MOLECULAR REPRODUCTION AND DEVELOPMENT 33:338-346 (1992)

In Vitro Decondensation of Mammalian Sperm and Subsequent Formation of Pronuclei-Like Structures for Micromanipulation MARKUS MONTAG, VERONICA TOK, SWEE-LIAN LIOW, ARIFF BONGSO, AND SOON-CHYE NG Department of Obstetrics and Gynecology, National University Hospital, National University of Singapore, Singapore

changes allow for the replication of pronuclear DNA during formation of the male pronucleus (Naish et al., 1987; Nonchev and Tsanev, 1990). To date, in vitro and in vivo studies of sperm decondensation and pronuclear formation provide greater insights into the complex organization of events that occur during early fertilization. Induction of nuclear decondensation of mammalian spermatozoa in vitro was shown to be successful only after the use of reducing agents to break the disulphide bonds of the protamines (for review, see Zirkin et al., 1985). More recently, Reyes et al. (1989) reported that the combined use of physiological agents like heparin and glutathione resulted in a very efficient decondensation of human sperm nuclei in vitro. The essential role of these agents during early fertilization is widely acknowledged (Calvin et al., 1986; Bellin and Ax, 1987; Perreault et al., 1988).Another breakthrough was the development of sperm pronuclei in vitro by components of a cell-free cytoplasmic preparation from amphibian eggs (Lohka and Masui, 1983). However, amphibian sperm protamines lack disulphide bonds; therefore, amphibian eggs provide no mechanism to reduce the -S-Sbonds of mammalian sperm. In other words, the formaKey Words: Microinjection, Protamine, Xenopus laevis, tion of pronuclei from human sperm after injection into Cell-free system, Male subfertility amphibian eggs or after incubation in an amphibian egg extract requires the use of disulphide-reducing INTRODUCTION agents (Iwao and Katagiri, 1984; Ohsumi et al., 1986). In eutherian mammals, mature spermatozoa are Under such conditions, the amphibian egg extract incharacterized by the presence of sperm-specific pro- duced a n immediate swelling of human sperm followed teins, the protamines, which are enriched in arginine by further decondensation and DNA synthesis (Gordon and cysteine residues. The latter are highly cross- et al., 1985). The amphibian egg extract, therefore, prolinked by disulphide bonds. During spermatogenesis, vides a n adequate means to investigate factors necesprotarnines replace the somatic histones and render sary for pronuclear formation and DNA synthesis greater stability to the mature sperm. The protamines (Blow and Laskey, 1986; Brown et al., 1991; Philpott et mediate the highest compaction and condensation of al., 1991). DNA known so far (Bellve, 1979; Ward and Coffey, The objective of this paper is to describe the opti1991). Following fertilization, cytoplasmic factors in mized transition of mammalian sperm into pronucleithe mammalian egg trigger major nuclear changes in like structures in vitro for micromanipulation. Microthe sperm (Longo and Kunkle, 1978; Yanagamichi, manipulation is commonly accepted in experimental 1978). First, the egg provides the mechanism to reduce the disulphide bonds of the sperm-specific protamines. This is recognized as a prerequisite for chromatin deFebruary 24,1992; accepted J u n e 4,1992. condensation and for the replacement of the protamines Received Address reprint requests to Dr. Markus Montag, Department of Obwith histone and non-histone proteins (Rodman et al., stetrics and Gynecology, National University Hospital, National Uni1981; Perreault et al., 1984,1987).Subsequently, these versity of Singapore, Singapore, 0511.

In this study, we describe an effiABSTRACT cient protocol for the formation of in vitro developed pronuclei for micromanipulation techniques. Our approach involved incubation of demembranated or permeabilized mammalian sperm in a phosphate buffer supplemented with heparin and p-mercaptoethanol.Under the prevailing conditions, we achieved a uniform and reliable synchronous decondensation of sperm nuclear DNA. This initial decondensation facilitated the removal of mammalian protamines upon subsequent incubation in an amphibian egg extract. The interchange of protamines for histones to stabilize the DNA structure is recognized as a prerequisite for pronuclear formation. Furthermore, immunocytochemical studies have revealed that pronuclear development is accompanied by the formation of a nuclear lamina with corresponding DNA synthesis. The method described gave a high yield of nuclei during pronuclear formation. Ultimately, our aim is to transfer the in vitro-developed pronuclei into mammalian oocytes by micromanipulation. This novel procedure may prove useful in alleviating severe male factor problems especially in oligozoospermic cases in our in vitro fertilization center. o 1992 WileyLiss, Inc.

0 1992 WILEY-LISS, INC.

IN VITRO FORMATION OF PROlNUCLEI FOR MICROMANIPULATION

339

Preparation of Egg Extract embryology. Recently, i t has been applied to human in vitro fertilization, especially in severe male factor paApproximately 21 h r prior to isolation of eggs, female tients (Gordon and Laufer, 1988; Fishel e t al., 1990; Xenopus frogs were injected with 100 IU and 500 IU Malter and Cohen, 1990; Ng et al., 1990). These pa- human chorionic gonadotropin (Intervet Int. B.V., tients usually have a very low sperm count (severe oli- Boxmeer, Holland), respectively, a t a n interval of 5 hr. gozoospermia) coupled with other male factors such as They were kept overnight in 100 mM NaC1, and the low motility (asthenozoospermia) and low normal forms eggs were collected the following morning by squeez(teratozoospermia). In clinical terms, a preselection of ing. Eggs were rapidly dejellied in 2% cysteine HC1 in sperm on the basis of developmental potential may 1x MMR, pH 7.9 (1x MMR; 100 mM NaCl, 2 mM KCl, prove to be beneficial. Our initial task therefore was to 2 mM CaCl,, 2 mM MgSO,, 5 mM Hepes KOH, pH 7.41, optimize the conditions necessary for pronuclear forma- washed several times in 0 . 2 5 ~MMR and electroactition in vitro. Here, we describe a protocol that first vated (12 V alternating current, 1 sec). After activaallowed for controlled decondensation of permeablized tion, the eggs were incubated for another 5 min before sperm followed by pronuclear formation using a cell- transfer into ice-cold egg extract buffer (prepared acfree system derived from activated eggs of Xenopus lae- cording to Blow and Laskey, 1986) for washing puruis. poses. Eggs were crushed by centrifugation a t 10,OOOg in a Sorvall HB-4 (Du Pont Company, Wilmington, DE) for 20 min at 4°C to yield a crude cytoplasmic extract MATERIALS AND METHODS (Lohka and Maller, 1985). This extract was carefully Preparation of Sperm removed and recentrifuged at 150,OOOg in a n SW-65 Semen samples were obtained from fertile donors in rotor in a Beckman ultracentrifuge (L8-70M; Beckman our subfertility center. After a liquefaction period of Instruments, Inc., Palo Alto, CA) to yield both purified 30-60 rnin at 37"C, the specimen was subjected to a soluble and membraneous fractions (Lohka and Maller, standard semen analysis (WHO, 1987) with emphasis 1985). The clear, soluble supernatant was removed, aligiven to sperm concentration, motility, and morphol- quoted, and immediately frozen in liquid nitrogen. The ogy. The semen was diluted with phosphate-buffered membrane vesicles fraction present in the fluffy layer saline (PBS; 137 mM NaC1, 2.7 mm KC1, 9.6 mM was diluted with egg extract buffer and concentrated by Na2HP04, 1.5 mM KH2P0,, pH 7.4) and filtered recentrifugation through a 0.5 M sucrose cushion. The through lens paper for removal of debris. The sperm final membrane vesicles suspension was stored a s desuspension was pelleted and pretreated employing ei- scribed (Pfaller et al., 1991). ther of the following protocols. 1. For permeabilization of sperm nuclear membrane, In Vitro Decondensation of Sperm Nuclei the sperm pellets were dissolved in a sucrose buffer (SMT; 250 mM sucrose, 5 mM MgCl,, 10 mM Tris HC1, To allow for a controlled and reproducible decondenpH 7.4) and adjusted to a concentration of 20 x 106/ml. sation, sperm prepared as described above were incuTen microliter aliquots were frozen by plunging the bated in a n in vitro decondensation buffer (DB) prior to tubes into liquid nitrogen. This freeze-thaw technique nuclear reformation in the egg extract. The buffer concaused damage to the sperm plasma membrane (Ma- sisted of salt medium as modified from Reyes et al. hadevan and Trounson, 1984); it is unknown whether (1989) (113 mM KCl, 12.5 mM KH,PO,, 2.5 mM such a treatment cause damage to the sperm chromatin Na2HP04,2.5 mM MgCl,, 20 mM Tris, pH 7.4) supple(Henry Sathananthan, personal communication). For mented with heparin (150 pM; Commonwealth Serum reference, frozen-thawed sperm nuclei will be referred Labs, Melbourne, Australia) and P-mercaptoethanol a t different concentrations (0.5, 1, and 2 mM). For initial to as permeabilized sperm. 2. For demembranation, the sperm suspension was decondensation studies, sperm suspensions were incusubjected to 1%Triton X-100 in SMT at room tempera- bated at a concentration of 2 x 106/mlin 25 p1 of DB at ture. After 15 min, lysolecithin was added (0.05% final 37°C with various concentrations of P-mercaptoethaconcentration), and incubation was continued for an- nol. Aliquots (1 p1) were removed at fixed time points other 5 min. Sperm were subsequently washed once in and placed onto a slide in a drop of fixation buffer (SMT SMT, 3% bovine serum albumine (BSA), and three containing 3.7% formalin, 200 mM sucrose and either 1 times in SMT, 0.4% BSA (Lohka and Masui, 1983; pg/ml propidiumiodide or 10 kg/ml Hoechst 33342). ExOhsumi et al., 1988). Demembranated sperm were fi- amination was done with a n inverted microscope (Zeiss ~ (Carl Zeiss, Obernally resuspended in SMT at the same concentration as IM35) and a 4 0 phase-objective kochen, Federal Republic of Germany). According to for method 1. Mature male golden hamsters were sacrificed, and the size and the shape of the decondensing sperm heads, the cauda epidydimis was removed. After capacitation each was given a decondensation value ranging from 0 for 30 min a t room temperature in PBS, the sperm to 3 (0 = condensed sperm, 1 = sperm head slightly desuspension was centrifuged and subjected to method 1 condensed without increase in size, 2 = partly deconor 2. Sperm from CBA/C57BC hybrid male mice were densed sperm head with a t least a two-fold increase in likewise treated. For prolonged storage, all sperm sus- size, 3 = fully decondensed sperm head with randomly distributed, phase-dense chromatin dots). For indexing, pensions were kept a t - 70°C.

340

M. MONTAG ET AL.

the values of 100 nuclei were scored and the sum was divided by the number of nuclei, giving a minimum value of 0 for condensed sperm and a maximum value of 3 for 100% decondensation of sperm nuclei.

protocol modified from Marushige and Marushige (1975). For each sample, 0.5-1 x lo6 sperm were applied for electrophoretic analysis. The sperdnuclear pellets were dissolved in 1.1M NaCl, 6 M urea, and 0.1 M 0-mercaptoethanol and incubated in this solution for Nuclear Assembly 2 h r a t 37°C. An equal volume of ice-cold 0.32 M HC1 For a typical nuclear assembly assay, decondensation was then added, and the solution was placed on ice for of frozen-thawed sperm was initiated in a volume of 25 30 min. After precipitation of the DNA by centrifugapl a s described above, with a suitable concentration of tion (13,00Og, 15 min), the supernatant was adjusted to 0-mercaptoethanol to effect a synchronous decondensa- 20% trichloroacetic acid (TCA) by addition of a 100% tion of almost all sperm nuclei within 1 hr. Subse- TCA stock solution (w/v) and incubated up to 48 h r in quently, free sulfhydryl groups were blocked by addi- the cold to precipitate proteins. Precipitates were recovtion of 10 mM N-ethylmaleimide (final concentration 2 ered by centrifugation (as above), washed twice with mM) and incubation for 10 min a t 4°C. In the mean- ice-cold 90% acetone, and dissolved in the sample buffer time, 100 p1 of the clear soluble fraction was thawed (0.9 N acetic acid, 1 M urea, 0.1 M P-mercaptoethanol, and supplemented with a n ATP regenerating system (1 15% sucrose). Basic protein samples were run on a Hoefer SE 250 mM ATP, 60 mM phosphocreatine, 150 pg/ml creatine phosphokinase, final concentrations; Sigma, St. Louis, vertical electrophoresis unit (Hoefer Scientific InstruMO). This solution was added to the decondensed sperm ments, San Francisco, CA) using acid-urea polyacrylheads, and incubation was continued a t room tempera- amide gels, which were prepared according to Zirkin et ture for 10 min; 30 pl of purified membrane fraction al. (1980), with the exception of a smaller gel size (6 cm was added, and the solution was gently mixed. For rep- separation gel; 0.4 cm separation gel). After the prelication studies, 20 pM bromodeoxyuridine (BrdUTP; runs, samples were applied and electrophoresed a t 13 Sigma) was included in the reaction. Aliquots were mA constant current. Gels were stained with taken at fixed time intervals up to 3 hr, stained as Coomassie brillant blue and destained with acetic acid described above, and subjected to light microscopy and methanol using standard methods. analysis.

Immunohistochemistry For immunocytochemical studies, we used monoclonal antibodies against Xenopus nuclear lamin LIII (Krohne and Benavente, 1986; gift from G. Krohne, German Cancer Research Centre, Heidelberg, Federal Republic of Germany) and BrdUTP (Serva, Heidelberg, Federal Republic of Germany). A rabbit antiserum raised against Bufo-protamine (Moriya and Katagiri, 1991), which reacted with human protamines on Western blots (Montag e t al., in preparation), was kindly donated by C. Katagiri (Hokaido University, Sapporo, Japan). Aliquots of 3 pl were withdrawn from the decondensation reaction or from the nuclear assembly reaction and allowed to settle into poly-L-lysine-coated coverslips (Mazia e t al., 1975). After fixation in ice-cold methanol (15 rnin), the coverslips were immediately transferred into PBS. Antibody reactions were done essentially as described by Montag e t al. (1991a), except that, for the detection of BrdUTP, DNA was denatured in 4 N HCl for 15 min prior to incubation with the primary antibody. Following the secondary antibody reaction, all preparations were stained with propidium iodide or Hoechst 33342 (concentration as above), washed in PBS, and mounted with a n antifading mounting solution (1,4-diazo-bicyclo-octane; Sigma; 100 mg/ml in 50% glycerol in PBS, pH 8.6). Electrophoresis Basic nuclear proteins were extracted from untreated frozen sperm, decondensed frozen-thawed sperm (stages 1-31, and newly formed pronuclei according to a

Microscopy Light microscopy was performed with a laser scanning microscope (LSM 10; Carl Zeiss) equipped with differential interference contrast (DIC) and phase optics, a n external blue argon laser working a t 488 nm, and another internal green helium neon laser working a t 543 nm, as described recently (Montag et al., 1991b). Transmitted and fluorescent images were recorded, averaged over four to eight frames, and stored in digitized form on hard disk. RESULTS In Vitro Sperm Nuclear Decondensation Decondensation of mammalian sperm is dependent mainly on the reduction of the strong disulphide bonds of the basic, sperm-specific nuclear proteins, the protamines. However, amphibian egg extracts used for nuclear reformation do not provide the means to reduce the -S-S- bonds of mammalian sperm. Therefore, permeabilization either by Triton X-100 or by lysolecithin, followed by pretreatment with reducing agents such as DTT, is necessary to allow for subsequent nuclear formation. Despite the intense pretreatments, Ohsumi and coworkers (1988) reported that the efficiency of decondensation and nuclear reformation of human sperm upon incubation in the amphibian extract was still very low (20-50%) compared to that of amphibian sperm (100%) in its homologous extract. Because similar results were obtained in our initial studies, we decided to develop a n alternative method based on a twostep process, namely, in vitro decondensation in a

IN VITRO FORMATION OF PRONUCLEI FOR MICROMANIPULATION

Fig. 1. Different stages of sperm nuclear decondensation are shown for human ( A stage 0; B: stage 1;C: stage 2, D stage 31,mouse (E: stage 0; F: stage 31, and hamster ( G stage 0; H: stage 3 ) . All images are from differential interference contrast (DIC)microscopy. Bars in D and H represent 10 pm for A-D and E-H, respectively.

buffer followed by nuclear reformation in the amphibian egg extract. In vitro sperm nuclear decondensation in a salt buffer supplemented with heparin and glutathione (GSH) had been recently described by Reyes and coworkers (1989). However, in our hands, decondensation with the use of GSH was successful only when the GSH was fresh. Later use of the same batch of GSH gave either a very delayed decondensation reaction or no reaction at all (see also Mahi and Yanagimachi, 1975). To overcome these difficulties, we substituted P-mercaptoethanol for GSH. Figure 1 shows the result of in vitro decondensation of frozen-thawed mammalian sperm using heparin and P-mercaptoethanol in combination. During incubation, human sperm nuclei exhibited a reproducible course of different stages of decondensation, which we classified as stages 0,1,2, and 3 (Fig. 1A-D, respectively) according to the compaction and the size of the nuclei (for details, see Materials and Methods). For comparison, the corresponding stages 0 and 3 for decondensing mouse (Fig. l E , F) and hamster (Fig. lG, H) sperm nuclei are also shown. In all species examined, stage 3 decondensed nuclei revealed a dotted appearance of the internal nuclear morphology, as seen by DIC. This finding proved to be characteristic of this stage of decondensation (Fig. l D , F, HI. However, the decondensation pattern, namely, the time required for decondensation up to stage 3, was found to be dependent on the pretreatment of the sperm as well a s on the concentration of P-mercaptoethanol. The results are summarized in the graphs in Figure 2. Demembranated human sperm (1% Triton X-100, 0.05% lysolecithin) showed a faster

341

decondensation reaction (Fig. 2A) compared to human sperm permeabilized by the freeze-thawing techcnique (Fig. 2B). For both reactions, higher concentrations of p-mercaptoethanol accelerated the overall time required for decondensation up to stage 3. In short, treated human sperm reached stage 3 after either 20 or 60 min using 2 or 0.5 mM P-mercaptoethanol, respectively, while frozen-thawed sperm nuclei need a much longer time to reach stage 3 at comparable concentrations of P-mercaptoethanol(45min a t 2 mM and 90 min at 0.5 mM). The results proved consistent with three repititions using the same sperm samples. However, slight variations in the timing of sperm nuclear decondensation could be observed with semen samples from different donors. I n addition, the findings indicated that the decondensation time was not dependent on the permeabilization technique used. Changes in the concentration of heparin caused a slower reaction only a t very low levels (

In vitro decondensation of mammalian sperm and subsequent formation of pronuclei-like structures for micromanipulation.

In this study, we describe an efficient protocol for the formation of in vitro developed pronuclei for micromanipulation techniques. Our approach invo...
1016KB Sizes 0 Downloads 0 Views