THE JOURNAL OF EXPERIMENTAL ZOOLOGY 254:305-312 (1990)
In Vitro Fertilization of Siberian Hamster Oocytes T.A. PARKENING Departments of Anatomy and Neurosciences and of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, Texas 77550 ABSTRACT Siberian hamsters were superovulated and various media were tested in an effort to fertilize the recovered oocytes in vitro. The highest percentage of fertilized ova was achieved by using a modified Tyrode's medium, designated MT (Bavister, J. Reprod. Fertil., 18.544-545, '69), previously formulated to fertilize Syrian hamster ova in vitro. Spermatozoa incubated in this medium in a concentrated state overnight (14 hr) and then diluted (1hr) fertilized 39% of the ova. Similar results (40%) were obtained with this medium by adding 20% human follicular fluid to fresh concentrated sperm for 30 min and then diluting the sperm for 2-3 h r prior to the addition of ova. Ova fertilized in vitro cleaved to the two-cell stage but failed to develop any further in culture. Two-cell embryos recovered from mated hamsters and cultured did not undergo additional cleavage. Four-cell embryos collected from mated females and cultured cleaved to the six- to eight-cell stage and stopped. Techniques and media used for fertilizing large numbers of Syrian and Chinese hamster ova in vitro will have to be modified to achieve the same degree of success in the Siberian hamster.
The Siberian hamster (Phodopus sungorus) is a seasonal breeder in the wild. For this reason, this species has received attention as a new laboratory animal because its gonadal development and function can be altered by varying the photoperiod (Hoffmann, '73, '78; Milette and Turek, '86; Stetson et al., '86; Yellon and Goldman, '84, '87). This has enabled researchers t o understand better the relationship of pineal, pituitary, and gonadal function in these rodents. Adult Siberian hamsters maintained under a normal rodent 14:lO (14 hr artificial light) light cycle, however, breed throughout the year, enabling colonies to be readily established. Syrian hamsters were one of the first laboratory rodents whose ova could be successfully fertilized in vitro (Yanagimachi and Chang, '63). Later it was shown that zona-free Syrian hamster ova could be penetrated by heterologous spermatozoa such as human sperm (Yanagimachi et al., '76). This unique feature boosted the research popularity of this species; many infertility laboratories now use the zona-free hamster ovum assay (also referred to as sperm penetration assay) as a test for evaluating the quality of human sperm. Surprisingly few studies have examined the fertilizing ability of ova from other hamster species. To my knowledge, the only other published reports concern the Chinese hamster. Austin and Walton ('60) and Pickworth et al. ('68)described aspects of fertilization in vivo, and Pickworth and Chang ('691, Yanagimachi et al. ('831,and Parkening and 0 1990 WILEY-LISS, INC.
Cisneros ('88) investigated in vitro fertilization. It was our recent success in culturing late two-cell and early four-cell Chinese hamster embryos to the blastocyst stage (Parkening and Cisneros, '88) that prompted the present study to investigate whether ova from Siberian hamsters could be fertilized in vitro and if early embryos would develop in culture.
MATERIALS AND METHODS Animals The Siberian hamsters used in these studies were born and raised at the University of Texas Medical Branch. The colony was established from 16 pairs obtained from Dr. A. Bartke (Southern Illinois University at Carbondale, Carbondale, IL), whose animals were descendants of a colony established by Dr. B. Goldman (Worcester Foundation for Experimental Biology, Shrewsbury, MA). The animals were housed under artificial lighting (14 hr light/lO h r dark) in clear plastic 48 x 27 x 13 cm cages (wood shavings as bedding) in a temperature controlled room (21-23"C). They were provided with laboratory rat chow and water ad libitum, with dried oatmeal provided twice per week as a food supplement. For breeding purposes, only one male and female should be paired per cage; housing multiple males or females with a member of the opposite sex seldom results in Received July 28, 1989; revision accepted December 6, 1989
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cauda epididymidis was excised from a male that had sired at least one litter, and a n incision was made to acquire a droplet of sperm. The sperm were placed in the drop of medium and left for Superovulation either 30 min or 14 hr in the incubator and then Females ranging in age from 1to 8 months (24- diluted in another 200 p1 drop to a concentration 34 g depending on age) were examined for their approximately 5 x 105/ml. The diluted sperm ability to be superovulated. If the animals were were preincubated for 1-3 h r before the addition exhibiting a n estrous cycle, they were given of ova. The oviducts from superovulated females Pregnant Mares' Serum Gonadotropin (PMSG) were then excised, carefully blotted on a piece of during diestrus. Injections were given either in- sterile filter paper, and placed in the paraffin oil traperitoneally or intramuscularly. Various regi- close to the drop of sperm suspension. Under a mens were given: 3 IU PMSG and 5 IU human dissection microscope, two sterile dissecting neechorionic gonadotropin (hCG), 5 IU PMSG and 5 dles were used to rupture the wall of the ampulla, IU hCG, 5 IU PMSG and 8 IU hCG, 5 IU PMSG allowing the ova within their cumulus mass t o and 10 IU hCG, 8 IU PMSG and 10 IU hCG, 10 IU exude into the oil. The cumulus mass was then PMSG and 10 IU hCG, 10 IU PMSG and 12 IU pushed into the droplet of sperm suspension, and hCG, 1IU PMSG and hCGI6 g body weight, and 1 the gametes were incubated for 7, 9, or 10 hr. For each experiment ova were pooled from sevIU PMSG and hCG18 g body weight. The time that elapsed between injections of PMSG and eral females, and the number was divided equally hCG was 48-52 hr. Oocytes were collected be- between three and six different media. Any unusual-appearing ova were discarded. Spertween 15 and 16 h r after the hCG injection. matozoa were likewise pooled from two males Culture media during each study, and the same dilution was Seven media were initially tested for the in used for each drop of medium. There were no exvitro fertilization of oocytes. These consisted of periments performed that did not result in the medium 199, Ham's F10, BWW (Biggers et al., fertilization of at least one ovum from one of sev'71), Brinster's ('71) medium, modified BWW eral media tested. This design helped t o reduce (MBWW) (Parkening and Cisneros, '881, and two variability of results due to possible differences in modified Tyrode's media-MT (Bavister, '69) and the gametes of the animals. TALP (Bavister and Yanagimachi, '77). All Examination of ova media except for medium 199 and Brinster's meThe ova were washed once in a fresh volume of dium were supplemented with 2.5 mg/ml bovine serum albumin (BSA). In some experiments, the medium selected for fertilization and mounted heat-inactivated fetal cord serum (10-20%) or hu- in a small drop on a slide beneath a coverslip conman follicular fluid (20%) was substituted for taining petroleum jelly at its corners. The ova BSA in the BWW, MBWW, MT, and TALP were compressed slightly by the coverslip t o premedia. The pH of the media ranged from 7.0 to vent movement, and 2% glutaraldehyde was irrigated under the coverslip. The slide was then 8.0. The medium of choice became the MT medium placed in a petri dish containing a filter paper (Bavister, '691, which was made by adding 30 mg soaked in 10% formalin and the eggs left oversodium bicarbonate (Sigma S-88751, 1.1 mg night in the refrigerator (4°C).The following sodium pyruvate (Sigma P-22561, 10 mg glucose morning, the eggs were stained by irrigating the (Sigma G-5000), and 25 mg BSA (Sigma A-4378) following solutions, in sequence, under the to 10 ml Tyrode's solution (Difco 5556-72). The pH coverslip: distilled water, 95% ethanol, 1%lacof the medium was 7.8 and the osmolarity 345 moid, and mounting medium (by volume 20% glycerol, 20% glacial acetic acid, 60% distilled wamOsm. ter). The coverslip was then sealed with clear nail In vitro fertilization polish, and the ova were examined for fertilizaA 200 p1 drop of one of the above media was tion with phase-contrast microscopy. placed under paraffin oil (Mallinckrodt, Inc; visIn vitro development cosity 125-135) in a Falcon culture dish (35 mm). Ova incubated with sperm for either 9 or 10 hr The medium was then equilibrated for 1-14 h r in an incubator (37°C) gassed with 5% COZ in air. A and considered to be fertilized from the appearany litters. From records collected on 151 litters in our colony, 762 pups were born, for a mean litter size of 5.1.
FERTILIZATION OF SIBERIAN HAMSTER OVA IN VITRO
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TABLE I . Superovulation of immature and mature Siberian hamsters using different regimens of gonadotropins
Hamsters
Gonadotropin regimen in IUs PMSG hCG
No. of hamsters ovulating (%)
No. of ova (mean 2 s.e.m.1
Immature' Immature Mature2 Mature Immature Mature Mature Mature Mature Immature Immature
3 5 5 5 5 5 5 8 5 10 5 10 8 10 10 10 10 12 1 IUI6 g body wt. 1IUI8 g body wt.
24 of 32 (75) 2 of 5 (40) 27 of 33 (82) 4 of 5 (80) 4 of 4 (100) 4 of 5 (80) 6 of 6 (100) 5 of 7 (71) 5 of 5 (100) 4 of 10 (40) 5 of 5 (100)
243 (7.6 1.1) 14 (2.8 1.7) 228 (6.7 0.9) 32 (6.4 2 2.0) 28 (7.0 3.0) 28 (7.0 5 2.7) 49 (8.2 & 0.5) 30 (5.0 & 1.3) 34 (4.8 1.3) 17 (1.7 5 0.9) 40 (8.0 5 1.7)
* * * * *
'Immature, ranged from 1 to 2 months of age. 'Mature, ranged from 3 to 8 months of age
RESULTS
were 2 months of age, and all weig,,ed over 30 g; hence they were approaching the weight commonly found in mature females. Mature females ovulated the most consistently when injected with 8 IU PMSG and 10 IU hCG (Table 1). The largest number of ova collected was 21 from an immature female (15 g) given 3 IU PMSG and 5 IU hCG. A 3-month-old female ovulated 21 ova and a 5-month-old female yielded 19 ova when given 5 IU PMSG and 5 IU hCG. Slightly more ova were ovulated when waiting 50-52 hr, instead of 48 hr, between injections of PMSG and hCG.
Superovulation The various regimens for superovulating Siberian hamsters are given in Table 1.This species of hamster, unlike the Syrian or the Chinese hamster, responds erratically t o gonadotropins. Females of the same weight and at the same stage of the estrous cycle, given a particular regimen of gonadotropins, may ovulate two or three oocytes or 10-15 oocytes or may not ovulate. Littermates responded with the same random pattern of ovulation. The greatest number of ova was acquired from immature hamsters (1-2 months of age) by injecting 3 IU PMSG and 5 IU hCG or by injecting 1 IU of gonadotropins/8 g body weight (Table 1). The two regimens were equivalent, since the hamsters receiving gonadotropins on a weight basis weighed 24-28 g. When a slightly higher amount of PMSG was administered to this group, it appeared t o have an inhibitory effect. Immature hamsters receiving 5 IU PMSG and 10 IU hCG responded t o a degree, but these animals
In vitro fertilization No fertilization was achieved when using medium 199, Ham's F10, Brinster's, or BWW (Table 2). Some ova became activated in Ham's F10 (70%) and Brinster's (29%). One of 31 ova was fertilized in modified BWW (MBWW) with BSA. The addition of 20% fetal cord serum or 20% human follicular fluid did not enhance the medium, since none of 77 ova were fertilized. The best results were achieved by using a modified Tyrode's medium with BSA or 20% human follicular fluid (Table 2). Twenty-one and twenty-nine percent of ova were fertilized, respectively, in MT and TALP when sperm were incubated for 30 min in a concentrated form and then diluted for 2 hr before adding ova. If the latter regimen was used and 20% human follicular fluid was added, fertilization was enhanced using MT (40%)and decreased using TALP (14%).The addition of 20% fetal cord serum t o MT also appeared t o be beneficial (32%). Spermatozoa incubated overnight (14 hr) with
ance of pronuclei or a fertilizing sperm tail were washed in fresh MT and transferred t o a new 200 ~1 droplet. Two- and four-cell embryos were collected from mated females on the second and third day after finding a copulatory sperm plug. All embryos were cultured in either MT (Bavister, '69) or MBWW (Parkening and Cisneros, '88) containing 4 mg/ml BSA or 10% fetal calf serum for 2 days. The eggs were periodically monitored for cleavage during the 2 day incubation. They were then mounted, fixed, and stained as above.
T.A. PARKENING
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TABLE 2 . I n vitro fertilization Media
of
Siberian hamster ova using various media
No. of experiments
Ova fertilized (%)
3 3 3 3 3 3 3 3 3 2 8 8 3 3
0140 (0) 0142 (0) 0139 (0) 0145 ( 0 ) 0143 (0) 1140 (3) 0138 (0) 0139 ( 0 ) 24182 (29) 6143 (14) 18187 (21) 541137 (39) 17143 (40) 12138 (32)
199 Ham’s F10 Brinster’s BWW BWW + 10% FCS’ MBWW MBWW + 20% HFF2 MBWW + 20% FCS TALP TALP + 20%HFF MT MT (sperm incubated 15 h)3 MT + 20% HFF MT + 20%FCS
Range of % fertilized
-
0-6 -
8-50 0-25 7-64 0-78 36-67 14-36
‘FCS, fetal cord serum. ‘HFF, human follicle fluid. 31n all the other experiments, sperm were incubated in a concentrated form for 30 min, then diluted and incubated for 2-3 hr before adding ova.
MT and diluted for 1 h r prior to adding ova fertilized as many ova in several experiments as MT with 20% human follicular fluid; however, there was greater variability within the experiments (Table 2). Spermatozoa remained motile in every medium during the length of time of the experiment. Spermatozoa in medium 199 did become markedly slower after 6 h r in culture compared with those in other media. However, all spermatozoa were active enough in each medium to readily remove the cumulus cells. In Ham’s F10 and Brinster’s spermatozoa did not make contact with the zona pellucida once the ova were denuded of cumulus cells. In the other media, sperm did make contact with the zona and appeared to attempt to penetrate it. Using MT and TALP, ova that were cultured with spermatozoa for 7 hr were often just being penetrated by sperm. Of the number of ova fertilized at this time period, 21 (42%) of 50 exhibited a swollen sperm head and the early formation of a female pronucleus (Figs. 1,2).In these experiments five of 46 unfertilized ova had one spermatozoon that had penetrated the zona pellucida, but the sperm head was not incorporated into the vitellus of the ovum. Of the ova with a swollen sperm head, 22% had the major portion of the sperm tail outside the zona pellucida (Fig. 11, and 78% had the sperm tail completely within the perivitelline space (Fig. 2). Ova that were fertilized 9 and 10 hr after incu-
bation had male and female pronuclei present (Figs. 3,4). Four of 60 ova (7%)were polyspermic. Two of these were dispermic, and two were trispermic. One of the dispermic ova had one fertilizing sperm with its tail within the perivitelline space and one fertilizing sperm with a major portion of its tail outside the zona pellucida. One of the trispermic ova also had one fertilizing sperm whose tail was largely outside the zona pellucida. Eleven (20%) of the remaining 56 monospermic ova had a fertilizing sperm with part of its tail outside the zona pellucida (Fig. 3).
In vitro culture Some additional ova fertilized in vitro in MT after 9 and 10 hr incubation were examined with a dissecting microscope. On the basis of the appearance of pronuclei and what in some instances appeared to be a fertilizing sperm tail, 26 ova were washed and placed in fresh medium. All of the ova had cleaved to the two-cell stage the following morning but failed after 2 days in culture to develop any further in the modified Tyrode’s medium. After these ova were fixed and stained, only 21 were considered to have been fertilized. Eight Siberian hamsters that mated naturally were killed 2 or 3 days after finding a copulatory plug to obtain early embryos. Four of the females killed between 0900 and 1000 hr on day 3 yielded 24 two-cell ova, and four females killed on day 3 between 1300 and 1400 h r yielded three two-cell and 20 four-cell embryos. One degenerating egg
FERTILIZATION OF SIBERIAN HAMSTER OVA IN VITRO
Fig. 1. A Siberian hamster ovum fertilized after a 7 hr incubation with spermatozoa. The swollen sperm head (S) and female pronucleus (P) are visible in the ooplasm. A major portion of the fertilizing sperm is outside the zona pellucida . and is visible to the right of the sperm head. ~ 5 4 0 The granular or filamentous appearance of the ova in Figures 1-4 results from the large number of paracrystalline inclusions found in their ooplasm. Fig. 2. An ovum incubated as in Figure 1, containing a swollen sperm head (upper periphery of ovum). A portion of the sperm tail visible against the ooplasm was completely enclosed within the perivitelline space. Two cumulus cells (C), slightly out of focus, remain attached to the zona pellucida. The female pronucleus (P) is just beginning to form. x 540.
was found in the 2 day group and four degenerating eggs in the 3-day group. The embryos were cultured in either MBWW or MT containing either BSA (4 mg/ml) or 10% fetal calf serum. None of the two-cell embryos cleaved any further in culture. Half of the four-cell embryos cleaved to
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Fig. 3. An ovum that had been in culture with spermatozoa for 9 hr. The fertilizing sperm still had a major part of its tail outside the zona pellucida. The male and female pronucleus and one polar body (near the upper periphery of ovum) are visible. x 540. Fig. 4. An ovum incubated for 10 hr with spermatozoa. The fertilizing sperm tail is visible against the vitellus and was incorporated into the perivitelline space. Both pronuclei (P) are visible. The other round profiles against the vitellus are cumulus cells (out of focus) still adhering to the zona pellucida. x 540.
the six- t o eight-cell stage and stopped. When the embryos were mounted, fixed, and stained, all but four (9%) showed evidence of a sperm tail inside the zona pellucida.
DISCUSSION If the Siberian hamster is to be used routinely for gamete research, it is essential that a better protocol for superovulation be developed. The
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method producing the most oocytes still resulted in sporadic ovulations and fewer ova than normally collected from using similar techniques on other laboratory rodents. Perhaps extending the time between PMSG and hCG injections would be beneficial. Increasing the time from 48 t o 50-52 hr between PMSG and hCG injections resulted in a slight increase in the number of oocytes ovulated. Waiting as long as 72 hr between injections proved to be the best regimen for superovulating immature Chinese hamsters (Roldan et al., 1987). Whereas a majority of immature Chinese hamsters respond by ovulating 15-30 ova after a protocol of 3 IU PMSG and 5 IU hCG given 48-50 hr apart (Parkening and Cisneros, 19881, unruptured ovarian follicles were often present. Roldan et a1 (1987) also reported many unruptured follicles in immature Chinese hamsters given 15 IU PMSG and hCG 72 hr apart. Similarly with the Siberian hamster, females that ovulated only two or three ova had several ripe follicles on their ovaries. Recently, some of these ovarian oocytes were recovered and tested for their ability t o fertilize in vitro. Several were penetrated and developed normal male and female pronuclei. Extending the time of collection from 15-16 t o 17-18 hr did not improve the rate of ovulation in the Siberian hamster, although some immature Chinese hamsters apparently do not complete ovulation until 20-21 hr after hCG (Roldan et al., 1987). Amounts of PMSG as low as 20 IU injected into immature rats have a detrimental effect on the maturation of some oocytes, presumably by altering the steroid environment of the follicle (Yun et al., 1989). Although it is possible that PMSG also had an adverse effect on oocytes in the Siberian hamster, rendering them infertile, this is doubtful, because the concentration given never exceeded 5 IU in immature or 10 IU in mature females. Seventy-five percent of ova recovered from superovulated immature Chinese hamsters using 15 IU PMSG and hCG were capable of fertilization in vitro (Roldan et al., 1987). The medium BWW (Biggers et al., '71) has proved an excellent medium for fertilizing rodent ova. It works well for the in vitro fertilization of both Syrian (Hirao and Yanagimachi, '78) and Chinese (Parkening and Cisneros, '88) hamsters. However, no Siberian hamster oocytes were fertilized in BWW. The only media found acceptable for fertilizing Siberian hamster oocytes were the modified Tyrode's media developed for fertilizing Syrian hamster oocytes (Bavister, '69; Bavister and Yanagamachi, '77). MT appeared to be
slightly better than TALP, although considerable variability existed within and between experiments. The variability was difficult to reconcile in that it existed when using the same sperm concentration from the same males. The amount of media remained uniform, and the oocytes were pooled, yet the number of ova fertilized would vary from one in ten to seven in ten with identical media in two identical droplets in the same culture dish. It appears that these media will have to be modified t o achieve a consistently high percentage of fertilized ova using this species. Incubating sperm overnight in a concentrated form using MT was beneficial for fertilization. However, adding 20% heat-inactivated human follicular fluid t o MT for a standard incubation of 2-3 hr achieved the same results. Follicular fluid has components necessary for the capacitation of sperm (Yanagimachi, '69). Speeding the induction of the acrosome reaction and capacitation may be necessary t o improve the rate of fertilization in the Siberian hamster. In one experiment, of the number of sperm adhering to the zona pellucida of ova, only one of 84 sperm had lost its acrosome. The addition of 20% human follicular fluid to TALP did not improve the percentage of oocytes fertilized in vitro. This follicular fluid, however, was not the same sample as that used with MT. Determining whether this caused the lower percentage of fertilized ova or was a reflection of the inherent variability in the experiments will require additional studies. It may be necessary to preincubate Siberian hamster spermatozoa for 5-6 hr, as is done for the fertilization of rat oocytes (Niwa and Chang, '74). Preliminary studies indicate that diluted suspensions of sperm, in the absence of ova, lose some of their motility after 6 hr in a manner similar t o that of diluted concentrations of rat sperm. Under these circumstances the addition of 2 mM caffeine (Gaddum-Rosse et al., '83) may be needed to enhance motility and fertilization. Sodium lactate may not be necessary in the medium t o achieve in vitro fertilization in the Siberian hamster. It was present in TALP and absent in MT. Sodium lactate and sodium pyruvate added t o a modified Krebs-Ringer bicarbonate medium increased fertilization in vitro and the number of sperm that had lost their acrosomes in the mouse (Miyamoto and Chang, '73a). Pyruvate can be converted to lactate, however, and pyruvate alone was sufficient t o induce the acrosome reaction in guinea pig sperm (Rogersand Yanagimachi, '75). Lactate may be playing a more important role in
FERTILIZATION OF SIBERIAN HAMSTER OVA IN VITRO
the early development of the embryo (Brinster, '65). Glucose, which is commonly added to media, has been found to have an inhibitory effect on inducing acrosome loss in guinea pig sperm (Rogers and Yanagimachi, '75) and also inhibits cleavage in attempts t o culture early embryos from the Syrian hamster (Schini and Bavister, '88). We are currently conducting studies t o investigate whether the presence of lactate and the absence of glucose in MT affects in vitro fertilization of ova and the development of early embryos from Siberian hamsters. Attempts were not made in these studies to alter the pH or the osmolarity of the different media. All the media ranged from pH 7.0 to pH 8.0, most in the range of 7.4-7.8, a range considered optimum for the fertilization of rodent ova (Miyamoto et al., '74). The MT used successfully for fertilizing Siberian hamster ova was found earlier (Bavister, '69) to be adequate at pH 7.6 or 7.8 for the in vitro fertilization of 83% and 78%, respectively, of Syrian hamster ova. The pH (7.8 and 7.6) and the osmolarity (345 and 300 mOsm) of MT and TALP media, respectively, was within the optimum range of pH (6.8-8.2) (Miyamoto et al., '74) and osmolarity (292-392) (Miyamoto and Chang, '73a,b) needed for fertilizing Syrian hamster ova in vitro. These levels also have been sufficient for fertilizing Chinese ova in vitro (Yanagimachi et al., '83; Parkening and Cisneros, '88). Still, the Siberian hamster, as a separate species, is quite different from the Syrian hamster in its response to short photoperiods (Yellon and Goldman, '87) and has a reproductive endocrine profile different from those of other hamsters. These animals are capable of becoming pregnant following a postpartum estrus, with the embryos developing in utero while the mother nurses her litter. If the female does not become pregnant during the postpartum estrus, she undergoes one additional estrous cycle while lactating and may subsequently mate and become pregnant (unpublished data). In the Syrian hamster the entire sperm tail enters the perivitelline space soon after the sperm head has fused with the vitellus of the ovum (Yanagimachi, '81). In contrast, studies in vitro and in vivo have shown that the flagellum of the fertilizing sperm in the Chinese hamster is not quickly incorporated and from later cleavage stages may not be completely incorporated (Yanagimachi et al., '83; Parkening and Cisneros, '88). From the present studies, the incorporation of the sperm tail into the perivitelline space of
311
Siberian hamster oocytes represents an intermediate stage between the other two species. Ninety-one percent of two-cell embryos from Siberian hamsters had evidence of a sperm tail inside the perivitelline space, compared with 45% of two- to four-cell Chinese hamster embryos (Parkening and Cisneros, '88) and 100% of two-cell Syrian hamster embryos (Yanagimachi and Chang, '64). Yanagimachi et al. ('83) speculated that the incorporation of the entire flagellum in fertilizing Chinese hamster ova may be dependent on the timing of gamete fusion. If the sperm fused with the vitellus soon after penetrating the zona pellucida, the motility of the sperm would decrease, and there may be little chance for oocyte rotation and movement of the whole tail through the penetration slit in the zona. However, if fusion did not occur immediately, the motility of the sperm would persist long enough for the entire sperm t o enter the perivitelline space. The length of the sperm does not appear to be an important factor in this process, since Siberian hamster sperm are the shortest of the three species (approximately 135 pm compared with 155 Fm and 250 pm, respectively, for Syrian and Chinese hamster sperm). Ova from three species of hamster have now been fertilized in vitro. From these studies, it is apparent that Tyrode's medium allows for fertilization, provided that minor modifications are made within the medium for the different species. Additional modifications are still necessary, however, t o produce an optimum medium for the fertilization of Siberian hamster ova. It also appears that there are some differences between the species in the process of fertilization based on the incorporation or nonincorporation of the entire tail of the spermatozoon within the perivitelline space. ACKNOWLEDGMENTS I thank Dr. B.D. Bavister, Department of Veterinary Science, University of Wisconsin, for his critical evaluation of the manuscript.
LITERATURE CITED Austin, C.R., and A. Walton (1960) Fertilization. In: Marshall's Physiology of Reproduction. A.S. Parkes, ed. Longmans, London, Vol. 1,pp. 310-416. Bavister, B.D. (1969) Environmental factors important for in vitro fertilization in the hamster. J. Reprod. Fertil., 18.544545. Bavister, B.D., and R. Yanagimachi (1977) The effects of sperm extracts and energy sources on the motility and acrosome reaction of hamster spermatozoa in vitro. Biol. Reprod., 16:228-237.
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Biggers, J.D., W.K. Whitten, and D.G. Whittingham (1971) The culture of mouse embryos in vitro. In: Methods in Mammalian Embryology. J.C. Daniel, ed. W.H. Freeman and Company, San Francisco, pp. 86-116. Brinster, R.L. (1965) Studies on the development of mouse embryos in vitro. IV. Interaction of energy sources. J . Reprod. Fertil., 10:227-240. Brinster, R.L. (1971) In vitro culture of the embryo. In: Pathways t o Conception. Charles C. Thomas, Springfield, 11linois, pp. 245-277. Gaddum-Rosse, P., L.B. Langley, and R.J. Blandau (1983) Studies on the fertilization of rat eggs in vitro. Anat. Rec., 205:59 (abstract). Hirao, Y., and R. Yanagimachi (1978) Temperature dependence of sperm egg fusion and post-fusion events in hamster fertilization. J. Exp. Zool., 205:433-438. Hoffman, K. (1973) The influence of photoperiod and melatonin on testis size, body weight, and pelage colour in the Siberian hamster (Phodopus sungorus). J. Comp. Physiol., 85:267-282. Hoffman, K. (1978) Effect of short photoperiods on puberty, growth and moult in the Siberian hamster (Phodopus sungorus). J . Reprod. Fertil., 54:29-35. Milette, J.J., and F.W. Turek (1986) Circadian and photoperiodic effects of brief light pulses in male Siberian hamsters. Biol. Reprod., 35:327-335. Miyamoto, H., and M.C. Chang (1973a) The importance of serum albumin and metabolic intermediates for capacitation of spermatozoa and fertilization of mouse eggs in vitro. J . Reprod. Fertil., 32:193-205. Miyamoto, H., and M.C. Chang (1973b) Effect of osmolarity on fertilization of mouse and golden hamster eggs in vitro. J. Reprod. Fertil., 33:481-487. Miyamoto, H., Y. Toyoda, and M.C. Chang (1974) Effect of hydrogen-ion concentration on in vitro fertilization of mouse, golden hamster, and rat eggs. Biol. Reprod., 10:487493. Niwa, K., and M.C. Chang (1974) Effects of sperm concentration on the capacitation of rat spermatozoa. J. Exp. Zool., 189:353-356. Parkening, T.A., and P.L. Cisneros (1988) Fertilization of Chinese hamster ova in vivo and in vitro and their subsequent development in culture. Biol. Reprod., 39:409-418. Pickworth, S., and M.C. Chang (1969) Fertilization of Chinese hamster eggs in vitro. J . Reprod. Fertil., 19:371-374. Pickworth, S., G. Yerganian, and M.C. Chang (1968) Fertil-
ization and early development in the Chinese hamster, Cricetulus griceus. Anat. Rec., 162:197-208. Rogers, B.J., and R. Yanagimachi (1975) Retardation of guinea pig sperm acrosome reaction by glucose: The possible importance of pyruvate and lactate metabolism in capacitation and the acrosome reaction. Biol. Reprod., 13.568-575. Roldan, E.R.S., T. Horiuchi, and R. Yanagimachi (1987) Superovulation in immature and mature Chinese hamsters. Gamete Res., 16:281-290. Schini, S.A., and B.D. Bavister (1988) Development of twocell hamster embryos in vitro is blocked by glucose and phosphate. Biol. Reprod., 38: [Suppl. 11 51. Stetson, M.H., E. Sarafidis, and M.D. Rollag (1986) Sensitivity of adult male Siberian hamsters (Phodopus sungorus) to melatonin injections throughout the day: Effects on the reproductive system and the pineal. Biol. Reprod., 35618623. Yanagimachi, R. (1969) In vitro capacitation of hamster spermatozoa by follicular fluid. J. Reprod. Fertil., 18:275-286. Yanagimachi, R. (1981) Mechanisms of fertilization in mammals. In: Fertilization and Embryonic Development In Vitro. J.D. Biggers and L. Mastroianni Jr., eds. Plenum, New York, pp. 81-180. Yanagimachi, R., and M.C. Chang (1963) Fertilization of hamster eggs in vitro. Nature, 200:281-282. Yanagimachi, R., and M.C. Chang (1964) In vitro fertilization of golden hamster ova. J. Exp. Zool., 156:361-376. Yanagimachi, R., Y. Kamiguchi, S. Sugawara, and K. Mikamo (1983) Gametes and fertilization in the Chinese hamster. Gamete Res., 8:97-117. Yanagimachi, R., H. Yanagimachi, and B.J. Rogers (1976) The use of zona-free animal ova as a test-system for the assessment of the fertilizing capacity of human spermatozoa. Biol. Reprod., 15:471-476. Yellon, S.M., and B.D. Goldman (1984) Photoperiod control of reproductive development in the male Siberian hamster (Phodopus sungorus). Endocrinology, 114:664-670. Yellon, S.M., and B.D. Goldman (1987) Influence of short days on diurnal patterns of serum gonadotrophins and prolactin concentrations in the male Siberian hamster (Phodupus sungorus). J . Reprod. Fertil., 80:167-174. Yun, Y.W., F.H. Yu, B.H. Yuen, and Y.S. Moon (1989) Effects of a superovulatory dose of pregnant mare serum gonadotropin on follicular steroid contents and oocyte maturation in rats. Gamete Res., 23:289-298.
In Vitro Fertilization of Siberian Hamster Oocytes T.A. PARKENING Departments of Anatomy and Neurosciences and of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, Texas 77550 ABSTRACT Siberian hamsters were superovulated and various media were tested in an effort to fertilize the recovered oocytes in vitro. The highest percentage of fertilized ova was achieved by using a modified Tyrode's medium, designated MT (Bavister, J. Reprod. Fertil., 18.544-545, '69), previously formulated to fertilize Syrian hamster ova in vitro. Spermatozoa incubated in this medium in a concentrated state overnight (14 hr) and then diluted (1hr) fertilized 39% of the ova. Similar results (40%) were obtained with this medium by adding 20% human follicular fluid to fresh concentrated sperm for 30 min and then diluting the sperm for 2-3 h r prior to the addition of ova. Ova fertilized in vitro cleaved to the two-cell stage but failed to develop any further in culture. Two-cell embryos recovered from mated hamsters and cultured did not undergo additional cleavage. Four-cell embryos collected from mated females and cultured cleaved to the six- to eight-cell stage and stopped. Techniques and media used for fertilizing large numbers of Syrian and Chinese hamster ova in vitro will have to be modified to achieve the same degree of success in the Siberian hamster.
The Siberian hamster (Phodopus sungorus) is a seasonal breeder in the wild. For this reason, this species has received attention as a new laboratory animal because its gonadal development and function can be altered by varying the photoperiod (Hoffmann, '73, '78; Milette and Turek, '86; Stetson et al., '86; Yellon and Goldman, '84, '87). This has enabled researchers t o understand better the relationship of pineal, pituitary, and gonadal function in these rodents. Adult Siberian hamsters maintained under a normal rodent 14:lO (14 hr artificial light) light cycle, however, breed throughout the year, enabling colonies to be readily established. Syrian hamsters were one of the first laboratory rodents whose ova could be successfully fertilized in vitro (Yanagimachi and Chang, '63). Later it was shown that zona-free Syrian hamster ova could be penetrated by heterologous spermatozoa such as human sperm (Yanagimachi et al., '76). This unique feature boosted the research popularity of this species; many infertility laboratories now use the zona-free hamster ovum assay (also referred to as sperm penetration assay) as a test for evaluating the quality of human sperm. Surprisingly few studies have examined the fertilizing ability of ova from other hamster species. To my knowledge, the only other published reports concern the Chinese hamster. Austin and Walton ('60) and Pickworth et al. ('68)described aspects of fertilization in vivo, and Pickworth and Chang ('691, Yanagimachi et al. ('831,and Parkening and 0 1990 WILEY-LISS, INC.
Cisneros ('88) investigated in vitro fertilization. It was our recent success in culturing late two-cell and early four-cell Chinese hamster embryos to the blastocyst stage (Parkening and Cisneros, '88) that prompted the present study to investigate whether ova from Siberian hamsters could be fertilized in vitro and if early embryos would develop in culture.
MATERIALS AND METHODS Animals The Siberian hamsters used in these studies were born and raised at the University of Texas Medical Branch. The colony was established from 16 pairs obtained from Dr. A. Bartke (Southern Illinois University at Carbondale, Carbondale, IL), whose animals were descendants of a colony established by Dr. B. Goldman (Worcester Foundation for Experimental Biology, Shrewsbury, MA). The animals were housed under artificial lighting (14 hr light/lO h r dark) in clear plastic 48 x 27 x 13 cm cages (wood shavings as bedding) in a temperature controlled room (21-23"C). They were provided with laboratory rat chow and water ad libitum, with dried oatmeal provided twice per week as a food supplement. For breeding purposes, only one male and female should be paired per cage; housing multiple males or females with a member of the opposite sex seldom results in Received July 28, 1989; revision accepted December 6, 1989
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cauda epididymidis was excised from a male that had sired at least one litter, and a n incision was made to acquire a droplet of sperm. The sperm were placed in the drop of medium and left for Superovulation either 30 min or 14 hr in the incubator and then Females ranging in age from 1to 8 months (24- diluted in another 200 p1 drop to a concentration 34 g depending on age) were examined for their approximately 5 x 105/ml. The diluted sperm ability to be superovulated. If the animals were were preincubated for 1-3 h r before the addition exhibiting a n estrous cycle, they were given of ova. The oviducts from superovulated females Pregnant Mares' Serum Gonadotropin (PMSG) were then excised, carefully blotted on a piece of during diestrus. Injections were given either in- sterile filter paper, and placed in the paraffin oil traperitoneally or intramuscularly. Various regi- close to the drop of sperm suspension. Under a mens were given: 3 IU PMSG and 5 IU human dissection microscope, two sterile dissecting neechorionic gonadotropin (hCG), 5 IU PMSG and 5 dles were used to rupture the wall of the ampulla, IU hCG, 5 IU PMSG and 8 IU hCG, 5 IU PMSG allowing the ova within their cumulus mass t o and 10 IU hCG, 8 IU PMSG and 10 IU hCG, 10 IU exude into the oil. The cumulus mass was then PMSG and 10 IU hCG, 10 IU PMSG and 12 IU pushed into the droplet of sperm suspension, and hCG, 1IU PMSG and hCGI6 g body weight, and 1 the gametes were incubated for 7, 9, or 10 hr. For each experiment ova were pooled from sevIU PMSG and hCG18 g body weight. The time that elapsed between injections of PMSG and eral females, and the number was divided equally hCG was 48-52 hr. Oocytes were collected be- between three and six different media. Any unusual-appearing ova were discarded. Spertween 15 and 16 h r after the hCG injection. matozoa were likewise pooled from two males Culture media during each study, and the same dilution was Seven media were initially tested for the in used for each drop of medium. There were no exvitro fertilization of oocytes. These consisted of periments performed that did not result in the medium 199, Ham's F10, BWW (Biggers et al., fertilization of at least one ovum from one of sev'71), Brinster's ('71) medium, modified BWW eral media tested. This design helped t o reduce (MBWW) (Parkening and Cisneros, '881, and two variability of results due to possible differences in modified Tyrode's media-MT (Bavister, '69) and the gametes of the animals. TALP (Bavister and Yanagimachi, '77). All Examination of ova media except for medium 199 and Brinster's meThe ova were washed once in a fresh volume of dium were supplemented with 2.5 mg/ml bovine serum albumin (BSA). In some experiments, the medium selected for fertilization and mounted heat-inactivated fetal cord serum (10-20%) or hu- in a small drop on a slide beneath a coverslip conman follicular fluid (20%) was substituted for taining petroleum jelly at its corners. The ova BSA in the BWW, MBWW, MT, and TALP were compressed slightly by the coverslip t o premedia. The pH of the media ranged from 7.0 to vent movement, and 2% glutaraldehyde was irrigated under the coverslip. The slide was then 8.0. The medium of choice became the MT medium placed in a petri dish containing a filter paper (Bavister, '691, which was made by adding 30 mg soaked in 10% formalin and the eggs left oversodium bicarbonate (Sigma S-88751, 1.1 mg night in the refrigerator (4°C).The following sodium pyruvate (Sigma P-22561, 10 mg glucose morning, the eggs were stained by irrigating the (Sigma G-5000), and 25 mg BSA (Sigma A-4378) following solutions, in sequence, under the to 10 ml Tyrode's solution (Difco 5556-72). The pH coverslip: distilled water, 95% ethanol, 1%lacof the medium was 7.8 and the osmolarity 345 moid, and mounting medium (by volume 20% glycerol, 20% glacial acetic acid, 60% distilled wamOsm. ter). The coverslip was then sealed with clear nail In vitro fertilization polish, and the ova were examined for fertilizaA 200 p1 drop of one of the above media was tion with phase-contrast microscopy. placed under paraffin oil (Mallinckrodt, Inc; visIn vitro development cosity 125-135) in a Falcon culture dish (35 mm). Ova incubated with sperm for either 9 or 10 hr The medium was then equilibrated for 1-14 h r in an incubator (37°C) gassed with 5% COZ in air. A and considered to be fertilized from the appearany litters. From records collected on 151 litters in our colony, 762 pups were born, for a mean litter size of 5.1.
FERTILIZATION OF SIBERIAN HAMSTER OVA IN VITRO
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TABLE I . Superovulation of immature and mature Siberian hamsters using different regimens of gonadotropins
Hamsters
Gonadotropin regimen in IUs PMSG hCG
No. of hamsters ovulating (%)
No. of ova (mean 2 s.e.m.1
Immature' Immature Mature2 Mature Immature Mature Mature Mature Mature Immature Immature
3 5 5 5 5 5 5 8 5 10 5 10 8 10 10 10 10 12 1 IUI6 g body wt. 1IUI8 g body wt.
24 of 32 (75) 2 of 5 (40) 27 of 33 (82) 4 of 5 (80) 4 of 4 (100) 4 of 5 (80) 6 of 6 (100) 5 of 7 (71) 5 of 5 (100) 4 of 10 (40) 5 of 5 (100)
243 (7.6 1.1) 14 (2.8 1.7) 228 (6.7 0.9) 32 (6.4 2 2.0) 28 (7.0 3.0) 28 (7.0 5 2.7) 49 (8.2 & 0.5) 30 (5.0 & 1.3) 34 (4.8 1.3) 17 (1.7 5 0.9) 40 (8.0 5 1.7)
* * * * *
'Immature, ranged from 1 to 2 months of age. 'Mature, ranged from 3 to 8 months of age
RESULTS
were 2 months of age, and all weig,,ed over 30 g; hence they were approaching the weight commonly found in mature females. Mature females ovulated the most consistently when injected with 8 IU PMSG and 10 IU hCG (Table 1). The largest number of ova collected was 21 from an immature female (15 g) given 3 IU PMSG and 5 IU hCG. A 3-month-old female ovulated 21 ova and a 5-month-old female yielded 19 ova when given 5 IU PMSG and 5 IU hCG. Slightly more ova were ovulated when waiting 50-52 hr, instead of 48 hr, between injections of PMSG and hCG.
Superovulation The various regimens for superovulating Siberian hamsters are given in Table 1.This species of hamster, unlike the Syrian or the Chinese hamster, responds erratically t o gonadotropins. Females of the same weight and at the same stage of the estrous cycle, given a particular regimen of gonadotropins, may ovulate two or three oocytes or 10-15 oocytes or may not ovulate. Littermates responded with the same random pattern of ovulation. The greatest number of ova was acquired from immature hamsters (1-2 months of age) by injecting 3 IU PMSG and 5 IU hCG or by injecting 1 IU of gonadotropins/8 g body weight (Table 1). The two regimens were equivalent, since the hamsters receiving gonadotropins on a weight basis weighed 24-28 g. When a slightly higher amount of PMSG was administered to this group, it appeared t o have an inhibitory effect. Immature hamsters receiving 5 IU PMSG and 10 IU hCG responded t o a degree, but these animals
In vitro fertilization No fertilization was achieved when using medium 199, Ham's F10, Brinster's, or BWW (Table 2). Some ova became activated in Ham's F10 (70%) and Brinster's (29%). One of 31 ova was fertilized in modified BWW (MBWW) with BSA. The addition of 20% fetal cord serum or 20% human follicular fluid did not enhance the medium, since none of 77 ova were fertilized. The best results were achieved by using a modified Tyrode's medium with BSA or 20% human follicular fluid (Table 2). Twenty-one and twenty-nine percent of ova were fertilized, respectively, in MT and TALP when sperm were incubated for 30 min in a concentrated form and then diluted for 2 hr before adding ova. If the latter regimen was used and 20% human follicular fluid was added, fertilization was enhanced using MT (40%)and decreased using TALP (14%).The addition of 20% fetal cord serum t o MT also appeared t o be beneficial (32%). Spermatozoa incubated overnight (14 hr) with
ance of pronuclei or a fertilizing sperm tail were washed in fresh MT and transferred t o a new 200 ~1 droplet. Two- and four-cell embryos were collected from mated females on the second and third day after finding a copulatory sperm plug. All embryos were cultured in either MT (Bavister, '69) or MBWW (Parkening and Cisneros, '88) containing 4 mg/ml BSA or 10% fetal calf serum for 2 days. The eggs were periodically monitored for cleavage during the 2 day incubation. They were then mounted, fixed, and stained as above.
T.A. PARKENING
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TABLE 2 . I n vitro fertilization Media
of
Siberian hamster ova using various media
No. of experiments
Ova fertilized (%)
3 3 3 3 3 3 3 3 3 2 8 8 3 3
0140 (0) 0142 (0) 0139 (0) 0145 ( 0 ) 0143 (0) 1140 (3) 0138 (0) 0139 ( 0 ) 24182 (29) 6143 (14) 18187 (21) 541137 (39) 17143 (40) 12138 (32)
199 Ham’s F10 Brinster’s BWW BWW + 10% FCS’ MBWW MBWW + 20% HFF2 MBWW + 20% FCS TALP TALP + 20%HFF MT MT (sperm incubated 15 h)3 MT + 20% HFF MT + 20%FCS
Range of % fertilized
-
0-6 -
8-50 0-25 7-64 0-78 36-67 14-36
‘FCS, fetal cord serum. ‘HFF, human follicle fluid. 31n all the other experiments, sperm were incubated in a concentrated form for 30 min, then diluted and incubated for 2-3 hr before adding ova.
MT and diluted for 1 h r prior to adding ova fertilized as many ova in several experiments as MT with 20% human follicular fluid; however, there was greater variability within the experiments (Table 2). Spermatozoa remained motile in every medium during the length of time of the experiment. Spermatozoa in medium 199 did become markedly slower after 6 h r in culture compared with those in other media. However, all spermatozoa were active enough in each medium to readily remove the cumulus cells. In Ham’s F10 and Brinster’s spermatozoa did not make contact with the zona pellucida once the ova were denuded of cumulus cells. In the other media, sperm did make contact with the zona and appeared to attempt to penetrate it. Using MT and TALP, ova that were cultured with spermatozoa for 7 hr were often just being penetrated by sperm. Of the number of ova fertilized at this time period, 21 (42%) of 50 exhibited a swollen sperm head and the early formation of a female pronucleus (Figs. 1,2).In these experiments five of 46 unfertilized ova had one spermatozoon that had penetrated the zona pellucida, but the sperm head was not incorporated into the vitellus of the ovum. Of the ova with a swollen sperm head, 22% had the major portion of the sperm tail outside the zona pellucida (Fig. 11, and 78% had the sperm tail completely within the perivitelline space (Fig. 2). Ova that were fertilized 9 and 10 hr after incu-
bation had male and female pronuclei present (Figs. 3,4). Four of 60 ova (7%)were polyspermic. Two of these were dispermic, and two were trispermic. One of the dispermic ova had one fertilizing sperm with its tail within the perivitelline space and one fertilizing sperm with a major portion of its tail outside the zona pellucida. One of the trispermic ova also had one fertilizing sperm whose tail was largely outside the zona pellucida. Eleven (20%) of the remaining 56 monospermic ova had a fertilizing sperm with part of its tail outside the zona pellucida (Fig. 3).
In vitro culture Some additional ova fertilized in vitro in MT after 9 and 10 hr incubation were examined with a dissecting microscope. On the basis of the appearance of pronuclei and what in some instances appeared to be a fertilizing sperm tail, 26 ova were washed and placed in fresh medium. All of the ova had cleaved to the two-cell stage the following morning but failed after 2 days in culture to develop any further in the modified Tyrode’s medium. After these ova were fixed and stained, only 21 were considered to have been fertilized. Eight Siberian hamsters that mated naturally were killed 2 or 3 days after finding a copulatory plug to obtain early embryos. Four of the females killed between 0900 and 1000 hr on day 3 yielded 24 two-cell ova, and four females killed on day 3 between 1300 and 1400 h r yielded three two-cell and 20 four-cell embryos. One degenerating egg
FERTILIZATION OF SIBERIAN HAMSTER OVA IN VITRO
Fig. 1. A Siberian hamster ovum fertilized after a 7 hr incubation with spermatozoa. The swollen sperm head (S) and female pronucleus (P) are visible in the ooplasm. A major portion of the fertilizing sperm is outside the zona pellucida . and is visible to the right of the sperm head. ~ 5 4 0 The granular or filamentous appearance of the ova in Figures 1-4 results from the large number of paracrystalline inclusions found in their ooplasm. Fig. 2. An ovum incubated as in Figure 1, containing a swollen sperm head (upper periphery of ovum). A portion of the sperm tail visible against the ooplasm was completely enclosed within the perivitelline space. Two cumulus cells (C), slightly out of focus, remain attached to the zona pellucida. The female pronucleus (P) is just beginning to form. x 540.
was found in the 2 day group and four degenerating eggs in the 3-day group. The embryos were cultured in either MBWW or MT containing either BSA (4 mg/ml) or 10% fetal calf serum. None of the two-cell embryos cleaved any further in culture. Half of the four-cell embryos cleaved to
309
Fig. 3. An ovum that had been in culture with spermatozoa for 9 hr. The fertilizing sperm still had a major part of its tail outside the zona pellucida. The male and female pronucleus and one polar body (near the upper periphery of ovum) are visible. x 540. Fig. 4. An ovum incubated for 10 hr with spermatozoa. The fertilizing sperm tail is visible against the vitellus and was incorporated into the perivitelline space. Both pronuclei (P) are visible. The other round profiles against the vitellus are cumulus cells (out of focus) still adhering to the zona pellucida. x 540.
the six- t o eight-cell stage and stopped. When the embryos were mounted, fixed, and stained, all but four (9%) showed evidence of a sperm tail inside the zona pellucida.
DISCUSSION If the Siberian hamster is to be used routinely for gamete research, it is essential that a better protocol for superovulation be developed. The
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method producing the most oocytes still resulted in sporadic ovulations and fewer ova than normally collected from using similar techniques on other laboratory rodents. Perhaps extending the time between PMSG and hCG injections would be beneficial. Increasing the time from 48 t o 50-52 hr between PMSG and hCG injections resulted in a slight increase in the number of oocytes ovulated. Waiting as long as 72 hr between injections proved to be the best regimen for superovulating immature Chinese hamsters (Roldan et al., 1987). Whereas a majority of immature Chinese hamsters respond by ovulating 15-30 ova after a protocol of 3 IU PMSG and 5 IU hCG given 48-50 hr apart (Parkening and Cisneros, 19881, unruptured ovarian follicles were often present. Roldan et a1 (1987) also reported many unruptured follicles in immature Chinese hamsters given 15 IU PMSG and hCG 72 hr apart. Similarly with the Siberian hamster, females that ovulated only two or three ova had several ripe follicles on their ovaries. Recently, some of these ovarian oocytes were recovered and tested for their ability t o fertilize in vitro. Several were penetrated and developed normal male and female pronuclei. Extending the time of collection from 15-16 t o 17-18 hr did not improve the rate of ovulation in the Siberian hamster, although some immature Chinese hamsters apparently do not complete ovulation until 20-21 hr after hCG (Roldan et al., 1987). Amounts of PMSG as low as 20 IU injected into immature rats have a detrimental effect on the maturation of some oocytes, presumably by altering the steroid environment of the follicle (Yun et al., 1989). Although it is possible that PMSG also had an adverse effect on oocytes in the Siberian hamster, rendering them infertile, this is doubtful, because the concentration given never exceeded 5 IU in immature or 10 IU in mature females. Seventy-five percent of ova recovered from superovulated immature Chinese hamsters using 15 IU PMSG and hCG were capable of fertilization in vitro (Roldan et al., 1987). The medium BWW (Biggers et al., '71) has proved an excellent medium for fertilizing rodent ova. It works well for the in vitro fertilization of both Syrian (Hirao and Yanagimachi, '78) and Chinese (Parkening and Cisneros, '88) hamsters. However, no Siberian hamster oocytes were fertilized in BWW. The only media found acceptable for fertilizing Siberian hamster oocytes were the modified Tyrode's media developed for fertilizing Syrian hamster oocytes (Bavister, '69; Bavister and Yanagamachi, '77). MT appeared to be
slightly better than TALP, although considerable variability existed within and between experiments. The variability was difficult to reconcile in that it existed when using the same sperm concentration from the same males. The amount of media remained uniform, and the oocytes were pooled, yet the number of ova fertilized would vary from one in ten to seven in ten with identical media in two identical droplets in the same culture dish. It appears that these media will have to be modified t o achieve a consistently high percentage of fertilized ova using this species. Incubating sperm overnight in a concentrated form using MT was beneficial for fertilization. However, adding 20% heat-inactivated human follicular fluid t o MT for a standard incubation of 2-3 hr achieved the same results. Follicular fluid has components necessary for the capacitation of sperm (Yanagimachi, '69). Speeding the induction of the acrosome reaction and capacitation may be necessary t o improve the rate of fertilization in the Siberian hamster. In one experiment, of the number of sperm adhering to the zona pellucida of ova, only one of 84 sperm had lost its acrosome. The addition of 20% human follicular fluid to TALP did not improve the percentage of oocytes fertilized in vitro. This follicular fluid, however, was not the same sample as that used with MT. Determining whether this caused the lower percentage of fertilized ova or was a reflection of the inherent variability in the experiments will require additional studies. It may be necessary to preincubate Siberian hamster spermatozoa for 5-6 hr, as is done for the fertilization of rat oocytes (Niwa and Chang, '74). Preliminary studies indicate that diluted suspensions of sperm, in the absence of ova, lose some of their motility after 6 hr in a manner similar t o that of diluted concentrations of rat sperm. Under these circumstances the addition of 2 mM caffeine (Gaddum-Rosse et al., '83) may be needed to enhance motility and fertilization. Sodium lactate may not be necessary in the medium t o achieve in vitro fertilization in the Siberian hamster. It was present in TALP and absent in MT. Sodium lactate and sodium pyruvate added t o a modified Krebs-Ringer bicarbonate medium increased fertilization in vitro and the number of sperm that had lost their acrosomes in the mouse (Miyamoto and Chang, '73a). Pyruvate can be converted to lactate, however, and pyruvate alone was sufficient t o induce the acrosome reaction in guinea pig sperm (Rogersand Yanagimachi, '75). Lactate may be playing a more important role in
FERTILIZATION OF SIBERIAN HAMSTER OVA IN VITRO
the early development of the embryo (Brinster, '65). Glucose, which is commonly added to media, has been found to have an inhibitory effect on inducing acrosome loss in guinea pig sperm (Rogers and Yanagimachi, '75) and also inhibits cleavage in attempts t o culture early embryos from the Syrian hamster (Schini and Bavister, '88). We are currently conducting studies t o investigate whether the presence of lactate and the absence of glucose in MT affects in vitro fertilization of ova and the development of early embryos from Siberian hamsters. Attempts were not made in these studies to alter the pH or the osmolarity of the different media. All the media ranged from pH 7.0 to pH 8.0, most in the range of 7.4-7.8, a range considered optimum for the fertilization of rodent ova (Miyamoto et al., '74). The MT used successfully for fertilizing Siberian hamster ova was found earlier (Bavister, '69) to be adequate at pH 7.6 or 7.8 for the in vitro fertilization of 83% and 78%, respectively, of Syrian hamster ova. The pH (7.8 and 7.6) and the osmolarity (345 and 300 mOsm) of MT and TALP media, respectively, was within the optimum range of pH (6.8-8.2) (Miyamoto et al., '74) and osmolarity (292-392) (Miyamoto and Chang, '73a,b) needed for fertilizing Syrian hamster ova in vitro. These levels also have been sufficient for fertilizing Chinese ova in vitro (Yanagimachi et al., '83; Parkening and Cisneros, '88). Still, the Siberian hamster, as a separate species, is quite different from the Syrian hamster in its response to short photoperiods (Yellon and Goldman, '87) and has a reproductive endocrine profile different from those of other hamsters. These animals are capable of becoming pregnant following a postpartum estrus, with the embryos developing in utero while the mother nurses her litter. If the female does not become pregnant during the postpartum estrus, she undergoes one additional estrous cycle while lactating and may subsequently mate and become pregnant (unpublished data). In the Syrian hamster the entire sperm tail enters the perivitelline space soon after the sperm head has fused with the vitellus of the ovum (Yanagimachi, '81). In contrast, studies in vitro and in vivo have shown that the flagellum of the fertilizing sperm in the Chinese hamster is not quickly incorporated and from later cleavage stages may not be completely incorporated (Yanagimachi et al., '83; Parkening and Cisneros, '88). From the present studies, the incorporation of the sperm tail into the perivitelline space of
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Siberian hamster oocytes represents an intermediate stage between the other two species. Ninety-one percent of two-cell embryos from Siberian hamsters had evidence of a sperm tail inside the perivitelline space, compared with 45% of two- to four-cell Chinese hamster embryos (Parkening and Cisneros, '88) and 100% of two-cell Syrian hamster embryos (Yanagimachi and Chang, '64). Yanagimachi et al. ('83) speculated that the incorporation of the entire flagellum in fertilizing Chinese hamster ova may be dependent on the timing of gamete fusion. If the sperm fused with the vitellus soon after penetrating the zona pellucida, the motility of the sperm would decrease, and there may be little chance for oocyte rotation and movement of the whole tail through the penetration slit in the zona. However, if fusion did not occur immediately, the motility of the sperm would persist long enough for the entire sperm t o enter the perivitelline space. The length of the sperm does not appear to be an important factor in this process, since Siberian hamster sperm are the shortest of the three species (approximately 135 pm compared with 155 Fm and 250 pm, respectively, for Syrian and Chinese hamster sperm). Ova from three species of hamster have now been fertilized in vitro. From these studies, it is apparent that Tyrode's medium allows for fertilization, provided that minor modifications are made within the medium for the different species. Additional modifications are still necessary, however, t o produce an optimum medium for the fertilization of Siberian hamster ova. It also appears that there are some differences between the species in the process of fertilization based on the incorporation or nonincorporation of the entire tail of the spermatozoon within the perivitelline space. ACKNOWLEDGMENTS I thank Dr. B.D. Bavister, Department of Veterinary Science, University of Wisconsin, for his critical evaluation of the manuscript.
LITERATURE CITED Austin, C.R., and A. Walton (1960) Fertilization. In: Marshall's Physiology of Reproduction. A.S. Parkes, ed. Longmans, London, Vol. 1,pp. 310-416. Bavister, B.D. (1969) Environmental factors important for in vitro fertilization in the hamster. J. Reprod. Fertil., 18.544545. Bavister, B.D., and R. Yanagimachi (1977) The effects of sperm extracts and energy sources on the motility and acrosome reaction of hamster spermatozoa in vitro. Biol. Reprod., 16:228-237.
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