Rabbit Eggs Research Articles

Effect of bacterial neuraminidase on the fertilization of rabbit eggs in vitro.

Effect of bacterial neuraminidase on the fertilization of rabbit eggs in vitro.

A comparison of in-vitro fertilization of rabbit eggs using spermatozoa recovered from the uterus or vagina

Spermatozoa were recovered from the uterine horns or vagina of mated rabbit does 12-20 hr p.c. and their fertilizing capacity tested in an in-vitro system. Uterine spermatozoa gave consistently high fertilization (93-100%) throughout. Vaginal spermatozoa gave good results (81-85%) 12-14 hr p.c. but fertilization then declined with time. Vaginal spermatozoa recovered from mated females with or without ligation of the uterine horns were equally able to fertilize eggs. Fragmentation of uncleaved eggs was noted in experiments with vaginal spermatozoa, but never with uterine spermatozoa. The former were slower in penetrating eggs and the zygotes were correspondingly slower in cleaving. Triploidy, possibly due to ageing, was found in approximately 15% of the embryos fertilized by vaginal spermatozoa.

Nuclear transplantation in the rabbit egg.

SINCE the experiments of Briggs and King1, in which nuclei from blastula cells were transplanted by microinjection into enucleated frogs' eggs, the technique of nuclear transplantation has been used extensively to study interactions between nucleus and cytoplasm during differentiation in amphibians2–5. But severe difficulties are encountered in attempts to apply this technique to the mammalian egg. The rabbit egg, with a diameter of 100 µm, is 1,000 times smaller in volume than a frog's egg, and the mouse egg has a volume one-third that of a rabbit's egg. An alternative, and technically easier method of transplanting a nucleus is by inducing fusion of an egg and a cell with Sendai virus. The technique of virus-induced fusion, originally developed for hybridising somatic cells6–7, has been adapted to fuse somatic cells with unfertilised or fertilised mouse eggs8–13. Although Sendai virus was shown not to harm mouse embryos in these experiments, and cells were successfully fused, the transplanted nuclei did not survive. Unfertilised mouse eggs which were fused with cells from embryos at the 2–8-cell stage failed to cleave8.

The relationship between zona digestion and cortical granule disappearance in rabbit eggs inseminated in vitro.

The relationship between zona digestion and cortical granule disappearance in rabbit eggs inseminated in vitro.

A reexamination of cleavage patterns in eutherian mammalian eggs: rotation of blastomere pairs during second cleavage in the rabbit.

The pattern of cleavage was examined during second and third furrowing of the rabbit egg. Two-cell eggs, collected just prior to onset of second cleavage, were continuously observed in a culture chamber, which was kept at 37 degrees C. Semi-cinematographic techniques were used to photograph progressive stages of cleavage. It was demonstrated that the pattern of cleavage in the rabbit differs from that in the sea urchin, because the blastomeres at the 4-cell stage are arranged crosswise in the former, while they are situated next to each other in the latter. The crosswise arrangement of the blastomeres in the rabbit at the 4-cell stage is a consequence of a 90 degree rotation of the polar axis in one hemisphere of the egg. Subsequently, due to the rotation of the original polar axis in one hemisphere, the third cleavage plane through one half of the egg is transverse to the third cleavage plane through the other half. Evidence is provided to show that the cross wise configuration of blastomeres at the 4-cell stage occurs in other eutherian mammals. It is proposed that this rotational cleavage pattern be recognized as distinct from those of radial, spiral and bilateral.

In Vitro Fertilization of Rabbit Eggs in Oviduct Secretions from Different Days Before and After Ovulation

In vitro fertilization of rabbit eggs in oviduct secretions has been studied through different days of the psudopregnant cycle. The appearance of the pronuclei and cleavage into two cells, which were the criteria of fertilization, occurred significantly less frequently when in vitro fertilization was attempted in the oviduct secretions obtained during the estrous (preovulatory) period of the cycle than it was in the secretions obtained during pseudopregnancy or in Brackett's medium plus 20% heated rabbit blood serum. Whether fertilization was attempted in ampulla secretions or in whole oviduct secretions had no effect on the success rate. The effects of oviduct secretions from the estrous period of the cycle is probably due mainly to an effect on the egg, involving the very first development processes during and after fertilization.

Electron microscope evaluation of rabbit eggs exposed to spermatozoa treated with capacitating agents.

Sperm capacitation (Austin, 1951; Chang, 1951) normally occurs in the female genital tract. The mechanism of capacitation is unknown. Agents considered to induce capacitation may be tested in vitro and the subsequent criteria for fertilization have been (1) the production of cleaved ova and, (2) the viability of embryos transferred to recipient females. In laboratory rodents, spermatozoa have been capacitated with enzymes, salt solutions or body fluids, but rabbit spermatozoa have not been easily capacitated in vitro. The evidence for amylase activity in rabbit uterine fluid (Kirton & Hafs, 1965), \g=b\-glucuronidasein rat uteri (Hayashi, 1964) and data indicating that \g=a\-and \g=b\-amylasedestroy capacitation factor (Dukelow, Chernoff & Williams, 1967) led to experiments with these enzymes on rabbit spermatozoa. Incubation with \g=b\-amylaseand uterine fluid shortened the time necessary for capacitation in vivo (Kirton & Hafs, 1965). In similar experiments, Johnson & Hunter (1972) concluded that \g=b\-amylase and \g=b\-glucuronidase initiated capacitation because they remove seminal antigens from the sperm surface. They attributed the action of blood sera on spermatozoa (Barros & Garavagno, 1970) to the presence of \g=b\-amylase and \g=b\-glucuronidase. Cleavage of rabbit eggs in vitro, however, was obtained after spermatozoa were exposed to postovulatory uterine fluid (Brackett & co-authors, 1972), McCoy's modified medium (Iwaki, 1971) and human follicular fluid (HFF) (Rosado, Hick, Reyes & Blanco, 1974). After sperm penetration, the fertilized ovum is devoid of cortical granules. The activated egg, although appearing to undergo normal cleavage, retains the granules (Longo, 1974). Cleaved cells resulting from incubation with 'capacitated' spermatozoa have not been previously assessed for cortical granules. In this report we investigated the effect of bovine follicular fluid (BFF), HFF, uterine fluid, McCoy's modified medium, /?-amylase, trypsin and chymotrypsin on rabbit spermatozoa in vitro. The treated spermatozoa were then exposed to eggs in vitro (Fraser, Dandekar & Vaidya, 1971). Cleaved cells were examined by electron microscopy for the presence of cortical granules. Trypsin and chymotrypsin were tested because of the observation that sperma¬ tozoa capacitated in vivo shed portions of the surface coat (Gordon, Dandekar ?5c

Polyadenylic acid-containing RNA in unfertilized and fertilized eggs of the rabbit

Molecular hybridization between 3H-polyuridylic acid and unlabeled RNA prepared from unfertilized rabbit eggs and 10-h postfertilization stage rabbit embryos has been used to measure the amount and subcellular localization of adenylated maternal RNA. The results reported indicate that there is poly (A)-containing RNA (putative messenger RNA) in unfertilized rabbit eggs. The amount of poly (A) in the RNA in rabbit eggs does not increase immediately after fertilization and is located primarily in the ribosomal fraction of the cell. The rate of protein synthesis in fertilized eggs is insensitive to α-amanitin at concentrations which inhibit RNA synthesis. These results suggest that maternal mRNA makes an important contribution to protein synthesis in early stages of cleavage in the rabbit embryo.

Ultrastructural analysis of artificially activated rabbit eggs.

Ultrastructural investigations have been carried out on parthenogenetic rabbit eggs in an effort to elucidate events occurring during artificial activation and their similarity to processes of fertilization and embryogenesis. Rabbit eggs were artificially activated by culturing at 10 degrees C for 24 hours followed by incubation at 37 degrees C for 2 to 24 hours. Examination of eggs immediately after incubation at 10 degrees C for 24 hours indicates that activation is initiated when the chromosomes coalesce to form a reticulum which is either surrounded completely by two parallel membranes or incompletely by cisternae of smooth endoplasmic reticulum. Aggregation of the chromosomes occurs as a result of a reduction in the number of microtubules making up the meiotic spindle. When cold treated ova are subsequently incubated at 37 degrees C a nucleus is formed which moves central where it may participate in the cleavage of the egg. Formation of a second polar body and release of the contents of the cortical granules as reported for inseminated eggs was not found to be a part of activation of the egg by cold treatment. Approximately 95% of the ova cultured at 10 degrees C for 24 hours followed by 37 degrees C for 12 hours were activated, i.e., they possessed a nucleus or they had cleaved. Many of the activated eggs cultured for short periods at 37 degrees C were structurally similar to fertilized ova, with further incubation fragmented eggs and abnormal multicellular stages predominated.

The dependence of annulate lamellae formation on the nucleus in parthenogenetic rabbit eggs.

Previously it has been shown that, in the rabbit, although annulate lamellae (AL) are absent in the follicular oocytes, they appear in the fertilized eggs after the formation of the pronuclei. Furthermore, neither pronuclei nor AL appear when unfertilized eggs are aged in vivo or in vitro. This study was undertaken to determine whether AL formation requires presence of an intact nucleus, or whether the sperm alone contains the stimulatory factors essential to AL synthesis. Rabbit eggs were exposed to 10 degrees C, then incubated for 24 hours. Control eggs were incubated without cold-treatment. Electron microscopic observations indicated that two-thirds of the eggs formed one to two 'pronuclei', or subnuclei. The remainder one-third of the cold-treated eggs and the control eggs failed to form 'pronuclei'. AL were present in large amounts only in those activated eggs (parthenogenones) which formed 'pronuceli.' AL were absent in the control and the non-activated experimental eggs, both of which failed to form a 'pronucleus.' A few small AL were observed in eggs with subnuclei. Condensed fine textured nucleoli appeared precociously during cold-treatment in some eggs and they were present in the 'pronuclei' of activated eggs. It was concluded that the sperm is not necessary for AL formation, but the presence of an intact nucleus is mandatory.

Influence of calcium ions on fertilization in the rabbit.

SummaryFertilization of rabbit eggs following oviductal insemination of uterine capacitated spermatozoa was shown to depend on Ca2+ levels in the sperm suspension. Decapacitation of rabbit spermatozoa by seminal plasma Fr I occurred even in the presence of 150 μg Ca2+/ml. The results indicate Ca2+ ions were important for expression of fertilizing capacity by rabbit spermatozoa, but they were not implicated in decapacitation.

Pincogenesis--parthenogenesis in rabbits by Gregory Pincus.

PINCOGENESIS—PARTHENOGENESIS IN RABBITS BY GREGORY PINCUS N. T. WERTHESSEN with R. C.JOHNSON* DwightJ. Ingle, previously editor, now advisory editor of thisjournal, wrote the biographical memoir of the late Gregory Pincus for the National Academy of Sciences [I]. A copy of that memoir reached my desk in the spring of 1973 and, since the man of whom he wrote had been my teacher, colleague, adviser, and dearest older friend from 1931 to 1967, I read it with great interest. I found that, of all the commentaries on Pincus I had heard or read, this one gave the most accurate picture of the man that I knew. But I was shocked when I read the section that dealt with Pincus's induction of "artificial" parthenogenesis in rabbit eggs ("Pincogenesis") in the 1930s. Ingle claimed that there was skepticism among some other workers in this field, since no one had repeated the work, and hence considered Pincus's claims to be questionable. In a mixed mood of appreciation for an excellent description of my mentor's career and anger at the fact that colleagues doubted an unequivocal fact of accomplishment on his part, I wrote to Ingle saying in effect that I was there, I helped make it happen. Perhaps the fact that in the preceding period I had learned that two other accomplishments of Pincus's laboratory prior to 1950 had not yet been repeated lent extra vehemence to my language.1 *Department of the Navy, Office of Naval Research, 495 Summer Street, Boston, Massachusetts 02210. Professor R. Christian Johnson, on leave from the University of Wisconsin—Green Bay, is writing a book, Science and Social Reform: The Searchfor a Scientific Contraceptive, in which the story of Pincus and the parthenogenic rabbits will be told in the context of Pincus's other work. We thank Professor Thomas Wegmann and Mr. James Reed of Harvard University for their advice. 'Pincus described in 1937 the culture of a fertilized rabbit egg from its earliest possible removal from the tubes through postimplantation stages. Thirty years later no one else had done this. The objective was to prove that development through the blastocyst stage and further required only a sufficiency of unknown growth requisites present in serum. The culture system devised permitted use of large quantities of flowing rabbit serum while retaining the growing embryo in the circumscribed area [2]. In a national conference [3, p. 393] several speakers had commented on the inability of maintaining organs in vitro for 24 hours let alone observing growth and repair. An appropriate system had been described in 1949 [4]. Pincus had initiated this work in 1935. A portion of human uterus bleeding from a curetted endometrium was observed to regrow endometrium under the influence of estrogen. From that point forward the system was used as a routine research tool. 86 I N. T. Werthessen with R. C. Johnson · Pincogenesis Despite the fact that Ingle and I had had many conversations at Laurentian Hormone Conference meetings since its earliest days, he did not know of my intimate involvement in these studies. He made a point of this in his reply to my letter and his invitation to prepare a rebuttal to the critics. It is important to explain why, although a major portion of my time was spent in this work over a 7-year period, my name appeared only in the "thank you" paragraph at the end of the papers describing parthenogenesis in rabbits. Pincus's research had three facets. One was genetics, the second was gamete behavior, and the third was regulation of the reproductive system . From the third developed his renown as an endocrinologist. This was my area. However, in those days at Crozier's department a student ordinarily did two separate lines of research. One was his professor's research, in which the student acted as a super-technician and received credit for research assistance. This was the price the student paid for support of his own thesis research. If his professor helped in this thesis work (guidance or collaboration at the bench), the professor's name appeared on the papers, usually as senior author. Ifnot, the papers were published under the name...

Development of preimplantation rabbit embryos in vivo and in vitro: II. A comparison of qualitative aspects of protein synthesis

Qualitative patterns of protein synthesis in preimplantation rabbit embryos grown in vivo and in vitro were examined by SDS polyacrylamide gel electrophoresis followed by autoradiography. The results demonstrate that (1) most qualitative changes in the pattern of protein synthesis occur during cleavage, (2) the blastocyst period of development is characterized by a remarkably uniform and constant pattern of protein synthesis, and (3) the qualitative pattern of protein synthesis in embryos cultured in vitro from the 1-cell to the blastocyst stage is essentially identical to the pattern of protein synthesis in embryos grown to a comparable stage in vivo. These results indicate that no “special” maternal factors, such as uterine proteins, are required in vitro either for the qualitative changes in the pattern of protein synthesis during cleavage, or for the initial expression of a pattern of protein synthesis characteristic of the entire blastocyst period. From these studies we conclude that, once fertilized, the rabbit egg proceeds through cleavage and blastocyst formation on its own endogenous developmental program.

Cortical granules in artifically activated (parthenogenetic) rabbit eggs.

AbstractThe fate of cortical granules was ascertained in cold‐shock activated rabbit eggs. Unfertilized oocytes were obtained 14 hours after intravenous injection of Human Chorionic Gonadotropin. Experimental oocytes were stored at 6 to 10°C for 24 hours in F10 medium containing 10% rabbit serum. Control oocytes, and the experimental ones subsequent to cold‐shock treatment, were incubated for 24 hours. Formation of ‘pronuclei’ occurred in 6% of the cold‐shock treated oocytes. None of the control oocytes were activated. Electron microscopic observations show that activation occurred without the dehiscence of cortical granule content into the perivitelline space. Cortical granules were dispersed in the cytoplasm or clumped together at the periphery. Occasionally, cortical granules were found discharged into the perivitelline space with the limiting membrane intact.

Ultrastructural changes in rabbit eggs aged in vivo.

Utilizing the techniques of light and electron microscopy, rabbit ova, aged for varying periods in vivo, were examined in order to determine the relation, if any, between the morphological changes of the unfertilized egg at various times following ovulation and the developmental defects that are known to occur as a result of delayed mating. Structural alterations of the second meiotic apparatus and the cytoplasm are noted as early as 14 h following the induction to ovulate (FITO) in approximately 20% of the eggs examined. The changes include an increase in cortical granules along the cortex, the structural alteration of microvilli, and the disruption of the meiotic spindle. With further aging, there is a progressive deterioration of the egg’s cytoplasm and meiotic spindle. By 20–30 h FITO, all of the eggs demonstrate alterations in the structure and orientation of the meiotic spindle. Later, the meiotic spindle is completely disrupted and the chromosomes are scattered throughout the cytoplasm. No nucleus is observed in aged eggs that have not fragmented. There is a gradual aggregation of the cytoplasmic organelles in specimens obtained approximately 30 h FITO, and, by 60 h, all of the particulate cytoplasmic components are located in one or several groups. During this period there is also a loss of cortical granules due to an apparent blebbing of the cortex of the egg. Dehiscence of the cortical granules as observed in inseminated eggs is not found.

Uridine incorporation and pyruvate metabolism in rabbit eggs cultured in vitro at normal and elevated temperatures.

Summary. Effects of elevated maternal body temperature upon incorporation of an RNA precursor by rabbit eggs were evaluated by culture in vitro at 39\m=.\0or 40\m=.\3\s=deg\C.temperatures comparable to rectal temperatures of rabbits in normal and elevated ambient temperatures. One-cell fertilized eggs incorporated significantly less [3H]uridine during culture at the higher temperature, indicating a depression of RNA synthesis. Uridine incorporation was not significantly decreased when two-cell ova were incubated with [3H]uridine after completion of the first cleavage, but was decreased in two-cell ova that had been subjected to the higher temperature only while in the one-cell stage of development. The observed reductions in uridine incorporation at the higher temperature were not accompanied by a decrease in energy production as determined by CO2 production from [14C]pyruvic acid. These results suggest that such alterations of regulatory function are associated with the sensitivity of the early zygote to thermal stress and is a mechanism by which elevated maternal body temperatures adversely influence subsequent embryonic development.

Memory and Transfer of Information

Memory and Transfer of Information

Egg transport in the macaque (Macaca fascicularis).

Egg transport was studied in 12 cycling crab-eating macaques (Macaca fascicularis) utilizing serial laparoscopy. Egg transport was evaluated by segmental flushing of the oviducts and uterus. Ovulated eggs, transferred rabbit eggs, and microspheres were transported to the ampullary-isthmic regicm by 48 hr, were retained in the isthmus between 48 and 96 hr, and entered the uterus between 96 and 120 hr after ovulation. These data suggest that the isthmus plays an important role in regulating the entry of eggs into the uterus.

Resistance to Taenia pisiformis larvae in rabbits—II. Temporal relationships and the development phase affected

The time required for the development of protective immunity to reinfection with T. pisiformis eggs in rabbits, and the stages of larval development affected by it, were examined using in vivo and in vitro systems. Protective immunity was apparent 9–12 days in vivo, and 12–15 days in vitro, after experimental immunisation. Full development of protective immunity resulted in the death of all oncospheres. Partial development of immunity appeared to damage reorganising oncospheres so that many were unable to complete larval development. Immune effects in vitro were assisted by the presence of complement. Secretions from the anterior region of developing larvae appeared to be immunogenic, but precipitations on developing larvae were not lethal in vitro, and may only slow the rate of migration through liver tissue in vivo.

The effects of aging on in vitro fertilization of rabbit eggs and subsequent embryonic development

AbstractRabbit eggs, with or without folliculai cells, are highly fertilizable in vitro (Fraser et al., »71). In vitro aging of intact, cumulus‐devoid, and corona‐devoid rabbit eggs did not appreciably lower their subsequent fertilizability in vitro with fresh sperm. The intact and cumulus‐devoid eggs were somewhat less fertilizable than the corona‐devoid eggs when aged for two and one‐half hours (70%, 68%, 91%, respectively), but this difference disappeared when the aging period was six hours (83%, 82%, 91%) or 12 hours (83%, 81%, 82%). Chromosome complements ranged from hypodiploid to polyploid in all groups, regardless of time of aging. Viability of embryos from the three groups, as determined by continued cleavage in vitro, was much reduced when compared to that of unaged eggs similarly cultured. There was no observable correlation between ploidy and development in culture; polyploid complements, particularly triploids, were found in both advanced and retarded stages.The possible effect of aging at an earlier time was noted in studies involving transfer of two to four cell embryos to pseudopregnant recipients. Embryos derived from eggs recovered 13 hours post‐LH and fertilized in vitro had low implantation rates and none of the implants developed to full term fetuses. However, the development of embryos from eggs recovered 12 hours post‐LH was not significantly different from embryos fertilized in vivo and cultured prior to transfer; 21% and 24%, respectively, of the embryos transferred developed to term.A number of the offspring have been raised to maturity. Those from the in vivo fertilized embryos have appeared normal and their eggs and sperm have had no obvious defects. From a similar sample size of in vitro fertilized offspring, 2/14 have displayed defects of the limbs resembling a condition termed splayleg, thought to be a recessive genetic trait. Whether conditions of in vitro fertilization might favor the union of two gametes carrying the recessive gene is not known.