Development 113, 779-788 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

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Human acrosome biogenesis: immunodetection of proacrosin in primary

spermatocytes and of its partitioning pattern during meiosis

DENISE ESCALIER1, JEAN-MARC GALLO2, MARTTNE ALBERT3, GERI MEDURI4, DIEGO

BERMUDEZ5, GEORGES DAVID1 and JOSEPH SCHREVEL2*

^Laboratoire de Biologie de la Reproduction et du Developpement, CHU Bicitre, 78 rue du Gtniral Leclerc, 94275 Le Kremlin Bicitre,France2URA CNRS 290, Laboratoire de Biologie Cellulaire, University de Poitiers, 4O Avenue du Recteur Pineau, 86022 Poitiers Cedex, Franceand the Museum d'Histoire Naturelle, 61 Rue Buffon, 75231 Paris Cedex 05, France^Laboratoire d'Embryologie, UER Biomidicale, University Paris v, 45 rue des Saints Peres, 75006 Paris, France''Laboratoire d'Anatomo-Pathologie, CHU Bicitre, 78 rue du Giniral Leclerc, 94276 Le Kremlin-Bicitre, France5Catedra de Histologia, Facultad de Medicina, Universidad de Malaga, 29080 Malaga, Espana

* Author for correspondence

Summary

Proacrosin biosynthesis timing during human spermato-genesis has been studied using the monoclonal antibody4D4 (mAb 4D4). Frozen and paraffin-embedded sectionsof testicular biopsies were labelled by standard indirectimmunofluorescence and avidin-biotin immunoperoxi-dase procedures. The labelling specificity was checkedby immunochemistry assays on unrelated tissues and bywestern blotting of testis extracts showing that only the50-55 xK^Mr proacrosin was recognized by mAb 4D4.Proacrosin was first observed in the Golgi region of mid-pachytene primary spermatocytes. In late pachyteneprimary spermatocytes, proacrosin was observed in tworegions located at opposite nuclear poles. During thesubsequent steps of the first meiotic division, the twobodies containing proacrosin were located: (i) onopposite sides of the equatorial plate during metaphase;

(ii) along the microtubular spindle during anaphase; and(iii) close to each chromosomal aggregate duringtelophase. Two bodies containing proacrosin were stillobserved in interphasic secondary spermatocytes. Thesingle labelled area observed in early spermatids wasfound to increase considerably in size during spermio-genesis. Anomalies of proacrosin scattering were ob-served in patients with Golgi complex partitioningfailure. These data' reveal proacrosin biosynthesisduring diploid and haploid phases of human spermato-genesis and the proacrosin partitioning pattern duringmeiosis.

Key words: proacrosin/acrosin, acrosome, meiosis, humanspermatozoa, spermatogenesis, testis.

Introduction

The acrosome is elaborated from vesicles stored in theGolgi apparatus center immediately after the com-pletion of meiosis. These vesicles fuse to give rise to alarge proacrosomal vesicle (Sandoz, 1970, 1972) whichforms the acrosomal vesicle over the nucleus (Dooherand Bennett, 1973; Burgos and Fawcett, 1955). Ad-ditional vesicles and granules originating from the Golgiapparatus contribute to the formation of the acrosome(Susi et al. 1971; Hermo et al. 1979, 1980; Burgos andGutierrez, 1986). Several hydrolases are sequesteredwithin the acrosomal matrix (Bellve' and O'Brien,1983), the major one had been identified as proacrosin(Meizel, 1972; Parrish and Polakoski, 1979).

Radioautographic methods have been used in studieson the synthesis of acrosomal glycoproteins during

spermatogenesis (Sandoz, 1972; Sandoz and Roland,1976; Tang et al. 1982; Clermont and Tang, 1985); whilerecently, germ cells have been investigated by means ofantibodies specific for acrosomal hydrolases. It hasbeen suggested that the expression of proacrosin duringmammalian spermatogenesis is initiated in spermatids.However, it is not clear whether proacrosin is presentfrom the beginning of spermiogenesis (FISrke et al.19S3a,b; Phi-Van et al. 1983; Mansouri et al.' 1983;Kallajoki and Su'ominen, 1984; Arboleda and Gerton,1988), or appears during the course of spermatiddifferentiation (Kallajoki et al. 1986).

We have previously demonstrated that the mono-clonal antibody 4D4 recognizes human proacrosin inmature spermatozoa and in ejaculated spermatids(Gallo et al. 1991). In the present study, labelling ofhuman testis sections with mAb 4D4 using several

780 D. Escalier and others

immunohistochemical procedures, revealed that proac-rosin was present as early as the prophase of the firstmeiotic division. It was detected during all thesubsequent steps of spennatogenesis and increased inquantity during acrosomogenesis. These data suggestmeiotic as well as postmeiotic synthesis of proacrosinduring the human spermatogenesis.

Materials and methods

TissuesHuman testicular biopsies (free from the tunica albuginea)were obtained from: (1) three autopsies and nine patients withan obstructive azoospermia, all presenting a normal germinalepithelium as revealed by light microscopy, (2) one patientwith macrocephalic spermatozoa known to originate frommeiotic division failure (Escalier, 1983, 1985) and (3) onepatient with oligozoospermia and multinucleate sperm cellsdue to cytokinesis impairment (Holstein et al. 1988). Biopsiesfrom human spleen, liver, kidney, ovary, breast, lung,pancreas, colon and dermoid kyst were used to check thespecificity of germ cell immunolabelling.

AntibodiesThe monoclonal antibody 4D4 was obtained after immuniz-ation of a mouse with a detergent-insoluble fraction fromhuman spermatozoa. mAb 4D4 specifically recognizes pri-mate proacrosin (Gallo et al. 1991). Two other monoclonalantibodies were used as controls: (1) a mAb generated at thesame time as mAb 4D4 that specifically recognizes a humanflagellar protein expressed during spermiogenesis (unpub-lished data) and (2) an anti-desmin antibody (Dakopatts,Santa Barbara, CA, USA). Secondary antibodies for immu-nocytochemistry were either biotinylated horse anti-mouseIgG (Vector Laboratories, Burlingame, CA, USA), or goatanti-mouse IgG conjugated to isothiocyanate fluorescein(GAM/FTTC, Nordic, The Netherlands). Secondary anti-bodies for western blotting were either goat anti-mouse IgGconjugated to peroxidase (BI 2413, Biosys SA, Compiegne,France) or sheep anti-mouse IgG conjugated to 125I (Amer-sham, Buckinghamshire, UK).

Electrophoresis and immunoblottingFrozen human testis autopsies and frozen human sperm withnormal parameters were thawed and homogenized in ice inphosphate-buffered saline (PBS pH7.2) containing 0.05%Triton X-100, 1 mM phenyl-methyl-sulfonyl-fluoride (PMSF)and lO/igml"1 a^-macroglobulin. After centrifugation at15 000 g for 2min, the pellets were sonicated in a buffercomposed of 10 mM Tris, 0.9% /V-lauroyl sarcosine, 2.5 Murea, 0.5% Triton X-100, lmM PMSF and 10/igml"1

Oi-macroglobulin and incubated in ice for 2h. Suspensionswere subsequently centrifuged at 15 000 g for 2min, andSDS-electrophoresis sample buffer was added to the super-natants. Proteins were separated by electrophoresis on 8.5%polyacrylamide gels according to Laemmli (1970). Transfer tonitrocellulose sheets and subsequent incubations with anti-bodies were performed according to Towbin et al. (1979) withmAb 4D4 culture supernatant as primary antibody. Antibodybinding was detected by either the immunoperoxidasemethod using diaminobenzidine as a chromogen or byautoradiogTaphy with the anti-mouse IgG conjugated to U5l.Controls were performed by omitting mAb 4D4 or bysubstituting it with the anti-human flagellum antibody (culturesupernatant).

Biopsy processing for immunocytochemistryFor paraffin embedding, tissue specimens were trimmed into4x2 mm blocks, then fixed in either Bouin's fixative or 7%formaldehyde at room temperature for a time not exceeding24 h. After the standard clarification procedures through xyloland alcohol, tissues were embedded in low melting pointparaffin (56°C). Paraffin sections (3/an thick) were placed onclear or neoprene-coated glass slides to avoid loosening of thesections during further manipulations. Sections were thendewaxed in toluene, rehydrated through graded alcohols andwashed with PBS at pH7.2.

For frozen testis sections, unfixed testis specimens wereembedded in Tissue-Tek (Myles Diagnostic Division, Elkart,USA), snap frozen in liquid nitrogen and stored at —80°Cuntil use. Frozen sections (3//m thick) were cut with aReichert cryomicrotome at -25CC, collected on slides asabove, air-dried at room temperature for 20min, fixed in—20°C acetone for 5min, then either immediately processedor stored in a cellulose and tinfoil wrapping at — 80 °C untiluse. Prior to immunostaining, sections were warmed to roomtemperature and then washed in PBS three times for 3min.Some frozen sections were additionally postfixed with 1 %formaldehyde in PBS, then washed again with PBS.

ImmunocytochemistryImmunolabelling was performed by standard indirect immu-nofluorescence or the three-step immunoperoxidase tech-nique using the biotin-avidin system (Vector Laboratories,Burlingame,CA, USA). Some sections were first treated witheither 7.5 % H2O2 in PBS for lOmin at room temperature orwith 0.1% trypsin in 0.05M Tris buffer (pH7.6) with 0.1%calcium chloride for a time not exceeding 15 min at 37 °C. Thereaction was stopped with 4°C PBS. To block nonspecificantibody binding, all washed sections were incubated at roomtemperature for 20 min in either 5 % normal horse serum or5 % BSA (grade V, Sigma, USA) in PBS. Sections were thenincubated with mAb 4D4 culture supernatant (diluted 1:2 and1:4) in a humid chamber at room temperature for lh. Afterwashing in PBS, sections were incubated for 30 min with thefiuorescein-conjugated antibody (diluted 1:40 with PBS/BSA) or the biotinylated antibody (diluted 1:250 withPBS/BSA) and washed again. Sections treated with thebiotinylated antibody were then incubated for 30 min withpreformed avidin-peroxidase complexes, and amino-ethyl-carbazole (AEC) was used as chromogen. For controls, mAb4D4 was substituted by PBS/BSA or irrelevant monoclonalantibodies (see Antibodies section).

Sections treated with the immunoperoxidase method werecounterstained with Harris's hematoxylin and mounted in anaqueous medium (Glycergel, Dakopatts, Santa Barbara,USA). Sections stained by indirect immunofluorescence werecounterstained with 0.02% Evans blue and mounted inCitifluor (glycerol/PBS, Citifluor Ltd, London, UK). Slideswere observed under an Olympus BH-2 photo-microscopefitted with standard light, epi-illumination and filters forFITC. Micrographs were taken at X1250 and X2500 usingeither Kodak Ektar 25 or T-MAX 400 films (Kodak, France)and automatic exposure.

Electron microscopyTestis biopsy specimens from the patients exhibiting atypicalsperm cells were fixed for 3 h in 1.2 % glutaraldehyde in 0.1 MSorensen's buffer and washed for 30 min in fresh buffercontaining 4 % (w/v) sucrose. Post-fixation was for 1 h in 1 %w/v osmic acid in 0.1M S6rensen's buffer with 4% sucrose.After dehydration through graded alcohols, biopsy specimens

Proacrosin biosynthesis in man 781

were embedded in Araldite. Sections obtained using aReichert OmU3 ultramicrotome were stained with uranylacetate and lead citrate and examined in a Siemens ElmiskopCT 150 transmission electron microscope.

Morphological identification of the spermatogenic cellsSpermatogenic stages were identified according to the criteriaof Clermont (1970), Holstein and Roosen-Runge (1981), andSchulze and Rehder (1984).

Spermatocytes were classified according to the followingcriteria: (1) leptotene stage: densely packed, regularlydistributed small round patches of chromatin; nuclei ofintermediate density (small, irregularly shaped nucleolieccentrically positioned); (2) zygotene stage: loosely distrib-uted cords or thread-like chromatin patches with a more orless tuft-like orientation; light nuclear area(s) near thenucleolemma; faded nuclear aspect (in favourable cellsections, one or two small darker masses are seen adjacent tothe nuclear margin); (3) early pachytene stage: thickerchromatin cords resulting in a denser nuclear aspect; poorlydiscernible nuclear margin; (4) mid-pachytene stage: scatteredaggregates of intensely stained and irregularly shaped coarsechromatin; marked contrast between the chromatin patchesand the surrounding karyoplasm; dense area underlying thenucleolemma (in favourable cell sections, three intenselystained masses including a prominent round central nucleolusand two smaller components are observed); (5) late pachytenestage: more diffuse large patches of intensely stained coarsechromatin (Guichaoua et al. 1986); dense nuclear contentsand margin; (6) diplotene stage: intensively stained coarsechromatin patches rather regularly disposed; (7) secondaryspermatocyte stage: round nucleus with sharply contrastednuclear envelope and homogeneous or slightly granular densechromatin (in favourable cell sections, three nucleoli or moreare discernible).

Main criteria for spermatid characterization were (1) earlyspermatids: small round nucleus with uniformly grainychromatin, acrosomal bleb; (2) mid-spermatids: more or lesselongated nucleus and presence of an acrosomal cap; (3)mature spermatids: highly condensed chromatin, elongatedand flattened nucleus, acrosome over the anterior nuclearregion.

Results

mAb 4D4 labelling in human testisIn testis sections stained by immunofluorescence orimmunoperoxidase, mAb 4D4 positively reacted withspermatocytes and spermatids while the other testicularcells were unstained (Figs 1 and 2). A similar mAb 4D4labelling pattern was found in spermatocytes andspermatids in sections pretreated with H2O2 to quenchendogenous peroxidase activity and stained with theimmunoperoxidase method. In controls incubated withPBS/BSA without mAb 4D4 (Fig. 2D) or with irrel-evant monoclonal antibodies, no staining was observedin spermatocytes or spermatids. When mAb 4D4 wasreplaced by a monoclonal antibody directed against asperm flageUar protein, only spermatids were stained atthe flagellar level (data not shown). Sections from apanel of other tissues (see Materials and methods)showed no staining with mAb 4D4 under the sameconditions.

The efficiency of the proacrosin immunolabelling was

found to be dependent on the technical proceduresused. Staining of germ cells with mAb 4D4 by standardindirect two-step immunofluorescence was faint, es-pecially in primary spermatocytes (Fig. 2A,F). Thethree-step avidin-biotin-immunoperoxidase techniquegave a more conspicuous labelling of both thespermatocytes and spermatids (Fig. 2C). In acetone-fixed frozen testis sections, only a few spermatocyteswere labelled with mAb 4D4. Moreover, proacrosinappeared to diffuse around the spermatocyte nuclei andoutside the spermatid acrosomes. Postfixation of frozensections with formaldehyde prior to immunolabellingslightly increased proacrosin preservation followingthawing as revealed by the greater number of labelledspermatids and the decrease of labelling diffusion(Fig. 2F). In testis sections fixed with Bouin's fluid priorto paraffin embedding, numerous spermatocytes andalmost all spermatids exhibited a strong and well-defined labelling (Fig. 1). A similar staining pattern wasobtained in sections fixed in Bouin's fluid and pre-treated with trypsin (Fig. 2E). When Bouin's fluid wasreplaced by formaldehyde, the incidence of labelledspermatocytes and spermatids was variable from onebiopsy to another, some of them exhibiting scarcelabelling (data not shown).

mAb 4D4 specificity to testis proacrosin determined byWestern blottingAs previously determined (Gallo et al. 1991), mAb 4D4recognized the 50 and 55X103 MT proacrosin in ejacu-lated human sperm cells (Fig. 3C, lane 3). Detection ofthe mAb 4D4 binding in testis extracts by theimmunoperoxidase method (which gave optimallylabelled spermatocytes in sections) (Fig. 3B) or theautoradiographic method (Fig. 3C) showed that onlythe 50 and 55 x 103 MT proacrosin polypeptides, with theexclusion of any other component, were recognized bymAb 4D4 in the human testis.

Cellular location of proacrosin during spermatogenesisZygotene and early pachytene primary spermatocyteswere unlabelled by mAb 4D4 (Fig. 1). In contrast, mid-pachytene primary spermatocytes exhibited a promi-nent more or less elongated labelling (being up to 3 /anin length) (Fig. 4C). Indirect immunofluorescentstaining revealed that this labelling was located at thelevel of the Golgi complex (Fig. 2A,B)- Inside thisstrongly labelled body, clear areas were observed(Fig. 5A). In some other cells, several protrusionsseemed to emerge from this stained mass (Fig. 5B)which, in some other cases, assumed the appearance ofa network of elongated elements with various thick-nesses (Fig. 5C).

Late pachytene spermatocytes as cells at an inter-mediate stage between mid- and late pachytene showedtwo distinct labelled bodies of equal size or not(Figs4A,E,F and 5D). They were located near thenucleus either close to each other (Fig. 4A), or atvarious distances from one another (Fig. 4E,F) includ-ing at opposite nuclear poles (Fig. 5D).

Metaphasic primary spermatocytes exhibited two

782 D. Escalier and others

Fig. 2. mAb 4D4 labelling of the human seminiferous epithelium according to different biopsies processings. (A) Bouin's-fixed, paraffin-embedded testis sections labelled by indirect immunofluorescence. Fluorescence is detected in 4 pachyteneprimary spermatocytes (arrows). (B) Phase-contrast micrograph of the same field'as-in A. Pachytene cells are labelled inthe Golgi region (arrows and insert). (C) Bouin's-fixed, paraffin-embedded testis sections labelled by the avidin-biotin-peroxidase complexes. A prominent labelling is observed in the pachytene primary spermatocytes (arrow) and spermatids(arrowhead). (D) Control testis section treated as in C except that mAb 4D4 has been substituted by PBS/BSA. Germcells are unlabelled. (E) Testis section processed as in C but treated with 0.1 % trypsin during lOmin prior toimmunolabelling. Pachytene primary spermatocytes are poorly labelled (arrows). (F) Frozen section labelled by indirectimmunofluorescence microscopy. The pachytene primary spermatocytes exhibit a faint and spotty fluorescence (arrows).A-D, x840; E, x425; F, x890. A-F, bar=20/m.

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Proacrosin biosynthesis in man 783

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labelled round bodies close to the metaphasic plate(Fig. 4G). At the anaphase stage, the labelled dots wereclose to the microtubular spindle and at variousdistances from the spindle poles (Fig. 4H,I). Finally, intelophase primary spermatocytes a labelled body wasobserved in the vicinity of each chromatin mass(Fig. 4J). Interphasic secondary spermatocytesexhibited two labelled bodies located close to thenuclear margin (Fig. 4K,L).

Additional smaller labelled dots were sometimesobserved in primary spermatocytes during chromosomemovement (Fig. 4H) and in secondary spermatocytes(Fig. 4K). Whether they correspond to scattered Golgivesicles could not be determined at the light microscopelevel. Several attempts to obtain mAb 4D4 immuno-gold labelling on Lowicryl-embedded testis sectionswere performed but the very low probability of

Fig. 3. Identification of the 4D4 epitope-bearingpolypeptides in human testis. (A) Coomassie-blue-stainedgel showing human testis proteins separated by SDS-PAGE under reducing conditions. Lane 1: fractioninsoluble in Triton X-100. Lane 2: fraction soluble inTriton X-100. (B,C) Corresponding nitrocellulose replicaincubated with mAb 4D4 and revealed either by theimmunoperoxidase method (B) or by autoradiography withanti-mouse IgG conjugated to 125I (C). The 4D4 antibodyrecognized polypeptides in the 50-55 x 103 Mr range in thetestis fraction insoluble in Triton X-100 (lanes 1) while thetestis fraction soluble in Triton X-100 were unlabelled by4D4 (lanes 2). The polypeptides recognized by mAb 4D4in testis were in the same molecular mass range that thoserecognized by 4D4 in sperm fraction insoluble in TritonX-100 (lane 3). The 55X103 Mr corresponds to humanproacrosin and the 50X103 MT to an intermediate formbetween proacrosin and acrosin which appears followingproacrosin cleavage during protein extraction (Gallo et al,1991). Lane 4 and left-hand side: relative molecular massmarkers.

observing spermatocytes undergoing meiotic divisionsas secondary spermatocytes in electron microscopyhave led us to tackle the question of the organellelocation of proacrosin by investigating pathologicalmodels (see below).

Early spermatids contained a single round labelledbody apposed to the nuclear outer border (Fig. 4L). Inmore differentiated spermatids, the labelled bodyappeared to be flattened on the side facing the nucleus(Fig. 4M). Still more differentiated spermatidsexhibited a crescent-shaped strongly labelled cap overthe anterior face of the nucleus (Fig. 4N). As spermatidmaturation proceeds, the amount of proacrosin con-siderably increased as revealed by the enlargement ofthe labelled area (Fig. 4L-N). In mature spermatids,the whole acrosomal region was labelled (Fig. 4B).

Proacrosin labelling in the case of spermatocytedivision anomaliesSince mAb 4D4-stained proacrosin appeared to belocated in the Golgi region of primary spermatocytes(Figs 2, 5) and partitioned during meiosis (Fig. 4),

Fig. 4. mAb 4D4 immunolabelling of human spermatocytesand spermatids (Bouin's-fixed, paraffin-embedded testisbiopsies labelled by the avidin-biotin-immunoperoxidaseprocedure). X1250. Bar=10//m. (A) The various labellingpatterns of late-pachytene primary spermatocytes. Twolabelled bodies are observed close to one another(arrowhead), or at various distances from one'another oreven at opposite poles. Arrows indicate bodies apparentlyless stained due to their location in another plane. (B) Latespermatids exhibiting labelling of the whole acrosome.(C-F): mAb 4D4-labelling patterns of the pachyteneprimary spermatocyte as a function of cell maturation:(C) single labelling being either round-shaped (left-handside) or elongated (right-hand side); (D) single labelledbody in which distinct subunits are discernible; (E) twolabelled bodies being distant from one another; (F) twolabelled bodies at opposite nuclear poles. (G) Metaphasicprimary spermatocyte showing two labelled bodies onopposite sides of the metaphase plate margin. (H) Early

anaphase primary spermatocyte exhibiting labelled bodiessituated each in one half of the microtubular spindle.(I) More advanced anaphase primary spermatocyte showinglabelled bodies close to the spindle poles. (J) Telophaseprimary spermatocyte exhibiting a labelled body close toeach chromatin mass (right-hand side). The arrow indicatesthe less discernible labelled body due to its location inanother plane. (K) Interphasic secondary spermatocytesexhibiting two labelled bodies (arrows). (L) Youngspermatids with a single labelled body (left-hand side).They can be distinguished from secondary spermatocytes(right-hand side) due to their smaller nuclear diameter.(M) More differentiated spermatids exhibiting mAb 4D4-labelling extension. Acrosomal vesicles that have releasedsome proacrosin contents exhibit clear centers. (N) Stillmore differentiated spermatids exhibiting labelling of thewhole acrosomal cap which extents over the anterior faceof the nucleus.

784 D. Escalier and others

Fig. 5. mAb 4D4-labelling patterns in pachytene primaryspermatocytes. (A) Mid-pachytene primary spermatocyteshowing clear areas inside the strongly labelled Golgi area(arrow). (B,C) Pachytene primary spermatocytes exhibitingvarious labelling patterns of the Golgi region. The labelledareas in B are suggestive of structures from which fourprotrusions seem to emerge (arrow). In C, labelling evokesa network of elements (arrowhead). (D) Late pachyteneprimary spermatocyte exhibiting labelling near each nuclearpole: one labelled area is prominent and assumes the sameaspect as in C (arrow), the other is smaller and roundshaped (arrowhead). (A-D) X2300. Bar=5/«n.

Fig. 6. mAb 4D4-labelling pattern in human testis bearingmeiotic division anomalies. (A, B, D, F, H) Bouin's-fixed,paraffin-embedded testis biopsies labelled by the avidin-biotin-immunoperoxidase procedure. (C, E, G, I) Electronmicrographs. (A-E) Sections from a biopsy characterizedby postmetaphase chromosome movement failure.(A,B) Metaphasic primary spermatocytes exhibiting anabnormally voluminous 4D4 labelled body on only one sideof the metaphase plate (arrowheads) as seen in polar (A)and lateral (B) views of the plate. X2000. Bar=5/an.(C) Metaphasic primary spermatocyte as seen in electronmicroscopy showing a monopolar half-spindle (ms). X6500.Bar=3/an. (D) Young spermatids exhibiting an abnormallyprominent labelled body (arrows). X1900. Bar=10/«n.(E) Early spermatid exhibiting Golgi units (arrows) whichabnormally extends all over the acrosome in formation(A). X12000. Bar=2/im. (F-I) Sections from a biopsycharacterized by spermatocyte cytokinesis failure. (F) Inthis early multinucleate spermatid, mAb 4D4 stainingreveals a single and prominent round labelled body whichis centrally placed (arrow). X3000. Bar=5/im. (G) Anunscattered round-shaped Golgi complex (GC) is presentin the space between the nuclei (N) of multinucleatespermatids. X13600. Bar=1.5/nn. (H, I) Spermatids moredifferentiated than in F exhibit a flattened labelled bodycomprising several extensions (arrows in H). This bodycorresponds to a single acrosome (a in I) spreadingbetween the nuclei (N). (H) X2600. Bar=5/xm.(I) X1950O. Bar=l/im.

failure, mAb 4D4 revealed a large labelled body in thespace between nuclei (Fig. 6F). In this cell region,electron microscopy showed the presence of a round-shaped Golgi complex (Fig. 6G) as normally found inpachytene primary spermatocytes. This Golgi complexgave rise to an acrosome with several extensions asrevealed by both 4D4 staining (Fig. 6H) and electronmicroscopy (Fig. 61).

biopsies from patients exhibiting different types ofmeiotic division anomalies were taken as models toelucidate whether proacrosin scattering depends on theGolgi complex partitioning.

In a patient with spermatocyte monopolar half-spindles (Fig. 6C), anaphase I-stage was not encoun-tered, which suggests post-metaphase chromosomemovement inability leading to giant spermatozoa(Escalier, 1983, 1985). Staining of testis sections withmAb 4D4 revealed two main anomalies. First, meta-phasic primary spermatocytes exhibited a labelled bodyon only one side of the equatorial plate (Fig. 6A,B)which was twice as large as in normal metaphasicspermatocytes (3/rni vs 1.5^m in length) (Fig. 4G).This labelled body was comparable to that observed innormal mid-pachytene spermatocytes (Fig. 4C).Second, early spermatids showed a labelled body beingup to 6.7 jan in length (Fig. 6D) vs 2.3/mi in normalspermatids (Fig. 4M). As shown in electron mi-croscopy, this spermatid region contained an abnor-mally prominent Golgi complex extending all over theacrosome in formation (Fig. 6E).

In another patient with spermatocyte cytokinesis

Discussion

Northern blot analysis of mouse mRNA has suggested aproacrosin biosynthesis in both meiotic and postmeioticspermatogenic cells (Kashiwabara et al. 1990). Dataobtained by means of the monoclonal antibody 4D4support this notion in human spermatogenesis and showthat proacrosin appears as early as the prophase of thefirst meiotic division. Positive reactivity with mAb 4D4was first observed in pachytene primary spermatocyteswhich exhibited an intensely labelled Golgi complex.During the subsequent meiotic steps, proacrosin label-ling was distributed among daughter cells. Finally, anincreased amount of labelled proacrosin was observedduring spermatid maturation.

Immunodetection of testicular proacrosinPrevious studies on proacrosin detection during mam-malian spermatogenesis using antibodies were per-formed on either frozen testis sections (Kallajoki andSuominen, 1984; Kallajoki et al. 1986) or isolated germcells (Florke et al. 1983a,b; Phi-Van et al. 1983;Mansoiiri et al. 1983) with a two-step labelling method.

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All these studies indicated that only spermatids werelabelled, except in the guinea pig, which showed a fewlabelled primary spermatocytes (Arboleda and Gerton,1988). Using mAb 4D4, we found that only the three-step immunolabelling on Bouin's-fluid-fLxed, paraffin-embedded testis sections enables the analysis ofproacrosin distribution in germ cells to be performed.

Other acrosomal constituents have also been foundto be first expressed in rodent primary spermatocytes(Fendersen et al. 1984; O'Brien et al. 1988; Toppari etal. 1985a,b), particularly the acrogranin, an acrosomalglycoprotein also present in pachytene spermatocytes(Anakwe and Gerton, 1990).

The immunodetection of proacrosin in germ cellsmay considerably be influenced by its moleculardiversity (Hardy et al. 1987). The anti-acrosin anti-bodies generated from acrosin (Florke et al. 1983a,b;Phi-Van et al. 1983) may not recognize the antigenicdeterminants of the primary proacrosin form or may bedirected against new determinants produced duringspermiogenesis (Arboleda and Gerton, 1988). It is thecase for monoclonal antibody C11H which has beenfound to recognize proacrosin only in late spermatids inthe mouse (Kallajoki et al. 1986). In contrast, this studyshows that mAb 4D4 is directed against an epitopepresent as soon as primary proacrosin is expressed inthe human testis.

Proacrosin and spermatogenic eventsThe mechanism of cytoplasmic organelles partitioningduring meiosis remains unknown (Suarez-Quian et al.1991). mAb 4D4 has allowed us to show how thelabelled body containing proacrosin in mid-pachytenespermatocytes gives rise to smaller bodies which aredistributed among daughter cells.

It has been suggested that forerunners of theproacrosomal vesicle first appear in zygotene primaryspermatocytes (Nicander and Ploen, 1969; Fawcett,1975; Holstein and Roosen-Runge, 1981). In pachytenespermatocytes, the Golgi body comprises several units(Kerr and de Kretzer, 1981) and its size increases due toan accumulation of trans Golgi during pachytenematuration (Suarez-Quian et al. 1991). The Golgi unitsscatter during diakinesis (Holstein and Roosen-Runge,1981). Finally, during the first three steps of spermio-genesis, the dense acrosomal matrix increases in sizeand electron density due to the incorporation ofadditional vesicles delivered by the Golgi apparatus(Burgos and Gutierrez, 1986; Thorne-Tjomsland et al.1988).

The ontogeny of proacrosin may, therefore, parallelthe biogenesis of acrosome-related Golgi vesicles andreflect the scattering of the Golgi complex units. Dataon two types of germ cell division failures are also infavour of this hypothesis, since, in these cases, both theGolgi complex and the 4D4 labelling failed to scatterduring spermatogenesis indicating that proacrosin wassequestered in the Golgi body. In one case, theformation of meiotic monopolar half-spindles led topostmetaphase chromosome movement inability(Escalier, 1985). Nevertheless, chromosome replication

and postmeiotic differentiation occurred, giving rise togiant spermatids (Escalier, 1983). In the other case, thenuclear events occurred normally but the absence ofspermatocyte cytokinesis led to the formation ofmultinucleate spermatids (Holstein et al. 1988).

Haploid expression of the preproacrosin gene hasbeen demonstrated in mammals (Adham et al. 1989;Klemm et al. 1990). Our data suggest a meiotic andpostmeiotic proacrosin synthesis in the human as in themouse in which acrosin mRNA, first detected inpachytene spermatocytes, has been found to increase inamount in round spermatids (Kashiwabara et al. 1990).Different mechanisms may be involved. First, the genemay be activated during meiosis and reexpressedpostmeiotically (Bellv6 and O'Brien, 1983; O'Brien,1987), as is the case for sperm cytochrome C (Golberg etal. 1977) and LDH-X (Hintz and Goldberg, 1977).Alternatively, meiotic and postmeiotic translationproducts may differ, as is the case for the sperm histoneH3 gene (Kennedy et al. 1985). Such stage-specificchanges leading to the synthesis of distinct proteinisoforms are known to occur also for sperm actin andtubulin (Hecht et al. 1984; Kim et al. 1989; Waters et al.1985; Slaughter et al. 1989).

In conclusion, the anti-proacrosin monoclonal anti-body 4D4 appears to be a useful probe for investigatingproacrosin biosynthesis and acrosome biogenesis inman as well as for studying the partitioning pattern ofthe Golgi apparatus during meiosis. Such propertiesmay be of particular interest in analysing variousspermatogenesis disorders or arrests in human infertilepatients.

We would like to thank Professor J. M. LUCIANI and DrM. R. GUICHAOUA (Laboratoire de Cytogdne'tique et deBiologie de la Reproduction, CHU Marseille, France) forhelpful discussions, Mrs N. PARSEGHIAN for technicalassistance, Dr A. VIELLEFOND (Laboratoire d'Anatomo-Pathologie, CHU BicStre, France) and the Deparment ofUrology (CHU Bicfitre, France) for providing tissues, Dr M.APPLANAT (U 135, INSERM, CHU Bicfitre, France) forhelp in autoradiography and Mrs F. SIRYANI for help intranslation. This work was supported by grants from theAgence Nationale de Valorisation de la Recherche (No x-84-07-039T-029-0) and from the Ministere de la Recherche et dela Technologie (MRT,No 85T-0873). Dr D. BERMUDEZwas supported by a fellowship from the DGICYT (Ministry ofthe Education, Spain).

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{Accepted 18 July 1991)