Studies on fertilization in the teleost. I. Dynamic responses of fertilized medaka eggs

In order to understand the mechanisms of fertilization in the teleost, the movements of the egg cortex, cytoplasmic inclusions and pronuclei were observed in detail in fertilized medaka Oryzias latipes eggs. The first cortical contraction occurred toward the animal pole region following the onset of exocytosis of cortical alveoli. The cortical contraction caused movement of oil droplets toward the animal pole where the germinal vesicle had broken down during oocyte maturation. The movement of oil droplets toward the animal pole region was frequently twisted in the right or left direction. The direction of the twisting movement has been correlated with the unilateral bending of non-attaching filaments on the chorion. The female pronucleus, which approached the male pronucleus from the vicinity of the second polar body, took a course to the right, left or straight along the s-p axis connecting the male pronucleus and the second polar body. The course of approach by the female pronucleus correlated with the bending direction of the non-attaching filaments that had been determined by rotation of the oocyte around the animal–vegetal axis during oogenesis. The first cleavage furrow also very frequently coincided with the axis. These observations suggest that dynamic responses of medaka eggs from fertilization to the first cleavage reflect the architecture dynamically constructed during oogenesis.     

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.     

Caffeine and artificial insemination

We studied the reactivation of cells and the repair of photomutagenic damage induced by xanthotoxin and visnagin plus NUV in arg-1 cells of Chlamydomonas reinhardii. Maintenance of liquid cultures in the dark resulted only in a slight reactivation of cells, even after 24 h. Repair of photomutagenic damage was more efficient: within 24 h the number of Arg⁺ revertants was reduced by 50% in cells cultured in the dark at 20°C. The repair was more efficient at 30°C. At the beginning of dark cultivation an after-effect could be observed. Cultivation in standard white light instead of dark after treatment resulted in a very strong after-effect. Therefore it was not possible to detect any photoreactivation.After treatment with xanthotoxin plus standard white light (24 h) neither reactivation of cells nor repair of photomutagenic damage was found. The after-effect was higher than after xanthotoxin plus NUV. It is possible that a small amount of repair could be masked by the after-effect.Treatment with visnagin yielded similar results. The photomutagenic effect of visnagin is described for the first time in this paper. The drug is a much less effective photomutagen than xanthotoxin. The photomutagenesis of visnagin may be attributable to photoproducts similar to those formed after treatment with furocoumarins.No definite conclusion can be drawn from the present results regarding the basis for the observed lack of repair (or reduced repair) after standard white light treatment; a possible cause might be a preferential formation of bi-adducts under these conditions.     

Fertility and ovum fertilization rate after laparoscopic or transcervical intrauterine artificial insemination of oxytocin-treated ewes

Based on our previous work, we found that exogenous oxytocin induces uterine tetany and cervical dilation, and permits transcervical access to the uterus. However, the oxytocin does not reduce sustained sperm transport from the uterus to the oviducts. Thus, we hypothesized that exogenous oxytocin may be a useful adjunct to transcervical intrauterine AI procedures for sheep: two experiments were conducted to test our hypothesis. In Experiment 1, purebred ewes (n = 75/group) were artificially inseminated intrauterine with either laparoscopic or oxytocin-transcervical (i.e., 200 USP units of oxytocin 30 min before AI) procedures. At 54 h after progestogenated pessaries were removed, ewes were inseminated with 200 x 10(6) sperm/0.25 ml of fresh, extended semen, which was collected from a purebred ram of the corresponding breed. Pregnancy rate was greater (P < 0.05) after laparoscopic (37.5%) than after transcervical AI (0%). Because of the disappointing results of Experiment 1, Experiment 2 was conducted to determine whether oxytocin or the AI procedure per se reduced ovum fertilization rate. Treatments were designed in a 2 x 2 factorial arrangement. At 60 h after norgestomet implant removal and 10 min before either laparoscopic or transcervical (cervical in a saline group) AI with 100 x 10(6) sperm/0.25 ml, ewes (n = 10/group) received an intravenous injection of either isotonic saline or 200 USP units of oxytocin. Fertilization rate, which was determined 72 h after AI, was greater (P < 0.05) after laparoscopic than after transcervical/cervical AI (92.5 vs 28%), but oxytocin treatment did not affect fertilization rate. The results indicate that exogenous oxytocin did not reduce ovum fertilization rate, but the transcervical AI procedure per se seemed to reduce fertilization rate.     

In vivo and in vitro fertilization of hamster, rat and mouse eggs after treatment with anti-hamster ovary antiserum.

Rabbit antiserum against hamster ovary was examined on agargel diffusion plates against several hamster tissues, and also against rat and mouse ovarian extracts. Unabsorbed anti-hamster ovary antiserum showed eight to nine precipitin bands for hamster ovary and four to eight bands for other tissue extracts, but no bands against sperm antigens. Anti-hamster ovary antiserum also showed three to four bands for rat and one to two bands for mouse ovarian extracts. The present experiments confirmed previous reports for the hamster and mouse that treatment of eggs with anti-ovary antiserum blocked in vitro fertilization and that the extent of the inhibition was related to the formation of a precipitate on the zone pellucida. A single injection of anti-hamster ovary antiserum inhibited fertilization in mice but not in rats. In vitro fertilization of mouse eggs was also inhibited in the presence of such antiserum.     

Kinetics of actin assembly attending fertilization or artificial activation of sea urchin eggs

Changes in the state of actin assembly triggered by fertilization or by artificial activation of sea urchin eggs were quantified using the DNase I inhibition assay. Insemination of Lytechinus pictus or Strongylocentrotus purpuratus eggs induces a cyclic variation in the level of G-actin as follows: between 0 and 30 s after insemination, the G-actin content decreases. This is followed by an increase in the amount of monomeric actin between 30 and 60 s, and then from 60 s to 5 min postinsemination there is a progressive decrease in the egg's level of G-actin. This latter decrease is more pronounced in S. purpuratus eggs than in L. pictus eggs. Using sperm mimetics that trigger an increase in intracellular calcium concentration (A23187 in sodium-free seawater), a cytoplasmic alkalinization (NH4Cl), a plasma membrane depolarization (seawater enriched with potassium ions), or all three of these phenomena (A23187 in normal seawater), each phase depicted at fertilization correlates with the following metabolic events accompanying egg awakening: phase 1, of uncertain origin (possibly related to plasma membrane depolarization); phase 2, elevation of intracellular calcium concentration; phase 3, alkalinization of the intracellular milieu but only if the transient intracellular calcium rise has taken place.     

In vitro fertilization and seed development inTrifolium

Summary Pistils ofTrifolium repens L. andT. ambiguum Bieb. were cultured on an agar-based modified Murashige-Skoog medium. Pistils with and without accessory floral parts were removed from flowers of selected clones ofT. repens, hand-pollinated under aseptic conditions, and planted on the medium. Pistils cultured without accessory floral parts showed no evidence of fertilization after 2 weeks. However, 52% of thoseT. repens pistils cultured with calyx lobes and pedicels contained ovules with maturing embryos 12 days after in vitro cross-pollination. Pistils fromT. ambiguum intraspecific cross-pollinations could not be cultured successfully under the same conditions; however, addition of various combinations of auxin, cytokinin, and gibberellic acid enhanced embryo growth. Fertilization and partial embryo development occurred in interspecific crosses betweenT. ambiguum andT. repens orT. hybridum only whenT. ambiguum was used as the pistillate parent. These results indicate that embryological development under in vitro conditions closely parallels in situ development although growth regulator requirements may vary among species. This work is Technical Contribution 1785 from the South Carolina Agricultural Experiment Station and was supported by SCAES-USDA Cooperative State Research Agreement No. 616-15-65.     

In vitro fertilization as a predictor of fertility from cervical insemination of sheep

The objective of this study was to determine if the quality of frozen-thawed ram semen could be effectively evaluated through in vitro fertilization (IVF) procedures prior to insemination as a means of improving pregnancy rate. In experiment 1, frozen semen from four Belclare rams was assessed using IVF and was used for cervical insemination of ewes (n = 181) in 13 pedigree Belclare flocks. There was a significant association between IVF score (proportion of oocytes cleaved at 48 h post insemination) and non-return rate (P < 0.001). For experiment 2, semen from nine Belclare rams was evaluated by IVF and semen from rams with the highest (n = 3) and lowest (n = 2) IVF scores was used for cervical insemination of ewes (n = 111) under experimental conditions. Differences in pregnancy rates between individual rams did not reach significance. Experiment 3 was designed to determine if differences detected between rams at field level could be accurately identified via IVF evaluation and involved frozen semen from eight Norwegian rams of known field fertility (non-return rates ranged from 45.7 to 73.8%). IVF score did not reflect the differences in field fertility. In the final experiment six of the eight Norwegian rams involved in experiment 3 were selected based on IVF score (three highest and three lowest) and their semen was used for cervical insemination (n = 90 ewes). While significant differences in pregnancy rate were found between individual rams (P < 0.02, range: 12.9-65.8%) they were not associated with IVF score. Ewe breed had a significant effect (P < 0.003) on pregnancy rate in both experiments 2 and 4. In conclusion, there was no evidence from this study that the evaluation of semen quality through IVF provided a useful predictor of pregnancy rate under field conditions. It may be that the IVF procedures as used routinely, which are essentially designed to maximize blastocyst yields rather than for detecting differences in fertilizing ability between batches of sperm, need to be modified.     

Egg laying and development of Neotropical trichogrammatid species in artificial eggs

The goal of this work was to assess the suitability of two artificial diets for egg laying and development of Trichogramma atopovirilia Oatman & Platner, Trichogrammatoidea annulata De Santis, and Trichogramma bruni Nagaraja (all Hymenoptera: Trichogrammatidae). Additionally, the quality of wasps reared in vitro was compared with those reared in vivo. The ‘standard’ diet consisted of Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae) pupal holotissues (65%), chicken egg yolk (18%), fetal bovine serum (8.5%), lactalbumin hydrolysate (8.5%), and anticontaminants (0.3%). The ‘modified’ diet differed from the standard one only in the D. saccharalis pupal holotissues, that were replaced with Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae) ones. Females of the three trichogrammatid species laid eggs in artificial eggs containing any artificial medium, but the modified one received more eggs. Although the standard diet was accepted for oviposition by the three wasp species, no development occurred. On the modified diet, only T. atopovirilia was able to develop to adult emergence. Adult F1 were of a quality that was similar to insects reared in vivo.     

In vitro fertilization of two species of deer mouse eggs by homologous or heterologous sperm and penetration of laboratory mouse eggs by deer mouse sperm

Newly ovulated eggs from immature deer mice (Peromyscus maniculatus and P. polionotus) and mature laboratory mice (Mus musculus) treated with PMSG and HCG were inseminated in vitro with spermatozoa recovered from the cauda epididymidis of mature males. The time required for capacitation of deer mouse sperm in culture was estimated to be about two to five hours based on the dispersal of sperm agglutination and increase of sperm motility. The rate of sperm penetration through the zona pellucida of deer mouse eggs by homologous or heterologous sperm was relatively high (72-91%) but that of laboratory mouse eggs by deer mouse sperm was low (20-21%). After penetration through the zona pellucida, a high proportion of deer mouse eggs (79-93%) were fertilized by homologous or heterologous deer mouse sperm but no laboratory mouse eggs were fertilized by sperm of two species of deer mice. The zona pellucida was dissolved in a higher proportion of laboratory mouse eggs cultured with P. maniculatus (45%) than with P. polionotus sperm (3.4%), but this did not happen by incubation of deer mouse eggs with homologous or heterologous sperm. It seems that there is little difference in sperm penetration and fertilization between these two closely related species of deer mice but the reactions between the mouse eggs and deer mouse sperm are quite different.     

Sperm distribution and fertilization after unilateral and bilateral laparoscopic artificial insemination with frozen-thawed goat semen

Generally, laparoscopic artificial insemination (LAI) provides a higher success rate than of cervical insemination in goats. However, the sperm distribution after LAI in goats remains unknown, particularly when frozen-thawed semen is used. This study evaluated the distribution of frozen-thawed goat spermatozoa after LAI and compared the effects of sperm numbers and deposition sites (unilateral and bilateral sites) on pregnancy rate. In experiment 1, the frozen-thawed spermatozoa were stained either with CellTracker Green CMFDA (CT-Green) or CellTracker Red CMPTX (CT-Red), and in vitro evaluations of viability and motility were performed. In experiment 2, the labeled spermatozoa were deposited via LAI into the left (CT-Green) and right (CT-Red) uterine horns (n = 4). After ovariohysterectomy (6 hours after insemination), the distributions of green- and red-colored spermatozoa were assessed via tissue section, flushing, and the oviductal contents were also collected. Experiment 3 was designed to test the pregnancy rates in a group of 120 does after LAI using different numbers of spermatozoa (60 and 120 × 10⁶ sperm per LAI) and different deposition sites. The results demonstrated that the fluorochromes used in this study did not impair sperm motility or viability. Frozen-thawed goat spermatozoa can migrate transuterinally after LAI, as evidenced by the observations of both CT-Green– and CT-Red–labeled spermatozoa in both uterine horns. Lower numbers of spermatozoa (60 × 10⁶) that are inseminated unilaterally (either ipsilateral or contralateral to the site of ovulation) can efficiently be used for LAI in goats (with a 56.67% pregnancy rate).     

Studies on fertilization in the teleost IV. Effects of aphidicolin and camptothecin on chromosome formation in fertilized medaka eggs

To clarify the mechanisms of fish fertilization, the effects of inhibitors of DNA polymerase-alpha and DNA topoisomerases on nuclear behavior before and after fertilization were examined in eggs of the medaka, Oryzias latipes. Eggs underwent the fertilization process from sperm penetration to karyogamy of pronuclei, even when inseminated and incubated in the continuous presence of aphidicolin (DNA polymerase alpha inhibitor), camptothecin (DNA topoisomerase I inhibitor), etoposide, or beta-lapachone (DNA topoisomerase II inhibitor). However, continuous treatment with aphidicolin or camptothecin during fertilization inhibited the formation of sister chromosomes that were normally separated into blastomeres at the time of the subsequent cleavage. Sister chromosome formation appeared concomitantly with an increase in histone H1 kinase activity at the end of DNA synthesis, 30 min post insemination. However, non-activated eggs that were inseminated in saline containing anesthetic MS222 and aphidicolin had high levels of histone H1 kinase and MAP kinase activities, and transformation of the penetrated sperm nucleus to metaphase chromosomes occurred even in the presence of aphidicolin or camptothecin. The male chromosomes were normally separated into two anaphase chromosome masses upon egg activation. These results suggest that DNA polymerase alpha or DNA topoisomerase I, but not DNA topoisomerase II, may be required for the process by which the mitotic interphase nucleus transforms to separable metaphase chromosomes while the activity of MAP kinase is low, unlike the situation in meiotic division, during which MAP kinase activity is high and DNA replication is not required.     

In vitro fertilization of sheep oocytes matured in vivo

Incubating washed ram spermatozoa in a modified Brackett's defined medium buffered with Hepes (DM-H) containing 20% of heat-inactivated sheep serum appears to be a reliable method of capacitating sperm for in vitro fertilization. Raising the Ca(++) concentration in the fertilization medium (DM-H-SS) to 10 mM stabilized the fertilization rate of various rams (2). This study was designed to determine if the developmental competence of the oocytes fertilized under such conditions was normal. Thirty-seven ewes, treated with progestagen sponges, were superovulated with porcine follicle stimulating hormone (pFSH: 16 mg). An intramuscular injection of gonadotropin releasing hormone (GnRH: 100 mug); given 24 to 26 h after sponge removal, induced the synchronization of ovulations 24 h later. Ovulated oocytes (n = 229) recovered with flushing of the oviducts were inseminated in vitro and 17 h later either fixed in acetic/alcohol (n = 115) to evaluate fertilization or transferred (n = 114) into 38 synchronized recipients (three oocytes/recipient) to evaluate their developmental competence. Of the fixed oocytes, 82.6% were fertilized and 61.7% were monospermic. Nineteen of the recipient ewes (50%) were pregnant at Day 18, and 16 ewes produced a total of 26 live young (mean: 1.63/ewe). The results showed a high efficiency of in vitro fertilization of ovulated oocytes in sheep following a pFSH-GnRH treatment and the in vivo developmental competence of oocytes fertilized in the presence of elevated Ca(++) concentration.