Rabbit Oviduct Research Articles

Survival of DNA-injected cow embryos temporarily cultured in rabbit oviducts

Holstein or Angus cows were superovulated, inseminated with fresh bull semen, and necropsied about 12 h after estimated time of ovulation. Ova were centrifuged at 15,600 G for 3 to 8 min to reveal pronuclei. In Experiment 1, pronuclear bovine embryos were transferred to ligated or unligated oviducts of 1-d pseudopregnant rabbits for 7 d; 30 of 32 embryos were recovered from ligated oviducts but only 2 of 26 from oviducts and uterine horns of unligated oviducts. In Experiment 2, a Rous sarcoma virus-chloramphenicol acetyl transferase fusion gene was injected into one pronucleus of about half of 404 fertilized bovine ova, using a micromanipulator and interference contrast optics. Injected and noninjected embryos were then transferred to opposite ligated rabbit oviducts. Embryos were recovered after 7, 8 or 9 d. Of 120 centrifuged but ininjected embryos recovered from rabbit oviducts, 66 (55%) were in the morula to hatching blastocyst stage of development. Of 105 embryos centrifuged and injected with foreign DNA, 55 (52%) were in the morula to hatching blastocyst stage. In Experiment 3, centrifuged bovine embryos, noninjected or DNA-injected, were cultured in rabbit oviducts for 7 d then transferred nonsurgically to the uterus of recipient cows. Embryos were also flushed from superovulated cows 8 d after estrus and transferred directly to recipient cows. After 7 d, the uterus of recipient cows was flushed nonsurgically to recover embryos. The proportion of transferred embryos recovered with normally elongated trophoblastic membranes and the proportion of recipient cows with developing embryos were 14 of 25 DNA-injected embryos, 5 of 8 cows; 6 of 15 centrifuged but noninjected embryos, 4 of 6 cows; and 11 of 29 embryos transferred directly, 5 of 8 cows. Results indicate that bovine embryos can be cultured in rabbit oviducts and survive after transfer to cow uteri and that injection of foreign DNA may not increase embryonic loss within the first 2 wk after injection.

Energy transport and cell polarity: Relationship of phosphagen kinase activity to sperm function

The energy required for motility of sea urchin sperm is transported from the mitochondrion to the flagellum by a phosphocreatine shuttle involving diffusion of phosphocreatine (PCr) between isozymes of creatine kinase (CrK) localized at the two sites (Tombes and Shapiro, Cell, 41:325, '85; Tombes et al., Biophys. J., 52:75, '87). The present studies demonstrate that high sperm CrK (various echinoderms; sea squirt, bristle worm, salmon) or arginine kinase (molusc, barnacle, moth) activity is seen in several species with sperm of a primitive morphology (mitochondrion at the base of the head, relatively long flagellum). In contrast, CrK activity is 10-100-fold less abundant in sperm of other species (frog, mouse, rooster, rabbit, bull, and human) that either possess a modified morphology (mitochondria that extend along the flagellum) and/or utilize glycolytic metabolism. We interpret these findings as support for the use of phosphagen kinase-dependent energy transport in cells in which the production of adenosine triphosphate (ATP) by the mitochondrion is distant from its utilization, leading to a form of metabolic polarization. Two other cell types, frog photoreceptors and rabbit oviduct cells, whose morphology and function also suggest that they exhibit metabolic polarization, contain relatively high CrK activity. The presence of high phosphagen kinase activity in metabolically polarized gametes and somatic cells further substantiates the role of such enzymes in facilitating energy transport.

Development to the late morula or blastocyst stage following in vitro maturation and fertilization of bovine oocytes

The developmental capacity of bovine oocytes matured and fertilized in vitro was investigated under five different culture systems (IVF-medium, BPM-G and TCM199 with bovine epithelial cells for in vitro culture; and rabbit and sheep oviducts for in vivo culture). Immature bovine oocytes were cultured for 24 h in TCM199 supplemented with 20% estrous cow serum and fresh granulosa cells. Fertilization in vitro was performed using pooled frozen-thawed semen from two bulls after swim-up and heparin treatments. Eighteen to 20 h after in vitro insemination, oocytes were transferred to rabbit or sheep oviducts, or were cultured in the three different media for 6 days. Recovery rates from the rabbit and sheep oviducts were 68.1% and 35.3%, respectively. In the oocytes recovered, the frequency of development to the late morula and blastocyst stages was not significantly different in rabbit ( 58 258 : 22.5%) and sheep ( 8 49 : 16.3%) oviducts. The frequency of in vitro development was higher in TCM199 with added bovine oviduct cells ( 44 361 : 12.2%) than in either IVF-medium ( 5 319 : 1.6%) or BPM-G ( 1 253 : 0.4%). Although the developmental capacity of oocytes in vitro was significantly ( P < 0.001) lower than in the rabbit (but not in the sheep) oviduct, the loss of oocytes in vivo counteracted this advantage of the oviduct system. The present results indicate that co-culture of embryos with bovine oviduct epithelial cells is an effective means of supporting the further development of bovine oocytes matured and fertilized in vitro.

Culture of agar-embedded one and two cell bovine embryos and embryos produced by nuclear transfer in the sheep and rabbit oviduct

Assisted reproductive technologies have played a major role in the genetic improvement of livestock as well as in the development of transgenic animal technologies. Artificial insemination (AI) was the first widely-accepted assisted reproductive technology for livestock, and this technology allows genetically superior males to produce more offspring than would be possible through conventional mating. Conventional AI has been enhanced in recent years with the use of sperm cells that have been flow cytometrically sorted into populations of X chromosome- and Y chromosome-bearing spermatozoa, facilitating production of offspring of a pre-determined genetic sex. A related genetic improvement technology, embryo transfer, involves non-surgical recovery of preimplantation embryos from one female and subsequent transfer into recipient females to enable genetically superior females to produce a greater number of offspring than would be possible through traditional mating. With the advent of ultrasound-guided procedures for recovery of oocytes from living females, coupled with advances in in vitro oocyte maturation, in vitro fertilization and in vitro embryo culture, the production of embryos in vitro is rapidly growing and may soon generate more embryos than are derived in vivo. Embryo micromanipulation procedures such as embryo bisection, microfertilization, and assisted embryo hatching are providing additional tools for reproductive biologists to produce live offspring. Embryo biopsy is permitting preimplantation genetic diagnosis not only for genetic sex determination but also for identification of embryonic genetic defects. Embryo culture and embryo coculture systems have also enabled proliferation of novel assisted reproductive technologies such as oocyte reconstruction and nuclear transfer.

Attempt to visualize an endogenous lectin recognizing α-L-fucose immunohistochemically in the rabbit oviduct

An immunohistochemical study was conducted in order to visualize an endogenous lectin, having specificity for alpha-L-fucose residues, in the ampullary and isthmic tracts of the rabbit oviduct. 2 different sequences (S1 and S2) were tested using antilectin UEA I antibody. Reactive sites towards S2 sequence were localized at ciliated cell level in the ampulla; in the isthmus, instead, the staining was present both in ciliated and secretory cells. A role in the sugar immobilization and gamete and zygote protection was postulated for the endogenous lectin evidenced.

Electromyographic activity and noradrenaline content of the rabbit oviduct under different hormonal states.

Spontaneous electromyographic (EMG) activity of the oviduct recorded in vivo in untreated, estrogen-treated, and progesterone-treated castrated rabbits was found to exhibit two main patterns: short spike bursts lasting 1-10 s and long trains of action potentials lasting several minutes, which constituted the major component of EMG activity. After estrogen treatment, both wet weight and noradrenaline (NA) content of the castrated rabbit oviduct were enhanced mainly at the ampullary-isthmic junction; long trains of discharges were significantly shorter (2.0-2.7 min vs 3.6-4.6 min) and appeared at more frequent intervals (9.8-12.2 min vs 14.2-22.6 min). After progesterone treatment, spontaneous EMG activity was not significantly different from that in untreated castrated rabbits (as was the NA content) except at the ampullary-isthmic junction. NA injection elicited a stimulatory response of the oviduct lasting 1-7 min in the three hormonal states. Phentolamine strongly depressed spontaneous EMG activity but the inhibition was more transient in castrated rabbits than in estrogen-treated and progesterone-treated animals. Propranolol had no effect on spontaneous EMG activity. These data and the high NA concentrations found in all parts of the isthmus support the hypothesis that adrenergic innervation plays a role in the organization of oviductal motility in the rabbit.

The mechanism and control of rabbit oviduct fluid formation.

The formation of rabbit oviduct fluid was monitored continuously by using an in situ vascular perfusion technique. Oviduct fluid was secreted linearly for at least 3 h at a mean rate of 20.8 +/- 1.5 microliter/h in estrous does. The rate more than doubled on Day 1 following mating, was similar to the value at estrus on Day 2, and dropped to 8.3 microliter on Day 3. Dibutyryl cyclic adenosine 3',5'-monophosphate (cAMP, 1 mM) added to the vascular medium abolished fluid secretion. The same response was obtained, after a lag period, following the addition of cholera toxin (1 mM), forskolin (1 mM), theophylline (1 mM), phorbol dibutyrate (40 microM), A23187 (2 micrograms/ml), 4-acetamido-4'-isothiocyonatostilbene-2,2'-disulphonic acid (SITS, 1 mM), and bumetanide (10 microM) to the vascular medium. N-ethylmaleimide (1 mM), which inhibits adenylate cyclase, stimulated oviduct fluid formation. The transmural potential difference (p.d.) across the oviduct was 5.46 +/- 1.01 mV. This was increased after cAMP addition to 8.7 +/- 1.22 mV. The p.d. in oviducts taken 3 days post-ovulation was 7.6 +/- 1.75 mV, and was increased by cAMP to 12.7 +/- 0.53 mV. Exposure to cholera toxin and forskolin almost doubled the cAMP content of the oviduct. The undirectional flux of chloride ions from the vascular compartment into the lumen was reduced by about 75% after the addition of cAMP, SITS, and bumetanide. A tentative model to account for the formation and regulation of rabbit oviduct fluid in terms of ion fluxes and cAMP and calcium ion concentrations is presented.

Early ovum and embryo loss after microsurgical anastomosis of the rabbit oviduct.

After resection and reanastomosis, ovum recovery and fertilization were studied in the rabbit oviduct. From these data the ovum and embryo loss was calculated. Loss was most pronounced in ampullary operated tubes and after a short recovery period.

Anatomical basis of a passive mechanism for ovum retention at the ampulloisthmic junction.

The unexplained behavior of egg retention at the ampulloisthmic junction (AIJ) is a mechanical phenomenon; therefore the key to this process must lie in the anatomy of the oviduct. To investigate the concept that simple anatomical differences between ampulla and isthmus cause AIJ retention, we quantified, in these two regions of the rabbit oviduct, the structural characteristics of the tubal wall that show marked differences: wall thickness and luminal caliber. The oviductal wall greatly increases in thickness and decreases in luminal caliber abruptly at the transition from ampulla to isthmus. These passive mechanical factors predict a dramatically reduced distensibility of the isthmic wall compared to that of the ampulla, and these factors provide us with a simple model to account for the apparent sphincteric activity of the AIJ. Actual measurements of tubal distensibility confirmed our prediction. In addition, egg transport studies following microsurgical removal of the AIJ showed normal transport, an indication that no special structure need exist at the AIJ. We conclude that the contrasting morphology of the ampulla and isthmus may suffice to explain AIJ retention, and no special active mechanism need be postulated.

Cytogenetic study of parthenogenetically activated bovine oocytes matured in vivo and in vitro.

Three experiments were performed to evaluate the potential for parthenogenetic activation of bovine oocytes in in vitro fertilization systems and to determine the chromosome complement of the resulting parthenogenotes. In the first experiment, immature oocytes from slaughtered cattles were matured in vitro in Defined Medium (DM) for 24 h to simulate in vitro fertilization conditions. Subsequently, a portion was fixed, and the remainder were transferred to rabbit oviducts. Oocytes were then cultured for 6-8 h or for 24 h with Colcemid present during the last 6 to 8 h and fixed on slides and examined. In the second experiment, mature oocytes were collected from the preovulatory follicles, and the oocytes were subjected to the same culture as in experiment I. In the third experiment, oocytes were treated as in experiment II, except that instead of transfer to rabbit oviducts, they were cultured an additional 48 h in vitro. In experiment I, 131 oocytes were fixed after culture in DM. Of the 79 oocytes analyzed in the pre-rabbit group, 71 (90%) were at the second meiotic metaphase (MII), and 8 (10%) were at pre-MII stage; none were activated. After transfer to rabbits, 291 were fixed. Of these, 80 were analyzed; 37 (46.3%) were MII, 7 (8.6%) were pre-MII, and 36 (45%) were activated. Of the 36 activated oocytes, 26 (72.2%) were haploid, 4 (11.1%) were diploid, 1 (12.8%) was tetraploid, and 5 (13.8%) were in the process of endoreduplication. In experiment II, 51 oocytes were fixed after culture in DM of which 36 (70.6%) could be analyzed; 30 (83.3%) were MII, and 6 (16.7%) were pre-MII. After culture in the rabbit, 68 were fixed of which 27 (39.7%) could be analyzed. Of these 27, 20 (74.1%) were MII, and 7 (25.9%) were activated; 6 were haploid, and 1 was endoreduplicating. In experiment III, 30 oocytes were fixed at the end of the culture period; only 10 could be analyzed of which 8 (80%) were MII and 2 (20%) were pre-MII. In all, 46% of in vitro and 26% of in vivo matured oocytes were activated, based on chromosomal analysis. Of those activated, the majority (74.4%) were haploid, suggesting that activation occurs at or after completion of MII. Endoreduplication appears to be one of the mechanisms leading to the formation of diploid and polyploid parthenogenotes.

Morphology and fertility after re-anastomosis of the rabbit fallopian tube with fibrin glue.

Morphology and fertility were studied in 20 female New Zealand White rabbits after re-anastomosis of the Fallopian tube with fibrin glue and conventional microsurgical techniques. All oviducts were patent postoperatively. No intraperitoneal adhesions were observed. There were no significant differences with regard to the number of corpora lutea, implantations and the nidation index. Morphological studies demonstrated a normal fold pattern and ciliation at the side of anastomosis in the sealed oviducts as well as in the sutured oviducts. No intraluminal fibrin deposits were found. For re-anastomosis of the Fallopian tube with fibrin glue, splinting is necessary. In some instances this may be related to a mucosal trauma. However, under optimal conditions the use of fibrin glue is equivalent to conventional microsurgical anastomosis of the oviduct. For tubocornual, ampullary--ampullary and isthmic--ampullary anastomoses with luminal disparity, fibrin glue seems to be inappropriate.

Development of centrifuged cow zygotes cultured in rabbit oviducts.

Zygotes from superovulated cows were centrifuged and pronuclei were detected by differential interference-contrast microscopy in 73% of 106 zygotes. Zygotes were then transferred to ligated oviducts of follicular-phase, 1-day pseudopregnant or 7-day pseudopregnant rabbits and recovered 5 days later. Their development did not differ from that of uncentrifuged zygotes transferred to the opposite oviduct: 41% of the embryos recovered from rabbit oviducts contained 17-32 nuclei and an additional 5% contained greater than 32 nuclei. In another experiment, 399 ova from unmated cows were transferred to rabbit oviducts to determine whether centrifugation induced parthenogenetic development. After 7 days, 257 ova were recovered; 16% of the recovered ova had developed parthenogenetically and contained 2-30 nuclei. Neither centrifugation of the ova nor reproductive status of the rabbits influenced the proportion of parthenogenotes found. Parthenogenetic development was also observed in 14 of 71 ova (20%) recovered on Day 7 from uninseminated superovulated cows. In an attempt to increase the probability of detecting treatment differences, centrifuged and control cow zygotes were incubated for 7 (rather than 5) days in opposite oviducts of fourteen 1-day pseudopregnant rabbits. Development was unaffected by centrifugation: 61% of the zygotes recovered had developed beyond the 16-cell stage, with 23, 24 and 15% containing 17-32, 33-64, and greater than 64 nuclei, respectively. Taking into account the percentage of zygotes in which pronuclei can be seen, the recovery rate from rabbit oviducts, and the proportion of embryos that develop to the morula stage or beyond, 26% of the original group of zygotes would be candidates for transfer into recipient cows.

Heterogenous and xenogenous fertilization of in vivo matured equine oocytes

Summary Forty-five in vivo matured equine oocytes were recovered from 63 follicular aspiration attempts (71.4%). HCG did not improve recovery rate (65% — 24/37 for treated vs 81% — 21/26 for nontreated mares). Fifteen oocytes were transferred into the oviduct of inseminated recipient mares (heterogenous fertilization) and 15 oocytes plus equine spermatozoa were transferred into rabbit oviducts (xenogenous fertilization). Ten oocytes (3 fertilized) were recovered from recipient mare oviducts following removal and flushing two days after transfer. Eight oocytes (nonfertilized) were recovered from rabbit oviducts. Oviductal transfer into separate recipient mares of three embryos produced from heterogenous fertilization resulted in two pregnancies. One mare produced a normal live foal and the other mare aborted at 20 days of gestation. Results from these studies suggest that: 1) a reliable method for collection of in vivo matured oocytes has been established, and 2) heterogenous fertilization is a technique that with refinement should be immediately applicable to obtain foals from valuable infertile mares that fail to get pregnant or produce embryos by standard methods.

Effects of RU 486 on the mechanical properties of rabbit fallopian tubes.

AbstractThe effects of RU 486 on the mechanical activities of the circular smooth muscles of the rabbit oviduct were investigated using an isometric tension recording method. Injection of human chorionic gonadotropin (hCG) markedly increased the frequency of the spontaneous contractions but simultaneous treatment with hCG and RU 486 did not increase the frequency. In the animals treated with hCG, the frequencies and amplitudes of the contraction increased, and the concentrations of progesterone in the sera increased. This action of hCG was suppressed when RU 486 was simultaneously injected. In oviductal smooth muscles simultaneously treated with progesterone and RU 486, the frequency of spontaneous contractions was markedly decreased as in the case of the simultaneous injection of hCG and RU 486. Thus, RU 486 alters the mechanical activities of the fallopian tube by preventing the release of progesterone induced by hCG and by acting against progesterone. This is the first report that RU 486 acts directly on tubal motility.

Adrenergic mechanisms of the contractile response of rabbit oviduct to electrical stimulation

1. 1. In rabbit oviduct electrical field stimulation evoked contraction in mature animals and contraction followed by relaxation in juvenile animals. 2. 2. Phentolamine (1 μM) inhibited the contraction. The relaxation was partly suppressed by propranolol (1–10 μM) and completely inhibited by guanethidine (5 μM). 3. 3. Cocaine (1 μM) increased the duration or the amplitude of contractions and abolished or did not change the relaxation. The contraction-increasing effect of cocaine was more pronounced in estradiol-treated mature rabbit. 4. 4. It is concluded that electrically evoked responses in rabbit oviduct are mediated by transmitters released from adrenergic terminals and that estradiol increases neuronal uptake of noradrenaline.

Leucine-enkephalin- and neuropeptide Y-modulation of [ 3H]noradrenaline release in the oviduct of mature and juvenile rabbits

1. The effects of leucine-enkephalin and neuropeptide Y (NPY) on [ 3H]noradrenaline release induced by electrical field stimulation were studied in the isthmic part of the oviduct of juvenile and mature rabbits. 2. [ 3H]noradrenaline and total tritium overflow in the presence of cocaine, corticosterone and hyoscine were determined by liquid scintillation spectrometry. 3. Tritium overflow evoked by electrical stimulation (1 or 4 Hz, 1 msec) was calcium dependent. [ 3H]noradrenaline content (measured by ion exchange chromatography) accounted for 85% of the total tritium overflow. 4. Leucine-enkephalin (1 μM) in the presence of the peptidase inhibitor bacitracin reduced the stimulation-evoked tritium overflow in mature rabbits by 26.1 ± 1.6% and in juvenile rabbits by 11.9 ± 1.9%. Naloxone (1 μM) antagonized the effect of leucine-enkephalin. 5. NPY (0.2 μM) reduced the evoked tritium overflow in mature rabbits by 23.4 ± 2.4% and in juvenile rabbits by 17.2 ± 4.3%. 6. It is concluded that leucine-enkephalin and NPY inhibited [ 3H]noradrenaline release in rabbit oviduct and the modulatory effect of leucine-enkephalin depends on maturity while NPY modulation is a more independent system.

Effects of two treatments on semen from different bulls on in vitro fertilization results of bovine oocytes

The semen of six different bulls was used to examine the effects of treatment with caffeine or caffeine plus Ca-ionophre on in vitro fertilization, cleavage and development into morulae of in vitro matured bovine oocytes. In vitro fertilization results (formation of both pronuclei, cleavage and development to ≥ four-cell stage were significantly (P<0.01) higher using caffeine plus Ca-ionophre than those using only caffeine. The rates of fertilization and first cleavage were only slightly variable among the bulls. However, the present data showed significant variability in formation of both pronuclei (36 to 75%) of fertilized ova and development to the ≥4cell stage (39 to 71%) by different bulls. Development into morulae of ova recovered from the rabbit oviduct did not show any significant differences in relation to sperm treatments or individual bulls.

Analysis of anti-implantational action of norethisterone-3-oxime

Mature rabbits received norethisterone-3-oxime (NETO) 2 mg/kg/day from day 2 through day 4 of pregnancy by gavage. Control rabbits were given vehicle. All animals were sacrified on day 5. The oviducts were removed and sections stained with HE. Morphological changes were evaluated. The outer and inner diameters of tubes, and the thickness of circular muscle layers were measured. The layers of the circular muscle cells and the mean thickness occupied by the cytoplasmic mass of the muscle cells were estimated. The fertilized ova flushed out from the oviduct were stained together with the section of ampullary-isthmic junction (AIJ) and isthmus by PAS. The intensity of the staining was observed microscopically and quantified with a microspectrophotometer. NETO treatment led to tortuosity of oviducts showing hyperplasia of epithelial cells and a mucosal edema. The circular muscle layer was thickened but no increase in the number of muscle cells was observed, indicating muscular contraction. Dilatated and congested capillaries appeared in the muscular and subserous layers. All these manifestations were so prominent that the lumen of AIJ and isthmus portion were significantly narrowed as shown by a decreased percentage of inner diameter to outer diameter at AIJ and isthmus. The amount of mucin secreted into the lumen was decreased, but there was an accumulation of mucin in the epithelial cells. The above-mentioned morphological changes lead to the suggestion that the anti-implantational action of NETO is exhibited by its effect on the oviduct in rabbits. Most probably, the action is mainly related to the estrogenic and anti-progestational activity Of NETO.

Ultrastructural and kinetic studies on uteroglobin secretion in the uterus and oviduct of the pseudopregnant rabbit.

The intracellular localization of uteroglobin, a progesterone-induced protein, was studied in uterus and oviduct by means of immunoelectron microscopy with the protein A-gold technique. In the uterus, uteroglobin was synthesized in the columnar epithelium of the endometrium where most of the cells were immunoreactive. The protein was localized mainly in small secretory granules which were seen in the process of release into the uterine lumen. The luminal microvilli were also heavily stained. In the oviduct, the secretory cells contained large immunoreactive granules at the apical zone, some of which were observed while discharging into the lumen. Within these secretory granules, uteroglobin accumulated particularly in lens-shaped patches at the periphery of the granules. In vitro kinetic studies on the secretion of newly synthesized uteroglobin indicated that the ability to store uteroglobin is greater in the oviduct than in the uterus; however, the rate of uteroglobin secretion is greater in the uterus than in the oviduct. Thus, there appears to be a good correlation between the microscopic and the functional observations.

Changes in physiological properties of rabbit oviduct by ovarian steroids.

Effects of estrogen and progesterone on electrical and mechanical properties of longitudinal smooth muscle cells of the rabbit fallopian tubes were investigated using microelectrode and isometric tension recording methods. In tissues from the estrogen-treated rabbits, there was a decrease in frequency of spontaneous contractions, whereas an increase was seen in tissues from the progesterone-treated rabbits. The membrane potential of smooth muscle cells of the fallopian tube was -58.5 mV. Following treatment with progesterone or estrogen, the membrane was either depolarized (-53.7 mV, P less than 0.05) or hyperpolarized (-63.2 mV, P less than 0.05), respectively. Spontaneous burst discharges were recorded from these smooth muscle cells, and changes in the frequency of the bursts induced by treatment with progesterone or estrogen corresponded well with changes in the spontaneous contractions. Passive electrical parameters such as the time and space constants were increased by treatment with progesterone and decreased by treatment with estrogen. Therefore endogenous ovarian steroids probably participate in transport of the ovum by controlling spontaneous membrane and mechanical activities of the smooth muscle cells of the fallopian tube.