1. Introduction

Spermatogenesis, a complex process, continuingthroughout the life of the male from puberty until oldage, consists of proliferation of spermatogonia,meiosis and spermatid differentiation.

Germ cell development within seminiferoustubules is organized in sequentially repeating cellassociations of spermatogonia, spermatocytes andspermatids specific to a given area (known as stagesof the seminiferous epithelium). In the goldenhamster, XIII stages have been described byClermont [1] based mostly on the morphologicalparameters and changes observed in spermatids.

Spermatogenesis and its regulation have beeninvestigated for decades. Various methods have beendeveloped to assess spermatogenesis and to distin-guish among different cell types in seminiferoustubules.

Laser Scanning Confocal Microscopy (LSCM) isa modern tool of cell analysis which has found wideapplication in various fields of biological research.This technique exhibits several advantages overconventional optical microscopy, the major one beingelimination of out-of-focus fluorescence originatingfrom regions of the specimen above and below theselected focal plane [4, 7, 8]. Thus, the observed

images contain only in-focus information that allowsproducing optical sections within fluorescent-labeledthick specimens without physical sectioning. Thisenables the generation of high-resolution 3D imagesof cells and tissues by reconstruction from serialoptical sections. Biostructures can be imaged underconditions that are close to the living state. Changesin cell size, shape, volume as well as modificationinside the individual cell, even at the level ofchromatin structure, may be observed by means ofLSCM [4, 7, 8].

In this study LSCM was used for studying sper-matogenesis inside the tubules under conditionsminimizing cell deformation by avoiding the multi-stage process of paraffin section preparation.Longitudinal optical sections were produced at thelayers of differentiating germ cells in seminiferoustubules and stage-specific changes were followedalong the tubule. In addition expression of variousproteins during different phases of spermatogenesiswas followed using LSCM.

Methods in Cell Science 24: 169–180 (2002). 2003 Kluwer Academic Publishers. Printed in the Netherlands.

Use of confocal microscopy for the study of spermatogenesis

Margarita Vigodner, Lawrence M. Lewin, Tova Glaser, Leah Shochat, Leonid Mittelman& Rachel GolanDepartment of Clinical Biochemistry, Sackler Medical School, Tel Aviv University, Ramat Aviv, Israel

Accepted in revised form 22 November 2002

Abstract. Spermatogenesis consists of spermatogo-nial proliferation, meiosis and spermatid differentia-tion. Laser scanning confocal microscopy (LSCM)may be used as an advanced analytical tool to followspermatogenesis inside the seminiferous tubuleswithout performing histological sections. For thispurpose, separated seminiferous tubules are fixed in0.5% paraformaldehyde, stained for DNA with pro-pidium iodide and analyzed by LSCM. By producinglongitudinal optical sections in the layer of sper-matogonia, spermatocytes and spermatids, stage-specific changes in their structure may be followedwithin the tubules by LSCM. Longitudinal z-sections

Key words: Confocal microscopy, Golden hamster, Spermatogenesis

Abbreviations: LSCM = laser scanning confocal microscopy

may be obtained to produce three-dimensionalimages of the seminiferous tubules. In addition, dif-ferent proteins may be followed during spermatoge-nesis in a stage specific manner within the tubule byincubation of the fixed seminiferous tubules withappropriate antibodies.

As an example of the spermatogenesis studiesusing described LSCM techniques, detailed exami-nation of spermatogonia, spermatocytes and sper-matids during golden hamster spermatogenesis ispresented. LSCM analysis of c-kit and SC3 proteinexpression at different stages of hamster spermato-genesis is demonstrated.

2. Materials

A. Chemicals– Albumin, Bovine Serum (Sigma, A7906)– Igepal, CA-630 (Nonidet p-40) (Sigma, I

3021)– Paraformaldehyde (Sigma, P-4170)– Propidium Iodide (Sigma, P-4170)– di-Potassium hydrogen phosphate-3-hydrate

(Riedel-de Haen 04243)– Potassium Chloride (Merck, #4936)– Sodium Hydroxide (Merck, #10462)– Sodium hydrogenphosphate dodecahydrate

(Merck A905879)– Sodium Chloride (Biolab, 69684)– Sodium azide (Sigma, S 8032)

B. Tubes and pipettes– Disposable Pasteur pipettes (Elkay, #127

P503000)– Drummond Dialamatric Microdispenser

(Drummond Scientific, catalog number 75)– Tubes (Sarstedt, #55.475)– Petri dishes (Falcon, #353652, Becton

Dickinson labware)C. Equipment

– Inverted laser scan microscope (Zeiss LSM410, 488 nm laser excitation)

3. Procedure

A. Preparation of solutions and buffers– Phosphate Buffered saline (PBS)

• 0.15M NaCl• 3.2 mM Na2HPO4(12 H2O)• 1.4 mM KH2PO4

• 2.6 mM KCl• pH = 7.45

– 0.2–0.5% Buffered Paraformaldehyde, pH 7.4• 0.2–0.5% gr of Paraformaldehyde is added

to 10 ml of PBS and heated to 60 °C.Then

• 1–3 drops of molar sodium hydroxide areadded until the solution clears.

– 0.2–0.5% Igepal in PBS solution• 20–50

µl of Igepal are transferred byDrummond dialamatric microdispenser toeach 10 ml of PBS and mixed well.

– PBS-3% BSA • 3 gr of BSA are added on the surface of the

PBS.B. Experimental animals

Golden hamsters (Mesocricetus auratus) are main-tained in at 22 ± 2 °C with a 10/14 hourslight/dark cycle and are supplied feed and waterad lib. After killing male hamsters by CO2

asphyxiation, testes are surgically excised, tunicaalbuginia and remains of fat and connective tissueare removed.

C. Sample preparationSeparate seminiferous tubules carefully withinPetri dishes in the presence of PBS to avoid tubuleaggregation during fixation. To fix tubules forconfocal microscopy, add to each 1 ml of sepa-rated seminiferous tubules 10 ml of 0.2–0.5%paraformaldehyde (depending upon the antibodyto be used, see below) overnight at 4 °C. Washthe tubules three times in PBS without centrifu-gation by leaving them for 5–7 minutes to letthem settle. Store tubules in 0.1% w/v Na azidein PBS solution at 0 °C until the analysis.

D. Confocal analysis of seminiferous tubulesBefore confocal microscopy, incubate the sepa-rated fixed seminiferous tubules with propidiumiodide (PI) (50 microgram/ml final concentration)to stain the DNA of cell nuclei to produce redfluorescence. Place portions of intact tubule on acover slip with a small amount of PBS solutionusing a pipet with a widened tip avoid drying.Examine tubules by inverted laser scan confocalmicroscopy from the layer of myoepithelial cells(outer layer) toward the lumen by producinglongitudinal optical sections at different layers inthe tube. For example, the first layer (aftermyoepithelial cells) will contain the nuclei ofspermatogonia and Sertoli cells. In order toobserve spermatocytes, focus more deeply.Spermatids are found closer to the tubule lumen.Determine the spermatogenic stage by comparingthe cell associations at each place in tubules withthose reported for the species by Clermont [1] (orother authors).

At different stages, three-dimensional imagesof seminiferous tubules may be obtained byreconstruction from the serial longitudinal z-sections obtained at 10 micron intervals startingfrom outer layer of spermatogonia and movingtoward the lumen.

E. General protocol for immunostaining of cellswithin seminiferous tubules followed by confocalmicroscopy analysis.In order to follow stage-specific expression ofvarious proteins during spermatogenesis a methodof antibody penetration within tubules was devel-oped in our laboratory. Using this technique stage-specific expression of c-kit protein was studiedduring spermatogenesis and synaptonemalcomplex structure was followed in different typesof spermatocytes using the antibody against it’saxial/lateral element [9, 11]. Here, the detailedgeneral protocol is described.

Fix tubules with 0.5% paraformaldehyde asdescribed in sample preparation. However therewere antibodies (e,g, against synaptonemalcomplex, [11]) that were sensitive to 0.5%paraformaldehyde fixation and 0.2% para-formaldehyde was used in this case. On the dayof the experiment, treat a portion (approximately

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0.5 ml) of separated fixed tubules with 5 ml of0.2–0.5% Igepal in PBS for 2–5 minutes. Thepercent and time of detergent treatment are criticaland must be validated for each antibody andfixation procedure to find the optimal conditionswhere the antibody reaches the cells in the layerof interest and tubule structure is not seriouslydisrupted because this is important for stage deter-mination. Separate most of the supernatant fluid,and transfer approximately 1 ml of the tubulepellet (containing some remaining medium withdetergent) into small Petri dishes. Transfermeasured portions of the tubule pellet to the newtubes with a pipet with a widened tip. On thetubule portions, add the first antibody diluted inPBS-3%BSA. The final concentration of antibodymust be determined for each procedure. It isrecommended to start with high antibody con-centration (examples for c-kit and SC3 inVigodner et al. [11]). After overnight incubationwith the first antibody, wash tubules in PBS(without centrifugation as described in samplepreparation) and incubate with fluorescein isoth-iocyanate (FITC)-conjugated secondary antibody(anti Ig-G-FITC) for 4–5 hours and wash threemore times. After the last wash, approximately0.5–1 ml of PBS is left in the tube and PI is addedto the final concentration of 50 micrograms/ml.LSCM analysis on the portions of tubules is per-formed as described above in D).

F. How LSCM may be used to follow differentphases of hamster spermatogenesis1. Spermatogonia and their regulation

The proliferative phase of spermatogenesisbegins with division of undifferentiated sper-matogonia and is terminated by division oftype B spermatogonia into preleptotene sper-matocytes. LSCM may be to observe sper-matogonia during stage transitions in thetubule.

Determine the stage of spermatogenesis ina certain tubule fragment (as described in D).Focus on the layer where only spermatogoniaand Sertoli cell nuclei are seen and photographthem. Diameters of spermatogonia may bemeasured at each stage and chromatin distrib-ution pattern may be described. The numberof Sertoli cells remains constant in matureanimals [3] and, therefore, can be used asinternal standard for evaluating spermato-gonia/Sertoli cell ratio at each defined stage.Count Spermatogonia and Sertoli cell from thephotograph of the given section (using a trans-parency prepared with a grid) and calculatespermatogonia/Sertoli cell ratio. Changes inthis ratio may indicate loci of spermatogonialdivisions.

In order to follow the process of spermato-gonial differentiation, c-kit protein (related to

the family of transmembrane tyrosine kinasereceptors) may be used as a marker for differ-entiated spermatogonia. In order to follow c-kit expression during spermatogenesis,perform confocal microscopy after incubationof fixed seminiferous tubules with anti c-kitantibodies followed by binding of FITC-labeled secondary antibody as described in Band in Vigodner et al. [9].

2. Meiotic phase of spermatogenesis.After the final DNA synthesis which occursin preleptotene spermatocytes the tetraploidcells undergo the series of changes denotedmeiosis. Confocal microscopy may be used asan analytical tool to observe spermatocytesinside the tubules following meiotic progres-sion consecutively at defined spermatogenicstages (see [11]). To study spermatocyte dif-ferentiation the structure of the synaptonemalcomplex may be studied at various stages ofspermatogenesis using an antibody against theprotein of its axial/lateral element as describedin B and in [11].

3. Spermiogenesis.Round spermatids, the products of meiosis,undergo further differentiation, leading even-tually to formation of spermatozoa. The layersof round and/or elongated spermatids arelocated closer to the lumen than are thr sper-matocytes. Photograph the spermatids charac-teristic of different stages of spermatogenesis.(Stages are determined as describe in D.)Changes in nuclear lengths and morphologyfollowing stage transition in the tubule may bedescribed., It must be taken into account thatthe cells may be sectioned at different angles.When measuring lengths of elongated sper-matids the maximal values (for sperm headscompletely lying in the given focal plane)represent the correct parameters.

4. Results and discussion

Confocal analysis of seminiferous tubules

Spermatogenesis consists of spermatogonial prolif-eration, meiosis and spermatid differentiation. In thisstudy, the use of laser scanning confocal microscopyto follow biochemical and morphological changes ingem cells at different stages of spermatogenesiswithin the seminiferous tubule is demonstrated. Thestages of spermatogenesis occur in sequence alongthe seminiferous tubule. Observing different types ofgerm cells by changing the focal plane in the tubuleallows determination of the spermatogenic stage inthe examined tubule fragment. Thus, transition fromone stage to another may be detected by moving thelaser beam along the tubule and following stage-

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specific changes in spermatogonia, spermatocytesand spermatids. The LSCM technique also enablesproducing three-dimensional images of seminiferoustubules thus providing information about the spatialarrangement of differentiating cell types in thetubules.

For example, in Figure 1A and Figure 1B a three-dimensional image of seminiferous tubules is pre-sented after reconstruction from serial longitudinalz-sections. Whereas the stereoscopic three-dimen-sional image of Figure 1A can only be viewed usingred/green spectacles, the image at Figure 1B was

obtained from Figure 1A by a ‘depth coding’ methodby which layers at different depths in the tubule werevirtually stained with different colors. With myo-epithelial cells set at the origin, nuclei of spermato-gonia and Sertoli cell were seen from approximately6 to 20 microns depth and no other cells were presentin this focal plane. Focusing deeper, from 15 to 40microns depth, spermatocytes were observed. Roundspermatids were found from 30 to 60 microns depthand elongated spermatids from 50 microns to thelumen (approximately 100 microns).

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Figure 1 A–D. Examples of use of confocal microscopy for study of spermatogenesis. A. Three-dimensional image ofthe propidium iodide stained nuclei from seminiferous tubule taken at the stage VII of hamster spermatogenesis. Displayof stereoscopy image which can be viewed with red/green spectacles. Scale bar = 20 µm. In the surface numerousresting primary spermatocytes are seen. Some A spermatogonia (arrow) and nucleoli of Sertoli cells (arrowhead) arealso present approximately at the same depth. More deeply, spermatocytes, followed by round spermatids are seen. Groupsof mature sperm cells are found close to the tubule lumen. B. ‘Depth coding’ composite of the image used in Figure 1A.Scale bar = 20 µm. In the surface (stained red) numerous resting primary spermatocytes are seen. Some A spermato-gonia (arrow) and nucleoli of Sertoli cells (arrowhead) are also present approximately at the same depth. More deeply,spermatocytes (stained yellow), followed by round spermatids (stained green) are seen. Groups of mature sperm cells(stained blue) are found close to the tubule lumen. C. C-kit expression at stages VII–VIII of golden hamster spermato-genesis. Green fluorescence represents c-kit expression; red fluorescence, staining of cell nuclei by propidium iodide.Scale bar = 20 µm. Numerous preleptotene spermatocytes surrounding Sertoli cells are strongly positive for c-kit.D. The structure of synaptonemal complex in mid-pachytene spermatocyte during meiosis in golden hamster. Display ofstereoscopy images which can be viewed with red/green spectacles. Scale bar = 5 µm.

Confocal analysis of spermatogonia

Proliferation in germinal cells of the testes occurs inspermatogonia. By focusing only at the layer of sper-matogonia and Sertoli cells, micrographs of differentspermatogenesis stages may be prepared. Using thenumber of Sertoli cells as internal standard, sper-matogonia/Sertoli cell ratio may be evaluated at eachdefined stage, thus indicating the stages wherespermatogonia divide during spermatogenesis. Forexample, in Figure 2A where the stages of goldenhamster spermatogenesis were determined as VIII–IX(according to the cell association at this place intubule), 8 spermatogonia and approximately 20

Sertoli cells are present, therefore the spermato-gonia/Sertoli cell ratio is 0.4. In a similar manner, atstages III-IV (Figure 2B), 36 spermatogonia and 24Sertoli cells may be counted. The spermatogonia/Sertoli cell ratio, therefore, is 1.5. Seven to tensections at the same stage were analyzed, where30–60 Sertoli cells were present in each section. Ateach stage diameters of 15–20 spermatogonia weremeasured and chromatin distribution patterns wasdescribed. Cell sizes and morphology, chromatin dis-tribution patterns, modes of cell division followed bychanges in the spermatogonia/Sertoli cell ratio duringhamster spermatogenesis are summarized in Table 1and Figure 3.

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Figure 2 A–D. Changes in the spermatogonia/Sertoli cell ratio during stage transition in seminiferous tubule of goldenhamster. Scale bars = 20 µm. A. Stage VIII–IX. A differentiated spermatogonia (A dif)/Sertoli cell (S) ratio is 0.4.L:leptotene spermatocytes. B. Stage III–IV. Intermediate spermatogonia (In )/Sertoli cell ratio is 1.5. C. Stage VI–VII.Spermatogonia/Sertoli cell ratio is changed from approximately 3 for B spermatogonia (B) in the lower-right part of thepicture to 6 for resting primary spermatocytes (RPS) in the upper-left part. Mit: mitosis of type B spermatogonia toproduce resting primary spermatocytes. D. Stage VII. Resting primary spermatocyte/ Sertoli cell ratio is 6. A undiffer-entiated spermatogonia (Aund)/Sertoli cell ratio is 0.2.

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Figure 3A–H. Confocal microscopy microphotographs of nuclei from different spermatogonia types observed duringstage transition in seminiferous tubules of golden hamster. Scale bars = 20 µm. A. Type A undifferentiated spermato-gonia (arrows) found at stage V; B. Type A differentiated spermatogonia at stages XI–XII; C. Intermediate spermato-gonia at stages IV–V. D. Mitosis (Mit) of intermediate spermatogonia (Int) to produce type B spermatogonia (B) at stagesIV–V; E. Type B spermatogonia at stage V. F. Mitosis (Mit) of type B spermatogonia (B) to produce resting primaryspermatocytes (RPS) at stage VI; G. Resting primary spermatocytes at stage VII. H. Preleptotene spermatocytes stage VIII.

In contrast with transverse paraffin sectionsusually used for spermatogenic studies, longitudinalsections obtained by LSCM also provided informa-tion about the cell arrangement in each layer. It isnotable from Figure 3 that ordered organizationaround the Sertoli cells is increased from interme-diate toward B spermatogonia and resting primaryspermatocytes. Such highly ordered organizationaround the Sertoli cells allows close Sertoli – germcell interaction at the premeiotic stages and may beimportant for ligand-receptor interactions such asbetween c-kit and its ligand ([9] Figure 1C, discussedbelow).

Analyses of many sections containing numerousspermatogonia and Sertoli cells allowed us toperform accurate spermatogonia/Sertoli cell ratioestimation. This parameter was calculated for eachstage of hamster spermatogenesis. The sites of celldivision were generally in agreement with thesefound by Clermont [1]. At stages XIII–II, Clermontdistinguished between two spermatogonial divisionsand found that after the second division, part of theA spermatogonia (one out of four cells) entered along period of rest becoming a type A stem cell(‘Stem cell renewal theory’), whereas others dividedinto Intermediate spermatogonia. Doubling of thespermatogonia/Sertoli cell ratio between stages XIIIto II may be explained by ‘Stem cell renewal theory’suggesting, however, that only half of A spermato-gonia (and not three of four) divided into interme-diate spermatogonia at stage I–II. Another possibilityis absence of the second division between stagesXIII–II. The last suggestion is supported by a studyof Miething [3] where, by following %S by bromode-oxyuridine incorporation in the golden hamster, the

author found only one peak in %S values betweenstages XIII-II. In spite of some variations in thenumber of spermatogonia at each stage, the sper-matogonia/Sertoli cell ratio can be helpful for stageidentification in the tubule, especially in the stagesII-VII where the number of spermatogonia is largeand the ratio is markedly changed from approxi-mately 1.5 to 3 and then to 6. It may also be used asa diagnostic criterion in some cases of testiculardisorders, where decrease in spermatogonia numbermay be obtained ([9], see below). In order to detectthe exact stage of impairment in spermatogonialproliferation it would be useful to follow stagetransitions along the tubule and to determine thespermatogonia/Sertoli cell ratio at each stage.

Immunostaining and LSCM analysis of c-kit proteinexpression during spermatogenesis

One of the systems regulating spermatogonial pro-liferation and differentiation is the c-kit receptor andits ligand, Steel factor. LSCM observation of longi-tudinal sections of seminiferous tubules followingstage transition along the tubule allowed us to followthe spermatogenic wave sequentially to determine inwhich stages of hamster spermatogenesis and inwhich cell types c-kit could be observed. It wasdemonstrated that c-kit150T system appears in dif-ferentiating hamster spermatogonia and remains asso-ciated with the cells until the pachytene stage ofspermatocytes, with maximal staining intensity at thepremeiotic stages. The results were summarized inVigodner et al. [9]. In Figure 1C the example ofimmunostaining with anti c-kit antibody at stage VIIof seminiferous epithelium is illustrated.

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Table 1. Appearance of spermatogonia (Sg) types during the spermatogenic stages in golden hamster. Cell sizes,morphology and ratio to the Sertoli cells. Aund – spermatogonia type A undifferentiate Adif – spermatogonia type Adifferentiated, In – intermediate spermatogonia, B – type B spermatogonia, RPS-resting primary spermatocytes

Sg type Stages Sg/sertoli Nuclear diameter Nuclear morphologycell ratio (microns)

A und I–VII < 0.4 8–11 Oval nuclei with uniformly distributed chromatin (Figure 3A)

A diff VII–VIII 0.4 ± 0.1 9–14 Dark nuclei containing condensed IX-X Cell divisions chromatin flattened in part a long XI–XII 0.7 ± 0.1 the nuclear membrane XIII–I Cell divisions (Figure 3B)

In II–IV 1.3 ± 0.4 9–11 Oval nuclei containing dots of IV–V Cell divisions (Figure 3D) heterochromatin (Figure 3C)

B V–VI 3.2 ± 0.3 8–9 Cell nuclei with central dot of VI Cell divisions (Figure 3F) heterochromatin and ring of

condensed chromatin along the nuclear membrane (Figure 3E, G)

RPS VII 6.0 ± 0.8 6–8

Confocal analysis of spermatocytes

After numerous spermatogonial divisions, cells entermeiosis. Focusing only at the layer of spermatocytes,micrographs of spermatocytes from different stagesof hamster spermatogenesis were prepared and theirsizes and morphology were described. The resultswere summarized in ‘Meiosis in the golden hamster.A flow cytometry and confocal microscopy study’[11]. To follow spermatocyte differentiation, thesynaptonemal complex was visualized in spermato-cytes of different stages inside the tubules using theantibody against the proteins of its lateral element[11]. In chosen spermatocytes from different stages,3-dimensional images were prepared to supplementthe information about synaptonemal complex struc-ture at these stages (see [11] and Figure 1D).

Confocal analysis of spermatids

Round spermatids, the products of meiosis, undergofurther differentiation, termed spermiogenesis.Dramatic changes in nuclei morphology (from roundspermatids to mature sperm) occurring during thisprocess may be studied by LSCM within the tubules.For example, in Figure 4 a gallery of spermatids fromdifferent stages is presented as observed by LSCManalysis of seminiferous tubules and their nuclearlengths are summarized in Table 2. Nuclear lengthsand morphology were recorded for 15–20 cells ateach spermatogenic stage.

Summary map of germ cell association duringhamster spermatogenesis as studied by means ofCLSM

LSCM observation of longitudinal sections of semi-niferous tubules provided useful information aboutsizes and morphology of spermatogonia, spermato-cytes and spermatids at different stages of sper-matogenesis in the golden hamster. Thus, extensiveinformation about stage-specific germ cell changesduring golden hamster spermatogenesis was obtainedwhich was summarized as a table of cell associationsthroughout different hamster spermatogenesis. Thisfacilitated relatively rapid and non- complicated stage

determination in the tubule by mean of LSCM(Figures 5, 6).

As can be seen from the figures, the stagedetermination can best be accomplished starting fromthe layer of spermatids. Whereas at stages I–VII,pachytene spermatocytes are associated with roundand then with elongated spermatids (Figure 5), noround spermatids may be found at the stages VII–XII.At these stages pachytene spermatocytes wereassociated with elongating spermatids (Figure 6).Dramatic changes in spermatid shape at stagesVIII–XIII simplify the determination of these stages(Figure 6). At stage XIII meiotic divisions andsecondary spermatocytes (differing in their sizes fromthe round spermatids) are found. The appearance ofround spermatids after meiotic division characterizesstage I. The Spermatogonia/Sertoli cell ratio is a goodmarker for distinguishing between stages II–IV, V–VIand VII (Figure 5). There are no significant changesin cell types during stages II–IV and, therefore, thesestages are difficult to determine exactly. However,some assessment may be done by following changesin sizes of spermatocytes and in size and morphologyof spermatids. Using LSCM to follow spermatogen-esis, observation of changes in one germ cell layer,is supplemented by information obtained from theother layers in the same tubule fragment as well asfrom the cell associations from both sides of thefragment in the tubule.

When LSCM analysis may be used for study spermatogenesis

Detailed characterization of spermatogenic stagesalong the tubule together with immunostaining withinseminiferous tubules without destruction of theirstructure gave us the possibility to study stage-specific expression of different proteins duringspermatogenesis by means of LSCM. Examples ofsuch proteins are c-kit protein on spermatogonia orSC3 protein in spermatocytes during meiosis. Stage-specific expression of many other proteins duringspermatogenesis may also be followed using thedescribed technique. Immunostaining within tubulesfollowed by LSCM analysis, however, is not alwayssuitable for the study of spermatids, especially, elon-

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Table 2. Nuclear diameters and form of spermatids as they appear during the spermiogenic steps in golden hamster

Steps Nuclear shape Nuclear length (microns)

1–3 Round (Figure 4A) 7–94–8 Gradual loss of round form (Figure 4B,C) 6–89–10 Triangular (Figure 4D) 5–810–11 Triangular-elongating (Figure 4E) 6–1012–13 Elongating (Figure 4F) 9–1214 Elongated (Figure 4G) 9–1215 Elongated 8.5–1016–17 Elongated (Figure 4H) 7–9

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Figure 4A–H. Confocal microscopy microphotographs of nuclei from different spermatid types observed during stagetransition in seminiferous tubules of golden hamster. Scale bar = 10 µm. A. Spermatids of steps 1–2. B. Spermatids ofsteps 4–5. C. Spermatids of steps 7–8. D. Spermatids of steps 9–10. E. Spermatids of steps 10–11. F. Spermatids ofsteps 12–13.G. Spermatids of step 14. H. Spermatids of step 17.

gated spermatids which are situated more deeply intubule. There are two reasons for this. Firstly, anti-bodies can reach spermatid layers only with aggres-sive detergent treatment which may disrupt the tubulestructure. Second, as the laser beam penetrates moredeeply within the tubules, its intensity decreases andthe sharpness of images decreases. In order to testthe antibody reactivity with the cells of inner layers,the edge of the tubule segment may be used as acontrol because antibody accessibility is equal for allcell layers at this interface.

Stage-specific changes in germ cell number and

morphology as a result of different failures in sper-matogenetic process may be detected using LSCManalysis. In order to detect the stage of impairmentin spermatogonial proliferation, meiotic progressionor spermatid differentiation in these cases, it wouldbe useful to follow stage transition along the tubule,comparing stage-specific parameters described fornormal spermatogenesis (such as spermatogonia/Sertoli cell ratio at each stage, germ cell nuclei sizesand morphology) with those found in abnormalspermiogenesis. This may be combined with theanalysis of protein expression, when stage specific

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Figure 5. Map of cell association during spermatogenesis in the golden hamster as studied by confocal microscopy.Stages I-VIII. Adif – type A differentiated spermatogonia; In – Intermediate type spermatogonia; B – type B spermato-gonia; RPS – resting primary spermatocytes; A undifferentiated spermatogonia (Figure 3 A) found in all spermatogenicstages are not shown.

changes for the given protein expression have beenstudied. Thus, for example, decreased germ/Sertolicell ratio was obtained at the premeiotic stages ofspermatogenesis in a group of old hamsters comparedto this ratio in younger ones. In addition, decreasedc-kit expression was found in some areas of semi-niferous tubules of old animals [9]. In a similarmanner, LSCM analysis of seminiferous tubules fromanimals which had undergone experimental cryp-torchidism [10], revealed a block in spermatid mat-uration at the stages IX–X of spermatogenesis at thebeginning stages of cryptorchidism.

Conclusions

LSCM analysis provided extensive information aboutproliferation, meiosis and spermatid maturationduring spermatogenesis in the golden hamster. Thisallowed studying spermatogenesis in a novel way byfocusing on individual cell types and followingchanges along the tubule. Tubule structure wasstudied under conditions that minimized the size andform deformation that paraffin section preparationmight cause. This technique could readily be adaptedto analysis of spermatogenesis in other species. It

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Figure 6. Continuation of Figure 5. Stages IX–XIII. Adif – type A differentiated spermatogonia; Pl – preleptotene sper-matocytes; L – leptotene spermatocytes; Z – zygotene spermatocytes; P early – early pachytene spermatocytes; P mid –mid pachytene spermatocytes; P late – late pachytene spermatocytes; Di – diplotene spermatocytes; Sec – secondaryspermatocytes. A undifferentiated spermatogonia (Figure 3A) found in all spermatogenic stages are not shown.

also may be used as a diagnostic tool in some casesof spermatogenic disorders. Rapid and relatively non-complicated methods for spermatogenesis assessmentmay be extremely useful in clinical practice foranalysis of testis biopsies from patients with varyingtesticular disorders.

Acknowledgements

The studies reported herein were supported, in part,by a grant from the office of the Chief ScientistMinistry of Health, State of Israel. The authors thankDr. C Heyting for her generous supply of SC3antibody.

Notes on suppliers

01. Becton Dickinson and Company, Franklin Lakes, NJ,USA

02. Biolab, Jerusalem, Israel03. Elkay Sales Inc, Oak Brook, IL, USA04. Merck, Darmstadt, Germany05. Riedel-de Haen, Seelze-Hannover, Germany06. Sarstedt Inc, Nurnberecht, Germany07. Sigma Chemical Co., Saint Louis, USA08. Carl Zeiss, Str. 22 73447 Oberkochen, Germany

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Address for correspondence: Dr. Patricia L. Morris (forM. Vigodner), Population Council, The RockefellerUniversity, Box 273, 1230 York Avenue, New York, NY10021, USAPhone: 1-212-327-8756; Fax: 1-212-327-7678E-mail: mvigodne@popcbr.rockefeller.edu

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