Department of Obstetrics and Gynecology, Fukushima Medical University, 1 Hikarigaoka Fukushima, Fukushima 960-1295, Japan
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: calcium oscillation/embryo development/intracytoplasmic spermatid injection/mouse/oocyte activation
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In basic studies to clarify parts of the fertilization mechanism involving round spermatids and elongated spermatids, we have focused on oocyte activation and Ca2+ oscillation during the process of fertilization.
The present study was intended to clarify four issues: (i) the oocyte activation and Ca2+ oscillation-inducing abilities of round spermatid, elongated spermatid and mature spermatozoa; (ii) the timing at which these abilities first appear (or become biologically active) during spermiogenesis; (iii) how the [Ca2+]i responses of injected oocytes change during maturation from spermatid to spermatozoa; and (iv) whether Ca2+ oscillation is essential for normal embryonic development to term.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of round/elongated spermatids and mature spermatozoa
Preparation of round/elongated spermatids
Testes were isolated from mature male B6D2F1 and ICR mice (810 weeks old). After removal of tunica, seminiferous tubules were placed in 1 ml of mHTF and cut into small pieces with a pair of fine scissors. One part of suspension containing fragments of seminiferous tubules was mixed thoroughly with one part of 0.9% NaCl containing 10% polyvinylpyrrolidone (PVP-360; Sigma, St Louis, MO, USA). The spermatogenic cells were released from tubular fragments and dispersed into the medium by repeated gentle pipetting. The final suspension contained spermatozoa as well as various developmental stages of spermatogenic cells. A 3 µl droplet of this suspension was placed in a plastic Petri dish (chamber for micro-injection) covered with mineral oil and kept for up to 2 h before the injection of the round/elongated spermatids into oocytes. The chamber was mounted on a stage of an inverted microscope equipped with micro-injection system and cooled to 17°C.
Preparation of mature spermatozoa
The cauda epididymis was isolated from mature male B6D2F1 and ICR (810 week old) mice. A dense mass of spermatozoa was obtained by puncturing the epididymal tubes with a 25-gauge needle. A small drop of the spermatozoa was placed into 3 ml of mHTF and incubated for 1015 min at 25°C to allow the spermatozoa to disperse evenly in the medium. This suspension was then lightly sonicated for 5 s at 5 W power output using an ultrasonic sonicator (Microson; Misonic Inc., Farmingdale, NY, USA) and washed by centrifugation for 5 min at 1000 g. By this treatment, >95% of spermatozoa were immobilized and decapitated (plasma membranes of spermatozoa were disrupted). All isolated sperm heads were diagnosed as `dead' after assessment with a sperm viability kit (Live/Dead Fertilight Sperm Viability Kit; Molecular Probes Inc., Eugene, OR, USA) (Kuretake et al., 1996). A 3 µl sample of this sperm suspension was mixed thoroughly with an equal volume of 0.9% NaCl containing 10% PVP and spermatozoa were injected into oocytes within 1 h after sonication.
Micoinjection of spermatids and spermatozoa
To investigate differences in fertilization mechanisms and the potential clinical use of round spermatids/elongated spermatids, we conducted detailed studies of oocyte activation and Ca2+ oscillation-inducing abilities in these immature cells and compared these abilities against those of mature spermatozoa by using micro-injection technique. Spermatids and spermatozoa were injected into oocytes using a micromanipulator with piezo-electric elements (Kimura and Yanagimachi, 1995).
Micro-injection of spermatids
Round spermatids (stage 17, Gorgi-phase to early cap phase) were easily recognized by their small size (~10 µm in diameter) and round nucleus with a centrally located nucleolar structure (Figure 1A) (Oakberk, 1956
; Ogura and Yanagimachi, 1993
; Sousa et al., 1999
). Elongated spermatids (stage 912) can be also easily identified by their distinctive morphology (Figure 1B
) (Oakberk et al., 1995).
|
|
Micro-injection of spermatozoa
A single epididymal sperm head (nucleus) was injected into oocytes in the same manner as described above. Injections of spermatozoa were performed just after sonication (within 1 h).
Control micro-injection
As a control, some oocytes were injected with a bolus (~5 µl) of mHTF without spermatids/spermatozoa in the same manner as described above.
As it is known that cooling is advantageous for micro-injection of mouse oocytes due to the more efficient healing of the pipette-made wound in the oolemma, all the procedures of intracytoplasmic injection were performed in 3 µl of mHTF on the stage cooled to 17°C. After injection, oocytes were kept at 17°C for 10 min and at room temperature for next 10 min to limit the oocyte degeneration (Kimura and Yanagimachi, 1995). Then these oocytes were washed three times in HTF and incubated under 5% CO2, 5% O2 and 90% N2 at 37°C.
During spermatid/sperm injection, no additional special procedures capable of inducing oocyte activation were performed, including vigorous cytoplasmic aspiration.
Examination of oocytes activation and embryo development
After injection, oocytes were incubated in HTF under mineral oil in a plastic Petri dish at 37°C under 5% CO2, 5% O2, 90% N2. After 45 h incubation, the oocytes were placed between slide and coverslip and fixed and stained for examination of the chromatin configuration of the spermatid/sperm and oocyte chromosome (Yanagida et al., 1991). Oocytes with a second polar body and a pronucleus (in the case of control injection) or pronuclei (spermatid/sperm injection) were considered `activated'.
In some experiments, spermatid/sperm-injected oocytes were cultured continuously in HTF for up to 96 h to examine embryo development to the morula and blastocyst stage.
Measurement of [Ca2+]i of spermatid/sperm-injected oocytes
Ca2+ responses of oocytes injected with spermatids/spermatozoa were examined by a fluorometric measurement of [Ca2+]i. Before injection, oocytes were loaded with the Ca2+-sensitive fluorescent dye fluo-3 acetoxymethyl ester (Fluo-3/AM; Molecular Probes Inc.) in dimeththylsulphoxide (final concentration 44 µmol/l in HTF) with 0.02% Pluronic F-127 for 30 min at 37°C. Loaded and injected oocytes were placed in a droplet (~3 µl) of mHTF covered with mineral oil on chambered coverglass (Lab-Tek, Nunc Inc., Naperville, IL, USA). The dish was mounted on the stage of a phase-contrast inverted microscope equipped with a Bio-Rad MRC-600 (Nippon Bio-Rad Lab., Tokyo, Japan) confocal laser scanning microscope system for fluorometrical mesurement. With most of the oocytes, measurements of Ca2+ responses were initiated 1520 min after injection, though some oocytes were measured starting immediately before the injection procedures. Ca2+ changes were sampled at 20 s intervals for ~60 min and in some cases for up to 180 min.
Examination of embryo transfer to foster mother
Morula-stage embryos of B6D2F1 mice that had developed from elongated spermatid injection and been cultured for 72 h were transferred into pregnant ICR (albino) females (1012 weeks old). The recipient females mated naturally with males (ICR) and the pregnancies were confirmed by vaginal plug on day 1. Morulae or blastocysts were transferred into the uteri of day 3 recipients. Foster mothers were allowed to deliver and raise their own pups (red eyes and white coat) as well as foster pups (black eyes and grey/brown/black coat). Some of their own pups were removed during lactation and all foster pups were allowed to develop and mature.
Statistics
Statistical significance was assessed using the 2-test; P < 0.05 was considered to be statistically significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Oocyte activation following injection of round/elongated spermatid and mature spermatozoa
Table I summarizes the results of experiments in which round/elongated spermatid or mature spermatozoa were injected into individual oocytes (B6D2F1). When round spermatids were injected, none of the oocytes were activated and all of the oocytes were arrested at metaphase II, with premature chromosome condensation of the spermatid nuclei. When electrical stimulation (1500 V/cm, 100 µs) was applied 1 h before round spermatid injection, the majority of the live oocytes (75%) were normally activated, as previously confirmed (Kimura and Yanagimachi, 1995
). When elongated spermatids were injected, most of the oocytes (87%) were normally activated without electrical stimulation, in contrast to round spermatid injection. When mature spermatozoa were injected, almost all of the oocytes were normally activated. None of the oocytes were activated with control micro-injection (i.e. injection of the same volume of medium as single round spermatid into oocytes).
|
When oocytes and spermatids/spermatozoa from ICR mice were used, 27% of oocytes injected with round spermatids were normally activated; however, almost all of the oocytes were activated when mature spermatozoa were injected and more than half when elongated spermatids were injected (Table II).
|
|
|
Nearly identical results were obtained using oocytes and spermatid/spermatozoa from ICR mice (Table IV). Although we have previously reported that oocyte activation and [Ca2+]i responses following round spermatid injection differed among species (Yazawa et al., 2000
), the oocyte activation and [Ca2+]i responses of spermatid/sperm-injected oocytes did not significantly different between B6D2F1 (F1 hybrid) and ICR (closed colony).
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In all animal species studied to date, a temporary increase of [Ca2+]i is observed after sperm-oocyte interaction, and this is believed to be a key factor of oocyte activation (Cuthbertson et al., 1981; Miyazaki and Igusa, 1981
). In mammals, unlike fish, echinoderms and frogs, the [Ca2+]i response of oocytes to the fertilizing spermatozoon exhibits a series of repetitive spikes known as `Ca2+ oscillation' (Miyazaki, 1991
; Kline and Kline, 1992
). It has been proved that intracytoplasmic sperm injection (ICSI) can induce Ca2+ oscillations similar to those of normal fertilization (Tesarik and Sousa, 1994
; Nakano et al., 1997
). However, the mechanisms by which spermatozoa generate [Ca2+]i transients and oscillations are not yet fully understood. It is currently believed that Ca2+ is released from intracellular stores (presumably endoplasmic reticulum), mainly through inositol 1,4,5-trisphosphate (IP3) receptors (Miyazaki et al., 1993
; Miyazaki, 1995
). In addition, the true importance of the oscillatory pattern of [Ca2+]i increases remains uncertain. Some authors have suggested that Ca2+ oscillation, rather than a single transient, is necessary for a complete inactivation of the MPF (Collas et al., 1993
, 1995
), and for composition of the resulting blastocyst (Hardy and Handyside, 1996
; Bos-Mikich et al., 1997
). It has been demonstrated that the developmental potential of oocytes activated with a monophasic Ca2+ transient was lower than that activated by treatment inducing Ca2+ oscillation by repetitive electrical stimulations (Ozil, 1990
). It was demonstrated that Ca2+ oscillation induced by Sr2+ has an effect on the number of ICM of blastocyst (Bos-Mikich et al., 1997
). They examined these phenomena using parthenogenetically activated rabbit and mice embryos. In general, oscillatory [Ca2+]i increases are considered essential for an optimal fertilization process of mammalian species (Raz and Shalgi, 1998
).
As discussed above, mammalian oocytes can be activated by some artificial chemical and physical stimuli such as Ca2+ ionophore, 7% ethanol, Sr2+, adenophostin (Sato et al., 1998), purified sperm factor (Sakurai et al., 1999
) and electrical stimulation. Some of these stimuli (Sr2+, adenophostin, purified sperm factor) can induce oscillatory [Ca2+]i increases, while others (Ca2+ ionophore, 7% ethanol and electrical stimulation) are known to induce only a single [Ca2+]i increase, unlike oscillatory spikes.
Recently, immature sperm cells such as round spermatids have been used for clinical treatment of men with non-obstructive azoospermia (Tesarik et al., 1996; Kahraman et al., 1998
) and for animal experiments (Ogura and Yanagimachi, 1993
; Ogura et al., 1994
; Kimura and Yanagimachi, 1995
). In mice (B6D2F1), microsurgically injected round spermatids could not induce oocyte activation at all, but normal fertilization was obtained through round spermatid injection with electrical stimulation before or after injection. After such fertilization, normal offspring were obtained after embryo transfer to the foster mother. [Ca2+]i responses of round spermatid-injected oocytes, however, was not observed. These findings suggest that Ca2+ oscillations may be induced by round spermatid injection applied with electrical stimulation; if not, Ca2+ oscillations may not be essential for normal embryonic development to offspring. This was the first question to consider before we started this experiment.
Oocytes cooling during the injection procedure and measurements of [Ca2+]i of injected oocytes
As explained in the Materials and methods section, it is advantageous for post-injection survival of mice oocytes to cool them to 17°C during and after the ICSI procedure. The oolemma of mouse oocytes is far more elastic and the `wound-healing' capacity after micro-injection is inferior to that of other animals (Kimura and Yanagimachi, 1995). All the micro-injection procedures were performed on a stage cooled to 17°C, and injected oocytes remained on the cooled stage of an inverted microscope for about 10 min. [Ca2+]i was then measured using another microscope equipped with an image processor. For these reasons, [Ca2+]i was measured for most of the spermatid/sperm-injected oocytes starting 15 min after injection in this experiment. It is known that a large [Ca2+]i increase occurs immediately after cell injection and follows oscillatory spikes in fertilizing oocytes (Swann, 1992
). This large initial [Ca2+]i increase seemed to occur within the first 15 min of incubation for cooling in most of the oocytes and it was not recorded. [Ca2+]i measurement of some oocytes was initiated before the injection procedure; the initial single increase of [Ca2+]i occurred in all of the oocytes within 10 min. That is to say, in all oocytes for which measurements began 15 min after injection, one more increase of [Ca2+]i might occur. For some oocytes, [Ca2+]i was measured for up to 180 min and no changes in the patterns of [Ca2+]i response observed during these observation periods. Based on these findings, we were able to confirm that our experimental set-up was appropriate for examination of [Ca2+]i responses of spermatid/sperm-injected oocytes.
Activation and Ca2+ oscillation-inducing abilities of round/elongated spermatids
When round spermatids (B6D2F1) were injected, none of the oocytes was activated and no [Ca2+]i responses were observed. These findings confirmed that round spermatids had no oocyte activation- and Ca2+ oscillation-inducing abilities. When elongated spermatid were injected without any artificial stimulation for activation, most of the oocytes (87%) were normally activated and most oocytes (94%) also exhibited transient patterns (type C) of [Ca2+]i, not oscillation patterns (type A). When mature spermatozoa were injected, almost all oocytes were normally activated and exhibited normal oscillation patterns (type A). These results confirm that the [Ca2+]i patterns of oocytes injected with immature sperm cells transform from a transient pattern (type C) to an oscillation pattern (type A) while maturing to spermatozoa. The results also confirm that oocyte activation-inducing ability is acquired at the stage of elongated spermatid and that Ca2+ oscillation-inducing ability is acquired at the mature-sperm stage of spermiogenesis. In addition, we observed a dissociation between the timing of the acquisition of the oocyte activation-inducing ability and that of Ca2+ oscillation-inducing ability of developing immature sperm cells. This may be the first report that there is such a situation as oocyte activation occurring without Ca2+ oscillation in mammalian fertilization without artificial stimulation. In another experiment, this dissociation between activation and Ca2+ oscillation was observed in the immature sperm cells of other species injected into mice oocytes [for example, round spermatids of hamster and rabbit, and elongated spermatids of rat (Yazawa et al., 2000)]. In this experiment, we were able to obtain some normal offspring after the transfer of embryos from elongated spermatid injection to foster mothers. We also confirmed that round spermatid injection applied with electrical stimulation 1 h before injection did not exhibit [Ca2+]i oscillation patterns. These two findings indicated that Ca2+ oscillation must not be always essential for normal embryo development to term, at least in the mice. This may be the answer to our first question.
When elongated spermatids were injected, the rate of embryo development to blastocysts was significantly lower than that of sperm-injected oocytes (Table V). Therefore, Ca2+ oscillation may play some role in fertilization and embryo development, as described above (Collas et al., 1993
, 1995
; Hardy and Handyside, 1996
; Bos-Mikich, et al., 1997
; Raz and Shalgi, 1998
).
It is not known whether sperm-borne oocyte-activating factor (SOAF) and Ca2+ oscillation-inducing factor (COIF) are identical, or how these factors change (e.g. structural transformation of molecules or changes of dosage; quality or quantity) while spermatogenic cells matured from spermatids to spermatozoa. We presume that these factors may be identical (i.e. sperm-borne oocyte activation and Ca2+ oscillation-inducing factor; `SOA-COIF') and, if so, this factor may increase as immature sperm cells mature, and the thresholds of the oocytes to induce oocyte activation and Ca2+ oscillation may differ (with the former threshold being less than the latter). When two elongated spermatids were injected into each oocyte, most of the oocytes exhibited normal oscillation patterns while oocytes injected with single elongated spermatids exhibited transient patterns. Perhaps a single elongated spermatid has insufficient SOA-COIF, whereas two elongated spermatids contain enough SOA-COIF to induce normal oscillation patterns. These findings partly confirm the above hypothesis.
In conclusion, we confirmed that SOA-COIF became gradually active and that the [Ca2+ ]i patterns of spermatid-injected oocytes changed from transient patterns to oscillation patterns during spermiogenesis. In addition, we directly observed that Ca2+ oscillation is not essential for development to normal offspring, which conflicts with the conventional notion that Ca2+ oscillation is essential in mammalian fertilization without any artificial stimulation.
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Collas, P., Chang, T., Long, C. and Robl, J.M. (1995) Inactivation of histone H1 kinase by Ca2+ in rabbit oocytes. Mol. Reprod. Dev., 40, 243258.
Collas, P., Sullivan, E.J. and Barnes, F.L. (1993) Histone H1 kinase activity in bovine oocytes following calcium stimulation. Mol. Reprod. Dev., 34, 224231.[ISI][Medline]
Cuthbertson, K.S.R., Whittingham, D.G. and Cobbold, P.H. (1981) Free Ca2+ increases in exponential phases during mouse oocyte activation. Nature, 294, 754747.[ISI][Medline]
Fishel, S., Green, S., Hunter, A. et al. (1997) Human fertilization with round and elongated spermatids. Hum. Reprod., 12, 336340.[Abstract]
Hardy, K. and Handyside, A.H. (1996) Metabolism and cell allocation during parthenogenetic preimplantation mouse development. Mol. Reprod. Dev., 43, 313322.[ISI][Medline]
Kahraman, S., Polat, G. and Samli, M. et al. (1998) Multiple pregnancies obtained by testicular spermatid injection in combination with intracytoplasmic sperm injection. Hum. Reprod., 13, 104110.[Abstract]
Kimura, Y. and Yanagimachi, R. (1995) Mouse oocytes injected with testicular spermatozoa or round spermatids can develop into normal offspring. Development, 121, 23972405.
Kline, D. and Kline, J.T. (1992) Repetitive calcium transients and the role of calcium in exocytosis and cell cycle activation in the mouse egg. Dev. Biol., 149, 8089.[ISI][Medline]
Kuretake, S., Kimura, Y., Hoshi, K. and Yanagimachi, R. (1996) Fertilization and development of mouse oocytes injected with isolated sperm heads. Biol. Reprod., 55, 789795.[Abstract]
Miyazaki, S. (1991) Repetitive calcium transients in hamster oocytes. Cell Calcium, 12, 205216.[ISI][Medline]
Miyazaki, S. (1995) Inositol trisphosphate receptor mediated spatiotemporal calcium signaling. Curr. Opin. Cell Biol., 7, 190196.[ISI][Medline]
Miyazaki, S. and Igusa, Y. (1981) Fertilization potential in golden hamster eggs consists of recurring hyperpolarizations. Nature, 290, 702704.[ISI][Medline]
Miyazaki, S., Shirakawa, H., Nakada, K. et al. (1993) Essential role of the inositol 1,4,5-trisphosphate receptor /Ca2+ release channel in Ca2+ waves and Ca2+ oscillations at fertilization of mammalian eggs. Dev. Biol., 158, 6278.[ISI][Medline]
Nakano, Y., Shirakawa, H., Mitsuhashi, N. et al. (1997) Spatiotemporal dynamics of intracellular calcium in the mouse egg injected with a spermatozoa. Mol. Hum. Reprod., 3, 10871093.[Abstract]
Oakberk, A.R. (1956) Description of spermatogenesis in the mouse and its use in analysis of the cycle of the seminiferous epithelium and germ cell renewal. Am. J. Anat., 99, 391413.[ISI]
Ogura, A. and Yanagimachi, R. (1993) Round spermatid nuclei injection into hamster oocytes form pronuclei and participate in syngamy. Biol. Reprod., 48, 219225.[Abstract]
Ogura, A., Matsuda, J. and Yanagimachi, R. (1994) Birth of normal young after electrofusion of mouse oocytes with round spermatids. Proc. Natl. Acad. Sci. USA, 91, 74607462.[Abstract]
Ozil, J.P. (1990) The parthenogenetic development of rabbit oocytes after repetitive pulsatile electric stimulations. Development, 109, 117127.[Abstract]
Pharrington, J., Lai, F.A. and Swann, K. (1998) A novel protein for Ca2+ signalling at fertilization. Curr. Top. Dev. Biol., 39, 215243.[ISI][Medline]
Raz, T., and Shalgi, R. (1998) Early events in mammalian egg activation. Hum. Reprod., 13 (Suppl.), 133145.[Medline]
Sagata, N. (1996) Meiotic metaphase arrest in animal oocytes: its mechanisms and biological significance. Trends Cell Biol., 6, 2228.[ISI]
Sakurai, A., Shoji, O., Kuwabara, Y. and Miyazaki, S. (1999) Fertilization, embryonic development, and offspring from mouse eggs injected with round spermatids combined with Ca2+ oscillation-inducing sperm factor. Mol. Hum. Reprod., 5, 132138.
Sasagawa, I. and Yanagimachi, R. (1996) Comparison of methods for activating mouse oocytes for spermatid nucleus transfer. Zygote, 4, 269274.[ISI][Medline]
Sato,Y., Miyazaki, S., Shikano, T. et al. (1998) Adenophostin, a potent agonist of inositol 1,4,5-trisphosphate receptor, is useful for fertilization of mouse oocytes injected with round spermatids leading to normal offspring. Biol. Reprod., 58, 867873.[Abstract]
Shultz, M.R. and Kopf, G.S. (1995) Molecular basis of mammalian egg activation. Curr. Top. Dev. Biol., 30, 2162.[ISI][Medline]
Sofikitis, N., Toda, T., Miyagawa, I. et al. (1996) Beneficial effects of electrical stimulation before round spermatid nuclei injections into rabbit oocytes on fertilization and subsequent embryonic development. Fertil. Steril., 65, 176185.[ISI][Medline]
Sousa, M., Barros, A., Takahashi, K. et al. (1999) Clinical efficacy of spermatid conception: analysis using a new spermatid classification scheme. Hum. Reprod., 14, 12791286.
Swann, K., (1992) Different triggers for calcium oscillations in mouse eggs, involved a ryanodine-sensitive calcium store. Biochem. J., 287, 7984.[ISI][Medline]
Swann, K. and Lai, F.A. (1997) A novel signaling mechanism for generating Ca2+ oscillations at fertilization in mammals. Bioessays, 19, 371378.[ISI][Medline]
Tesarik, J. and Sousa, M. (1994) Composition of Ca2+ responses in human oocytes fertilized by subzonal insemination and by intracytoplasmic sperm injection. Fertil. Steril., 62, 11971204.[ISI][Medline]
Tesarik, J., Rolet, F., Brami, C. et al. (1996) Spermatid injection into human oocytes? Clinical application in the treatment of infertility due to non-obstructive azoospermia. Hum. Reprod., 11, 780783.[Abstract]
Vanderzwalmen, P., Zech, H., Birkenfeld, A. et al. (1997) Intracytoplasmic injection of spermatids retrieved from testicular tissue: influence of testicular pathology, type of selected spermatids and oocyte activation. Hum. Reprod., 12, 12031213.[ISI][Medline]
Yanagida, K., Yanagimachi, R., Perreault, S.D. et al. (1991) Thermostability of sperm nuclei assessed by micro-injection into hamster oocytes. Biol. Reprod., 44, 440447.[Abstract]
Yazawa, H., Yanagida, K., Katayose, H. et al. (2000) Comparison of oocyte activation and Ca2+ oscillation-inducing abilities of round/elongated spermatids of mouse, hamster, rabbit, rat and human assessed by mouse oocyte activation assay. Hum. Reprod., 15, 25822590.
Submitted on September 14, 2000; accepted on February 14, 2001.