J. Anat. (1969), 104, 2, pp. 253- 262 253With 13 figuresPrinted in Great Britain

A histochemical study of human seminal vesicle epithelium

A. RIVA* AND R. A. STOCKWELLt

Department of Anatomy, St Thomas's Hospital Medical School, London

(Received 1 May 1968)

INTRODUCTION

Biochemical investigations have shown (Mann, 1964) that the seminal vesiclesplay an important role in the formation of seminal plasma. In man, constituents ofthe semen such as fructose (Mann, 1946) and sialic acid (Warren, 1959) have beenattributed to seminal vesicle secretion. In spite of this, no studies concerning thehuman seminal vesicle have been found in the histochemical literature apart fromincidental reports (Glenner, Folk & McMillan, 1962) and scattered data mentionedby Lillie (1965).The present histochemical investigation of the epithelium, which supplements an

examination of its fine structure (Riva, 1967), has been particularly focused on sub-stances and enzymes related to fructose metabolism; other constituents of seminalvesicle epithelium such as ribonucleoproteins, lipids, lipofuscins, and sialic acid con-taining mucins have also been studied.

MATERIALS AND METHODS

Specimens of seminal vesicle from ten patients aged 53, 54, 55, 56, 57, 57, 59, 62, 72and 78 years were collected at cystectomy or retropubic prostatectomy and immedi-ately fixed or processed. Care was taken to avoid subjects with a clinical history ofprostato-vesiculitis. Routine histological techniques were carried out to ascertain thatinflammation or neoplastic infiltration were absent in the organs studied.

Ribonucleic acid. Tissue fixed in Carnoy's fluid was stained by the methyl green-pyronin method (Pearse, 1960). Control sections were treated with 1% crystallineribonuclease prior to staining.

Fats. Lipids were demonstrated in formol-calcium-fixed frozen sections with Sudanblack in propylene glycol and with Nile blue sulphate. Phospholipids were investi-gated with the acid haematein technique using pyridine-extracted control material.These techniques were performed according to Pearse (1960).

Lipofuscins. The following histochemical techniques (Pearse, 1960) were performedon tissue fixed in formol-calcium, Bouin's or Carnoy's fluid and embedded in paraffinwax: Schmorl, Ferrous iron (Lillie), Masson-Fontana silver, Nile blue (Hueck),Alternative Nile blue (Lillie), and Sudan black in propylene glycol. Bleaching re-actions and the long Ziehl Neelson method were also used. In addition unstainedmounted sections were examined in ultra-violet light.

* Present address: Instituto di Anatomia Umana Normale, Via Porcell 2, Cagliari, Italy.t Present address: Department of Anatomy, University Medical School, Edinburgh.

254 A. RIVA AND R. A. STOCKWELL

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Histochemistry ofhuman seminal vesicleGlycogen. Glycogen was studied in paraffin-embedded material fixed in Gendre,

Rossman or Carnoy's fluids. To avoid a possible loss of glycogen during preparativestages (Filotto, 1962) 95 % ethanol was employed, in some instances, to float sectionsbefore mounting on slides. Similarly, an alcoholic periodic acid solution was used inperforming the periodic acid-Schiff (PAS) technique. Besides PAS, Best's carmineand Carleton's iodine methods (Pearse, 1960) were carried out; controls were digestedwith diastase or with saliva.

Mucins. The following staining techniques were used on paraffin wax sections oftissue fixed in 10 % formalin (phosphate buffered, pH 7 2) or in Gendre fluid: PAS;Alcian Blue at pH 2 5, 1 0 and 0-5; Alcian Blue at different critical electrolyte con-centrations (Scott & Dorling, 1965); Azure A, 0-02% at pH 3 0 and 2-0; the Halereaction (Mowry, 1958). Control sections were treated with neuraminidase (CholeraFiltrate 240 units/ml SIGMA) for 24 h at 37 °C before staining with PAS, Alcianblue at pH 2-5 and Hale's colloidal iron.Enzymes. Acid and alkaline phosphatase were investigated with a modification

(Burstone, 1962) of the Gomori metal precipitation methods. Diazo methodsemploying a-naphthyl phosphate (Pearse, 1960) were also carried out. Non-specificesterases were demonstrated using naphthol AS-LC acetate as substrate (Burstone,1962). Controls were incubated in substrate-free medium; in the case of acid phos-phatase, a complete medium containing 0 01 M sodium fluoride as inhibitor was alsoused. Neutral formalin-fixed frozen sections were employed.For the demonstration of glucose and fructose-6-phosphatase activity, unfixed

frozen sections were incubated either in Wachstein & Meisel's (1956) or in a modified(Fouquet & Wegmann, 1964) Chiquoine medium. Controls were prefixed in formalinfor 30 min or were incubated in a substrate-free medium. A study of phosphatases wasalso carried out both on unfixed and formalin fixed material in Gomori lead-containing media at pH 5, 5-5, 6, 6-5, 7, 7.5, 8, 8-5, 9, 9*5 containing x-glyceropho-sphate (0-01 M), ,-glycerophosphate (0 01 M), glucose-6-phosphate (Lights, 0 002 M)or fructose 6-phosphate (Boehringer, 0 002 M) as substrates. Acetate (pH 5, 5-5), tris-maleate (pH 6 to 8) and tris-HCl (pH 8 5 to 9-5) buffers were used at 0'05 Mconcentration.

Glucose-6-phosphate and 6-phosphogluconate dehydrogenases were demonstratedon unfixed frozen sections (Wegman & Gerzeli, 1961); lactate and succinate dehydro-genases were studied (Pearse, 1960) using Nitro-blue tetrazolium (Nitro BT; G. T.Gurr) as electron acceptor with an incubation time of 20 min. Controls were incubatedin a substrate-free medium.

Fig. 1. Diffuse sudanophilia in epithelial cell cytoplasm. Sudan black.Fig. 2. Note the intense positive reaction to the acid haematein technique. Compare with Fig. 1and with Fig. 3. Acid haematein.Fig. 3. Pigment granules are stainable with acid haematein even after pyridine extraction. Acidhaematein after pyridine extraction.Fig. 4. Persistence of sudanophilia due to pigment in paraffin embedded material. Sudan black.

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Histochemistry of human seminal vesicle 257

RESULTS

The cytoplasm of epithelial cells assumes a purple red colour with the methylgreen-pyronin method. This pyroninophilia disappears after ribonuclease extractionand results therefore from the presence of ribonucleoproteins.Many sudanophilic droplets (Fig. 1) of varying size are scattered throughout all

parts of the epithelium. Many of them are removed by pyridine extraction and appearto be mainly phospholipids, since most of them are stained blue by both the acidhaematein (Fig. 2) and nile blue methods.

In addition to the lipid granules large irregular bodies are observed which are

Table 1

RNAMethod Result

Methyl green-pyronin Y +Methyl green-pyronin Y withribonuclease digestion

Carbohydrates and mucinsReactions Result

PAS +PAS with diastase digestion +Best's carmineCarleton's iodineAlcian blue at pH 2-5, and at pH 1 -

Alcian blue at 0-2, 0 4, 0 7 MgC12 -

Hale's colloidal iron +Hale with neuraminidase digestion +Azure A at pH 3, and at pH 2

LipidsMethod Result

Sudan black BNile blue sulphateAcid haemateinAcid haematein after pyridineextraction

resistant to pyridine extraction

Blue

PigmentsMethod Result

Bleaching methodsSudan black in paraffin sections +Hueck's nile blue +Lillie's nile blue + for

lipofuscinsLong Ziehl-Neelson method +Schmorl's methodFerrous IronMasson-Fontana silver

EnzymesReaction Result

Acid phosphataseAlkaline phosphataseGlucose-6-phosphataseFructose-6-phosphataseNon-specific esteraseGlucose-6-phosphate dehydrogenase6-phosphogluconate dehydrogenaseLactate dehydrogenaseSuccinate dehydrogenase

+

+++++

(Fig. 3) and stainable with sudan dyes in paraffinsections (Fig. 4). These bodies have a pale yellow colour in unstained sections andexhibit a strong yellow fluorescence when examined in ultraviolet light. They give apositive result with the Ziehl-Neelson (Fig. 5) and nile-blue (Fig. 6) methods for

Fig. 5. Acid fast pigment granules. Long Ziehl-Neelson method.Fig. 6. The pigment granules show a strong positivity to nile blue. Hueck nile blue method.Fig. 7. Acid phosphatase reaction. Particulate activity in the cytoplasm of epithelial cells. Notethe activity in sites corresponding to pigment granules. Azo dye method.Fig. 8. Esterase activity. Compare with Fig. 7. Nuclei have been counterstained with haemalum.Naphthol-AS-LC method.Fig. 9. Alkaline phosphatase reaction. Note that the activity is confined to blood vessels. Naphthylphosphate method.

Anat. I0417

258 A. RIVA AND R. A. STOCKWELL

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Histochemistry of human seminal vesiclelipofuscins; negative results are obtained with the Schmorl's ferrous iron and Masson-Fontana silver reactions. Acid phosphatase and non-specific esterases are alsodemonstrable in them.No glycogen has been detected. Amorphous or globular material stainable with

PAS is present in the apical portions of the cells and sometimes discernible also inthe luminar contents; this is resistant to diastase and salivary digestion and is notsoluble in water. The Best's carmine and iodine methods give negative results. Thematerial behaves as a neutral mucin in that it is not stainable with cationic dyes,although a positive result is found using the Hale method; this is not abolished byprior neuraminidase digestion.Numerous reactive granules of various size are seen in the epithelium with methods

for acid phosphatase (Fig. 7); similar results are obtained for non-specific esterase(Fig. 8). Non-specific alkaline phosphatase is confined to blood vessels and noactivity can be detected either in the epithelium or the stroma (Fig. 9). Glucose-6-phosphatase and fructose-6-phosphatase activities are also absent.A study of phosphatase was carried out on unfixed frozen tissue using x-glycero-

phosphate, ,-glycerophosphate, glucose-6-phosphate and fructose-6-phosphate assubstrates, over a wide range of pH. Activity in granular form is present in theepithelium only between pH 5-2 and 5 5, particularly with ,3-glycerophosphate as sub-strate. The pattern of this latter reaction is not changed when formalin-fixed tissue isemployed. It seems possible therefore that the moderate activity observed in freshtissue with glucose and fructose-6-phosphate as substrates may be caused by non-specific acid phosphatase activity.

Glucose-6-phosphate and 6-phosphogluconate dehydrogenase activities are easilydemonstrable in the apical cytoplasm of the epithelial cells (Figs. 10, 11). Markedreactions are obtained for both enzymes, but that for glucose-6-phosphate dehydro-genase is always slightly stronger. The enzyme 6-phosphogluconate dehydrogenaseappears to be less resistant to denaturation, since a brief period of storage (2-3 h) ofthe tissue in the cryostat cabinet is enough to produce an appreciable loss of activity.

Positive results are also obtained with lactate (Fig. 12) and succinate (Fig. 13)dehydrogenases. These enzymes are both located uniformly throughout all the cyto-plasm of the epithelial cell.

DISCUSSION

The strong pyroninophilia observed in the cytoplasm of the epithelial cells is inagreement with the ultrastructural finding (Riva, 1967) of an abundant rough-surfaced endoplasmic reticulum. These results are also in support of the concept(Porter & Melampy, 1952) that the seminal vesicle is concerned with the synthesis ofprotein.

Phospholipids are a constant feature in the seminal vesicle: they have been demon-strated, together with neutral fats, in the corresponding organs of many mammals,notably in the rat (Cavazos, Porter & Melampy, 1954) and in the bull (Mann,

Fig. 10. Intense glucose-6-phosphate dehydrogenase activity in the epithelial cells. Nitro BT.Fig. 11. 6-phosphogluconate dehydrogenase. Compare with Fig. 10. Nitro BT.Fig. 12. Lactate dehydrogenase. Nitro BT.Fig. 13. Succinate dehydrogenase. Nitro BT.

I7

259

A. RIVA AND R. A. STOCKWELL

Davies & Humphrey, 1949; Cons, 1957). Both in the rat (Cavazos et al. 1954) andthe bull (Cons, 1957) they disappear in the castrate animal. In man, however, theyform only a minimal constituent of the secretion product since there is biochemicalevidence (Mann, 1964, chapter IX) that the bulk of seminal phospholipids originatefrom the prostate.The yellowish bodies present in the cytoplasm of epithelial cells are positive for

lipofuscins, the negativity of reactions for iron being characteristic (Lillie, 1965) ofthe human pigment. It is possible that their presence is associated with the age of thesubjects. Although they are referred to simply as pigment granules, the presence ofacid phosphatase and of esterase activity seems to relate them to lysosomes. Thelysosomal nature of human seminal vesicle pigments cannot then be excluded.

In spite of the fact that seminal vesicle fluid has the highest sialic acid concentrationfound in any human tissue (Warren, 1959) or fluid, no acid mucins and particularlysialoproteins are demonstrable in the seminal vesicle epithelium. A neutral substanceonly, strongly PAS-positive, can be detected in the apical cytoplasm of epithelial cellsand sometimes in the contents of the lumen. Positive results obtained with the Halereaction cannot be taken as indicative of the presence of an acid mucin, for thespecificity of this method is in doubt (Davies, 1952; Zaccheo & Viale, 1957); it is saidto stain also neutral mucopolysaccharides and polyphosphates (Zugibe, 1963). Onthe other hand, neuraminidase has no appreciable effect on the substance. It isextremely difficult to interpret this finding because sialic acid in seminal fluid ismostly present in a bound form (Warren, 1959); thus it should be histochemicallydetectable in the epithelium.Problems relating to fructose metabolism in the male accessory organs have been

reviewed recently by Mann (1964). Although blood glucose is regarded as the pre-cursor of seminal fructose, two different metabolic pathways have been proposed forfructose formation.On the basis of biochemical investigations carried out mainly on bull and ram

seminal vesicle, Mann & Lutwak-Mann (1951) believe that blood glucose is con-verted to fructose according to the system:

Glucose .-- Glycogen phnephoryl Glucose-l-phosphate.-.phosphoglucomutase phosphoglucose isomeraseGlucose-6-phosphate Uoe8ers

Fructose-6-phosphate alkalinephosphatase FructoseAccording to Mann & Lutwak-Mann (1951) glucose-6-phosphate is only partiallyconverted into fructose-6-phosphate, the rest being hydrolysed to glucose by alkalinephosphatase. Glucose is then re-phosphorylated by the glandular tissue and isremoved from the semen. This pathway is supported by results obtained by Parr andWarren (1951) in the fructose-forming organs of the rat, and those of Kellermann(1955), working with guinea-pig seminal vesicle.

Hers (1957) suggests a second metabolic pathway, finding evidence in the ramseminal vesicle for the presence of aldose reductase (nicotinamide-adenine dinucleo-tide phosphate (NADP)-dependent) and ketose reductase (NAD-dependent).

Glucose +NADPH +H aldose reductase Sorbitol +NADP+So+NAD + ketose reductaseSorbitol +ND-+ Fructose +NADH + H+

260

Histochemistry ofhuman seminal vesicleHers's observations have been confirmed and extended to other species (Williams-Ashman, Banks & Wolfson, 1957).

Histochemical investigations fail to demonstrate a parallelism between the presenceof abundant glycogen and the secretion of fructose (Cons, 1957; Filotto, 1960;Fouquet & Wegmann, 1964; Mann, 1964) thus lending no support to the phosphory-lative pathway. Enzyme histochemical studies of rodent male accessory organs(Fouquet, 1964; Fouquet & Wegmann, 1964; Johnson, 1965) are more compatiblewith Hers's (1957) hypothesis. Samuels, Harding & Mann (1962) assert that bothmetabolic pathways can coexist and that the problem is to find out which is the moreimportant under physiological conditions.The present findings in the human seminal vesicle are in agreement with the

oxidative rather than the phosphorylative mechanism. No enzymes active specificallyon glucose- and fructose-6-phosphate could be demonstrated in the epithelium.Although non-specific acid and alkaline phosphatase have been detected, the firstenzyme shows the granular localization typical of lysosomal enzymes, and the secondis confined to blood vessels. Both enzymes hydrolyse fl-glycerophosphate morereadily than other substrates, and a role in the mechanism of fructose secretion seemsunlikely. On the other hand the pronounced activity of glucose-6-phosphate and6-phosphogluconate dehydrogenases of the pentose shunt pathway is in accord withthe presence of an NADPH-dependent enzyme such as aldose reductase. Hers (1957)has demonstrated that the concentration of reduced NADP is the limiting factor forthe pentose pathway; thus a very active pentose cycle should accompany a strongaldose reductase activity. The presence of lactate dehydrogenase and succinate de-hydrogenase in human seminal vesicle epithelium show that the glycolytic pathwayand citric acid cycle respectively are active also, in agreement with the ultrastructuralfinding of large numbers of mitochondria in the epithelial cells (Riva, 1967).

Finally, it should be stressed that these studies have been made on organs frompatients of 53 years and more. An estimate of the extent to which senile changes mayhave affected the results largely depends on the examination of material yet to beobtained from young healthy adults.

SUMMARY

1. A histochemical investigation has been made of human seminal vesicle epi-thelium.

2. The cytoplasm of the epithelial cells exhibits a strong pyroninophilia attributedto ribonucleoprotein.

3. Many lipid and notably phospholipid droplets are present.4. Pigment granules are stainable with methods for lipofuscins and esterase and

acid phosphatase are also positive in them.5. No glycogen or acid mucin has been detected.6. Enzymes capable of hydrolysing glucose- and fructose-6-phosphate preferen-

tially are not demonstrable.7. Various dehydrogenases are present; in particular, glucose-6-phosphate and

6-phosphogluconate dehydrogenase give strong reactions.8. The significance of these results is discussed, mainly in relation to problems of

fructose secretion.

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A. RIVA AND R. A. STOCKWELL

We are indebted to Mr K. E. D. Shuttleworth of St Thomas's Hospital and toMessrs J. P. Williams and D. Williams of St Paul's Hospital for access to surgicalmaterial. We thank Mr J. S. Fenton for assistance with the photomicrographs.Finally, we are extremely grateful to Professor D. V. Davies for suggesting thisproblem, for his advice during the work and for reading the text.

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