaces. The present epididymis; Bartsch et al. (1978) were the first to uti- study was undertaken using isotropic uniform random lise stereology as a tool to describe epididymal struc- sections to investigate the differential and absolute ture. Subsequently several reports (Aire, 1979; Djak- growth of the various epididymal compartments during iew and Jones, 1982; Miller and Killian, 1987; Oke et normal development. al., 1988) have described stereological aspects of the MATERIALS AND METHODS epididymis in a variety of species. Common to all these Animals studies is the assumption that the classical stereologiPregnant Sprague-Dawley rats (17-20 days gestacal methods for surfaces are applicable to the epididyma1 epithelium. Jiang et al. (1992) have recently con- tion) were obtained from the Central Animal House a t firmed that this methodology is valid in the adult. However it remains more rigorous to use isotropic uniReceived July 23, 1993; accepted August 17, 1993. form random sections as it avoids assumptions about 0 1994 WILEY-LISS, INC. 192 F.X. JIANG Monash University and maintained at a temperature of 19 t 1"C, 14 h light and 10 h dark with food and water ad libitum. The day of birth was defined as day 0. Litters were sexed and female pups discarded. Male pups were pooled into litters of ten for each nursing mother until use or weaning a t the end of the fourth week after birth. Groups of five animals were used at each time point. Fixation and Tissue Sampling Epididymides were fixed by perfusion and subsequent immersion with a glutaraldehyde-formaldehyde fixative (Jiang et al., 1992). Following fixation the epididymides were divided into an initial segment, caput, corpus and cauda (Robaire and Hermo, 1988) and weighed. In older animals (230 days) the segments were sliced into a series of slabs approximately 1.5 mm thick, the slabs were then subdivided into bars measuring approximately 1.5 x 1.5 mm2 in cross section; the bars were arranged end to end and further divided into blocks measuring 1.5 x 1.5 x 1.5 mm3. Four of these blocks were then selected for stereological analysis using a systematic uniform random scheme (Gundersen et al., 1988). In the younger animals the segments were divided into three to four blocks and all blocks were processed. The sampled blocks were postfixed in 2% osmium tetroxide in 0.2 M cacodylate buffer stained en bloc with uranyl acetate in 0.2 M maleate buffer, dehydrated in a series of graded concentrations of ethanol, and embedded in Epon-Araldite resin with no specific orientation. Sections (1 pm) were cut with glass knives, stained with 1%toluidine blue in 1% borax, and examined using light microscopy. The specific gravity was determined by estimating the buoyant density in a series of sucrose solutions (Bertram et al., 1986). The shrinkage due to processing was determined for representative blocks of each segment by measuring the tissue volume before and after processing as previously described by Zhengwei et al. (1990). Where 'CPinter, 'CPepith, and 'CPlumare the total number of points hitting interstitial, epithelial, and duct luminal compartments; CPTot,epid = CPinter + ZPepith + CP,,,; Ilum and denote intersections between a test line and the luminal and basement membrane aspects of the epididymal epithelium; Up) is the length of the test line associated with a test point hitting the epididymis, and M is the magnification. The luminal aspect of the epithelium was defined as the apical margin of the cytoplasm deep to any surface specialisation (cilia or microvilli). The basement membrane aspect of the epithelium was defined as the base of the cytoplasm which is adjacent to the basement membrane (refer to Fig. 1). The diameters of both the tubule and its lumen were calculated from the ratio of the volume to surface density assuming a cylindrical model. The diameter of the duct and its lumen were also directly measured on a digitizing tablet using Sigmascan 3.9 (Jandel Scientific, Corte Madera, CA) in conjunction with a Leitz Orthoplan microscope equipped with a drawing tube. Since sections of the epididymis contain both circular and elliptical profiles of the tubule the diameter and epithelial height were measured across the lesser of the long and short axes of the profiles. Length density of the ductus epididymidis was calculated from volume and the directly measured diameter data assuming a cylindrical model. Absolute data (volume, surface, and length) were calculated as the product of the density data and reference volume of the relevant segment. Statistics Data were analysed using the Peritz F test for mulStereologicalAnalysis Volume density (fraction) of interstitium (VVinterst tiple comparisons (Harper, 19841, programmed on a computer. Differences were considered to be epid), epithelium (VVepith,epid),and lumen ( v ~ , ,id)~of, ~ personal the epididymis together with surface density of tge lu- significant when P < 0.05. Data are reported as mean minal (SVlum,epjd) and basement membrane t SEM in both tables and figures. (Sn.mem,epid) aspects of the epitheiium in the rat epiRESULTS didymis were determined on sections obtained from the Histological Material arbitrarily orientated tissue blocks using classical Fixation of the tissues used in this study was good, methods for surfaces (Jiang et al., 1992). The section was located a t low magnification (93 x ) a random field some minor separation artefact was apparent in the was then examined a t higher magnification (284 x ) interstitium especially in the peritubular regions. Repusing a projection microscope (Leitz Neopromar) and resentative sections are shown in Figure 1. probed with a coherent square test system [l(p) = 10 Shrinkage and Specific Gravity mm, a(p) = 100 mm21. Stereological parameters were Shrinkage of the epididymal segments from collecthen calculated using the following standard formulae tion (after perfusion) to final sectioning varied from 12 (Weibel, 1979): to 22% (by volume). There were no significant differences between segments. Specific gravity in all segCPinterst ments was approximately 1.068 at day 15. It declined VVinter.epid = xpTot,epidl (1 to 2%)to a minimum at day 45 before rising again a t day 60; it then slowly declined to the value observed at day 120. In the 120 day animals specific gravity was 1.0593 t: 0.0009 in the initial segment, 1.0578 t ~ DEVELOPMENTAL STEREOLOGY OF RAT EPIDIDYMIS 193 Fig. 1. Representative micrographs of the initial segment of the epididymis from a rat aged 15 days (A) and the cauda epididymidis from a rat aged 45 (B)days. Epithelial, interstitial, and luminal compartments together with the luminal and basement membrane aspects of the epithelium are readily discerned. Printed magnification is x 200. 0.0010 in the caput, 1.0595 rt 0.0004 in the corpus, and 1.0670 0.0002 in the cauda (P< 0.001 compared to other segments). 0.031 on day 45 and then remained relatively constant to 120 days. In the caput it fell rapidly from 0.558 +0.022 on day 30 to 0.258 rt 0.028 on day 60 and remained constant thereafter. In the corpus the fall in Postnatal Growth of Epididymal Segments volume fraction was greatest between day 45 (0.476 It All segments grew rapidly in the period from 15 to 90 0.018) and day 60 (0.328 rt 0.018); it then slowly fell to days (Table 1) with the most rapid growth occurring 0.220 If: 0.011 on day 120; a similar pattern was obbetween days 15 and 45 (8- to 15-fold);growth was less served in the cauda. In contrast to the pattern observed with volume fracmarked thereafter. Growth in the proximal segments had essentially ceased by 90 days but continued in the tion data, growth in absolute volume of the epithelium distal segments which increased by 1.7- and 1.6-fold (Table 2) was continuous from 15 to 90 days in all segbetween 90 and 120 days in the corpus and cauda, re- ments. Between 90 and 120 days there was no significant change in epithelial volume in the initial segment spectively. and the caput but substantial growth was still apparEpithelium ent in both the corpus (1.5-fold) and the cauda epiThe epithelium was a relatively minor component of didymides (1.75-fold). all segments of the epididymis at day 15, representing 25 to 31% of the tissue volume (Fig. 2). Rapid differen- lnterstitium tial growth occurred in this component between 15 and The interstitium was the dominant compartment in 30 days and a t 30 days it constituted in excess of 5010 all segments of the epididymis at day 15 representing of tissue volume in the initial segment, caput, and cor- approximately 70% of the tissue volume (Fig. 3). In all pus; however this growth was less marked in the cauda segments interstitial volume fraction fell as developwhere the epithelium constituted 33%of the tissue vol- ment progressed, this was most marked in the initial ume on day 30. After day 30 the volume of the epithe- segment where the less vigorous growth, relative to lium relative to the segmental volume decreased in all that of the whole segment, produced a rapid decrease in segments (i.e., it grew less than the segment as a the volume fraction from 0.746 rt 0.011 on day 15 to whole). In the initial segment it decreased from a vol- 0.410 It 0.020 on day 30 and plateaued a t 0.320 rt 0.013 ume fraction of 0.564 +- 0.023 on day 30 to 0.476 It on day 60. A similar pattern was observed in more 194 F.X. JIANG TABLE 1. Segmental volumes (mm3)of the rat epididymis from day 15 to day 120 Caput Initial segment 1.37 t 0.17 4.18 t 0.27' 12.4 0.6l 25.5 t 2.6l 37.2 t 3.42 39.9 i 2.9 Day 15 Day 30 Day 45 Day 60 Day 90 Dav 120 2.72 t 0.18 8.37 t 0.74l 41.9 2 3.01 97.1 t 3.9' 173 i 8l 195 10 * * corpus 1.42 0.07 6.60 2 0.63l 16.2 t 1.5l 33.1 1.7l 49.5 2 3.71 83.1 t 4.7l * * Cauda 3.90 i 0.47 11.6 i 0.9' 32.5 It 1.3l 67.5 i 7.5l 148 3l 237 t 13l * 'P < 0.01 vs. the same segment in previous age stage. 'P < 0.05 vs. the same segment in previous age stage. *** 06 E 2 *** 06 05 05 04 04 0.3 03 - 50-60% of the epididymis in the caput corpus and cauda but only 20% in the initial segment. Luminal volume (Table 4) increased in all segments up to 90 days with the maximum rate of growth occurring between days 45 and 60 in the initial segment, caput, and corpus. There was no change in the absolute volume of the lumen in the initial segment and caput after 90 days. Maximum rate of growth in the luminal compartment of the cauda was observed between days 60 and 90 and luminal volume continued to increase in both the corpus and cauda between 90 and 120 days. 0.7 07 d 0.2 02 (Initial Segment) - 01 .+. 00 (Caput) 01 W ll 00 0 15 30 45 60 n 90 15 30 45 60 120 1 ~ .... .. .-. . .. 90 120 O7 06 05 04 03 03 (Corpus) 02 i 0 15 30 45 60 90 120 _ _0 15 30 45 60 90 120 Days after B i r t h Fig. 2. Epithelial volume fraction as a function of age in each segment of the rat epididymis from day 15 to day 120. *P < 0.05 and ***P < 0.005 vs previous age stage. Bar is mean t SEM. distal segments although the rate of change was less marked with distal progression. Interstitial volume (Table 3) increased in all segments throughout the study period. However there was no significant change in volume between 90 and 120 days in the initial segment, whereas there was significant increase in the caput (1.3-fold) and a doubling in the corpus and cauda. Lumen The lumen of the epididymal duct system was minimal and represented less than 2% of the epididymal volume a t 15 days (Fig. 4). There was a rapid increase in volume fraction from 30 days; in the initial segment and caput the majority of the increase occurred between days 30 and 60, with minimal change thereafter. The volume fraction of the lumen continued to increase in the more distal regions until 90 days with little change thereafter. Definitively the lumen constituted Surface Area of the Luminal Border The surface density of the luminal border in 15-dayold animals ranged from 2.83 +- 0.91 mm-' (in the initial segment) to 3.91 rt 0.78 mm-' (in the corpus) with no significant differences between segments. Surface density increased between 15 and 60 days and was stable between 8 and 10 mm-' from 60 to 120 days in all segments. The total surface area of the luminal border (Table 5 ) in the initial segment increased from 4.18 1.50 mm2 on day 15 to 355 rt 42 mm2 on day 90, with no further increase to day 120; a similar pattern was observed in the caput. In contrast in the corpus and cauda the surface area continued to increase to 120 days, in the corpus there was a 48% increase from 491 rt 67 to 727 rt 54 mm2 between 90 and 120 days, while in the cauda there was a 75% increase 1,024 f 54 to 1,797 rt 193 mm2 in the same period. Surface Area of the Basement Membrane Aspect of the Epithelium Surface density of the basement membrane aspect of the epithelium, on day 15 was 27.2 rt 1.9 mm-', 26.7 rt 1.1 mm-', 21.0 +- 0.9 mm-l, and 17.1 rt 1.2 mm-' in the initial segment, caput, corpus, and cauda, respectively. There was little change in surface density from 15 to 30 days, but the period from 30 to 60 days was characterised by a relatively rapid decline in all segments. Definitively (120 days) surface density was 16.6 rt 1.1 mm-' in the initial se ent, 10.1 f 0.9 mm-' in the caput, 10.6 rt 0.5 m m - c the corpus and 8.40 +- 0.90 mm-' in the cauda. The total surface area of the basement membrane (Table 6) showed a continuous increase from 15 to 90 days in the initial segment and caput, there was a further small but not significant increase (22%) in the initial segment between 90 and 120 days, no change was observed in the caput over the same period. In the corpus and cauda there was a continuous increase from 195 DEVELOPMENTAL STEREOLOGY OF RAT EPIDIDYMIS TABLE 2. Epithelial volume (mm3)of each segment of the rat epididymis from day 15 to day 120 Initial segment 0.336 t 0.038 2.32 t 0.07l 5.97 t 0.64l 12.7 t 1.3l 17.5 t 1.3l 19.3 t 2.8 Dav 15 D& 30 Day 45 Day 60 Day 90 Day 120 Caput 0.808 t 0.01 4.73 t 0.56l 15.8 rt 0.54l 25.3 t 3.2l 44.5 t 4.8l 46.4 t 1.9 corpus 0.438 t 0.054 3.29 t 0.31' 7.60 t 0.501 10.8 rt 0.4l 12.1 t 1.2 18.4 2 1.7l Cauda 0.956 t 0.110 3.66 rt 0.25l 10.2 t 2.7l 15.8 t 2.2l 20.7 t 0.9 36.1 t 3.9l lP < 0.01 vs. the same segment in previous age stage day 90 and 120. In the corpus and cauda total length increased throughout the period 15 to 120 days with a significant increase between 90 and 120 days in the cauda. 0 75 4 0 60 Epithelial Height \**% 0 45 0 30 0 15 - p /, 1 (Initial Segment) i_ii_iii_i-L 0 0c "l 0 * 0 15 "" 15 10 45 60 90 15 30 45 60 120 90 120 0 60 0 30 (Cauda) 0 15 1 0151 ' 0 15 30 45 60 ,--AJ 90 000 '20 Epithelial height is readily calculated from the difference between the duct and lumen diameters (Table 7). In the initial segment it increased rapidly from 13.3 rt 2.1 pm to 41.0 3.7 pm between 15 and 45 days and showed little change thereafter. In the caput it increased from 18.3 It 5.5 pm to 33.7 k 2.0 pm on day 30 followed by a slight but not significant decrease. In the corpus there was again a rapid increase from 19.1 k 1.7 pm on day 15 to 49.6 k 5.1 pm on 45 followed by a decrease to 42.7 rt 7.0 pm a t day 60, with stable values thereafter. A similar pattern was observed in the cauda with a maximum of 27.4 k 4.7 pm on day 45 decreasing to 18.0 I 7.6 pm on day 90. Direct measurement of epithelial height gave similar values to those calculated from the density data, the only exception to this was in the cauda on day 45 when the lumen appears very irregular (Fig. 1B) and direct measurement is difficult. 0 15 30 45 60 90 I20 Days a f t e r Birth DISCUSSION Shrinkage and Specific Gravity In this work stereological data was estimated in Epon embedded tissue, whilst reference volume was determined on the freshly perfused tissue. Absolute data is reported as the product of the stereological ratio and the reference volume of the freshly perfused seg15 to 120 days with increases of 46% and 71% respec- ment. Volume shrinkage due to processing was uniform between segments and varied between 12% and tively between 90 and 120 days. 21%. Bertram et al. (1986) have demonstrated that tisDiameter and Length of the Epididymal Tubule sue shrinkage varies with tissue type, shrinkage, and Tubule diameter (Table 7) in the initial segment in- swelling occurring within the same block when multicreased from 37.8 k 2.2 pm at 15 days to 175 k 7 at 60 ple tissue types were present. Bolender (1983) has sugdays with little change between 60 and 120 days. In the gested that volume and surface may well behave difcaput corpus and cauda the diameter increased from 15 ferently during tissue shrinkage since surface can to 90 days and was stable between 90 and 120 days. accommodate volume shrinkage by becoming crenated. Definitively the diameter was lowest in the initial seg- Thus the application of shrinkage corrections in a ment (165 k 11 pm), intermediate in the caput (298 I study concerned with volume and surface area is not 35 pm), and corpus (276 12 pm) and largest in the justifiable. In this work density data derived from procauda (333 k 32 pm). A similar pattern of age depen- cessed tissue have been combined with the reference dent increase was observed with the luminal diameter. volume measured on freshly perfused tissue to yield Definitively the luminal diameter was smallest in the absolute volume and surface data. The reporting of the initial segment (87.1 k 4.0 pm), intermediate in the data for volume change associated with processing will caput (239 I29 pm) and corpus (232 8 pm), and facilitate the comparison between this and other studies. largest in the cauda (286 It 38 pm). Specific gravity peaked on day 30 and fell to a minTotal length of the epididymal tubule (Table 8 ) increased continuously from day 15 to day 90 in the ini- imum on day 45, probably reflecting the formation of tial segment and corpus, with little change between the tubular lumen. Definitively the specific gravity Fig. 3. Interstitial volume fraction as a function of age in each segment of the rat epididymis from day 15 to day 120. *P < 0.05 and ***P < 0.001 vs previous age stage. Bar is mean 2 SEM. * 196 F.X. JIANG TABLE 3. Interstitial volume (mm3)of each segment of the rat epididymis from day 15 to day 120 Initial segment 1.02 t 0.13 1.69 i 0.162 5.41 lr: 0.35l 8.10 t 0.94' 11.1i 1.5 12.9 i 0.5 Day 15 Day 30 Day 45 Day 60 Day 90 Day 120 Caput 1.87 i 0.09 3.43 t 0.24' 12.5 t 1.6l 24.6 i 2.8l 38.4 t 5.3' 51.2 t 5.2' corpus 0.968 i 0.098 3.12 i 0.32l 6.89 rt 0.73l 9.75 t 1.10 11.5 2 1.5 22.8 t 0.9l Cauda 2.80 t 0.35 7.06 t 0.92l 17.0 2 1.4l 26.2 t 3.7 39.2 t 1.9' 74.7 2 8.5' 'P < 0.01 vs. the same segment in previous age stage. ' P < 0.05 vs. the same segment in previous age stage. O 0 65 g 3 % ? i (Initial Segment) _I/ ;p 01 00 I&'&,, 0 15 30 45 60 , , , 90 120 I 0 15 30 45 60 90 120 1 0 **J 15 30 45 60 90 l?O Days a f t e r Birth Fig. 4. Luminal volume fraction as a function of age in each segment of the rat epididymis from day 15 to day 120. **P< 0.01 and ***P < 0.005 vs previous age stage. Bar is mean 2 SEM. was significantly higher in the cauda than the other segments on day 120 and this probably reflects the high sperm content in this region a t this time. Postnatal Changes in Stereological Parameters of the Epididymis In this work both ratio (volume density, surface density, and length density) and absolute data have been determined. Examination of the volume density data gives an indication of the development and differentiation of the epididymis, whilst absolute data gives an indication of its growth. The volume density data reported in this study clearly shows a progressive development from proximal to distal regions of the rat epididymis. Volume density of the epithelium reached adult levels in the initial segment on day 45, in the caput on day 60, and in the corpus and cauda on day 90. Volume density of the lumen also approached adult levels in the initial segment and caput on day 60 and in the corpus and cauda on day 90. Growth in the absolute volume of the initial segment and caput essentially ceased on day 90, whilst considerable growth occurred in the corpus and cauda in the period 90 to 120 days. In all segments the rapid increase in both the absolute and volume fraction of the luminal compartment in the peripubertal period (30-60 days) was the dominant factor influencing epididymal maturation. In this study surface density estimates were performed on isotropic uniform random sections (IUR) rather than vertical sections (Baddeley et al., 1986). The use of vertical sections is the preferred method for the estimation of surface density. However it is difficult to obtain sections with a well-defined vertical axis from small irregularly shaped pieces of tissue, such as the developing epididymis. In a previous study (Jiang et al., 1992)we have demonstrated using both IUR and fixed orientations sections that the surface of the epididymal tubule is isotropic. In other studies (unpublished data) on adult animals we have used vertical sections for surface density estimation and obtained results consistent with those from IUR sections. In the present study we used IUR sections to ensure that surface estimates were valid a t all ages. Surface and length density have dimensions mm-' and mm-2, respectively; changes in either parameter are not as simple to interpret as changes in volume density which is dimensionless. In contrast to the density data estimates of total surface area and length are readily interpreted in terms of growth. Surface area of both the luminal and basement membrane aspects of the epithelium did not change after 90 days in the initial segment and caput but substantial growth occurred between 90 and 120 days in the corpus and cauda. Surface areas reported in this study are an underestimate; the true surface area is amplified by the presence of surface specialisations which have been estimated to increase the luminal surface area 2.3-fold in the adult rat (Djakiew and Jones, 1982). If the epididymal tubule is assumed to be a convoluted cylinder, surface, and length density are simply functions of volume density and diameter. Diameter data is absolute and readily interpreted, for a constant volume density a smaller diameter tubule has a greater surface density. The diameter of the epididyma1 tubule can be determined by either direct measurement or calculated from the ratio of the volume to surface density (subject to the assumption of a cylindrical model); in the present study the latter method was used. There was a close agreement between calculated and directly measured values in all segments of the epididymis, except in the cauda on day 45 when epithelial hypertrophy produced irregularities of the 197 DEVELOPMENTAL STEREOLOGY OF RAT EPIDIDYMIS TABLE 4. Luminal volume (mm3)of each segment of the rat epididymis from day 15 to day 120 Day 15 Day 30 Day 45 Day 60 Day 90 Dav 120 Initial segment 0.008 t 0.004 0.112 t 0.0212 1.04 t 0.17l 4.61 t 0.59l 8.59 t 0.68l 7.68 t 0.87 Caput 0.028 t 0.011 0.226 t 0.046l 13.6 t 1.6l 47.1 t 4.7l 92.9 t 7.5l 97.6 t 14.0 corpus 0.016 t 0.007 0.176 t 0.047l 1.68 t 0.401 12.6 t l.ll 25.9 t 2.01 42.0 t 2.6l Cauda 0.082 t 0.035 0.870 t 0.16l 5.32 t 0.38l 25.6 t 2.3l 87.8 t 2.5l 126 t 15l ' P < 0.01 vs. the same segment in previous age stage. ' P < 0.05 vs. the same segment in previous age stage. TABLE 5. Surface area (mm') of the luminal border of each segment of the rat epididymis from day 15 to day 120 Day 15 Day 30 Day 45 Day 60 Day 90 Dav 120 Initial segment 4.18 t 1.50 22.7 t 3.9l 79.6 t 8.5l 215 t 15l 355 t 42l 350 t 35 Caput 10.4 rt 1.7 45.8 t 5.6l 433 2 32l 903 t 401 1,627 t 134l 1.620 t 52 corpus 5.79 t 1.60 30.6 t 5.2l 105 t 18l 320 t 16l 491 t 672 727 rt 54' Cauda 11.9 t 2.1 45.7 t 4.41 144 t 26l 542 2 56l 1,024 t 54l 1.797 t 193l 'P < 0.01 vs. the same segment in previous age stage. 2P< 0.05 vs. the same segment in previous age stage. TABLE 6. Surface area (mm') of the basement membrane aspect of the epithelium of each segment of the rat epididymis from day 15 to day 120 Day 15 Day 30 Day 45 Day 60 Day 90 Day 120 Initial secrment 36.7 t 3.9 111 t 8l 212 t 27l 402 t 52l 542 rt 51 660 rt 57 Camt 72.3 t 3.1 228 t 26l 675 t 8l 1,153 t 10l1 1,917 t 2552 1,957 t 81 Comus 29.7 t 3.8 136 t lo1 227 t 19l 386 t 22l 606 t 68l 882 rt 8l Cauda 67.2 rt 11 172 t 8l 349 t 16l 682 t 1101 1,152 t 87l 1,974 t 177l lP < 0.01 vs. the same segment in previous age stage. 'P < 0.05 vs. the same segment in previous age stage. TABLE 7. Tubular and luminal diameters (pm) of the ductus epididymis calculated from surface density and volume fraction data Day 15 Day 30 Day 45 Day 60 Day90 Day 120 Initial segment Tubule Lumen 8.6 t 4.4 37.8 t 2.2 87.8 t 1.8l 22.3 t 4.8l 51.4 t 5.3l 133 rt 6l 85.2 t 9.2' 175 t 7l 194 t 7 98.6 t 7.6 165 t 11 87.1 t 4.0 Caput Tubule 46.6 t 3.6 86.8 t 2.2l 174 t 111 258 t 23' 294 t 20 298 5 35 Lumen 9.9 t 1.8 19.4 t 2.4l 124 t 8l 212 t 25l 229 rt 17 239 t 29 corpus Tubule Lumen 16.8 2 6.9 62.2 t 3.0 24.8 t 7.6 102 t 4l 64.7 t 10' 164 2 6l 160 t 20' 246 rt 19l 217 rt 18' 256 rt 18 276 2 12 232 2 8 Cauda Tubule 63.4 It 3.0 105 t 4l 178 rt 5l 255 t 28l 380 2 25' 333 2 32 Lumen 27.0 rt 11.0 74.4 t 9.1' 168 t 301 193 t 18 345 t 111 286 t 38 'P < 0.01 vs. the same segment in previous age stage. 'P < 0.05 vs. the same segment in previous age stage. tubule lumen which influenced the estimation of it's diameter. All segments of the epididymis increased in diameter of both the luminal and basement membrane aspects of the tubule from 15 to 90 days, with little change between 90 and 120 days. Between 60 and 90 days the increase in luminal diameter was 12% (not significant) in the initial segment, 22% in the caput, 37% in the corpus, and 74%in the cauda. This is again consistent with a progressive differentiation from proximal to distal segments. Definitively the luminal diam- eter was smallest (high luminal surface to volume ratio) in the proximal segment consistent with a transport function and highest in the distal segments consistent with a storage function. Epithelial height showed a consistent pattern in the more distal segments; a small peak at 30 days in the caput decreased to the mature value a t day 45. In the corpus and cauda it peaked on day 45 before decreasing to mature values on days 60 and 90, respectively. Definitively epithelial height was greatest in the proxi- 198 F.X.JIANG TABLE 8. Length of the duct (mm) of each segment of the rat epididymis from day 15 to day 120 Day 15 Day 30 Day 45 Day 60 Day 90 Day 120 Initial segment 374 t 45 476 t 55 579 rt 63 884 rt 80’ 1,201 t 128 1,141 rt 81 Caput 702 rt 204 1,041 rt 105 1,259 rt 86 1,943 rt 183l 2,597 rt 342 2,466 t 279 corpus 265 +- 34 505 t 57 533 t 53 727 rt 432 754 rt 90 1,065 t 87 Cauda 504 t 99 491 t 52 662 rt 81 1,117 rt 129l 1,350 2 141 1,938 2 111’ ‘P < 0.01 vs. the same segment in previous age stage. ’P < 0.05 vs. the same segment in previous age stage. ma1 segment, consistent with a transport function and lowest in the distal segments consistent with a storage function. Cell number and volume are important indicators of organ maturation which have not been addressed in the present study. The development of the “Optical Disector,” (Braendgaard et al., 1989) which is applicable to thick methacrylate sections, will facilitate the derivation of such data. It would be appropriate in a future study to combine stereology with either tritiated thymidine uptake or stathmokinetic techniques to yield data on cell proliferation and cell number, particularly in the epithelial compartment. Such data would clarify the question of when and if growth changed from a hyperplastic to a hypertrophic mode. Although there are no other developmental stereological studies of the rat epididymis, it is of interest to compare these observations with other work. Sun and Flickinger (1979) investigated the qualitative changes in the rat epididymis with age. Cytological differentiation was the major feature of the period from 15 to 45 days, whereas the period from 45 to 90 days represented a period of expansion without further differentiation. 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