Functional maturation of the epididymis in the rat

Transcription

1 Functinal maturatin f the epididymis in the rat B. S. Setty and Q. Jehan Divisin fendcrinlgy, Central Drug Research Institute, Lucknw, India Summary. Weight, histlgical and bichemical changes in the rat epididymis were investigated during prepubertal, pubertal and pstpubertal perids. The phase f mst rapid grwth f the epididymis cmmenced at 21 days and extended t 60 days f age; this perid crrespnded clsely t the nset f andrgen prductin at 3 weeks and stabilizatin f the Leydig cell number at 60 days. Histlgical differentiatin in the caput epididymidis started befre sperm entry and was cmplete in the cauda nly several days after the spermatza had appeared. The presence fappreciable quantities f glycerylphsphrylchline (GPC) and sialic acid in the epidiymis f 21dayld rats suggests inherent secretry ability f the epididymal epithelium. The cncentratins f GPC, sialic acid, phsphlipids and glycgen in the epididymis gradually increased with age, but each came under the influence f andrgen at a different age. There was n evidence t suggest that the presence f spermatza has a stimulatry effect n the epididymis. Maximal secretry activity f the epididymis became established nly by 90 days f age. Intrductin Althugh histlgical and bichemical changes that ccur during testicular maturatin are well dcumented, little is knwn abut the preparatry changes that take place in the epididymis in the prepubertal rat. The epididymis, like the accessry sex glands, is an andrgendependent rgan (Bishp, 1961), and ther testicular prducts, e.g. rete testis fluid and/r spermatza, may als affect epididymal physilgy (Ellit, 1965; Waites & Setchell, 1969). Reid (1959) has studied the histgenesis f the rat epididymis frm birth t puberty and Brks (1976) has examined the changes thrughut the whle excurrent duct system. We have investigated the functinal maturatin f the epididymis while critical events are ccurring in the gametgenic and endcrine cmpartments f the testis. The present study cvers the perid frm the time the epididymis first becmes influenced by andrgens t that when the histlgical differentiatin in all epididymal regins is cmplete. Epididy mal maturatin at different ages was als cmpared with that f the accessry sex glands. Materials and Methds Clnybred male rats f knwn birth date were killed at 21, 35 and 42 days (prepubertal) and 60,90 and 120 days (pubertal) f age. T examine the respnse f the epididymis t exgenus andrgen, prepubertal animals were treated i.m. with 100 r 150 pg teststerne prpinate/day frm the 14th r 28th day f age, respectively, daily fr 7 days (Table 1). All the rats were reared under unifrm husbandry cnditins in aircnditined (24 ± 1 C) quarters. At autpsy the testes, caput and cauda prtins f the epididymis, seminal vesicles, ventral prstate and cagulating gland were excised and weighed. The testes and epididymal segments were fixed in Buin's fluid and the sectins were stained with haematxylin and esin. Levels f ttal prtein (Umbreit, Burris & Stauffer, 1959), sialic acid (Aminff, 1961), glycgen (Mntgmery, 1957), phsphlipid (Hawk, Oser & Summersn, 1957) and glycerylphsphrylchline (GPC) (White, 1959) were measured in epididymal tissue. Alkaline phsphatase activity in the ventral prstate and fructse cncentratin f the cagulating gland were taken as indices f secretry

2 activity f the accessry sex glands. The activity f alkaline phsphatase was measured accrding t the prcedure f Hawk et al. (1957) using sdium ßglycerphsphate as substrate and is expressed as mg phsphrus released/h at 37 C. The technique f Mann (1946) was used t determine fructse cncentratin. Fr the 21dayld grup, tissues frm 3 r 4 animals were pled fr each estimatin and each cnstituent was determined frm 68 such pls. Fr the ther grups, each cnsisting f 68 rats, bth epididymides f each animal were cmbined fr the estimatins. The results were assessed by Student's t test and a level f 5 % was taken t be significant. Weight changes Results A significant increase in weight f the testis, epididymis and accessry sex glands was fund at all the ages studied (Table 1); maximum increase (200%) ccurred fr the testis between 21 and 35 days, and fr the epididymis between 35 and 60 days. Treatment with teststerne prpinate fr 7 days at 14 and 28 days f age caused an increase in the weight f the caput and cauda epididymidis and in the weight and secretry activity f the accessry genital rgans. Histlgical changes Spermatgenesis had reached the stage f pachytene spermatcytes by 21 days, thrugh the early steps f spermatid develpment by 35 days and was cmplete by 60 days f age. In the interstitium there was a prepnderance f fibrblastlike cells at 21 days but nly a few fully develped Leydig cells. There was a gradual increase in the number f fully differentiated Leydig cells with age, reaching a maximum at 60 days. In 21dayld rats the tubular diameter f the epididymis was narrw and the lumen was lined by cubidal r clumnar cells which were arranged in ne r tw layers. Sterecilia had appeared in sme tubules f the caput but nne was present in the cauda epididymidis. By 35 days there was an increase in the tubular diameter and the initial segment f the caput epididymidis shwed distinct adult features althugh the epithelial cell height was relatively lw. In the distal caput regin a strati fied epithelium was seen nly in a few areas and the lumen cntained a few spermatids. In the cauda regin the epithelium was 24 cells thick and the cells were f the clumnar type. In 60dayld rats the caput epididymidis shwed all the adult features, including spermatza, but it was nt until 90 days that the cauda had differentiated int the adult type and scalled light r clear cells culd be seen in the epithelium. In the prximal cauda epididymidis the size f the tubules was greater than that in the caput and the epithelial cells were lw and clumnar, while in the distal cauda the tubular diameter was maximal and the lumen was lined by cubidal epithelium. Treatment with teststerne prpinate resulted in a marked increase in tubular size and there was evidence f hypertrphy and hyperplasia. The number f mittic figures increased by abut 65 % at 21 days and by 100 % at 35 days cmpared with the respective untreated cntrls (mittic figures/ 100 tubules in the cntrls at 21 days: caput 12, cauda 6; at 35 days: caput 4, cauda 3). Andrgen treatment failed t induce differentiatin f the epithelium in the initial segment at 21 days but hastened the appearance fsterecilia in the caput and cauda regins. Bichemical changes There was a significant increase in sialic acid cncentratin in the caput epididymidis up t 42 days fllwed by a decline at 60 days and the level remained unaltered during puberty. In cntrast, there was a gradual but insignificant rise with age in the cauda regin. In all the age grups (excepting 42 days) sialic acid cncentratin was higher in the cauda epididymidis (P < 001 ). A marked increase in glycgen level was seen up t 90 days in bth the segments f the epididymis. The first significant rise in phsphlipid cntent was seen at 60 days and the level was maintained until 120 days. At 35 days and later, the cauda epididymidis cntained higher cncentratins f

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4 phsphlipids. The cncentratin f GPC rse slightly at 35 days; a marked rise was bserved at 42 days but peak values were attained nly by 90 days. In the prepubertal rats GPC cncentratin was relatively higher in the cauda epididymidis (P < 005) but with age the difference between the tw segments became negligible. Treatment with teststerne prpinate (Table 1) resulted in a significant rise in the phsphlipid and GPC cncentratins f the epididymis but it failed t alter the levels f glycgen and sialic acid. There was a marked increase in the secretry activity f the accessry sex glands in immature rats fllwing andrgen therapy. Discussin The present study shws that the functinal maturatin f the epididymis and accessry sex glands f prepubertal rats ccur tgether. The first phase f rapid grwth f the epididymis (up t 60 days) crrespnds clsely t the perid between the nset f andrgen prductin and the attainment f high circulating levels f teststerne and dihydrteststerne (Knrr, VanhaPerttula & Lipsett, 1970; Gupta, Zarzycki & Rager, 1975). The increase in accessry rgan weight up t 60 days was equivalent t thse f the epididymis and testes and thereafter the grwth f the accessry sex glands exceeded that f the epididymis. The weight changes in the accessry rgans and epididymis up t 60 days f age prbably reflect true tissue grwth, whereas in sexually mature rats they reflect bth the tissue grwth and an increase in their secretry prducts. This suppsitin is substantiated by the present bservatins n teststernetreated prepubertal rats and thse n adult castrated rats (Rajalakshmi & Prasad, 1968 ; Brks, Hamiltn & Mallek, 1974). The Leydig cells are functinal in 21dayld rats (Knrr et al, 1970; Pdestà & Rivarla, 1974) althugh the epididymis is still histlgically undifferentiated. At this age, hwever, dihydrtes tsterne and cytplasmic receptrs appear (Calandra, Pdestà, Rivarla & Blaquier, 1974), tgether with increased levels f andrgenbinding prtein (ABP) (Hanssn et al, 1974), in the caput regin, suggesting that the epididymis is already under the influence f andrgens. The number f fully differentiated Leydig cells increases by 35 days when plasma cncentratins f teststerne and dihydrteststerne are reprted t be rising (Gupta et al, 1975). Histlgical differentiatin was at an advanced stage in the caput regin while the cauda epididymidis cntinued t shw infantile features. The presence f spermatids in the epididymal ducts at this age suggests that testicular fluid, pssibly cntaining andrgen (Cper & Waites, 1974), was entering the epididymis in significant quantities (see Setchell, 1970), thus facilitating early differentiatin f the caput regin. The develp ment f sterecilia appears t be andrgenmediated since their appearance in prepubertal rats culd be hastened by exgenus teststerne. Spermatza were present in the epididymal tubules f 60dayld rats. Peak levels f ABP in the caput are seen arund this age (Hanssn et al, 1974) and the cncentratin f dihydrteststerne within the epididymal tissue is abut 5fld mre than at 35 days (Calandra etal, 1974). These findings suggest that the rat attains puberty by 60 days f age, bth in terms f gametgenic and endcrine activities. The delay f histlgical differentiatin f the cauda epididymidis int the adult type until 90 days culd be due t a lw receptr cncentratin in the cauda epididymidis f grwing animals r t a higher requirement f andrgen by this regin (Gupta, Rajalakshmi & Prasad, 1974). The presence f appreciable quantities f GPC and sialic acid in the epididymis f 3weekld rats, befre its histlgical differentiatin and testicular andrgen prductin, suggests inherent secretry ability f the epithelium. At this age the epididymis and the accessry rgans have the capacity t respnd t andrgen stimulatin. It is, hwever, nly in the 6th pstnatal week that andrgeninduced stimulatin f epididymal secretry activity becmes evident (Hall & Gmes, 1973 ; Gupta et al, 1975), and this is well in advance f sperm entry. A secnd rise in GPC level between 6090 days is pssibly due t a further increase in the cncen tratin f andrgens in the circulatin (Ghanadian, Lewis & Chishlm, 1975) and in the epididymal tissue itself (Calandra et al, 1974). The significant increase in epididymal GPC cncentratin in the andrgentreated prepubertal rats supprts this view, because spermatza d nt participate in the

5 accumulatin f GPC in the epididymis (Dawsn & Rwlands, 1959; Brks et al, 1974). Until 60 days f age the cauda epididymidis cntained relatively higher cncentratins f GPC. Althugh the caput epididymidis is reprted t be mre active in GPC synthesis (Dawsn & Rwlands, 1959), a higher cncentratin is t be expected in the cauda if GPC is even partly respnsible fr maintaining smtic pressure ffluid in this regin (Riar, Setty & Kar, 1973). A rise in the sialic acid level in the caput epididymidis until Day 42 fllwed by a significant fall at 60 days suggests a dilutin impsed by testicular fluid, and ligatin f the efferent ducts befre the nset f puberty prevents this fall in sialic acid (Rajalakshmi & Prasad, 1969). Since nearly 80 % fthe testicular fluid is resrbed in the prximal epididymis (Hwards, Jhnsn & Jessee, 1975), n dilutin in the sialic acid level is t be expected in the cauda epididymidis at puberty ; at all the ages examined the cauda generally cntained higher sialic acid cncentratins. Previus investigatrs have reprted that the sialic acid cncentratin in the epididymis is andrgendependent (Peyre & Laprte, 1966; Rajalakshmi & Prasad, 1968). The present study, hwever, has shwn that andrgen des nt influence the sialic acid level in the epididymis f prepubertal rats : its cntinued rise in the epididymis even befre sperm entry indicates that this cnstituent is lcally synthesized. Brks (1976) has als reached a similar cnclusin. Our findings n the amunts f prtein and glycgen in the epididymis f maturing rats are cmparable t thse f Brks (1976). Glycgen in the epididymal epithelium has been cnsidered as an energy reserve (Brks, 1976), and as a surce f the glucse6phsphate needed fr pentse phsphate activity in this rgan (Shll & Leathern, 1973). The rise in phsphlipid cncentratin at 60 days f age des nt seem t be due t sperm entry because efferent duct ligatin is reprted nt t alter epididymal phsphlipid level (Turner & Jhnsn, 1971) ; the elevated andrgen cncentratins in the mature rat are prbably the stimulating factr. This investigatin was supprted by a grant frm the Ministry f Health and Family Planning, Gvernment f India. We thank Dr Nitya Nand, Directr fr his interest in the study, Prfessr G. M. H. Waites fr his hlepful suggestins in the preparatin f the manuscript and Mrs S. F. Daltn fr typing it. References Aminff, D. (1961) Methds fr the quantitative estimatin f Nacetylneuraminic acid and their applicatin t hydrlysates f sialmucids. Bichem. J. 81, Bishp, D.W. (1961) Bilgy f spermatza. In Sex and Internal Secretins, 3rd edn, Vl. II, pp Ed. W. C. Yung. Williams and Wilkins, Baltimre. Brks, D.E. (1976) Changes in the cmpsitin f the excurrent duct system f the rat testis during pst natal develpment. /. Reprd. Fert. 46,3138. Brks, D.E., Hamiltn, D.W. & Mallek, A.H. (1974) Carnitine and glycerylphsphrylchline in the reprductive tract f the male rat. /. Reprd. Fert. 36, Calandra, R.S., Pdestà, E.J., Rivarla, M.A. & Blaquier, J.A. (1974) Tissue andrgens and andrphilic prteins in rat epididymis during sexual develpment. Sterids 14, Cper, T.G. & Waites, G.M.H. (1974) Teststerne in rete testis fluid and bld f rams and rats. /. Endcr. 62, Dawsn, R.M.C. & Rwlands, I.W. (1959) Glyceryl phsphrylchline in male reprductive rgans f rats and guinea pigs. Q. Jl exp. Physil. 44, Ellit, P.R. (1965) Effect f efferentiectmy and rchidectmy n the metablism f the epididymis f the muse. /. cell. cmp. Physil. 66, Ghanadian, R., Lewis, J.G. & Chishlm, G.D. (1975) Serum teststerne and dihydrteststerne changes with age in rat. Sterids 25, Gupta, D., Zarzycki, J. & Rager, K. (1975) Plasma teststerne and dihydrteststerne in male rats during sexual maturatin and fllwing rchidectmy and experimental bilateral cryptrchidism. Sterids 25,3342. Gupta, G., Rajalakshmi, M. & Prasad, M.R.N. (1974) Reginal differences in andrgen threshlds f the epididymis f the castrated rat. Sterids 24, Hall, R.W. & Gmes, W.R. (1973) Teststerne levels in the serum and testes f grwing rats fllwing prenatal expsure t Busulphan. /. Reprd. Fert. 35, Hanssn, V., Trygstad, O., French, F.S., McLean, W.S., Smith, A.A., Tindall, DJ., Weddingtn, S.C, Petrusz, P., Nayfeh, S.N. & RiTzéN,.. (1974) Andrgen transprt and receptr mechanisms in testis and epididymis. Nature, Lnd. 250,

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