The ability of freeze-dried bull spermatozoa to induce calcium oscillations and resumption of meiosis

The objective was to investigate the ability of freeze-dried (FD) bull spermatozoa to induce calcium oscillations in mouse oocytes and meiosis resumption in in vitro-matured bovine oocytes after intracytoplasmic sperm injection (ICSI). Bull spermatozoa were freeze-dried and stored for 1 y at +25, +4, or -196 degrees C. In the first experiment, rehydrated sperm heads were microinseminated into hybrid mouse oocytes loaded with fluo-3/AM, and the kinetics of intracellular calcium concentration was monitored for 1h. Repetitive increases of intracellular calcium concentration were recorded in the majority of injected oocytes, with exception of a few oocytes injected with FD sperm heads stored at +4 degrees C (11%) and +25 degrees C (8%) that exhibited a single increase or no response (non-oscillated). The proportion of oocytes that oscillated with high frequency (>or=10 spikes/h) was higher in the non-dried control group (79%; P<0.05) than in the FD groups (58, 55, and 54% for storage at -196, +4, and +25 degrees C, respectively). In the second experiment, control and FD spermatozoa were microinseminated into in vitro-matured, denuded bovine oocytes. The oocytes were fixed and stained 12h after ICSI. A higher proportion of bovine oocytes injected with control spermatozoa (70%; P<0.05) resumed meiosis than those injected with +25, +4 and -196 degrees C stored FD spermatozoa (53, 48, and 57%, respectively). The proportion of ICSI oocytes that developed to the pronuclear stage (complete activation) was higher in the control group (64%; P<0.05) than those in all the FD groups (34, 27, and 28% for storage at -196, +4, and +25 degrees C, respectively). Thus, the ability of bull spermatozoa to induce frequent intracellular calcium spikes in mouse oocytes was impaired by the process of freeze-drying, without differences among storage at +25, +4 or -196 degrees C, probably resulting in a lower proportion of bovine oocytes that resumed meiosis and/or developed to the pronuclear stage.     

Activity of a sperm-borne oocyte-activating factor in spermatozoa and spermatogenic cells from cynomolgus monkeys and its localization after oocyte activation

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 Ca(2+) oscillations in a pattern similar to that seen following normal fertilization. Most spermatocytes did not produce typical intracellular Ca(2+) 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.     

Direct differentiation of human pluripotent stem cells into haploid spermatogenic cells

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been shown to differentiate into primordial germ cells (PGCs) but not into spermatogonia, haploid spermatocytes, or spermatids. Here, we show that hESCs and hiPSCs differentiate directly into advanced male germ cell lineages, including postmeiotic, spermatid-like cells, in?vitro without genetic manipulation. Furthermore, our procedure mirrors spermatogenesis in?vivo by differentiating PSCs into UTF1-, PLZF-, and CDH1-positive spermatogonia-like cells; HIWI- and HILI-positive spermatocyte-like cells; and haploid cells expressing acrosin, transition protein 1, and protamine 1 (proteins that are uniquely found in spermatids and/or sperm). These spermatids show uniparental genomic imprints similar to those of human sperm on two loci: H19 and IGF2. These results demonstrate that male PSCs have the ability to differentiate directly into advanced germ cell lineages and may represent a novel strategy for studying spermatogenesis in?vitro.     

Albumin-bound lipids induce free cytoplasmic calcium oscillations in human osteoblast-like cells

[Ca(2+)](i) oscillations were found in human osteoblast-like cells (hOB cells) exposed to high-lipid bovine serum albumin (BSA), but not when exposed to low-lipid BSA. These [Ca(2+)](i) oscillations were inhibited by heptanol and suramin, which implies that gap junctions and purinergic signalling may be important for these [Ca(2+)](i) oscillations. The high-lipid BSA preparation that was used contains arachidonic acid. [Ca(2+)](i) oscillations could be induced by low lipid albumin with arachidonic acid added. The albumin-bound lipids were also important for osteoblast growth since DNA synthesis and the total cell protein content was higher in hOB cells exposed to high-lipid BSA. The effect of arachidonic acid on hOB cell proliferation was bone-donor dependent; both stimulatory and inhibitory effects were observed. The physiological importance of albumin-bound lipids is unclear; given that albumin has only minimal contact with osteoblasts under normal conditions. Only when bone capillaries are disrupted, e.g. during a fracture, would significant amounts of albumin reach osteoblasts. Albumin-bound lipids could therefore contribute to stimulation of osteoblast proliferation during fracture healing.     

Porcine oocyte activation: differing roles of calcium and pH

Intracellular pH has recently been shown to increase during parthenogenetic activation of the porcine oocyte. In the following set of experiments, intracellular pH was monitored during activation and pronuclear development was assessed following activation treatments with calcium, in the absence of calcium, and in oocytes loaded with the calcium chelator BAPTA-AM in calcium-free medium. Intracellular pH increase was not different among groups when treating with 7% ethanol or 50 microM calcium ionophore, or during treatment with thimerosal for 12 or 25 min. Activation with thimerosal (200 microM, 12 min) followed by 8 mM dithiothreitol (DTT, 30 min) resulted in a decreased pronuclear development in calcium-free medium with or without BAPTA-AM loaded oocytes as compared to controls. Activation with 50 microM calcium ionophore resulted in pronuclear development that was different between the calcium-free and BAPTA-AM loaded oocytes in calcium-free medium. Similar incidences of pronuclear formation were observed in all ethanol treatment groups. It was concluded that external calcium as well as large changes in intracellular free calcium are not necessary for the increase in intracellular pH, but normal intracellular calcium signaling is critical for normal levels of pronuclear development. Finally, oocytes were measured for intracellular pH changes for 30 min following subzonal sperm injection. Intracellular pH did not increase, although pronuclear formation was observed 6 hr post SUZI. This suggested that major differences were still present between sperm-induced and parthenogenetic activation of the porcine oocyte.     

Local anesthetics and phenothiazine tranquilizers induce parthenogenetic activation of the mouse oocyte

Incubation of recently ovulated mouse oocytes in various concentrations of local anesthetics and phenothiazine tranquilizers initiates the completion of meiosis and the formation of pronuclei. The potency with which these drugs induce oocyte activation is comparable to their relative ability to displace Ca²⁺ from artificial lipid membranes. Oocyte activation induced by these drugs is inhibited by Ca²⁺. Perturbation of the plasma membrane of the oocyte by these drugs may displace Ca²⁺, which is responsible for the integrity of the microtubule-microfilament system that normally maintains the ovulated mammalian oocyte blocked at the Metaphase II of meiosis until sperm penetration.     

The fertilising ability of spermatogenic cells derived from cultured mouse immature testicular tissue

There are many reports about the in vitro culture of spermatogenic cells, but no-one has succeeded in inducing the differentiation from spermatogonia to intact sperm. Also the study of in vitro testicular tissue culture has hardly advanced. We studied the culture of mouse immature testicular tissue derived from 5-day-old mice. We aimed to achieve the differentiation of spermatogenic cells in order to observe spermatogenesis in testicular tissue in vitro. We also froze mature testicular tissue and immature testicular tissue cultured for 2 weeks. Furthermore, spermatogenic cells differentiated by culturing were injected into metaphase II oocytes to determine whether these differentiated cells and frozen-thawed testicular tissue have fertilising and developmental ability. Under the culture conditions employed, secondary spermatocytes and a few round spermatids differentiated from spermatogonia were observed in the immature testicular tissue cultured for 2 weeks. When spermatogenic cells derived from cultured immature testicular tissue, cultured frozen immature testicular tissue and frozen-thawed mature testicular tissue were injected into ooplasm, the oocytes were fertilised and fertilised oocytes developed to the 8-cell stage. We suggest that spermatogenic cells derived from cultured immature testicular tissue have fertilising and developmental abilities equivalent to that of sperm. Also these abilities of spermatogenic cells obtained from cultured frozen immature testicular tissue and frozen-thawed mature testicular tissue were better than those of the same cells before freezing.     

Myofilament-generated tension oscillations during partial calcium activation and activation dependence of the sarcomere length-tension relation of skinned cardiac cells

During partial Ca2+ activation, skinned cardiac cells with sarcoplasmic reticulum destroyed by detergent developed spontaneous tension oscillations consisting of cycles (0.1-1 Hz) of rapid decrease of tension corresponding to the yield of some sarcomeres and slow redevelopment of tension corresponding to the reshortening of these sarcomeres. Such myofilament-generated tension oscillations were never observed during the full activation induced by a saturating [free Ca2+] or during the rigor tension induced by decreasing [MgATP] in the absence of free Ca2+ or when the mean sarcomere length (SL) of the preparation was greater than 3.10 microm during partial Ca2+ activation. A stiff parallel elastic element borne by a structure that could be digested by elastase hindered the study of the SL--active tension diagram in 8-13-microm-wide skinned cells from the rat ventricle, but this study was possible in 2-7-microm-wide myofibril bundles from the frog or dog ventricle. During rigor the tension decreased linearly when SL was increased from 2.35 to 3.80 microm. During full Ca2+ activation the tension decreased by less than 20% when SL was increased from 2.35 to approximately 3.10 microm. During partial Ca2+ activation the tension increased when SL was increased from 2.35 to 3.00 microm. From this observation of an apparent increase in the sensitivity of the myofilaments to Ca2+ induced by increasing SL during partial Ca2+ activation, a model was proposed that describes the tension oscillations and permits the derivation of the maximal velocity of shortening (Vmax). Vmax was increased by increasing [free Ca2+] or decreasing [free Mg2+] but not by increasing SL.     

The human cytomegalovirus protein pUL38 suppresses endoplasmic reticulum stress-mediated cell death independently of its ability to induce mTORC1 activation

As obligate intracellular parasites, viruses not only hijack cellular machinery, they also deregulate host stress responses for their infection. Human cytomegalovirus (HCMV) modulates the endoplasmic reticulum (ER) stress response, due at least in part to the viral protein pUL38, and one of the consequences is to maintain the viability of infected cells. Consequently, pUL38-deficient virus induces premature cell death during infection. In addition, pUL38 activates mammalian target of rapamycin complex 1 (mTORC1), which may also antagonize other detrimental cellular stresses (N. J. Moorman et al., Cell Host Microbe 3:253-262, 2008). It remains elusive how pUL38 inhibition of cell death is related to mTORC1 activation. In this study, we defined the interplay of the two pUL38 activities. We constructed a series of pUL38 truncation mutants based on the secondary structure prediction and evolutionary conservation of its sequence. We found that the N-terminal 239 residues of pUL38 were necessary and sufficient to block cell death induced by pUL38-deficient virus or by the ER stress inducer tunicamycin. However, this pUL38 domain was unable to activate mTORC1 when expressed alone. Importantly, small-molecule inhibitors of mTORC1, rapamycin or torin 1, did not compromise pUL38 activity to block cell death in isolation or in virus infection. Expression of a constitutively active variant of an mTORC1 activator, Rheb (Ras homolog enriched in brain), could not prevent cell death induced by pUL38-deficient virus. Collectively, we provide genetic and biochemical evidence that pUL38 prevents ER stress-induced cell death independent of its role in mTORC1 activation.     

Development of isothiocyanate-enriched broccoli,and its enhanced ability to induce phase 2 detoxification enzymes in mammalian cells

Broccoli florets contain low levels of 3-methylsuphinylpropyl and 4-methylsulphinylbutyl glucosinolates. Following tissue disruption, these glucosinolates are hydrolysed to the corresponding isothiocyanates (ITCs), which have been associated with anticarcinogenic activity through a number of physiological mechanisms including the induction of phase II detoxification enzymes and apoptosis. In this paper, we describe the development of ITC-enriched broccoli through the introgression of three small segments of the genome of Brassica villosa, a wild relative of broccoli, each containing a quantitative trait locus (QTL), into a broccoli genetic background, via marker-assisted selection and analysis of glucosinolates in the florets of backcross populations. Epistatic and heterotic effects of these QTLs are described. The ITC-enriched broccoli had 80-times the ability to induce quinone reductase (a standard assay of phase II induction potential) when compared to standard commercial broccoli, due both to an increase in the precursor glucosinolates and a greater conversion of these into ITCs.     

ATP autocrine/paracrine signaling induces calcium oscillations and NFAT activation in human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hMSCs) have the potential to differentiate into several types of cells. Calcium ions (Ca(2+)) play an important role in the differentiation and proliferation of hMSCs. We have demonstrated that spontaneous [Ca(2+)](i) oscillations occur without agonist stimulation in hMSCs. However, the precise mechanism of its generation remains unclear. In this study, we investigated the mechanism and role of spontaneous [Ca(2+)](i) oscillations in hMSCs and found that IP(3)-induced Ca(2+) release is essential for spontaneous [Ca(2+)](i) oscillations. We also found that an ATP autocrine/paracrine signaling pathway is involved in the oscillations. In this pathway, an ATP is secreted via a hemi-gap-junction channel; it stimulates the P(2)Y(1) receptors, resulting in the activation of PLC-beta to produce IP(3). We were able to pharmacologically block this pathway, and thereby to completely halt the [Ca(2+)](i) oscillations. Furthermore, we found that [Ca(2+)](i) oscillations were associated with NFAT translocation into the nucleus in undifferentiated hMSCs. Once the ATP autocrine/paracrine signaling pathway was blocked, it was not possible to detect the nuclear translocation of NFAT, indicating that the activation of NFAT is closely linked to [Ca(2+)](i) oscillations. As the hMSCs differentiated to adipocytes, the [Ca(2+)](i) oscillations disappeared and the translocation of NFAT ceased. These results provide new insight into the molecular and physiological mechanism of [Ca(2+)](i) oscillations in undifferentiated hMSCs.     

c-erbB2 and c-myb induce mouse oocyte maturation involving activation of maturation promoting factor

Proto-oncogenes are involved in cell growth, proliferation, and differentiation. In the present study, we investigated the roles and mediating pathways of proto-oncogenes c-erbB(2) and c-myb in mouse oocyte maturation by RT-PCR, real-time quantitative PCR, western blot, and recombinant proto-oncogene protein microinjection. Results showed that both c-erbB(2) and c-myb antisense oligodeoxynucleotides (c-erbB(2) ASODN and c-myb ASODN) inhibited germinal vesicle breakdown and the first polar body extrusion in a dose-dependent manner. However, microinjection of recombinant c-erbB(2) or c-myb protein into germinal vesicle stage oocytes stimulated oocyte meiotic maturation. In addition, the expression of c-erbB(2) and c-myb mRNA was detected in oocytes; and c-erbB(2) ASODN and c-myb ASODN inhibited c-erbB(2) mRNA and c-myb mRNA expression, respectively. Maturation promoting factor (MPF) inhibitor roscovitine did not affect the expression of c-erbB(2) mRNA and c-myb mRNA, but blocked the effects of recombinant c-erbB(2) and c-myb protein-induced oocyte maturation. Further, cyclin B1 protein expression in oocytes was remarkably inhibited by c-erbB(2) ASODN, c-myb ASODN, and roscovitine. Nonsense tat ODN had no effect on the expression of c-erbB(2), c-myb, and cyclin B1. These results suggest that c-erbB(2) and c-myb may induce oocyte maturation through mediating a pathway involving the activation of MPF.     

Generation of genetically modified mice by oocyte injection of androgenetic haploid embryonic stem cells

Haploid cells are amenable for genetic analysis. Recent success in the derivation of mouse haploid embryonic stem cells (haESCs) via parthenogenesis has enabled genetic screening in mammalian cells. However, successful generation of live animals from these haESCs, which is needed to extend the genetic analysis to the organism level, has not been achieved. Here, we report the derivation of haESCs from androgenetic blastocysts. These cells, designated as AG-haESCs, partially maintain paternal imprints, express classical ESC pluripotency markers, and contribute to various tissues, including the germline, upon injection into diploid blastocysts. Strikingly, live mice can be obtained upon injection of AG-haESCs into MII oocytes, and these mice bear haESC-carried genetic traits and develop into fertile adults. Furthermore, gene targeting via homologous recombination is feasible in the AG-haESCs. Our results demonstrate that AG-haESCs can be used as a genetically tractable fertilization agent for the production of live animals via injection into oocytes.     

Effects of urocortin on T-type calcium currents in mouse spermatogenic cells

Urocortin (UCN), a newly isolated peptide, has been found to play an important role mainly in female reproductive system. In order to investigate the effect of UCN on T-type calcium currents (I(Ca,T)), exploring the mechanisms of UCN's role in male reproductive system, especially in acrosome reaction, we directly measured the I(Ca,T) in mouse spermatogenic cells exposed to UCN using standard whole-cell patch-clamp recording technique. Our results showed that UCN reversibly inhibited the T-type Ca(2+) currents in the cells in a concentration-dependent manner. The current density was inhibited by about 19% after exposure of the cells to UCN (0.1 microM) for 5 min, from the control value of 6.75+/-1.17 to 5.26+/-0.82pA/pF. UCN up-shifted the current-voltage (I-V) curve. Frequency-dependence of UCN's effects on I(Ca,T) was also observed. Moreover, UCN at 0.1 microM did not markedly affect the activation of I(Ca,T) but shifted the inactivation curve of I(Ca,T) to the left. The inhibitory effect of UCN on the T-type Ca(2+) current was not affected by Astressin, the CRF receptor blocker. Since T-type calcium channels are a key component in acrosome reaction, our data suggest that UCN might be a significant factor in male reproductive action and a potential contraceptive agent.     

The follicular oocyte and its granulosa cells in domestic pig

Summary Pig oocytes and their surrounding granulosa cells obtained from mature Graafian follicles at a stipulated time near to ovulation were studied in some details electronmicroscopically. Particular emphasis is given to the corona radiata cell processes and to the heterogeneous population of mitochondria in the oocyte.The corona radiata cell processes contain various components such as filaments, mitochondria, multivesicular bodies and lipid droplets in their matrix. The contact relationship of the corona radiata cell processes to the oocytes is maintained by desmosomes. Usually, the two parallel surface membranes forming the desmosome are separated by a space of about 200 Å. Occasionally, the two membranes approximate each other to form a junction having a gap of about 70 Å. Apparently the membranes become fused in some regions.Of particular interest is the distribution and structural characteristics of the single-membrane-bounded structures, and their relationship to the cytomembranes and the mitochondria. On the basis of the present and earlier (Norberg, 1972) observations, the question arises whether the formation and development of mitochondria of pig oocytes depend, at least partly, on a metamorphosis of single-membrane-bounded structures derived from less complex membraneous elements. Final conclusions concerning this problem demand integrated morphological and biochemical investigation regarding the biosynthesis of mitochondria.This work was supported by the Agricultural Research Council of Norway.     

A single-pool model for intracellular calcium oscillations and waves in the Xenopus laevis oocyte.

We construct a minimal model of cytosolic free Ca2+ oscillations based on Ca2+ release via the inositol 1,4,5-trisphosphate (IP3) receptor/Ca2+ channel (IP3R) of a single intracellular Ca2+ pool. The model relies on experimental evidence that the cytosolic free calcium concentration ([Ca2+]c) modulates the IP3R in a biphasic manner, with Ca2+ release inhibited by low and high [Ca2+]c and facilitated by intermediate [Ca2+]c, and that channel inactivation occurs on a slower time scale than activation. The model produces [Ca2+]c oscillations at constant [IP3] and reproduces a number of crucial experiments. The two-dimensional spatial model with IP3 dynamics, cytosolic diffusion of IP3 (Dp = 300 microns 2 s-1), and cytosolic diffusion of Ca2+ (Dc = 20 microns 2 s-1) produces circular, planar, and spiral waves of Ca2+ with speeds of 7-15 microns.s-1, which annihilate upon collision. Increasing extracellular [Ca2+] influx increases wave speed and baseline [Ca2+]c. A [Ca2+]c-dependent Ca2+ diffusion coefficient does not alter the qualitative behavior of the model. An important model prediction is that channel inactivation must occur on a slower time scale than activation in order for waves to propagate. The model serves to capture the essential macroscopic mechanisms that are involved in the production of intracellular Ca2+ oscillations and traveling waves in the Xenopus laevis oocyte.     

The effect of spermatogenic disruption on the ability of testicular fluid to stimulate androgen production by normal Leydig cells

Reports from this and other laboratories have concluded that unilateral disruption of spermatogenesis induces a predominantly ipsilateral increase in the responsiveness of Leydig cells to stimulation with luteinizing hormone (LH) and have suggested that if such effects were mediated by locally produced hormones then such "factors" should be detectable in testicular interstitial fluid. We sought to demonstrate such factors in testicular fluid from gonads subjected to a variety of treatments that disrupt gametogenesis. Fluid (TF) was drained from testes of adult rats that had been sham treated, irradiated, or treated with busulfan in utero, made unilaterally or bilaterally cryptorchid, or were unilaterally or bilaterally efferent-duct-ligated. Leydig cells obtained from normal rats basally produced 8 +/- 1 ng androgen/10(6) Leydig cells/2 h and, when maximally stimulated with LH, produced 66 +/- 3 ng. The addition of the various TFs to the incubations significantly increased both basal and LH-stimulated androgen production. TF from lesioned testes was more effective in increasing androgen production than TF from control rats. Unilateral lesions caused an increase in the ability of TF from the disrupted testes to increase the androgen production by normal Leydig cells, as compared to TF from contralateral testes. Thus, locally produced "factor(s)" do appear to modify Leydig cell function. Additional studies using TF from control and bilaterally cryptorchid animals suggest that the "factor' in TF is heat-labile; has a molecular size between bovine serum albumin and ovalbumin; exerts a portion of its action independently of cAMP formation; and does not appear to be LH, follicle-stimulating hormone, prolactin, or gonadotropin-releasing hormone.     

The differential ability of splenic adherent cells from B6.lpr mice to induce suppressor T cells

Hapten-coupled splenic adherent cells or resident peritoneal cells from autoimmune B6.lpr mice that are over 5 mo of age fail to induce first-order inducer suppressor T cells (Ts1). However, the same population of hapten-coupled cells can induce both delayed-type hypersensitivity responses and third-order effector suppressor T cells (Ts3). Thus, splenic and peritoneal antigen-presenting cells from B6.lpr mice display a defined defect in the ability to induce certain suppressor T cell responses. The cellular defect in Ts1 induction is controlled by the lpr gene, since age-matched congenic B6 mice do not display this defect. The splenic adherent cell defect is temporarily correlated with the autoimmunity that develops in B6.lpr animals. The antigen-presenting defect in the B6.lpr splenic adherent population for Ts1 induction is reversible by culturing the cells in interferon-gamma. The results are discussed as an illustration of the relationship between experimental models of autoimmunity and defects in a suppressor T cell cascade.     

DRP1 interacts directly with BAX to induce its activation and apoptosis

The apoptotic executioner protein BAX and the dynamin‐like protein DRP1 co‐localize at mitochondria during apoptosis to mediate mitochondrial permeabilization and fragmentation. However, the molecular basis and functional consequences of this interplay remain unknown. Here, we show that BAX and DRP1 physically interact, and that this interaction is enhanced during apoptosis. Complex formation between BAX and DRP1 occurs exclusively in the membrane environment and requires the BAX N‐terminal region, but also involves several other BAX surfaces. Furthermore, the association between BAX and DRP1 enhances the membrane activity of both proteins. Forced dimerization of BAX and DRP1 triggers their activation and translocation to mitochondria, where they induce mitochondrial remodeling and permeabilization to cause apoptosis even in the absence of apoptotic triggers. Based on this, we propose that DRP1 can promote apoptosis by acting as noncanonical direct activator of BAX through physical contacts with its N‐terminal region.     

Appearance of the oocyte activation ability of mouse round spermatids cultured in Vitro

This study was undertaken to determine the expression time of fertilization and oocytes activation abilities of spermatids in the mouse. When elongating or elongated spermatids isolated from fresh testes of adult males (B6D2F1) were injected into mature mouse oocytes, both spermatids could activate the mature oocytes and occur fertilization. On the one hand, the round spermatids could not activate mature oocytes, when microinjected into oocytes. In some experiments, recovered round spermatids were cultured under co-culture systems using Sertoli cells as a feeder cell. Under the co-culture system, developed elongating spermatids could stimulate and fertilized mature oocytes. These results indicate that the start of oocyte activation appearance is between the stage of round spermatid and elongating spermatids and the activation ability increases with the advance of spermiogensis. On the other hand, round spermatids isolated from males of ICR strain mouse already have the oocyte activation ability and the fertilizing ability. The result obtained suggests that the expression time of the oocyte activating ability is difficult between the mouse strain.