ABSTRACT
Mice which are heterozygous for two complementary lethal t mutations (t6/tw32) exhibit complete male sterility. Experiments have been conducted to determine if spermatozoa from these heterozygous males can fertilize oviducal ova in vitro. The experiments have been designed so that specific barriers which are present during in vivo fertilization can be sequentially removed and the fertilizing ability of the spermatozoa tested after each barrier is eliminated. Our results show that spermatozoa from t6/tw32 males are unable to effect fertilization even after all of the barriers normally imposed by the female reproductive tract have been removed.
INTRODUCTION
Male mice heterozygous for two lethal but complementary t mutations, tLx/tLv, are sterile while females are fertile. Previous investigators have noted that intercomplement males exhibit normal mating behavior and produce near normal numbers of spermatozoa (Bryson, 1944; Braden & Gluecksohn-Waelsch, 1958; Bennett & Dunn, 1967). Although the intercomplement males contain a high percentage of morphologically abnormal spermatozoa (Bryson, 1944; Rajasekarasetty, 1951; Braden & Gluecksohn-Waelsch, 1958; Dooher & Bennett, 1977; Nadijcka & Hillman, 1980), the number of aberrant gametes cannot be correlated with the sterility of these animals (Rajasekarasetty, 1951 ; Braden & Gluecksohn-Waelsch, 1958; Nadijcka & Hillman, 1980).
Previous reports have also noted that spermatozoa obtained from tLx/tLv males exhibit a reduced level of motility when compared with corresponding gametes from fertile controls (Bryson, 1944; Bennett & Dunn, 1967; McGrath & Hillman, unreported observations). Unlike control spermatozoa which exhibit forward motility, the mutant spermatozoa remain in place and ‘twitch’.
Braden & Gluecksohn-Waelsch (1958) proposed that sterility in tLx/tLv males results from the inability of the mutant spermatozoa to traverse the uterotubal junction and reach the site of fertilization. Although dissimilar findings were reported by Bennett & Dunn (1967), Olds (1970) confirmed the observation that virtually no spermatozoa from tLx/tLv males are able to enter the female oviduct. Moreover, Olds (1971) found that the failure of spermatozoa to reach the site of fertilization is not the sole cause of intercomplement male sterility. She injected spermatozoa from tw18/tw22 males into the ovarian bursae following ovulation. Despite the close proximity of the gametes, mutant spermatozoa were still unable to effect fertilization. From this study, however, it is not possible to determine the factor(s) impeding fertilization. For example, it cannot be determined if the spermatozoa are unable to become capacitated, to traverse the cumulus cells, to undergo the acrosome reaction, to penetrate the zona pellucida, or to fuse with the vitelline membrane.
The limitations imposed by these in vivo studies can be circumvented by studying the ability of spermatozoa from intercomplement males to fertilize oviducal ova in vitro. We have used in vitro techniques to determine if the spermatozoa obtained from tLx/tLv (t6/tw32) males can, first, disperse the cumulus cells; second, undergo the acrosome reaction; and third, fuse with the ovum. This report presents the results of these studies.
MATERIALS AND METHODS
Both the balanced lethal T/t6 and T/tw32 mouse stocks were maintained by brother-sister matings, t6/tw(tm) males were obtained from reciprocal crosses of T/t6 x T/tw32 and were distinguished from their littermates by the presence of a normal tail. Sterility was determined by mating t6/tw32 males to BALB/c females for the duration of ten mating units (Bennett & Dunn, 1967). Using this mating regimen, none of the t6/tw32 males employed in this study sired offspring.
Spermatozoa were obtained from both the cauda epididymides and vasa deferentia of mutant (t6/tw32) and control ( + / + ; Swiss Albino) males. Spermatozoa from these regions in wild-type mice are referred to as mature spermatozoa since they are capable of fertilizing ova either in vivo or in vitro (Bedford, 1966; Brackett, Hall & Oh, 1978). Immature spermatozoa were obtained from the caput epididymides of the control males. The cauda epididymides and vasa deferentia from each male were placed together into 1 ml of modified Tyrode’s medium (Fraser & Drury, 1975). The control cauda epididymides and vasa deferentia were gently teased to allow the spermatozoa to disperse. Since their motility is reduced, spermatozoa from the mutant animals, and from the wildtype caput epididymides, were obtained by gently applying pressure to the ducts.
Spermatozoan concentrations were calculated from hemocytometer counts of aliquots of individual suspensions. Any spermatozoon evidencing spontaneous movement was scored as motile. The percentage of immotile spermatozoa in each suspension was determined, and the number of gametes added to the insemination dishes was adjusted to give equivalent numbers of motile spermatozoa. Final concentrations ranged from 1·2 to 8·0 x105 spermatozoa for mature gametes from +/+ males, and from 0·45 to 4·2 x 106 spermatozoa for gametes from either t6/tw32 males or the caput epididymides of the wild-type males.
Cumulus cell-surrounded mouse ova were obtained from Fx hybrid females (T/t” x C57BL/6J). These ova were used in fertilization studies requiring either cumulus cell-surrounded or zona pellucida-surrounded ova (Barkening & Chang, 1976). The hybrid females were superovulated with an intraperitoneal injection of pregnant mare serum gonadotrophin (PMSG; 5 IU), followed 48 h later with a second injection of human chorionic gonadotrophin (HCG; 5 IU), (Edwards & Gates, 1959).
In contrast with the cumulus cell-surrounded and zona-surrounded ova, denuded ova are readily fertilized in vitro regardless of their genotype (Wolf, Inoue & Stark, 1976). Therefore, for those studies which required removal of the zona pellucida, ova were obtained from outbred Swiss Albino females. These females were superovulated with 10 IU PMSG and 10 IU HCG. All ova were obtained (13-14 h post HCG injection) by puncturing the distended ampullae of the oviducts with a pair of watchmaker’s forceps. Cumulus cells were removed from the ova by means of a brief incubation (3-5 min) in fertilization medium containing 300 IU hyaluronidase/ml (Cross & Brinster, 1973). Zonae pellucidae were mechanically removed with a small bore glass pipette (Wolf et al., 1976).
Following hyaluronidase treatment and zona removal, the ova were washed through four drops of fertilization medium. These ova, as well as cumulus cell-surrounded ova, were co-incubated with the spermatozoa in 0·2 ml drops of modified Tyrode’s medium (Fraser & Drury, 1975) under silicone oil (Dow-Corning 200 Fluid; 50 cs viscosity). The silicone oil had been previously equilibrated with fertilization medium lacking bovine serum albumin (BSA). The gametes were incubated for 6 h at 37 °C under an atmosphere of 5 % O2, 5 % CO2, and 90 % N2. At the completion of the incubation period, the ova were removed, washed through four drops of modified Tyrode’s medium (4 mg/ml BSA) and reincubated. Four hours later, the ova were fixed (2-5% glutar-aldehyde followed by overnight fixation in cold neutral buffered formalin), stained with aceto-lacmoid (Toyoda & Chang, 1974) and examined with phase optics. Ova were scored as fertilized if they contained at least two pronuclei and a spermatozoan tail (Wolf et al., 1976).
Quantitative estimates of the number of spermatozoa from t6/tw32 males bound to fertilized and unfertilized eggs were performed as follows: zona-less ova were obtained and divided into two groups. The first group was incubated for 2 h without spermatozoa. The second group was incubated for 2 h with mature wild-type spermatozoa which had been previously incubated for 2 h in fertilization medium. At the end of this co-incubation period the fertilized ova were removed and thoroughly washed to remove any bound spermatozoa (the absence of spermatozoa was ascertained by a microscopic examination of a sample of the ova). The unfertilized (group 1) and fertilized (group 2) zona-less ova were then separately incubated for 4 h with spermatozoa from a t6/tw32 male. Both groups of ova were then combined (to insure identical manipulations), washed through four drops of fertilization medium, fixed and then stained for light microscopic analyses. Ova were classified as fertilized and unfertilized, and the number of ova which had bound spermatozoa plus the number of bound spermatozoa/egg were determined for each group.
Electron microscopic studies were performed on ova fixed in either 2-5% glutaraldehyde or picric acid-formaldehyde (Stefanini, DeMartino & Zamboni, 1967). Ova were postfixed with 1 % osmium tetroxide, dehydrated through an ethanol series, and embedded in Epon 812. Ultrathin serial sections were cut and stained with uranyl acetate (Watson, 1958). The sections were examined with a Philips 300 electron microscope.
RESULTS
In the first series of experiments, spermatozoa from the cauda epididymides and vasa deferentia of either t6/tw32 or +/+ males were incubated with cumulus cell-surrounded ova. At the completion of the 6 h incubation period, ova which had been co-incubated with either the mutant or wild-type spermatozoa had lost their surrounding cumulus cells. Control ova incubated for 6 h in the absence of spermatozoa retained their cumulus cells. Thus, spermatozoa from t6/tw32 males appear to contain qualitatively normal levels of hyaluronidase activity. This result supports the findings by Erickson & Krzanowska (1974). Ninety-three percent of the ova incubated with wild-type spermatozoa were fertilized. All of those ova incubated with spermatozoa from the intercomplement males remained unfertilized (Table 1).
In the second series of experiments, either mutant or wild-type spermatozoa from the cauda epididymides and vasa deferentia were added to insemination dishes which contained ova previously treated to remove their surrounding cumulus cells. The results (Table 2) show that fertilization was effected in 78 % of those ova incubated with wild-type spermatozoa, while all of the ova incubated with the mutant spermatozoa remained unfertilized. In the final experiment of this series, t6/tw32 and +/+ spermatozoa from the cauda epididymides and vasa deferentia and + / + spermatozoa from the caput epididymides were separately incubated with zona-less mouse ova. Table 3 shows that the percentage of zona-less + / + mouse ova fertilized by mature wild-type spermatozoa was high (93 %). Conversely, none of the ova were fertilized by the mutant spermatozoa despite the removal of all of the barriers normally present during fertilization. In addition, none of 111 denuded ova incubated with immature wild-type spermatozoa were fertilized.
Light microscopic analyses showed that the zona-less ova which had been incubated with either mutant spermatozoa or immature wild-type spermatozoa for six hours, although unfertilized, had numerous spermatozoa bound to the egg plasma membrane (Figs 1-3). Many of these spermatozoa were ‘twitching’ at the time of examination and most were morphologically normal.
An unwashed ovum after 6 h in vitro incubation with spermatozoa from the cauda epididymides and vasa deferentia of a t6/tw32 male. Note the movement of the spermatozoan tails and the normal morphology of most of the attached spermatozoa. Arrows indicate the abnormal gametes, x 1600.
A micrograph of a washed zona-less ovum following a 6 h incubation period with mutant spermatozoa. The washing procedure causes the tails of the spermatozoa to be sheared from the bound heads, x 1300.
A micrograph of a washed zona-less ovum that had been incubated with immature spermatozoa from a wild-type male. Like mutant spermatozoa, the tails of the immature gametes are lost during the washing procedure (cf. Fig. 2). Mature wild-type spermatozoa remain intact under the same conditions, x 1300.
A micrograph of a washed zona-less ovum that had been incubated with immature spermatozoa from a wild-type male. Like mutant spermatozoa, the tails of the immature gametes are lost during the washing procedure (cf. Fig. 2). Mature wild-type spermatozoa remain intact under the same conditions, x 1300.
In order to determine if there is specificity in the binding capacity of the mutant gametes, spermatozoa from the intercomplement males were incubated with either fertilized or unfertilized zona-less ova (see Methods). The results (Table 4) show that 33 % of the fertilized ova and 90% of the unfertilized ova had bound mutant spermatozoa. Furthermore, of those ova which had bound gametes, the mean number of bound spermatozoa/egg was approximately four times greater before fertilization (5·7 spermatozoa/egg) than after fertilization (1·4 spermatozoa/egg). Thus, spermatozoa from t6/tw32 males preferentially bind to the plasma membranes of unfertilized mouse ova.
Since morphologically normal, motile spermatozoa from mutant males will bind to, but not fertilize, denuded ova, it was of interest to determine whether mutant spermatozoa undergo the acrosome reaction. At the ultrastructural level, spermatozoa which have bound to the vitelline membrane have lost their acrosomes. This observation does not, however, prove that a physiologically normal acrosome reaction occurred.
Ultrastructural observations of serial sections of ova with attached mutant spermatozoa also show that while the two gametic membranes are frequently in close apposition (Figs 4 A and B), there is no indication of extensive membrane fusion between the two gametes. This is in contrast to the sequence of events which normally accompanies mammalian fertilization (Yanagimachi & Noda, 1970; Anderson, Hoppe, Whitten & Lee, 1975; Noda & Yanagimachi, 1976). With few exceptions, the mutant spermatozoa remained entirely outside the ovum. In the exceptional cases, short cytoplasmic extensions from the egg partially engulfed the mutant spermatozoon.
(A) An electron micrograph showing a portion of a denuded ovum and a bound mutant spermatozoon. The gametes had been co-incubated for 6 h. Note the absence of the acrosome from the spermatozoon, x 42600. (B) A higher magnification of the area circumscribed in Fig. 4(A). Note the close association of the gametic membranes. No membrane fusion has occurred, x 81000. AM, inner acrosomal membrane; NM, spermatozoan nuclear membrane; PM, egg plasma membrane.
(A) An electron micrograph showing a portion of a denuded ovum and a bound mutant spermatozoon. The gametes had been co-incubated for 6 h. Note the absence of the acrosome from the spermatozoon, x 42600. (B) A higher magnification of the area circumscribed in Fig. 4(A). Note the close association of the gametic membranes. No membrane fusion has occurred, x 81000. AM, inner acrosomal membrane; NM, spermatozoan nuclear membrane; PM, egg plasma membrane.
DISCUSSION
Mammalian fertilization is a complex process which requires the fertilizing spermatozoon to: (1) acquire the ability to fertilize (mature) while in transit through the epididymis (Bedford, 1975; Orgebin-Crist, Danzo & Davies, 1975), (2) undergo certain biochemical changes known as ‘capacitation’ (Bedford, 1970; Chang & Hunter, 1975; Chang, Austin, Bedford, Brackett, Hunter & Yanagimachi, 1977), and (3) arrive at the site of fertilization (which is ultimately the ovum plasma membrane).
In the current series of experiments we have sequentially eliminated those barriers which in vivo restrict the number of spermatozoa which contact the egg plasma membrane. We have therefore insured that spermatozoa from tQ/tw32 males arrive at the site of fertilization and, by so doing, have eliminated this parameter as the primary cause for intercomplement male sterility. Our results do not, however, allow us to determine if the sterility is caused by a failure of the spermatozoa to mature within the epididymides or by their inability to become capacitated.
There are several lines of evidence which indicate that tLx/tLv male sterility results from a lack of spermatozoan maturation. Bryson (1944) and Bennett & Dunn (1967) reported that spermatozoa from tLx/tLy males exhibit reduced levels of motility. We have also observed that spermatozoa from the cauda epididymides and vasa deferentia of t6/tw32 males show minimal forward progression and, in addition, their movement closely resembles the motility pattern of immature spermatozoa obtained from the caput epididymides of + / + males. Analogously, wild-type spermatozoa obtained from the caput region of the epididymis will bind to, but not fertilize, zona-less mouse ova in vitro. These similarities between the spermatozoa obtained from the cauda epididymides and vasa deferentia of the i6/(w32 males and the immature spermatozoa obtained from fertile + / + males suggest that sterility results from the failure of the mutant spermatozoa to undergo the necessary maturational events which normally occur in the male reproductive tract.
The hypothesis that t alleles interfere with spermatozoan development can also explain the different forms of sterility that have been noted in tLx/tLv and tSL/tSL males. Sterility in tSL homozygotes results from the developmental arrest of spermatids (Bennett & Dunn, 1971; Dooher & Bennett, 1974). The present observations indicate that tLx/tLv sterility is caused by incomplete spermatozoan maturation. The combined observations suggest that both types of sterility are caused by a deficiency in the normal program of differentiation of the spermatozoon. The extent or time of this deficiency in the sterile males would be dependent upon the specific combination of the recessive tn mutations.
Acknowledgement
This research was supported by United States Public Health Service Grants nos. HD 00827 and HD 09753 and by a Biomedical Research Support Grant, SO7-RR07115. The authors would like to thank Marie Morris and Geraldine Wileman for their technical assistance and Dr Don P. Wolf for his advice on obtaining zona-less mouse ova.