Oocyte activation and Ca2+ oscillation-inducing abilities of mouse round/elongated spermatids and the developmental capacities of embryos from spermatid injection

Hiroyuki Yazawa1, Kaoru Yanagida and Akira Sato

Department of Obstetrics and Gynecology, Fukushima Medical University, 1 Hikarigaoka Fukushima, Fukushima 960-1295, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To investigate differences in fertilization mechanisms and the potential clinical use of round/elongated spermatid, we conducted detailed studies of oocyte activation and Ca2+ oscillation-inducing abilities in these immature sperm cells and compared these abilities against those of mature spermatozoa. When round spermatids from B6D2F1 mice were injected, none of the oocytes was activated and no intracellular Ca2+ ([Ca2+]i) increases were observed. Elongated spermatids could induce activation normally in 87% of injected oocytes, but Ca2+ oscillation could not be induced at all and most of the oocytes (94%) exhibited only several transient [Ca2+]i rises (transient patterns). Because normal offspring could be obtained when embryos through elongated spermatid injection were transferred to foster mothers, it seems that a normal oscillation pattern of [Ca2+]i is not essential for normal fertilization and embryo development. [Ca2+]i patterns of injected oocytes changed from transient patterns to oscillation patterns while the injected immature sperm cells were maturing to spermatozoa. Dissociations were seen between the timing of appearance of oocyte activation and that of Ca2+ oscillation-inducing abilities in maturing sperm cells. These dissociations may be due to differences in the thresholds to oocyte activation and Ca2+ oscillation-inducing factor for inducing oocyte activation and Ca2+ oscillation.

Key words: calcium oscillation/embryo development/intracytoplasmic spermatid injection/mouse/oocyte activation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Since fertilization by round spermatid nucleus was first reported (Ogura and Yanagimachi, 1993Go), many animal experiments using immature spermatogenic cells (spermatid and spermatocyte) have been conducted (Ogura et al., 1994Go; Kimura and Yanagimachi, 1995Go; Sasagawa and Yanagimachi, 1996Go; Sofikitis et al., 1996Go). Clinical therapies for non-obstructive azoospermia have also been investigated (Tesarik et al., 1996Go; Fishel et al., 1997Go; Vanderzwalmen et al., 1997Go; Sousa et al., 1999Go). Fertilization and pregnancy rates after injection of immature sperm cells have tended to be lower than those of mature or testicular spermatozoa. It is thought that spermatids have immature function of fertilization. For example, mice round spermatids could not induce oocyte activation when injected without any artificial induction of oocyte activation, but round spermatid-injected oocytes treated with electrical stimulation could develop normally to offspring (Kimura and Yanagimachi, 1995Go). The mechanisms of fertilization with immature sperm cells are still subject to debate.

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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Preparation of oocytes
B6D2F1 and ICR female mice (6–8 weeks old) were superovulated with i.p. injection of 8 IU pregnant mare's serum gonadotrophin (PMSG; Teikokuzouki Co., Tokyo, Japan) followed by 8 IU human chorionic gonadotrophin (HCG; Mochida Pharmaceutical Co., Tokyo, Japan) 48 h later. Mature oocytes were collected from the oviducts ~16 h after HCG injection. They were freed from cumulus cells by treatment with 0.1% hyaluronidase in HEPES-buffered human tubal fluid medium (mHTF; Irvine Scientific, Santa Ana, CA, USA) supplemented with 10% synthetic serum substitute (SSS; Irvine Scientific). The cumulus-free oocytes were rinsed thoroughly and placed in drops of human tubal fluid medium (HTF; Irvine Scientific) with 10% SSS covered with mineral oil for up to 2.5 h at 37°C under 5% CO2, 5% O2 and 90% N2.

Preparation of round/elongated spermatids and mature spermatozoa
Preparation of round/elongated spermatids
Testes were isolated from mature male B6D2F1 and ICR mice (8–10 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 (8–10 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 10–15 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., 1996Go). 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, 1995Go).

Micro-injection of spermatids
Round spermatids (stage 1–7, 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 1AGo) (Oakberk, 1956Go; Ogura and Yanagimachi, 1993Go; Sousa et al., 1999Go). Elongated spermatids (stage 9–12) can be also easily identified by their distinctive morphology (Figure 1BGo) (Oakberk et al., 1995).



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Figure 1. Round spermatids (A) and elongated spermatids (B) retrieved from mouse testicular tissue and suspended in medium. Scale bar = 10 µm.

 
A round or elongated spermatid was sucked into an injection pipette (5–6 µm inner diameter at the tip) attached to a piezo-electric driving unit (model PMM-MB-A; Prime Tech Ltd, Tuchiura City, Japan) (Figure 2A and BGo). A mature unfertilized oocyte was held by a holding pipette and its zona pellucida was penetrated by applying several piezo pulses (Figure 2CGo). After the needle had been advanced deeply into the ooplasm, the oolemma was punctured with one piezo pulse and the entire round/elongated spermatid was slowly expelled into the ooplasm before the pipette was gently withdrawn (Figure 2D and EGo). In some experiments, two round or elongated spermatids were injected into a single oocyte to examine the dose of oocyte activation and Ca2+ oscillation-inducing substances contained in a round/elongated spermatid.



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Figure 2. Round spermatid (ROS) injection into oocyte using piezo micromanipulator. A round spermatid was sucked into an injection pipette attached to a piezo-electric pipette driving unit (A, B). The zona pellucida of the mature unfertilized oocyte was penetrated by applying several piezo pulses (C). The needle was advanced deeply into the ooplasm without piezo pulse and then the oolemma was punctured with one piezo pulse (D) before the pipette was gently expelled (E). Scale bar = 10 µm.

 
As it was known that mouse (B6D2F1) round spermatids could not activate oocytes, some oocytes were injected with round spermatids 1 h after electric stimulation (1500 V/cm, 100 µs, Model FTC-03; Shimazu Co., Tokyo, Japan) to induce oocyte activation.

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, 1995Go). 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 4–5 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., 1991Go). 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 15–20 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 (10–12 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 {chi}2-test; P < 0.05 was considered to be statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this experiment, we observed oocyte activation and Ca2+ oscillation following spermatid/sperm injection, using homologous oocytes and spermatids/spermatozoa of two different strains of mice (B6D2F1 and ICR).

Oocyte activation following injection of round/elongated spermatid and mature spermatozoa
Table IGo 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, 1995Go). 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).


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Table I. Activation of mouse (B6D2F1) round spermatid (ROS)/elongated spermatid (ELS)- and sperm-injected oocytes
 
When two round spermatids were injected into individual oocytes (B6D2F1), none of the oocytes was activated. Control injections (using the same volume of medium as two round spermatids) produced the same results as above.

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)Go.


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Table II. Activation of mouse (ICR) round spermatid (ROS)/elongated spermatid (ELS)- and sperm-injected oocytes
 
Ca2+ oscillation following injection of round/elongated spermatids and mature spermatozoa
We classified [Ca2+]i response patterns of injected oocytes into four groups as shown in Figure 3Go. Normal oscillation patterns (type A) consisted of repetitive spike-shaped [Ca2+]i increases with intervals at 2–10 min. Abnormal oscillation patterns (type B) consisted of continuous [Ca2+]i elevation for 20–30 min. before the normal oscillation pattern. This pattern was rare in intact (i.e. not degenerated after the injection procedure) oocytes, and, in this experiment, we observed only 2 type B patterns out of 183 [Ca2+]i measured oocytes. Transient patterns (type C) consisted of several (1–4) transient [Ca2+]i increases. Durations of these [Ca2+]i transients were usually prolonged for 3–5 min and could be distinguished from the spike-shaped [Ca2+]i increases of type A patterns. No-response patterns (type D) consisted of no [Ca2+]i increases during the observation period.



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Figure 3. Intracellular calcium ([Ca2+]i) patterns of spermatid/sperm-injected oocytes were classified into four groups (Types A-D). Type A: normal oscillation pattern, consists of repetitive spike-shaped [Ca2+]i rises which intervals are 2–10 min. Type B: abnormal oscillation pattern, consists of a combination of continuous [Ca2+]i rise and oscillation pattern. Type C: transient pattern, consists of several (1–4) transient [Ca2+]i rises. Type D: no-response pattern, consists of no [Ca2+]i rises during observation periods. x-axis: time after starting measurement of [Ca2+]i; y-axis: intensity.

 
Table IIIGo summarizes the results of [Ca2+]i measurements of spermatid/sperm-injected oocytes (B6D2F1). All of the round spermatid-injected oocytes exhibited type D patterns. In the case of round spermatid injection with electrical stimulation, none of the oocytes exhibited oscillation patterns; most exhibited type D patterns. No Ca2+ oscillation patterns were produced and almost all of the oocytes exhibited transient patterns (type C) after injection of elongated spermatids, even though most of them were normally activated (Tables I and IIIGoGo). All of the oocytes injected with spermatozoa exhibited normal oscillation patterns.


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Table III. [Ca2+]i responses of mouse (B6D2F1) round spermatid (ROS)/elongated spermatid (ELS)- and sperm-injected oocytes
 
When two round spermatids were injected into each oocyte (B6D2F1), some of the oocytes (29%) exhibited transient patterns (type C); when two elongated spermatids were injected into each oocyte, most of the oocytes (86%) exhibited normal oscillation patterns (type A) even though none had exhibited any oscillation pattern after single-elongated spermatid injection.

Nearly identical results were obtained using oocytes and spermatid/spermatozoa from ICR mice (Table IVGo). Although we have previously reported that oocyte activation and [Ca2+]i responses following round spermatid injection differed among species (Yazawa et al., 2000Go), the oocyte activation and [Ca2+]i responses of spermatid/sperm-injected oocytes did not significantly different between B6D2F1 (F1 hybrid) and ICR (closed colony).


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Table IV. [Ca2+]i responses of mouse (ICR) round spermatid (ROS)/elongated spermatid (ELS)- and sperm-injected oocytes
 
Culture and transfer of embryos obtained by injection of elongated spermatid into oocytes (B6D2F1)
Tables V and VIGoGo summarize results of the culture and transfer to the foster mother of embryos derived from the oocytes fertilized by injection of elongated spermatid (B6D2F1). One hundred and fifty-one oocytes were injected with elongated spermatids: 90% of surviving oocytes were fertilized and 43% of fertilized oocytes developed into blastocyst stage when cultured for 96 h. The rate of embryo development to blastocyst stage (43%) was significantly lower than that of sperm-injected embryos (61%, P < 0.02). The results of embryo transfer indicate that elongated spermatid-injected oocytes can develop into live offspring (Table VIGo). All the newborns grew into normal adults and all proved to be fertile.


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Table V. In-vitro development of mouse oocytes fertilized by injection of elongated spermatids (ELS) or spermatozoa (fertilized oocytes were cultured for 96 h after micro-injection)
 

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Table VI. Term development of embryos derived from the oocytes fertilized by injection of elongated spermatids
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background of the experiments
The exact mechanisms of oocyte activation and [Ca2+]i response during fertilization remain subject to debate. One of the principal events of oocyte activation is thought to be the inactivation of metaphase-promoting factor (MPF) which has blocked the cell cycle of oocytes at the metaphase of second meiotic division (Sagata, 1996Go). Some researchers have suggested that the oocyte is activated either by binding a spermatozoon to receptors on the oocyte plasma membrane to initiate a signal transduction pathway (Miyazaki et al., 1993Go; Shultz and Kopf, 1995Go), or by introducing sperm-borne oocyte-activating factors (SOAF) into the oocyte cytoplasm (Swann and Lai, 1997Go; Pharrington et al., 1998Go).

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., 1981Go; Miyazaki and Igusa, 1981Go). 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, 1991Go; Kline and Kline, 1992Go). It has been proved that intracytoplasmic sperm injection (ICSI) can induce Ca2+ oscillations similar to those of normal fertilization (Tesarik and Sousa, 1994Go; Nakano et al., 1997Go). 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., 1993Go; Miyazaki, 1995Go). 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., 1993Go, 1995Go), and for composition of the resulting blastocyst (Hardy and Handyside, 1996Go; Bos-Mikich et al., 1997Go). 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, 1990Go). It was demonstrated that Ca2+ oscillation induced by Sr2+ has an effect on the number of ICM of blastocyst (Bos-Mikich et al., 1997Go). 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, 1998Go).

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., 1998Go), purified sperm factor (Sakurai et al., 1999Go) 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., 1996Go; Kahraman et al., 1998Go) and for animal experiments (Ogura and Yanagimachi, 1993Go; Ogura et al., 1994Go; Kimura and Yanagimachi, 1995Go). 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, 1995Go). 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, 1992Go). 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., 2000Go)]. 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 VGo). Therefore, Ca2+ oscillation may play some role in fertilization and embryo development, as described above (Collas et al., 1993Go, 1995Go; Hardy and Handyside, 1996Go; Bos-Mikich, et al., 1997Go; Raz and Shalgi, 1998Go).

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
 
1 To whom correspondence should be addressed. E-mail: h-yazawa{at}fmu.ac.jp Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on September 14, 2000; accepted on February 14, 2001.





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