MOLECULAR REPRODUCTION AND DEVELOPMENT 27:168-172 (1990)
Chromosome Constitution of Highly Motile Mouse Sperm A. ESTOP,’ V. CATALA,’;” AND J. SANTAL02 ‘Department of Medical Genetics, Western Pennsylvania Hospital, Pittsburgh, Pennsylvania, ‘Department of Cell Biology, Autonomous University of Barcelona, Barcelona, Spain In this study, we address the ABSTRACT relationship between motility and genetic content of mouse sperm. The chromosome complements of highly motile mouse sperm, selected using the swim-up technique, were analyzed after in vitro fertilization, at the first cleavage state. They were compared to those of unselected sperm. Identification of male and female chromosome sets was possible because of their differential condensation at the first mitotic division. In vitro fertilization, swim-up separation, chromosome preparation, and staining were carried out using standard techniques. The results indicate that highly motile mouse sperm did not differ in types and frequencies of chromosomal abnormalities from those not selected for motility. Moreover, separation of motile sperm does not deviate the sex ratio from the theoretical 1 : 1.
Key Words: Motility, Genetics, Sex chromosome ratio
INTRODUCTION In the female mammal, millions of sperm are ejaculated and deposited into the female genital tract to ensure the fertilization of a few ova. However, very few of them will reach the fertilization site, and those that will are highly motile. The motility characteristics of a semen sample are considered determinant in judging its quality. The ability of the human cervix to exclude many of the morphologically abnormal spermatozoa present in the semen of fertile men has long been recognized (Bergman, 1955). The selection could be due to the quality of the motility of the spermatozoa or to the existence of a physical filter (Moghissi, 1977). Mortimer et al. (1982) revealed that spermatozoa with a n abnormal head form have a higher incidence of other defects such a s midpiece, tail, and other defects that might impair their motility, suggesting that the apparent selection of morphologically normal spermatozoa is a consequence of their higher motility. Makler (1980) studied the distribution of normal and abnormal forms among motile and nonmotile sperm. According t o this study, the percentage of abnormal forms was much lower in motile sperm (22%)compared with 42% among
0 1990 WILEY-LISS, INC.
unselected sperm. On the other hand, when Martin and Rademaker (1988) studied the sperm chromosomes of human males with different frequencies of morphologically abnormal sperm forms, they found no correlation between abnormal morphology and chromosome abnormalities. An important question is whether highly motile sperm are likely to be genetically superior to less motile sperm. There is evidence that, in in vivo conditions, in the mouse, genetically imbalanced sperm take part in the fertilization process, producing abnormal zygotes subject to postzygotic loss (Redi e t al., 1984). Therefore, genetically abnormal sperm may retain motility at levels sufficient to affect fertilization. However, morphologically grossly abnormal sperm are preferentially lost before they reach the fallopian tube (Krazanowska, 1974; Redi et al., 1984). In humans, separation of highly motile sperm is being used in assisted reproductive techniques such as in vitro fertilization and artificial insemination to enhance the efficiency of the system. Although the in vitro fertilization rate of human mature ova with unselected sperm is high (80-90%), the implantation rate is rather low. This could be due to fertilization by defective sperm due to the higher chances for less than optimal sperm to affect fertilization in vitro compared with the in vivo situation. In fact, i t has been shown that diploid mouse spermatozoa have higher chances to fertilize under in vitro conditions than in vivo (Santalo e t al., 1986). In this study we address the relationship between motility and genetic content in mouse sperm. The chromosome complements of highly motile sperm, selected using the swim-up technique, were analyzed after in vitro fertilization, a t the first cleavage stage. They were compared with those of unselected sperm. Identification of male and female chromosome sets was possible because of their differential condensation a t the
Received January 18, 1990; accepted March 26, 1990. Address reprint requests to A. Estop, Department of Medical Genetics, Western Pennsylvania Hospital, Pittsburgh, PA 15224.
CHROMOSOME CONSTITUTION OF HIGHLY MOTILE MOUSE SPERM first mitotic division. This difference in chromosome condensation between male- and female-derived chromosomes is a direct consequence of the asynchrony in the development of the male and female pronuclei (Fraser and Maudlin, 1979).
MATERIALS AND METHODS Animals CBA x C57 Bli6J hybrid mice were used as gamete donors. Females were 24-42 days old and males 2-3 months old. Females were injected intraperitoneally with 5 IU of pregnant mare serum gonadotropin (PMSG, Sigma), followed by 5 IU of human chorionic gonadotropin, HCG, Sigma) 48 h later. All animals were killed by cervical dislocation immediately before removal of their gametes.
169
Chromosome Preparations Chromosome preparations were made following Tarkowski’s method (Tarkowski, 1966). Slides were routinely stained with Leishman’s stain. If some overlapping of chromosomes was present, the chromosome spreads were C-banded (Sumner, 1972). Heterochromatin staining helped in avoiding ambiguity in chromosome number. The sex ratio was calculated according to the presence of the Y chromosome. Statistical Analysis discontinuity correction was
x2 test with the Yates used.
RESULTS (TABLE 1) In the control group, the total number of recovered eggs was 599. The number of fertilized eggs was 480 (80.13%), and 119 were unfertilized. Four hundred Culture Media twenty-seven allowed cytogenetic analysis; however, The medium used for handling and culturing the 53 could not be studied due to overlapping or scattering eggs was M16 Wittingham’s medium (Whittingham, of the chromosomes. Spreads with fewer than 38 chro1971) containing 4 mg bovine serum albumin ([BSAI, mosomes were not included in the study. If only one crystallized and lyophilized, SigmaPml. For capacita- genome was analyzable, the spread was also discarded. tion and in vitro fertilization we used Tyrode’s (T6) Male and female genomes were analyzed separately medium containing 15 mg BSAiml (Fraction V; when possible. In cases when both genomes were Sigma). For collection of motile sperm in swim-up ex- mixed, the source of a chromosome anomaly could not periments, we used HT6 also supplemented with BSA be identified. In such case, it was recorded a s of “un(Whittingham, 1971). Media were overlaid with paraf- known origin.” In the experimental group we recovered fin oil and equilibrated overnight in a 5% CO, incuba- 536 eggs, of which 437 were fertilized (81.52%) and 99 tor before use. unfertilized. Three hundred ninety-seven were of sufficient quality for cytogenetic analysis. Separation of Motile Sperm Aneuploidy Sperm of two cauda epididymis from the same male A conservative estimate of aneuploidy is twice the donor were used simultaneously in each experiment. One epididymis was used for selection of motile sperm frequency of hyperhaploidy, the reason being that the and the other a s a control. The sperm suspensions were number of hypohaploid complements exceeds that of prepared in T6 without BSA. After 20 min of disper- hyperhaploid, probably because of a technical artifact sion, the cells were centrifuged at 600g for 6 min, then caused by the spreading technique. The total frequency they were allowed to swim up in 0.75 ml of HT6 with of hyperhaploidy was 3.5% in the embryos produced BSA during 30 min at 37°C. Motile sperm were col- with unselected sperm and 4.3% in those where the lected and aliquoted a t a concentration of 1,000,000- highly motile sperm was used. If we look a t the male 1,500,000 spermiml in 0.4 ml drops of the same media and female genomes separately, the frequency of male hyperhaploidy is 1.2% in the control group and 1.3% in for capacitation and fertilization. the experimental group. The rate of hyperhaploidy in the female sets was 1.2% in the control group and 2.5% In Vitro Fertilization in the experimental group. In the control and experimental groups, the sperm Polyploidy suspensions were preincubated for 50 min to achieve Of the 427 analyzed zygotes in the control group, 73 capacitation. The concentration of whole sperm used was the same as in the experimental group. For each showed triploid complements (17%) and in the experimale, two females were sacrificed and their oviducts mental group, of 397 analyzed, 56 were triploid placed directly into the capacitated sperm suspension, (14.1%). The most important contribution to triploidy where the oocytes were released. After a n incubation of was fertilization by two sperm or dispermy (7.5% and 5 h, the eggs were removed, washed once in fresh me- 7.3%, respectively, for controls and experimentals),foldium, and kept overnight (18-20 h) in a 0.4 ml drop of lowed by fertilization of a diploid oocyte by a haploid culture medium containing M vinblastine sulfate sperm (6.1% in controls and 5% in experimentals). Immature mouse females ovulate more diploid oocytes (Sigma).
170
A. ESTOP ET AL. TABLE 1. Results of the Chromosome Analysis of First Cleavage Mouse Embryos Produced In Vitro by Sperm Selected by the Swim-Up Technique and by Unselected Sperm Fertilized An a1y zed Unfertilized Percent fertilized Unfertilized MI1 Partheno Hyperhaploids Female Male Unknown Polyploids Female 2n Dispermy Male 2n Unknown Structural rearrangements Male Unknown C-bands Male Female
Selected 437 397 99
(?A)
(81.52) 99 82 17 17
loa
5b 2 56 20 29
(4.3) (2.5) (1.3) (0.5) (14.1) (5.0) (7.3)
7 (1.8) 5 (1.3) 2 Frag 1 Ring 1 Frag 1 Triradial 150 78 (52.0) 72 (48.0)
Unselected 480 427 119
(%)
(80.13) 119 105 14 15 (3.5) 5 (1.2) 5 (1.2) 6‘ (1.4) 73 (17.0) 26d (6.1) 32d (7.5) 3 (0.7) 12 (2.8) 3 (0.7) 1 Frag 1 Pulverized 1 Ring 178 83 95
(46.6) (53.4)
“Includes one hyperhaploidy, which is also included in dispermy. b T ~ embryos o with 22 chromosomes. “One embryo with 42 chromosomes. tetraploid: 2 d n + P 2n.
than younger adults or older females (Catala et al., 1988; Estop, 1989). Also strain differences have been described in the production of diploid oocytes (Hansmann and El-Nahass, 1979). Included in the triploids are haploid oocytes fertilized by diploid sperm. Three of those (0.7%) were found in the control group. The absence of fertilizing diploid sperm in the experimental group may suggest that diploid sperm tend to have lower motility. There was one tetraploid zygote. The source of tetraploidy could be established as being a diploid oocyte fertilized by two sperm.
Structural Rearrangements Only three male genomes were identified a s carriers of a structural rearrangement in the control group. One complement had a n acentric fragment. Another male genome had multiple breaks resembling “pulverized” chromatin. There was a ring chromosome of unknown origin in one zygote. The total frequency of structural rearrangements in this group was 0.7%. In the experimental group the frequency was 1.3%, including two fragments and one ring chromosome in three different male complements. One fragment and a nonhomologous exchange figure both of unknown origin were also present in two different zygotes.
Sex Ratio One hundred seventy-eight zygotes in the controls and 150 in the experimentals were analyzed with Cbanding. The presence of a Y chromosome was necessary to identify a male genome in a n embryo. If only one complement was analyzable, the cell was excluded from the study. In experiments using the whole unselected sample, the percentage of Y-bearing sperm was 46.6% and t h a t of X-bearing sperm was 53.4%. When highly motile sperm was used for in vitro fertilization, these frequencies varied slightly to 52% of Y-bearing sperm vs. 48% of X-bearing sperm, increasing the number of male embryos (Fig. 1). This increase is not statistically significant. DISCUSSION The percentages of chromosome abnormalities in the one-cell zygotes obtained by highly motile sperm did not differ from those obtained with unselected sperm. Although structural rearrangements and hyperhaploidy are slightly increased in embryos where selected sperm was used, those differences are not statistically significant. Male hyperhaploidy remains constant in highly motile and in unselected sperm. Polyploidy was high in both controls and experimentals, dispermy be-
CHROMOSOME CONSTITUTION OF HIGHLY MOTILE MOUSE SPERM 60
53.4%
52.0% 46.6O/o
171
y////////1
c
c
a,
P a, a 20 10
0 Male Unselected
Female Selected
Fig. 1. Percentage of male and female first cleavage mouse embryos produced by unselected sperm and by sperm selected by the swim-up technique.
ing the most common cause for a triploid zygote. Both dispermy and diploid oocytes may be considered fertilization anomalies. Polyspermy may be higher in in vitro fertilization because of the alteration of the spermiegg ratio. The increase in polyspermy and in the number of diploid oocytes may be a consequence of the degree of maturation of the oocyte at the time of retrieval. Triploidy has been reported to occur more frequently in immature human oocytes cultured in vitro prior to fertilization (Van der Ven et al., 1985), in human oocytes aged in vitro (Plachot et al., 1988), and in immature and overmatured mouse oocytes (Badenas et al., 1989). It has been suggested that procedures to separate motile sperm may have clinical application in human in vitro fertilization and intrauterine insemination by increasing the probability of fertilization by a normal sperm and consequently the chances of normal embryonic development (Berger et al., 1985). Various procedures have been described to separate motile sperm (Berger et al., 1985; Wiklaud e t al., 1987). Sperm selected in Percoll gradients, and found a t the level of 100% Percoll, penetrate hamster eggs at higher frequencies than unselected sperm (Forster et al., 1983). Wiklaud e t al. (198'7) found that sperm collected by a self-migration method in a medium containing hyaluronic acid achieved more pregnancies than sperm separated by other methods. The fact that sperm displaying high motility does not differ from unselected sperm as far a s chromosome abnormality is concerned cannot be completely established. Fractions of sperm separated by swim-up are enriched with very motile sperm and are clean of dead cells and debris, whereas whole sperm contains different populations of sperm, i.e., mo-
tile, less motile, nonmotile plus dead cells. In a n in vitro situation, it is not clear which sperm will fertilize. Capacitation and acrosome reaction are strict requirements; however, the quality of the motility of the sperm might not be a s determinant for in vitro fertilization a s i t is in a n in vivo situation. Another question is the possible relationship between separation of motile sperm and its consequences in altering the sex ratio. Both in the mouse and in humans, Y-bearing sperm are thought to be more motile because they carry slightly less DNA than Xbearing sperm. Based on this assumption, different systems and techniques have been described and applied to sex prediction in humans. We have shown that swim-up and centrifugation a s a means for separating highly motile sperm do not affect the sex ratio in mouse in vitro fertilized embryos. There is a slight shift towards male embryos in those fertilized by highly motile sperm; however, these differences are not significant, and the sex ratio is maintained in both populations of embryos. Brandriff et al. (1986) studied the sperm chromosomes of two human males where sperm was selected for motility using the swim-up system after hamsteregg penetration and consequent formation of pronuclear chromosomes. In their system, chromosome anomalies, both numerical and structural, were similar in motile and whole semen; however, the X- and the Y-bearing sperm were not reported in these experiments. These data together with our own data in the mouse demonstrate that selection of motile sperm does not improve the quality of the genetic content of the sperm a s far as chromosome anomalies is concerned ianeuploidy and structural abnormalities). Moreover,
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we have demonstrated that, in the mouse, separation of motile sperm does not deviate the sex ratio from the theoretical 1:l.
ACKNOWLEDGMENTS This research was funded by the West Penn Hospital Foundation of West Penn Hospital, Pittsburgh, project 8022. V.C. was partly supported by a fellowship from the Cirit (Generalitat de Catalunya), Spain. REFERENCES Badenas J, Santalo J, Calafell JM, Estop AM, Egozcue J (1989):Effect of the degree of maturation of mouse oocytes at fertilization: A source of chromosome imbalance. Gamete Res 24:205-218. Berger T, Harris RP, Moyer DL (1985): Comparison of techniques for selection of motile sperm. Fertil Steril 43268-273. Bergman P (1955): Sperm migration and its relation to the morphology and motility of spermatozoa. Int J Fertil 1:45-54. Brandriff BF, Gordon LA, Haendel S, Ashworth LK, Cariano AV (1986):The chromosomal constitution of human sperm selected for motility. Fertil Steril 46:686-690. Catala V, Estop AM, Santalo J, Egozcue J (1988): Sexual immaturity and maternal age. Incidence of aneuploidy and polyploidy in first cleavage mouse embryos. Cytogenet Cell Genet 48233-237. Estop A (1989): Nondisjunction and maternal age in the mouse. In Hassold T (eds): “Molecular and Cytogenetic aspects of Nondisjunction.” New York: Anal R. Liss, pp 221-244. Fraser LR, Maudlin I (1979): Analysis of aneuploidy in first cleavage mouse embryos fertilized in vitro and in vivo. Environ Health Perspect 31:141-149. Foster MS, Smith WD, Lee WI, Berger RE, Karp LE, Stenchever MA (1983): Selection of human spermatozoa according to their relative motillity and their interaction with zona-free hamster eggs. Fertil Steril 40:655. Hansmann I, El-Nahass E (1979): Incidence of nondisjunction in mouse oocytes. Cytogenet Cell Genet 24:115-121. Krazanowska H (1974): The passage of abnormal spermatozoa
through the interotubal junction in the mouse. J Reprod Fertil38: 81-90. Makler A (1980): Distribution of normal and abnormal forms among motile, non-motile, live and dead human spermatozoa. Int J Androl 3:620-628. Martin RH, Rademaker A (1988): The relationship between sperm chromosomal abnormalities and sperm morphology in humans. Mutat Res 207:159-164. Moghissi KS (1977): sperm migration through the human cervix. In Insler V, Bettendoy G (eds): “The Uterine Cervix in Reproduction.” Stutgart: Thieme Publishers, pp 146-165. Mortimer D, Leslie EE, Kelly RW, Templeton AA (1982):Morphological selection of human spermatozoa in vivo and in vitro. J Reprod Fertil 64:391-399. Plachot M, de Grouchy J, Junca AM, Mandlebaum J, Salat-Bardoux J , Cohen J (1988): Chromosome analysis of human oocytes and embryos: Does delayed fertilization increase chromosome imbalance? Hum Reprod 3:125-127. Redi CA, Garagua S, Pellicciari C, Manfredi Romanini MG, Capanna E, Winking H, Gropp A (1984): Spermtozoa of chromosomally heterozygous mice and their fate in male and female genital tracts. Gamete Res 9273-286. Santalo J, Estop AM, Egozcue J (1986):The Chromosome complement of first cleavage mouse embryos after in vitro fertilization. J In Vitro Fertil Embryo Transfer 3:99-105. Sumner AT (1972): A simple technique for demonstrating embryonic heterochromatin. Exp Cell Res 15:304-308. Tarkowski AK (1966): An air drying method for chromosome preparation of mouse eggs. Cytogenetics 5:394-400. Van der Ven HH, Al-Hasani S, Diedrich K, Hamerich V, Lehmann F, Krebs D (1985): Polyspermy in in vitro fertilization of human oocytes: Frequency and possible causes. In Sepala M, Edwards RG (eds): “In Vitro Fertilization and Embryo Transfer.” Ann NY Acad Sci 44288-95. Whittingham DG (1971):Culture of mouse ova. J Reprod Fertil Suppl 14:7-21. Wiklaud M, Wik 0, Quist K, Soderlund B, Janson PO (1987): A selfmigration method for preparation of sperm for in vitro fertilization. Hum Reprod 2191-195.
Chromosome Constitution of Highly Motile Mouse Sperm A. ESTOP,’ V. CATALA,’;” AND J. SANTAL02 ‘Department of Medical Genetics, Western Pennsylvania Hospital, Pittsburgh, Pennsylvania, ‘Department of Cell Biology, Autonomous University of Barcelona, Barcelona, Spain In this study, we address the ABSTRACT relationship between motility and genetic content of mouse sperm. The chromosome complements of highly motile mouse sperm, selected using the swim-up technique, were analyzed after in vitro fertilization, at the first cleavage state. They were compared to those of unselected sperm. Identification of male and female chromosome sets was possible because of their differential condensation at the first mitotic division. In vitro fertilization, swim-up separation, chromosome preparation, and staining were carried out using standard techniques. The results indicate that highly motile mouse sperm did not differ in types and frequencies of chromosomal abnormalities from those not selected for motility. Moreover, separation of motile sperm does not deviate the sex ratio from the theoretical 1 : 1.
Key Words: Motility, Genetics, Sex chromosome ratio
INTRODUCTION In the female mammal, millions of sperm are ejaculated and deposited into the female genital tract to ensure the fertilization of a few ova. However, very few of them will reach the fertilization site, and those that will are highly motile. The motility characteristics of a semen sample are considered determinant in judging its quality. The ability of the human cervix to exclude many of the morphologically abnormal spermatozoa present in the semen of fertile men has long been recognized (Bergman, 1955). The selection could be due to the quality of the motility of the spermatozoa or to the existence of a physical filter (Moghissi, 1977). Mortimer et al. (1982) revealed that spermatozoa with a n abnormal head form have a higher incidence of other defects such a s midpiece, tail, and other defects that might impair their motility, suggesting that the apparent selection of morphologically normal spermatozoa is a consequence of their higher motility. Makler (1980) studied the distribution of normal and abnormal forms among motile and nonmotile sperm. According t o this study, the percentage of abnormal forms was much lower in motile sperm (22%)compared with 42% among
0 1990 WILEY-LISS, INC.
unselected sperm. On the other hand, when Martin and Rademaker (1988) studied the sperm chromosomes of human males with different frequencies of morphologically abnormal sperm forms, they found no correlation between abnormal morphology and chromosome abnormalities. An important question is whether highly motile sperm are likely to be genetically superior to less motile sperm. There is evidence that, in in vivo conditions, in the mouse, genetically imbalanced sperm take part in the fertilization process, producing abnormal zygotes subject to postzygotic loss (Redi e t al., 1984). Therefore, genetically abnormal sperm may retain motility at levels sufficient to affect fertilization. However, morphologically grossly abnormal sperm are preferentially lost before they reach the fallopian tube (Krazanowska, 1974; Redi et al., 1984). In humans, separation of highly motile sperm is being used in assisted reproductive techniques such as in vitro fertilization and artificial insemination to enhance the efficiency of the system. Although the in vitro fertilization rate of human mature ova with unselected sperm is high (80-90%), the implantation rate is rather low. This could be due to fertilization by defective sperm due to the higher chances for less than optimal sperm to affect fertilization in vitro compared with the in vivo situation. In fact, i t has been shown that diploid mouse spermatozoa have higher chances to fertilize under in vitro conditions than in vivo (Santalo e t al., 1986). In this study we address the relationship between motility and genetic content in mouse sperm. The chromosome complements of highly motile sperm, selected using the swim-up technique, were analyzed after in vitro fertilization, a t the first cleavage stage. They were compared with those of unselected sperm. Identification of male and female chromosome sets was possible because of their differential condensation a t the
Received January 18, 1990; accepted March 26, 1990. Address reprint requests to A. Estop, Department of Medical Genetics, Western Pennsylvania Hospital, Pittsburgh, PA 15224.
CHROMOSOME CONSTITUTION OF HIGHLY MOTILE MOUSE SPERM first mitotic division. This difference in chromosome condensation between male- and female-derived chromosomes is a direct consequence of the asynchrony in the development of the male and female pronuclei (Fraser and Maudlin, 1979).
MATERIALS AND METHODS Animals CBA x C57 Bli6J hybrid mice were used as gamete donors. Females were 24-42 days old and males 2-3 months old. Females were injected intraperitoneally with 5 IU of pregnant mare serum gonadotropin (PMSG, Sigma), followed by 5 IU of human chorionic gonadotropin, HCG, Sigma) 48 h later. All animals were killed by cervical dislocation immediately before removal of their gametes.
169
Chromosome Preparations Chromosome preparations were made following Tarkowski’s method (Tarkowski, 1966). Slides were routinely stained with Leishman’s stain. If some overlapping of chromosomes was present, the chromosome spreads were C-banded (Sumner, 1972). Heterochromatin staining helped in avoiding ambiguity in chromosome number. The sex ratio was calculated according to the presence of the Y chromosome. Statistical Analysis discontinuity correction was
x2 test with the Yates used.
RESULTS (TABLE 1) In the control group, the total number of recovered eggs was 599. The number of fertilized eggs was 480 (80.13%), and 119 were unfertilized. Four hundred Culture Media twenty-seven allowed cytogenetic analysis; however, The medium used for handling and culturing the 53 could not be studied due to overlapping or scattering eggs was M16 Wittingham’s medium (Whittingham, of the chromosomes. Spreads with fewer than 38 chro1971) containing 4 mg bovine serum albumin ([BSAI, mosomes were not included in the study. If only one crystallized and lyophilized, SigmaPml. For capacita- genome was analyzable, the spread was also discarded. tion and in vitro fertilization we used Tyrode’s (T6) Male and female genomes were analyzed separately medium containing 15 mg BSAiml (Fraction V; when possible. In cases when both genomes were Sigma). For collection of motile sperm in swim-up ex- mixed, the source of a chromosome anomaly could not periments, we used HT6 also supplemented with BSA be identified. In such case, it was recorded a s of “un(Whittingham, 1971). Media were overlaid with paraf- known origin.” In the experimental group we recovered fin oil and equilibrated overnight in a 5% CO, incuba- 536 eggs, of which 437 were fertilized (81.52%) and 99 tor before use. unfertilized. Three hundred ninety-seven were of sufficient quality for cytogenetic analysis. Separation of Motile Sperm Aneuploidy Sperm of two cauda epididymis from the same male A conservative estimate of aneuploidy is twice the donor were used simultaneously in each experiment. One epididymis was used for selection of motile sperm frequency of hyperhaploidy, the reason being that the and the other a s a control. The sperm suspensions were number of hypohaploid complements exceeds that of prepared in T6 without BSA. After 20 min of disper- hyperhaploid, probably because of a technical artifact sion, the cells were centrifuged at 600g for 6 min, then caused by the spreading technique. The total frequency they were allowed to swim up in 0.75 ml of HT6 with of hyperhaploidy was 3.5% in the embryos produced BSA during 30 min at 37°C. Motile sperm were col- with unselected sperm and 4.3% in those where the lected and aliquoted a t a concentration of 1,000,000- highly motile sperm was used. If we look a t the male 1,500,000 spermiml in 0.4 ml drops of the same media and female genomes separately, the frequency of male hyperhaploidy is 1.2% in the control group and 1.3% in for capacitation and fertilization. the experimental group. The rate of hyperhaploidy in the female sets was 1.2% in the control group and 2.5% In Vitro Fertilization in the experimental group. In the control and experimental groups, the sperm Polyploidy suspensions were preincubated for 50 min to achieve Of the 427 analyzed zygotes in the control group, 73 capacitation. The concentration of whole sperm used was the same as in the experimental group. For each showed triploid complements (17%) and in the experimale, two females were sacrificed and their oviducts mental group, of 397 analyzed, 56 were triploid placed directly into the capacitated sperm suspension, (14.1%). The most important contribution to triploidy where the oocytes were released. After a n incubation of was fertilization by two sperm or dispermy (7.5% and 5 h, the eggs were removed, washed once in fresh me- 7.3%, respectively, for controls and experimentals),foldium, and kept overnight (18-20 h) in a 0.4 ml drop of lowed by fertilization of a diploid oocyte by a haploid culture medium containing M vinblastine sulfate sperm (6.1% in controls and 5% in experimentals). Immature mouse females ovulate more diploid oocytes (Sigma).
170
A. ESTOP ET AL. TABLE 1. Results of the Chromosome Analysis of First Cleavage Mouse Embryos Produced In Vitro by Sperm Selected by the Swim-Up Technique and by Unselected Sperm Fertilized An a1y zed Unfertilized Percent fertilized Unfertilized MI1 Partheno Hyperhaploids Female Male Unknown Polyploids Female 2n Dispermy Male 2n Unknown Structural rearrangements Male Unknown C-bands Male Female
Selected 437 397 99
(?A)
(81.52) 99 82 17 17
loa
5b 2 56 20 29
(4.3) (2.5) (1.3) (0.5) (14.1) (5.0) (7.3)
7 (1.8) 5 (1.3) 2 Frag 1 Ring 1 Frag 1 Triradial 150 78 (52.0) 72 (48.0)
Unselected 480 427 119
(%)
(80.13) 119 105 14 15 (3.5) 5 (1.2) 5 (1.2) 6‘ (1.4) 73 (17.0) 26d (6.1) 32d (7.5) 3 (0.7) 12 (2.8) 3 (0.7) 1 Frag 1 Pulverized 1 Ring 178 83 95
(46.6) (53.4)
“Includes one hyperhaploidy, which is also included in dispermy. b T ~ embryos o with 22 chromosomes. “One embryo with 42 chromosomes. tetraploid: 2 d n + P 2n.
than younger adults or older females (Catala et al., 1988; Estop, 1989). Also strain differences have been described in the production of diploid oocytes (Hansmann and El-Nahass, 1979). Included in the triploids are haploid oocytes fertilized by diploid sperm. Three of those (0.7%) were found in the control group. The absence of fertilizing diploid sperm in the experimental group may suggest that diploid sperm tend to have lower motility. There was one tetraploid zygote. The source of tetraploidy could be established as being a diploid oocyte fertilized by two sperm.
Structural Rearrangements Only three male genomes were identified a s carriers of a structural rearrangement in the control group. One complement had a n acentric fragment. Another male genome had multiple breaks resembling “pulverized” chromatin. There was a ring chromosome of unknown origin in one zygote. The total frequency of structural rearrangements in this group was 0.7%. In the experimental group the frequency was 1.3%, including two fragments and one ring chromosome in three different male complements. One fragment and a nonhomologous exchange figure both of unknown origin were also present in two different zygotes.
Sex Ratio One hundred seventy-eight zygotes in the controls and 150 in the experimentals were analyzed with Cbanding. The presence of a Y chromosome was necessary to identify a male genome in a n embryo. If only one complement was analyzable, the cell was excluded from the study. In experiments using the whole unselected sample, the percentage of Y-bearing sperm was 46.6% and t h a t of X-bearing sperm was 53.4%. When highly motile sperm was used for in vitro fertilization, these frequencies varied slightly to 52% of Y-bearing sperm vs. 48% of X-bearing sperm, increasing the number of male embryos (Fig. 1). This increase is not statistically significant. DISCUSSION The percentages of chromosome abnormalities in the one-cell zygotes obtained by highly motile sperm did not differ from those obtained with unselected sperm. Although structural rearrangements and hyperhaploidy are slightly increased in embryos where selected sperm was used, those differences are not statistically significant. Male hyperhaploidy remains constant in highly motile and in unselected sperm. Polyploidy was high in both controls and experimentals, dispermy be-
CHROMOSOME CONSTITUTION OF HIGHLY MOTILE MOUSE SPERM 60
53.4%
52.0% 46.6O/o
171
y////////1
c
c
a,
P a, a 20 10
0 Male Unselected
Female Selected
Fig. 1. Percentage of male and female first cleavage mouse embryos produced by unselected sperm and by sperm selected by the swim-up technique.
ing the most common cause for a triploid zygote. Both dispermy and diploid oocytes may be considered fertilization anomalies. Polyspermy may be higher in in vitro fertilization because of the alteration of the spermiegg ratio. The increase in polyspermy and in the number of diploid oocytes may be a consequence of the degree of maturation of the oocyte at the time of retrieval. Triploidy has been reported to occur more frequently in immature human oocytes cultured in vitro prior to fertilization (Van der Ven et al., 1985), in human oocytes aged in vitro (Plachot et al., 1988), and in immature and overmatured mouse oocytes (Badenas et al., 1989). It has been suggested that procedures to separate motile sperm may have clinical application in human in vitro fertilization and intrauterine insemination by increasing the probability of fertilization by a normal sperm and consequently the chances of normal embryonic development (Berger et al., 1985). Various procedures have been described to separate motile sperm (Berger et al., 1985; Wiklaud e t al., 1987). Sperm selected in Percoll gradients, and found a t the level of 100% Percoll, penetrate hamster eggs at higher frequencies than unselected sperm (Forster et al., 1983). Wiklaud e t al. (198'7) found that sperm collected by a self-migration method in a medium containing hyaluronic acid achieved more pregnancies than sperm separated by other methods. The fact that sperm displaying high motility does not differ from unselected sperm as far a s chromosome abnormality is concerned cannot be completely established. Fractions of sperm separated by swim-up are enriched with very motile sperm and are clean of dead cells and debris, whereas whole sperm contains different populations of sperm, i.e., mo-
tile, less motile, nonmotile plus dead cells. In a n in vitro situation, it is not clear which sperm will fertilize. Capacitation and acrosome reaction are strict requirements; however, the quality of the motility of the sperm might not be a s determinant for in vitro fertilization a s i t is in a n in vivo situation. Another question is the possible relationship between separation of motile sperm and its consequences in altering the sex ratio. Both in the mouse and in humans, Y-bearing sperm are thought to be more motile because they carry slightly less DNA than Xbearing sperm. Based on this assumption, different systems and techniques have been described and applied to sex prediction in humans. We have shown that swim-up and centrifugation a s a means for separating highly motile sperm do not affect the sex ratio in mouse in vitro fertilized embryos. There is a slight shift towards male embryos in those fertilized by highly motile sperm; however, these differences are not significant, and the sex ratio is maintained in both populations of embryos. Brandriff et al. (1986) studied the sperm chromosomes of two human males where sperm was selected for motility using the swim-up system after hamsteregg penetration and consequent formation of pronuclear chromosomes. In their system, chromosome anomalies, both numerical and structural, were similar in motile and whole semen; however, the X- and the Y-bearing sperm were not reported in these experiments. These data together with our own data in the mouse demonstrate that selection of motile sperm does not improve the quality of the genetic content of the sperm a s far as chromosome anomalies is concerned ianeuploidy and structural abnormalities). Moreover,
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we have demonstrated that, in the mouse, separation of motile sperm does not deviate the sex ratio from the theoretical 1:l.
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