Received November 1, 1978
andrologia 11 (3): 197-210 (1979)
Laboratory of Embryology, Institute of Biological Sciences Pontifical Catholic University o f Chile, Santiago, CHILE and Department of Cellular Biology and Genetics, School of Medicine University of Chile, Santiago, CHILE.
Human Sperm Penetration into Zona-Free Hamster Oocytes as a Test to Evaluate the Sperm Fertilizing Ability C. BARROS, J . GONZALEZ, E. HERRERA and E. BUSTOS-OBREGON
Introduction Ejaculated mammalian spermatozoa need to undergo physiological as well as morphological changes in the female tract, which render them fertile, prior to entry into the oocyte (see Barros - 1974 and Barros et al. - 1974 for reviews). The study of the environmental conditions for the spermatozoon to undergo capacitation, has been greatly enhanced due to the use of in vitro fertilization techniques. There are many mammalian species in which the use of homologous oocytes to test the fertilizing ability of the spermatozoa is not desired due to logistic type of problems and/or due to ethical problems. The use of in vitro cross-fertilization allows the scientist to overcome these problems. Oocytes of many different rodent species devoid of the zona pellucida have already been used to this purpose (Hanada and Chang - 1972-1976). The knowledge of the conditions under which human spermatozoa can be rendered fertile is of paramount importance due to the clinical implications. It is also of great importance when there is a need to evaluate the semen characteristics in cases of male infertility. Overstreet and Hembree (1976) used human oocytes recovered from deceased women to test the ability of human spermatozoa to cross the zona pellucida. They found that spermatozoa from semen sampleqof fertile men penetrated the zona pellucida of cadaver oocytes at an apparently higher rate than those from infertile individuals. Yanagimachi et al. (1976) showed that human spermatozoa can fuse with zonafree hamster oocytes in vitro. Later, Barros et al. (1978) reported a bioassay which consisted in the actual human sperm entry into zona-free hamster oocytes to evaluate the intrinsic fertile ability of human spermatozoa from patients attending an Infertility Clinic. Seventy-six percent of individuals with normal spermiogram were positive on the sperm entry test as compared to only thirty-four percent of suspected infertile individuals. The purpose of the present work is to give additional evidence of the validity of the bioassay. Evidence will be presented to show that at the ultrastructural level the behaviour of the gamete membranes does not differ basically from that of normal fertilization. Furthermore, we show that failure of human sperm fusion to the hamster oocytes is related to the human sperm characteristics and not to the quality of hamster oocytes.
Materials and Methods Human semen samples were obtained from patients attending an Infertility Clinic at the University of Chile. From each sample, the spermiogram was prepared and a sample of the semen was given a code number and then transferred to the Catholic Key-words:Human spermatozoon - cross fertilization - bioassay
-
zona-free oocytes
198
C. Barros, J. Gonzales, E. Herrera and E. Bustos-Obregon
University of Chile, where bioassay was performed without knowledge of the semen characteristics. At the end of the experiments, the results of the bioassay were compared with those of the spermiogram. Two ways were used to clean spermatozoa of seminal plasma. The first one was based on the technique described by Lopata et al. (1976) which consisted in placing the semen sample within a test tube and then a Pasteur pipette filled with TMPA medium (Table 1) was dipped in the semen sample. To avoid evaporation, the open end of the test tube was sealed with Parafilm and placed in the incubator at 37' C for 30 minutes. The spermatozoa swim up into the Pasteur pipette and from there they were recovered to be used in the bioassay. The second technique used was to layer the semen sample on the top of a 10% ficoll solution prepared in TMPA medium. The sample was spun down for 10 minutes at 300 g and from the pellet the spermatozoa were obtained to be used in the bioassay. Table 1: Chemical composition of TMPA medium gll
NaCl KCI CaCI, . 2 H, 0 MgCI, . 6 H, 0 NaHCO, Glucose ' H, 0 Na Pyruvate HSA
8.000 0.20 0.260 0.212 3.000 1.008 0.036 35.000
One hundred microliters of TMPA solution were placed at the center of a tissue culture Petri dish (Falcon 3001), covered with liquid parafin and then the spermatozoa to be assayed added to the drop of culture medium. The dish was incubated for a period of one hour at 37' C. At the end of this incubation period, zona-free hamster oocytes were added to the sperm suspension and the dish incubated for an additional period of three hours. The oocytes were examined with the phase-contrast microscope for evidence of sperm chromatin dispersion within the hamster oocyte. In a series of experiments, the viability of the hamster oocytes was tested as follows: two and a half hours after the start of incubation, capacitated hamster spermatozoa were added to the mixture that contained the zona-free hamster oocytes and human spermatozoa. When the oocytes were examined with the phase-contrast microscope, human and hamster spermatozoa could be clearly distinguished. Recovery of hamster oocytes Adult female hamsters were injected with 25 i.u. of Pregnant Mare's Serum Gonadotrophin (PMSG) in the morning of the day of the post-estrous discharge (Orsini - 1961) and with 25 i.u. of Human Chorionic Gonadotrophin (HCG) 56 hours later. Ovulation begins 12 hours after the HCG injection and is completed about 17 hours later. The oocytes were recovered from the oviducal ampulla and treated with a 0.01% solution of hyaluronidase to eliminate the granulosa cells. Then the oocytes were washed at least three times in fresh TMPA medium. Then they were treated with a 0.01% solution of trypsin to eliminate the zona pellucida and washed for at least three times in fresh TMPA medium. The zona-free oocytes were then added to the test sperm suspension. andrologia 11 (1979)
Human Sperm Penetration
199
Electron microscope studies Hamster oocytes inseminated in vitro with human spermatozoa were fKed as described by Jones (1973), dehydrated in a series of increasing concentrations of acetone and embedded in a low viscosity epoxy resin (Spurr - 1969). Ultrathin sections were obtained in the MT2B Porter Blum ultramicrotome. The grids with sections were stained with uranyl acetate and lead citrate.
Results In vitro capacitation of human spermatozoa
Human spermatozoa preincubated in TMPA medium became able to fuse with zonafree hamster oocytes. The incubation time required was about sixty minutes, even though shorter times were sometimes enough for the spermatozoa to fuse with the oocytes. When the medium used in the present work contained human serum albumin at lower concentration than 35 mg/ml, the capacitation of spermatozoa was not consistently repeatable. The criteria used in order to judge sperm capacitation and spermegg fusion was to observe with the phase-contrast microscope dispersion of the sperm chromatin within the hamster oocytes (Fig. 1). In order to make sure that the changes taking place in the human sperm head and later interaction with the oocyte could be equated to the changes occurring in homologous fertilization, we undertook a study with the electron microscope. The observations showed that the human spermatozoon (Fig. 2) after being incubated in TMPA medium undergoes changes in the main portion of the acrosome (Fig 3) that is, the acrosome reacts exposing the inner acrosomal membrane in the anterior portion of the sperm head. The equatorial segment of the acrosome remains at this point intact, thus the sperm plasma membrane becomes continuous with the outer acrosomal membrane of the equatorial segment region. Spermatozoa preincubated for longer times showed that the equatorial segment region had also reacted and the sperm plasma membrane becomes continuous with the inner acrosomal membrane at the caudal end of the place originally occupied by the equatorial segment (Fig. 4).Further stages of interaction showed that the spermatozoon becomes closely associated with the oocyte microvilli. Initial stages of membrane fusion are observed between the post-acrosomal sperm plasma membrane and the oocyte surface. Soon after gamete membrane fusion, the sperm chromatin begins to disperse within the egg cytoplasm (Fig. 5). One to one and a half hour after the sperm and oocytes were mixed, the sperm chromatin appeared fully dispersed and the nuclear membrane of the male pronucleus was reorganized. The sperm tail remains in close apposition to the developing male pronucleus (Fig. 6 , 7 , 8 ) and the surface of the oocyte appeared devoid of cortical granules. The male pronucleus continued its development and nucleoli became evident within it (Fig. 9,lO). In the course of our observations, we found spermatozoa in which the chromatin appeared abnormally condensed. Spermatozoa with similar characteristics were found within the oocyte cytoplasm (Fig. 13).
Bioassay of human semen samples A total of 179 human semen samples of the same number of patients were studied in the present work. The semen samples were classed as normals and abnormals using andrologia 11 (1979)
200
C. Barros, J. Gonzales, E. Herrera and E. Bustos-Obregon
andrologia 11 (1979)
Human Sperm Penetration
20 1
Table 2: Summarized data of normal and abnormal spermiograms
Normal Abnormal
Sperm count* (10' cells/ml)
Motility**
Sperm morphology***
(% 11I-IV)d
70 normal sperm
131.99 f 79.6Sa 50.07 f 40.64b
62.29 48.42
* 8.44a * 16.86b
69.72 57.57
f f
7.98a 10.8lC
an = 84 Mean f SD (1 semen sample per subject) b n = 95 Mean t SD (1 semen sample per subject) cn = 90 Mean t SD (1 semen sample per subject) d defined by Sans (1976) AJp < 0.0001 **p < 0.0001 ***p < 0.0001 Table 3: Spermentry test of semen samples from patients with normal and abnormal spermiograms
Spermiogram No. of patients Normal Abnormal
84 95
No. of samples tested 84 95
Sperm entry test positive negative
70 62 (74) 30 (32)
22 (26) 65 (68)
the parameters of the spermiogram, specially, sperm count, percentage of progressive motility and percentage of normal sperm morphology. In Table 2 we present the mean values of the parameters mentioned above for all the semen samples studied. A given sample was considered to have an abnormal spermiogram when the cell count was less than 40 x lo6 cells/ml, the progressive sperm motility was below sixty percent or when less than 70% of the sperm had a normal morphology. From the analysis of the data we obtained 84 semen samples with normal spermiogram and 95 with abnormal spermiogram. Sixty-two (74%) of 84 normal semen samples were positive on the sperm entry test as compared to only thirty (32%) of 95 abnormal semen samples (Table 3). The bioassay was considered positive regardless of the number of oocytes fused with human spermatozoa in each experiment. In 54 normal and in 71 abnormal semen samples, the percentage of penetrated oocytes was recorded (Table 4). Eight hundred and ten oocytes were inseminated with spermatozoa from normal semen samples, resulting in 205 (25%) penetrated oocytes, while 1046 oocytes were inseminated with spermatozoa from abnormal semen samples, resulting in 76 (7%) penetrated oocytes. When calculated as the mean of the percentage of the different samples, spermatozoa from normal samples penetrated 29.4% f 3.99 S.E., as compared to 7.66% f 1.96 S.E. in the abnormal samples. Fig. 1: phase contrast micrograph of a human spermatozoon within the hamster oocyte cytoplasm. The sperm chromatin has already dispersed (arrow). Fig. 2: Human spermatozoon from a normal semen sample. The main portion (a) and the equatorial segment (es) are intact. Fig. 3: Human spermatozoa after incubation in TMPA medium. The main portion of the acrosome has already reacted. At the equatorial segment region of the acrosome, the sperm plasma membrane is continuous with the outer acrosome membrane (arrow). andrologia 11 (1979)
202
C. Barros, J. Gonzales, E. Herrera and E. Bustos-Obregon
Fig. 4: Human spermatozoon in close contact with the surface of the zona-free hamster oocyte. Note that in the spermatozoon the equatorial segment has reacted. The sperm plasma membrane is now continuous with the inner acrosomal membrane (arrows). Fig. 5: Human spermatozoon already fused with the zona-free hamster oocyte. The sperm-chrornatin dispersion is underway. Fig. 6: The human spermatozoon has already formed the male pronucleus (P). Tail (t) elements are near the pronucleus. andrologia 11 (1979)
Human Sperm Penetration
203
Table 4: Percentage of penetrated zona-free hamster oocytes with human spermatozoa from normal and abnormal semen samples
No. of samples Normal Abnormal
54 71
No. of inseminated oocytes 810 1046
Penetrated oocytes No. %
Mean percentage of penetration ? S.D.*
205 76
29.41 1.66
25 I
f
29.32
* 16.52
*p < 0.0001 Table 5: Hamster sperm penetration into zona-free hamster oocytes contemporary exposed to human spermatozoa
No. of samples Normal Abnormal
I I
Total No. of inseminated oocytes 165 228
*Mean percentage fertilization per sample Ap > 0.1
No. of penetrated oocytes by hamster spermNo./ % % x* 110166.7 171175
66.16 72.47
* *
30.06A 31.58.
* SD
In order to make sure that the lack of penetration of human spermatozoon into hamster oocytes was not due to abnormal hamster oocytes, toward the end of the incubation capacitated hamster spermatozoa were added to the oocytes. The size and morphology of both types of spermatozoa are clearly different, so that when evaluating gamete fusion, eggs penetrated by either sperm or by both were clearly differentiated (Fig. 1 1 , 12). One hundred and sixty five oocytes were inseminated with human spermatozoa (Table 5) from semen samples with normal spermiogram. Hamster spermatozoa were added half an hour before the end of the incubation. The percentage of penetration of hamster oocytes with human spermatozoa from normal semen samples was 24.2 and the same oocytes were penetrated in 66.7 percent with hamster spermatozoa. When the same experiments were carried out with spermatozoa from abnormal semen samples, the percentage of penetration was 7.5 with human spermatozoa, but with the hamster spermatozoa it was 75. The attachment of human and hamster spermatozoa from normal and abnormal human semen samples and its relation to the sperm concentration is presented in Table 6. Semen samples giving positive results in the bioassay had a mean number of sperm attached of 21.3 f 3.2 for the normal samples and 16.78 f 4.7 for the abnormal semen samples. On the other hand, the mean number of sperm in the incubation medium was 19.017 f 1.972 sperm/ml for the normal samples giving positive results in the bioassay. In the abnormal samples, the mean sperm concentration was 4.995 k 1S66. Normal and abnormal semen samples giving negative results in the bioassay did not differ in the mean number of human sperm attached, while the sperm concentration in the incubation medium was different, 14.239 f 3.494 in the normal samples as compared to 3.939 k 1.394 in the abnormal samples. andrologia 11 (1 979)
204
C. Barros, J. Gonzales, E. Herrera and E. Busros-Obregon
andrologia 11 (1 979)
Human Sperm Penetration
205
Table 6: Mean percentage of human and hamster spermatozoa attached to the surface of zona-freehamster oocytes of the bioassayed semen samples Type of sample
BioHuman sperm assay Attachment Cells/ul n +
Normal Abnormal Normal Abnormal
-
av/s b = p
> 0.1
+
14 15 5 22
x
kSE
21.3 +3.2a 16.78 k4.7b 4.1 k 1.3c
4.0
av/sc= p
t
1.6d
< 0.0001
n 9 7 5 16
Hamster sperm Cells/ul
Attachment kSE
19017t 1972 4995k 1566 14239k3494 3 9 3 9 t 1394
bv/sd = p
n
z
kSE
n
6 4 3 5
19.65 18.7 39.9 22.6
k2.3 k 3.8 k9.9 k5.8
4 4 2 3
< 0.01
kSE 3250 t 1934 1887 k 848 1406 k 281 2238 ~ 6 . 9 1
c v/s d = p > 0.1
The contemporary hamster sperm attachment was not different in the four groups and the hamster sperm concentration was lower than the lowest human sperm concentration. Furthermore, in the normal-negative group, hamster sperm attachment was the highest and the sperm concentration was the lowest.
Discussion It has been established beyond any doubt that in normal fertilization the acrosomal reaction is an essential prerequisite for sperm passage through the egg investments (Franklin et al. - 1970). On the other hand, when the zona pellucida is removed from the oocyte by enzymatic treatment, the normal unreacted sperm can attach but never fuse with the oocyte plasma membrane (Barros and Berrios - 1977). It has been suggested that the acrosome reaction occurs not only to allow diffusion of the acrosomal enzymes but also to allow gamete membrane fusion (Barros and Herrera - 1977). In the present work we have established that with an in v i m preincubation of human sperm in TMPA for a period of about 1 hour, the spermatozoa are rendered able to undergo the acrosome reaction as well as to fuse with zona-free hamster oocytes. Previously, it had been reported that human spermatozoa required a period of about 7 hours in vitro to become capacitated (Yanagimachi et al. - 1976). The difference in time is most probably associated with the different culture conditions employed in the present work, as compared with previous ones. The importance of serum albumin for the occurrence of the acrosome reaction has been shown in spermatozoa of different species (Miyamoto and Chang - 1973). It is probable that the high albumin concentration used here might have enhanced the sperm capacitation ability of TMPA. The ultrastructural observations showed that human spermatozoa undergo the acrosome reaction as described previously for other mammalian spermatozoa as well as for human (Barros et al. - 1967, Soupart and Strong - 1974). That is, the main portion of the acrosome reacts first leaving intact the equatorial segment. We also found spermatozoa in close association with the surface of the hamster oocyte, in which the equaFig. 7: Cross section of the human sperm tan The sperm mitochondria (s) are clearly distinguishable from the egg mitochondria (e). Male pronudeus (P). Fig. 8: Male pronucleus (F') in close asSociation with the sperm tail. The egg cortex appears devoid of cortical granules. Fig. 9: Male pronudeus (P) with three nucleoli. A cross section of the sperm tail (t) is near the pronucleus. Fig. 10: Male and female pronuclei. andrologia 11 (1979)
206
C. Barros, J . Gonazles, E. Herrera and E. Bustos-Obregon
andrologia 11 (1979)
H u m a n Sperm Penetration
207
torial segment had also reacted. The meaning of the equatorial segment in fertilization has been debated. Bedford (1972) believes that sperm and egg membrane fusion occurs between the sperm plasma membrane overlying the anterior portion of the equatorial segment and the oocyte plasma membrane. Recently, Moore and Bedford (1978) have presented ultrastructural evidence to show that gamete membrane fusion in the hamster occurs between the sperm plasma membrane overlying the equatorial segment and the oocyte plasma membrane. Thus the “desmosome septate-like” structure originally described by Barros and Franklin (1968) would correspond to the whole equatorial segment. A somewhat different view point has been suggested by Barros (1977) based on ultrastructural studies of guinea-pig sperm entry into zona-free hamster oocytes (Barros and Herrera - 1977). Guinea-pig sperm consistently lacked the equatorial segment when fusing with the zona-free hamster oocytes. Moreover, the ultrastructural evidence showed that the initial fusion point was between oocyte microvilli and the sperm plasma membrane in the region originally occupied by the equatorial segment. The above mentioned experimental evidence then suggested that the equatorial segment needed to react for gamete membrane fusion to occur. It is very likely that both mechanisms of gamete fusion can occur, which would indicate that the necessary trigger for gamete membrane fusion is the occurrence of the acrosomal reaction, regardless of whether the equatorial segment is present or not. Furthermore, the initial gamete fusion in the present work was observed between the post-acrosomal region of the sperm and the oocyte plasma membrane. Subsequent events of fertilization such as sperm chromatin dispersion and male pronuclear nucleoli development, are essentially those of normal fertilization, therefore at this point we can conclude that the way gametes fuse in the system used in the present work does not differ from that observed in normal fertilization. The facts mentioned above give strong support to validity of the results of the bioassay. We are well aware that an important event of fertilization, namely sperm passage through the cumulus oophorus and through the zona pellucida, is not being tested. Due to the ethical and practical problems associated to the work with human oocytes, it seems to us that our approach at the present time is the best. The finding of abnormally looking spermatozoa within the oocyte cytoplasm deserves a special comment. It has not been established whether morphologically abnormal spermatozoa are genetically abnormal. Nonetheless, since the in vitro fertilization technique is at present being used in the treatment of infertility in humans, we believe that special precautions should be used in order to avoid the possibility of fertilization by abnormal spermatozoa. Bioassay
The use of the spermiogram as a tool to predict the fertilizing ability of any single sperm cell has a limited value, since the parameters used are rather flexible and of statistical value only. The boundaries between normal and abnormal values for sperm count, motility and morphology are those most commonly accepted in the literature and follow closely the criteria of Schirren (1972). However, widely different opinions may be found in Fig. 11: Golden hamster and human spermatozoa shown for comparison. Fig. 12: Human and golden hamster spermatozoa on the surface of a zona-free hamster oocyte. Fig. 13: Human spermatozoon with abnormally condensed chromatin lying at the surface of the oocyte (arrow). A similarly abnormal spermatozoon within the oocyte. Note that the nuclear membrane is still visible (arows). andrologia 11 (1 979)
208
C. Barros, J. Gonzales, E. Herrera and E. Bustos-Obregon
different publications, mostly regarding sperm morphology (Joel and Botella-Llusia 1971). Recently, some useful practical guidelines for evaluating seminal samples have been advanced by Eliasson (1 976). Clearly, a lot more work is needed to standardize the criteria for evaluation of semen and complementary tests to ascertain the fertilizing ability of a given semen sample with reasonable confidence are sorely needed. Since our experiments were blind 84 of 179 were classed as normal and 95 of 179 as abnormal. The test resulted in 74% of penetration in the normal as compared to 32%in the abnormal, which correlates well with their being fertile and unfertile. It might appear surprising not to have obtained percentages closer to 100% in the normals. One possibility to explain this is to blame the technique, however, during the course of the work the percentage has been highly consistent, which makes it unlikely attributable to technical problems. A more likely explanation could be that abnormal spermatozoa - found in normal semen samples as defined in “Results” in a relatively high proportion - may account for the negative penetration tests with otherwise normal semen. In the case of those individuals classed as abnormals, it should not be surprising to have 32%of positive results in the bioassay, because we have to keep in mind that under in vitro conditions we are artificially avoiding to the sperm many problems it should encounter in in vivo fertilization, such as sperm motility and sperm concentration. Obviously, motility in an in vitro system needs not to follow the pattern found in the cervical or uterine fluids. This issue may be of relevance since according to some authors motility is the single most important parameter accepted to determine the quality of human semen (Mac Leod - 1971). Fertility impairment related to cervical mucus interference with the sperm function or number of spermatozoa that passes the cervical barrier, are circumvented by placing a considerable number of spermatozoa in close proximity to the oocyte in the in vitro procedure. In this sense the relatively low percentage of sperm penetration becomes more meaningful. When we examine the absolute of the mean percentage of human sperm penetration into the zona-free hamster oocytes, the discrimination between normal and abnormal spermiograms becomes more apparent. The data of sperm attachment as related to sperm concentration in the assayed sample showed that the sperm attachment is not related to the sperm concentration. The negative results in the abnormal sperm samples cannot be attributed to the low sperm concentration, since in normal and abnormal samples the sperm attachment was significantly low as compared to the samples with positive results and independent of the sperm concentration. Moreover, the lack of sperm penetration cannot be attributed to the detrimental effect of human sperm or human seminal plasma to the hamster oocytes, since in all the samples tested, contemporary hamster sperm penetration was over 70% and sperm attachment was about 20 sperm/egg irrespective again of the sperm concentration. The above evidence gives strong support to the validity of the bioassay as a true discriminating system to assay the actual penetrating ability of human semen samples, which in turn it is hoped will prove to be correlated with their fertilizing ability. It is also evident that the bioassay would have more use when considered from the negative point of view, that is, if a given semen sample is negative on the sperm entry test, it is very likely that those same spermatozoa will never fertilize a human oocyte in vivo. On the other hand, if an abnormal sample gives a positive test one cannot be sure that they will fertilize human oocytes in vivo even though they would have more possibilities than those from samples giving negative results. andrologia 11 (1979)
Human Sperm Penetration
209
summary Human sperm entry into zona-free hamster oocytes was used t o test the fertile ability of spermatozoa from semen samples of men attending an Infertility Clinic. Sperm chromatin dispersion as seen under the phase-contrast microscope, was used as criterion for sperm entry. The ultrastructural study showed that the behaviour of the gamete membranes during fusion did not basically differ from that of normal fertilization. Eighty four samples were classed as normal (according t o t h e spermiogram) but only 6 2 (74%) gave a positive test, as compared t o only 30 (32%) positive test of 95 samples with abnormal spermiograms. Eight hundred and ten oocytes inseminated with spermatozoa from normal samples gave a 25% penetration, while 1046 oocytes inseminated with spermatozoa from abnormal samples gave a 7% penetration. The normality of hamster oocytes was demonstrated b y contemporary insemination with human and hamster spermatozoa. While the percentages of human sperm penetration remained low, hamster sperm penetration was over 70%. The present bioassay is recommended as a n additional parameter t o the spermiograms for the study of male fertility.
Penetration menschlicher Spermatozoen in Zona pellucida-freie Hamster-Oozyten. Test zur Uberpriifung der Fertilisation von Spermatozoen
Zusammenfassung Der Eintritt menschlicher Spermatozoen in pellucidafreie Oozyten von Hamstern wurde verwandt, u m die Fertilitat menschlicher Samenproben einer andrologischen Klinik zu untersuchen. Die Chromatin-Dispersion des Spermas, im Phasenkontrastmikroskop festgestellt, wurde als Kriterium f i den Spermatozoeneintritt genommen. Die Ultrastrukturstudie zeigte, d a 8 das Verhalten der Gametenmembran wahrend der Verschmelzung sich nicht wesentlich von normalen Fertilitatsvorgangen unterscheidet. Vierundachtzig Proben wurden als normal (bezogen auf das Spermiogramm) eingeschatzt, aber nur 6 2 (74%) erzielten ein positives Ergebnis im Vergleich zu 30 (32%) positiven Ergebnissen von 95 Proben pathologischer Spermiogramme. Achthundertzehn Oozyten, die mit Spermatozoen von normalen Proben befruchtet wurden, erreichten eine Penetration von 2576, wahrend 1046 Oozyten, die mit pathologischen Samenproben befruchtet wurden, eine Penetration von 7% erlangten. Der Normalzustand der Hamsteroozyten wurde durch eine gleichzeitige Befruchtung mit Spermatozoen von Menschen und Hamstern nachgewiesen. Wahrend der Prozentsatz menschlicher Spermatozoenpenetration niedrig blieb, war die der Hamster iiber 70%. Der vorliegende Bioassay wird als zus3tzlicher Parameter zu den Spermiogrammen zur Untersuchung menschlicher Zeugungsfahgikeit empfohlen.
References Barros, C. 1974. Capacitation of mammalian spermatozoa. In: E M. Coutinho and F. Fuchs (Eds.) Physiology and Genetics of Reproduction. Plenum Publishing Corp., New York. Part B, pp. 3-24. Barros, C. 1977. The fertile mammalian spermatozoon. Rev. de Microsc. Electr. 4,107-113. Barros, C., J.M. Bedford, L.E. Franklin and C.R. Austin. 1967. Membrane vesiculation as a feature of the mammalian acrosome reaction. J. Cell Biol. 34,Ol-05. Barros, C. and M. Berrios. 1977. Is the activated spermatozoon really capacitated? J. Exp. Zool. 201,65-72. Barros, C. and L.E. Franklin. 1968. Behaviour of the gamete membranes during sperm entry into the mammalian egg. J. Cell Biol. 37, C13-Cl8. andrologia 11 (1979)
210
C. Barros, J. Gonazles, E. Herrera and E. Bustos-Obregon
Barros, C., A. Garavagno and M. Berrios. 1974. What is capacitation? Excerpta Medica. Int. Congr. Ser. No. 394, pp. 123-127. Barros, C., J. Gonzalez, E. Herrera and E. Bustos-Obregon. 1978. Fertilizing capacity of human spermatozoa evaluated by actual penetration of foreign eggs. Contraception 17, 87-92. Barros, C. and E. Herrera. 1977. Ultrastructural observations on the incorporation of guinea-pig spermatozoa into zona-free hamster oocytes. J. Reprod. Fert. 49,47-50. Bedford, J.M. 1972. An electron microscopic study of sperm penetration into the rabbit egg after natural mating. Am. J. Anat. 133, 213-254. Eliasson, R. 1976. Semen analysis and laboratory workup. In: A.T.K. Cockett and R.L. Urry (Eds.): Male Infertility. Grune and Stratton, New York, pp. 169-188. Franklin, L.E., C. Barros and E.N. Fussell. 1970. The acrosomal region and the acrosome reaction in sperm of the golden hamster. Biol. Reprod. 3, 180-200. Hanada, A. and M.C. Chang. 1972. Penetration of zona-free eggs by spermatozoa of different species. Biol. Reprod. 6, 300-309. Hanada, A. and M.C. Chang. 1976. Penetration of hamster and rabbit zona-free eggs by rat and mouse spermatozoa, with special reference to sperm capacitation. J. Reprod. Fert. 46, 239241. Jones, R.C. 1973. Preparation of spermatozoa for electron and light microscopy. J. Reprod. Fert. 33,145-149. Joel, C.A. and J. Botella-Llusia. 1971. Principles of modern semen examination and Botella-Casares test. In: C.A. Joel (Ed.): Fertility Disturbances in Men and Women. Karger, Basel, pp. 75106. Lopata, A., M.J. Patullo, A. Chang and 9. James. 1976. A method for collecting motile spermatozoa from human semen. Fert. Steril. 27,677-684. Mac Leod, J. 1971. The semen quality in relation to male fertility. In: C.A. Joel (Ed.): Fertility Disturbances in Men and Women. Karger, Basel, pp. 127-154. Miyamoto, H. and M.C. Chang. 1973. The importance of serum albumin and metabolic intermediates for capacitation of spermatozoa and fertilization of mouse eggs in vitro. J. Reprod. Fert. 32, 193-205. Moore, H.D.M. and J.M. Bedford. 1978. Ultrastructure of the equatorial segment of hamster spermatozoa during penetration of oocytes. J. Ultrastruct. Res. 62, 110-1 17. Orsini, M.W. 1961. The external vaginal phenomena characterizing the stages of the estrous cycle, pregnancy, pseudopregnancy, lactation and the anestrous hamster Mesocricetus auratus, Waterhouse. Proc. Anim. Care Panel 11, 193-206. Overstreet, J.W. and W.C. Hembree. 1976. Penetration of the zona pellucida of non living human oocytes by human spermatozoa in vitro. Fert. Steril. 27: 815-831. Schirren, C. 1972. Praktische Andrologie. Berlin: Briider Hartmann. Soupart, P. and P.A. Strong. 1974. Ultrastructural observations on human oocytes fertilized in vitro. Fert. Steril. 25, 11-43. Spurr, A.R. 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 3 1-43. 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, 47 1-476. Acknowledgements: This work has been supported by Grants from PLAMIRH (64.142.1.77), UNESCO (31/77) and from the Pontifical Catholic University of ChCe Research Fund (92/78) to Dt. C. Barros and Grant 62.138.1.77 from PLAMIRH to Dr. E. Bustos-Obregon. We are also indebted to Dr. A. McLaren for her valuable comments on the manuscript. Address: Prof. Dr. E. BUSTOS-OBREGON, Universidad de Chile, Facultad de Medicina, Santiago Norte. Dept. de Biologia Celulary Genetica, Casilla 6556, Santiago 7/Chile.
andrologia 11 (1979)
andrologia 11 (3): 197-210 (1979)
Laboratory of Embryology, Institute of Biological Sciences Pontifical Catholic University o f Chile, Santiago, CHILE and Department of Cellular Biology and Genetics, School of Medicine University of Chile, Santiago, CHILE.
Human Sperm Penetration into Zona-Free Hamster Oocytes as a Test to Evaluate the Sperm Fertilizing Ability C. BARROS, J . GONZALEZ, E. HERRERA and E. BUSTOS-OBREGON
Introduction Ejaculated mammalian spermatozoa need to undergo physiological as well as morphological changes in the female tract, which render them fertile, prior to entry into the oocyte (see Barros - 1974 and Barros et al. - 1974 for reviews). The study of the environmental conditions for the spermatozoon to undergo capacitation, has been greatly enhanced due to the use of in vitro fertilization techniques. There are many mammalian species in which the use of homologous oocytes to test the fertilizing ability of the spermatozoa is not desired due to logistic type of problems and/or due to ethical problems. The use of in vitro cross-fertilization allows the scientist to overcome these problems. Oocytes of many different rodent species devoid of the zona pellucida have already been used to this purpose (Hanada and Chang - 1972-1976). The knowledge of the conditions under which human spermatozoa can be rendered fertile is of paramount importance due to the clinical implications. It is also of great importance when there is a need to evaluate the semen characteristics in cases of male infertility. Overstreet and Hembree (1976) used human oocytes recovered from deceased women to test the ability of human spermatozoa to cross the zona pellucida. They found that spermatozoa from semen sampleqof fertile men penetrated the zona pellucida of cadaver oocytes at an apparently higher rate than those from infertile individuals. Yanagimachi et al. (1976) showed that human spermatozoa can fuse with zonafree hamster oocytes in vitro. Later, Barros et al. (1978) reported a bioassay which consisted in the actual human sperm entry into zona-free hamster oocytes to evaluate the intrinsic fertile ability of human spermatozoa from patients attending an Infertility Clinic. Seventy-six percent of individuals with normal spermiogram were positive on the sperm entry test as compared to only thirty-four percent of suspected infertile individuals. The purpose of the present work is to give additional evidence of the validity of the bioassay. Evidence will be presented to show that at the ultrastructural level the behaviour of the gamete membranes does not differ basically from that of normal fertilization. Furthermore, we show that failure of human sperm fusion to the hamster oocytes is related to the human sperm characteristics and not to the quality of hamster oocytes.
Materials and Methods Human semen samples were obtained from patients attending an Infertility Clinic at the University of Chile. From each sample, the spermiogram was prepared and a sample of the semen was given a code number and then transferred to the Catholic Key-words:Human spermatozoon - cross fertilization - bioassay
-
zona-free oocytes
198
C. Barros, J. Gonzales, E. Herrera and E. Bustos-Obregon
University of Chile, where bioassay was performed without knowledge of the semen characteristics. At the end of the experiments, the results of the bioassay were compared with those of the spermiogram. Two ways were used to clean spermatozoa of seminal plasma. The first one was based on the technique described by Lopata et al. (1976) which consisted in placing the semen sample within a test tube and then a Pasteur pipette filled with TMPA medium (Table 1) was dipped in the semen sample. To avoid evaporation, the open end of the test tube was sealed with Parafilm and placed in the incubator at 37' C for 30 minutes. The spermatozoa swim up into the Pasteur pipette and from there they were recovered to be used in the bioassay. The second technique used was to layer the semen sample on the top of a 10% ficoll solution prepared in TMPA medium. The sample was spun down for 10 minutes at 300 g and from the pellet the spermatozoa were obtained to be used in the bioassay. Table 1: Chemical composition of TMPA medium gll
NaCl KCI CaCI, . 2 H, 0 MgCI, . 6 H, 0 NaHCO, Glucose ' H, 0 Na Pyruvate HSA
8.000 0.20 0.260 0.212 3.000 1.008 0.036 35.000
One hundred microliters of TMPA solution were placed at the center of a tissue culture Petri dish (Falcon 3001), covered with liquid parafin and then the spermatozoa to be assayed added to the drop of culture medium. The dish was incubated for a period of one hour at 37' C. At the end of this incubation period, zona-free hamster oocytes were added to the sperm suspension and the dish incubated for an additional period of three hours. The oocytes were examined with the phase-contrast microscope for evidence of sperm chromatin dispersion within the hamster oocyte. In a series of experiments, the viability of the hamster oocytes was tested as follows: two and a half hours after the start of incubation, capacitated hamster spermatozoa were added to the mixture that contained the zona-free hamster oocytes and human spermatozoa. When the oocytes were examined with the phase-contrast microscope, human and hamster spermatozoa could be clearly distinguished. Recovery of hamster oocytes Adult female hamsters were injected with 25 i.u. of Pregnant Mare's Serum Gonadotrophin (PMSG) in the morning of the day of the post-estrous discharge (Orsini - 1961) and with 25 i.u. of Human Chorionic Gonadotrophin (HCG) 56 hours later. Ovulation begins 12 hours after the HCG injection and is completed about 17 hours later. The oocytes were recovered from the oviducal ampulla and treated with a 0.01% solution of hyaluronidase to eliminate the granulosa cells. Then the oocytes were washed at least three times in fresh TMPA medium. Then they were treated with a 0.01% solution of trypsin to eliminate the zona pellucida and washed for at least three times in fresh TMPA medium. The zona-free oocytes were then added to the test sperm suspension. andrologia 11 (1979)
Human Sperm Penetration
199
Electron microscope studies Hamster oocytes inseminated in vitro with human spermatozoa were fKed as described by Jones (1973), dehydrated in a series of increasing concentrations of acetone and embedded in a low viscosity epoxy resin (Spurr - 1969). Ultrathin sections were obtained in the MT2B Porter Blum ultramicrotome. The grids with sections were stained with uranyl acetate and lead citrate.
Results In vitro capacitation of human spermatozoa
Human spermatozoa preincubated in TMPA medium became able to fuse with zonafree hamster oocytes. The incubation time required was about sixty minutes, even though shorter times were sometimes enough for the spermatozoa to fuse with the oocytes. When the medium used in the present work contained human serum albumin at lower concentration than 35 mg/ml, the capacitation of spermatozoa was not consistently repeatable. The criteria used in order to judge sperm capacitation and spermegg fusion was to observe with the phase-contrast microscope dispersion of the sperm chromatin within the hamster oocytes (Fig. 1). In order to make sure that the changes taking place in the human sperm head and later interaction with the oocyte could be equated to the changes occurring in homologous fertilization, we undertook a study with the electron microscope. The observations showed that the human spermatozoon (Fig. 2) after being incubated in TMPA medium undergoes changes in the main portion of the acrosome (Fig 3) that is, the acrosome reacts exposing the inner acrosomal membrane in the anterior portion of the sperm head. The equatorial segment of the acrosome remains at this point intact, thus the sperm plasma membrane becomes continuous with the outer acrosomal membrane of the equatorial segment region. Spermatozoa preincubated for longer times showed that the equatorial segment region had also reacted and the sperm plasma membrane becomes continuous with the inner acrosomal membrane at the caudal end of the place originally occupied by the equatorial segment (Fig. 4).Further stages of interaction showed that the spermatozoon becomes closely associated with the oocyte microvilli. Initial stages of membrane fusion are observed between the post-acrosomal sperm plasma membrane and the oocyte surface. Soon after gamete membrane fusion, the sperm chromatin begins to disperse within the egg cytoplasm (Fig. 5). One to one and a half hour after the sperm and oocytes were mixed, the sperm chromatin appeared fully dispersed and the nuclear membrane of the male pronucleus was reorganized. The sperm tail remains in close apposition to the developing male pronucleus (Fig. 6 , 7 , 8 ) and the surface of the oocyte appeared devoid of cortical granules. The male pronucleus continued its development and nucleoli became evident within it (Fig. 9,lO). In the course of our observations, we found spermatozoa in which the chromatin appeared abnormally condensed. Spermatozoa with similar characteristics were found within the oocyte cytoplasm (Fig. 13).
Bioassay of human semen samples A total of 179 human semen samples of the same number of patients were studied in the present work. The semen samples were classed as normals and abnormals using andrologia 11 (1979)
200
C. Barros, J. Gonzales, E. Herrera and E. Bustos-Obregon
andrologia 11 (1979)
Human Sperm Penetration
20 1
Table 2: Summarized data of normal and abnormal spermiograms
Normal Abnormal
Sperm count* (10' cells/ml)
Motility**
Sperm morphology***
(% 11I-IV)d
70 normal sperm
131.99 f 79.6Sa 50.07 f 40.64b
62.29 48.42
* 8.44a * 16.86b
69.72 57.57
f f
7.98a 10.8lC
an = 84 Mean f SD (1 semen sample per subject) b n = 95 Mean t SD (1 semen sample per subject) cn = 90 Mean t SD (1 semen sample per subject) d defined by Sans (1976) AJp < 0.0001 **p < 0.0001 ***p < 0.0001 Table 3: Spermentry test of semen samples from patients with normal and abnormal spermiograms
Spermiogram No. of patients Normal Abnormal
84 95
No. of samples tested 84 95
Sperm entry test positive negative
70 62 (74) 30 (32)
22 (26) 65 (68)
the parameters of the spermiogram, specially, sperm count, percentage of progressive motility and percentage of normal sperm morphology. In Table 2 we present the mean values of the parameters mentioned above for all the semen samples studied. A given sample was considered to have an abnormal spermiogram when the cell count was less than 40 x lo6 cells/ml, the progressive sperm motility was below sixty percent or when less than 70% of the sperm had a normal morphology. From the analysis of the data we obtained 84 semen samples with normal spermiogram and 95 with abnormal spermiogram. Sixty-two (74%) of 84 normal semen samples were positive on the sperm entry test as compared to only thirty (32%) of 95 abnormal semen samples (Table 3). The bioassay was considered positive regardless of the number of oocytes fused with human spermatozoa in each experiment. In 54 normal and in 71 abnormal semen samples, the percentage of penetrated oocytes was recorded (Table 4). Eight hundred and ten oocytes were inseminated with spermatozoa from normal semen samples, resulting in 205 (25%) penetrated oocytes, while 1046 oocytes were inseminated with spermatozoa from abnormal semen samples, resulting in 76 (7%) penetrated oocytes. When calculated as the mean of the percentage of the different samples, spermatozoa from normal samples penetrated 29.4% f 3.99 S.E., as compared to 7.66% f 1.96 S.E. in the abnormal samples. Fig. 1: phase contrast micrograph of a human spermatozoon within the hamster oocyte cytoplasm. The sperm chromatin has already dispersed (arrow). Fig. 2: Human spermatozoon from a normal semen sample. The main portion (a) and the equatorial segment (es) are intact. Fig. 3: Human spermatozoa after incubation in TMPA medium. The main portion of the acrosome has already reacted. At the equatorial segment region of the acrosome, the sperm plasma membrane is continuous with the outer acrosome membrane (arrow). andrologia 11 (1979)
202
C. Barros, J. Gonzales, E. Herrera and E. Bustos-Obregon
Fig. 4: Human spermatozoon in close contact with the surface of the zona-free hamster oocyte. Note that in the spermatozoon the equatorial segment has reacted. The sperm plasma membrane is now continuous with the inner acrosomal membrane (arrows). Fig. 5: Human spermatozoon already fused with the zona-free hamster oocyte. The sperm-chrornatin dispersion is underway. Fig. 6: The human spermatozoon has already formed the male pronucleus (P). Tail (t) elements are near the pronucleus. andrologia 11 (1979)
Human Sperm Penetration
203
Table 4: Percentage of penetrated zona-free hamster oocytes with human spermatozoa from normal and abnormal semen samples
No. of samples Normal Abnormal
54 71
No. of inseminated oocytes 810 1046
Penetrated oocytes No. %
Mean percentage of penetration ? S.D.*
205 76
29.41 1.66
25 I
f
29.32
* 16.52
*p < 0.0001 Table 5: Hamster sperm penetration into zona-free hamster oocytes contemporary exposed to human spermatozoa
No. of samples Normal Abnormal
I I
Total No. of inseminated oocytes 165 228
*Mean percentage fertilization per sample Ap > 0.1
No. of penetrated oocytes by hamster spermNo./ % % x* 110166.7 171175
66.16 72.47
* *
30.06A 31.58.
* SD
In order to make sure that the lack of penetration of human spermatozoon into hamster oocytes was not due to abnormal hamster oocytes, toward the end of the incubation capacitated hamster spermatozoa were added to the oocytes. The size and morphology of both types of spermatozoa are clearly different, so that when evaluating gamete fusion, eggs penetrated by either sperm or by both were clearly differentiated (Fig. 1 1 , 12). One hundred and sixty five oocytes were inseminated with human spermatozoa (Table 5) from semen samples with normal spermiogram. Hamster spermatozoa were added half an hour before the end of the incubation. The percentage of penetration of hamster oocytes with human spermatozoa from normal semen samples was 24.2 and the same oocytes were penetrated in 66.7 percent with hamster spermatozoa. When the same experiments were carried out with spermatozoa from abnormal semen samples, the percentage of penetration was 7.5 with human spermatozoa, but with the hamster spermatozoa it was 75. The attachment of human and hamster spermatozoa from normal and abnormal human semen samples and its relation to the sperm concentration is presented in Table 6. Semen samples giving positive results in the bioassay had a mean number of sperm attached of 21.3 f 3.2 for the normal samples and 16.78 f 4.7 for the abnormal semen samples. On the other hand, the mean number of sperm in the incubation medium was 19.017 f 1.972 sperm/ml for the normal samples giving positive results in the bioassay. In the abnormal samples, the mean sperm concentration was 4.995 k 1S66. Normal and abnormal semen samples giving negative results in the bioassay did not differ in the mean number of human sperm attached, while the sperm concentration in the incubation medium was different, 14.239 f 3.494 in the normal samples as compared to 3.939 k 1.394 in the abnormal samples. andrologia 11 (1 979)
204
C. Barros, J. Gonzales, E. Herrera and E. Busros-Obregon
andrologia 11 (1 979)
Human Sperm Penetration
205
Table 6: Mean percentage of human and hamster spermatozoa attached to the surface of zona-freehamster oocytes of the bioassayed semen samples Type of sample
BioHuman sperm assay Attachment Cells/ul n +
Normal Abnormal Normal Abnormal
-
av/s b = p
> 0.1
+
14 15 5 22
x
kSE
21.3 +3.2a 16.78 k4.7b 4.1 k 1.3c
4.0
av/sc= p
t
1.6d
< 0.0001
n 9 7 5 16
Hamster sperm Cells/ul
Attachment kSE
19017t 1972 4995k 1566 14239k3494 3 9 3 9 t 1394
bv/sd = p
n
z
kSE
n
6 4 3 5
19.65 18.7 39.9 22.6
k2.3 k 3.8 k9.9 k5.8
4 4 2 3
< 0.01
kSE 3250 t 1934 1887 k 848 1406 k 281 2238 ~ 6 . 9 1
c v/s d = p > 0.1
The contemporary hamster sperm attachment was not different in the four groups and the hamster sperm concentration was lower than the lowest human sperm concentration. Furthermore, in the normal-negative group, hamster sperm attachment was the highest and the sperm concentration was the lowest.
Discussion It has been established beyond any doubt that in normal fertilization the acrosomal reaction is an essential prerequisite for sperm passage through the egg investments (Franklin et al. - 1970). On the other hand, when the zona pellucida is removed from the oocyte by enzymatic treatment, the normal unreacted sperm can attach but never fuse with the oocyte plasma membrane (Barros and Berrios - 1977). It has been suggested that the acrosome reaction occurs not only to allow diffusion of the acrosomal enzymes but also to allow gamete membrane fusion (Barros and Herrera - 1977). In the present work we have established that with an in v i m preincubation of human sperm in TMPA for a period of about 1 hour, the spermatozoa are rendered able to undergo the acrosome reaction as well as to fuse with zona-free hamster oocytes. Previously, it had been reported that human spermatozoa required a period of about 7 hours in vitro to become capacitated (Yanagimachi et al. - 1976). The difference in time is most probably associated with the different culture conditions employed in the present work, as compared with previous ones. The importance of serum albumin for the occurrence of the acrosome reaction has been shown in spermatozoa of different species (Miyamoto and Chang - 1973). It is probable that the high albumin concentration used here might have enhanced the sperm capacitation ability of TMPA. The ultrastructural observations showed that human spermatozoa undergo the acrosome reaction as described previously for other mammalian spermatozoa as well as for human (Barros et al. - 1967, Soupart and Strong - 1974). That is, the main portion of the acrosome reacts first leaving intact the equatorial segment. We also found spermatozoa in close association with the surface of the hamster oocyte, in which the equaFig. 7: Cross section of the human sperm tan The sperm mitochondria (s) are clearly distinguishable from the egg mitochondria (e). Male pronudeus (P). Fig. 8: Male pronucleus (F') in close asSociation with the sperm tail. The egg cortex appears devoid of cortical granules. Fig. 9: Male pronudeus (P) with three nucleoli. A cross section of the sperm tail (t) is near the pronucleus. Fig. 10: Male and female pronuclei. andrologia 11 (1979)
206
C. Barros, J . Gonazles, E. Herrera and E. Bustos-Obregon
andrologia 11 (1979)
H u m a n Sperm Penetration
207
torial segment had also reacted. The meaning of the equatorial segment in fertilization has been debated. Bedford (1972) believes that sperm and egg membrane fusion occurs between the sperm plasma membrane overlying the anterior portion of the equatorial segment and the oocyte plasma membrane. Recently, Moore and Bedford (1978) have presented ultrastructural evidence to show that gamete membrane fusion in the hamster occurs between the sperm plasma membrane overlying the equatorial segment and the oocyte plasma membrane. Thus the “desmosome septate-like” structure originally described by Barros and Franklin (1968) would correspond to the whole equatorial segment. A somewhat different view point has been suggested by Barros (1977) based on ultrastructural studies of guinea-pig sperm entry into zona-free hamster oocytes (Barros and Herrera - 1977). Guinea-pig sperm consistently lacked the equatorial segment when fusing with the zona-free hamster oocytes. Moreover, the ultrastructural evidence showed that the initial fusion point was between oocyte microvilli and the sperm plasma membrane in the region originally occupied by the equatorial segment. The above mentioned experimental evidence then suggested that the equatorial segment needed to react for gamete membrane fusion to occur. It is very likely that both mechanisms of gamete fusion can occur, which would indicate that the necessary trigger for gamete membrane fusion is the occurrence of the acrosomal reaction, regardless of whether the equatorial segment is present or not. Furthermore, the initial gamete fusion in the present work was observed between the post-acrosomal region of the sperm and the oocyte plasma membrane. Subsequent events of fertilization such as sperm chromatin dispersion and male pronuclear nucleoli development, are essentially those of normal fertilization, therefore at this point we can conclude that the way gametes fuse in the system used in the present work does not differ from that observed in normal fertilization. The facts mentioned above give strong support to validity of the results of the bioassay. We are well aware that an important event of fertilization, namely sperm passage through the cumulus oophorus and through the zona pellucida, is not being tested. Due to the ethical and practical problems associated to the work with human oocytes, it seems to us that our approach at the present time is the best. The finding of abnormally looking spermatozoa within the oocyte cytoplasm deserves a special comment. It has not been established whether morphologically abnormal spermatozoa are genetically abnormal. Nonetheless, since the in vitro fertilization technique is at present being used in the treatment of infertility in humans, we believe that special precautions should be used in order to avoid the possibility of fertilization by abnormal spermatozoa. Bioassay
The use of the spermiogram as a tool to predict the fertilizing ability of any single sperm cell has a limited value, since the parameters used are rather flexible and of statistical value only. The boundaries between normal and abnormal values for sperm count, motility and morphology are those most commonly accepted in the literature and follow closely the criteria of Schirren (1972). However, widely different opinions may be found in Fig. 11: Golden hamster and human spermatozoa shown for comparison. Fig. 12: Human and golden hamster spermatozoa on the surface of a zona-free hamster oocyte. Fig. 13: Human spermatozoon with abnormally condensed chromatin lying at the surface of the oocyte (arrow). A similarly abnormal spermatozoon within the oocyte. Note that the nuclear membrane is still visible (arows). andrologia 11 (1 979)
208
C. Barros, J. Gonzales, E. Herrera and E. Bustos-Obregon
different publications, mostly regarding sperm morphology (Joel and Botella-Llusia 1971). Recently, some useful practical guidelines for evaluating seminal samples have been advanced by Eliasson (1 976). Clearly, a lot more work is needed to standardize the criteria for evaluation of semen and complementary tests to ascertain the fertilizing ability of a given semen sample with reasonable confidence are sorely needed. Since our experiments were blind 84 of 179 were classed as normal and 95 of 179 as abnormal. The test resulted in 74% of penetration in the normal as compared to 32%in the abnormal, which correlates well with their being fertile and unfertile. It might appear surprising not to have obtained percentages closer to 100% in the normals. One possibility to explain this is to blame the technique, however, during the course of the work the percentage has been highly consistent, which makes it unlikely attributable to technical problems. A more likely explanation could be that abnormal spermatozoa - found in normal semen samples as defined in “Results” in a relatively high proportion - may account for the negative penetration tests with otherwise normal semen. In the case of those individuals classed as abnormals, it should not be surprising to have 32%of positive results in the bioassay, because we have to keep in mind that under in vitro conditions we are artificially avoiding to the sperm many problems it should encounter in in vivo fertilization, such as sperm motility and sperm concentration. Obviously, motility in an in vitro system needs not to follow the pattern found in the cervical or uterine fluids. This issue may be of relevance since according to some authors motility is the single most important parameter accepted to determine the quality of human semen (Mac Leod - 1971). Fertility impairment related to cervical mucus interference with the sperm function or number of spermatozoa that passes the cervical barrier, are circumvented by placing a considerable number of spermatozoa in close proximity to the oocyte in the in vitro procedure. In this sense the relatively low percentage of sperm penetration becomes more meaningful. When we examine the absolute of the mean percentage of human sperm penetration into the zona-free hamster oocytes, the discrimination between normal and abnormal spermiograms becomes more apparent. The data of sperm attachment as related to sperm concentration in the assayed sample showed that the sperm attachment is not related to the sperm concentration. The negative results in the abnormal sperm samples cannot be attributed to the low sperm concentration, since in normal and abnormal samples the sperm attachment was significantly low as compared to the samples with positive results and independent of the sperm concentration. Moreover, the lack of sperm penetration cannot be attributed to the detrimental effect of human sperm or human seminal plasma to the hamster oocytes, since in all the samples tested, contemporary hamster sperm penetration was over 70% and sperm attachment was about 20 sperm/egg irrespective again of the sperm concentration. The above evidence gives strong support to the validity of the bioassay as a true discriminating system to assay the actual penetrating ability of human semen samples, which in turn it is hoped will prove to be correlated with their fertilizing ability. It is also evident that the bioassay would have more use when considered from the negative point of view, that is, if a given semen sample is negative on the sperm entry test, it is very likely that those same spermatozoa will never fertilize a human oocyte in vivo. On the other hand, if an abnormal sample gives a positive test one cannot be sure that they will fertilize human oocytes in vivo even though they would have more possibilities than those from samples giving negative results. andrologia 11 (1979)
Human Sperm Penetration
209
summary Human sperm entry into zona-free hamster oocytes was used t o test the fertile ability of spermatozoa from semen samples of men attending an Infertility Clinic. Sperm chromatin dispersion as seen under the phase-contrast microscope, was used as criterion for sperm entry. The ultrastructural study showed that the behaviour of the gamete membranes during fusion did not basically differ from that of normal fertilization. Eighty four samples were classed as normal (according t o t h e spermiogram) but only 6 2 (74%) gave a positive test, as compared t o only 30 (32%) positive test of 95 samples with abnormal spermiograms. Eight hundred and ten oocytes inseminated with spermatozoa from normal samples gave a 25% penetration, while 1046 oocytes inseminated with spermatozoa from abnormal samples gave a 7% penetration. The normality of hamster oocytes was demonstrated b y contemporary insemination with human and hamster spermatozoa. While the percentages of human sperm penetration remained low, hamster sperm penetration was over 70%. The present bioassay is recommended as a n additional parameter t o the spermiograms for the study of male fertility.
Penetration menschlicher Spermatozoen in Zona pellucida-freie Hamster-Oozyten. Test zur Uberpriifung der Fertilisation von Spermatozoen
Zusammenfassung Der Eintritt menschlicher Spermatozoen in pellucidafreie Oozyten von Hamstern wurde verwandt, u m die Fertilitat menschlicher Samenproben einer andrologischen Klinik zu untersuchen. Die Chromatin-Dispersion des Spermas, im Phasenkontrastmikroskop festgestellt, wurde als Kriterium f i den Spermatozoeneintritt genommen. Die Ultrastrukturstudie zeigte, d a 8 das Verhalten der Gametenmembran wahrend der Verschmelzung sich nicht wesentlich von normalen Fertilitatsvorgangen unterscheidet. Vierundachtzig Proben wurden als normal (bezogen auf das Spermiogramm) eingeschatzt, aber nur 6 2 (74%) erzielten ein positives Ergebnis im Vergleich zu 30 (32%) positiven Ergebnissen von 95 Proben pathologischer Spermiogramme. Achthundertzehn Oozyten, die mit Spermatozoen von normalen Proben befruchtet wurden, erreichten eine Penetration von 2576, wahrend 1046 Oozyten, die mit pathologischen Samenproben befruchtet wurden, eine Penetration von 7% erlangten. Der Normalzustand der Hamsteroozyten wurde durch eine gleichzeitige Befruchtung mit Spermatozoen von Menschen und Hamstern nachgewiesen. Wahrend der Prozentsatz menschlicher Spermatozoenpenetration niedrig blieb, war die der Hamster iiber 70%. Der vorliegende Bioassay wird als zus3tzlicher Parameter zu den Spermiogrammen zur Untersuchung menschlicher Zeugungsfahgikeit empfohlen.
References Barros, C. 1974. Capacitation of mammalian spermatozoa. In: E M. Coutinho and F. Fuchs (Eds.) Physiology and Genetics of Reproduction. Plenum Publishing Corp., New York. Part B, pp. 3-24. Barros, C. 1977. The fertile mammalian spermatozoon. Rev. de Microsc. Electr. 4,107-113. Barros, C., J.M. Bedford, L.E. Franklin and C.R. Austin. 1967. Membrane vesiculation as a feature of the mammalian acrosome reaction. J. Cell Biol. 34,Ol-05. Barros, C. and M. Berrios. 1977. Is the activated spermatozoon really capacitated? J. Exp. Zool. 201,65-72. Barros, C. and L.E. Franklin. 1968. Behaviour of the gamete membranes during sperm entry into the mammalian egg. J. Cell Biol. 37, C13-Cl8. andrologia 11 (1979)
210
C. Barros, J. Gonazles, E. Herrera and E. Bustos-Obregon
Barros, C., A. Garavagno and M. Berrios. 1974. What is capacitation? Excerpta Medica. Int. Congr. Ser. No. 394, pp. 123-127. Barros, C., J. Gonzalez, E. Herrera and E. Bustos-Obregon. 1978. Fertilizing capacity of human spermatozoa evaluated by actual penetration of foreign eggs. Contraception 17, 87-92. Barros, C. and E. Herrera. 1977. Ultrastructural observations on the incorporation of guinea-pig spermatozoa into zona-free hamster oocytes. J. Reprod. Fert. 49,47-50. Bedford, J.M. 1972. An electron microscopic study of sperm penetration into the rabbit egg after natural mating. Am. J. Anat. 133, 213-254. Eliasson, R. 1976. Semen analysis and laboratory workup. In: A.T.K. Cockett and R.L. Urry (Eds.): Male Infertility. Grune and Stratton, New York, pp. 169-188. Franklin, L.E., C. Barros and E.N. Fussell. 1970. The acrosomal region and the acrosome reaction in sperm of the golden hamster. Biol. Reprod. 3, 180-200. Hanada, A. and M.C. Chang. 1972. Penetration of zona-free eggs by spermatozoa of different species. Biol. Reprod. 6, 300-309. Hanada, A. and M.C. Chang. 1976. Penetration of hamster and rabbit zona-free eggs by rat and mouse spermatozoa, with special reference to sperm capacitation. J. Reprod. Fert. 46, 239241. Jones, R.C. 1973. Preparation of spermatozoa for electron and light microscopy. J. Reprod. Fert. 33,145-149. Joel, C.A. and J. Botella-Llusia. 1971. Principles of modern semen examination and Botella-Casares test. In: C.A. Joel (Ed.): Fertility Disturbances in Men and Women. Karger, Basel, pp. 75106. Lopata, A., M.J. Patullo, A. Chang and 9. James. 1976. A method for collecting motile spermatozoa from human semen. Fert. Steril. 27,677-684. Mac Leod, J. 1971. The semen quality in relation to male fertility. In: C.A. Joel (Ed.): Fertility Disturbances in Men and Women. Karger, Basel, pp. 127-154. Miyamoto, H. and M.C. Chang. 1973. The importance of serum albumin and metabolic intermediates for capacitation of spermatozoa and fertilization of mouse eggs in vitro. J. Reprod. Fert. 32, 193-205. Moore, H.D.M. and J.M. Bedford. 1978. Ultrastructure of the equatorial segment of hamster spermatozoa during penetration of oocytes. J. Ultrastruct. Res. 62, 110-1 17. Orsini, M.W. 1961. The external vaginal phenomena characterizing the stages of the estrous cycle, pregnancy, pseudopregnancy, lactation and the anestrous hamster Mesocricetus auratus, Waterhouse. Proc. Anim. Care Panel 11, 193-206. Overstreet, J.W. and W.C. Hembree. 1976. Penetration of the zona pellucida of non living human oocytes by human spermatozoa in vitro. Fert. Steril. 27: 815-831. Schirren, C. 1972. Praktische Andrologie. Berlin: Briider Hartmann. Soupart, P. and P.A. Strong. 1974. Ultrastructural observations on human oocytes fertilized in vitro. Fert. Steril. 25, 11-43. Spurr, A.R. 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 3 1-43. 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, 47 1-476. Acknowledgements: This work has been supported by Grants from PLAMIRH (64.142.1.77), UNESCO (31/77) and from the Pontifical Catholic University of ChCe Research Fund (92/78) to Dt. C. Barros and Grant 62.138.1.77 from PLAMIRH to Dr. E. Bustos-Obregon. We are also indebted to Dr. A. McLaren for her valuable comments on the manuscript. Address: Prof. Dr. E. BUSTOS-OBREGON, Universidad de Chile, Facultad de Medicina, Santiago Norte. Dept. de Biologia Celulary Genetica, Casilla 6556, Santiago 7/Chile.
andrologia 11 (1979)
