phase-contrast and electron- microscopic …€¦ · in those cases where culture ma conditiony hav...

y.CellSci. 18, 1-17(1975)Printed in Great Britain

PHASE-CONTRAST AND ELECTRON-

MICROSCOPIC OBSERVATIONS ON A

MEMBRANOUS COMPLEX IN PRIMARY

SPERMATOCYTES OF THE MOUSE

A. PLESHKEWYCH* AND L. LEVINE

Department of Biology, Wayne State University,Detroit, Michigan 48202, U.S.A.

SUMMARY

A prominent cytoplasmic inclusion present in living mouse primary spermatocytes has beenobserved by both light and electron microscopy. It began to form at prometaphase and con-tinued to increase in thickness and length as the cells developed. By metaphase it was a distinctsausage-shaped boundary that enclosed a portion of the cytoplasm between the spindle and thecell membrane. At the end of metaphase, the inclusion reached its maximum length. At telo-phase, it was divided between the daughter secondaries. The inclusion persisted as a circularcontour in the interphase secondary spermatocyte.

Electron microscopy of the same cultured cells that were previously observed with lightmicroscopy revealed that the inclusion was a distinctive formation of membranes. It consistedof agranular cisternae and vesicles, and was therefore a membranous complex. Many of thesmaller vesicles in the membranous complex resembled those found in the spindle. The cis-ternae in the membranous complex were identical to the cisternal endoplasmic reticulum ofinterphase primary spermatocytes. Neverthless, the organization of vesicles and cisternae intothe membranous complex was unique for the primaries in division stages, since such anorganization was not present in their interphase stages.

INTRODUCTION

Several references to studies on cultured mammalian germ cells may be cited, butonly a few of these described the detailed cytology of the living primary spermatocyte.In those few cases where detailed descriptions were given (Fawcett & Ito, 1958;Bryan, 1971), the spermatocytes failed to engage in or complete their meiotic divisions.In those cases where culture conditions may have permitted spermatogenesis tooccur, organ cultures were used (Steinberger & Steinberger, 1966 a), thereby necessi-tating that fixation and sectioning precede cytological observations. In other studies,cultures were initiated with previously dispersed cells (Jordan, Katsh & de Stackel-burg, 1961; Kodani & Kodani, 1966; Steinberger & Steinberger, 19666), but germinalcells did not become sufficiently flattened for phase-contrast microscopy. In a recentstudy on Chinese hamster germ cells (Ellingson & Yao, 1970), the seminiferous tubuleswere flattened under dialysis membranes to allow the continuous observation of thecultures as they developed. Primary and secondary spermatocytes were observed to

• Present address: Natural Sciences Concentration, Rosary Hill College, Buffalo, New York14226, U.S.A.

I CEL 18

2 A. Pleshkeioych and L. Levine

have completed their divisions in these cultures, but the detailed cytology of the livingprimary spermatocyte in division was not described.

In the present study, mouse primary spermatocytes were cultured under conditionsthat allowed a detailed cytological analysis to be made of them while they completedthe meiotic divisions. They were found to develop a prominent cytoplasmic structureduring division that was not present in interphase. This structure was subsequentlystudied with the electron microscope and shown to be composed of membranes,forming a membranous complex.

METHODS

Materials

The spermatocytes were cultured in medium F, the composition of which is shown in Table i.Most substances were obtained commercially, except for the mouse serum and testes extract.Mouse serum was prepared as follows. Juvenile male mice, weighing approximately 20 g, wereanaesthetized with ether. The orbital regions were wiped with 70 % ethanol. They were thenbled from the orbital artery into centrifuge tubes that were refrigerated at 4 °C for about 1 h.The blood was then centrifuged at 270g for 7 min to pack the clot; the serum was decanted,immediately frozen, and stored at — 22 °C.

Testes extract was prepared from adult male mice. These were killed by cervical dislocation.Their testes were immediately removed and placed in equal volumes of sterile Puck's saline G.After the tunica albuginea was removed, the seminiferous tubules were cut into i-mm lengths;

Table 1. Composition of medium F

A. Concentrations in 100 ml Puck's saline G*, fig

Thyroxin" 21Testosterone" 200Vitamin Ad 20Vitamin C° 144Vitamin E' 2000

B. Concentrations in 10 ml medium F, ml, fig

Puck's saline G (A, above) 3'6 —Puck's medium N-15* 40 —NCTC-135' o-4 —Mouse serum 1-5 —Mouse testes extract 0-5 —Follicle-stimulating hormone' — 700Luteinizing hormone11 — 500Insulin' — 50

* Grand Island Biological Co., New York.b L-thyroxine pentahydrate, Nutritional Biochemicals Corp., Ohio (NBC).0 Mann Research Laboratories Inc., New York (MRL).d Vitamin A, crystalline alcohol, NBC.* L-ascorbic acid, Fisher Scientific Co., New Jersey.' D-a-tocopherol, acid succinate, NBC.' Containing testosterone, vitamins A, C, H.11 Procine, MRL.1 Equine, MRL.

Membranous complex in mouse spermatocytes 3

equal volumes of cold Puck's saline G were added and the tissue was thoroughly homogenized(5-10 min) in an ice bath. Centrifugation (30,ioog) followed for 2 h, the supernatant wasremoved, frozen immediately, and stored at — 22 °C.

Preparation of spermatocytes

Vigorous 4-week-old males were killed by cervical dislocation. The scrotal sac was surface-sterilized with 70 % ethanol and a testicle removed to a drop of medium, previously placed undermineral oil (Squibb, U.S.P.) contained in a glass Petri dish. The tunica albuginea was removedand 3 samples of seminiferous tubules, about 3 mm long, were cut from 3 different tubulesegments.

Further dissection was carried out in a special culture observation chamber. This was madeof a 2-5 X7'S cm aluminium plate with a central rectangular 26 x 15 mm hole. A carefullycleaned sterilized coverslip was sealed over the hole with melted paraffin-petrolatum (3:1).The resulting well was filled with mineral oil and a drop of medium F was placed on thecoverslip under the oil. The tubule segments were transferred into the droplet. Removal ofsome medium compressed the tubules and caused expulsion of the germinal cells; furtherslight pressure on the tubules, applied with stainless steel needles, aided in further emptying thetubules and spreading the cells on the coverslip. The tubules were then removed, and anothercoverslip was placed over the well and sealed with the paraffin-petrolatum. This method wasessentially modelled after Levine (1972).

The culture was incubated on the microscope stage at 31 °C by means of an air curtain. Thiswas the temperature measured at the surface of scrotum with a banjo thermistor. Observationsof the cultures were made with the following optics: Zeiss Neofluar x 100, 1*3 n.a. phase-contrast objective, Zeiss Achro-aplan 14 n.a. condenser with a Zeiss Fal Series 549 greenfilter and KG-i W3rmschutzfilter in the light path. Data were recorded both in writing and bytime-lapse photography. In the latter case a specimen-shielding shutter protected the cellsbetween exposures.

Electron microscopy

A modified culture method was used when electron microscopy followed light microscopy.Gelatin was incorporated into the culture medium to make the spermatocytes adhere to thecoverslips. The gelatin-maintenance medium was prepared by dissolving 1 g of gelatin (Bakerand Adamson, U.S.P.) in 1 ml of hot glass-distilled water. After cooling and solidification thegelatin was cut into 4-mm squares and placed in a sterile screw-cap flask with 3-17 ml mediumcontaining 152 ml Puck's saline G, 1-50 ml Puck's Medium N-15 and 0-15 ml NCTC-135.Mineral oil (Squibb, U.S.P.) was added over the suspension, and the flask and its contentswere equilibrated at 4 °C for about 12 h with occasional shaking. Then 4 ml of completemedium F were added under the mineral oil and equilibration continued at 4 °C for another12 h. After this time, the medium was decanted and the gelatin blocks transferred intoa graduated centrifuge tube. Sufficient mineral oil was added to cover the gelatin and it wasmelted at 40 °C to record its volume and calculate the new concentration of gelatin that resultedfrom imbibition during equilibration. The new concentration usually fell between 11 and 12 %.Before use enough maintenance medium F must be added to dilute the gelatin to 8 0 % inorder for it to be at optimal tonicity.

The maintenance medium with gelatin was used instead of medium F for the spermatocytesdestined for electron microscopy. In addition, the culture-observation chamber was preparedwith a carbon-coated coverslip before filling it with mineral oil in order to facilitate eventualseparation of the cells from the coverslip after Epon embedding (Robbins & Gonatas, 1964).

The chamber was placed in a refrigerator at — 6 °C for 5 min, upon completion of its pre-paration, to induce gelation of the medium. Following this the culture was placed on themicroscope stage and a field with dividing spermatocytes was encircled and observed until thecells reached the desired division stage. At this time, the spermatocytes were photographed,and the carbon-coated coverslip was removed from the aluminium slide and immersed rapidly,cell side down, at a 45° angle into 2% glutaraldehyde (Sabatini, Bensch & Barrnett, 1963)buffered with o-i M s-collidine at pH 7-4 (Luft, 1956) at o °C. The coverslips were immersed

4 A. Pleshkewych and L. Levine

at this angle for 20 min to ensure thorough removal of the oil. Following this they were placedin Coplin jars, rinsed 5 times in 01 M j-collidine buffer (pH 7-4); four of these rinses were for5 min each, the fifth lasted 12 h. Postfixation followed at 25 °C for 1 h in 2 % osmium tetroxide(Palade, 1955) buffered with o-i M s-collidine (pH 74). After postfixing, the cultures weredehydrated in an ethanol series, and infiltrated through propylene oxide: Epon to Epon alone.

The method of Robbins & Gonatas (1964) was used for removal of the infiltrated cells fromthe coverslips. That is, the coverslips were remounted cell side up and the previously photo-graphed encircled field was relocated in order to scratch and break the carbon coat with a diamondobjective marker. A plastic capsule filled with Epon was then inverted over the encircled cellson the coverslip before polymerization.

The capsule was snapped off the coverslip when polymerization was complete. Examinationof the capsule face revealed the circular break in the carbon coat, so that it could be trimmedas close to the cells as possible. Five hundred 70-nm sections were cut with a diamond knife onan LKB Ultratome. The sections were picked up on 200-300 mesh copper grids and stainedfor 15 min with uranyl acetate (Watson, 1958) followed by 15 min with lead citrate (Reynolds,1963) before observation in an RCA EMU-3H electron microscope operating at 50 kV.

RESULTS

Maximum survival time for primary spermatocytes in medium F was 27 + 1 h at31 +1 °C. The survival time was affected by several conditions; one of the mostimportant of these was brief exposure to air for even a fraction of a second, for thisresulted in irreversible lethal hypertonic effects (Levine, 1972). Another importantfactor was the medium itself. The medium used resulted from a number of trials atcombining Puck's saline G (Puck, Cieciura & Robinson, 1958) with various supple-ments. The variations of media and results obtained are described elsewhere (Plesch-kewych, 1970). Suffice it to say, that in a medium which consisted of 41% Puck'ssaline G, 40% Puck's N-15, 4% NCTC 135 and 15 % foetal calf serum, withoutaddedvitamins, hormones or testes extract, the primary spermatocytes remained normalin appearance for about 2 h. Very soon thereafter degenerative signs became apparent.These were swelling of chromosomes, erratic and uneven chromosome motions andfailure of cells to divide. Another important factor affecting spermatocyte survivaltime was the incubation temperature. Cultures deteriorated rapidly at 36 + 2 °C or26 + 2 °C. Using all of the optimal culture conditions described, primary spermato-cytes survived long enough to complete normal meiosis I from the diakinetic stage ofprophase to interkinesis of the daughter secondary spermatocyte.

A prominent sausage-shaped structure is found in the cytoplasm of metaphaseprimary spermatocytes (Fig. 1, me), between the outer spindle margin and the cellmembrane. It consists of a relatively refractile contour which encloses a lighter centralarea. Its outer boundary is composed of several layers of fine linear structures on whichmitochondria and the large dark granules are found; occasionally, granular materialis also present in the light central area.

This inclusion was never found in any of the interphase primary spermatocytes.The earliest stage at which it was observed was in prometaphase, when the inclusionappeared as several thin parallel lines lying close to one another. As prometaphaseprogressed these thin lines fused to form the more prominent structure seen at meta-phase I. Its subsequent behaviour is shown in Figs. 2-8, which are frames printedfrom the time-lapse film from which Fig. 1 was taken. The inclusion lengthened during


metaphase (Figs. 1-3); its ends extended towards the spindle poles and curved aroundthem to form the letter ' C . The region of the inclusion juxtaposed to the spindleinterzone became straightened and narrowed during anaphase (Figs. 4, 5). Duringcytokinesis the inclusion was divided between the 2 daughter secondary spermatocytes(Figs. 6-8). Bridge-like connexions were formed between the inclusion and the con-densed chromatin masses in late telophase (Fig. 7). These connexions remained untilthe nuclear envelope was uniformly reconstructed (Fig. 8). The inclusion persistedinto late interphase of the secondary spermatocyte and became smaller as thesecondaries aged.

When the time-lapse films are projected the large dark granules are seen to moveparallel to the outer boundary of the inclusion. During telophase the movement ofthese granules becomes localized in the area between the inclusion and the newlyformed nuclear envelope or on the surface of the condensed chromatin, before thenew nuclear envelope is formed. Also apparent on projection is the slow progressiveelongation of the inclusion as the cell completes metaphase.

The sausage-shaped inclusion is present after fixation and embedding. Fig. 9A isa field of testicular cells in Epon. A metaphase spermatocyte in the field (Fig. 9 A, box)is enlarged in Fig. 9B. This cell is normal in appearance as is evidenced by the lateralseparation of chromosomes and their regular alignment on the spindle equator. Thespindle is normally shaped. Just outside one spindle margin is the sausage-shapedinclusion. It is imaged as a definite boundary near the bottom of the cell, but becausethe cell is tipped slightly, the inclusion cannot be focused sharply along its entirelength. Nevertheless, it is outlined by the aligned granules and the relatively clearspace that it delimits.

Electron micrographs showed that the sausage-shaped structure was a formationof membranes, a membranous complex. Fig. IOA shows a living primary spermato-cyte in metaphase that was cultured in gelatin maintenance medium: a typical inclu-sion is present. Fig. IOB is an electron micrograph of this spermatocyte. Rows ofmembranous profiles occupy the position of the inclusion in the living cell. One rowof membranous elements is adjacent to and concentric with the plasma membrane(Figs, IOB, 11), and another row runs next to the spindle (Figs, IOB, I I ) . Between therows is a space approximately 1-5 /tm wide (Figs, IOB, I I , 12). Within this space aremitochondria and vesicles in a matrix that is identical to the cytoplasmic ground sub-stance outside the borders of the membranous complex (Fig. 12).

There are 2 forms of membranes in the membranous complex: cisternal andvesicular. The cisternal forms are parallel profiles, 40-80 nm apart, which are closedat either end to form flattened sacs 1-3 /tm in length (Figs. 11, 12). The other elementsare round or elliptical vesicles, 100-200 nm in diameter (Figs, IOB, I I , 12) or largerones in the i-/*m size range. In some cases, the vesicular form predominated, whilein others the cisternal form was more common. Both the vesicular and cisternal formsare agranular (Fig. 12).

Long lamellar cisternae are occasionally present as single isolated units in thecytoplasm of metaphase primary spermatocytes at some distance from the mem-branous complex. Most of these lamellae, however, are arrayed in the membranous


complex (Fig. IOB), oriented in stacks with their long axes parallel to the centriolaraxis. Occasionally, however, one of these cisternal units crosses the space within themembranous complex (Fig. 12). The cisternal units contain a very fine granular sub-stance that differs in texture, but not in density, from the cytoplasmic ground sub-stance (Figs. 11, 12). The cisternal units present very little evidence of branching,although some of the lamellar units are irregular in outline because of local swellings.

The vesicular structures of about 1 /tm diameter interrupt the rows of cisternae(Figs, IOB, 11, 12). They have clear interiors and are frequently continuous with thecisternae (Fig. 12). The 100—200 nm vesicles are interspersed among the lamellae.They may be aligned in groups along the axis defined by the cisternae (Fig. 11),aggregated in irregular masses adjacent to the membranous complex (Fig. IOB), orscattered at random in the cytoplasmic matrix enclosed by the membranous complex(Figs. 11, 12). Most of the vesicles have clear, electron-transparent interiors, althougha few of them contain an amorphous, smoothly textured substance; a very smallnumber contain a substance similar to the matrix within the cisternae. Vesiclessimilar to those that associate with the membranous complex are also found withinthe spindle (Figs, IOB, I I ) .

An organelle that appeared to be a multivesicular body was found in one of thesections (Fig. 12). It was situated very close to the membranous complex and seemedto be embedded in an aggregated mass of vesicles. Another association that was foundinfrequently was between chromatin and cisternae of the membranous complex(Fig. 11).

The cytoplasmic membranes in interphase primary spermatocytes (Fig. 13) formedstructures characteristic of the cisternal agranular endoplasmic reticulum. Individualcisternae tended towards parallel alignment with the plasma membrane or the nuclearenvelope. They were continuous with the outer membrane of the nuclear envelope(Pleshkewych, 1970). The width of the cisternae (30-50 nm) was in the same sizerange as the width of the perinuclear cisternal space. The cisternae contained a finelygranular substance. Vesicles were found scattered at random through the cytoplasm,aggregated in masses, or closely associated with the Golgi complex (Pleshkewych,1970).

In no case were cisternal or vesicular formations found in the interphase spermato-cytes that were as distinctive as those of the membranous complex. However, theindividual lamellar and vesicular units of the membranous complex were very similarto those found in the interphase spermatocytes.

DISCUSSION

The culture method used in this study involved isolation of the spermatocytes fromthe seminiferous tubule in a thin layer of maintenance medium under a vapour sealof mineral oil. The spermatocytes were sufficiently compressed by the medium topermit observation with phase-contrast microscopy, and the mineral oil preventedevaporation of water and the consequent development of lethal hypertonic effects.This method is essentially that of Levine (1972) except for the culture medium. The


medium, which consisted of Puck's saline G and NCTC-135 was supplemented withvitamins, hormones, homologous serum and testes extract. These supplements wereessential, as the spermatocytes became abnormal within a few hours without them.The importance of hormones and vitamins in mammalian male germ cell culture wasdemonstrated by Steinberger, Steinberger & Perloff (1964), who showed that follicle-stimulating hormone (FSH) was needed to maintain spermatocytes for extendedperiods of time. In addition vitamins A, C and E promoted differentiation of primaryspermatocytes from the germinal epithelium, although reduction divisions were notobserved (Steinberger & Steinberger, 1966a). Studies by Basu, Nandi & Nandi (1966)on the culture of frog testes showed that FSH, luteinizing hormone, insulin andtestosterone resulted in maintenance of germ cells and stimulation of spermatogenesisthrough mciosis I.

Ellingson & Yao (1970) described extended maintenance of Chinese hamster germcells in Rose chambers under dialysis membranes. They reported that primaryspermatocytes in metaphase, when cultures were initiated, completed their meioticdivision, and remained normal for 3-4 days. In addition, spermatogonia lived for2-3 weeks and underwent frequent mitoses. Their medium did not contain supple-mental vitamins and hormones, but the dialysis membrane which was used to com-press the seminiferous tubules may have concentrated macromolecules or slowed thediffusion of these or other critical substances away from the tubules. In our culturemethod tubule fragments were removed from the chamber after their cellular contentswere spread on the coverslip, so that only the isolated cells remained. It is suggestedthat the absence of the seminiferous tubules made supplementation by the variousfactors necessary. Although the effects of each of the individual factors were nottested separately, the culture method we describe provides a system in which theseand other effects may be determined.

In the present study, the survival time in vitro was of sufficient duration to permitcompletion of normal meiosis I from diakinesis through telophase. However, the cellsdid not survive very long when compared to mammalian somatic cell culture. It issuggested that the minimal amount of medium that was necessary to flatten the cellsfor phase-contrast observation contributed to their shorter lives. Supporting this isthe observation that thicker preparations tended to live longer. These thicker pre-parations, however, were unsuitable for phase-contrast microscopy, and the poten-tiality for longer life spans was compromised in order to obtain detailed observationsof spermatocyte cytology as they developed.

Stabilization of cell position was essential for the electron microscopy. A specialmethod was needed for this as other methods (Mole-Bajer & Bajer, 1967), whichinvolved application of an agar-gelatin membrane over the cells, could not be used.The presence of mineral oil in the culture chamber, an absolute requirement initself, prevented placement of such a membrane.

The method for cell stabilization that was used here was originally developed tocause cricket spermatocytes to adhere to coverslips for cytochemical experiments(Levine & Shifrin, 1973). The method involved incorporation of gelatin into themaintenance medium and its solidification by chilling after the cells were isolated


in it. The cells were stabilized therefore by the gel that encased them. In theory,the method may introduce abnormalities, for exposure to low temperatures isknown to disrupt spindle organization (Rebhun, Rosenbaum, Lefebvre & Smith,1974). But the cold-induced spindle disruption is fully reversible. Reorganization ofspindle microtubules and reappearance of spindle birefringence commences im-mediately upon return to normal temperatures and is complete after a short time -2-4 min at 23 °C for giant amoeba spindle microtubules (Roth, 1967), and 8 minat 27 °C for spindle birefringence in pollen mother cells (Inoue, 1963). It wouldappear that the mouse spermatocytes recovered from possible harmful effectsof the low temperature required to initiate gelation. Cells that were isolated andstabilized in gelatin medium and exposed briefly to the lower temperaturesformed normally shaped spindles and metaphase plates upon their return to incuba-tion temperatures, and they underwent normal anaphases. Furthermore, sufficienttime must have elapsed between gel initiation and fixation, since most sectionsthrough the spindle contained relatively long bundles of parallel microtubules thatspanned more than two thirds the distance between kinetochore and centriole (Fig. 1 o B,and Pleshkewych, 1970).

That the formation of cisternae and vesicles found in electron micrographs is theinclusion in living cells is supported by the following. The inclusion is present afterfixation and embedding, albeit with less contrast than in living cells, perhaps as aresult of suboptimal refractive index differences between it and the Epon matrix.In low-magnification micrographs the distinct organization of stacked cisternae andvesicles that are designated as the membranous complex appear in the same positionas the inclusion in living cells. Such a distinct organization was not found in any otherlocation nor was it present at interphase.

The lamellar membranes were agranular; they formed sacs which were practicallyidentical to the cytoplasmic membranes found in the interphase primary spermato-cytes. The membranes in interphase spermatocytes were typical of agranular endo-plasmic reticulum of the rat (Palade, 1955), guinea pig (Fawcett & Ito, 1958), crayfish(Kaye, Pappas, Yasuzurin & Yamamoto, 1961) and locust (Barer, Joseph & Meek,i960) spermatocytes. In the mouse spermatocyte continuities were found betweenthe lamellated cisternal elements and the outer membrane of the nuclear envelope(Pleshkewych, 1970), an observation that further strengthens their designation asendoplasmic reticulum (Watson, 1955). Furthermore, in living cells, the membranouscomplex interacted with the telophase chromatin by means of bridge-like extensions.Although the latter may represent adventitious connexions, we cannot discount thepossibility that these extensions represent the transfer of membrane material on tothe telophasic chromatin for the assembly and/or further synthesis of the new nuclearenvelope around the daughter nucleus. This behaviour would be consistent withthat of the endoplasmic reticulum, which is believed to contribute to nuclear envelopeformation after cell division (Barer et al. i960; Merriam, 1961).

Most of the vesicles in the membranous complex had clear interiors that dif-ferentiated them from cross-sections of the cisternae, which enclosed a characteristicmatrix. It is not possible to determine the exact origins of these vesicles. Some


probably arise by vesiculation of lamellae brought on by inadequate fixation. Never-theless, the largest of the vesicles in the membranous complex resembled those foundin close association with the Golgi lamellae (Pleshkewych, 1970), or scattered aboutthe cytoplasm of interphase spermatocytes. The vesicles in the small-to-intermediatesize range resembled those present in the spindle. The membranous complex, there-fore, does not appear to contain unique components. It is rather the organization ofthese components during the meiotic divisions that distinguishes it.

Distinct formations of smooth endoplasmic reticulum that differ from those ininterphase occur during mitosis in somatic cells. In regenerating rat liver (Dougherty& Lee, 1967), aggregations of smooth endoplasmic reticulum were followed throughmitosis. The changing pattern of location of the smooth endoplasmic reticulum sug-gested that displacement or flow of these membranes had occurred. A possible rolefor the aggregates in regulation of cytokinesis was suggested by their position at lateanaphase. Distinctive aggregation of smooth endoplasmic reticulum and spindlemicrotubules occurred in the spore mother cells of Tradescantia during prometaphase(Wilson, 1970). This formation had the morphological properties of a polar centreand was postulated to be analogous to the centrioles of animal cells. These formationsof smooth endoplasmic reticulum differ from the membranous complex of mousespermatocytes in several respects, but they serve to illustrate that the endoplasmicreticulum may become differentially organized during cell division in a variety of celltypes.

A predoctoral fellowship awarded to A.P. by the Graduate School, Wayne State University,is gratefully acknowledged. This work was submitted by A.P. in partial fulfilment of theDoctor of Philosophy degree.

REFERENCES

BARER, R., JOSEPH, S. & MEEK, G. A. (i960). The origin and fate of the nuclear membrane inmeiosis. Proc. R. Soc. B 152, 353-366.

BASU, S. L., NANDI, J. & NANDI, S. (1966). Effects of hormones on adult frog (Rana pipiens)testes in organ culture. J. exp. Zool. 162, 243-256.

BRYAN, J. M. D. (1971). Spermatogenesis revisited. I. On the presence of multinucleatespermatogenic cells in the seminiferous epithelium of the mouse. Z. Zellforsch. mikrosk. Anat.ii2, 333-349-

DOUGHERTY, W. J. & LEE, M. MCN. (1967). Light and electron microscope studies of smoothendoplasmic reticulum in dividing rat hepatic cells. J. Ultrastruct. Res. 19, 200-220.

ELLINGSON, D. J. & YAO, T. S. (1970). Growth and observations of Chinese hamster semini-ferous epithelium in vitro, jf. Cell Sci. 6, 195-205.

FAWCETT, D. W. & ITO, S. (1958). Observations on the cytoplasmic membranes of testicularcells, examined by phase-contrast and electron microscopy. J. biophys. biochem. Cytol. 4,•35-142.

INOUE, S. (1963). Organization and function of the mitotic spindle. In Primitive MotileSystems in Cell Biology (ed. R. N. Allen & N. Kamiya), pp. 549-594. New York andLondon: Academic Press.

JORDAN, R. T., KATSH, S. & DE STACKELBURG, N. (1961). Spermatogenesis with possiblespermiogenesis of guinea pig testicular cells grown in vitro. Nature, Lond. 192, 1053-1055.

KAYE, G. I., PAPPAS, G. D., YASUZURIN, G. & YAMAMOTO, H. (1961). The distribution andform of the endoplasmic reticulum during spermatogenesis in the crayfish, Camabaroidesjaponicus. Z. Zellforsch. mikrosk. Anat. 53, 159-171.

io A. Pleshkewych and L. Levine

KODANI, M. & KODANI, K. (1966). The in vitro cultivation of mammalian Sertoli cells. Proc.•natn. Acad. Sci. U.S.A. 56, 1200-1206.

LEVINE, L. (1972). Preparation and maintenance of living house cricket (Aclieta domesticus)spermatocytes. Biol. Reprod. 7, 211-222.

LEVINE, L. & SHIFRIN, N. (1973). Distribution of adenosine triphosphatase during anaphase incricket primary spermatocytes. Nature, Lond. Z49, 256-258.

LUFT, J. H. (1956). Permanganate - new fixative for electron microscopy, jf. biophys. biochein.Cytol. 2, 799-802.

MERRIAM, R. W. (1961). Nuclear membrane structure during cell division in Cliaetopterouseggs. Expl Cell Res. 22, 93-107.

MOLE-BAJER, J. & BAJER, A. (1967). Studies of selected endosperm cells with the light andelectron microscope. The technique. Cellule 67, 257-265.

PALADE, G. E. (1955). Studies on the endoplasmic reticulum. II. Simple dispositions in cellsin situ. J. biophys. biochem. Cytol. 1, 567-582.

PLESHKEWYCH, A. (1970). Phase and Electron Microscopic Studies of Mouse Primary Spermato-cytes Maintained in vitro. Ph.D. Thesis, Wayne State University, Detroit, Michigan.

PUCK, T. T., CIECIURA, S. J. & ROBINSON, A. (1958). Genetics of somatic mammalian cells.III. Long-term cultivation of euploid cells from human and animal subjects. J. exp. Med.108, 945-955-

REBHUN, K. I., ROSENBAUM, J., LEFEBVRE, P. & SMITH, G. (1974). Reversible restoration ofthe birefringence of cold-treated isolated mitotic apparatus of surf clam eggs with chick braintubulin. Nature, Lond. 249, 113-115.

REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain inelectron microscopy. J. Cell Biol. 17, 208-212.

ROBBINS, E. & GONATAS, N. K. (1964). In vitro selection of the mitotic cell for subsequentelectron microscopy. J. Cell Biol. 20, 356-358.

ROTH, L. E. (1967). Electron microscopy of mitosis in amoebae. III. Cold and urea treatments:a basis for tests of direct effects of mitotic inhibitors on microtubule formation. J. Cell Biol.34. 47-59-

SABATINI, D. D., BENSCH, K. & BARRNETT, R. J. (1963). Cytochemistry and electron micro-scopy. The preservation of cellular ultrastructure and enzymatic activity by aldehydefixation. J. Cell Biol. 17, 19-58.

STEINBERGER, A. & STEINBERGER, E. (1966a). Stimulatory effect of vitamins and glutamineon the differentiation of germ cells in rat testes organ culture in chemically denned media.Expl Cell Res. 44, 429-435.

STEINBERGER, A. & STEINBERGER, E. (19666). In vitro culture of rat testicular cells. Expl CellRes. 44, 443-452-

STEINBERGER, E., STEINBERGER, A. & PERLOFF, W. (1964). Initiation of spermatogenesis in vitro.Endocrinology 74, 788-792.

WATSON, M. L. (1955). The nuclear envelope. Its structure and relation to cytoplasmic mem-branes. J. biophys. biocliem. Cytol. 1, 257-270.

WATSON, M. L. (1958). Staining of tissue sections for electron microscopy with heavy metals.J. biophys. biocliem. Cytol. 4, 475-478.

WILSON, H. J. (1970). Endoplasmic reticulum and microtubule formation in dividing cellsof higher plants - a postulate. Planta 94, 184-190.

[Received 16 November 1973 -Revised 15 October 1974)


Figs. i—8. Selected frames from a time-lapse motion film showing the growth andbehaviour of the cytoplasmic inclusion as the primary spermatocyte developedthrough meiosis I, from early metaphase to interkinesis. Magnification marker isio/tm and applies to all figures, x 2000.

Fig. 1. The cytoplasmic inclusion (me) is situated in the cytoplasm between thelateral spindle (s) margin and cell membrane of the late prometaphase primaryspermatocyte. Mitochondria (m) and various-sized granules (gr) are apparent. Somegranules tend to align parallel with the contour of the membranous complex.

Fig. 2. Early metaphase, 35 min later. The cytoplasmic inclusion (me) is slightlyelongated.

Fig. 3. Late metaphase, 34 min later. The cytoplasmic inclusion (tnc) is elongatedin the polar axis. A large granule is associated with its surface.

Fig. 4. Very early anaphase, 2 min later. The cytoplasmic inclusion (me) isC-shaped.

Fig. 5. Late anaphase, 4 min later. The cytoplasmic inclusion (me) appears com-pressed in its mid-section adjacent to the spindle interzonal region.

Fig. 6. Early telophase, 5 min later. The cytoplasmic inclusion (me) is beingdivided as cytokinesis occurs (arrows).


F i g s . 1-6. F o r l e g e n d see p . I J .

Membranous complex in mouse spermatocytes

Figs. 7-9. For legend see p. 14.

14 A. Pleshkervych and L. Levine

Fig. 7. Telophase, 5 min later. The cytoplasmic inclusion (me) is present in eachof the daughter spermatocytes. A connexion (arrow) is apparent between the cyto-plasmic inclusion (me) and the condensed chromatin of the newly forming nucleus (n).

Fig. 8. Interkinesis, 10 min later (95 min after Fig. 1). The cytoplasmic inclusionis present in both daughter spermatocytes. It is adjacent to the newly formednuclear envelope (nmb) in the bottom spermatocyte. A large granule (jgr) is presenton the nuclear envelope in the upper cell.Fig. 9. A, Phase-contrast micrograph of a representative field of fixed and embeddedgerm cells. The carbon coat is broken at X where it was scribed marking the field foreventual separation and electron microscopy. Magnification marker is 10 fim;x 700. B, Enlargement of the boxed area in A. The cytoplasmic inclusion (me) ispresent as a boundary on which granules are aligned, c, chromosomes. Magnificationmarker is io/im; x 1100.

Fig. 10. A, Phase-contrast micrograph of primary spermatocytes in metaphase andpromeraphase cultured in maintenance medium containing gelatin to adhere themto the coverslip, for subsequent electron microscopy. Arrows point to thecytoplasmic inclusion (me), c, chromosome; s, spindle area. Magnification marker is10/4m; x 1330. B, Electron micrograph of the same cells in Fig. IOA, fixed 15 minafter the living cells were photographed. The region in Fig. IOA to which the arrowspoint (me) corresponds to a formation of stacked lamellae (nil), the cisternal elementsof the membranous complex, interspersed with vesicles (ve) of different sizes, me,membranous complex; mt, microtubules; sve, spindle vesicles: magnification markeris 1 /im; x 8000.

Fig. 11. Section through a chromosome (c) adjacent to the membranous complex(me). Arrows point to the close association, or possible intermingling between thechromatin and cisternae (ml) of the membranous complex. A long cisternal elementobliquely crosses the space between the inner and outer boundaries of the mem-branous complex. The spindle vesicles (sve) are similar to those that are near orwithin the membranous complex (ve). vi, mitochondrion. Magnification marker is1 /im; x 10800.


Fig. 12. Higher magnification of a region from the membranous complex of aprimary spermatocyte in metaphase showing that the profiles of its cisternalelements (mt) and vesicles (ve) are agranular. In the cytoplasmic matrix between thecisternal formations (arrow) are smaller vesicles. A multivesicular body (mvb) and anaggregation of vesicles (small arrow) are also present. They are closely associated witheach other and with the cisternae (ml) of the membranous complex. Magnificationmarker is i /tm; x 20 500.

Fig. 13. Section through an interphase primary spermatocyte showing the nuclearenvelope (ne), cisternae of agranular endoplasmic reticulum (er) and vesicles (ye).Note the similarity between the cisternae of this figure and those of the membranouscomplex in Fig. IOB and others, p, pore complex. Magnification marker is 1 /tin;x68oo.


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