Bid Cell (1996) 88, 23-28 0 Elsevier, Paris

23

Original article

The motility and fertility of golden hamster sperm cultured in BSA-free medium

Norihiko Uto, Yumi Yamahama

Department of Biology, Hamamutsu University School of Medicine, Handa-cho, Hamamatsu-City, Shizuoka 431-31, Japan

(Received 11 November 1996; accepted 30 November 1996)

Summary - Before fertilization, capacitation and the acrosome reaction in mammalian spermatozoa must be completed. The motility and fertility of hamster sperm were examined in four kinds of modified Tyrode’s solution with or without bovine serum albumin (BSA). Since the presence or absence of polyvinylalcohol (PVA) in the media was another variable, its effect on the sperm motility and fertility was also studied. Sperm were incubated in four different media for up to 6 h at 375°C. After 4 h of incubation in the media containing BSA alone or BSA and PVA, sperm were hyperactivated, showing a high sperm motility index (SMI) and were able to fertilize more than 80% of eggs. However, their fertility rapidly decreased during further incubation. In contrast, sperm in the medium containing PVA and no BSA showed low SMI scores after 4 h. However, during the following 2-h period, the SMI progres- sively increased and sperm were hyperactivated. Furthermore, the hyperactivated sperm in the PVA containing medium were able to effectively fertilize eggs. Our results indicate that hamster sperm can be capacitated in BSA-free medium and that capacitation occurs much more slowly in such a medium. We suggest that PVA is a reasonable alternative to BSA in in vitro fertilization and that this slowly progressing system may be a good model for studying various steps in sperm activation.

sperm motility / BSA-free medium / in vitro fertilization / hamster

Introduction

It is known that in mammalian in vitro fertilization (IVF), sperm just collected from the cauda epididymus never pene- trate into the egg. However, when sperm are pre-incubated in vitro in a chemically defined medium for a few hours, they undergo capacitation and acquire the ability to fertilize eggs [15]. In hamsters, the media commonly used for IVF are slightly modified Tyrode’s solutions containing bovine serum albumin (BSA) (eg, Ham F-10, TALP, HECM-3 with 1 mM hypotaurine [2]). However, a BSA-free medium, TL-PVA (a modified Tyrode’s solution containing sodium lactate and polyvinylalcohol (PVA)) is also used for IVF [4] and the sperm motility bioassay for quality-control testing of culture conditions [Sj, and HECM-3 with 1 mM hypotaurine medium without BSA is one of the suitable media for IVF and embryo culture [2]. Both of these latter media contain PVA but no proteins.

PVA is used as a replacement for BSA because it has been shown to support sperm motility and embryo develop- ment [3]. However, sperm preincubated in TL-PVA for 3-3.5 h cannot penetrate into oocytes [3] even though they are motile. These observations appear to suggest that BSA is a key factor in the processes of sperm capacitation [ 1 l] and acrosome reaction [1, 81 in vitro. However, BSA-con- taining media hyperactivate sperm, making it difficult to use when trying to evaluate subtle sperm quality and to optimize various conditions for sperm activation. It is, therefore, necessary to continue our search for a better cul- ture medium for the purpose of IVF.

Toward this goal, we have analyzed the motility and fer- tility of hamster sperm that were incubated in various media with or without BSA. We found that sperm were activated

more slowly in the media without BSA and that after 6 h of incubation, they were able to fertilize oocytes.

Materials and methods

Sperm preparation

Cauda epididymal contents (CEC) were collected from mature male golden hamsters (Mesocricentus aura&s, 4-6 months old) as described by Bavister [4]. To examine the quality of the sperm, CEC were diluted into the TL-HEPES-PVA medium [4], and sperm motility was quickly checked. CEC containing highly active sperm (motility ~70%) were used for the experiments.

For sperm preincubation, 2 ml each of the sperm preincuba- tion media in a 35-mm Petri dish was overlaid with 2 ml silicone oil and equilibrated overnight at 375°C with 10% CO, in air. Two ~1 of CEC were added to each dish along with 20 ~1 of sperm motility factors (PHE, penicillamine-hypotaurine-epine- phrine) [4]. The sperm suspensions were incubated at 375°C under 10% CO, in air for 2-6 h. Sperm were usually capacitated within 3-3.5 h in HECM-3 with 1 mM hypotaurine.

Culture media

Hamster embryo culture medium, HECM-3 with 1 mM hypotau- rine ([2], table I) was used for insemination and embryo culture. Four kinds of sperm preincubation media were used: a medium without BSA was TL-PVA, and three media containing BSA were HECM-3 with I mM hypotaurine, TAL-PVA and TAL. The chemical composition of these media is described in table I. BSA (catalog no A-9647) and PVA (P-8136) were purchased from Sigma Chemical Co (ST Louis, MO, USA). These media were prepared immediately before use with deionized water obtained using a Milli-Q water system (Millipore Co, Bedford, MA), and sterilized by passing through Millex-GV filters (Millipore).

24

TabIe I. The composition of the four kinds of media.

Component

--__l__l--------.--...----..-._--- ---...-- -.-_-.-.- ._.. TL- PVA HEKN-3 TATAL-PVA n1.

I with ImM hypotaurirw)

PVA NaCl KC1 CaC1,a2Hz0 MgCl,.6H,O Glucose NaH,PO, NaHCO, Na-lactate HCl(1 M) Hypotaurine Glutamine BSA

0.10 mg/ml 114.00

3.16 2.00 0.50 5.00 0.35

25.00 10.00

0.10 mg/ml 113.80

3.00 1.90 0.46 5.00”

25.00 3.70 1.40 @/ml 1.00 0.20 3.00 mg/ml*

0. IO mg/ml 114.00

3.16 2.00 0.50 5.00 0.35

‘5.00 IO.00

3 .OO mg/ml

I il.00 i?h : 00 !I.50 s.00 0.35

25.00 10 00

Concentrations are expressed in mM unless indicated otherwise. * Added only to the sperm preincubation medium.

Sperm motility assay

After 2, 4 and 6 h of incubation. sperm in each dish were exam- ined under a dark-field stereo dissecting microscope to obtain data necessary for calculating the sperm motility index (SMI) [S]. The experiments were repeated at least four times with sperm from different males. The SMI was determined-from the following two .quantities: 1) the percentage of motile sperm; and 2) the quality of sperm motility,.expressed by a number between 1 and 5. Motility quality grades 1 to 4 were arbitrary increments of increasing vigor and progressive sperm motility. Grade 4.5 was given to sperm preparations containing 40 to 70% hyperacti- vated sperm and the rest showing grade 4 motility. Grade 5 was given to preparations in which greater than 70% of the motile sperm showed hyperactivation. The SMI was computed as (per- centage of motile sperm) x (motility grade) x (motility grade). For example, a sperm preparation with 80% of the sperm matile and a motility grade of 4.5 will have an SMI of 1620.

Assayfor sperm penetration into the egg

The egg cumulus complexes (ECC) were collected from the ovi- duct of superovulated females (3-4 months old) 15-17 h after hCG injection [14]. Both the insemination and embryo culture dishes were prepared by arranging 100 ~1 drops of HECM-3 with 1 mM hypotaurine in 60-mm Petri dishes overlaid with 10 ml sili- cone oil and equilibrated overnight at 37S”C with 10% CO, in air [2]. ECC were evenly divided and placed intQ.the drops in the insemination dish, and then 10 fl of preincubated sperm suspen- sion and 1 ,~l of PHE were added. Sperm and eggs were coincu- bated for 2 h at 37.5X in 10% CO, in air. After 2 h, eggs were washed with HECM-3 with 1 mM hypotaurine and transferred into the culture dish. The eggs were incubated for an additional 2 h and examined for the rate of sperm penetration. An oocyte was considered penetrated if it contained one OF more sperm within the perivitelline space or in the egg itself.

Examination of sperm motility pattern

After incubation in various media for 3 h, sperm motility was examined under a phase-contrast microscope, and their move- ment was recorded on videotape. One cycle of sperm beating was traced on the worksheet from successive video frames.

Assessment qf acrosome reaction

At 2-h intervals, sperm were removed from the dish, mounted between a slide and cover&p. and examined with a phase-con- trast microscope (X 400) as described by Uto er al [12]. Motile sperm were carefully examined and those with lost or collapsed

acrosome caps were scored as ‘acrosome reacted’. 1mmotil.e sperm were not considered. The percentage of acrosome-reacted sperm was determined by dividing the number of acrosome- reacted sperm by the number of total motile sperm.

Results

Characteristics of hamster sperm motility in four kinds c.?j media

Figure 1 shows typical temporal changes in sperm motility in four kinds of media. When sperm were incubated-in HECM-3 with 1 mM hypotaurine, TAL-PVA orTAt, they exhibited highly increased motility by the 2 h pdint of incu- bation. After 4 .h of incubation in these media, .SM1 often reached a peak and most of the active sperm were hyperae-

2000 1 I ! / 1500

1 i i !

5 1000 /

cn ~

1 500 }

j 1 1

01

0 2 4 6 8

INCUBATION TtME ( h >

Fig 1. Changes.in sperm motility in four kinds of media. SMI calculated as described in Materials and methods; Results. are typical patterns of for each of the four media. SMIs in HECM-3 with 1mM hypotaurlne(l)), TAL-PVA (A) and T&L(+) were high after 2 h of incubation. In TL-PVA(U), SMI was low at 2 b, but it gradually~ increased.

Motility and fertility of hamster sperm 25

Fig 2. Typical fields of free-swimming sperm in four kinds of media after 2 h of incubation. A. Sperm incubated in TL-PVA. B. Sperm incubated in HECM-3 with 1 mM hypotaurine. C. Sperm incubated in TAL-PVA. D. Sperm incubated in TAL. The formation of sperm clusters was seen in B, C, D, but not in A. Scale, 100 m.

tivated. These high SMI scores diminished during the next 2 h of incubation. In contrast, sperm in TL-PVA showed low SMI scores after 2 h of incubation, but they began to exhibit higher scores during further incubation, reaching the highest value after 6 h. As shown in figure 2, sperm in the BSA containing media for 2 h formed clusters of head-to- head associated sperm and each cluster contained 3 to 20 sperm (fig 2B, C, D). The sperm in these clusters were usu- ally moving vigorously, showing a forward progressing movement pattern. After 6 h of incubation, most of the clus- ters had dissociated. Although most of the sperm in TL- PVA medium did not form clusters after 2 h of incubation (fig 2A), they were hyperactivated within 6 h of incubation.

Figure 3 shows typical patterns of sperm movements after 1 h and 3 h of incubation. Most of the sperm in TL-PVA for 1 h only twitched (fig 3A, B), while many sperm showed advancing movements in HECM-3 with 1 mM hypotaurine (fig 3D), TAL-PVA and TAL. After 3 h of incubation, most of the sperm in HECM-3 with 1 mM hypotaurine showed the ‘figure eight’ looping movement, that is the typical ‘hyperactivation’ movement (fig 3E). This pattern of sperm movement was observed also in TAL-PVA and TAL. Sperm in TL-PVA for 3 h did not show the ‘hyperactivation’ move- ment, but they exhibited very active forward progressing movement (fig 3C) which was similar to the pattern observed after 1 h in the media containing BSA.

The results presented in table II show the relationship between sperm motility and the status of the acrosome during 3 h of incubation. Regardless of the kind of media used, most of the sperm from which the acrosome had fallen off were immotile. The percentages of immotile no-acrosome sperm (IM-nA), ie completely acrosome reacted sperm, were 25-35% in all four media after 3 h of incubation. During 1 h

Fig 3. Typical patterns of sperm movement during the capacita- tion process in four kinds of media. A, B, C. Sperm movement in TL-PVA after 1 h (A, B) and 3 h (C) of incubation. D, E. Move- ment in HECM-3 with 1mM hypotaurine after 1 h (D) and 3 h (E) of incubation. In E, The ‘figure eight’ looping movement that is the typical ‘hyperactivation’ movement is illustrated. Tbis pattern of sperm movement was also observed in TAL-PVA and TAL. Scale, 20 pm.

of incubation, many clusters of sperm appeared in HECM-3 with 1 mM hypotaurine, TAL-PVA and TAL, while there was no cluster formation in TL-PVA. Immotile-acrosome intact sperm (IM-A) in TL-PVA was about 20% at 1 h, but it gradually decreased and the proportion of motile-acrosome intact sperm (M-A) increased with time.

26 N Uto. Y- Yamaharna

Table II. Relationship between sperm motility and the acrosome status during 3 h of sperm preincubation in four kinds of media.

Mediu

TL-PVA

HECM-3

TAL-PVA

TAL

Incubation Total no No of sperm in various stasep fime (h) of sperm IM-A (%) IM-nA (%) M-A {W) M-nA (%) 34CL ($1;)

--_- 1 337 67 (19.9) 137 (40.7) 123(36.5) 0 (0.0) 10<3.Oj 2 397 42(10.6) 164(41.3) lLsO(45.3) 0 (0.0) 11 12.8, 3 345 24 (7.0) 124 (35.9) 186 (53.9) 0 (0.0) 11 (3.2) 1 593 27 (4.6) I39 (23.4) 100(16.9) 0 (O.Oj 327 iis.:; 2 394 15 (3.8) 95 (24.1) 87 (22.1) 0 (0.0) 197 (50.0) 3 382 I2 (3.1) 106(27.7) 103 (27.0) 0 (0.0) “33 (42.1 i 1 384 21(.5.5) 86(22.4) 48 (12.5) 0 (0.0) 2'9 (59.6,

2 371 19 (5.1) 62(16.7) 121(32.6) 0 (0.0) 169 ($5.6) 3 510 26 (5.1) 135 (26.5) ilfS(22.7) 0 (0.0) 233 145.73

I 358 12 (3.4) 73 (28.0) 65 (18.2) 0 (0.0) 208 (58.P) 2 542 14t2.6) 152(28.0) 83 (15.3) 0 (0.0) 293 (54.1 i 3 368 4(1.1) 105 (28.5) 65 (17.7) 0 (0.0) 194 (,S2.7)

a Progressive grade of sperm motility and the state of the acrosome were divided into five groups: innnotile-acrosome intact (IM-Ai. immotile-no acrosome (IM-nA), motile-acrosome intact (M-A), motile-no actosome (M-nA) and motile-cluster (M-CL). All percentages derived from the total sperm examined

50

0

0 2 4 6 INCUBATION TIME (h)

Fig 4. Incidence of acrosome reaction in sperm incubated in four media. Only motile sperm were carefully examined-and those which had lost or collapsed acrosome caps were recorded as ‘acrosome reacted’. Immotile sperm were not considered. The media used were HECM-3 with 1 n&f hypotaurine (O), TAL- PVA(A), TAL(+), and TL-PVA(O).

Acrosome reaction

Figure 4 shows the incidence of acrosome-reacted sperm over a 6-h period in the four media. The-percentage of acro- some-reacted sperm in the total number of motile sperm is shown. During 2 h of incubation, most of the motile sperm did not undergo acrosome reaction, but about 50% and 70% of motile sperm in TAL and HBCM-3 with 1 mM hypotau- rine were acrosome-reacted after 4 h and 6 h of incubation, respectively. The percentage of acrosome-reacted sperm in TAL-PVA increased to 50% in 6 h. In contrast, only 10% of the sperm incubated in TL-PVA were the acrosome-reacted type. in spite of vigorous movement after 6~ h of incubation. The medium without BSA (ie TL-PVA) scarcely induced acrosome reaction.

The fertility of sperm incubated infbur kinds of medid

When sperm were incubated in HECM-3 with 1 n&r nypo- taurine, they usually showed hyperactivation within 3 hoof incubation. In an ordinary IVF, preincubation in this medium for 3 h is often used, providing good results in IVF- experiments in hamsters. Table III shows the results of fer- tilization using sperm preincubated for 3 h in the four media. Indeed, about 80% of eggs were penetrated by sperm incubated in HBCM-3 with 1 mM hypotaurine. Simi- lar results were obtained. with TAL-PVA (71 .l%) and TAL 178.2%). On the other hand, sperm incubated in TL-PVA penetrated only about 40% of the eggs,

Although the total number of eggs used for each case was small, we also obtained similar data after 2, 4, and 6 h of incubation (table IV). Sperm in the media with BSA (HECM-3 with 1 mM hypotaurine, TAL-PVA, and TAL) for 2 and 4 h appeared to have fertilizability comparable to that obtained after 3 h of incubation. However, after 6 h of incubation, sperm in these media showed greatly reduced fertilizability. .In fact, none out of seven eggs could be ferti- lized by sperm incubated for 6 h in HECM-3 with 1 mM hypotaurine. Sperm in TL-PVA increased their abil- ity to fertilize eggs as the incubation time became longer. Ten out of 11 eggs were fertilized by sperm in this medium for 6 h, while only a third of the eggs were fertilized by sperm incubated in the same medium for 2 h.

Discussion

When hamster sperm were incubated in various media com- monly used for IVF. they exhibited different motile proper- ties depending of the presence or absence of BSA in the media. We quantitated the overal-sperm motility by using SMI, an index that reflects both sperm activity and sperm viability [l]. High SMI values indicate that the sperm exhibit high motility and high viability. Sperm incubated in the three media containing BSA consistently showed higher SMIs at early times -&an those treated with the BSA-free medium. In particular, the sperm incubated in the .media containing BSA and PVA (TAL-PVA and HECM-3 with 1 mM hypotaurine) revealed increasing SMIs during the

Motility and fertility of hamster sperm 27

Table III. Fertilizability of sperm preincubated for 3 h in four kinds of mediaa.

Sperm preincubation media

Total no ofems

Attached by spermb Not Zona

attached (%) attached (%)

Penetrating by spermb Zona ES@

penetrated (%) penetrated (%)

TL-PVA 37 11 (29.7) 12 (32.4) 4 (10.8) 10 (27.0) HECM-3 45 7 (15.6) 2 (4.4) 4 (8.9) 32 (71.1) TAL-PVA 38 5 (13.2) 6 (15.8) 2 (5.3) 25 (65.8) TAL 32 2 (6.3) 5 (15.6) 2 (6.3) 23 (71.9)

a Sperm were preincubated for 3 h in given media and then coincubated with ECC in HECM-3 with 1 mM hypotaurine (BSA-free) for 2 h. Eggs were cultured for an additional 2 h. b Values given are the numbers of eggs which were attached or penetrated by sperm 4 h after insemination. All percentages derived from the total eggs inseminated.

Table IV. Fertilizability of sperm preincubated for 2,4, or 6 h in four kinds of median.

Sperm preincubation Total no

Attached by spermb Not Zona

Penetrated by spermb Zona Egg

media Time (h) ofem attached (%) attached (O/O) penetrated (%) penetrated (%)

2 12 3 (25.0) 5 (41.7) 0 (0.0) 4 (33.3) TL-PVA 4 9 l(ll.1) 2 (22.2) 0 (0.0) 6 (66.7)

6 11 l(9.1) 0 (0.0) 0 (0.0) 10 (90.9)

2 9 0 (0.0) 2 (22.2) l(ll.1) 6 (66.7) HECM-3 4 4 0 (0.0) 0 (0.0) 0 (0.0) 4 (100.0)

6 7 4 (57.1) 3 (42.9) 0 (0.0) 0 (0.0) 2 9 0 (0.0) 0 (0.0) l(ll.1) 8 (88.9)

TAL-PVA 4 11 1 (9.1) l(9.1) 0 (0.0) 9 (81.8) 6 8 0 (0.0) 4 (50.0) 0 (0.0) 4 (50.0) 2 9 0 (0.0) 0 (0.0) 0 (0.0) 9 (100.0)

TAL 4 12 0 (0.0) 1 (8.3) 1 (8.3) 10 (83.3) 6 5 1 (20.0) 4 (80.0) 0 (0.0) 0 (0.0)

a Sperm were preincubated for 2,4 and 6 h in given media and then coincubated with ECC in HECM-3 with 1 mM hypotaurine (BSA- free) for 2 h and eggs were cultured for an additional 2 h. b Values given are the numbers of eggs which were attatched or penetrated by sperm 4 h after insemination. All percentages derived from the total eggs inseminated.

first 4 h of incubation, and the high SMI scores were main- tained beyond the intial 4-h period. On the other hand, the sperm in the BSA-free medium (TL-PVA) had a signifi- cantly lower SMI score during the first 4 h of incubation. However, the score rapidly increased during the next 2-h period, reaching the highest SMI level obtained by using BSA-containing media. These results indicate that PVA is an effective substitute for BSA in sustaining vigorous ham- ster sperm motility in a gamete culture system. They further suggest that PVA may be satisfactorily used for the purpose of IVF, thus eliminating some of the problems associated with the use of BSA.

To more closely study the effects of different media on sperm behavior, we analyzed sperm motility patterns. Many sperm in the media containing BSA showed the active for- ward progressing movement pattern after 1 h of incubation, and within 3 h, they showed the hyperactivated pattern. In contrast, most of the sperm in the BSA-free medium showed a high frequency beat with small amplitude after 1 h of incubation, and even after 3 h of incubation, their motile activity was similar to that exhibited by sperm in the media with BSA for 1 h. The sperm in the BSA-free medium for 3 h did not exhibit the hyperactivation move- ment. These observations appear to suggest that BSA plays a role in sperm hyperactivation.

In mammalian IVF, the capacitated sperm can undergo the acrosome reaction and then fertilize eggs. According to Corselli and Talbot [6], only capacitated acrosome-intact sperm can penetrate into the ECC while uncapacitated sperm and capacitated acrosome-reacted sperm cannot. During capacitation, the acrosome membrane of the sperm undergoes many changes, and the sperm also undergoes hyperactivation, a qualitative change in sperm motility [ 131. Yanagimachi [13] reported that hyperactivation was not necessary for sperm to fuse with the egg membrane, indi- cating that hyperactivated sperm are already capacitated.

To investigate if hyperactivated sperm are indeed capaci- tated, we examined the fertility of sperm that were hyperac- tivated. Sperm incubated in the media containing BSA (HECM-3 with 1 n-N hypotaurine, TAL-PVA, TAL) for 3 h were hyperactivated and a large proportion of sperm pene- trated eggs. However, a substantially reduced number of sperm incubated in the BSA-free medium (TL-PVA) for the same length of time were able to penetrate eggs. This results is consistent with that of Bavister [3]; that the sperm incubated in the BSA-free medium for 3-3.5 h poorly pene- trated the cumulus-free egg. Although the proportion of the acrosome-reacted sperm in TL-PVA was low, some sperm did undergo the acrosome reaction. We suggest that eggs were penetrated by sperm that became capacitated prior to

becoming acrosome-reacted. Sperm in the BSA-free medium show a high SMI score after 6 h of incubation and their fertility also increased. This result is consistent with the idea that hyperactivated sperm are capacitated. When sperm were kept in the BSA-containing media for 6 h, their fertility was considerably lost. This may be due to an increased propotion of acrosome-reacted sperm and/or to decreased sperm motility in such cultures. Cummins and Yanagimachi [7] suggested that the ability for sperm to enter the cumulus matrix coincides with physical changes that occur during capacitation preceding complete loss of the acrosomal cap. Sperm that have completed the acro- some reaction in vitro penetrate the matrix poorly, whereas those with ‘modified’ acrosome caps can enter the niatrix.

The way by which BSA induces capacitation and acrosome reaction in sperm is not clear at present. Peters [IO] and Mei- zel [9] suggested the possibility that a variety of small mole- cules, such as steroids, fatty acids and amines, associated with BSA may be released from it and act on the sperm plasma membrane, thus hyperactivating them. It has been reported that a synthetic polymer, PVA is able to sustain sperm viabil- ity but failes to effectively induce sperm capacitation and acrosome reaction [3]. However, the experiments described in this report have demonstrated that after prolonged incubation in the BSA-free, PVA-containing medium (TL-PVA), some sperm undergo acrosome reaction and, during this process, become capable of fertilizing eggs. This result indicates that sperm capacitation can occur, albeit slowly, without the action of BSA (and small molecules associated with~it) in vitro. Thus, it is possible that factors such as viscosity, pH, and 0, concentration in the media also play some role in sperm acti: vation, and such studies are now in progress in our laboratory.

Acknowledgment

We thank Professor Emeritus Hidemi &to, University of Nagoya, for his valuable advice and proofreading of this manuscript.

References

1 Andrews JC, Bavister BD (1989) Hamster zonae peilucidae cannot induce physiological acrosome. reactions in chemi-

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3

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1 3

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<ally capacitated hamster spermatozoa in the absence oi albumin. Biol Reprod 40, 117-122 Barnett DK. Bavister BD (1992) Hypotaurine requiremen: for in virro development of golden hamster one-cell embryos into morulae and blastocysts, and production of term off- spring from in vitro fertilized ova. Biol Reprod 47, 297~-304 Bavister BD (1981) Substitution of a synthetic polymer for protein in a mammalian gamete culture system. J Exp Zen! 217,45-51 Bavister BD (1989) A consistently successful procedure 10: in vitro fertilization of golden hamster eggs-. Gamete &.r 13 139-158 Bavister BD, Andrews JC (1988) A rapid sperm rnotiiirq bioassay procedure for quality-control testing of water and culture m&a. J Vitro Fertil Embryo Transfer 5, 67-7.5 Corselli J. Talbot P (1987) In vitro penetrgtion of hamster oocyte cumulus complexes using physiological numbers;- (of sperm. Dt% Biol 122,227-242 Cummins JM, Yanagimachi R (1986) Development ot‘ abii- ity to penetrate the cumulus oophorus by hamster spermato- zoa capacitated in vitro, in relation to the timing of ~the acro- some reaction. Gamate Res 15, 187-212 Dow MPD, Bavister BD (1989) Direct contact iz require<! between serum albumin and hamster spermatozoa for capa citation in vitro. Gamete Res 23, 171-180 Meizel S (I 978) The mammalian sperm acrosome reaction, 4 biochemica! approach. In: Development in Mammals Vol ;i (Johnson MH, ed). North-Holland Publishing Amsterdam, I-+& Peters TT ( 1975) Serum albumin. In: The Plasma Proteins StructLtre. Function, and Genetic Control (Putnam FW ed? Academic Press New York, 1133-l 181 Stewart-Savage J (1993) Effect of bovine serum albumr~ concentration and source on sperm capacitation in thz golden hamster. Biol Reprod 49, 74-8 1 1Jto N, Yoshimatsu N, Lopata A, Yanagimachl R ( 1988 I The zona-induced acrosome reaction of hamster spermato roa. J Enp zoo1 248. 113-120 Yanagimachi R (198 1) Mechanisms of fertilization in mani mals In: F’ertitizarion and embryonic development in vitro. (Mastroianni L, Riggers JD, eds) Plenum Press. New York. 8 l-182 Yanagimachi R (1984) Developmental ability of precu ciously superovulated golden hamster eggs. Gamete. Res 9. ?I+--237 Yanagimachi R (1988) Mammalian fertilization. ir:. 777:. physiology of reproduction (Knobil E, Neil1 JD, edsj Rave? Press, New York. 135-185