Vascular permeability to effectors of the immune system in the male rat reproductive tract at puberty

Journal of Reproductive Immunology 28 (1995) 85-109

Vascular permeability to effecters of the immune system in the male rat reproductive tract at puberty P. P611iinen*a7b,T.G. Coopera aInstitute of Reproductive Medicine, Westfalian Wilhelm+University, D-48149 Miinster, Germany bDepartment of Anatomy, Institute of Biomedicine, University of Turku, FIN-20520 Turku, Finland Received 6 June 1994; revision received 18 September 1994; accepted 21 September 1994

Abstract

The net movements and distribution volumes of IgG in the pubertal rat testis and epididymis were measured using physiological techniques. A new method to calculate vascular permeabilities was developed. The volume of testicular extracellular fluid accessible to IgG ( Veq,$g) increased between 20 and 30 days of age and remained above 20-day-old levels. Estimates of the surface areas of the exchange vessels (S, cm*) increased with age, most between 20 and 30 days of age. The speed at which equilibrium between tissue extracellular fluid and serum was reached (K, min-‘) increased between 20 and 30 days and decreased between 44 and 60 days of age. The lymph flow (&) and estimates of permeabilities (P) to IgG increased between 20 and 30 days of age and remained high. In the caput epididymidis Veq increased between 30 and 60 days and S between 44 and 60 days of age, but K increased between 30 and 44 days and decreased between 44 and 60 days of age. Q, and the estimates of P increased between 20 and 44 days of age and remained at that level. In the corpus epididymidis, Vq did not change with age, but S increased between 30 and 60 days and K, QL and P between 20 and 44 days of age. In the cauda epididymidis, there were no obvious changes in Vcgwith age, but the estimates of S increased and K values decreased between 44 and 60 days of age. QL and P increased between 30 and 44 days and then decreased. After 30 days of age the values for Vq, K, QL and S were larger in the testis than in the cauda, whereas P at 44 days of age was not different. The number of perivascular mononuclear cell infiltrate profiles per 1000 epididymal tubule cross-sections increased with age in the caput epididymidis, but no infiltrates were observed in the corpus or cauda before 60 days of age. l

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86

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of Reproductive Immunology 28 (1995) 85-109

It is concluded that the observed increases in vascular permeability to IgG with age in the testis, caput and corpus reflect the function of the microvessels rather than an increase in the size of the vascular bed. Keywords: Testis; Epididymis; Circulation;

Permeability; Immunoglobulin;

Leukocyte

1. Introduction

The presence of autoantigenic spermatozoa in the male reproductive tract demands mechanisms to prevent an antisperm autoimmune response from occurring (see Polllinen and Cooper, 1994). The blood-testis and bloodepididymis barriers act to restrict the entry of immunoglobulins to the lumen of seminiferous (Koskimies et al., 1971; Johnson, 1972; PGllanen and Setchell, 1989) and epididymal (Wong et al., 1983; Yeung et al., 1991) tubules, but in the testis, autoantigens are also present outside the barrier in the basal compartment of the seminiferous tubules (Salomon et al, 1982; Yule et al, 1988; Saari et al., 1994), suggesting that additional protective mechanisms are needed. Because experimental immunization against sperm autoantigens leads to mononuclear leucocyte infiltrates in the epididymal interstitium while the tubule is intact (Teuscher et al., 1987, 1989; Zhou et al., 1989; Itoh et al., 1992), there is good evidence that the segregation of autoantigens is incomplete in the epididymis as well. Clearly, it is important to determine the extent to which effecters of the immune system pass through the vascular endothelium in these tissues and how this is regulated with age. Although the transport of IgG to the epididymal lumen is restricted in many species (Wong et al., 1983; Weininger et al., 1982; Yeung et al., 1991), the amounts of IgG in epididymal interstitial fluid are not known. Little is known of the vascular components of the blood-epididymis barrier (Hoffer and Hinton, 1984) and no quantitative data on the vascular permeability to IgG in the epididymis exist, although penetration of substances through the epididymal endothelium is believed to be relatively free (Kormano, 1967a,b, 1968a). In the present study, the net movements of ‘251-IgGwere measured in the pubertal rat testis and epididymis using physiological techniques and the appearance of perivascular mononuclear cell infiltrates was studied in the epididymides of rats of various ages to determine whether the endothelial cells play a role in the immunology of these organs. 2. Materials and methods 2.1. Animals

Sprague-Dawley

rats were used at 20, 30,44 and 60 days of age (n = 60).

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of Reproductive Immunology 28 (1995) 85-109

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2.2. Experimental protocol Rats were weighed before starting the experiment and anaesthetised with pentobarbitone sodium. ‘251-iodinated sheep IgG (0.2 &i in 0.2 ml 0.9% NaCVlOO g body weight), was injected into the right jugular vein through a 27 G needle. At various times (3, 75, 150, 300 and 1200 min) after injection, the testes and epididymides were removed, a blood sample was collected from the posterior vena cava and the sera obtained. The epididymides were divided into caput, corpus and cauda regions and weighed. The testes were decapsulated and the capsule (with the testicular artery and veins) and the parenchyma were weighed separately. Radioactivity in the segments of epididymides, testis parenchyma, testicular capsules and sera were measured in a y-counter. 2.3. Determination of volumes of distribution The volumes of distribution were calculated from the formula: volume of distribution in $g =

tissue counts/mm per gram tissue serum countslmin per ~1

The interstitial proportion of the testis and the extraluminal proportion of the epididymal weight were used in the calculation of volumes of distribution (@g), because the blood-testis and blood-epididymis barriers prevent IgG from reaching the luminal space and therefore using whole tissue weights would not fulfill the requirements of even tissue distribution (Amtorp, 1980). The proportions of interstitial tissue of the whole rat testis as measured by Setchell et al. (1987) were used and the extraluminal proportions of the various segments of the rat epididymis at different ages were calculated from the data of Jiang et al. (1994). Estimates of the extraluminal proportions at 20 days were calculated from the values for 15- and 30-day-old rats, assuming that the development of the extraluminal proportion between 15 and 30 days of age was linear. The extraluminal proportions of the various segments of the epididymis in the 20-, 30-, 45 and 60-day-old rats in the proximal epididymis were 98.02%, 97.3 l%, 73.04% and 57.82%, in the middle segment of the epididymis 98.87%, 97.33%, 89.63% and 61.93% and in the terminal segment of the epididymis 97.80%, 92.50%, 83.63% and 62.07%. 2.4. Calculation of the permeability x surface area (PS) product (lymph flow)

The permeability (P) x surface area (s) products were calculated as previously described (Amtorp, 1980) by multiplying the maximal space (V,,) by the slope of the line of In (1 - V,/Vecl>against time, fitted by the least squares method. V, is the distribution volume at time t and V,, the maximal distribution volume. If ‘251-IgG leaves the tissue intact and only via lym-

88

P. Piillhen, T.G. Cooper /Journal of Reproductive Immunology 28 (1995) 85-109

phatics, the PS products are estimates of lymph flow (Q), because the t,,2 of accumulation in equilibration is the same as the t,n of clearance and when the formula: QL = 0.693 VeqltllZ(1) (Maddocks et al., 1987) and the equation: P = 0.693 VeglSflR(2) (Wittmers et al., 1976) are incorporated, the equation: QL = PS (3) is obtained. 2.5. Calculation of microvascular permeability The permeability x surface area product of a substance is PS = x (4). The surface area (S) of a microvessel can be calculated from the equation: S = 2~rl(5), where 2nr = perimeter, I= length of the vessel and r = radius of the vessel. The volume of a vessel can be calculated from the equation: V = ?rr21 (6), where rr 2 = the area of cross-section of the vessel and 1= the length of the vessel. Therefore, the length of the vessel is 1 = V/ur2 (7). Assuming that the part of the circulation where IgG penetrates through the endothelium (the exchange vessels) is composed of vessels of the same diameter, the total length of these vessels can be calculated using the volume of the exchange vessels (Vex): 1 = V&r2 (8). Because at 3 min after injection, most of the marker is still in the intravascular space, the 3 min space (VjmiJ = volume of blood vessels in the tissue (pllg). Therefore, the volume of the exchange vessels is: Vex= cVs/3min (9), where c = proportion of the exchange vessels of the total volume of blood vessels in the organ. Then: 1 = cV3,,.&rr2 (10). Eqs. 4 and 5 can be combined: 2nPrl= x (11). From Eqs. 10 and 11: P = xr/2cVl,,,i, (12), where x = PS, r = radius of the exchange vessels in the model and V3minthe volume of organ circulation in @g. From Eqs. 4 and 12: P = PS r/2cV,,, (13). Because PS = KV,, (Johnson and Wilson, 1966; Wittmers et al., 1976; Amtorp, 1980), Eq. 13 can be reformulated to P, = 0.5 rKV~,l(cV~,i,) (14). Because in this theoretical model it is assumed that the exchange vessels are of the same diameter, r is a constant and can be given a value r pm. The unit of P in this equation is [(~llg/min)~m]l(~l/g) = pm/min. This is a unit of velocity, expressing the speed at which the marker moves to the extravascular tissue. The same end result is obtained, if the PS product is multiplied by tissue weight to get ~m3/min and divided by S,, in pm2 (Eq. 15). 2.6. Calculation of exchange vessel surface area as a function of c and r

For calculation of the surface area of the exchange vessels, the following equation can be derived from Eq. 13: S TC= 2V3min~r-’(15). This gives S,, in units of surface area per gram. 2.7. Estimation of c and r and absolute values for P and S The volume of the exchange vessels has been estimated morphometrically in the adult rat testis, being 5.8 pi/g whole tissue (Damber et al., 1985).

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Because the 3-min space, which reflects the volume of circulation, was 7.8 pi/g whole tissue in the adult rat testis in the present study, the proportion of the exchange vessels of the whole volume of circulation (Vjmin) is estimated to be -74%. If it is assumed that this is the proportion of the volume of exchange vessels of the volume of whole circulation in all the tissues studied, Eq. 17 can be put in the form: P, = 0.67 rKFQJ’3min (16) and Eq. 15 in the form: S, = 1.5 V3minr-l(17). To obtain an estimate of r, S, (cm2/g) for the 60-day-old rat testis was divided by the morphometrically obtained surface area of the exchange vessels in the adult rat testicular interstitium (278.4 cm2/g interstitial tissue Damber et al., 1985). The proportion of the interstitial tissue of the whole testicular weight was calculated using the morphometrical data of Setchell et al. (1987). The obtained value for the estimate of r was 4.3. Using this value, Eq. 16 can be rewritten to: P = 2.9 KVeqIV3min (18) and Eq. 17 to: S = 0.34 k’jmin(19). This gives S in units of surface area per gram. Estimates for surface area per gram interstitial tissue for testis and epididymis were obtained from V3minvalues calculated using interstitial tissue proportions of the testicular and epididymal weights (Setchell et al., 1987; Jiang et al., 1994). These proportions were 52.58%, 40.86%, 32.98% and 26.67% for the proximal epididymis, 53.50%, 47.27%, 42.53% and 29.46% for the middle epididymis and 65.22%, 60.86%, 52.3 1% and 38.8 1% for the distal epididymis at 20, 30, 44 and 60 days of age, respectively. 2.8. Histology The epididymides of three 20-, 30-, 44- and 60-day-old rats were removed immediately after sacrifice with CO2 and fixed by immersion in Bouin’s fixative. They were embedded in paraffin and sections 5 pm in thickness were cut and stained with haematoxylin and eosin. The number of focal perivascular mononuclear cell infiltrate profiles was counted and expressed per number of tubule cross-sections in the same tissue slices. The criterion for a perivascular mononuclear cell infiltrate was the presence of > 10 mononuclear leukocytes around a blood vessel. 2.9. Statistical analysis Differences in the distribution volumes, in the estimates of exchange vessel surface areas and in the incidences of perivascular mononuclear cell infiltrate profiles per 1000 epididymal tubule cross-sections were analysed statistically using analysis of variance (ANOVA) and Tukey’s test. The equality of the regression lines at different ages and in different tissues was tested using the F-test and the differences of regression lines from zero were tested using Student’s t-test as described (Sachs, 1984). The parallelism of pairs of regression lines was analysed using Student’s t-test with Bonferroni correction (Sachs, 1984).

45.3 f 97.7 + 197.3 f 293.3 zt

(days)

20 30 44 60

I.9 (15) 2.3 (15)a 2.8 (15)“~~ 2.7 (15)e,b,c

13.5 l 20.4 l 46.2 f 110.1 f

Caput (mg)

1.2 (30) 4.8 zt I.1 (30)a 9.7 zt 1.4 (30)“~~ 40.6 zt 3.1 (28)a~b~c 93.6 zt

corpus (mg) 0.6 (30) 1.0 (29) 1.3 (30)“,b 3.9 (28)a,b,c

Testicular parenchyma (me)

I I .O * 0.6 (30) 98.2 f 13.8 (30) 19.0 l 0.9 (30)a 457.7 ze 8.5 (30)s 45.6 zt 1.1 (30)“*b 1073.2 f 14.3 (30)“*b 119.2 f 3.2 (28)s~~~~ 1464.0 + 2.8 (28)a.bsc

Cauda (mg)

The values given are mean A S.E.M. The numbers in parentheses refer to the number of samples. “p < 0.01 vs. 20-day-old. bp c 0.01 vs. 3&day-old. “p < 0.01 vs. 44-day-old.

MY

(g)

Age

29.6 zt 30.0 f 51.8 i 84.2 f

2.0 (30) 2.9 (30) 2.2 (30)“*b 3.1 (28)a.b*c

Testicular capsule (mg)

Table I Body weights of rats of various ages and weights of their caput, corpus and cauda epididymidis, the testicular parenchyma and capsule

P. PM&en,

T. G. Cooper/Journal

of Reproductive Immunology 28 (1995) 85- IO9

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3. Results 3. I. Body and tissue weights The body weights of the intact non-treated rats increased steadily with age (Table 1) and corresponded well to those reported earlier (Setchell et al., 1987; Piilllnen and Setchell, 1989). The testis parenchyma weights were quite similar to those in the earlier studies, but the testes of the 30- and 44-day-old rats were slightly heavier than reported before. The weights of the testicular capsules increased with age to reach a maximum in the 60-day-old rats. The weights of the epididymal heads, bodies and tails increased steadily with age. 3.2. Distribution volumes of IgG Testis. In the testis parenchyma, the IgG spaces calculated per interstitial tissue weight increased with time in all the age groups (Table 2). These spaces in the 20-day-old rats were smaller than those in the other age groups at all time points after injection. The maximal spaces (spaces at equilibrium, VW, pi/g) were reached in 1200 min in all the age groups, except in the 30-day-old rats, in which they were reached at 300 min. In the 30-day-old rats, the maximal spaces, reflecting the volume of extracellular fluid in the interstitial tissue, were higher than in the other age groups. The 3-min spaces (pi/g, reflecting the volume of circulation in the tissue) in the mid and late puberty were greater than those in early puberty, demonstrating a pubertal increase in the vascular bed size (see Table 2). The volumes of distribution of IgG in the testicular capsule increased steadily with time in all the age groups (Table 2). The maximal space was reached in 1200 min in all the age groups, except in the 30-day-old rats, in which it was reached at 300 min. Both the volume of circulation and the volume of extracellular fluid were higher in the early pubertal than in the mid-pubertal rats, but there were no differences in these volumes between the mid and late pubertal age groups. Epididymis. In all the segments of epididymis and in all the age groups, the IgG spaces per extraluminal weight increased with time (Table 2). The maximal spaces were reached in 1200 min in all the age groups in all the segments of the epididymis, except in the corpus of 30-day-old rats, in which they were reached at 300 min. No significant differences in the volumes of circulation or the volumes of extracellular fluid could be localized between the age groups in the caput and the cauda, but in the corpus epididymidis, the volume of circulation was significantly higher in early puberty than in late puberty. Differences in maximal spaces between organs. In the early pubertal rats the maximal spaces, reflecting the volume of extracellular fluid accessible to i.v. injected *251-IgG,in the testicular capsule were higher than those in the ex-

36.9 A 115.1 * 81.3 f 78.1 f

Caput epididymidis 20 40.3 zk 9.0 30 23.2 zt 7.3 44 30.9 zt 2.9 60 31.7 f 1.7

(6) (6) (6)

(6)

56.2 35.4 62.4 76.7

295.9 207.2 144.9 138.3

170.1 502.2 406.5 297.3

zt f f f

f f f zt

zt f f f

13.7 (6) 4.8 (6) 5.4 (6) 2.8 (6)b

11.2 (6) 42.8 (6) 7.7 (6) 11.9 (6)

36.6 (6) 26.9 (6)a 5.4 (6)“*b 12.0 (6)‘~~.~

G spaces &l/g) 75 min

3.9 (6)* 5.0 (6)“.b 3.4 (6)“~~

13.0 (6)b

Immunoglobulin 3 min

Testicular rap&e 20 96.6 f 7.0 (6)b 30 44.3 f 13.6 (6) 44 65.2 zt 7.1 (6) 60 68.1 f 4.1 (6)

30 44 60

Testis 20

(days)

Age

42.7 73.0 95.0 99.5

453.8 288.9 196.6 169.7

203.1 757.9 579.4 453.8

(6) (6)” (6)” (6)“,b

149.4 (6) 38.2 (6) Il.6 (6) 17.8 (6)

48.1 88.7 20.4 59.3

f 19.8 (6) f 9.1 (6) +z 11.9 (6) + 4.4 (6)”

zt zt zk f

f f f f

150 min

71.3 61.5 155.5 141.4

455.7 366.1 314.8 214.7

57.3 9869.0 630.2 545.1

49.4 73.0 22.7 16.5

21.9 28.4 28.9 12.8

(6)d (6) (6) (4)

(6) (6)” (6)“,b (4)“,b

f 14.0 (6) f 19.1 (6) i 4.5 (6)“.b f 6.4 (4)“~~

f f f f

f f f f

300 min

*104.5 *99.8 ‘168.0 *I85.0

594.4 338.7 378.8 368.5

24.3.6 715.2 sh664.3 sh597.4

zt zt f f

f f + f

89.0 51.7 36.4 40.8

15.0 41.8 36.7 83.8

(6)b (6) (6) (6)

(6) (6)” (6)” (6)6

10-3d lO_Zd” lO_jd.” 10-j

-1.996 -1.617 e-1.007 e-4.261

x x x x

10-4d lO-3 lo-= 10-s

e-4.492 x 1O-3 ‘-4.665 x 1O-3 e-5.734 x 10-s e-2.2B2 x lO-3

r-1.075 e-1.094 e-7.790 e-7.910

x x x x

tissue of caput.

K (min-‘)

and the extraluminal

f 11.8 (6) f 23.7 (6) +z 26.3 (6) zt 27.7 (6)

1200 min

Table 2 bmnunoglobulin spaces, K values and PS products (Qt) in the interstitial tissue of the testis, testicular capsule cauda epididymides of rats of various ages and statistically significant differences between the age groups

and

0.02 I 0.161 1.692 0.788

2.172 0.841

2.670 1.708

5.175 4.726

0.262 9.503

PS(Q,) (ctl/g/min)

corpus

S

7

z ZS 2

%

S P

$ z

8 :: ;. a

a

2 i, 8

L 2

31.4 zt 2.7 (6) 17.0 zt 5.5 (6) 24.4 f 3.5 (6)

30 44 60

63.7 (6) 34.3 (6) 3.4 (6) 2.1 (6)

108.4 zt 14.7 (6) 70.0 + 14.1 (6) 97.0 zt 8.3 (6) 77.9 zt 2.3 (6)

155.2 zt 79.6 f 84.3 l 76.9 f

23.4 (6) 41.3 (6) 5.8 (6) 3.3 (4)

122.3 f 20.0 (6) 125.9 z+z13.9 (6) 159.3 f 3.7 (6) 119.8 f 5.0 (4)

112.1 f 151.9 f 111.2 f 107.3 f

100.5 (6) 19.1 (6) 15.0 (6) 15.8 (6)

150.3 + 28.8 (6) 181.8 f 14.9 (6) 231.0 f 15.9 (6) 196.1 f 31.0 (6)

167.7 f 140.3 f 116.4 f 132.7 f

r-3.797 e-3.156 e-3:777 e-2.643

-1.033 ‘-5.063 e-7.365 e-4.802

x x x x

x x x x

10-3 lO-3 lo-3d IO-’

lo-4C.d 10-s 10-3d lO-3

0.571 0.574 0.872 0.518

0.015 0.769 0.857 0.637

The values given are mean * S.E.M. The values in parentheses refer to the number of epididymides per group. Maximal spaces are given in itah. Superscripts: For differences of K values from zero, superscripts on the left side of the K values. For parallelism of K values, superscripts on the right side of the K values. “p < 0.05 vs. 20-day-old. bp < 0.05 vs. 30-day-old. “p < 0.05 vs. 44-day-old. dp < 0.05 vs. 60-day-old. ‘p < 0.001 vs. zero. fp < 0.05 vs. zero. gp < 0.01, Ves vs. Yes in 20-day-old. “p < 0.05, Veg vs. Vq in 30-day-old. + V values in the various age groups significantly different in ANOVA. but no differences in Tukey’s test.

17.2 zk 11.1 (6)

Car&r epididymidis

20

f 9.3 (6) f 9.4 (6) zt 2.5 (6) zrz2.0 (6)

54.5 f 10.5 (5) 30.8 f 6.3 (6) 59.9 zt 3.1 (6)

l36.9 *37.8 *52.0 *64.5

23.8 f 14.0 (6)

30 44 60

40.5 * 13.6 (6) 23.8 f 1.4 (6)a 27.4 f 2.0 (6)’

Corpus epididymidis 20 83.6 zt 11.2 (6)

L!?

S

2

2 2 g z!

z S 3 P

$ z R

2 2 \ b a”

3

:

Q 8 B

;

P

2 3 L

3

83.8 (6)‘c~caP~cor~cau-7,910 -2.282 40.8 (6)=” 27.7 (6) -4.261 15.8 (6) -4.802 31.0 (6) -2.643

-7.790 -5.734 -1.007 -7.365 -3,777

x lO-3 x lO-3 x 10-3cor

~0-3lc.cap.cor.cau ]0-3CaP.Cor

cap. COT

x

IO-3’C.

x

x x x x x

lo-sea” 10-3car 10-2 lO-3

~0-21c.caP.cor.cau

x x x x

lO-3 lO-3 lO-3 IO-’

lO-4 10-d 10-j

,0-3=P.Cor

10-3

_ 1,094 x

x x x x x

-4.665 -1.617 -5.063 -3.156

-1.075 -4,492 -1.996 -1.033 -3.797

K (min -’ )

4.726 0.841 0.788 0.637 0.518

5.175 2.172 1.692 0.857 0.872

9.503 1.708 0.161 0.769 0.574

0.262 2.670 0.021 0.015 0.571

p.9 (QL) (NglmW (6) (6) (6) (6)ti,cau (6)

277.0 f 234.4 zt 236.8 zt 198.4 zt 134.1 f

279.5 f 224.2 f 235.6 f 172.4 f 93.5 f

11.9 (6) 14.1 (6) 12.4 (6) 14.5 (6)” 19.2 (6)“Jc,car.cor

17.3 (6) 24.5 (6) 22.1 (6) 10.2 (6)” 30.4 (6)ti*‘c.cap

414.2 zt 13.9 (6)‘c,‘“” 152.6 f 46.9 (6) 189.8 + 65.8 (6) 287.3 f 105.4 (6) 164.4 + 15.4 (6)

45.0 23.9 65.7 76.9 93.0

(cm2/g interstitium)

128.0 f 332.5 f 268.8 f 525.4 zt 131.3 zt

S*

175.5 35.8 72. I 67.4 61.6

184.6 96.6 158.8 104.5 148.8

239.5 111.8 20.2 55.1 53.0

20.6 80.5 1.5 0.6 96.2

P* (nntlmin)

The values given are mean f S.E.M. The values in parentheses refer to the number of epididymides per group. PS and P were calculated using group means. Consequently, no standard errors or statistical significances can be provided. “p < 0.05 vs. testis. “p < 0.05 vs. testicular capsule. “Qp < 0.05 vs. caput. carp < 0.05 vs. corpus. CB”- / nnc ..^ _^..A”

597.4 + 368.5 zt 185.0 f 132.7 f 196.1 f

664.3 * 36.7 (@tC.CaP.COLW 378.8 +z 36.4 (6)cap~cor~ca” 168.0 f 26.3 (6) 116.4 zt 15.0 (6) 231.0 zt 15.9 (6),“’

44 &ys Testis Testicular capsule Caput corpus Cauda

60 &ys Testis Testicular capsule Caput corpus Cauda

869.0 * 366.I + 99.8 f 151.9 * 181.8 f

30 aklys Testis Testicular capsule Caput corpus Cauda

28.4 (@WPWW 73.0 (6)cap.cor.cau 23.7 (6) 41.3 (6) 14.9 (6)

243.6 f 15.0 (6) 594.4 * 89.0 (fpaP.cor.cau 104.5 + 11.8 (6) 167.7 AZ100.5 (6) 150.3 f 28.8 (6)

20 days Testis Testicular capsule Caput corpus Cauda

We)

yes

Table 3 Maximal volumes of distribution (Yes). K values, PS (QL) values (= K x Ves), exchange vessel surface areas per gram interstitial tissue (S*) and microvascular permeabilities (P*) in the testis and epididymis in the various age groups

P. Piilhen,

T.G. Cooper/Journal of Reproductive Immunology 28 (1995) 85-109

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traluminal tissue of segments of epididymis. In the 30-day-old rats, the volume of accessible extracellular fluid in the testicular interstitium was higher than those in the extraluminal tissue of the segments of epididymis or testicular capsule. In the late pubertal rats, the accessible extracellular fluid volumes in testicular interstitium were higher than the volumes in extraluminal tissue of the segments of epididymis or in the testicular capsule and that in the testicular capsule was higher than that in the extraluminal tissue of corpus epididymidis (Table 3). 3.3. The regression curves for penetration of ‘2SI-IgG into the testis and the various segments of epididymis

In early puberty the regression curves (slope and constant) for penetration of “‘1-1gG into the various parts of the testis and the epididymis were equal, but from mid-puberty onwards differences between organs could be observed @ < 0.05). Only testis and the caput and the corpus epididymidis showed differences in the regression curves between ages. All the slopes of the regression curves (K, mm-‘) except those for the 20- and 30-day-old caput epididymidis and for the 20-day-old corpus epididymidis were different from zero. Testis. In the testis parenchyma, the slopes of the regression curves (K), reflecting the speed at which equilibrium between extracellular fluid and serum was reached (Table 2), increased in early puberty, remained thereafter at the same level in mid-puberty and then decreased towards the end of pubertal development. In the testicular capsule, the slopes remained in the same range of magnitude throughout puberty. Epididymis. In the caput epididymidis, the slopes of the regression curves were not different from zero before late puberty. Towards the end of puberty the slope decreased. In the corpus, the slopes were not different from zero before mid-puberty. The slopes in late puberty were greater than in early puberty, but decreased towards the end of pubertal development. In the cauda, all the K values were significantly different from zero. The K values did not change significantly before late puberty, when they decreased. Differences between the organs. Significant differences in the slopes of the regression curves were observed between the organs in the various age groups (Table 3). In early puberty, the slope in the testicular capsule was higher than that in the testis, caput and the corpus epididymidis. In midpuberty, the slope in the testis was larger than those in the capsule and the segments of epididymis. In late puberty, the slope in the testis was higher than that in the cauda, the slope in the testicular capsule was greater than that in the caput and the slopes in the caput and corpus were higher than that in the cauda. At the end of puberty, the slope in the testis was larger than

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those in the testicular capsule and the segments of epididymis, the slope in the testicular capsule smaller than those in the caput and corpus and the slope in the corpus higher than that in the cauda. 3.4. PS products (lymph flow) in the testis and its excurrent ducts Clear differences in the pattern of development of the PS products could be observed between the various parts of the male reproductive tract (Table 2). In the testicular interstitial tissue and the corpus epididymidis, there was an increase in the PS products between 20 and 30 days of age, whereafter the PS products decreased slowly or remained stable. This was not so in the caput epididymidis, where the PS products increased to - loo-fold values between 20 and 44 days of age and slightly decreased thereafter, or in the testicular capsule or the cauda epididymidis, where the PS product remained relatively stable during pubertal development. 3.5. Surface area of exchange vessels in the testis and its excurrent ducts Testis. The exchange vessel surface area per testis increased markedly in early puberty and continued to increase throughout pubertal development. The estimates of surface area in cm2/g interstitial tissue increased in the testis in early puberty and decreased then in mid-puberty. In the testicular capsule, the exchange vessel surface area per the whole tissue remained unchanged in early pubertal development, but increased then in the mid- and late puberty. The estimates of exchange vessel surface area in cm2/g tissue decreased in early puberty and remained unchanged thereafter. Epididymis. In both the caput and cauda epididymidis, the exchange vessel surface area per whole organ increased in late puberty. There were no signilicant differences in exchange vessel surface area in cm2/g interstitial tissue between the age groups in these tissues. In the corpus epididymidis, the exchange vessel surface area per the whole tissue remained unchanged in early puberty and increased in mid- and late puberty (Table 4). Differences between tissues. Comparison of exchange vessel surface areas per whole organ in the various tissues showed that there was more surface area for transport of IgG in the testicular interstitium and capsule than in any of the epididymal segments and that the various segments of the epididymis were not different from each other in this respect in any of the age groups studied. In early puberty, however, exchange vessel surface area in cm2/g interstitial tissue (Table 3) in the corpus epididymidis was higher than that in the cauda or in the testis parenchyma. In mid-puberty, the exchange vessel surface area in cm2/g interstitial tissue was higher in the testis than in the cauda and in the connective tissue of the testicular capsule. In late puberty, the exchange vessel surface area in cm2/g interstitial tissue was smaller in

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Table 4 Exchange vessel surface areas (S,) as a function of the proportion (c) of exchange vessels of the whole circulation of the tissue and as a function of the exchange vessel radius (r) with estimates (S) of absolute values Age (days) TestiF 20 30 44 60

s* (cm*)

S, (cm’)

21.4 f 112.8 f 178.8 zt 234.2 f

3.7 f 1.9 (6) 19.4 +z 0.9 (6)a 30.7 f 2.0 (6)“,b 40.3 z’z1.3 (6)a,bUc

128.0 f 414.2 f 279.5 f 277.0 f

9.8 3.5 II.7 21.1

+ + f zt

1.4 (6) I.0 (6)a 1.0 (6)b 2.0 (6)a*b.c

332.5 * 23.9 (6)b 152.6 z’z46.9 (6) 224.2 zt 24.5 (6) 234.4 f 14.1 (6)

cr-’ cr-’ cr-’ cr-’

2.7 I.2 3.4 7.2

f + + f

I.1 0.3 0.3 0.5

(6) (6) (6) (6)a.b*c

268.8 f 189.8 f 235.6 f 236.8 f

65.7 (6) 65.8 (6) 22.1 (6) 12.4 (6)

11.1 (6) CT-’ 5.1 (6) CT-’ II.4 (6) CT-’ 7.8 (6) cr-’

Testicularcapsule 20 57.3 f 7.9 (6) CT-’ 30 20.6 f 5.8 (6) cr-’ 44 68.2 zt 5.7 (6) cr-’ 60 122.9 f II.7 (6) cr-’ Caputepididymidis 20 15.5 * 30 6.7 f 44 19.7 f 60 42.0 f Corpusepididymidis 20 6.0 30 4.5 44 20.3 60 38.5

6.3 (6) 1.9 (6) 1.7 (6) 3.0 (6)

S* (cm*/g interstitium)

45.0 (6)b 13.9 (6)” 17.3 (6)a.b II.9 (6)a.b

f 0.9 f I.1 f I.1 z’z2.8

(6) (6) (6) (6)

cr-’ cr-’ cr-’ CT-’

1.0 f 0.8 f 3.5 f 6.6 zt

0.2 0.2 0.2 0.5

(6) (6) (6)“*b (6)a.b,c

l4l2.4 f l287.3 f *172.4 zt *198.4 f

60.4 (6) 105.4 (6) 10.2 (6) 14.5 (6)

Cauda epididymidis 20 3.6 f 2.5 30 9.2 h 0.4 44 14.0 -+ 4.7 60 38.6 f 4.9

(6) (6) (6) (6)

CT-’ cr-’ cr-’ cr-’

0.6 f 1.6 f 2.4 f 6.6 +

0.4 0.1 0.8 0.8

(6) (6) (6) (6)a*b.c

131.3 f 164.4 f 93.5 f 134.1 f

93.0 (6) 15.4 (6) 30.4 (6) 19.2 (6)

The values are mean f S.E.M. The values in parentheses refer to number of epididymides per group. S,, permeability as a function of the proportion (c) of the exchange vessels of the whole circulation and as a function of the exchange vessel radius (r); F, estimate of exchange vessel surface areas. when c = 0.74 and r = 4.3. “p < 0.01 vs. 20-days-old. bp < 0.01 vs. 30-days-old. “p c 0.01 vs. 44-days-old. *p c 0.01 in ANOVA, no differences in Tukey’s test.

the cauda than in the testis, testicular capsule, caput epididymidis and corpus epididymidis. The surface area of the exchange vessels in cm2/g interstitial tissue was also smaller in the corpus epididymidis than in the testicular parenchyma in late puberty. At the end of puberty, the surface area of the

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Table 5 Permeabilities of the testicular and epididymal microvessels to immunoglobulin G as a function of the proportion of exchange vessels of the whole circulation of the tissue and as a function of the exchange vessel radius with estimates of absolute values

Age

PCC

P*

(days)

(nm/min)

(nmmin)

3.5 rc-’ 41.3 rc-’ 31.8 rc-’ 30.3 rc-’

20.6 239.5 184.6 175.5

13.8 rc-’ 19.3 rc-’ 16.7 rc-’ 6.2 rc-’

80.5 III.8 96.6 35.8

0.3 rc-’

3.5 rc-’ 21.4 rc-’ 12.4 rc-’

1.5 20.2 158.8 72.1

Corpus epididymidis 20 0.1 rc-’ 30 9.5 rc-’ 44 18.0 rc-’ 60 1I.6 rc-’

0.6 55. I 104.5 67.4

Testis

20 30 44 60 Testicular capsule 20

30 44 60 Caput epididymidk 20 30

44 60

Cauah epididymidis 20 16.6 rc-’ 30 44 60

9.1 rc-’ 25.7 rc-’ 10.6 rc-’

96.2 53.0 148.8 61.6

P,, permeability as a function of the proportion (c) of the exchange vessels of the whole circulation and as a function of the exchange vessel radius (r). p, estimate of permeabilities, when c = 0.74 and r = 4.3.

vessels involved in substance exchange in cm*/g interstitial tissue was smaller in the cauda than in the testis parenchyma, testicular capsule, caput and the corpus epididymidis, and the exchange vessel surface area (cm2/g) in the corpus epididymidis was smaller than in the testis parenchyma. 3.6. Microvascular permeability to IgG in the testis and epididymis Changes with age. The microvascular permeability to IgG in the testis rose lo-fold in early puberty and thereafter remained at the same level (Table 5).

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In the testicular capsule, an increase in microvascular permeability in mn/min was observed in early puberty, whereafter the permeability gradually decreased. In the caput and corpus epididymidis, the microvascular permeability in mn/min increased > IO-fold during puberty to reach a maximum in late puberty and then decreased slightly towards the end of pubertal development. In the cauda the values for microvascular permeability remained in the same range of magnitude throughout puberty (Table 5). Disferences between tissues. Comparison of the microvascular permeabilities in the various organs (Table 3) suggested differences in the permeability in nm/min between the organs studied. In early puberty, the microvascular permeability in the testicular interstitium, capsule and the cauda epididymidis were clearly higher than those in the caput or corpus epididymidis. In mid-puberty, these values were in the same range of magnitude in all the tissues studied, except for smaller values in the caput epididymidis. In late puberty, the microvascular permeabilities were in the same range of magnitude in all the studied tissues, except for smaller values in the testicular capsule of 60-day-old rats. 3.7. Perivascular mononuclear cell infiltrates in the epididymis The number of focal perivascular mononuclear cell infiltrate profiles (Fig. 1) per 1000 epididymal tubule cross-sections increased with age in the caput epididymidis (Fig. 2). No infiltrates were observed in the corpus or cauda before 60 days of age. The perivascular infiltrates were usually found in the superficial or septal connective tissue, but occasional infiltrates deeper in the epididymis were also observed. 4. Discussion Methodological considerations. To estimate vascular permeabilities it is necessary to take into account at least the following: (1) blood flow or perfusion rate, (2) penetration rates through the capillary wall and diffusion through the extracellular space, (3) cell penetration rates, movement within the cell and chemical rate processes, and (4) lymph removal rate (Wittmers et al., 1976). Because i2%IgG does not penetrate into the cell and 20 h after injection 93% of radioactivity in the tissue can be precipitated by trichloroacetic acid (PiillHnen and Setchell, 1989), the error caused by factors mentioned in point three is considered to be small. Because the distribution volumes were expressed per weight of the extratubular tissue of the testis (Setchell et al., 1987) and per extraluminal tissue of the epididymis (Jiang et al., 1994) instead of the whole tissue weights, the restriction of ‘251-IgGdiffusion (point 2) by the blood-testis and blood-epididymis barriers (Koskimies et al., 1971; Johnson, 1972; Wong et al., 1983; PiillPnen and Set-

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Fig. 1. Focal perivascular mononuclear cell infiltrate in the caput epididymidis of a 60-day -old rat. x 143.

Fig. 2. Incidence of mononuclear cell infiltrate profiles per 1000 epididymal tubule crosssections in histological sections of (a) caput and (b) corpus and cauda epididymides of three rats of different ages. The figures are mean f S.E.M. The figures in parentheses refer to number of tubule cross-sections per group. Infiltrate is > 10 mononuclear leukocytes around a blood vessel.

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chell, 1989; Yeung et al., 1991) can be excluded. The half-times of diffusion were not measured in the various tissues in the present study, but because the observed half-time for accumulation of ‘*‘I-IgG to the tissues is orders of magnitude higher than the usual diffusion half-times (Wittmers et al., 1976), the contribution of diffusion rates to the present observations is probably small. The half-time of the flow-limited process is - 1 min for ‘*‘I-IgG in the adult rat testis as calculated from the equation tl12= 0.693 V,,IF (Wittmers et al., 1976) using the present V, (pi/g) and the blood flow (F, &g/min) measured by Damber et al. (1985), demonstrating that the role of the flowlimited process is negligible in transfer of *251-IgGto the testis. This is in accord with the electron microscopical observations that only 14.9% of interendothelial junctions have expanded clefts, possibly allowing substance penetration (Holash et al., 1993). Because the blood flow values in the testis are higher than those in the epididymis (Setchell et al., 1964; Waites and Setchell, 1966; Jaffe and Free, 1979) and the maximal volumes of distribution in the testis are higher than in the epididymis as observed in the present study, the half-time of flow-limited processes may be in the same range in the epididymis as in the testis, i.e. much smaller than the observed half-time of accumulation of ‘251-IgGto these tissues. Because the role of the flow-limited process is small in transfer of ‘251-IgGto the testis, possible heterogeneity of flow to the various parts of the testis (Desjardins, 1989) would not significantly affect the obtained permeability values. The effect of lymph flow on the present estimates cannot be excluded, because a possible factor affecting lymph flow in the proximal epididymis is the absorption of water from the lumen by the epithelial cells. Since at equilibrium influx equals efflux, the lymph flow reflects changes in the influx of fluid and if the volume of fluid absorbed from the lumen is significant in comparison with the amount arriving in the caput epididymal interstitial tissue from the blood circulation, lymph flow in the caput epididymidis may be higher than that estimated from Eq. 3 and therefore, the measured accumulation of marker to the tissue may be slower. However, the small permeabilities obtained in the early pubertal caput epididymidis are unlikely to reflect absorption of water from the lumen, because testicular fluid secretion has not started at this age (Setchell and Waites, 1971) and therefore the absorptive function of the epididymal epithelial cells is probably low. The increasing K values with age in the caput and corpus also do not reflect arrival of marker in the luminal fluid, because in the adult rat the transport of a marker from the rete testis to the terminal caput epididymidis takes 48 h (English and Dym, 1984). A mathematical model for expressing permeability (units of distance/unit of time) as a function of exchange vessel radius (r) and proportion (c) of the

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whole circulation made up by the exchange vessels was developed in the present study. Although in this model the r of the exchange vessels was assumed to be constant, which will not exactly be the case in nature, this does not affect the estimation of permeabilities (P) and surface areas (S), because the standardization of r affects the values of microvascular length (Z) in the model (Eq. 8) and the estimates of P and S were calculated as a function of 1(Eqs. 10, 14 and 15). Therefore, the estimates obtained for P in nm/min and S in cm* are real in spite of the standardization of r. Errors in the absolute values of S and P may, however, be created if the proportions of the exchange vessels of the whole circulation are different from testis in the other tissues studied. With this reservation the model should provide valid estimates. The standardized r of exchange vessels in the present model (4.3 pm) is similar to morphometrical estimates of r for capillaries in the testis (3.8 pm; Holash et al., 1993) and epididymis (caput, 4.6 pm; corpus, 4.4 pm; cauda, 4.1 pm; Markey and Meyer, 1992). The present estimates of P in run/mm are the first to be reported for testicular and epididymal microvessels. In comparison to other models for estimation of microvascular permeability, the equation given in the present study, P = 0.5rKVeq/cV3tin (P= KVeqIS) is of the same form as that of Wittmers et al. (1976) for non-flowlimited transport of test molecules (P= 0.693f1,2-’ VJS). From these, K = 0.693 t,,* -’ (K= ln[l - V,lV,,]lt), demonstrating that equations for calculation of P used in the present study and in the study of Wittmers et al. (1976) are similar. The surface areas of testicular and epididymal microvessels were estimated for the first time in rats of different pubertal ages in the present study. The adult testicular capillary and postcapillary pericytic venule surface area has been estimated previously (Damber et al., 1985) and this estimate was used in calculation of the value for standardized r in the model used. It is correct to use the surface area of these two types of microvessels for estimation of r, because both types of microvessels are involved in exchange of substances from blood to the interstitial space (Wiedeman, 1963). The estimates of the exchange vessel surface areas in the present study were derived mathematically from the data on 3-min volumes of distribution, r and c. The surface area per gram interstitial tissue obtained in this way decreased similarly along the epididymis as did the surface area per pm3 tissue obtained using morphometry (Markey and Meyer, 1992). The absolute values of PS to IgG obtained in the adult rat testicular interstitium were comparable in size with those in implantation and nonimplantation sites of the mouse uterus (Bany and McRae, 1992), but those in the testicular capsule, caput, corpus and cauda epididymidis were smaller by a range of magnitude, suggesting that there is less flow of IgG through

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the epididymal than the testicular interstitium. The value of lymph flow obtained from the data of Galil et al. (1981) for the adult rat testis per gram interstitial tissue using the equation QL = 0.693 V&r0 (tin = the half-time of clearance of i.t. injected 1251-albumin, Ves= the volume of distribution of i.v. injected ‘*%albumin in the testis at equilibrium) was similar (4.465 &nin/g interstitial tissue) to the value obtained using Eq. 3 in the present study (4.726 @nin/g interstitial tissue), demonstrating that QL = PS in the testis. However, the value for testicular lymph flow given by Maddocks et al. (1987) for rats weighing 280-570 g was 6.804 ~l/rnin/g interstitial tissue, suggesting that the flow through the testis may still change after 60 days of age. The precondition for estimation of lymph flow using Eq. 3, that all the marker leaves the testis via lymphatics, is fulfilled, because almost all albumin injected into the testis is recovered from lymph draining the organ (Setchell and Zupp, unpublished observation, cited in Maddocks et al., 1987). Physidogicd significance. The present results clearly indicate that there are differences in the speed of penetration of IgG into the peritubular fluid of the testis and epididymis between early and late pubertal rats. The observed differences between tissues and ages may reflect structural or functional differences in the microvascular wall in the various locations, and may be due to changes in the hormonal environment during puberty (Lee et al., 1975; Foldesy and Leathem, 1981). For example, androgen receptors are expressed in the smooth muscle cells of rat testicular small arteries (Bergh and Damber, 1992), suggesting that regulation by testosterone of the proportion of capillaries closed by the terminal arteriolar muscle might occur in the testis, because the permeability surface area (PS) product increases between 20 and 30 days of age, when testosterone synthesis increases (Lee et al., 1975). However, if only androgen-induced opening of more capillaries were to explain the increase in PS product of testicular interstitial vessels between 20 and 30 days of age, the PS for testicular interstitial tissue should decrease sharply between 30 and 44 days of age, because the estimates of surface area of testicular exchange vessels per gram interstitial tissue decreased significantly after 30 days of age. As PS did not decrease in the testicular interstitial tissue after 30 days of age, androgen-regulated opening and closure of capillaries cannot solely be responsible for the observed changes in PS. Therefore, active regulation of substance transfer by testicular endothelial cells may take place, as suggested by the present estimates of permeabilities in the testis of 20- and 30-day-old rats. Although androgen receptors are expressed by a testicular endothelial cell line in vitro (Nakhala et al., 1984), they cannot be demonstrated immunohistochemically in vivo (Bergh and Damber, 1992), suggesting that if androgen regulation of testicular endothelial cell function occurs, it is probably indirect. The present observations also suggest an active role for endothelial cells

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in the proximal parts of the epididymis in the transfer of substances to the peritubular fluid, because the pattern of pubertal development of the penneabilities in these tissues is clearly different from that in the cauda or testicular capsule. The increase in estimates of microvascular permeability and lymph flow between 20 and 44 days of age in the proximal and middle thirds of the epididymis may not be related to fenestrations in the subepithelial capillaries, because these are present in every segment of the intact adult rat epididymis (Hoffer and Hinton, 1984). However, it may reflect the different source of vasculature to these parts of the epididymis (Kormano, 1968b; Chubb and Desjardins, 1982), namely that the caput epididymidis receives its vascular supply from the spermatic artery (MacMillan, 1952) and levels of testosterone in the superior epididymal branch of this artery in the rat are higher than those in the general circulation owing to transfer of testosterone from the testicular veins in the pampiniform plexus (Free and Tillson, 1975). The initial segment may also be exposed to higher concentrations of testosterone because of the anastomoses of veins in this region to the testicular capsular venous plexus (Chubb and Desjardins, 1982) and in rabbits the capillaries in the initial segment depend on androgen binding protein-related androgens reaching the epididymis through the luminal route (Clavert et al., 1981). Because the cauda epididymidis only receives its vascular supply from the internal iliac artery (MacMillan, 1952), where concentrations of testosterone are the same as in the peripheral circulation, it is possible that a different pattern of development is seen in the microvessels of this part of the male reproductive tract due to differences in local testosterone concentrations. The present studies do not exclude the possibility that the arrival of spermatozoa in the epididymis affects vascular permeability by modulating secretion of vasoactive factors by the epithelial cells, but evidently the bioactive peptides derived from pro-enkephalin, the expression of which by the epididymal epithelial cells is regulated by luminal sperm (Garrett et al., 1990), do not increase vascular permeability, but rather decrease it, because opioid receptor agonists inhibit extravasation (Khalil and Helme, 1990). Further studies are required to reveal the agents involved in regulation of microvessels in the epididymis. The significant increase in volumes of distribution at equilibrium, i.e. the volume of extratubular extracellular fluid accessible to IgG in the testis in early puberty, probably reflects the formation of the testicular interstitial lymphatic sinusoids. In the caput epididymidis, the volume of extraluminal extracellular fluid (V,,) increased between 30 and 60 days of age, suggesting that the volume of extracellular fluid in the extraluminal part of the caput epididymidis may increase when spermatozoa appear in the lumen. Whether this reflects an increase in fluid absorption by the epididymal epithelium and the related increase in lymph flow or an immune response to the appearance

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of autoantigenic sperm in the lumen and an increase in capillary permeability remains to be clarified. Interestingly, it was also observed in the present study that the incidence of perivascular mononuclear cell infiltrate profiles per 1000 tubule cross-sections doubled in parallel with the increase in extraluminal extracellular fluid volume between 30 and 60 days of age. This observation may be significant in relation to endothelial function, because activated macrophages and lymphocytes secrete cytokines that are able to increase vascular permeability (Burke-Gaffney and Keenan, 1993). The present finding that focal perivascular mononuclear cell infiltrates are present in the normal rat caput epididymidis and that their incidence per 1000 tubule cross-sections slightly increases with age during puberty is surprising. Some small perivascular infiltrates could already be observed in the epididymal connective tissue before appearance of mature spermatozoa in the lumen, suggesting that their presence may be involved in the normal turnover of epididymal epithelial lymphocytes (Dym and Romrell, 1975), which appear to the epithelium at the same age (Leeson and Leeson, 1964; Sun and Flickinger, 1979). In this regard, the basal cells of the epididymal epithelium have recently been shown to express a tissue-fixed macrophage-specific antigenie determinant (Yeung et al., 1994), suggesting that the basal cells, like the lymphocytes, may also migrate to the epididymal epithelium from the bloodstream. As cells resembling morphologically immature spermatocytes could occasionally be seen in the lumen of the caput of the early pubertal rats, an immune response against germ cell autoantigens cannot be excluded. All the infiltrates were perivascular and focal, however, which suggests that the lymphocytes were well under control in the extratubular epididymal environment. The contribution of the endothelial cells of the microvasculature surrounding the male reproductive tract to the blood-testis and blood-epididymis barriers has repeatedly been discussed (Goldacre and Sylven, 1962; Kormano, 1967a,b; Setchell et al., 1987, 1990; Poll&en and Setchell, 1989; Holash et al., 1993) and not without reason. The testicular endothelial cells express several transporter and pump molecules known to be specific for endothelia that do not permit passive diffusion (Niemi and Setchell, 1986; Holash et al., 1993) and only 14.9% of inter-endothelial cell junctions have expanded clefts (Holash et al., 1993) that may permit paracellular transport of molecules < 10 nm in diameter. The physiological blood-testis barrier also does not develop as rapidly as the formation of the inter-Sertoli cell tight junctions (Setchell et al., 1987) and different amino acids are transported through the endothelium at different rates (Bustamante et al., 1982; Setchell et al. 1984), suggesting an active contribution of microvessels to the regulation of substance transfer to the tissue surrounding the seminiferous tubules. The present results support the suggestion of an active role of endothelial

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cells in regulation of substance transfer to the peritubular environment at puberty, but they also indicate that the testicular endothelium as a whole is not an immunological barrier in regard to transfer of ‘*‘I-IgG in normal conditions. However, it remains open as to whether IgG is transported through expanded inter-endothelial cell clefts (Holash et al., 1993) or by transcytosis (Weihe et al., 1979), or both. Because the Stokes radius of immunoglobulin G is 5.6 nm, steric exclusion will allow at most one, if any, IgG molecule to go through the expanded inter-endothelial junctional cleft (width 10 nm; Holash et al., 1993) at one time. If the testicular endothelium is similar to others in this respect, the intercellular spaces make up only < 1% of the endothelial surface area (Deetjen and Speckmann, 1994) and if only 14.9% of the inter-endothelial cell junctions are open (Holash et al., 1993) less than 0.15% of the exchange vessel surface area in the testis is composed of open inter-endothelial cell junctions. Using the dimensions for open inter-endothelial cell junctions given by Holash et al. (1993) it can then be calculated that there are <4 x 10” open interendothelial cell junctions/g interstitial tissue in the adult rat testis and that > 2 x 1O-25 m3 (- 10 IgG molecules) flow through one open interendothelial cell junction per second, if transcytosis is not involved in transport of IgG to the testicular interstitium. In conclusion, the present study suggests that the rat testicular and epididymal endothelial cells may play an active role in regulation of transfer of IgG and other circulating proteins to the interstitial connective tissue of the male reproductive tract and that this transfer function of the endothelial cells is subject to regulation at puberty. Further investigation of the factors regulating endothelial cell functions in the male reproductive tract is essential to understand the role of these cells in the immunology and endocrinology of the male reproductive tract. Acknowledgments The authors thank Professor E. Nieschlag (Institute of Reproductive Medicine, Westfalian Wilhelms-University, Miinster) for critical review of the manuscript. P. Poll&ten’s work was supported by the Alexander von Humboldt Foundation, the University of Turku Foundation, the Jalmari and Rauha Ahokas Foundation and the Deutsche Forschungsgemeinschaft (grant no: Ni 130/11-3C, TGC). References Amtorp, 0. (1980) Estimation of capillary permeability of inulin, sucrose and mannitol in rat brain cortex. Acta Physiol. Stand. 110, 331-342.

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