Effect of albumin on fertilization of mouse ova in vitro

Serum albumin is an obligatory component of the incubation medium for the fertilization of mouse ova. Normal, untreated bovine serum albumin supports high rates of fertilization of cumulus-free ova both with and without their zonae pellucidae. Heat-treated or trichloroacetic acid-extracted bovine serum albumin is unable to support the fertilization of a majority of zona-intact ova but fertilization of zona-free ova is unimpaired. Spermatozoa incubated in medium containing heated bovine serum albumin fertilize zona-intact ova when 2 mM caffeine is present but the progress of sperm head decondensation is delayed when compared to normal controls. Trichloroacetic acid extracted BSA preferentially and irreversibly inhibits zona penetration by spermatozoa, but this effect is not mediated by an inhibition of spermatozoal motility or zona-binding ability. This effect occurs after only a 10-min preincubation of the spermatozoa in the extracted BSA or when the medium contains only a 10% (v/v) proportion of this albumin. It is estimated that mouse spermatozoa under the conditions used take 2 hr to penetrate the zonae pellucidae of 50% of ova and effect fertilization.     

Penetration in vitro of zona-free pig oocytes by homologous and heterologous spermatozoa

We examined the penetrability of pig, rat and bull spermatozoa into zona-free pig oocytes. Frozen-thawed boar spermatozoa penetrated into both zona-intact and zona-free oocytes with similar efficacy in a modified Tris-buffered medium (mTBM) supplemented with BSA and caffeine, but not in medium without caffeine. Rat epididymal spermatozoa did not readily penetrate into zona-free pig oocytes in mTBM with BSA. However, when a modified Krebs-Ringer bicarbonate solution was used, penetration rate varied with sperm concentrations at insemination: 79% of the oocytes were penetrated at 1.0 x 10(6) cells/ml, but very few at 0.1 x 10(6) and 10.0 x 10(6) cells/ml. In all oocytes penetrated, no activation was observed and the sperm nucleus was fully decondensed but did not transform into a male pronucleus. Frozen-thawed bull spermatozoa were also found to penetrate into zona-free pig oocytes in mTBM with BSA, caffeine and heparin: higher penetration rates were obtained with 1.0 x 106 and 10.0 x 10(6) spermatozoa/ml compared with 0.1 x 10(6) spermatozoa/ml. The penetration rate with 1.0 x 10(6) spermatozoa/ml was stable in five different bulls. All oocytes penetrated were activated and male pronuclear formation was observed in 57-79% of the penetrated oocytes. These results suggest that capacitation or the acrosome reaction is required for boar, rat, and possibly, bull spermatozoa to penetrate into zona-free pig oocytes. Bull spermatozoa can easily induce activation of pig oocytes and form male pronuclei, but rat spermatozoa cannot do so, indicating species differences in the ability of spermatozoa to activate pig oocytes and to transform to male pronuclei in the ooplasm.     

The effect of sera, bovine serum albumin and follicular cells on in vitro maturation and fertilization of porcine oocytes

The effects of fetal calf serum (FCS), estrus gilt serum (EGS) BSA, dispersed granulosa cells, hemi-sections of follicular wall, and replacement of medium after 24 hours on in vitro maturation and fertilization of porcine oocytes were studied. The results indicate that the use of BSA for 24 or 48 hours inhibited the expansion of cumulus cells and the maturation of oocytes. An incubation of 24 hours culture in FCS followed by a second 24 hours in BSA containing medium did not decrease the rate of maturation but significantly decreased the polyspermy and mean number of spermatozoa penetrated/oocyte. Renewing the medium with or without removal of cumulus cells during the second incubation increased the maturation rate. Removal of cumulus cells decreased the penetrability, the polyspermy rates of the oocyte and the mean number of spermatozoa/oocyte penetrated. The EGS-supplemented medium, dispersed granulosa cells or hemi-sections of follicular wall did not affect the maturation or fertilization rates. In conclusion, BSA, a protein supplement in maturation medium, inhibited cumulus cell expansion and maturation of porcine oocytes. After resumption of meiosis triggered by FCS, BSA did not influence maturation. The FCS-BSA treatment reduced the incidence of polyspermy and the mean number of spermatozoa penetrated/oocyte without decreasing the rate of maturation and fertilization.     

Penetration of the zona-free or intact eggs by foreign spermatozoa and the fertilization of deer mouse eggs in vitro

Zona-free eggs were introduced to fresh or preincubated sperm suspensions and the penetration of eggs by foreign spermatozoa was examined, as evidenced by enlargement of the sperm head and formation of the male pronucleus. It was found that zona-free hamster eggs can be penetrated by guinea-pig, deer mouse and rabbit spermatozoa but zona-free rat, mouse and rabbit eggs cannot be penetrated by guinea-pig spermatozoa. Furthermore, zona-free rat and mouse eggs cannot be penetrated by spermatozoa from two species of deer mice and the Mongolian gerbil. The zona pellucida of a few intact rat eggs can be penetrated by mouse (6%) and by P. leucopus spermatozoa (14%) but enlargement of the sperm head and formation of pronuclei were observed in the former but not in the latter. It seems that (1) sperm capacitation is required for the penetration of zona-free eggs, (2) the attachment of foreign spermatozoa to eggs may indicate their potential ability of penetration in some cases, (3) there is a certain affinity between the vitellus of one species and spermatozoa from another species, (4) the block to the entry of foreign spermatozoa is not only in the zona pellucida but also in the vitelline membrane, (5) zona-free hamster eggs can be penetrated by spermatozoa of six species, (6) mouse spermatozoa can penetrate zona-free eggs of three species, and (7) fertilization of intact P. maniculatus eggs can be achieved in vitro.     

Direct contact is required between serum albumin and hamster spermatozoa for capacitation in vitro

A dialysis unit was used to test whether direct physical contact between serum albumin and hamster spermatozoa is required for capacitation and/or the acrosome reaction. Sperm and bovine serum albumin (BSA) were incubated cither together (direct incubation) or separated by a dialysis membrane (indirect incubation). Sperm viability was supported with “sperm motility factors” (hypotaurine and epinephrine) and polyvinylalcohol (PVA). Spermatozoa became capacitated and underwent acrosome reactions when directly incubated in medium containing BSA (TALP-PVA), but did not undergo acrosome reactions when indirectly incubated with BSA (medium TLP-PVA). When sperm were first incubated for 4 hr indirectly with BSA, followed by 4 hr direct incubation with BSA, capacitation did not occur during indirect incubation. These findings indicate that an “intimate association” is necessary between serum albumin and spermatozoa to support capacitation under in vitro incubation conditions. The data are consistent with the concept of direct transfer of compounds from sperm to albumin and/or vice versa during sperm capacitation.     

Nitric oxide interacts with the cAMP pathway to modulate capacitation of human spermatozoa

This study aimed to demonstrate nitric oxide production by human spermatozoa and to characterize the interaction between nitric oxide and cAMP-related pathway in the control of human sperm capacitation and protein tyrosine phosphorylation. Spermatozoa were incubated in Tyrode's medium with or without bovine serum albumin (BSA), and nitric oxide was measured with the spin trap sodium N-methyl-D-glucamine dithiocarbamate. Under noncapacitating conditions, spermatozoa produced low levels of nitric oxide. However, under capacitating conditions, prominent nitric oxide adduct signals were obtained and a time-dependent increase of nitric oxide production was observed. When spermatozoa were incubated in Tyrode+BSA medium with nitric oxide-releasing compounds, intracellular cAMP concentrations increased to levels higher than those of spermatozoa incubated in Tyrode+BSA alone. In contrast, incubation with nitric oxide synthase inhibitors (N(G)-nitro-L-arginine methyl ester or N(G)-monomethyl L-arginine) decreased intracellular sperm cAMP concentrations. The inhibitory effect observed with N(G)-nitro-L-arginine methyl ester on capacitation and tyrosine phosphorylation of two sperm proteins (105, 81 kDa) was overcome by the presence of cAMP analogs or of a phosphodiesterase inhibitor. These results indicate that nitric oxide is produced by capacitating human spermatozoa and that it may act as a cellular messenger by modulating the cAMP pathway involved in capacitation and protein tyrosine phosphorylation.     

In vitro fertilization of rat and mouse eggs by ejaculated sperm and the effect of energy sources on in vitro fertilization of rat eggs.

In vitro fertilization of rat and mouse eggs by ejaculated or epididymal spermatozoa in chemically defined media was studied. Penetration rates by ejaculated sperm was very low (0 to 8%) in the rat, but 11 to 41% of eggs were penetrated by ejaculated sperm in the mouse. The optimal concentration of sperm for in vitro fertilization appears to be similar whether ejaculated or epididymal sperm were used. The time of sperm penetration in the mouse eggs, however, was delayed for one-half to one hour when ejaculated sperm were used. The importance of sodium pyruvate, sodium lactate and glucose in the medium containing bovine serum albumin for in vitro fertilization of rat eggs was examined. When rat eggs in cumulus clot were exposed to epididymal sperm preincubated for five hours, the presence of sodium pyruvate, sodium lactate and glucose was found to play an important role. When exposed to non-incubated epididymal sperm sodium pyruvate could be omitted without much decline of the fertilization rate. When the denuded eggs were exposed to non-incubated sperm, penetration rates were very low (0 and 5%) in the absence of pyruvate. It appears that although lactate, pyruvate and glucose are all important for in vitro fertilization of rat eggs, pyruvate can be supplied by the follicular cells surrounding the eggs.     

Early nuclear events of in vitro fertilization in the domestic fowl (Gallus domesticus)

In vitro fertilization in the domestic fowl (Gallus domesticus) was investigated by observation of the early nuclear events. Ova retrieved from the fimbria following ovulation were inseminated in vitro with 10(6)-10(7) spermatozoa in Dulbecco's modified Eagle's medium (DMEM) for 10 min and then further incubated in DMEM + albumen for 1, 2, 3, or 4 hr. These eggs were histologically examined by epifluorescent microscopy after staining with 4',6'-diamidino-2-phenylindole (DAPI). Nuclei of spermatozoa at various stages of transformation were observed in the ova incubated for 1-3 hr. Close pairing of two pronuclei, presumed to be male and female juxtaposition, was detected in ova incubated for 4 hr. These data provide direct evidence for the in vitro fertilization of fowl eggs and suggested that the early process of in vitro fertilization is comparable to that of in vivo fertilization.     

The effect of various capacitation active compounds and capacitation time on the in vitro fertility and protein tyrosine phosphorylation profiles of bovine sperm

In this paper the effects of capacitation and fertilisation stimulating compounds (heparin, caffeine, glucose, D-penicillamine, bovine serum (BOS), bovine serum albumin (BSA), polyvinyl alcohol (PVA)) were analysed in several in vitro fertilisation protocols. Attention was paid to the rate of penetrated oocytes, kinetics of penetration and to polyspermic fertilisation. Cryopreserved bovine sperm and in vitro matured bovine oocytes were used throughout all the fertilisation experiments. As detected in the first 8 h fertilisation experiment with non-incubated sperm, the supplementation of medium with heparin, BOS and glucose supported the fertilisation rate most effectively (100%), including the kinetics of pronuclei formation (52.4%). The absence of BOS resulted in a decreased fertilisation rate (62.7%) as well as a delay in pronuclei formation (13.6%), similar to that after substitution of heparin with caffeine (73.0% and 25.4%, respectively). The penetration rate in the control medium with BOS (without heparin and caffeine) was surprisingly high, especially in medium without glucose (62.2%). The positive effect of glucose on sperm penetration was observed mainly in a chemically defined medium with PVA. High polyspermy rates were observed throughout all experiments in the media containing heparin or caffeine and BOS as the macromolecular component. D-Penicillamine was not shown to be a fertilisation-stimulating molecule. However, as detected in the second experiment in which oocytes were fertilised with 5 h incubated sperm, its positive effect on the prolongation of a fertile life span of cryopreserved spermatozoa was significant. The presence of either caffeine or heparin in the fertilisation medium (FM) with BOS during sperm incubation induced tyrosine phosphorylation of an approximately 90 kDa protein, detected after 5 h of sperm incubation. The absence of BOS reduced tyrosine phosphorylation of this protein in fertilisation medium with heparin. The percentage of motile spermatozoa and those with intact acrosomes were monitored throughout all experiments.     

Ability of in vitro maturing bovine oocytes to transform sperm nuclei to metaphase chromosomes.

Bovine oocytes at the germinal vesicle stage were inseminated in Brackett & Oliphant's medium with bovine serum albumin, caffeine and heparin. Eight hours after insemination, oocytes were transferred into tissue culture medium-199 containing 10% fetal calf serum and cultured for 5-40 h at 39 degrees C in 5% CO2 in air. The proportions of unpenetrated and penetrated oocytes reaching metaphase II increased as the time of examination increased, reaching 70 and 65% 40 h after transfer, respectively. When oocytes were penetrated by more than four spermatozoa, meiotic maturation was greatly retarded. Sperm nuclei were decondensed in most (81%) penetrated oocytes 5 h after transfer. The decondensed sperm nuclei were recondensed and then transformed to metaphase chromosomes which were morphologically compacted at first but became slightly dispersed later. The formation of the metaphase chromosomes was observed in 86% of penetrated oocytes examined 40 h after transfer, and occurred in all metaphase II oocytes at that time. In oocytes penetrated by more than nine spermatozoa, no such transformation of sperm nuclei was observed. Well-developed male and female pro-nuclei were observed in only three (6%) of 51 oocytes penetrated 40 h after transfer.     

Ultrastructure of in vivo fertilization in the goat

In vivo fertilization of goat eggs has been studied by electron microscopy. Eggs were recovered from superovulated or natural cyclic goats, 32 to 52 hours after the onset of oestrus; only eggs recovered between 46 and 52 hours were fertilized. Spermatozoa penetrated the zona pellucida tangentially leaving vesiculated products of the acrosome reaction at the zona surface. As sperm penetrated into the ooplasm, the second meiotic division completed and cortical granule exocytosis occurred. However a few unreacted cortical granules usually remained in the cortex of the two fertilized eggs, adjacent to the plasma membrane. After swelling the two pronuclei presented similar ultrastructural morphology: they contained small, compact, agranular nucleoli and unevenly distributed chromatin. The cytoplasm in close vicinity to the apposed pronuclei contained large stacks of annulate lamellae, smooth endoplasmic reticulum, prominent Golgi complexes, as well as dense areas of unidentified material. The abundance of cytoplasmic organelles near the pronuclei might be the expression of intensive metabolic activity. Conversely, in the cortex of fertilized ova several large organelles-free cytoplasmic areas were randomly distributed.     

Taurine and human spermatozoal capacitation

The effect of taurine at various concentrations (0.01, 0.1 and 1 mM) on the in vitro motility and fertilizing capacity of human spermatozoa was studied. Spermatozoa collected from 10 normal men were washed in BWW medium and incubated with taurine for 5 hours, the period required for spermatozoal capacitation. The percent motilities were recorded at 0 and 5 hours during capacitation preincubation with taurine. After incubation, the spermatozoa were washed with BWW medium to remove taurine before insemination of the zona-free hamster ova for an assessment of the fertilizing capacity. Taurine caused a significant dose-dependent increase in the penetration of the zona-free hamster ova in comparison to the control (p less than 0.05). Taurine did not have any significant effects on the spermatozoal motility during capacitation preincubation. The results suggest that there may be a physiological role for this beta-amino acid in human spermatozoal capacitation in vivo.     

Intracytoplasmic injection of porcine, bovine, mouse, or human spermatozoon into porcine oocytes

We determined the incidence of activation, male pronuclear formation, and apposition of pronuclei in porcine oocytes following intracytoplasmic injection of various porcine sperm components and foreign species spermatozoa, such as that of cattle, mouse or human. The porcine oocytes were activated by injection of a spermatozoon or an isolated sperm head. In contrast, injection of either sperm tail or a trypsin- or NaOH-treated sperm head failed to induce oocyte activation. Because injection of mouse, bovine, or human spermatozoon activated porcine oocytes, the sperm-borne activation factor(s) is not strictly species-specific. Male pronuclear formation and pronuclear apposition were observed in porcine oocytes following injection of porcine, bovine, mouse or human spermatozoa. Electrical stimulation following sperm cell injection did not enhance the incidence of male pronuclear formation or pronuclear apposition compared with sperm cell injection alone (P > 0.1). Following porcine sperm injection, the microtubular aster was organized from the neck of the spermatozoon, and filled the whole cytoplasm. In contrast, following injection of bovine, mouse, or human spermatozoon, the maternal-derived microtubules were organized from the cortex to the center of the oocytes, which seems to move both pronuclei to the center of oocytes. Cleavage to the two-cell stage was observed at 19-21 hr after injection of porcine spermatozoon. However, none of the oocytes following injection of mouse, bovine, or human spermatozoa developed to the mitotic metaphase or the two-cell stage. These results suggested that the oocyte activating factor(s) is present in the perinuclear material and that it is not species-specific for the porcine oocyte. Self-organized microtubules seemed to move the pronuclei into center of oocytes when foreign species spermatozoa were injected into porcine oocytes.     

Fertilization by sperm injection in the rabbit

Whole rabbit spermatozoa and isolated sperm nuclei were microinjected directly into the ooplasm of hamster and rabbit ova. These injected sperm decondensed and formed male pronuclei during subsequent in-vitro culture. Injection of whole spermatozoa and sperm nuclei prepared by a protocol known to allow in-vitro capacitation of ejaculated spermatozoa yielded a significantly higher (P < 0.01) number of activated rabbit ova containing male pronuclei than did injection of uncapacitated epididymal sperm nuclei or ejaculated sperm nuclei. Rabbit ova fertilized by sperm injection were capable of undergoing normal-appearing cleavage division during 22 h of culture.     

In vitro capacitation of stallion spermatozoa in calcium-free Tyrode's medium and penetration of zona-free hamster eggs

Capacitation of stallion spermatozoa in Tyrode's calcium-free (TCF) medium was assessed. Twelve gel-free ejaculates were collected. After removal from the seminal plasma, cells were washed three times with 0.85% saline containing 0.1% bovine serum albumin (BSA) and resuspended in TCF. Both washing and incubation media were adjusted to pH 8 and 300 to 310 mOsm. Final sperm concentration during incubation was 2 x 10(6) cells/ml. The diluted ejaculates were incubated for 2, 4, 6, 8 and 10 h at 37 degrees C in an atmosphere containing 5% CO(2). Acrosomes were stained with naphthol yellow and erythrocin B initially and after each incubation period and evaluated microscopically. Transmission electron microscopy was used to verify whether normal acrosome reaction was occurring or if cells were degenerating. Penetration of zona-free hamster oocytes was evaluated using 10(3) to 10(4) sperm/ml suspension and coincubating eggs for 3.5 to 4 h with sperm. Penetration tests were done for wash and incubation treatments and recorded positive when swollen sperm heads or male pronuclei were present. Incubation time affected acrosome integrity (P<0.001). Incubation for 8 to 10 h significantly improved acrosome reaction (P<0.001) and the percentage of reacted acrosomes increased sharply after 6 h of incubation (P<0.001). None of the washed sperm penetrated zona-free eggs at zero time, but sperm from all incubation treatments penetrated eggs. A peak penetration rate of 29.9% was observed at 8 h (P<0.001). Results indicate that under the conditions used, the requirement for Ca(++) in the medium for the process of capacitation and acrosome reaction can be substituted for by elevated pH.     

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.     

Studies on Ergothioneine. IX. Ergothioneine Stimulates Mouse Spermatozoa and Fertilization in vitro

The effects of ergothioneine on spermatozoa and ova were investigated in vitro and in vivo. Spermatozoa were treated with ergothioneine in vitro , and injected into the uterine cavity of female mice immediately after the induction of superovulation. The ova were recovered 24 hr later and assessed for fertilization. Preincubation of spermatozoa with ergothioneine resulted in a significant increase in the fertilization rate. When ova were inseminated in the same manner in vitro with spermatozoa treated with 0.1 or 1.0 mM of ergothioneine, the penetration rate was significantly increased. These results suggest that ergothioneine is effective in inducing both capacitation and the acrosome reaction of mouse spermatozoa. Ergothioneine at concentrations of 0.1 and 1.0 mM in the preincubation medium was also effective in inducing the acrosome reaction of guinea pig spermatozoa. However, it had no significant effect on the development of 2-cell ova in vitro .     

In vitro fertilization of horse follicular oocytes matured in vitro

In vitro fertilizing ability of stallion spermatozoa was assessed using horse follicular oocytes matured in vitro. After collection, stallion spermatozoa were either: 1) washed and incubated in TALP medium with 3 mg/ml bovine serum albumin (BSA) and 10 micrograms/ml heparin for 4h, 2) washed and incubated in TALP with 3 mg/ml BSA for 3 h and cultured for a further 1 h with 1 mM caffeine and 5 mM dbcAMP, 3) washed and incubated in TALP medium with 3 mg/ml BSA at pH 7.9-8.2 for 2-4 h, or 4) diluted and incubated in TALP medium with 10 mg/ml BSA and 7.14 microM calcium ionophore A 23187 for 5-10 min followed by washing. After a given pretreatment, suspensions were diluted into B2 medium to a concentration of 5 x 10(6) sperm/ml and co-incubated with oocytes for 12 h or 24-48 h. In the ionophore-treated group, 18 of 54 oocytes (33%) were fertilized by 12 h, and 11 of 45 (24%) cleaved by 24-48 h. Evidence of fertilization was not found in the oocytes incubated with spermatozoa from other treatment procedures.     

The effects of washing and protein supplementation on the acrosome reaction of ram spermatozoa in vitro

Ram spermatozoa were incubated for 12 h at a concentration of 1–5 × 10⁶ ml^?1 in a modified BWW medium with TRIS (pH 8.0 in air, 308 mOsm) and a variety of pre-treatments were examined. These included washing in hypertonic (360 mOsm) medium by centrifugation and also supplementing with purified bovine serum albumin (BSA), with fatty acid-depleted BSA and with 5% v/v heat-inactivated sheep serum. Motility was assessed by phase contrast microscopy, and the incidence of acrosome reactions among live spermatozoa was estimated from nigrosin-eosin live-dead smears. All treatments showed a steady decline in sperm motility with time, and progressive increases in the proportion of live, acrosome-reacted spermatozoa (% ARS). However, there were no significant differences between washing regimes except for the treatment with hypertonic medium., where % ARS was elevated significantly after 9 and 12 h incubation. No differences were seen in % ARS between the various protein supplementations, although serum promoted significantly better survival.     

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.