Cell Tissue Res (1995) 282:291-296 Cell&Tissue

Research �9 Springer-Verlag 1995

Electron-microscopic localisation of thiol and disulphide groups by direct monomaleimido-nanogold labelling in the spermatozoa of a marsupial, the tammar wallaby (Macropus eugenii) Minjie Lin, Yulia Sistina*, John C. Rodger

Department of Biological Sciences, University of Newcastle, NSW 2308, Australia

Received: 29 February 1995 / Accepted: 8 June 1995

Abstract. This study utilised a commercially available monomaleimido-nanogold reagent to directly label cel- lular thiol groups (SH) of marsupial (tammar wallaby) spermatozoa before and after reduction of disulphides (S-S) with mercaptoethylamine hydrochloride (MEA). The sperm surface, mitochondrial membranes, axoneme and tail fibres were all labelled with gold particles be- fore MEA treatment and the label intensity was in- creased after S-S reduction. The acrosomal membranes and matrix of spermatozoa contained no detectable SH prior to MEA treatment. However, after moderate MEA treatment (1 mg/ml) gold label was associated with the acrosomal membrane and invaginated acrosomal mem- brane within the acrosomal matrix. After exposure to 5 and 10 mg/ml MEA, gold particles heavily labelled the acrosomal matrix. Thus, the acrosomal membranes and matrix of tammar wallaby spermatozoa both contain S-S cross-linked structures, and this may contribute to the unusual stability of the marsupial acrosome. Under all treatment conditions the nucleus remained unlabelled. This is consistent with early studies which indicated that cysteine was absent from the nuclear protamines. The study also demonstrated that monomaleimido-nanogold can be used to resolve SH- and S-S-rich cellular struc- tures directly, in addition to its use to label antibodies and Fab fragments for immunochemical localisation.

Key words: Spermatozoa - Acrosome - Thiols - Di- sulphides - Electron microscopy - Monomaleimido nanogold - Macropus eugenii (Marsupialia)

This study was supported by a grant to J.C.R. from the Australian Research Council. Y.S. was the recipient of an Australian Inter- national Development Program Fellowship. *Permanent address: Faculty of Biological Sciences, General Soedirman University, Purwokerto, Indonesia Correspondence to: M. Lin

Introduction

The spermatozoa of marsupials are characterised by un- usually stable acrosomes which resist disruption by freeze thawing, detergent or lysophosphatidylcholine treatments, which disrupted the plasma membrane (Mate and Rodger 1991; Sistina et al. 1993a, b). This contrasts with the situation for most eutherian mammals where the acrosome is a fragile structure and its loss or alteration is often used as an early indicator of cell damage (Watson 1979; Parks and Graham 1992). The spermatozoa of some eutherian mammals such as bats (Krutzsch and Crichton 1991) and the shrew Suncus murinus (Bedford et al. 1994) depart from this pattern of acrosomal fragili- ty or exhibit evidence of acrosome stabilisation. Al- though the marsupial sperm acrosome is relatively sta- ble, the nucleus is disrupted by treatments such as air drying, high concentrations of divalent cations and mild detergents, which do not effect the spermatozoa of most eutherian mammals (Bedford and Calvin 1974; Cum- mins 1980). The fragility of the marsupial sperm nucleus is due to the absence of cysteine in the nuclear prota- mines, which precludes disulphide (S-S) stabilisation (Balhorn et al. 1988; Fifis et al. 1990; Balhorn, personal communication).

To explore the basis of marsupial acrosome stability, spermatozoa of the common brushtail possum Tricho- surus vulpecula (Mate et al. 1994) and the tammar wal- laby Macropus eugenii were examined for the presence of S-S groups using thiol (SH)-specific penetrating (monobromobimane) and non-penetrating (monobromo- trimethyl ammoniobimane) labels followed by fluores- cence microscopy and electrophoretic analysis. Free SH and S-S revealed by treatment with the reducing agent dithiothreitol (DTT) were present in the acrosome, mid- piece and principal-piece of the tail, but absent in the nu- cleus of the spermatozoa of both species of marsupial (Mate et al. 1994). Labelling of the acrosome was only seen after treatment with the penetrating agent indicating that the SH and S-S were not present on the surface of the spermatozoa but on, or in the acrosome. Although

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this approach localised S-S to the acrosome, it did not allow resolution o f whether S-S bonds were associated with the acrosomal membrane, acrosomal matrix or both.

The present study carried our examination of the mar- supial acrosome further by using the relatively new thi- ol-specific gold labelling agent, monomale imido-nano- gold, to examine the ultrastructural location of SH and S-S groups in tammar wallaby spermatozoa. Al though well characterised as a means to covalently label anti- bodies for immuno-go ld electron-microscopic localisa- tion (Hainfeld and Furuya 1992; Vandre and Burry 1992; Burry et al. 1992), this agent has not been previously used to localise sub-cellular thiols and disulphides di- rectly.

Materials and methods

Tammar wallaby (Macropus eugenii) semen was collected by electro ejaculation, allowed to coagulate and the spermatozoa ob- tained after swimming-up into phosphate-buffered saline (PBS) as previously described (Sistina et al. 1993b). These spermatozoa were diluted 1:1 with c~-mercaptoethylamine hydrochloride (MEA) in PBS to achieve a final concentration of 0, 1, 5 or 10 mg/ml. The sperm-MEA mixture was then incubated for 60 rain at room temperature to reduce S-S bonds to reactive SH groups. MEA was used on the recommendation of the manufacturer of the monomaleimido-nanogold probe because nanogold particles de- grade in concentrated thiols such as dithiothreitol (DTT) and ~- mercaptoethanol (Nanogold Product Information, Nanoprobes Inc, Stony Brook, N,Y.).

Light microscopy: Free SH and reduced S-S detected by mBBr staining

The procedure was essentially as described previously using DTT as the reducing agent (Mate et al. 1994). After MEA incubation spermatozoa were washed by centrifugation at 750 g for 5 min and resuspended in 1 ml PSB. Thiol groups were fluorescently la- belled by addition of an equal volume of 0.5 mM monobromobi- mane (mBBr; Calbiochem, La Jolla, Calif.; cat # 596105) for 15 min in the dark. All subsequent procedures were carried out with minimal exposure to light. The spermatozoa were then diluted to 10 ml with PBS, washed by centrifugation (750 g, 5 rain), resus- pended in 1 ml PBS and fixed by addition of 4 ml 2% paraformal- dehyde in PBS. Cytospin preparations of the fixed spermatozoa were assessed by fluorescence microscopy (Axiophot photomicro- scope, Carl Zeiss; exciter filter 365 nm, dichroic element 390 rim, barrier filter 395 nm). Spermatozoa were photographed with a standardised exposure time, processing and printing conditions to allow semi-quantitative comparison between treatments.

Electron microscopy: Free SH and reduced S-S detected by monomaleimido-nanogold labelling

After incubation in MEA the spermatozoa were centrifuged at 750 g for 5 min, resuspended in 4 ml PBS, then gently fixed for 15 min by adding 4 ml of 1.0% glutaraldehyde in PBS to achieve a final concentration of 0.5% glutaraldehyde. To remove the fixa- tive, spermatozoa were washed three times by centrifugation at 750 g for 5 rain and resuspended in 4 ml PBS. After the third cen- trifugation the spermatozoa were resuspended in 2 ml PBS, then diluted with 2 ml of 0.04 M glycine in PBS added and left at RT for 5 min. Following three further washes by centrifugation with PBS and resuspension in 2 ml PBS, the contents of one vial (6

nmol) of monomaleimido-nanogold (Nanoprobes Inc, Stony Brook, N.Y.; cat # 2020) was dissolved in 0.5 ml PBS and added to the sperm suspension (about 1.2x106 sperm/per nmol Nano- gold) and left at RT for 30 min. Nanogold-treated spermatozoa were diluted to 4 ml with PBS and then washed three times by centrifugation and resuspension in 4 ml of PBS. Fixation was completed by addition of 4 ml 2% glutaraldehyde in PBS to achieve a final concentration of 1% glutaraldehyde. After fixation for 30 min, spermatozoa were washed by centrifugation; three times with PBS and twice with deionised water. The gold label was developed with HQ Silver reagent (Nanoprobes Inc, Stony Brook, N.Y.; cat # 2012) for 2.5 rnin, and then the spermatozoa were washed three times by centrifugation and resuspension in de- ionised water. Finally, the spermatozoa were dehydrated and em- bedded in Spurr's resin (three days curing at RT, two days at 40 ~ C and overnight at 60 ~ C to harden fully). This cautious hardening protocol was used because nanogold particles are unstable above 50~ (Nanogold Product Information, Nanoprobes Inc, Stony Brook, N.Y.). Unstained ultrathin sections were examined in a JEOL-1200EX electron microscope operating at 80 kV.

Animal experimentation was approved by the University of Newcastle Animal Care and Ethics Committee and the New South Wales National Parks and Wildlife Service, Australia.

Results

Non-MEA-treated spermatozoa

Fluorescence light microscopy of mBBr-treated sperma- tozoa showed that the entire tail including the connect- ing piece of the neck and mitochondrial sheath were brightly fluorescent but the head was unlabeled except for a faint periacrosomal ring (Fig. la).

Electron-microscopic examination o f monomale imi- dogold-treated spermatozoa revealed moderate nanogold labelling present over the entire plasma membrane, the connect ing piece of the neck, the mitochondrial sheath (but not the mitochondrial matrices) of the mid-piece (Fig. 2) and the axoneme, and between the ribs of the fi- brous sheath o f the tail. Only very rarely were gold par- ticles found in the acrosome or associated with the acro- somal membrane. No gold particles were seen in the nu- cleus.

MEA-treated spermatozoa

Fluorescence microscopy of spermatozoa treated with 1 mg/ml M E A prior to mmBr revealed little change in la- belling over non -MEA controls except for a faint fluores- cence over the entire acrosome (Fig. lb). Acrosomal flu- orescence was more marked after treatment with 5 mg/ml M E A (Fig. 1 c) and the entire spermatozoa were brightly labelled after treatment with 10 mg/ml M E A (Fig. ld).

Electron microscopy revealed moderate gold labelling o f the acrosomal membrane after treatment with 1 mg/ml M E A (Fig. 3). The invaginated acrosomal mem- brane was also labelled. The intensity of the labelling of the plasma membrane was slightly higher than that in non-MEA-trea ted spermatozoa. However, no labelling could be detected in the matrix of the acrosome and the nucleus o f 1 mg/ml MEA-treated spermatozoa.

The acrosomal matrices were densely labelled after 5 mg/ml M E A treatment (Fig. 4a) and very intensely la-

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Fig. la-d. Fluorescence micro- graphs of tammar wallaby sperma- tozoa treated with the thiol-specific label monobromobimane (mBBr) after pre-treatment with the reduc- ing agent MEA. a 0 mg/ml MEA, b 1 mg/ml MEA, e 5 mg/ml MEA, d 10 mg/ml MEA. Arrowheads indi- cate location of the acrosome. Bars: 10 gM

belled after 10rag/m1 MEA treatment (Fig. 5a). The nucleus, however, remained unlabelled in both treat- ments.

In all three groups of MAE-treated spermatozoa, the mid-piece and the principal piece were heavily labelled. The label was mainly located on the plasma and mito- chondria membranes, the axoneme, the surface of the dense fibres and the space between the ribs of the fi- brous sheath (Figs. 4b, 5b). Only very rarely were gold particles found within the matrices of mitochondria, the dense fibrous and fibrous sheath.

Discussion

When eutherian spermatozoa pass through the epididy- mis, the nuclear chromatin, perinuclear material of the head, the mitochondrial sheath and dense fibers of the tail are all "hardened" by an increase in S-S bonds with- in these organelles (Calvin 1975). This stabilisation in eutherian spermatozoa is engendered by oxidation of protein-bound SH to S-S bonds (see reviews: Gordon 1977; Bedford 1979). In contrast, stabilisation of the nu- clear structure by S-S bonds did not occur in marsupial

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spermatozoa (Bedford and Calvin 1974), presumably due to the lack of cysteine-rich polypeptides or proteins in the nucleus (Balhorn et al. 1988; Fifis et al. 1990). The absence of SH and S-S groups in the nucleus of marsupial spermatozoa was demonstrated by a recent fluorescence-microscopic study using SH-specific label mBBr (Mate et al. 1994). This characteristic of marsupi- al spermatozoa was confirmed by the present study which found no gold labelling in the nuclei of any MEA- treated tammar wallaby spermatozoa (see Figs. 3-5). Absence of S-S groups in the marsupial sperm nucleu s is undoubtedly a major reason for the fragility of the sperm head (Mate and Rodger 1991) and its rapid decondensa- tion in high concentration cation solutions (Cummins 1980).

Although the nucleus is fragile, earlier detergent stud- ies had suggested that the acrosomes of marsupial sper- matozoa were unusually stable (Sistina et al. 1993a). Studies using the SH-specific fluorescent label mBBr showed that SH and S-S groups were located in the acro- somal region of wallaby and possum spermatozoa (Mate etal . 1994). The ultrastructural localisation obtained with monomaleimido-nanogold label demonstrated that significant concentrations of S-S were located in the acrosomal membrane and the acrosomal matrix. These results strongly support the hypothesis that the stability of the marsupial acrosome is due to disulfide stabilisat- ion of the acrosome (Mate et al. 1994). However, the physiological significance of this unusual stability, par- ticular for capacitation and fertilization, requires further study.

(

Fig. 2a, b. Unstained sections of non-MEA-treated tammar walla- by spermatozoa treated with the thiol-special label monomaleimi- do-nanogold, a Longitudinal section of the head and b cross-sec- tion of the mid-piece. A moderate, but specific localisation of par- ticles is evident on the plasma membrane (PM) and the mitochon- drial membrane (M). However, the acrosome (A), acrosomal mem- brane (AM), mitochondria matrices (Mx) and the nucleus (N) are particle free

Fig, 3a, b. Unstained sections of the head of tarm-nar wallaby spermatozoa treated with 1 mg/ml MEA prior to monomaleimido- nanogold, a Longitudinal section of the head and b cross-section of the head and a fraction of the mid-piece. Gold particles label the acrosomal membrane (AM) and the invaginated acrosomal membrane vesicles (b, V). The matrix of the acrosome (A) and the nucleus (P0 are unlabelled. The density of the labelling of the sperm surface is slightly higher than that in non-MEA-treated spermatozoa (see Fig. 2)

Fig. 4a, b. Unstained sections of tammar wallaby spermatozoa treated with 5 mg/ml MEA prior to monomaleimido-nanogold, a Longitudinal section of the head and b cross-sections of head and mid-piece. There is intense gold particle labelling of the acrosome (A), and mid-piece (MP) structures. The nuclear contents (PO re- main unlabelled

Fig. 5a, b. Unstained sections of tammar wallaby spermatozoa treated with 10 mg/ml MEA prior to monomaleimido-nanogold, a Longitudinal section of head and b longitudinal sections of princi- pal piece. There is very intense particle labelling of the acrosome (A), the axoneme (Ax) and the space between the ribs of the fi- brous sheath (F). Nuclear contents (N) remain unlabelled

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The present study used MEA, a relative mild reduc- ing agent, rather than DTT which has commonly been used for studies of SH and S-S localisation in mammal spermatozoa (Bedford and Calvin 1974; Calvin 1975). It was found that 1 mg/ml MEA treatment reduced a sig- nificant amount of acrosomal membrane S-S bonds to SH groups that could be revealed by weak mBBr fluo- rescence (Fig. lb) or monomaleimido-nanogold label- ling (Fig. 2). However, 5 and 10 mg/ml MEA treatments led tO strong light-microscopic acrosomal fluorescence (Figs. lc, d) and electron-microscopic gold labelling of the acrosomal matrix (Figs. 4a, 5a). The pattern of fluo- rescence labelling after 5 mg/mI MEA treatment for 60 rain was similar to that after 5 mM DTT treatment for 20 rain (Mate et al. 1994).

No gold label was found in the nucleus of any MEA- treated tammar wallaby spermatozoa (see Figs. 3-5). However, nuclear fluorescence labelling with mBBr was seen after treatment with 10 mg/ml MEA. This was pre- sumably due to MEA reduction revealing reactive SH on the sperm head surface rather than within the nucleus (compare Figs. ld and 5). In the earlier light-microscop- ic study no surface SH or S-S was detected using the non-penetrating bimane qBBr (Mate et al. 1994). This difference between the two studies may be due to the use of MEA rather than DTT as the reducing agent.

This ultrastructural study provides further evidences to support our earlier suggestion that the stability of the acrosome and the fragility of the nucleus are related to the distribution of the disulfide groups in the marsupial sperm. In addition we have demonstrated that mono- maleimido-nanogold can be used as a direct subcellular SH label using conventional electron-microscopic tech- niques.

Acknowledgements. Dr. Karen Mate's critical eye and comments assisted us in preparing the manuscript.

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