Identification and cellular localisation of voltage-operated calcium channels in immature rat testis

Molecular and Cellular Endocrinology 162 (2000) 25–33

Identification and cellular localisation of voltage-operated calciumchannels in immature rat testis

Alessandra Fragale b, Salvatore Aguanno b, Matthew Kemp a, Matthew Reeves a,Kerry Price a, Ruth Beattie a, Peter Craig a, Steve Volsen a, Emanuele Sher a,

Angela D’Agostino b,*a Eli Lilly and Company, Erl Wood Manor, Windlesham, Surrey, GU20 6PH, UK

b Department of Histology and Medical Embryology, Uni6ersity ‘La Sapienza’, 6ia A. Scarpa 14, 00161 Rome, Italy

Received 20 October 1999; accepted 7 February 2000

Abstract

Sertoli cells regulate the spermatogenic process mainly through the secretion of a complex fluid into the lumen of theseminiferous tubules behind the blood–testis barrier, containing many of the essential proteins necessary for maintenance andmaturation of male germ cells. Thus, the study of Sertoli cell secretory processes is strictly correlated with the understanding ofthe regulatory mechanisms of spermatogenesis. In this work the authors have explored the voltage-sensitive calcium channelvariety in the immature rat testis, their localisation and distribution within the seminiferous epithelium and peritubular andinterstitial tissues as well as the possible role in the control of Sertoli cell secretion. The results reported in this paper, obtainedby in situ hybridisation, immunohistology of rat testicular sections and Western blot analysis of Sertoli cell plasma membranes,show that mammalian Sertoli cells express mRNA encoding for several voltage-operated calcium channel subunits and expresssuch proteins on their surface. Experiments performed on Sertoli cell monolayers cultured in the presence of specific toxinsindicate that both N and P/Q-type Ca2+ channels are involved in the regulation of protein secretion. © 2000 Elsevier ScienceIreland Ltd. All rights reserved.

Keywords: Sertoli cells; Rat testis; Ca2+; Voltage-operated Ca2+ channels; FSH

www.elsevier.com/locate/mce

1. Introduction

The testis can be divided morphologically and func-tionally into interstitium and seminiferous tubules,which are responsible for the production of androgensand spermatozoa, respectively. In the seminiferoustubules of the mammalian testis, Sertoli cells are re-sponsible for creating a unique environment in whichmale germ cells divide and differentiate into spermato-zoa (Bardin et al., 1988). Sertoli cells maintain thestructural integrity of the seminiferous tubular epithe-lium by means of unique junction specialisations, whichconstitute a component of the blood–testis barrier.This barrier segregates the epithelium of the tubule intoan exterior or basal compartment containing sper-

matogonia as well as preleptotene spermatocytes, andan interior or adluminal compartment that contains themore differentiated spermatocytes and the spermatids.Seminiferous tubules are surrounded by a basal laminaunder which peritubular myoid cells are present.

It is well known that many cellular processes includ-ing hormone and neurotransmitter secretion, cytoskele-tal function, ion channel and enzyme activities, cellproliferation and gene expression are regulated by thelevel of intracellular Ca2+ concentration [Ca2+]i. Amajor pathway for Ca2+ influx into the cells is repre-sented by voltage-operated Ca2+ channels (VOCCs)which, in neurons and many other cell types, differ insub-cellular location, biophysical and pharmacologicalproperties and modulation. In order to define the vari-ous classes of Ca2+ channels several classes of polypep-tide ligands, collectively referred as v-toxins, have beenused: a number of v-conotoxins, isolated from Conus

* Corresponding author. Tel.: +39-6-49766803; fax: +39-6-4462854.

E-mail address: [email protected] (A. D’Agostino)

0303-7207/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.PII: S 0 3 0 3 -7207 (00 )00213 -6

A. Fragale et al. / Molecular and Cellular Endocrinology 162 (2000) 25–3326

snails, which, as exemplified by v-conotoxin GVIA,specifically and irreversibly block the so-called N-typehigh voltage-activated VOCCs and v-agatoxins, morerecently isolated from the funnel-web spider Agelenopsisaperta, which are, like v-agatoxin IVA, highly specificfor P/Q-type VOCCs (Olivera, 1994). Both N- andP/Q-types VOCCs are involved in the regulation ofneurotransmitter release from nerve terminals (Kamiyaet al., 1988; Obaid et al., 1989; Rieke and Schwartz,1994). On the other hand recent studies have alsodemonstrated that both N and P/Q-type VOCCs partic-ipate in the control of exocytosis and hormone secre-tion in a number of non-neuronal cell types (Sher et al.,1988, 1990; Codignola et al., 1993; von Ruden andNeher, 1993).

A study performed on confluent monolayers of cul-tured rat Sertoli cells loaded with the intracellularfluorescent probe fura-2 showed the presence ofvoltage-gated calcium channels sensitive to nifedipine,nicardipine and v-conotoxin (D’Agostino et al., 1992).A further report investigating the possible role of suchchannels in protein secretion from cultured Sertoli cellsshowed that the block of N-type VOCCs by v-conotoxin GVIA resulted in a conspicuous inhibition ofprotein secretion in the culture medium, while totalRNA and protein synthesis were not affected (Tarantaet al., 1997).

The crucial role of Sertoli cells in the regulation ofthe spermatogenic process is mainly due to the secretionof a complex fluid into the lumen of the seminiferoustubules behind the blood–testis barrier, containingmany of the essential proteins necessary for mainte-nance and maturation of germ cells in the adluminalcompartment. Thus, the study of Sertoli cell secretoryprocesses is strictly correlated with the understanding ofthe regulatory mechanisms of spermatogenesis.

In the present study we explored the voltage-sensitivecalcium channel heterogeneity in the immature rattestis, their localisation and distribution within the sem-iniferous epithelium and peritubular and interstitial tis-sues as well as their possible role in the regulation ofSertoli cell secretion.

2. Materials and methods

2.1. Chemicals

v-Conotoxin GVIA and v-agatoxin were purchasedfrom Bachem UK and Peptide Institute, Louisville,KY, respectively.

Ovine FSH was a gift of the NIADDK Hormonedistribution Program (Baltimore, MD). Other chemi-cals, unless otherwise specified, were of the purest gradeavailable from Sigma (St Louis, MO).

2.2. Animals

Male Wistar rats (Charles River Breeding Laborato-ries, Wilmington, MA) were used in all the experiments.Animals were housed in accordance with guidelines foranimal care of University of Rome ‘La Sapienza’ andwere killed humanely by asphyxiation with CO2 beforeorgan removal. The testes were excised from immatureWistar rats aged from 18 to 21 days, where the sper-matogenesis was still uncompleted, washed and fixed in4% paraformaldehyde for in situ hybridisation analysisor in Bouin’s solution for the immunolocalisation ex-periments. Samples were then dehydrated and paraffinembedded. For the Sertoli cell cultures, the testes wereimmediately processed after excision, as describedbelow.

2.3. Immunolocalisation of calcium channel a subunits

For the general mapping study, a1A, a1B, and a1E

sub-unit proteins, corresponding to P/Q-type, N-type,and R-type calcium channels, respectively, were lo-calised in paraffin sections by a immunoperoxidasemethod with a commercially available kit (VectastainElite ABC Kit, Vector Laboratories, Bretton, Peterbor-ough, UK), following the manufacturer’s instructions.Briefly, after dewaxing and rehydration through gradedalcohols, sections were treated for 30 min with 0.3%(v/v) hydrogen peroxide, washed and incubated with1.5% (v/v) goat serum in PBS to block the non-specificbinding sites. Sections were covered with 200 ml ofanti-a1A, a1B, and a1E serum (2.5 mg IgG/ml in PBS) for30 min (Volsen et al., 1995). Non-specific staining wasdetermined by replacement of the primary antibodieswith a solution of 2.5 mg/ml normal rabbit im-munoglobulin. After washing, all the sections wereincubated for 30 min in biotinylated goat anti-rabbitsecondary antibody (5 mg/ml in PBS/1.5% (v/v) goatserum). Following washing, sections were treated withstreptavidin/biotin complex for 30 min, washed andthen incubated with the diaminobenzidine tetrahy-drochloride/nickel chloride substrate. Colour develop-ment was allowed to proceed for 7 min, then sectionswere washed in tap water. Following dehydration ingraded alcohols, sections were cleared in xylene andthen mounted in DPX (BDH, Eastleigh, Hants., UK).

2.4. Riboprobe generation

Polymerase chain reaction (PCR) primers were de-signed for the generation of templates for rat a1A, a1B,a1E riboprobes (Krieg and Melto, 1987). Sense andantisense oligonucleotide primers, for a1A sub-unit cor-responding to nucleotide positions 3200–3220 and3366–3386 (Starr et al., 1991), for a1B sub-unit corre-sponding to 6668–6688 and 6871–6891 (Dubel et al.,

A. Fragale et al. / Molecular and Cellular Endocrinology 162 (2000) 25–33 27

1992), and for a1E to 3000–3021 and 3320–3341(Soong et al., 1993), were synthesised. The primers forthe antisense strand were modified by addition to the 5%end of a 20 nucleotide extension containing the pro-moter sequence for T7 DNA dependent RNA poly-merase (Milligan et al., 1987). These were used with anunmodified sense primer to generate by PCR a templatefor an antisense riboprobe. Similar reactions were per-formed with a sense primer containing the T7 promoterand an unmodified antisense primer to generate a tem-plate for a sense control riboprobe.

Restriction digest analyses were performed on thePCR products to confirm that the desired sequence hadbeen generated. To generate fluorescein-labelled senseand antisense cRNA probes, a commercially availablekit (RNA colour kit, Amersham International, Amer-sham, UK) was used. The in vitro transcription methodwas performed following the manufacturer’s instruc-tions. Briefly, 1 mg of template was used in a 20 mlreaction comprising 20 mM dithiothreitol; 6 mMMgCl2; 2 mM spermidine; 0.01% bovine serum albumin(BSA); 20 units human placental ribonuclease inhibitor;0.125 mM fluorescein-11-UTP; 0.5 mM ATP; 0.5 mMCTP; 0.5 mM GTP; 0.375 mM UTP; 25 units T7 RNApolymerase; 40 mM Tris–HCl, pH 7.6. Reactions wereperformed at 37°C for 4 h. Probes were analysed byblotting an aliquot of the reaction product onto anitro-cellulose membrane (Hybond N, Amersham In-ternational) and by comparing the fluorescent intensity,observed when illuminated with a 254 nm UV lightsource, with that obtained from a known standard.Probes were stored at −20°C and used in hybridisationexperiments without further purification.

2.5. In situ mRNA hybridisation

In situ hybridisation was performed following Pringleet al. (1990). Briefly, sections were dewaxed in xylene,rehydrated in graded alcohols and digested with 25mg/ml proteinase K (Boehringer Mannheim, Lewes,East Sussex, UK). Sections were prehybridised for 2 hat 55°C in 25 ml of a solution comprising 2 X SSC, 1 XDenhardt’s solution (0.02% [w/v] Ficoll, 0.02% [w/v]polyvinylpyrrolidone, 0.02% [w/v] BSA), 300 mg/ml her-ring testes DNA, 50% [v/v] deionised formamide. Theprehybridisation solution was then replaced with thesame solution in which fluorescein labelled riboprobewas added to a final concentration of 0.5 ng/ml. Hy-bridisation was allowed to proceed overnight at 55°C.Testis sections were then washed sequentially at 55°Cfor 2×5 min in 4 X SSC, 2 X SSC and finally 0.2 XSSC, each wash solution containing 30% (v/v) for-mamide. Probe/RNA hybrids were immunolocalisedwith a sheep anti-fluorescein alkaline phosphatase con-jugated antibody (Amersham International). Alkalinephosphatase activity was finally stained with the nitro-

blue tetrazolium/bromochloroindolyl phosphate tech-nique (Bland et al., 1991). Non-specific hybridisationwas evaluated by using a fluorescein-labelled sense ribo-probe or by pre-treatment of sections with RNase.Omission of the riboprobe allowed the specificity of thedetection system to be confirmed.

2.6. Sertoli cell in 6itro cultures

Sertoli cell monolayers were prepared from the testesof 18–21-day-old Wistar rats with the procedure ofDorrington et al. (1975) used according to previouslydescribed methods (Galdieri et al., 1981; D’Agostino etal., 1992; Taranta et al., 1997). Briefly, testes wereremoved, cleaned from the tunica albuginea, mechani-cally reduced in small fragments and resuspended in anequal volume of Eagle’s minimum essential medium(MEM). Fragments were digested with 99% trypsin1:250, 1% DNAase for 30 min at 32°C, then washedwith Hank’s solution. Enzymatic digestion was re-peated twice; the cell suspension was then centrifugedat 1500×g for 5 min. Pellets were resuspended 1:10 inserum-free MEM with Earle’s salt’s. Cells were thenplated in 3.5 cm Petri dishes and incubated at 32°C ina controlled atmosphere of 95% air: 5%CO2. On day 3of culture, cell monolayers were subjected to hypotonictreatment (Galdieri et al., 1981) to completely removeendogenous germ cells and then allowed to recover for24 h. Experiments were performed on day 4 of culture.

2.7. Metabolic labelling

Rat Sertoli cell monolayers were labelled for 6 h with50 mCi/ml/MEM of [35S]-methionine (New EnglandNuclear, specific activity 1175 Ci/mmol) in the presenceor in the absence (control) of v-conotoxin GVIA (5mM) and v-agatoxin (1 mM). Labelling with [35S]-me-thionine was performed in methionine-free medium.After labelling, the culture medium was collected, cen-trifuged at 1000×g for 5 min in order to eliminate celldebris then trichloroacetic acid (TCA)-precipitated.Cells were washed with MEM, lysed in 10 mM Tris-HCl, pH 7.4 containing protease inhibitors (leupeptin,200 mg/ml, phenylmethylsulfonylfluoride, 1 mM) thenTCA-precipitated.

2.8. SDS-PAGE and immunoblotting

Enriched-membrane preparations were preparedfrom Sertoli cell cultures. Cells were scraped off theculture dishes, pelleted and washed with PBS. Thepellet was resuspended in lysis buffer (150 mM NaCl,1% SDS, 20 mM Tris–HCl, 1 mM EDTA, mammalianProtease Inhibitor Cocktail (Sigma), pH 8), and incu-bated for 10 min at room temperature. DNA wassheared by passage through a 19-G needle, before


centrifugation at 10 000×g for 10 min. The superna-tant was diluted in an equal volume of 2% BSA in PBS(w/v) and centrifuged at 100 000×g for 45 min. Super-natants from this were retained.

Reducing Laemlli buffer was added and the prepara-tion was boiled for 3 min. Samples containing 20–40 mgprotein were loaded onto pre-cast 10% Tris–GlycineBis–Acrylamide gels (Novex), and blotted onto 0.45mm nitrocellulose membrane (Novex). After anovernight block with 5% (w/v) skimmed milk in PBS,the blots were immunostained with either polyclonalrabbit anti-human a1A (500 ng/ml) or polyclonal rabbitanti-human a1B (250 ng/ml). Following incubation in250 ng/ml horseradish peroxidase conjugated goat anti-rabbit IgG, immunoreactive bands were detected usingenhanced chemiluminescence methodology using ECL-Plus, according to manufacturer’s instructions (Amer-sham). All antibodies were diluted in 2% skimmed milkin PBS (w/v) and the blot was washed extensively withPBS between each stage.

2.9. Statistics

Statistical analysis was performed by analysis of vari-ance (ANOVA). PB0.05 was conventionally consid-ered to indicate statistical significance.

3. Results

3.1. Immunolocalisation analysis

The identification and cell localisation of voltage-op-erated Ca2+ channels on paraffin sections has beenperformed by using antibodies specific for the differentsubunits of several VOCC types, as described in Section2. Intense staining with the a1A antibody (Fig. 1),specific for P/Q-type calcium channels, was obtained atthe level of the basal compartment of the seminiferousepithelium, where contact surfaces between Sertoli andimmature germ cells (spermatogonia and early sperma-tocytes) were well decorated. Furthermore, a1A im-munoreactivity was also localised between Sertoli cellsand basal lamina, and between the basal lamina andperitubular myoid cells. In the interstitial compartment,Leydig cells appeared intensely stained.

Immunolocalisation of N-type channels has been per-formed with specific a1B antibodies (Fig. 2). Althoughthe intensity of staining was generally lower than thatfound with the a1A antibodies, clear immunostainingwas present at the level of contact surfaces betweenSertoli cells and germ cells in the adluminal compart-ment as well as between Sertoli cells and basal lamina.In the interstitium some blood vessels also appeareddecorated. Fig. 3 shows control experiments with nonspecific IgG.

a1E Antibodies, specific for R-type channels, did notshow any stained testicular tissue (not shown).

3.2. In situ hybridisation

In order to detect which cells in immature rat testesexpress the a1A, a1B and a1E subunit mRNAs, weperformed in situ hybridisation analysis on seminifer-ous epithelium paraffin sections of 18–21-day-old ratswith the appropriate riboprobes as described in Section2.

The experiments with the a1A antisense riboprobe,coding for P/Q-type calcium channels, showed (Fig. 4)that this subunit was strongly expressed by the basalcompartment of the seminiferous tubules, where the a1A

mRNA expression pattern co-localised with the profileof the a1A protein assayed by immunolocalisation stud-ies. A weak but specific staining was found in theperitubular myoid cells. A positive staining was foundalso in the Leydig cells.

Fig. 1. Microphotograph of a testis from 21-day-old Wistar rat. Thearrow indicates the contact surface areas between Sertoli cells andimmature germ cells in the basal compartment of the seminiferousepithelium, where intense staining by a1A antibody, specific for P/Qtype calcium channels, is well observable. The double arrow indicatesa1A immunoreactivity localised between basal lamina and peritubularmyoid cells (1000 X).


Fig. 2. Microphotograph of 21-day-old rat testis. Immunolabelling bya1B antibody, specific for N-type calcium channels, is specificallypresent at level of contact surfaces between Sertoli cells and germcells, in the adluminal compartment (arrow) as well as betweenSertoli cells and basal lamina (double arrow) (1000 X).

3.3. Western blotting

Western blots of Sertoli cell plasma membrane en-riched-preparations confirmed the immunolocalisationresults. Specific bands of approximately 210 and 240kDa were detected with the a1A and a1B specific anti-bodies, respectively (Fig. 7). In the case of a1A a lowerband around 95 kDa was also recognised by the anti-bodies as often reported in both brain and recombinanta1A preparations (Scott et al., 1998).

3.4. Metabolic labelling

Sertoli cell cultures were metabolically labelled with35S-methionine for 6 h in the presence of 100 nM FSH,in the presence or absence of v-agatoxin (1 mM), andv-conotoxin GVIA (5 mM). Protein secretion in theculture medium showed an inhibition of 35–50%, de-pending on the confluence state of cultured cells, fol-lowing the treatment with v-conotoxin and of 25–35%following the treatment with v-agatoxin (Fig. 8).

Fig. 3. Microphotograph of 21-day-old rat testis. Negative control:primary antibodies have been replaced with a solution of 2.5 mg/mlnormal rabbit IgG (400 X).

A strong positive signal was detected in the peritubu-lar compartment with the antisense riboprobe of thea1B subunit (coding for the N-type calcium channels). Alighter widespread signal was present in the cytosol ofSertoli cells, particularly in the contact areas betweenSertoli and germ cells, as well as between Sertoli andperitubular myoid cells (Fig. 5). These data correlatewell with the immunostaining obtained with the antia1B subunit antibody, confirming that Sertoli cells ex-press a significant amount of this subunit. No signalwas detected in the in situ hybridisation experimentswith the appropriate riboprobe coding for the R-typechannels (not shown), according to the results obtainedwith the anti- a1E antibodies.

Hybridisations performed by using the fluorescein-la-belled sense riboprobes (Fig. 6) or after pre-treatmentof sections with RNAse (not shown), were completelynegative.


Fig. 4. Microphotograph of 21-day-old rat testis. The basal compart-ment of the seminiferous tubules shows strong hybridisation with thea1A antisense riboprobe, specific for P/Q channel mRNA transcripts(arrow). A specific staining is also present in the peritubular myoidcells (double arrow) and in the interstitium (+ ) (800 X).

The presence of N-type VOCCs in the adluminal com-partment of the seminiferous epithelium, between Ser-toli cells and pachytene spermatocytes, could have arole in the regulation of tubular fluid secretion which islargely elaborated by Sertoli cells. Sertoli cells appear tobe able to secrete many of the essential proteins neces-sary for maintenance and maturation of germ cells inthe adluminal compartment (Fawcett, 1975; Bardin etal., 1988). Such proteins are signalling molecules, forcell-to-cell communication, as well as extracellularmolecules, which establish and maintain the microenvi-ronment in the adluminal compartment of the seminif-erous tubule. This hypothesis is in agreement with theinhibition of Sertoli cell protein secretion following thespecific block of N-type channels by v-conotoxin, al-ready shown in a previous work (Taranta et al., 1997)and confirmed in this paper. As shown by the resultsobtained with metabolic labelling experiments, theblock of v-agatoxin-sensitive channels also partiallyinhibits protein secretion in Sertoli cells labelled for 6 h.The presence of VOCCs between Sertoli cells and base-ment membrane appears very intriguing also on the

Fig. 5. Microphotograph of 21-day-old rat testis. a1B Antisenseriboprobe, specific for N-type VOCCs, hybridised to mRNA tran-scripts in the cytosol of Sertoli cells, particularly in the adluminalcompartment (where Sertoli cells contact pachytene spermatocytes)(arrow). A strong positive signal is also present in the basal compart-ment (double arrow) (400 X).

4. Discussion

The results reported in this paper, obtained by in situhybridisation, immunohistology of rat testicular sec-tions and Western blot analysis of Sertoli cell plasmamembranes, show that mammalian Sertoli cells expressmRNA encoding for several VOCC a1 subunits andexpress such Ca2+ channel proteins on their surface.

The identification of N-type and P/Q type VOCC’s inthe rat testis is particularly interesting because it addsnew evidence to the general scenario suggesting thatseveral endocrine cell types express these particularCa2+ channels, still often believed to be expressed onlyin the nervous system. Pancreatic cells (Davalli et al.,1996), pituitary cells (Sher and Clementi, 1991), adrenalmedullary chromaffin cells (Kim et al., 1995) as well assmall cell lung carcinoma cells (Codignola et al., 1993)and insulin secreting cell lines (Sher et al., 1988; Mag-nelli et al., 1995) express both N- and P/Q Ca2+

channels that participate in the control of their secre-tory activity.

The specific localisation of the different types ofVOCC subtypes raises several issues worth discussing.


Fig. 6. Microphotograph of 21-day-old rat testis. Sense a1A (a) anda1B (b) riboprobes (400 X).

Fawcett, 1967; Fawcett et al., 1970). The blood–testisbarrier is not, in fact, located in the walls of theinterstitial vessels but consists of two components: (1)

Fig. 7. Western blots of Sertoli cell membrane-enriched preparations.The figure shows, on the left, the a1A predominant 210 kDa band andthe less abundant 190 kDa band. Also present is a 95 kDa band(truncated form). On the right is shown the a1B predominant band atca. 240 kDa.basis of very recent data showing that the extracellular

matrix proteins (i.e. laminin) rapidly increase the intra-cellular concentration of cytosolic Ca2+ [Ca2+]i incultured rat Sertoli cells (Taranta et al., 1999) viavoltage gated calcium channels. This laminin effect iscontrolled by FSH.

The localisation of P/Q channels in the plasma mem-branes of Sertoli cells adjacent to the basal lamina andbetween basal lamina and peritubular myoid cells couldsuggest a role at the level of the blood–testis barrier,which selects the substances that can reach the lumen ofthe seminiferous tubule. Near the base of the seminifer-ous epithelium, the adjoining Sertoli cells possess exten-sive junction complexes, involving two Sertoli cells(Flickinger and Fawcett, 1967), in which gap junctions,as well as multiple focal tight junctions, are present.These unique intercellular junctions probably have mul-tiple functions. In addition to the obvious general func-tion of helping to maintain the structural integrity ofthe epithelium, the areas of very close proximity to themembranes (gap junctions) probably constitute low-re-sistance pathways providing cell-to-cell communication,whereas the linear tight junctions effectively seal theintercellular clefts and constitute the intraepithelialcomponent of the blood–testis barrier (Flickinger and

Fig. 8. Effect of v-conotoxin GVIA and v-agatoxin on FSH-stimu-lated protein secretion of cultured rat Sertoli cells. Sertoli cell cultureswere labelled with 35S-methionine for 6 h in the presence of 100 nMFSH with or without (controls) v-conotoxin GVIA (5 mM) andv-agatoxin (1 mM). Protein secretion in the culture medium shows aninhibition of about 50% following treatment with v-conotoxin GVIAand of 25–35% following treatment with v-agatoxin. *, Statisticallydifferent from the control group. One-way analysis of variance wasperformed on four to six experiments.


the peritubular contractile layer, retarding but not com-pletely preventing access of substances to the seminifer-ous epithelium; and (2) the intraepithelial component,due, as already mentioned, to contacts between adja-cent Sertoli cells. Wherever electron-opaque markersreach the base of the seminiferous epithelium, they alsoenter the intercellular clefts surrounding the sper-matogonia and the pre-leptotene spermatocytes, butthey do not penetrate more deeply into the epithelium.Therefore, since the extracellular route from the inter-stitium is barred by occluding junctions between thesupporting cells, substances would have to pass throughthe Sertoli cell cytoplasm to reach germ cells in theadluminal compartment. To elucidate a possible role ofP/Q channels in the formation and/or maintenance ofthe blood–testis barrier, work is in progress to studywhen such channels are expressed on Sertoli cell mem-brane (whether or not contemporary to the barrierappearance) and the effects of the block by v-agatoxinsbefore the barrier is formed (from 15th to 18th postna-tal day).

Acknowledgements

This work was supported by the Ministero dell’Uni-versita e della Ricerca Scientifica e Tecnologia* and byEli Lilly and Company Limited.

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