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Regular Article |
a Department of Veterinary Science, National Institute of Infectious Diseases, Tokyo 162-8640, Japan
b Tsukuba Primate Center, National Institute of Infectious Diseases, Ibaraki 305-0843, Japan
c Laboratory Animal Research Center, University of Tsukuba, Ibaraki 305-8575, Japan
d Department of Physiology, Tokyo Women's Medical University of Medicine, Tokyo 162-8666, Japan
e Laboratory of Intracellular Metabolism, National Institute for Physiological Sciences, Okazaki 444-8585, Japan
ABSTRACT
It is widely accepted that mature mammalian oocytes are induced to resume meiosis by a sperm-borne oocyte-activating factor(s) (sperm factor, SF) immediately after normal fertilization or intracytoplasmic sperm injection. The SF is most likely a soluble factor that is localized within the cytoplasm of mature spermatozoa, but the exact stage at which it appears during spermatogenesis and its localization after oocyte activation is not fully understood, except in the mouse. First, we injected mature spermatozoa and spermatogenic cells from cynomolgus monkeys into mouse oocytes to assess their oocyte-activating capacity. More than 90% of mouse oocytes were activated after injection of monkey spermatozoa. Round spermatids and primary spermatocytes (late pachytene to diplotene) also activated oocytes (93% and 79%, respectively). Injection of monkey spermatozoa and spermatids induces intracellular Ca2+ oscillations in a pattern similar to that seen following normal fertilization. Most spermatocytes did not produce typical intracellular Ca2+ oscillations. Second, we transferred pronuclei or cytoplasts from mouse oocytes that had been activated by monkey spermatozoa or spermatids into intact mature mouse oocytes by electrofusion in order to examine the localization of the SF after pronuclear formation. Some of the SF was localized within the pronuclei, but some stayed in the ooplasm. This study demonstrated that spermatogenic cells of cynomolgus monkeys acquire oocyte-activating capacity at much earlier stages than those of mice, and that the monkey SF has a pronucleus-directing nature, although to a lesser extent than the mouse SF.
calcium, fertilization, sperm, spermatid, spermatogenesis
INTRODUCTION
The fertilizing spermatozoon delivers a male genome into an oocyte and activates the oocyte, leading to the resumption of meiosis and then the initiation of normal embryonic development. Two possible pathways of oocyte activation have been proposed: a transmembrane receptor mechanism involving G-proteins and a sperm-borne oocyte-activating factor (SF) mechanism [1, 2]. The recent data on intracytoplasmic sperm injection (ICSI) favors the latter hypothesis, because ICSI bypasses the sperm–egg fusion process [3]. Active SF has been demonstrated in the cytosolic fraction of spermatozoa from rabbits [4], hamsters [5–7], boars [5, 8], rhesus monkeys [9], and humans [10]. The SF molecules must be produced and stored in spermatogenic cells some time before the completion of spermiogenesis, because mature spermatozoa are translationally inactive. A recent study showed that human SF is already present at the round spermatid stage, as demonstrated by their oocyte-activating capacity and the generation of intracellular Ca2+ oscillations following intracytoplasmic injection [11]. By contrast, in the mouse, round spermatids eventually have no oocyte-activating capacity, while the capacity for elongating spermatids is intermediate between that of round spermatids and that of mature spermatozoa [12]. Therefore, the stage at which SF first appears or becomes biologically active during spermatogenesis may vary with species. The active SF is gradually accumulated into elongating spermatids in other laboratory rodents, including Mongolian gerbils (Meriones unguiculatus) and mastomys (Praomys coucha) (unpublished results). Other than humans, however, we do not know of any animals in which round spermatids have full oocyte-activating capacity [11]. The first purpose of this study was to determine whether the relatively early expression of SF reported in humans is common to other nonhuman primates. We chose the cynomolgus monkey (Macaca fascicularis) as a model, because their testicular cells are easily collected in biopsy samples [13]. Because there is very little, if any, specificity in the SF activity between species [4–10, 12], we injected monkey spermatogenic cells into mouse oocytes to assess their oocyte-activating capacity. It has already been demonstrated that this experimental system is effective for human spermatozoa [14].
At fertilization, repetitive increases in intracellular Ca2+ concentration ([Ca2+]i) called Ca2+ oscillations occur in all mammalian species studied to date, due to Ca2+ release from the endoplasmic reticulum mainly through the inositol 1,4,5-trisphosphate receptor [2]. Injection of sperm extract into eggs induces intracellular Ca2+ oscillations similar to those seen at fertilization [7, 8, 10, 11, 15, 16]. Intracellular Ca2+ oscillations cause cortical granule exocytosis and resumption of meiosis [17], which is then followed by pronucleus formation and DNA synthesis. Because the Ca2+ oscillations reappear at metaphase of the first mitosis [18], SF could be stored somewhere within oocytes during interphase (pronuclear stage) until release. By transferring either pronuclei or cytoplasts of fertilized mouse oocytes into homologous metaphase (M) II-arrested oocytes, Kono et al. [19] clearly demonstrated that SF is incorporated into the male and female pronuclei as intracellular Ca2+ oscillation attenuates and stays there until nuclear membrane breakdown at the first mitosis. This finding facilitated understanding of the biochemical nature of SF, while every attempt by biochemical analysis has failed to characterize the SF molecule(s) [20, 21]. This unique behavior of SF after pronuclear formation has been confirmed only in the mouse, and we do not know if this is common in other mammalian species. The second purpose of this study was to trace the monkey SF after oocyte activation, by transferring each component of fertilized oocytes into intact M II oocytes.
MATERIALS AND METHODS
Preparation of Mouse Oocytes
B6D2F1 female mice (7- to 10-wk-old) were injected with 5 IU of eCG followed 48 h later by 5 IU of hCG. Mature oocytes were collected from oviducts 15–17 h after hCG injection and were freed from cumulus cells by treatment with 0.1% hyaluronidase in CZB medium [22]. The oocytes were transferred to fresh CZB medium and incubated at 37°C in an atmosphere of 5% CO2:95% air until micromanipulation.
Collection of Spermatozoa and Spermatogenic Cells
Cynomolgus monkey (Macaca fascicularis) spermatozoa and spermatogenic cells were collected from sexually mature males that were bred and maintained in the Tsukuba Primate Center (TPC), National Institute of Infectious Diseases, Japan. Each male was anesthetized using 10–15 mg ketamine hydrochloride per kilogram of body weight (Ketalar; Sankyo, Tokyo, Japan) [23]. Epididymal spermatozoa were collected from surgically extruded caudae epididymides. The caudae were minced in 10 ml of TYH medium [24] and incubated for 30 min in an atmosphere of humidified 5% CO2 in air. The tissue was then layered onto 3 ml of 90% Percoll (Pharmacia, Uppsala, Sweden) and centrifuged at 800 x g for 10 min. After confirming that the sedimented spermatozoa had good motility, they were then frozen in cryostraws, according to the method of Sankai et al. [25]. On the day of experiments, the frozen sperm samples were thawed using a rapid warming method (i.e., a water bath at 37°C for 30 sec), gently layered onto 2 ml of 90% Percoll, and centrifuged at 800 x g for 10 min. Spermatozoa with good motility were recovered from the pellet.
Monkey spermatogenic cells were obtained from the testis by testicular needle biopsy [13]. The biopsy samples were treated to collect spermatogenic cells mechanically as reported previously for the golden hamster [26]. In brief, samples containing seminiferous tubules were transferred into Hepes-CZB medium and cut into small pieces with fine scissors. The pieces of seminiferous tubules were pipetted gently to release spermatogenic cells into the medium. The cell suspension was filtered through a 50-µm nylon mesh, then centrifuged at 700 x g for 5 min at 4°C. The cells were resuspended in Hepes-CZB medium and washed twice by centrifugation. The cell suspension was stored at 4°C until use. Some of the monkey spermatogenic cells were cryopreserved for later use, according to the methods of Ogura et al. [27].
To ascertain the reliability of our techniques for intracytoplasmic injection and nuclear transfer, we also conducted control experiments using mouse sperm and spermatogenic cells. Mouse spermatozoa were collected from the cauda epididymides and spermatogenic cells from the testes of mature C57BL/6 or B6D2F1 male mice.
Microinjection of Sperm and Spermatogenic Cells
Microinjection was carried out using a micromanipulation system equipped with a Piezo micropipette driving unit (Prime Tech, Ibaraki, Japan). The cover of a plastic dish (Falcon no. 1006; Becton Dickinson, Franklin Lakes, NJ) was used as a microinjection chamber. Several small drops (1–2 µl), each containing Hepes-CZB (for oocytes) or 12% polyvinylpyrrolidone (PVP), were placed on the bottom and covered with silicone oil (Aldrich Chemical Co., Milwaukee, WI). Spermatozoa and spermatogenic cells were suspended in one of the PVP drops. A spermatozoon of apparently normal morphology was inserted tail-first into an injection pipette. A monkey spermatozoon, or mouse sperm head separated from the tail by applying a few Piezo pulses, was then injected into an oocyte. A round spermatid, spermatocyte, or early spermatocyte was drawn into the injection pipette and injected into the ooplasm after breaking the plasma membrane. The procedure of intracytoplasmic injection using the Piezo micromanipulator was exactly as reported previously [28, 29].
Examination of Oocytes
In this study, we employed two criteria for activation of injected oocytes. One was the presence of pronuclei discernible by an inverted microscope 4–5 h after sperm/spermatogenic cell injection. The other was the presence of pronuclei within whole-mounted oocytes observed by phase-contrast microscopy 2.5–3 h after injection [30]. The latter contained pronuclei that were smaller than those of the former, but our preliminary experiments confirmed that these two criteria were ultimately equivalent for determining oocyte activation.
Measurement of [Ca2+]i
[Ca2+]i was measured by a Ca2+-imaging method described previously [31, 32]. In brief, oocytes were loaded with the Ca2+-sensitive fluorescent dye fura-2 acetoxymethylester (fura-2 AM; Molecular Probes Inc., Eugene, OR). The stock solution contained 2 mM fura-2 AM in dimethylsulfoxide. It was freeze-stored and diluted to 5 µM in Hepes-CZB before use. Oocytes were kept in the medium containing fura-2 AM for 7–9 min at room temperature and then rinsed thoroughly with Hepes-CZB. A group of oocytes (three to five) was successively injected with sperm or spermatogenic cells as described above. Those eggs were transferred to a dish for Ca2+ imaging in another microscope system that was connected to an image processor (Argus 200; Hamamatsu Photonics, Hamamatsu, Japan). They were placed in the same optic field, and [Ca2+]i was measured simultaneously. The time lag between injection and the start of [Ca2+]i measurement was between 2 min (the last injected oocyte) and 12 min (the first injected oocyte). To excite fura-2 AM fluorescence, UV light at wavelengths of 340 or 380 nm (UV340 or UV380) was produced by a xenon lamp and a 340 ± 10 or 380 ± 10 nm narrow bandpass filter, respectively, and shone on the oocyte through a x40 objective lens (Fluor 40; Nikon, Tokyo, Japan). The emission fluorescence (F) passed to a silicon intensifier target camera through a 510 ± 10-nm bandpass filter. The Ca2+ images were sampled at 20-sec intervals by applying UV340 for 0.25 sec, followed 0.8 sec later by UV380 for 0.25 sec for 60–120 min. Data sets were later processed to calculate the ratio R = F340/F380. A calibration curve between R and [Ca2+]i was obtained by measuring R for Ca2+-EDTAOH (N-[2-hydroxyethyl]ethylenedinitrilotriacetic acid) buffer solutions. After measurement of [Ca2+]i, the oocytes were examined for the presence of pronuclei by whole-mount preparation as described above [30].
Nuclear/Cytoplasm Transfer
To localize the sperm/spermatogenic cell-derived SF in the activated oocytes, their pronuclei, cytoplast, or second polar body were transferred into freshly recovered mouse mature oocytes by electrofusion 4–5 h after injection with spermatozoa or spermatogenic cells. Mouse pronuclear oocytes that had been fertilized by natural mating or parthenogenetically activated by SrCl2 [19] were used as donors in the control nuclear transfer experiments. The media used for oocytes before and during nuclear transfer were CZB and Hepes-CZB, respectively. This contained 20 mg/ml sucrose and 6.6 µg/ml cytochalasin B, so that micromanipulation could be performed more safely. The pronuclei, second polar body, and cytoplast removed from the donor oocytes were inserted into the perivitelline space of the recipient oocytes using a Piezo micromanipulator. About 20 min later, the oocytes that had received a donor karyoplast or cytoplast were placed in fusion medium containing 300 mM mannitol, 100 µM MgSO4, and 0.1 mg/ml polyvinylalcohol, and exposed to a single direct current pulse (3000 V/cm, 10 µsec). After 2 h in culture, oocytes that fused with the donor karyoplast or cytoplast were examined for activation by whole-mount preparation [30]. Oocytes that proceeded to telophase II or further were considered to be activated.
Statistical Analysis
The results were evaluated using Fisher exact probability test, and a P value less than 0.05 was considered statistically significant.
RESULTS
Oocyte-Activating Capacity of Spermatozoa and Spermatogenic Cells
First, we injected either a single mouse spermatozoon or round spermatid into a mature mouse oocyte. As has been already reported [29, 32], mouse spermatozoa successfully activated homologous oocytes, but round spermatids and primary spermatocytes did not (Table 1). As shown in Figure 1a, an oocyte that received a mouse round spermatid stayed at M II and induced premature condensation of the spermatid chromosomes. Thus, we confirmed that our injection technique was reliable enough to be applied to the next series of experiments using monkey spermatozoa and spermatogenic cells. The heads of the monkey spermatozoa were about 6 µm long and 4 µm wide, similar to those of human sperm (Fig. 2a). The monkey round spermatids and primary spermatocytes (late pachytene to diplotene) were approximately 12 µm and 20 µm in diameter, respectively (Fig. 2b). When monkey epididymal and testicular spermatozoa were injected, 90–95% of mouse oocytes were activated (Table 1). Round spermatids activated oocytes as efficiently as mature spermatozoa (93%) (Fig. 1b) and primary spermatocytes activated oocytes to a lesser extent (79%) (Table 1). When early primary spermatocytes (mostly early pachytene and leptotene speramtocyes) were injected, the percentage of oocyte activation was remarkably reduced (24%) (Table 1). No oocytes were activated by monkey lymphocytes (Table 1). There was a marked tendency for oocytes activated by monkey spermatozoa or spermatogenic cells to form a single pronucleus as compared with those activated by mouse spermatozoa (Table 1).
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[Ca2+]i Response in Oocytes Injected with Spermatozoa or Spermatogenic Cells
We examined whether injection of monkey spermatozoa or spermatogenetic cells induces Ca2+ oscillations in mouse oocytes. Figure 3a shows Ca2+ oscillations after injection of a mouse spermatozoon. The zero time is the time of injection, and the recording was started about 8 min later. It has been shown that the first Ca2+ transient after ICSI as well as at fertilization is usually larger and longer than succeeding Ca2+ transients [31, 33, 34]. This pattern was reproduced in the present experiments (Fig. 3a). The interval between Ca2+ transients was 10–20 min. For injection of monkey spermatozoa, 37 out of 41 oocytes (90%) showed Ca2+ oscillations. The frequency of Ca2+ transients, particularly at the early phase of Ca2+ oscillations, tended to be higher than that in the case of mouse spermatozoa (Fig. 3b). Ca2+ oscillations lasted for at least 60–90 min. For injection of monkey round spermatids, 14 of 22 oocytes (64%) showed Ca2+ responses: Ca2+ oscillations in 10 oocytes (see Fig. 3c) and a single large Ca2+ transient alone in four oocytes. Other eight oocytes showed no Ca2+ response (Fig. 3d). Of the 22 oocytes, 17 oocytes were subjected to nucleus staining, and the nuclear stage could be determined in 12 of 17 oocytes, although not examined with exact one-to-one correspondence to the occurrence of the Ca2+ response. Formation of two pronuclei (2PN) was observed in six oocytes, telophase (T) II in two oocytes, and M II in four oocytes. Thus, the percentage of oocyte activation was 8 of 12 (67%) in the experiments of [Ca2+]i measurement. For injection of monkey spermatocytes, no Ca2+ oscillations were observed except 1 of 25 oocytes examined. In seven oocytes, the basal [Ca2+]i was slightly elevated at the start of [Ca2+]i measurement and gradually declined to a lower level (see Fig. 3e). Seventeen of the 25 oocytes were subjected to nucleus staining, and the nuclear stage could be determined in 14 of 17 oocytes. Two pronuclei was observed in 1 oocyte, 1PN in 2 oocytes, T II in 1 oocyte, and M II in 10 oocytes. The percentage of oocyte activation was 4 of 14 (30%). For injection of buffer alone, all (13) oocytes showed no elevation of [Ca2+]i at all throughout recording.
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Localization of SF after Oocyte Activation
To examine SF in the oocytes that were activated by sperm or spermatogenic cells, we transferred their karyoplast, cytoplast, or second polar body into freshly recovered unfertilized oocytes by electrofusion. The numbers of oocytes activated after nuclear/cytoplast transfer is summarized in Table 2. In our preliminary study, application of a fusion pulse alone did not activate oocytes when the fusion medium contained no Ca2+ (data not shown). The male and female pronuclei, but not the second polar body or the cytoplast, from oocytes activated by mouse spermatozoa induced activation in most recipient oocytes. Some of the cytoplasts at T II activated recipient oocytes, indicating that SF was distributed throughout the cytoplasm shortly after fertilization. When oocytes that had been activated by monkey spermatozoa, spermatids, or spermatocytes were used as donors, not only the pronuclei, but also the cytoplasts, could activate recipient oocytes (11%–60% and 10%–39%, respectively). Here again, spermatocytes have less, if any, oocyte-activating capacity as compared with more mature sperm cells. In every experimental group, there was a tendency for the male pronuclei to show better oocyte-activating capacity than the female pronuclei or cytoplast. The female pronucleus of oocytes parthenogenetically activated by Sr2+ treatment activated only a few oocytes (12%).
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DISCUSSION
This study supports the notion that mouse oocytes are a useful model for assessing SF in male gametes. The advantage of using mouse oocytes is the very low activation rate following sham injection or application of electric pulse in the absence of Ca2+. Using this experimental system, we demonstrated that monkey spermatozoa readily activated mouse oocytes and formed a well-developed male pronucleus at a rate similar to homologous fertilization. This study also revealed that monkey round spermatids that have not commenced spermiogenesis have full oocyte-activating capacity, like mature spermatozoa. This contrasts with mouse round spermatids that completely lack this capacity [12, 29, 32]. Thus, the stage at which SF first appears or becomes active depends on the species, although the SF activity is not strictly species specific, as has been demonstrated by previous heterologous experiments [4–10, 12].
The presence of active SF at the round spermatid stage has been reported only in humans [11]. The activation of human oocytes induced by homologous round spermatids is associated with Ca2+ oscillations [11], as was also observed with monkey round spermatids in our study. This indicates that human and monkey round spermatids have already matured as spermatozoa in the sense of oocyte-activating capacity. In fact, normal babies have been born in human clinics following intracytoplasmic injection of round spermatids without the aid of artificial stimuli to activate oocytes [35]. Recently, we also found that cynomolgus monkey oocytes that had been injected with round spermatids developed to the morula/blastocyst stage (unpublished results) at a rate similar to that seen after epididymal sperm injection [36].
Furthermore, we showed that the majority (79%) of oocytes injected with monkey spermatocytes formed well-developed pronuclei. The possibility that oocytes might have been stimulated by the medium coinjected with monkey spermatocytes can be excluded because injection of primary spermatocytes from mice did not eventually activate oocytes (see Table 1). However, monkey spermatocytes did not induce typical Ca2+ oscillations except one case. Very recently, Yazawa et al. [37] have demonstrated that there is a dissociation between the timings of appearance of oocyte-activating capacity and that of Ca2+ oscillation-inducing capacity during spermatogenesis, using several mammalian species as models. Before these immature sperm cells acquire the full Ca2+ oscillation-inducing capacity, they can induce only one to four transient [Ca2+]i rises in oocytes [37]. In our experiments, 7 out of 25 oocytes injected with monkey spermatocytes initially showed a slight elevation of the basal [Ca2+]i that gradually declined to a lower level. Whether the monkey SF is really expressed in spermatocytes will be determined more precisely when the SF molecule is identified.
In this study, a considerable number of mouse oocytes injected with monkey spermatozoa or spermatogenic cells formed a single pronucleus. According to Rybouchkin et al. [14], mouse oocytes injected with human spermatozoa formed a single pronucleus that was shown to be 2n diploid. Therefore, it is most probable that the chromosomes from monkey spermatozoa or spermatogenic cells intermingled with mouse chromosomes to form a single chromosome mass, rather than failing to be decondensed.
Our nuclear transfer experiments using mouse oocytes activated by mouse spermatozoa confirmed the previous study by Kono et al. [19], who demonstrated that 1) the oocyte-activating activity is exogenously introduced into the oocytes by spermatozoa but not caused by parthenogenetic activation with ethanol or strontium, and that 2) after pronuclear formation, the activity is localized within the male and female pronuclei but not in the cytoplasm [19]. We further showed that the oocyte-activating activity exists in the ooplasm shortly after fertilization (T II) but is absent in the second polar body. These findings suggest that SF diffuses over the ooplasm from the incorporated or injected spermatozoon and then is concentrated to the pronuclei after the second meiotic division. Correspondingly, long-lasting Ca2+ oscillations in fertilized mouse oocytes cease about the time of pronuclear formation [38]. Subsequently, low-frequency Ca2+ oscillations reappear after nuclear envelop breakdown at the first mitosis [18]. Because the transcription activity of oocytes is very low during the first cell cycle [39], it is reasonable to consider that Ca2+ oscillation-inducing SF itself, rather than newly expressed molecules, stay within the pronuclei, and that the SF is liberated again from the pronuclei into the ooplasm and induces repetitive Ca2+ release. It will be interesting to examine whether those dynamics of SF are common in mammalian species.
The present study showed that the SF liberated from the injected monkey spermatozoa or spermatids is capable of directing to the pronuclei in mouse oocytes, as the mouse SF. The oocyte-activating activity remained in the ooplasm in some cases, suggesting that some species specificity may exist in the process of concentration of SF into the pronuclei (see Table 2). Thus, it is probable that each component (the pronuclei, cytoplast, and second polar body) contained only a part of the total monkey SF introduced into oocytes, leading to lower activation rates after nuclear transfer than the pronuclei of oocytes injected with mouse spermatozoa. Some pronuclei and cytoplasts from oocytes injected with monkey primary spermatocytes activated recipient oocytes, but the rate was not significantly different from that of the control nuclear transfer using strontium-treated oocytes.
Although the biological and biochemical properties of mammalian SF have been studied extensively, the SF molecule is not identified yet. As intracytoplasmic injection experiments strongly suggest that SF with Ca2+-releasing activity exists in the cytosolic fractions of mammalian spermatozoa, the search for the true SF should be continued. When the SF protein is identified, its expression during spermatogenesis, its species-specificity in the oocyte-activating capacity, and its localization in activated oocytes will be determined in a more direct, precise manner.
FOOTNOTES
First decision: 22 January 2001.
1 Supported by grants from the Ministry of Education, Science, Sports, and Culture, Japan, and the Ministry of Health and Welfare, Japan. 
2 Correspondence: Atsuo Ogura, Department of Veterinary Science, National
Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo
162-8640, Japan. FAX: 81 3 5285 1111; aogura{at}nih.go.jp
Accepted: March 12, 2001.
Received: December 11, 2000.
REFERENCES
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 N. Ogonuki, H. Tsuchiya, Y. Hirose, H. Okada, A. Ogura, and T. Sankai Pregnancy by the tubal transfer of embryos developed after injection of round spermatids into oocyte cytoplasm of the cynomolgus monkey (Macaca fascicularis) Hum. Reprod., June 1, 2003; 18(6): 1273 - 1280. [Abstract] [Full Text] [PDF]
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N. Ogonuki, K. Mochida, K. Inoue, J. Matsuda, Y. Yamamoto, K. Takano, and A. Ogura Fertilization of Oocytes and Birth of Normal Pups Following Intracytoplasmic Injection with Spermatids in Mastomys (Praomys coucha) Biol Reprod, May 1, 2003; 68(5): 1821 - 1827. [Abstract] [Full Text] [PDF]
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