Effect of hyaluronidase, beta-glucuronidase and beta-N-acetylglucosaminidase inhibitors on sperm penetration of the mouse oocyte

The role of hyaluronidase, beta-glucuronidase and beta-N-acetylglucosaminidase in the penetration by mouse spermatozoa through the layers surrounding the oocyte was investigated by in vitro techniques. Myocrisin, fenoprofen, phosphorulated hesperidin and PS53 (a hydroquinone-sulfonic acid-formaldehyde polymer) inhibited fertilization when incubated with capacitated spermatozoa before the treated spermatozoa were mixed with intact oocytes but not when the inhibitor-treated, capacitated spermatozoa were added to oocytes free of follicle cells. The antifertility activity did not appear to be due to an effect on sperm motility or on the oocytes. These 4 compounds are known hyaluronidase inhibitors and, of the acrosomal enzymes tested, only share inhibition of hyaluronidase. Kinetic studies indicated that myocrisin is a reversible inhibitor of mouse sperm hyaluronidase whereas the other three are irreversible inhibitors. Adding saccharolactone, a beta-glucuronidase inhibitor, or N-acetylglucosaminolactone and N-acetylgalactosaminolactone, beta-N-acetylglucosaminidase inhibitors, to capacitated spermatozoa under the same conditions as the hyaluronidase inhibitors did not decrease fertilization. This was the case even though the beta-glucuronidase or beta-N-acetylglucosaminidase activities of the spermatozoa were completely inhibited, at least at the time that the inhibitor-treated, capacitated spermatozoa were mixed with the oocytes. The hyaluronidase activity of mouse spermatozoa remained unaltered during the incubation period required for capacitation; however, prolonged incubation caused a significant decrease in hyaluronidase. Untreated mouse spermatozoa caused hydrolysis of hyaluronic acid more effectively than did sperm extracts obtained by detergent extraction. These results are consistent with the theory of an essential role of hyaluronidase in mouse fertilization. At least in this species, the enzyme appears to be specifically involved in sperm penetration through the follicle cell layer. The data do not support an essential role for beta-glucuronidase and beta-N-acetylglucosaminidase in the penetration by mouse spermatozoa through the oocyte's investments. In contrast to some other species, sperm capacitation in mice does not result in a loss of hyaluronidase although part of the enzyme activity is lost on prolonged incubation. Mouse spermatozoa appear to be able to digest substrate (hyaluronic acid) even though hyaluronidase is not released.     

Effect of hypophysectomy on mouse oocyte maturation in vitro.

There was no difference in frequency of maturation of oocytes obtained from mice hypophysectomized for 2 weeks compared to those from sham-operated or untreated (control) animals of the same age. By 7 weeks, and also at 12 and 17 weeks, the incidence of polar body formation in vitro was significantly reduced. The number of oocytes which remained meiotically inactive in culture was increased at 7, 12 and 17 weeks after hypophysectomy. This decrease in spontaneous oocyte maturation in vitro could be partly overcome by administering exogenous PMSG, oestradiol-17beta or PMSG + oestradiol-17beta, but not progesteron or hCG, to hypophysectomized mice.     

Mammalian oocyte activation: lessons from the sperm and implications for nuclear transfer.

Events after fertilisation have been carefully studied in the last decades. However, there are still several questions to be clarified in relation to the signalling pathway initiated by the sperm, the identification of proteins or factors involved in the activation of the arrested oocyte, and the inactivation of specific molecules involved in the meiotic arrest. The present state of knowledge in mammalian fertilisation allows the development of activation protocols that closely mimic the events initiated by the sperm according to certain major factors (MPF activity and MAPk activity). These protocols are successfully used for the activation of oocytes after NT giving rise to offspring. Few cloned animals have yet been produced. However, the pregnancy and the survival rates after birth are not significantly different when different activation protocols are compared. This fact argues fora major reason forthe low success in the efficiency of NT. Eventually, factors related to the recipient oocyte, the donor cell or the culture conditions are part of these major problems that the reconstructed embryo has to overcome to develop into a normal offspring. Nonetheless, the development of activation protocols that closely imitate the mechanism of activation initiated by the sperm are of special interest to improve the developmental potential of cloned embryos.     

Multiple activation of mouse sperm motility

To investigate the activation mechanism of mouse sperm motility, the intact sperm in various activities were further investigated after demembranation. When dry sperm was diluted into sucrose solution, the sperm exhibited low motility with the swimming velocity of 13.5 ± 3.8 μm/s and the beat frequency of 1.5 ± 0.4 Hz. The demembranated sperm were immotile in the reactivation solution lacking cAMP. Meanwhile, when dry sperm was diluted into the solution containing either high concentration of NaCl or Ca²⁺, they exhibited the beat frequency of about 9 Hz. The demembranated ones exhibited the intermediate motility in the absence of cAMP. When dry sperm were diluted into the sucrose solution containing HCO₃, the sperm exhibited a vigorous motility with the swimming velocity of 181.2 ± 10.1 μm/s and the beat frequency of 11.3 ± 1.2 Hz. The demembranated sperm exhibited the high reactivation motility (90%) and flagellar beat frequency (9 Hz) in the absence of cAMP. These values were almost equivalent to those obtained in the demembranated sperm pretreated with sucrose or Ca²⁺ or NaCl and reactivated in the presence of cAMP. The activation induced by bicarbonate was considered complete in comparison with the activation by Ca²⁺ or NaCl. It was likely that the activation of mouse sperm motility took multiple states. © 1993 Wiley-Liss, Inc.     

Intracytoplasmic sperm injection in bovine: effects of oocyte activation, sperm pretreatment and injection technique

Intracytoplasmic sperm injection (ICSI) is a very important technique for treating male subfertility and for basic research. The efficiency of ICSI in bovine is very limited because of the necessity for additional oocyte activation before or after the ICSI procedure. In this study, we compared the effects of seven different protocols on activation and fertilization rates of bovine oocytes after ICSI and on their subsequent development under in vitro conditions. The protocols include 1) different chemical activation of oocytes, 2) pretreated or nonpretreated sperm, and 3) conventional or Piezo-driven injection techniques. In all three groups, ICSI, sham-injected, and noninjected, the highest activation rates were obtained after treatment of oocytes with ionomycin followed by 6-dimethylaminopurine (6-DMAP). Using this treatment for oocyte activation, 59% of oocytes were activated and 31% of oocytes were fertilized using dithiothreitol (DTT) pretreated spermatozoa and Piezo-driven injection. Using the protocols with the same oocyte activation or activation with calcium ionophore (Ca-I) and cycloheximide (CHX), nonpretreated sperm, and conventional injection technique, early cleavage rate (79.6% and 77.6%, respectively) were significantly (P <0.01) higher when compared with all other protocols. The latter protocol resulted in 8% blastocyst and 90% of the obtained blastocysts were found to be diploid. Our results demonstrate that activation of oocytes, sperm treatment, and injection technique separately or together could improve the success of bovine ICSI.     

Mammalian phospholipase Czeta induces oocyte activation from the sperm perinuclear matrix

Mammalian sperm-borne oocyte activating factor (SOAF) induces oocyte activation from a compartment that engages the oocyte cytoplasm, but it is not known how. A SOAF-containing extract (SE) was solubilized from the submembrane perinuclear matrix, a domain that enters the egg. SE initiated activation sufficient for full development. Microinjection coupled to tandem mass spectrometry enabled functional correlation profiling of fractionated SE without a priori assumptions about its chemical nature. Phospholipase C-zeta (PLCzeta) correlated absolutely with activating ability. Immunoblotting confirmed this and showed that the perinuclear matrix is the major site of 72-kDa PLCzeta. Oocyte activation was efficiently induced by 1.25 fg of sperm PLCzeta, corresponding to a fraction of one sperm equivalent (approximately 0.03). Immunofluorescence microscopy localized sperm head PLCzeta to a post-acrosomal region that becomes rapidly exposed to the ooplasm following gamete fusion. This multifaceted approach suggests a mechanism by which PLCzeta originates from an oocyte-penetrating assembly--the sperm perinuclear matrix--to induce mammalian oocyte activation at fertilization.     

Zona pellucida modifications in the mouse in the absence of oocyte activation

Mouse oocytes arrested in metaphase II exhibit zona hardening and a reduced fertilization rate after exposure to the cryoprotectant dimethylsulfoxide (Johnson J, In Vitro Fertil Embryo Transfer 6:168-175, 1989) but do not undergo parthenogenetic activation (Johnson and Pickering, Development 100:313-324, 1987). This paper shows that dimethylsulfoxide causes proteolytic modification of the zona pellucida glycoprotein ZP2 and inhibition of sperm binding. These effects of dimethylsulfoxide are caused by premature exocytosis of the cortical granules, a process that is initiated usually on fertilization. A model for the mechanism of action of dimethylsulfoxide is proposed based on the combined effects of cytoskeletal modification and osmotic shock. The presence of serum before and during the exposure to dimethylsulfoxide was found to reduce significantly these deleterious effects on the mouse zona pellucida without inhibiting the cortical granule release. These results highlight the suitability of dimethylsulfoxide as a tool to study the mechanisms leading to cortical granule release. Use of dimethylsulfoxide allows the separation of oocyte parthenogenetic activation from cortical granule release, and addition of serum allows separation of cortical granule release from the action of the cortical granule contents. Their use allows a dissection of the mechanisms underlying each of these three related events.     

Effects of oocyte activation and sperm preparation on the development of porcine embryos derived from in vitro-matured oocytes and intracytoplasmic sperm injection

The objective was to determine the effects of various methods of oocyte activation and sperm pretreatment on development of porcine embryos derived from in vitro-matured oocytes and intracytoplasmic sperm injection (ICSI). The second polar body was extruded in the majority (>78.4%) of in vitro-matured (IVM) oocytes 4h after electrical pulse activation. In embryos generated by ICSI and sham-ICSI, a combination of an electrical pulse, with various chemical activators 4 h later, improved (P < 0.05) blastocyst formation rate compared to activation only with a pulse. Treatment with 6-dimethylaminopurine (DMAP) after electrical activation significantly increased the oocyte activation rate. The effects of exposure of sperm to repeated freeze-thaw cycles (without cryoprotectant) on oocyte activation and the effects of sperm pre-incubated with dithiothreitol (DTT) or Triton X-100 on early embryo development were also examined. Blastocyst formation rates after ICSI did not differ between motile sperm and those rendered immotile by one-time freezing and thawing without cryoprotectant. However, sperm rendered immotile by three cycles of freezing/thawing without cryoprotectant had a significantly lower blastocyst formation rate. Although oocytes injected with sperm pre-incubated with Triton X-100 had a higher normal fertilization rate than those pre-incubated with DTT or one-time frozen/thawed sperm, rates of blastocyst formation and cell numbers were similar among the three groups. In conclusion, various methods of oocyte activation and sperm preparation significantly affected the developmental capacity of early porcine embryos derived from IVM and ICSI.     

Space asymmetry directs preferential sperm entry in the absence of polarity in the mouse oocyte

Knowledge about the mechanism that establishes embryonic polarity is fundamental in understanding mammalian development. In re-addressing several controversial claims, we recently proposed a model in which mouse embryonic polarity is not specified until the blastocyst stage. Before fertilization, the fully differentiated oocyte has been characterized as “polarized,” and we indeed observed that the sperm preferentially enters the polar body half. Here we show that preferential sperm entry is not due to an intrinsic polarity of the oocyte, since fertilization takes place uniformly when the zona pellucida is removed. We suggest that the term “asymmetry” denotes morphological differences, whereas “polarity” in addition implies developmental consequences. Thus, the mouse oocyte can be considered “asymmetric” but “non-polarized.” The penetration through the zona pellucida is also random, and a significant proportion of sperm binds to the oocyte membrane at a point distant from the zona penetration site. Time-lapse recordings confirmed that sperm swim around the perivitelline space before fertilization. Experimental enlargement of the perivitelline space in the non-polar body half increased the regional probability of fertilization. Based on these experiments, we propose a model in which the space asymmetry exerted by the first polar body and the zona pellucida directs sperm entry preferentially to the polar body half, with no need for oocyte polarity.     

Effect of in vivo oocyte aging on sperm chromatin decondensation in the golden hamster

Aged spontaneously activated hamster oocytes recovered from adult females 18 and 24 hours after ovulation were at the pronuclear stage. These oocytes and fresh controls were inseminated in vitro with capacitated hamster spermatozoa and observed with the phase-contrast microscope. The percentage of fertilization in fresh control oocytes was 98%, as compared to 36% and 18% when the oocytes were recovered 18 and 24 hours after ovulation, respectively. The mean number of sperm decondensations per egg in control oocytes was 10, and in the aged ones it was 0.69 and 0.12 when the oocytes were recovered 18 and 24 hours after ovulation, respectively. When similarly treated oocytes were studied with scanning and transmission electron microscopy, it was found that the degree of gamete membrane fusion was greater than that observed with the phase-contrast microscope, but that most of the spermatozoa failed to decondense the chromatin. We suggest that parthenogenetic oocytes at the pronuclear stage are in a similar stage of the cell cycle as in fertilized eggs, in which the cytoplasm does not have the ability to decondense the sperm chromatin.     

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.     

Parthenogenetic activation pattern and microtubular organization of the mouse oocyte after exposure to 1,2-propanediol.

We have studied the effect of 1,2-propanediol (PROH) on cumulus-oocyte complexes from the mouse. We determined the morphological survival rate, the pattern of parthenogenetic activation, and the microtubular and chromosomal organization. Cumulus-oocyte complexes were collected at 16 h post hCG from superovulated female hybrid mice. These cumulus-intact oocytes were exposed to 1.5 or 3 M PROH for 6, 12, or 18 min at 0, 22, or 37 degrees C. The cryoprotectant was diluted out in a 1 M sucrose solution at 22 degrees C. After 5-6 h at 37 degrees C, oocytes were denuded and examined under Nomarski optics. The results show that PROH can induce degeneration and parthenogenetic activation in the mouse oocyte in a concentration, temperature, and time-dependent way. As the activation stimulus was strengthened, an increasing proportion of oocytes shifted from parthenogenetic activation with polar body extrusion to parthenogenetic activation with polar body retention and even to immediate cleavage. Nontoxic and nonactivating conditions involved mainly exposure to 1.5 M PROH at 0 degrees C. Spindle integrity and chromosomal organization were analyzed for exposure to 1.5 and 3 M PROH for 12 min at 0 degrees C. The separate effect of cooling and exposure to 1 M sucrose were also evaluated. Microtubules were visualized by monoclonal anti-alpha-tubulin labeling followed by immunogold-silver staining. Cooling and exposure to 1 M sucrose or to 1.5 M PROH did not induce major abnormalities in the microtubular or chromosomal organization. On the other hand, a significant percentage of deformities such as spindle size reduction and loss of bipolarity were observed after exposure to 3 M PROH. The results of the present study demonstrate that the use of PROH as a single cryoprotectant for the freezing of mature unfertilized oocytes cannot be recommended in procedures involving ambient temperature or concentrations exceeding 1.5 M PROH. On the other hand, the potential beneficial effect of low temperatures may outweigh the effect of concentration at subzero temperatures and could be explored further in the tailoring of conditions for slow controlled freezing.     

Repetitive calcium stimuli drive meiotic resumption and pronuclear development during mouse oocyte activation.

Freshly ovulated (12 hr post hCG) F1 (C57BL/6 x CBA) hybrid mouse oocytes were parthenogenetically activated by repetitive elevation of Ca2+ induced by carefully controlled electrical pulses. Different patterns of stimulation were employed to examine the role of repetitive calcium changes on meiotic resumption and pronuclear development. In the first series of experiments oocytes received 33 electrical pulses of 1.8 kV/cm delivered every 4 min. The pulse duration decreased according to a negative exponential equation from a 900-microseconds first pulse to give a total pulse duration of 18.721 msec. The strength of calcium stimuli was varied by changing the concentration of CaCl2 in the medium. Ninety-eight percent of the oocytes stimulated with 12 microM calcium extruded the second polar body by the end of treatment and 92% completed pronuclear formation between 3.5 and 8 hr after the first pulse. For higher or lower Ca2+ concentrations the proportion of oocytes developing pronuclei decreased; the timing of pronuclear formation was retarded and the majority of oocytes failed to form a pronucleus after extrusion of the second polar body. In the second series of experiments, the strength of the calcium stimuli was modulated by changing the duration of the 33 electrical pulses given in the presence of 12 microM calcium. By increasing the total pulse duration to 33.958 msec, 100% of the oocytes activated and completed pronuclear formation between 3 and 5 hr after the first electric pulse. Stimulation protocols of lower total pulse duration (less than 18.721 msec) gave rise to high rates of partial activation (up to 95%). Examination of these partially activated oocytes showed metaphases with haploid sets of chromatids characteristic of third meiotic metaphase arrest. The results indicate that repetitive calcium stimuli can regulate the rate and extent of meiotic resumption and the time course of pronuclear formation during mouse oocyte activation. They suggest that meiotic resumption in mammalian oocytes is regulated by the amplitude and frequency of cytosolic calcium oscillations induced by the activating stimulus.     

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.     

Characterization of glycolysis and pentose phosphate pathway activity during sperm entry into the mouse oocyte

Studying the events that occur during gamete fusion and sperm decondensation in the oocyte remains difficult because sperm-oocyte fusion and subsequent sperm decondensation represent a short part of the fertilization process, and their exact timing is difficult to determine. There is therefore a need for greater understanding of the events that occur during this period. The main purpose of this study was to examine the metabolic aspects of this time frame by characterizing glucose metabolism (glycolytic and pentose phosphate pathway [PPP] activities) during sperm fusion and decondensation into zona-free oocytes in mice. The metabolism of glucose through both glycolysis and the PPP was measured in ovulated MII oocytes, free of cumulus cells, and the levels of glucose metabolized were found to be low. Upon sperm entry, both glycolytic and PPP activity increased substantially. To determine whether this elevation in glucose metabolism was part of the activation process, the metabolism of parthenogenetically activated oocytes was measured, and no increase in metabolism was observed. The characterization of glucose metabolism during sperm fusion and decondensation into the oocyte, and comparison to parthenogenetically activated oocytes, showed that the fertilizing sperm is responsible for an increase in both glycolytic and PPP activity during fusion and/or decondensation. The significance of this observation during the fertilization process and for the developing embryo is as yet unclear and warrants further investigation.     

Pachytene pairing in relation to sperm and oocyte numbers in a male-fertile reciprocal translocation in the mouse

In adult males carrying the male-fertile reciprocal translocation T(2;4)13H, body weights, testis weights, and sperm counts were higher in heterozygotes than in homozygotes. Heterozygotes whose mothers were C3H/He exceeded their reciprocal counterparts in the same criteria. At 3-4 days of age, no significant differences between homozygous and heterozygous females were found in body weight, ovarian volume, or oocyte numbers, although mean oocyte volumes were somewhat larger in heterozygotes than in homozygotes. In homozygous males and females the synaptonemal complexes of rearranged chromosomes appeared as bivalents that were indistinguishable from normal bivalents. In most gametocytes of heterozygotes, the translocation was present in the form of a quadrivalent. The degree of pairing failure was greater in oocytes than in spermatocytes. Terminal asynapsis of quadrivalents was very rare in spermatocytes, but it affected one quarter of the oocytes. Only very few translocation configurations were associated with the XY bivalent. It is concluded that the number of sperm produced in male heterozygotes can match the general increase in vigor by the formation of a high level of fully paired quadrivalents, whereas a greater degree of terminal asynapsis in the quadrivalents of oocytes may indicate a slightly more deleterious effect of this translocation on oogenesis.     

雷公藤多甙对小鼠卵母细胞成熟和体外受精的影响

采用超排卵技术研究雷公藤多甙（GTW）对小鼠卵母细胞的成熟和体外受精以及脏器等的影响,GTW对小鼠卵母细胞生发泡破裂没有影响,但可以抑制卵母细胞第一极体的释放,影响卵母细胞的存活率并可降低体外受精率和超排卵的卵母细胞数量。GTW可以破坏卵母细胞成熟,降低卵母细胞的体外受精能力,影响小鼠的正常生殖功能。     

Effect of the in vitro conditions of mouse oocyte maturation on their capacity for parthenogenetic activation as affected by cycloheximide and ethanol

The efficiency of 7% ethanol and cycloheximide (CH) as activators of parthenogenesis was compared on the in vitro matured oocytes of the CBA/C57BL (F1) mice. CH is a more potent activator but the oocytes are activated by ethanol more synchronously. The oocytes matured in the F12, F10 and RPMI 1640 media were activated by ethanol reliably better than those matured in the Minimum Essential Medium Eagle or in the M3 medium. Addition of five amino acids (arginine, alanine, serine, aspartic and glutamic acids), only ornithine or arginine, as well as prolongation of oocyte cultivation in M3 from 19.5 to 26.5 h reliably increased the percentage of activated oocytes.