Immunohistochemical localization of prostaglandin synthase in the rat uterus and embryo during the peri-implantation period

Prostaglandins (PGs) appear to have a role in the appearance of the increased uterine vascular permeability and subsequent decidualization observed at implantation in many species. However, the sites of production of these PGs have not been clearly established. To clarify the PG synthetic capacity of the blastocyst and the various types of cells in the uterus at implantation, we have studied the immunohistochemical localization of PG synthase in the rat blastocyst on Days 5 to 7 and uterus on Days 1, 4, 5, 6, and 7 of pregnancy. Labeling of PG synthase was negligible in the uterus on Day 1 of pregnancy. On Day 4, there was increased labeling in the luminal and glandular epithelium, in stromal cells adjacent to the luminal epithelium, and in blood vessels and some leukocytes. PG synthase was detected in the blastocysts on Days 5 to 7, but there was a gradual loss of label in the luminal and glandular epithelial cells during this period. Early differentiating stromal cells adjacent to the luminal epithelium in the implantation site on Day 5 showed bright labeling, whereas peripheral stromal cells were only slightly labeled. By Day 7, the differentiated cells of the primary decidual zone showed little or no label, but cells in the secondary decidual zone were brightly labeled. These results indicate that PG synthase is present in the rat blastocyst and in several kinds of uterine cells, and that its localization in uterine cells changed markedly during the implantation process.     

Comparative effects of 6-hydroxydopamine and alpha-adrenoceptor antagonists on intrauterine migration and spacing of blastocysts in the rat

Sympathetic nerve terminals were destroyed by administration of 6-hydroxydopamine (2 x 50 mg/kg) at 10:00 h on Days 4 and 5 of pregnancy in the rat. In the myometrium, this treatment markedly decreased noradrenaline concentrations (by 99%, P less than 0.001), demonstrating that myometrial noradrenaline is mainly originated from sympathetic nerves; therefore after 6-hydroxydopamine, the distribution and spacing of blastocysts remain unaffected throughout the uterus. Administration of phenoxybenzamine (2 x 6 mg/kg) in the morning of Days 4 and 5, or prazosin (4 x 3 mg/kg) from 12:00 h on Day 4 until 12:00 h on Day 5 disorganized the even distribution of blastocysts from the tubal end to the cervical end of the uterine horns. These results provide evidence that a noradrenergic transmission via action on myometrial post-synaptic alpha 1-adrenoceptors is involved as a regulatory mechanism of uterine motility for distribution and spacing of blastocysts in the rat uterus.     

Differential expression of ezrin/radixin/moesin (ERM) and ERM-associated adhesion molecules in the blastocyst and uterus suggests their functions during implantation

Development of the blastocyst to implantation competency, differentiation of the uterus to the receptive state, and a cross talk between the implantation-competent blastocyst and the uterine luminal epithelium are all essential to the process of implantation. In the present investigation, we examined the possibility for a potential cross talk between the blastocyst and uterus involving the ezrin/radixin/moesin (ERM) proteins and ERM-associated cytoskeletal cross-linker proteins CD43, CD44, ICAM-1, and ICAM-2. In normal Day 4 blastocysts and after rendering dormant blastocysts to implantation-competent by estrogen in vivo (activated), the outer surface of mural trophectoderm cells showed much higher levels of radixin as compared to those in the polar trophectoderm cells, inner cell mass (ICM), and primitive endoderm. In contrast, ezrin was present on both the mural and the polar trophectoderm cell surfaces of normal Day 4 and activated blastocysts at higher intensity than dormant blastocysts. A distinct localization was noted in the primitive endoderm of dormant blastocysts that was not apparent in activated or normal Day 4 blastocysts. The expression of moesin was modestly higher at the mural trophectoderm of implantation-competent blastocysts, while the localization appeared to be present primarily on the polar trophectoderm cell surface of Day 4 blastocysts. The localization of ERM-associated adhesion molecules CD43, CD44, and ICAM-2 was more intense in the implantation-competent blastocysts compared with the dormant blastocysts. However, while CD44 was present both in the trophectoderm and in ICM, CD43 and ICAM-2 were localized primarily to the trophectoderm. The signal for ICAM-1 was very intense in the ICM but was modest in the trophectoderm. No significant changes in fluorescence intensity were noted between activated and dormant blastocysts. In the receptive uterus on Day 4 of pregnancy, ERM proteins were localized to the uterine epithelium, while on Day 5 the localization, especially of radixin and moesin, extended to the stroma surrounding the implantation chamber. With respect to ERM-associated adhesion molecules, while CD44 and ICAM-1 were exclusively localized in the stroma on Day 4, CD43 and ICAM-2 were localized to the epithelium. On Day 5, the localization of CD44 and ICAM-1 became highly concentrated in the antimesometrial stroma of the implantation chamber. The localization of CD43 and ICAM-2 remained mostly epithelial, although some stromal localization of CD43 was noted on Day 5. These results suggest that differential expression and distribution of ERM proteins and ERM-associated adhesion molecules are involved in the construction of the cellular architecture necessary for blastocyst activation and uterine receptivity leading to successful implantation.     

Development of pig blastocysts in a uterine environment advanced by exogenous oestrogen

Routine embryo transfer techniques were used to establish recipient groups in which blastocysts were either asynchronous (blastocysts 24 h behind recipient uterus) or synchronous with their uterine environment. Oestradiol valerate (5 mg) was administered on Day 11 of the recipient's cycle to stimulate release of uterine secretion in the synchronous gilts (Group SE) and one group (AE) of asynchronous gilts. The gilts in the other asynchronous group (Group AC) were injected with vehicle (sesame oil). Embryos recovered on Day 14 by hysterectomy and flushing were evaluated for morphological development. Oestradiol treatment resulted in a failure of blastocyst development in Group AE gilts only. Recoverable oestradiol in the uterine flushings was increased in gilts in Groups AC and SE which contained elongated blastocysts. Plasmin inhibitor levels were lower in Groups AC and SE while PGF tended to be increased. Acid phosphatase activity was higher and recoverable Ca2+ was lower in Groups AE and SE. Failure of blastocyst development in Group AE is believed to have resulted from a failure to undergo trophoblastic elongation due to premature alteration of the uterine environment at a critical period of blastocyst development or from the presence of an unfavourable uterine environment for blastocyst attachment and development shortly after Day 12.     

Survival of equine embryos co-cultured with equine oviductal epithelium from the four- to eight-cell to the blastocyst stage after transfer to synchronous recipient mares

In this study we examined the ability of equine oviductal epithelial cells (OEC) to support the development of four- to eight-cell equine embryos in vitro and investigated the ability of co-cultured embryos to continue normal development after transfer to synchronous recipient mares. Equine embryos obtained at Day 2 after ovulation were cultured with or without OEC for 5 days. Those OEC co-cultured embryos that reached the blastocyst stage and embryos recovered from the uterus at Day 7 were surgically transferred to synchronous recipient mares. Co-culture with OEC improved (P < 0.01) development of four- to eight-cell embryos to blastocysts compared to medium alone (11/15 vs 0/6) during 5 days in vitro. Embryos co-cultured with OEC were smaller (P < 0.05) and more delayed in development than Day-7 uterine blastocysts. There was no difference in the Day-30 survival rate of co-cultured blastocysts (3/8) or Day-7 uterine blastocysts (5/8) after transfer to recipient mares. These results indicate that co-culture with OEC can support development of four- to eight-cell equine embryos in vitro and that co-cultured embryos can continue normal development after transfer to recipient mares.     

A timetable of embryonic development, and ovarian and uterine changes during pregnancy, in the stripe-faced dunnart, Sminthopsis macroura (Marsupialia: Dasyuridae)

Aged stages (63) were available for establishment of a timetable of embryonic development of the stripe-faced dunnart. On Day 0 oocytes reaching maturity were found in the ovary. Within +/- 24 h of time 0 (time of minimum morning weight) polymorphonuclear leucocytes appeared and spermatozoa were last detected in the urine of 70% of females. Embryos were collected at intervals during pregnancy by hemihysterectomy and the embryos in the contralateral uterus either were examined at a later stage of pregnancy or allowed to develop to term. Cleavage to the unilaminar blastocyst stage with around 32 cells took 3 days with a cleavage arrest of 24 h at the 4-cell stage. Expansion of the unilaminar blastocyst occurred over the next 3 days. Primitive endoderm cells appeared on Day 6, fully bilaminar blastocysts by the end of Day 7 and trilaminar blastocysts on Day 8. Shell loss and implantation of 13-15-somite stage embryos occurred on Day 8 and organogenesis over the next 2-3 days. The gestation period was 9.5-12.0 days with most births occurring between 10.5 and 11.0 days. Major steps in embryonic development were correlated with stages in the development of the corpora lutea, which were maximal in size, and possibly in secretory activity, when the embryos were at the bilaminar blastocyst stage. Regression commenced when the embryos were at the primitive streak stage. At the time the corpora lutea were maximal the uterine epithelium reached its greatest height and the endometrium was thick and folded. Later in pregnancy villous-like projections of the epithelium formed, and the luminal epithelial cells became rounded. Two cell populations, a tier of 8 smaller cells above the yolk mass and a tier of 8 larger cells around the sides of the yolk mass appeared at the 16-cell stage. From the 16-cell stage to the blastocyst stage, with 150-200 cells, two cell populations distinguished by size, cell cycle time, cytoplasmic appearance and position relative to the yolk mass were present. The two populations were indistinguishable in blastocysts with greater than 200 and less than 2000 cells. They reappeared in blastocysts with greater than 2000 cells, as the darker cells of the embryoblast, and as the paler cells of the trophoblast. The darker cells lay in the yolky hemisphere and the paler cells in the non-yolky hemisphere.     

A Globosus amorphus from an in vitro fertilized embryo transferred to a Japanese black cow

A Globosus amorphus along with a living calf was encountered following the transfer of blastocysts obtained by in vitro fertilization of in vitro-matured follicular oocytes in Japanese black cattle. Two embryos obtained 9 days after in vitro fertilization developed into either a hatched blastocyst with distinct inner cell mass or an expanded blastocyst with indistinct inner cell mass. The embryos were loaded into a 0.25-ml plastic straw and were nonsurgically transferred to the uterus of a heifer on Day 8 (Day 0 = estrus). On Day 75, a twin pregnancy was ultrasonically diagnosed in the right uterine horn, in which a live fetus with distinct limbs and a concomitant ovoid mass were detected. On Day 287, the dam developed parturient paralysis with dropsy of the fetal membranes. By palpation per rectum an ovoid mass was detected in the body of the uterus [corpus uteri] and a larger live fetus was in the uterine horn. A cesarean section was performed to extract a live fetus and a Globosus amorphus. The live fetus was female with the 60, XX female complements.     

Effect of the in vitro culture system on the kinetics of blastocyst development and sex ratio of bovine embryos

Bovine blastocysts were produced using 6 different systems: 5 commonly used in vitro culture systems (synthetic oviduct fluid medium - SOF- without fetal calf serum, SOF supplemented with 10% serum for the entire culture period, SOF supplemented with 10% serum from Day 4 of culture, M199 coculture with bovine oviduct epithelial cells, M199 coculture with granulosa cell monolayer) and 1 in vivo culture system involving collection of blastocysts from superovulated bovine donors at Day 7. Zygotes obtained from IVM/IVF were assigned randomly to 1 of the 5 systems tested and were cultured for 9 d (Day 0= day of insemination). Cleavage, development to the blastocyst stage and blastocyst sex ratio were assessed in all treatments. In addition, the effect of the IVC system on the kinetics of blastocyst development and sex ratio was assessed on Days 6, 7, 8, and 9. The presence of fetal calf serum in SOF not only resulted in faster development (19.1% of blastocysts in SOF supplemented with serum vs 7.1% in absence of serum at Day 6; P < 0.05) and increased blastocyst production (47.5% of blastocysts in SOF supplemented with serum vs 34.4% in absence of serum; P < 0.05) but it also enhanced overall male survival. The coculture systems produced fewer blastocysts than culture in SOF (27.6 to 28.3% in coculture vs 47.5% in SOF supplemented with serum; P < 0.05), but similar to SOF without fetal calf serum, they had no effect on blastocyst sex ratio.     

Survival of small and large littermate blastocysts in swine after synchronous and asynchronous transfer procedures

Thirty-two crossbred sows were assigned to synchronous and asynchronous embryo transfer procedures to determine if, within a litter, small blastocysts were as viable as large blastocysts. Synchronous embryo transfers were established when donors and recipients displayed the onset of estrus (Day 0) within 6 h of each other. Asynchronous transfers were established when recipients displayed the onset of estrus 18 to 24 h after that of donors. An equal number (four or five) of the smallest and largest diameter blastocysts, from a Day 7 donor, were transferred to separate uterine horns of a Day 7 (synchronous) or a Day 6 (asynchronous) recipient. Each recipient's uterine horns were ligated at the external bifurcation to prevent transuterine embryonic migration. The percentage of blastocysts surviving was determined 300 h (12.5 d) after donors exhibited estrus. Small as well as large Day 7 blastocysts survived following asynchronos transfer to a Day 6 recipient. However, fewer (P<0.01) small blastocysts survived synchronous transfer than large blastocysts. These data suggested that small blastocysts were lost due to asynchrony with the uterine environment; however, when transferred to a less advanced environment, small blastocysts were equally viable as large blastocysts.     

Conceptus-mediated integrity of endometrial epithelial cells and maintenance of relaxin synthesis in pregnant rabbits: effects of unilateral oviduct ligation

A previous study indicated rabbit endometrial relaxin synthesis is stimulated by blastocyst (Lee VH, Fields PA, Biol Reprod 1990; 40:737-745). To evaluate this hypothesis, unilateral oviduct ligations were placed (A) at the oviduct isthmus on Day 1 post-copulation and (B), in a separate group of rabbits, at the infundibulum before copulation. Blastocysts migrate into and implant in the uterine horn contralateral to the ligated oviduct only (conceptus-bearing uterus). The uterine horn ipsilateral to the ligated oviduct will be referred to as the non-conceptus-bearing uterus. Uteri and ovaries were removed on Days 4-28 of pregnancy and were evaluated for relaxin using guinea pig anti-porcine relaxin serum and avidin-biotin light microscopy immunohistochemistry. Results were identical for both models. Blastocysts first attach to the antimesometrial uterine surface by Day 7 post-copulation. Implantation on the mesometrial surface occurs on Days 8-11. Relaxin was observed in antimesometrial endometrial glands of both conceptus and non-conceptus-bearing uteri on Days 4-7 of pregnancy. Beyond Day 7, relaxin was observed in antimesometrial and mesometrial endometrial glandular and luminal epithelial cells at implantation sites of the conceptus-bearing uterus only. Relaxin was not found between implantation sites. Endometrial epithelial cells of the non-conceptus-bearing uterus were regressing by Day 9. These data indicate a conceptus-mediated maintenance of endometrial epithelial cells. Furthermore, the data suggest a paracrine maintenance of epithelial cell integrity and relaxin synthesis since these parameters are preserved only in the conceptus-bearing uterus. Cell-cell communication between conceptus and endometrium appears to be specific since endometrium between implantation sites does not contain relaxin. Uterine tissue from pseudopregnant rabbits (Days 1-16) was evaluated. Relaxin was observed in the antimesometrial glands on Day 7 only. Like the endometrium in the ligation model, endometrial epithelial cells of the pseudopregnant rabbit uterus were regressing by Day 9. These results indicate that pregnancy is not required for, but may enhance, relaxin synthesis. In addition, endometrial epithelial cells regress in the absence of pregnancy. Regression of endometrial epithelial cells on Day 9 suggests that maternal recognition of pregnancy occurs during the preimplantation period (Days 4-8).     

Effects of cervical dilation and intrauterine infusions on the timing of oviductal transport of equine embryos

Equine embryos spend 5 to 6 d in the oviduct before entering the uterus as expanded blastocysts, and cannot be consistently collected nonsurgically until Day 7. Technologies such as cryopreservation and embryo splitting, which are most successful with embryos at the morula or early blastocyst stage, have not been used in mares because equine morulae and early blastocysts are located in the oviduct and cannot be recovered nonsurgically. These experiments test the hypothesis that transport of equine embryos through the oviduct can be hastened by cervical dilation or by acute, sterile endometritis induced by intrauterine oyster glycogen treatment. Cervical dilation with or without intrauterine infusion of 0.5 ml PBS on Day 4 did not appear to hasten the transport of embryos into the uterus since Day 5 uterine embryo recovery rates were not higher (P > 0.1) for mares with cervical dilation or cervical dilation plus PBS infusion vs mares receiving no treatments (0 of 5 and 0 of 5 vs 0 of 10, respectively). Intrauterine infusions of 40 ml of 1% oyster glycogen or 40 ml of PBS on Day 3 did not appear to hasten the transport of embryos into the uterus since Day 5 uterine embryo recovery rates were not higher (P > 0.1) for oyster glycogen- or PBS-treated vs untreated mares (2 of 12 and 3 of 11 vs 0 of 10, respectively). Cervical and uterine treatments on Day 3 or Day 4 and uterine lavages on Day 5 decreased (P < 0.05) Days 11 to Day 15 pregnancy rates compared with that of untreated mares. Day 11 to Day 15 pregnancy rates were 1 of 5 for mares with Day 4 cervical dilation and Day 5 uterine lavage, 1 of 5 for mares with Day 4 PBS infusion and Day 5 uterine lavage, 2 of 12 for mares with Day 3 oyster glycogen infusion and Day 5 uterine lavage, and 3 of 11 for mares with Day 3 PBS infusion and Day 5 uterine lavage vs 7 of 10 for mares that received no treatment or lavage. Cervical and uterine manipulations on Day 3 or 4 and uterine lavage on Day 5 appeared to decrease pregnancy rates by Days 11 to 15. The results of these experiments do not support the hypothesis that cervical dilation or uterine infusion hasten oviductal transport, since neither cervical manipulation nor transcervical infusion of oyster glycogen or PBS into the uterus significantly hastened the rate of embryo transport into the uterus.     

Genetic and environmental determinants of interferon-tau secretion by in vivo- and in vitro-derived bovine blastocysts

Several experiments were conducted to assess the effects of genotype and various culture media on interferon-tau secretion by in vitro-derived bovine blastocysts and to compare these values with interferon released by blastocysts flushed from superovulated cows. In experiment 1, oocytes were inseminated with semen from three different bulls. While paternal genotype had no effect on cleavage rate, the size or hatching ability of blastocysts, it was a significant determinant of the embryo's ability to develop to the blastocyst stage and of subsequent interferon-tau secretion. In the second experiment, embryos were cultured in synthetic oviductal fluid containing either polyvinyl alcohol, bovine serum albumin or fetal bovine serum. While there was no effect of supplement on the percentage of embryos developing to the blastocyst stage, blastocysts which formed in medium with polyvinyl alcohol had significantly fewer cells, were older at blastocyst formation and produced significantly more interferon-tau. In the third experiment, embryos were cultured to the blastocyst stage in either TCM199 alone or in co-culture with buffalo rat liver, bovine oviductal or bovine uterine epithelial cells. Culture with oviductal or buffalo rat liver cells increased blastocyst cell number, although secretion of interferon-tau was not affected. In the final experiment, bovine blastocysts were flushed from superovulated cows on Day 7 following insemination. Overall, secretion of interferon-tau by in vivo-produced blastocysts did not differ from that of age-matched blastocysts produced in vitro.     

Expression and estradiol regulation of Wnt genes in the mouse blastocyst identify a candidate pathway for embryo-maternal signaling at implantation

Implantation of mammalian embryos depends on differentiation of the blastocyst to a competent state and of the uterine endometrium to a receptive state. Communication between the blastocyst and uterus ensures that these changes are temporally coordinated. Although considerable evidence indicates that the blastocyst induces expression of numerous genes in uterine tissue, potential signaling mechanisms have yet to be identified. Moreover, whereas a surge of maternal estradiol occurring on Day 4 of pregnancy in the mouse is critically required for many of the peri-implantation uterine changes, whether this surge also affects blastocyst gene expression has not been established. We show here that mouse morulae express genes encoding several members of the Wnt family of signaling molecules. Additional Wnt genes are newly expressed following development to blastocyst. Unexpectedly, Wnt5a and Wnt11 are expressed in embryos that undergo the morula-to-blastocyst transition in vivo, but only weakly or not at all in embryos that do so in vitro. Upregulation of Wnt11 is temporally coordinated with the surge of maternal estradiol on Day 4. Wnt11 fails to be upregulated in blastocysts obtained from mice ovariectomized early on Day 4 or from mice treated with the estradiol antagonist, ICI 182,780. Administration of estradiol-17beta or its metabolite, 4-OH-estradiol, to ovariectomized mice restores Wnt11 expression. Moreover, Wnt11 expression is not upregulated when blastocysts are trapped in the oviduct following ligation of the utero-tubal junction, nor when estradiol-17beta or 4-OH-estradiol are administered to blastocysts in vitro. These results establish a comprehensive profile of Wnt gene expression during late preimplantation development, demonstrate that estradiol regulates gene expression in the blastocyst via uterine factors, and identify Wnts as potential mediators of embryo-uterine communication during implantation.     

Aromatase activity in individual day-11 pig blastocysts

Blastocysts were flushed from both uterine horns of 10 gilts on Day 11 of pregnancy. In these gilts 15.1 +/- 2.3 (mean +/- s.d.) corpora lutea were present and 12.7 +/- 3.1 spherical blastocysts were recovered. In all the gilts variation in blastocyst diameter was observed. Aromatase activity was measurable in 118 of 121 examined blastocysts and varied from 0.005 to 19.330 pmol [1 beta-3H]androstenedione converted into 3H2O in 20 min (mean 1.31). This variation in aromatase activity reflected a difference between and within gilts. Of the total variation between all blastocysts, 67% was due to differences between gilts. A positive exponential relationship existed between blastocyst diameter and aromatase activity, but this relationship was different between gilts (P less than 0.0001). The observed variation in aromatase activity may be caused by differences in germ layer differentiation of the blastocysts.     

Progesterone regulates FGF10, MET, IGFBP1, and IGFBP3 in the endometrium of the ovine uterus

Progesterone (P4) is unequivocally required to maintain a uterine environment conducive to pregnancy. This study investigated the effects of P4 treatment on expression of selected growth factors (fibroblast growth factor 7 [FGF7], FGF10, hepatocyte growth factor [HGF], and insulin-like growth factors [IGF1 and IGF2]), their receptors (MET, FGFR2(IIIB), and IGF1R), and IGF binding proteins (IGFBPs) in the ovine uterus. Ewes received daily injections of corn oil vehicle (CO) or 25 mg of P4 in vehicle from 36 h after mating (Day 0) to hysterectomy on Day 9 or Day 12. Another group received P4 to Day 8 and 75 mg of mifepristone (RU486, a P4 receptor antagonist) from Day 8 through Day 12. Endometrial FGF10 mRNA levels increased between Day 9 and Day 12 and in response to P4 on Day 9 in CO-treated ewes, which had larger blastocysts, and were substantially reduced in P4+RU486-treated ewes, which had no blastocysts on Day 12. Endometrial FGF7 or HGF mRNA levels were not affected by day or reduced by RU486 treatment, but MET mRNA levels were higher in P4-treated ewes on Day 9 and Day 12. Levels of IGF1, IGF2, and IGF1R mRNA in the endometria were not affected by early P4 treatment. Although stromal IGFBPs were unaffected by P4, levels of IGFBP1 and IGFBP3 mRNA in uterine luminal epithelia were increased substantially between Day 9 and Day 12 of pregnancy in CO-treated ewes and on Day 9 in early P4-treated ewes. Therefore, FGF10, MET, IGFBP1, and IGFBP3 are P4-regulated factors within the endometrium of the ovine uterus that have potential effects on endometrial function and peri-implantation blastocyst growth and development.     

The ovine uterus as a host for in vitro-produced bovine embryos

A series of experiments were conducted to determine whether bovine blastocysts would develop beyond the blastocyst stage in the ovine uterine environment. In Experiment 1, in vitro matured, fertilized and cultured (IVM/IVF/IVC) expanded bovine blastocysts were transferred into uteri of ewes on Day 7 or 9 of the estrous cycle and collected on Day 14 or 15 to determine if the bovine blastocysts would elongate and form an embryonic disk. Springtime trials with ewes that were synchronized with a medroxyprogesterone acetate (MAP) sponge resulted in a 78% blastocyst recovery rate, and 68% of the recovered spherical or elongated embryos had embryonic disks. In Experiment 2, transfer of 4-cell bovine embryos to the oviducts of ewes at Day 3 resulted in a lower recovery (47 vs 80%) than the transfer of blastocysts at Day 7 when embryos were recovered at Day 14. However, the percentage of embryos containing embryonic disks was higher for embryos transferred at the 4-cell stage (71%) than for embryos transferred as blastocysts (50%). In Experiment 3, IVF embryos from super-ovulated cows or Day 8 in vitro produced embryos transferred to cows were collected at Day 14 and were found to be similar in size to those produced by transfer to ewes in Experiment 2. In Experiment 4, the transfer of bovine blastocysts to ewes did not prolong the ovine estrous cycle. In Experiment 5, extension of the ovine estrous cycle by administration of a MAP releasing intravaginal device allowed bovine embryos to elongate extensively and to become filamentous. In Experiment 6, uterine flushings on Day 14 or Day 16 contained elevated levels of interferon-tau when bovine blastocyst were transferred on Day 7. Transfer of bovine embryos to the reproductive tract of a ewe allows some embryos to develop normally to advanced perimplantation stages and may be a useful tool for studying critical stages of embryo development and the developmental capacity of experimental embryos.     

Endoscopic embryo collection and embryo transfer into the oviduct and the uterus of pigs

We describe the first complete embryo transfer program, including flushing of embryos from the oviducts via the uterine horns, transfer of embryos into the Fallopian tubes or the uterine horns and recording of the number of piglets born live. The described procedure is minimally invasive and allows the use of pigs simultaneously for embryo collection and production of normal pregnancies. A 30 degrees forward oblique endoscope provided optimal visualization of the reproductive organs and free access to the organs for embryo flushing and transfer. In contrast to surgical and nonsurgical methods, endoscopy allows to pre-examine the genital tract for reproductive abnormalities and successful ovulation. A total of 95 prepuberal gilts or cyclic sows were used in this trial. Embryos or oocytes were collected from hormonally treated pigs via endoscopy(n = 17) on Day 3 and via laparotomy or post mortem after slaughter (control group, n = 38) on Day 3 and 6 after insemination. One (unilateral collection, n = 7) or both oviducts (bilateral collection, n = 10) were flushed endoscopically. We recovered 114 (average 16/pig) and 279 (average 28/pig) oocytes or embryos with fertilization rates of 89% and 72%, respectively. In the control group 834 oocytes or embryos were collected at Day 3 and 6 after insemination (fertilization rate 64%, total 534 embryos, 33 at 2-, 367 at 4-, 2 at 8-cell stage, 24 morulae and 108 blastocysts). Of 836 embryos recovered by endoscopy, surgery or slaughter 528 Day 3 embryos at 2- to 4-cell stage were transferred into (one) oviducts (n = 27 pigs, about 20/pig) resulting in 9 pregnant pigs diagnosed at Day 28 by sonography. Of the 9, 8 carried a total of 49 piglets to term. A total of 195 Day 6 embryos were transferred into uterine horns (n = 12 pigs, about 16/pig), resulting in 5 pregnant pigs carrying a total of 38 offspring to term. The use of endoscopy in assisted reproduction of pigs has the advantages of allowing easy access to the ovary, oviduct and uterus, clear view of the organ manipulation without exposure and exteriorization of viscera during surgery.     

Inequality in function of the right and left ovaries and uterine horns of the mouse

Inequality in function of the left and right ovaries and uterine horns of mice was evaluated in three separate experiments. In Exp. 1, the effect of position in the reproductive tract on various reproductive characteristics was evaluated in 158 pregnant hybrid mice. Ovulation rate, number of fetuses, total fetal weight and total placental weight were higher (P less than 0.05) on the right than the left on Day 18 of pregnancy (vaginal plug = Day 1). In Exp. 2, the effect of previous sham or unilateral ovariectomy (right or left) in mated Swiss-Webster mice was compared with unoperated mated controls (N = 17-24/treatment). In control mice, ovulation rate, total fetal weight and ovarian weight were higher (P less than 0.05) on the right than left side. Surgery (sham or unilateral, ovariectomy) decreased (P less than 0.005) ovulation rates, number of fetuses, ovarian weights, total fetal weight and total placental weight on Day 18 of pregnancy. Unilateral ovariectomy decreased (P less than 0.05) ovulation rates and ovarian weights more than did sham operation. Ovulation rates were higher (P less than 0.01) when the left ovary was manipulated or removed rather than the right ovary. For Exp. 3, pairs of 8 hybrid mouse embryos each (morulae and blastocysts) were surgically transferred to the left and right uterine horns of the same (bilateral, N = 15) or different (unilateral, N = 28) Swiss-Webster recipients. In almost all incidences, embryo survival (to Day 18 of pregnancy) was twice as high (P less than 0.05) in right than left uterine horns. We conclude that the left and right ovaries and uterine horns are not equal in function in Swiss-Webster and a hybrid strain of mice.     

Ultrasonic evaluation of uterine involution in Bulgarian Murrah buffalo after administration of oxytocin

The aim of this study was to determine the time taken for complete uterine involution in Bulgarian Murrah buffaloes following normal parturition and oxytocin stimulated milking; and to establish the time course of the change in size of the uterine horns, the cervix and caruncles between parturition and involution by means of ultrasonography. There were 17 animals in the study aged 3-6 years and average parity of 2.17 ± 0.18. They were administered 20 IU oxytocin 15 min before each milking. Rectal palpation and transrectal ultrasonography were performed at 3 d intervals from Days 1 to 34 post partum. The involution of the non-gravid and gravid uterine horns, and the cervix was complete by Days 22 and 25 post partum when their diameters were 2.7 ± 0.4 cm, 2.8 ± 0.3 cm and 3.12 ± 0.4 cm, respectively. Caruncles underwent rapid regression until Day 10 post partum. It was not possible to determine the dimensions of the caruncles after that time. The cumulative percentage of animals whose uterus was located in the pelvic cavity increased from 24% at Day 10 post partum to 100% at Day 34 post partum. The combination of rectal palpation and transrectal ultrasonography provided a reliable method of evaluating changes in the uterus over time and determining the time of uterine involution. The present study showed that complete uterine involution, with the uterus located in the pelvic cavity, was achieved by Day 34 after parturition in all 17 Bulgarian Murrah buffaloes treated with oxytocin before milking.     

Ultrastructural studies on maternal-embryonic cell interaction during experimentally induced implantation of rat blastocysts to the endometrium of the mouse.

Rat blastocysts collected around noon on Day 5 of pregnancy were transferred to the uterus of the mouse on Day 3 of pseudopregnancy. Out of 430 rat blastocysts transferred, a total of 37 were recovered as xenogeneic implants from the recipient mice killed 36, 48, 52, 58, 72, 96, and 120 hr after transfer. None of the transferred blastocysts was found surviving in the host uterus beyond 96 hr after transfer. Electron microscopic examination of the recovered implants revealed that rat blastocysts can successfully undergo the stages of ovum implantation in the mouse uterus from the early attachment to the initial phase of the trophoblastic invasion of the endometrium. During the late attachment stage, desmosomes (maculae adhaerentes), intermediate junctions (zonulae adhaerentes), and gap junctions (nexuses) were formed xenogeneically between the foreign trophoblast and the uterine epithelial cells of the host. Trophoblast cells of xenogeneic implants were destroyed shortly after the penetration of the basement membrane of the luminal epithelium of the host endometrium, leading to the degeneration and sloughing off of the transferred embryos.