Journal of in Vitro Fertilization and Embryo Transfer, Vol. 7, No. 3, 1990
Effect of Removal of Cumulus Cells from One-Cell Mouse Embryos on in Vitro Development MAHENDRA DE SILVA, 1'2 KAREN STRACHER, 1 SUZANNE SAUER, 1 PETER M. HORVATH, 1 and WILLIAM J. BUTLER 1
Submitted: October 21, 1989 Accepted: February 6, 1990
KEY WORDS: cumulus; streptomycin; embryo; in vitro development.
The effects o f the removal o f cumulus cells from fertilized mouse oocytes (one-cell embryos) and the presence o f streptomycin in culture medium on in vitro development were studied. H a m ' s F-IO medium with (0.075 g/liter) or without streptomycin was supplemented with human serum (15%). Cumulus-intact embryos were harvested from oviducts after mice were superovulated with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Hyaluronidase (300 IU/ml) was used to remove the cumuli. Embryos were cultured (i) with cumulus~without streptomycin (n = 238), (ii) with cumulus~with streptomycin (n = 185), (iii) without cumulus~with streptomycin (n = 210), and (iv) without cumulus~without streptomycin (n = 218). Embryonic development was assessed 24, 96, and 120 hr after initiation o f culture. Percentage two cells and percentage small or expanded blastocysts were not different (P > 0.05) among experimental groups. Percentages (X +- SE) hatched blastocysts f o r the four groups were (i) 36 +- 8 and 54 +7, (ii) 35 +- 8 and 55 +- 6, (iii) 19 +. 5 and 42 +- 6, (iv) 23 +5 and 47 +- 5 at 96 and 120 hr, respectively. Percentages all (small, expanded, and hatched combined) blastocysts were (i) 74 +- 5 and 74 + 5, (ii) 74 +- 9 and 72 +- 5, (iii) 56 +- 6 and 63 +--5, and (iv) 61 +- 5 and 63 +- 5 at 96 and 120 hr, respectively. A greater (P < 0.05) percentage o f embryos developed to blastocysts and hatched by 96 and 120 hr, when they were cultured with the cumulus intact. There was no effect o f streptomycin on embryonic development. Cumulus cells may act as "nurse" cells during a critical stage o f the development o f the embryo.
INTRODUCTION Mammalian oocytes are connected to cumulus cells by gap junctions (1-3). These junctions facilitate communication and serve as channels for providing nutrients between the cumulus and the oocyte (4). When cumulus cells are removed from mouse oocytes, they fail to grow in culture. When oocytes are grown in coculture with the cumulus cells, there is also no increase in growth (5). When mouse oocytes are freed of attached cumulus cells and grown on fibroblast monolayers, growth is arrested, but isolated oocytes survive in vitro for several days (6). These studies clearly show that cumulus cells are important for the growth of the oocyte and that they must be in contact with the oocyte by means of membrane specializations. The frequency of in vitro fertilization is lower in mouse oocytes that are matured without the cumulus. However, the number of fertilized oocytes that develop to live offspring is similar, whether the cumulus is removed or not (7). The beneficial effects of cumulus cells on oocyte development have thus far been examined only with oocytes collected from ovaries following treatment with pregnant mare's serum gonadotropin (PMSG) but prior to administration of human chorionic gonadotropin (hCG). Such oocytes are in meiotic arrest, with their cumuli tightly bound to the oocyte. Following an endogenous surge of gonadotropins or following administration of hCG, oocytes resume meiosis, and the cumulus undergoes expansion (8). Just prior to ovulation, gap junctions are markedly
1 Division of Reproductive Endocrinology and Genetics, Department of Obstetrics and Gynecology,AlbanyMedicalCoUege,
Albany, New York 12208. 2 To whom correspondence shouldbe addressed at Divisionof Reproductive Endocrinologyand Genetics: A-75, Department of Obstetrics and Gynecology,Albany Medical College, 47 New ScotlandAvenue, Albany,New York 12208. 129
0740-7769/90/0600-0129506.00/0 9 1990PlenumPublishingCorporation
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DE SILVA, STRACHER, SAUER, HORVATH, AND BUTLER
reduced and the oocyte no longer communicates with the cumulus cells (3). Therefore, it is conceivable that oocytes collected after the endogenous surge of gonadotropins or administration of hCG would not depend on the cumulus cells to obtain nutrients for growth and development. Experiments have not been conducted previously to verify this. Therefore, one objective of the present investigation was to study the effects of removal of cumulus cells from fertilized oocytes (one-cell embryos) obtained from mice superovulated with PMSG and hCG on in vitro development. The second objective of the investigation was to study development in vitro of one-cell mouse embryos cultured with or without their cumuli in Ham's F-10 medium in the presence or absence of streptomycin. Streptomycin has been shown to inhibit protein synthesis in many other cell systems (9). Although streptomycin (0.075 g/liter) is routinely used when preparing Ham's F-10 medium, its effects on embryonic development at this specific concentration had not been studied previously.
MATERIALS AND METHODS Animals Virus antibody-free B6C3F 1 female mice, 28 days old, and breeding-age male mice, 8-10 weeks old, were obtained from Taconic farms (Germantown, NY). They were housed in a pathogen-free environment and allowed free access to feed and water. Lighting was provided for 12 hr daily.
Collection of One-Cell Embryos Female mice were injected with 7 IU PMSG (Sigma Chemical, St. Louis, MO) intraperitoneally (ip) at 1400 on a random day of their estrous cycle. Forty eight hours later, they were injected with 7 IU hCG (Sigma Chemical, St. Louis, MO) ip, (10) and each female was immediately paired with a male mouse. About 18-20 hr after hCG, mice were checked for vaginal plugs and killed by cervical dislocation. Oviducts were collected using sterile techniques and the swollen ampullae were gently incised with a fine pair of forceps and a 26-G hypodermic needle to allow escape of the one-cell embryos. The embryos were always in clumps surrounded by a cumulus mass.
Preparation of Culture Media Ham's F-10 nutrient mixture (Gibco, Grand Island, NY) in powder form was dissolved in 18-MfL Milli-Q water (Millipore, Bedford, MA) and was supplemented with Ca lactate (0.2452 g; Baker Chemical, Phillipsburg, NJ), sodium bicarbonate (2.1 g; Mallinckrodt, Paris, KY), and penicillin G (benzylpenicillin; 0.075 g; Sigma Chemical, St. Louis, MO). After the contents were brought up to a liter, osmolarity was checked and adjusted to 280 mOsm/Kg. Ham's F-10 solution was then divided into two equal halves. To one half, streptomycin sulfate (0.037 g; Sigma Chemical, St. Louis, MO) was added and thoroughly mixed. Both halves (with and without streptomycin) were filter sterilized using a 0.2-~m filter, prior to use in embryo cultures. To make growth media for embryo cultures, heatinactivated human serum was mixed at a 15% concentration with Ham's F-10 medium, with and without the streptomycin. Growth media as well as a small quantity of the plain Ham's F-10 medium were preequilibrated for 18-20 hr at 37~ under a humidified atmosphere of 5% CO2 and 95% air. Plain Ham's F-10 medium was used for the moats of culture dishes and for monitoring pH. Embryos were cultured in 1 ml preequilibrated growth medium in organ culture dishes (Becton Dickinson, Lincoln Park, N J).
Experimental Protocol The experiment was a 2 • 2 factorial design. Twelve replicates of the experiment were conducted. A fresh batch of Ham's F-10 medium with and without streptomycin was made for each replicate. One-cell embryos from three or four mice were pooled for each replicate. From this pool, clumps of embryos surrounded by their cumulus masses were randomly allocated to growth media, with and without streptomycin. The remaining embryos in the pool were treated with I0 drops of freshly prepared hyaluronidase (300 IU/ml; type II from sheep testes; Sigma Chemical, St. Louis, MO) (10). Hyaluronidase was able to easily strip off the cumuli within 1 or 2 rain. The naked embryos were then washed twice by transferring to fresh growth media without streptomycin before they were placed in dishes containing growth medium with and without streptomycin. At least 10 embryos (i.e., 10-36) were assigned to each treatment corn-
Journal of in Vitro Fertil&ation and Embryo Transfer, Vol. 7, No. 3, 1990
INFLUENCE OF THE CUMULUS ON EMBRYONIC DEVELOPMENT
bination in a single replicate. Embryos were observed 24, 96, and 120 hr after initiation of cultures to assess development to two cells and small, expanded, or hatched blastocysts, respectively. To determine if hyaluronidase had direct effects on embryonic development, a group of one-cell embryos surrounded by their cumulus masses was cultured for 24 hr. When they became two cells (n = 44) and were completely devoid of the surrounding cells, they were exposed to the same concentration of hyaluronidase as one cells. After 1 to 2 min of exposure to hyaluronidase, they were washed twice and transferred to growth medium. Another group of two-cell embryos served as controls (n = 45). Embryonic development was assessed 72 and 96 hr after initiation of culture.
Statistical Analyses Percentage development of one-cell embryos to two cells was calculated. Percentage development to small, expanded, hatched, and all (small, expanded, and hatched combined) blastocysts by 96 and 120 hr after culture was calculated based on the number of two cells. Data were first examined for homogeneity of variance. Since there was no evidence for lack of homogeneity, angular transformations of percentages were not performed prior to statistical analyses. Differences in percentage development to two cells or blastocysts were evaluated using an analysis of variance for a completely randomized block design. In this analysis, replicates were considered blocks, embryos with and embryos without the cumulus, and antibiotics (absence or presence of streptomycin) were considered the main effects. The interactions of main effects were also included in the statistical model (11).
131
Table I. Development of One-Cell Mouse Embryos with or without Cumuli, to Two Cells, When Cultured for 24 hr, in the Presence ( + ) or Absence ( - ) of Streptomycin Cumulus
Streptomycin
% Two-cell embryos (X _+ SE) a
+ +
- (n = 238) b + (n = 185) + ( n = 210) - ( n = 218)
90 --- 3 85- 3 84-4 82---3
-
a No significant difference among means. b n = embryos tested in 12 replicates.
RESULTS Percentage development of one-cell embryos to two cells is shown in Table I. Percentage development to blastocysts by 96 and 120 hr is shown in Tables II and III, respectively. There were no differences (P > 0.05) among the four treatment groups in percentage development to two cells. Similarly, differences were not observed in the development to either small or expanded blastocysts. However, percentages hatched and A L L blastocysts at 96 and 120 hr were greater (P < 0.05) when one-cell embryos were cultured with the cumulus intact. There was no effect of streptomycin or cumulus • streptomycin interaction on development to either two cells or blastocysts. Development of two-cell embryos exposed to hyaluronidase was similar to those not exposed to hyaluronidase (data not shown).
DISCUSSION The beneficial effects of cumulus cells on oocyte development have been previously documented (3). As mentioned earlier, these studies were conducted
Table II. Development of One-Cell Mouse Embryos with or without Cumuli, to Blastocysts, When Cultured for 96 hr in the Presence ( + ) or Absence ( - ) o f Streptomycin % blastocysts (X" -+ SE) Cumulus
Streptomycin
Small
Expanded
Hatched
All a
+ +
18-+2 14-+4 15--3
20+-4 25-+6 22+5
36-+8" 35+8"
74-+5' 74-+9"
-
- ( n = 238) b + (n = 185) + ( n = 210)
19+5
56+6
-
-
16--2
22-+4
23-+5
61-+5
(n
=
218)
All = small, expanded, and hatched stages combined. b n = embryos tested in 12 replicates. * Means for hatched and all blastocysts greater (P < 0.05) for embryos cultured with the cumulus than for embryos cultured without the cumulus. No effects (P > 0.05) of streptomycin. a
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DE SILVA, STRACHER, SAUER, HORVATH, AND BUTLER
Table HI. Development of One-Cell Mouse Embryos with or without Cumuli, to Blastocysts, When Cultured for 120 hr, in the Presence ( + ) or Absence ( - ) of Streptomycin % blastocysts (X" --- SE) Cumulus
Streptomycin
Small
Expanded
Hatched
All~
+ +
9--3 8 --- 3 13---4
11-4 9 --- 3
-
- (n = 238)b + (n = 185) + ( n = 210)
8•
54• 55 --- 6* 42---6
74• 72 +-- 5* 63-+5
-
-
10•
6---3
47---5
63-+5
(n =
218)
= small, expanded, and hatched blastocysts combined. b n = embryos tested in 12 replicates. * Means for hatched and all blastocysts greater (P < 0.05) for embryos cultured with the cumulus than for embryos cultured without the cumulus. No effects (P > 0.05) of streptomycin.
a All
with oocytes obtained from ovaries following PMSG treatment but prior to administration of hCG. The cumuli in these oocytes are tight (i.e., gap junctions are intact), enabling transfer of nutrients. In the present study, one-cell embryos were obtained after the mice were treated with PMSG and hCG. These one-cell embryos were surrounded by expanded cumuli. Previous studies indicated that following hCG administration the physical integrity of the cumulus and the oocyte is disrupted. It is therefore reasonable to assume that the oocyte would not be able to obtain nutrients from the cumulus and would not depend on the cumulus for growth and development. Certainly, the one-cell embryos in the present study were able to develop without the cumulus, but the presence of the cumulus further enhanced their development to more advanced stages. Therefore, our findings have shown that the cumulus, even when completely expanded, continues to nurture the embryo. Pyruvate and lactate are considered important sources of energy for the development of the early cleavage stages of the mouse embryo (12) and rat cumulus cells were shown to produce lactate (13). Since the oocyte is embedded in the cumulus matrix for a prolonged period after ovulation, it is possible that the oocyte may be utilizing cumulus substrates as sources of energy. Further studies are needed to understand how the cumulus promotes the development of the young emb r y o in spite of the fact that an important communication pathway via gap junctions is lost. Our findings are useful when one considers human in vitro fertilization. Here, oocytes are obtained from preovulatory follicles after administration of follicle stimulating hormone alone or in combination with luteinizing hormone followed by hCG. A majority of these oocytes have completed meiotic
maturation (metaphase II) and have well expanded cumuli. Following insemination, these oocytes are stripped of their cumuli to check for fertilization. Although, they are capable of successful development (14), based on the present evidence with the mouse embryo, their chances of development may be significantly diminished when the cumuli are removed. The earliest detectable effects of streptomycin are thought to be on membrane functions and potassium efflux from the cell. More importantly, streptomycin inhibits polypeptide synthesis (9). In the present study, we were interested in finding out whether the concentration of streptomycin that is routinely used for preparing Ham's F-10 medium would lessen the chances of development of the embryo. It appears from our study that streptomycin can safely be used at 0.075 g/liter as an antibiotic supplement, when preparing Ham's F-10 medium for mammalian embryo cultures.
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INFLUENCE OF THE CUMULUS ON EMBRYONIC DEVELOPMENT
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