Gonadotropin exposure,salt storage and storage duration affect penetration of domestic cat oocytes by homologous spermatozoa

Salt-stored domestic cat oocytes are routinely used to study sperm function in domestic and nondomestic felids. Our objectives were to assess the effects of in vitro maturation (IVM), salt storage and storage duration on penetration of domestic cat oocytes by homologous spermatozoa. In Experiment 1, domestic cat spermatozoa were coincubated with fresh immature oocytes, salt-stored (2-3 weeks) immature oocytes, or salt-stored (2-3 weeks) IVM oocytes matured in Minimum Essential Medium containing 0.1IU FSH and 0.1IU LH/ml (IVM1) or 0.5IU FSH and 2.2IULH/ml (IVM2). In Experiment 2, all oocytes were matured (IVM2) and inseminated fresh or after salt storage for 2-3 weeks, 2-3 months or 9 months. In Experiment 1, penetration of the outer zona pellucida (OZP) was greater (P<0.05) in salt-stored IVM2 oocytes than in salt-stored immature oocytes, whereas penetration of salt-stored IVM1 oocytes was intermediate (P>0.05). In Experiment 2, penetration of the OZP and inner zona pellucida (IZP) was higher (P<0.05) in fresh IVM2 oocytes than in salt-stored oocytes, and a higher (P<0.05) proportion of oocytes had IZP sperm after 2-3 weeks of storage than after 2-3 months. Penetration of the perivitelline space was higher (P<0.05) in fresh IVM2 oocytes than in oocytes stored for 2-3 weeks or 2-3 months. These results suggest that oocyte penetration is improved by IVM, but is impaired by exposure to salt-storage solution and prolonged storage duration.     

Atrial natriuretic peptide induces an acrosome reaction in giant panda spermatozoa and enhances their penetration of salt-stored porcine oocytes

Atrial natriuretic peptide (ANP) is a vasodilator peptide primarily produced in the heart. Locally synthesized ANP has been found in reproductive tissues of various mammals and humans, and plays an important role in rat oocyte maturation and human sperm function. The objective of the present study was to determine the effects of ANP on the function (acrosome reaction and zona penetration) of giant panda spermatozoa. In fresh and frozen-thawed spermatozoa that had been preincubated for 2.5h, treatment with ANP (for 60 min) significantly increased the proportion of acrosome-reacted spermatozoa; maximal response (an acrosome reaction in 18.3 and 21.8% of fresh and frozen-thawed spermatozoa, respectively) was detected at 1 nM ANP. Treatment with C-ANP-(4-23), an analogue of ANP and specific binder to natriuretic peptide receptors-C (NPRC), had no significant effect on the acrosome reaction. However, the cyclic guanosine 5'-monophosphate (cGMP)-dependent protein kinase (PKG) inhibitor KT5823 completely abolished the effect of ANP on acrosome reaction. The effects of ANP, caffeine and heparin on frozen-thawed sperm function were studied by insemination of porcine salt-stored oocytes in a modified Tris-buffered medium (mTBM). The presence of ANP, caffeine or heparin in the insemination medium resulted in a higher proportion (P < 0.05) of oocytes with spermatozoa in the zona and perivitelline space (PVS), and a higher average number of spermatozoa/oocyte (P < 0.05) in the zona and PVS. However, in the absence of ANP, caffeine and heparin, there were no oocytes with a spermatozoon in the PVS. There were no differences among ANP, caffeine or heparin treatments for the proportion of oocytes penetrated or average number of spermatozoa/oocyte in the zona and PVS. In conclusion, we inferred that ANP induced the acrosome reaction of preincubated giant panda spermatozoa by a PKG pathway. Furthermore, ANP enhanced the penetrability of porcine salt-stored oocytes by frozen-thawed giant panda spermatozoa.     

Sperm capacitation in the domestic cat (Felis catus) and leopard cat (Felis bengalensis) as studied with a salt-stored zona pellucida penetration assay.

The ability of domestic cat or leopard cat spermatozoa to penetrate zonae pellucidae (ZP) of salt-stored, domestic cat oocytes was examined as an assay for sperm capacitation. Ovarian oocytes were recovered after ovariectomy and matured in vitro for 18-36 h. Following removal of cumulus cells, the oocytes were used fresh, or stored (4 degrees C, 0.5-24 weeks) in a HEPES-buffered hypertonic salt solution. Electroejaculated, washed sperm (2-4 x 10(6) sperm/ml) were preincubated for 1.0 h (38 degrees C, 5% CO2 in air) and then co-incubated (2 x 10(5) sperm/ml) with fresh or stored oocytes for 6.0 h. Gametes were incubated in a protein-free, modified Tyrode's solution (TLP-PVA) or in the same medium containing 4.0 mg/ml bovine serum albumin (BSA; TALP-PVA). Treatments were compared for percentage ZP penetration (defined as sperm heads reaching more than halfway through the ZP) as an index of sperm capacitation. In both the domestic cat and leopard cat, there was no difference (P greater than 0.05) in sperm penetration of fresh ZP (domestic cat, 42.5 +/- 5.4%; leopard cat, 38.6 +/- 2.8%) or stored ZP (domestic cat, 32.4 +/- 4.2%; leopard cat, 27.6 +/- 2.3%). Sperm incubated in protein-free medium (TLP-PVA) were less capable (P less than 0.05) of ZP penetration (domestic cat, 14.6 +/- 5.9%; leopard cat, 7.9 +/- 3.0%) than sperm incubated in medium TALP-PVA containing BSA (domestic cat, 60.3 +/- 5.9%; leopard cat, 58.4 +/- 3.0%).(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of oocyte storage and cumulus cell presence on canine zona penetration by domestic dog spermatozoa

Zona penetration assays (ZPAs) have been developed in numerous species to evaluate sperm fertilizing potential. Preservation methods to stockpile oocytes would be beneficial because of the difficulty in obtaining sufficient numbers of fresh oocytes. Using a canine ZPA, the objectives of this study were to evaluate: (1) two methods of storing canine oocytes (salt storage and intrafollicular cooling) and (2) the effects of cumulus cells on oocyte penetration. In experiment 1. oocytes from fresh ovaries were assigned at random to 3 categories: fresh control (FRE), salt storage in solution 1 (1.5 M MgCl2.6H2O; SS1) and salt storage in solution 2 (0.5 M (NH4)2SO4, 0.75 M MgCl2.6H2O, 0.2 mM ZnCl2; SS2). Each category was subdivided into two treatments: cumulus cells intact (intact) and cumulus cells removed (denuded), resulting in a total of six treatments with n > 15 oocytes per treatment for each ejaculate. Fresh (FRE) intact oocytes demonstrated greater sperm-oocyte interaction than other treatments, including FRE denuded oocytes (11.7 +/- 0.6 versus <4.1 +/- 0.5 sperm-oocyte and 94.9 versus <55.6% penetration; P < 0.01). Poor sperm-oocyte interaction was demonstrated with all salt-stored oocytes (< or = 1.6 +/- 0.2 sperm-oocyte and < 51% penetration), but was further attenuated in the absence of cumulus cells. In experiment 2, oocytes obtained from fresh (FRESH) or cooled (24 h COOL, 48 h COOL) ovaries were used with cumulus cells intact for a total of three treatments with n > 15 oocytes per treatment for each ejaculate. No significant difference was observed in sperm interaction between oocytes from fresh, 24 and 48 h COOL ovaries ( 12.3 +/- 0.5 to 13.1 +/- 0.4 sperm-oocyte and 92.2-97.7% penetration; P > 0.01). These results indicate that salt storage may cause damage to canine oocytes, subsequently impairing sperm penetration, whereas short-term intrafollicular cooling does not affect the oocyte's penetrability. Furthermore, greater sperm interaction in oocytes with an intact cumulus suggests a possible role for cumulus cells in canine gamete interaction.     

Sperm binding capacity and ultrastructure of the zona pellucida of stored canine oocytes

Sperm binding to the zona pellucida is a prerequisite for fertilization, and tests that evaluate this function have been described for several species. When carrying out such tests in the canine species, ovaries or oocytes have to be stored to obtain a sufficient number of oocytes at the time of testing. In the present study, the sperm binding capacities of salt-stored oocytes and oocytes from deep frozen ovaries were measured and compared with that of fresh oocytes. Two different procedures for washing the sperm-oocyte complexes (gentle and tough) were used before evaluating the number of bound spermatozoa. The total number of oocytes that bound spermatozoa was significantly lower for both salt-stored and deep frozen oocytes compared with fresh oocytes. Significantly fewer spermatozoa bound to stored oocytes than to fresh oocytes (P 相似文献   

Use of salt-stored zonae pellucidae for assessing rabbit sperm capacitation for in vitro fertilization

Zonae pellucidae of tubal and follicular oocytes were collected and prepared for salt storage. Cumulus and corona radiata cells were removed from oocytes with hyaluronidase and a small bore pipette. The oocytes (referred to as zonae since vitelli were rendered nonfunctional) were stored in a salt solution at 4°C. Using in utero capacitated sperm, the penetrability of zonae from tubal and follicular oocytes stored immediately after collection was compared to controls, i.e., in vitro development of tubal ova to the 4-cell stage within 24 hr. The penetration rates were 100% (8 penetrated/ 8 inseminated), 77.8% (7 penetrated/ 9 inseminated), and 100% (10 fertilized/10 inseminated), respectively, and these were not statistically different. The mean (x ) numbers of sperm able to penetrate the zonae, into the perivitelline space (PVS) for tubal (34.0) and follicular (1.1) oocytes were significantly different (P < 0.01). However, following maturational incubation before salt storage, zonae of tubal and follicular origin showed no significant differences in penetrability of in utero capacitated sperm when assessed by percent penetration, or mean numbers of sperm cells reaching the PVS: tubal zonae, 100% (15/15), and follicular zonae, 100% (18/18), and mean number of sperm in the PVS (x [tubal zonae] = 12.4, and x [follicular zonae] = 11.8). The penetrability of tubal zonae with and without maturational incubation was compared, and no significant differences in penetrability by in utero capacitated sperm were present when assessed by percent penetration nonmatured 92.6% (25/27) and matured 93.3% (28/30) and mean number of sperm in the PVS (x [nonmatured] = 3.33 and x [matured] = 2.41). In vitro capacitation of ejaculated rabbit sperm by serum treatment was assessed by the penetration of salt-stored zonae, zonae-free hamster oocytes (ZFHO), and in vitro fertilization of freshly collected tubal oocytes. None of 60 salt-stored zonae and none of 31 tubal oocytes were penetrated, and these values were significantly (P < 0.005) smaller than the 9 of 78 (12%) zona-free hamster ova that were penetrated by sperm cells from the same sample. In vitro capacitation of ejaculated rabbit sperm by washing and preincubation was assessed by the penetration of salt-stored zonae, zona-free hamster oocytes (ZFHO), and fertilization of freshly collected tubal oocytes. Seventy-six of 80 salt-stored zonae were penetrated, and this was significantly (P < 0.005) greater than the 67 of 87 tubal oocytes fertilized and 30 of 35 ZFHO penetrated, which were not significantly different. The salt-stored zonae were more readily penetrated by capacitated sperm when compared to tubal oocytes. However, the ZFHO are more penetrable than salt-stored zonae and tubal oocytes when incompletely capacitated sperm is used. A useful role for this approach in studies dealing with sperm fertilizing ability is anticipated.     

Developmental capacity of vitrified immature porcine oocytes following ICSI: effects of cytochalasin B and cryoprotectants

In the present study, effects of concentration and pretreatment time of cytochalasin B (CB), and of two types of cryoprotectant solutions on the nuclear maturation of vitrified-warmed porcine oocytes were examined. Also, the developmental capacity of vitrified immature porcine oocytes following intracytoplasmic sperm injection (ICSI) was investigated. The nuclear maturation rate (46.8%) of the vitrified-warmed oocytes treated with 7.5 microg/mL CB for 30 min was significantly higher (P < 0.05) than those (13.9-39.2%) of the vitrified-warmed oocytes treated with 0, 2.5, or 5.0 microg/mL CB for 10 or 30 min. Additionally, the nuclear maturation rate of oocytes treated with CB and vitrified in ethylene glycol (EG) (37.1%) was significantly higher (P < 0.05) than that of EG + dimethyl sulfoxide (Me(2)SO) (23.9%). However, no significant differences were observed in the cleavage and blastocyst development rates among the control (45.2 and 20.0%, respectively), the EG group (37.8 and 13.5%, respectively) and the EG + Me(2)SO group (39.3 and 14.3%, respectively). These results demonstrated that: (1) pretreatment with 7.5 microg/mL CB was beneficial for the vitrification of immature porcine oocytes; (2) the combination of EG and Me(2)SO as a cryoprotectant was not advantageous for in vitro maturation (IVM) of vitrified immature porcine oocytes; and (3) vitrified-warmed porcine oocytes matured after IVM, developed to the blastocyst stage without distinct differences compared to fresh oocytes following ICSI.     

In-vitro penetration of pig oocytes matured in culture by frozen-thawed ejaculated spermatozoa.

Pig oocytes matured in culture were inseminated with frozen-thawed ejaculated spermatozoa without preincubation in modified tissue culture medium (TCM) 199. High penetration rates (85-89%) and increased incidence of polyspermy were obtained at 25-100 x 10(6) spermatozoa/ml. Wide variation in penetration rates (16-89%) was observed in oocytes inseminated in medium containing 5mM caffeine and at 25-50 x 10(6) spermatozoa/ml obtained from 6 boars, regardless of sperm motility. At 25-50 x 10(6) spermatozoa/ml, penetration rates of oocytes were dependent upon the concentration of caffeine in the medium: there was no penetration without caffeine, but penetration was highest (89%) with 5mM caffeine. None of the oocytes was penetrated in the medium supplemented with heparin at 5-40 micrograms/ml. When heparin was included in the medium with 5mM caffeine, it inhibited the efficacy of caffeine to promote sperm penetration of oocytes.     

In vitro sperm penetration through the zona pellucida of immature and in vitro matured oocytes using fresh, chilled and frozen canine semen

The aim of this study was to evaluate the effect of sperm cryopreservation and the maturation state of the oocyte on the time course of canine gamete interaction during co-culture for periods of 1-10 h. Semen samples were obtained by digital stimulation and ejaculates processed as fresh, chilled and frozen samples. Sperm were co-cultured with immature or in vitro mature bitch oocytes for up to 10 h. At hourly intervals, oocytes were evaluated for sperm penetration with epifluorescence microscopy. The results were analyzed statistically using generalized linear models. Spermatozoa treatments had a significant effect on the total percentage of oocyte penetration for both types of oocytes; fresh spermatozoa showed the highest average penetration rate, while frozen sperm showed the lowest value (p<0.05). At the 1st hour of co-culture, chilled and frozen dog sperm had a higher penetration percentage (p<0.05) of in vitro matured canine oocytes (43.6% and 45.7%, respectively) than the fresh sperm had (33.8%). Sperm penetration was directly proportional to the time of incubation, when fresh or chilled sperm were used (P<0.05); in contrast, frozen dog sperm did not change penetration rates with either immature or in vitro matured oocytes over time. There was a significant difference in the average of penetration rate between immature (47.3%) and in vitro matured oocytes (56.6%) throughout the 10h of culturing; irrespective of sperm treatment. The optimal incubation time in terms of maximizing penetration rates probably are dependent on how spermatozoa were processed prior to fertilization.     

Effects of caffeine and/or heparin in a chemically defined medium with or without glucose on in vitro penetration of bovine oocytes and their subsequent development

Bovine cumulus-enclosed oocytes were matured in culture for 22 to 24 h, freed from cumulus cells and inseminated with frozen-thawed spermatozoa in a chemically defined medium containing 1 mg polyvinylalcohol/ml with or without 5 mM caffeine and/or 10 micrograms heparin/ml and 13.9 mM glucose. Penetration of oocytes was observed only in the medium containing caffeine and/or heparin. Regardless of the presence of glucose, similar proportions of oocytes were penetrated in the medium containing heparin with (73 and 83%) or without (36 and 41%) caffeine. However, when the medium was supplemented with caffeine only, a higher penetration rate was observed in the presence (41%) than in the absence (27%) of glucose. When oocytes inseminated in medium containing caffeine and heparin with or without glucose were cultured in a chemically defined, protein-free medium, 72 and 90% and 9 and 21% of inseminated oocytes developed to the > or = 2-cell and blastocyst stages 48 and 192 h post insemination, respectively. These results, obtained using chemically defined conditions, indicate that glucose is required for stimulating fertilization in vitro of bovine oocytes and that synergistic action of caffeine and heparin appears independently of the reversing activity of caffeine on the inhibition of heparin-induced sperm capacitation by glucose.     

In vitro maturation, fertilization and development of follicular oocytes from buffalo (Bubalus bubalis).

Cumulus-oocyte complexes, 5596, were cultured for 24 h in either TCM-199 or Ham's F-10 with or without gonadotrophins and supplemented with either 20% buffalo oestrous serum (BES) or fetal calf serum (FCS). The maturation rates of oocytes cultured in TCM-199 or Ham's F-10 medium supplemented with 20% BES were 47.4 +/- 17.8 and 44.8 +/- 25.6, respectively. Addition of luteinizing hormone (LH) (5 micrograms ml-1) significantly improved the maturation rate in the Ham's F-10 medium supplemented with 20% BES (76.8 +/- 18.3), but follicle-stimulating hormone (FSH) (0.5 micrograms ml-1) and oestradiol (1 microgram ml-1) failed to synergize with LH (71.7 +/- 19.5). In the TCM-199 system, LH failed to enhance the maturation rate but the addition of FSH and oestradiol significantly enhanced the proportion of mature oocytes (42.7 +/- 1.4 and 81.7 +/- 14.5, respectively; P less than 0.05). Frozen-thawed spermatozoa prepared in Bracket and Oliphant (BO) medium and treated with 5 mmol caffeine 1(-1) + 10 micrograms heparin showed a higher fertilization rate (29.8%) than those treated in Hepes-Talp and treated with 10 micrograms heparin ml-1 (19.6%). Fertilization rate was significantly improved when fresh ejaculated spermatozoa treated with 5 mmol caffeine 1-1 and 10 micrograms heparin in BO medium (50%) was used. Rate of cleavage and development were also higher when in vitro fertilization was carried out with fresh ejaculated spermatozoa treated with caffeine and heparin (34.1 and 36.8%, respectively) than with frozen-thawed spermatozoa (27.0 and 22.0%, respectively). Development rate was enhanced when fertilized ova were cultured in ligated rabbit oviduct (28.0%) than when co-cultured on oviductal cell monolayers (8.2%). The results indicate that oocytes cultured in medium supplemented with BES and gonadotrophins reveal high rates of maturation and development to the blastocyst stage after fertilization with fresh ejaculated spermatozoa.     

Synergistic effect of caffeine and heparin on in-vitro fertilization of cattle oocytes matured in culture

Frozen-thawed spermatozoa obtained from six different bulls were suspended in Brackett and Oliphant's (BO) medium (14), with or without 10 mM caffeine, after washing. A 50-mul aliquot of the sperm suspension was added to the 50-mul BO medium supplemented with bovine serum albumin (BSA, 20 mg/ml) and heparin (20 mug/ml) in which the bovine follicular oocytes matured in culture had been introduced previously. The proportion (35%) of oocytes penetrated in the presence of heparin alone 20 to 24 h after insemination was not significantly different from those (32%) penetrated in the presence of caffeine alone as reported previously (1). When heparin was added to the caffeine in the fertilization medium, the penetration rate of oocytes increased significantly to 68% (P < 0.001), indicating that both chemicals act sinergistically to induce capacitation and/or acrosome reaction of spermatozoa and stimulate in vitro fertilization of cattle oocytes. However, great variation in penetration rates (35 to 96%) was observed among the different bulls. The optimal concentration of heparin in the suspension medium in which the highest rate of oocyte penetration took place was 10 mug/ml.     

Purification of hyperthermophilic archaeal amylolytic enzyme (MJA1) using thermoseparating aqueous two-phase systems

Purification of a recombinant, thermostable alpha-amylase (MJA1) from the hyperthermophile, Methanococcus jannaschii, was investigated in the ethylene oxide-propylene oxide random copolymer (PEO-PPO)/(NH(4))(2)SO(4), and poly(ethylene glycol) (PEG)/(NH(4))(2)SO(4) aqueous two-phase systems. MJA1 partitioned in the top polymer-rich phase, while the remainder of proteins partitioned in the bottom salt-rich phase. It was found that enzyme recovery of up to 90% with a purification factor of 3.31 was achieved using a single aqueous two-phase extraction step. In addition, the partition behavior of pure amyloglucosidase in polymer/salt aqueous two-phase systems was also evaluated. All of the studied enzymes partitioned unevenly in these polymer/salt systems. This work is the first reported application of thermoseparating polymer aqueous two-phase systems for the purification of extremophile enzymes.     

Application of a zona pellucida binding assay (ZBA) in the domestic cat benefits from the use of in vitro matured oocytes

Background

Zona pellucida binding assays (ZBAs) have proven useful in determining the fertilising ability of spermatozoa in several species. Most ZBAs use fresh or salt-stored oocytes collected from fresh ovaries but because ovaries are not easy to obtain on a regular basis, chilled and frozen-thawed ovaries have been tested, with varying results. The present study tested the hypothesis that cat spermatozoa, either fresh or frozen-thawed, can bind to homologous zona pellucida of oocytes retrieved from frozen-thawed queen ovaries to a similar extent as they can bind to the zona pellucida of fresh, in vitro matured oocytes.

Methods

Ovaries were collected from queens after routine ovario-hysterectomy and either stored in NaCl at -20°C until use (treatment animals), or used fresh (controls). Cumulus-oocyte complexes (COCs) were retrieved by ovarian slicing from either source and used directly (immature oocytes from frozen-thawed ovaries; treatment animals) or after in vitro maturation (IVM) (fresh ovaries; controls) for 24 hours in TCM 199, supplemented with 1 IU hCG/mL and 0.5 IU eCG/mL and 0.5% bovine serum albumin (BSA). The oocytes were incubated for 4 hours in 5% CO₂ in air at 38°C and 100% humidity in the presence of 5 × 10⁶ fresh or frozen-thawed spermatozoa/mL. Representative samples of oocytes were processed for scanning electron microscopy (SEM).

Results

Both fresh and frozen-thawed spermatozoa bound to the in vitro matured zona pellucida but significantly fewer, or no, spermatozoa bound to frozen-thawed, immature zona pellucida (P < 0.001). Also, more fresh spermatozoa than frozen-thawed spermatozoa bound to the zona pellucida (P < 0.001). The zona pellucida surface differed in morphology (SEM), with in vitro matured oocytes showing a dense surface with few fenestrations in contrast to their frozen-thawed, immature counterparts, where fenestrations were conspicuously larger.

Conclusion

In conclusion, under the conditions of the present study, immature oocytes recovered from ovaries frozen immersed in NaCl at -20°C are less suitable for use in feline ZBA.     

Effect of co-culture of porcine sperm and oocytes with porcine oviductal epithelial cells on in vitro fertilization

This study was designed to determine the effect of co-culture with porcine oviductal epithelial cell (POEC) monolayers on in vitro fertilization of pig oocytes. The in vitro penetrability of mature (experiment 1) or immature (experiment 2) oocytes was studied in presence or absence of POEC during IVF with fresh semen. In experiment 3, boar and POEC effects were analyzed but in this case with frozen-thawed spermatozoa. In experiment 4, the spermatozoa were pre-incubated before IVF with or without POEC in order to assess their effect on IVF sperm-related parameters. In experiment 5, the effect of POEC was studied by co-culturing them with oocytes before IVF to determine if monospermy was improved. The results showed that high sperm concentration and POEC increase oocyte penetrability (P<0.01) and decrease monospermy rate (P<0.01), in both mature and immature oocytes (P<0.01) with fresh semen and a 18 h culture time. With frozen semen was detected a boar and POEC effect (P<0.01) on penetration rate. The sperm pre-culture 2 h with POEC also resulted in an increase of sperm penetration in terms of number of sperm per oocyte (P<0.01) and this treatment did not increase monospermy when contact time between gametes was limited to 6 h although monospermy was higher when POEC were present during IVF. Finally, exposure of oocytes to POEC for 4 h before IVF facilitated monospermic penetration to over 70% (P<0.01). In conclusion, the use of POEC in porcine IVF systems provides the possibility of working with low sperm concentrations and the effect of POEC on monospermy depends on sperm concentration, boar and contact time between gametes. Moreover, the exposure of oocytes to POEC before IVF improves the rate of monospermy.     

IN VITRO FERTILIZATION OF IN VITRO MATURED MINKE WHALE (BALAENOPTERA ACUTOROSTRATA) FOLLICULAR OOCYTES

In vitro fertilization of follicular oocytes harvested from ovaries and matured in vitro was attempted for 55 minke whales ( Balaenoptera acutorostrata ) captured for Japanese research purposes in the Antarctic Ocean during the period from November 1995 to March 1996. In Experiment 1, effects of culture duration (96 h or 120 h) on maturation of follicular oocytes and addition of caffeine (5 mM) and/or heparin (100 pg/ml) on sperm penetration and pro-nuclear formation were investigated. Spermatozoa recovered from the vasa deferentia of four mature males were diluted (5-fold) and frozen at - 80°C. The post-thawed and pooled spermatozoa were used for in vitro insemination. A higher ( P < 0.05) proportion of the oocytes cultured for 120 h (34.2% of 260) progressed beyond the second metaphase stage than of the oocytes cultured for 96 h (26.0% of 262). For the matured oocytes, higher rates of penetration ( P < 0.05) and pronuclear formation ( P < 0.01) were obtained in the oocytes cultured for 120 h (55.1% and 40.4%) than in those cultured for 96 h (32.4 % and 20.6%). Addition of caffeine and heparin did not show a significant effect. In Experiment 2, follicular oocytes matured for 120 h and then inseminated were cultured to examine the subsequent development in two culture systems (with and without co-cultured cumulus cells). Of 448 inseminated oocytes, cleaved embryos (2–16 cells) were observed with (5.8%) and without (4.9%) co-cultured systems. No cleavage was observed in 54 ova without insemination. These results indicate that in vitro fertilization of minke whale in vitro matured follicular oocytes with cryopreserved spermatozoa is possible, yielding cleaved embryos.     

Penetration in vitro of bovine oocytes during maturation by frozen-thawed spermatozoa

Bovine immature oocytes cultured for various times in TC-199 medium were inseminated with frozen-thawed spermatozoa in Medium BO with caffeine (5 mM) and heparin (10 micrograms/ml). Very high penetration rates (95-100%) were obtained in all oocytes which had been cultured for 0-20 h. When oocytes cultured for 0 and 4 h were inseminated, 100% of them were penetrated and had a decondensing sperm head and most of the oocytes remained at the stage of condensed germinal vesicle (GV) to telophase-I 20-22 h after insemination. The formation of male and female pronuclei was first observed in oocytes inseminated 8 h after culture. The proportions of polyspermy and average number of spermatozoa in penetrated oocytes gradually decreased as oocyte maturation proceeded. Penetration of at least one spermatozoon with a decondensing head into oocytes at the GV stage (without culture) was almost completed up to 8 h after insemination and at that time most of the penetrated oocytes were still at the stage of GV or condensed GV. These results indicate that maturation of bovine oocytes is not required for sperm penetration into the vitellus or for sperm nuclear decondensation under the in-vitro conditions used.     

Development of the competence of bovine oocytes to release cortical granules and block polyspermy after meiotic maturation

Bovine immature oocytes do not have the ability to block polyspermic penetration. The present study was conducted to determine whether this is correlated to cortical granule (CG) distribution and the competence of oocytes to release CG upon sperm penetration, and whether the ability of bovine oocytes to release CG develops during in vitro maturation. Fluorescein isothiocyanate-conjugated Lens culinaris agglutinin was used for detecting CG in immature and mature oocytes before and after sperm penetration and electric stimulation. The labeled oocytes were examined with laser confocal and fluorescent microscopes. The results show that CG exist as clusters in all immature oocytes. The CG were not released from immature oocytes exposed to electric pulse or penetrated by spermatozoa, resulting in 94% of oocytes being polyspermic. When immature oocytes were cultured for 22h in vitro , 81% extruded the first polar body and reached metaphase II. In mature oocytes, 25% of oocytes showed CG clusters, 42% and 33% of oocytes showed partial and complete CG dispersion, respectively. When mature oocytes were inseminated in vitro , only 15% of oocytes were polyspermic. Cortical granule exocytosis occurred in 97% of oocytes after sperm penetration and 84% of oocytes released all of the CG 18 h after insemination. Electric pulse induced all of the mature oocytes to release CG but only 55% released all of their CG 18 h post stimulation. These results indicate that polyspermy in immature bovine oocytes is the result of the complete failure of the oocyte to release CG after sperm penetration. Bovine oocytes became competent to release CG by sperm penetration and electric stimulation after meiotic maturation. These results provide evidence that CG exocytosis plays an important role(s) in the establishment of the block to polyspermy in bovine oocytes.     

Effect of sperm penetration in vitro on completion of first meiosis by bovine oocytes arrested at various stages in culture.

Bovine oocytes cultured for 12-20 h in TC-199 were incubated for 24 h in fertilization medium, Brackett and Oliphant medium with bovine serum albumin (10 mg ml-1), caffeine (5 mmoll-1) and heparin (10 micrograms ml-1), with or without frozen-thawed spermatozoa. High penetration rates (93-96%) and significantly (P < 0.001) higher maturation rates were obtained in oocytes incubated with (93-100%) than without (62-72%) spermatozoa. However, when oocytes at the germinal vesicle stage were cultured for 44 h fertilization medium, maturation of oocytes to metaphase II was reduced. However, all oocytes that were first cultured for 20 h and further for 24 h with spermatozoa were penetrated and 40% of the penetrated oocytes reached metaphase II. All of the remaining oocytes that did not mature arrested at the stages of condensed germinal vesicle (39%) or prometaphase I (22%). These results indicate that oocytes at metaphase I at and after sperm penetration are stimulated by sperm penetration to complete maturation.     

The Hemolymph of the ascidian Styela plicata (Chordata-Tunicata) contains heparin inside basophil-like cells and a unique sulfated galactoglucan in the plasma

The hemolymph of ascidians (Chordata-Tunicata) contains different types of hemocytes embedded in a liquid plasma. In the present study, heparin and a sulfated heteropolysaccharide were purified from the hemolymph of the ascidian Styela plicata. The heteropolysaccharide occurs free in the plasma, is composed of glucose ( approximately 60%) and galactose ( approximately 40%), and is highly sulfated. Heparin, on the other hand, occurs in the hemocytes, and high performance liquid chromatography of the products formed by degradation with specific lyases revealed that it is composed mainly by the disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4)) (39.7%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(6SO(4)) (38.2%). Small amounts of the 3-O-sulfated disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4)) (9.8%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4))(6SO(4)) (3.8%) were also detected. These 3-O-sulfated disaccharides were demonstrated to be essential for the binding of the hemocyte heparin to antithrombin III. Electron microscopy techniques were used to characterize the ultrastructure of the hemocytes and to localize heparin and histamine in these cells. At least five cell types were recognized and classified as univacuolated and multivacuolated cells, amebocytes, hemoblasts, and granulocytes. Immunocytochemistry showed that heparin and histamine co-localize in intracellular granules of only one type of hemocyte, the granulocyte. These results show for the first time that in ascidians, a sulfated galactoglucan circulates free in the plasma, and heparin occurs as an intracellular product of a circulating basophil-like cell.