Developing embryo and cyclic changes in the uterus of Peripatus (Macroperipatus) acacioi (Onychophora,Peripatidae)

Female reproductive tracts of the viviparous neo-tropical onychophoran Peripatus acacioi have been examined at different times throughout the year, and the altering relationship between the developing embryo and the uterus is described. Depending on her age and time of year, the female may have one or two generations of embryos within her uterus. The uterine wall consists of a thin outer epithelium and basal lamina, three layers of muscles, and a thick basal lamina beneath an inner epithelium lining the uterus lumen. These layers are consistent along the length of the uterus apart from the inner epithelial lining, which varies according to position in the uterus and the developmental stage of embryos contained in the uterus. Early embryos are positioned along the length of the uterus and therefore have space in which to grow. During cleavage and segment formation, each embryo is contained within a fluid-filled embryo cavity that increases in size as the embryo grows. Morulae and blastulae are separated by lengths of empty uterus in which the epithelial lining appears vacuolated. Until the process of segment formation is complete, the embryos are attached to a placenta by a stalk and remain in the same part of the upper region of the uterus. As these embryos grow, the lengths of vacuolated cell-lined uterus between them decrease. Each embryo cavity is surrounded by the epithelial sac, the maternal uterine epithelium, which becomes overlaid by a thin layer of cells, the embryo sac, which is believed to be of embryonic origin. The placenta is a syncytial modification of the epithelial sac located at the ovarian end of each embryo cavity covered by the embryo sac and is analogous to the mammalian noninvasive epitheliochorial placenta. Segment-forming embryos have their heads directed toward the ovary. As the embryo gets longer during segment formation, its posture changes from coiled to flexed. Once segment formation is complete, the embryo loses contact with its stalk, an embryonic cuticle forms, and the embryo turns around so that its head is directed toward the vagina. The embryo escapes from its embryo sac and moves to the lower part of the uterus. In the lower part of the uterus, the straightened fetuses are first unpigmented but subsequently become pigmented as the secondary papillae on the body surface form and an adult-type cuticle forms beneath the embryonic cuticle. While the embryos are contained within their embryo cavities, nutrients are supplied by the placenta. Throughout development the mouth is open and in the mature fetus the gut is lined by peritrophic membrane and material is present in the gut lumen. Trachea have been observed only in fetuses that were ready for birth. Insemination, cyclical changes in the uterine epithelium, and the nature of the cuticle shed at parturition are discussed. © 1995 Wiley-Liss, Inc.     

Evidence of increased endometrial vascular permeability at the time of implantation in the short-nosed fruit bat, Cyanopterus sphinx

Early embryonic development and implantation were studied in tropical short-nosed fruit bat Cyanopterus sphinx. We report preimplantation development and embryo implantation. Different stages of cleavage were observed in embryo by direct microscopic examination of fresh embryos after retrieving them either from the oviduct or the uterus at different days, up to the day of implantation. Generally, the embryos enter the uterus at the 8-cell stage. Embryonic development continued without any delay and blastocyst were formed showing attachment to the uterine epithelium at the mesometrial side of the uterus. A distinct blue band was formed in the uterus. The site of blastocyst attachment was visualized as a blue band following intravenous injection of pontamine blue. Implantation occurred 9+/-0.7 days after mating. This study reports that bat embryonic development can be studied like other laboratory animals and that this bat shows blue dye reaction, indicating the site and exact time of implantation. This blue dye reaction can be used to accurately find post-implantational delay. We prove conclusively that this species of tropical bat does not have any type of embryonic diapause.     

Hormonal asynchrony and embryonic development

Luteinizing hormone (LH), progesterone and estradiol profiles in peripheral blood serum were compared among parous and nonparous females with normal, abnormal or no embryonic development. Hormonal profiles between parous and nonparous females of the same embryonic status did not differ and the data were combined. Estrous cycle length was longer (P<.05) in parous (22.3±.4 days) than nonparous females (21.0±.4 days). Females with normal developing embryos had a higher serum progesterone concentration at Days 3 and 6 and a lower ratio of estradiol to progesterone than did females with abnormal embryonic development. Females with a normal embryo had higher (P<.05) preovulatory LH peaks than females with abnormal development or no recovery of an oocyte or embryo (34.3±4.7, 11.8±6.8 and 13.3±2.5 ng/ml, respectively). The interval from onset of estrus to LH peak was 8.9±2.1, 13.7±3.7 and 13.5±6.2 hr for females with normal, abnormal or no recovery of an embryo. The lower LH peak, the longer interval from onset of estrus to LH peak, and lower progesterone concentration in peripheral blood serum in females with abnormal embryos or no recovery indicated that these females had a hormonal asynchrony. The hormonal asynchrony may produce an undesirable uterine environment for male and female gametes or embryos which resulted in fertilization failure or embryonic death. In the second experiment, more transferable embryos were obtained when superovulated females received prostaglandin F_2α (PGF_2α) intravenously rather than intramuscularly. Administering PGF₂α intravenously rather than intramuscularly may have caused the demise of the corpus luteum sooner and thereby produced a more normal uterine environment which allowed more embryos to develop normally.     

Factors affecting pregnancy rates and early embryonic death after equine embryo transfer

In the present study, 638 embryo transfers conducted over 3 yr were retrospectively examined to determine which factors (recipient, embryo and transfer) significantly influenced pregnancy and embryo loss rates and to determine how rates could be improved. On Day 7 or 8 after ovulation, embryos (fresh or cooled/transported) were transferred by surgical or nonsurgical techniques into recipients ovulating from 5 to 9 d before transfer. At 12 and 50 d of gestation (Day 0 = day of ovulation), pregnancy rates were 65.7% (419 of 638) and 55.5% (354 of 638). Pregnancy rates on Day 50 were significantly higher for recipients that had excellent to good uterine tone or were graded as "acceptable" during a pretransfer examination, usually performed 5 d after ovulation, versus recipients that had fair to poor uterine tone or were graded "marginally acceptable." Embryonic factors that significantly affected pregnancy rates were morphology grade, diameter and stage of development. The incidence of early embryonic death was 15.5% (65 of 419) from Days 12 to 50. Embryo loss rates were significantly higher in recipients used 7 or 9 d vs 5 or 6 d after ovulation. Embryos with minor morphological changes (Grade 2) resulted in more (P<0.05) embryo death than embryos with no morphological abnormalities (Grade 1). Between Days 12 and 50, the highest incidence of embryo death occurred during the interval from Days 17 to 25 of gestation. Embryonic vesicles that were imaged with ultrasound during the first pregnancy exam (5 d after transfer) resulted in significantly fewer embryonic deaths than vesicles not imaged until subsequent exams. In the present study, embryo morphology was predictive of the potential for an embryo to result in a viable pregnancy. Delayed development of the embryo upon collection from the donor or delayed development of the embryonic vesicle within the recipient's uterus was associated with a higher incidence of pregnancy failure. Recipient selection (age, day after ovulation, quality on Day 5) significantly affected pregnancy and embryo loss rates.     

Ultrastructural adaptations for viviparity in the female reproductive system of gyrodactylid monogeneans

The female reproductive system of viviparous monogeneans (Gyrodactylus and Macrogyrodactylus) has been examined using fluorescence microscopy and transmission electron microscopy. The female system is tubular, made up of a thin-walled proximal seminal receptacle/ootype and a distal uterus, separated by a complex cellular region. Both parts have a continuous syncytial cytoplasmic lining. Maturing oocytes in the seminal receptacle/ootype are in intimate contact with the receptacle lining. The uterus cytoplasmic lining completely surrounds the developing embryo, and is continuous with anterior and posterior cell bodies which fluoresce strongly when stained with bisBenzimide. This lining is most extensive around small embryos, when it contains specialised organelles including star-shaped configurations of electron-dense membranes and multilamellate bodies. Pits in the uterus wall bridged by membranous structures connect the cytoplasmic lining to parenchyma or digestive cells. The cytoplasmic lining regresses as the embryo develops, but remains continuous and in intimate contact with the embryonic tegument (at least until the near-term embryo begins independent movement). Numerous ribosomes, membranes and mitochondria in the uterine cytoplasmic layer indicate a high metabolic rate, and exo/endocytotic vesicles in the F1 tegument suggest transfer of materials occurs between parent and embryo. Putative vitelline cells in the posterior of the body contain abundant RNA, ribosomes and membrane-bound secretory bodies, and are filled with an electron-lucent secretion. However, there are no ducts associated with these cells, and their function remains unknown. The cytoplasmic lining of both the seminal receptacle/ootype and the uterus appears to regulate oocyte/embryo nutrition. Similar syncytial layers occur in rotifers, but are unlike the nutritive epithelia of most other viviparous organisms.     

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.     

PGF2α levels in Day 8 blood plasma are increased by the presence of one or more embryos in the uterus

In cattle, the detection of very early endometrial responses is considered to be hampered by the presence of only a single embryo. Therefore, we have previously developed a model of multiple embryo transfer to circumvent this hindrance. In this work, we analysed embryo–maternal interactions in the bovine uterus on day 8 of development while comparing the presence of multiple v. single embryos using embryo transfer and artificial insemination, respectively. Concentration of proteins (β-actin, NFkB, clusterin and immunoproteosome 20S β5i subunit–i20S), by western blot, and hexoses (glucose and fructose) were measured in paired samples of uterine fluid (UF) from the same animal with and without embryos in the uterus and were compared with UF obtained after artificial insemination. Prostaglandin (PG) F₂α and PGE₂ concentrations were also analysed in blood plasma. The four proteins analysed and hexoses were unaffected by the presence of one or more embryos in the uterus. However, blood PGF₂α showed similar, significant increases with one or more embryos over cyclic animals; such changes were not observed in blood PGE₂. Although multiple embryo transfer may appear to be non-physiological, we showed that the uterus, at the very early embryonic stages, does exhibit physiological reactions. Multiple embryo transfer can, therefore, be used for studies of very early embryo–maternal interactions in vivo in monotocous species.     

Dysregulated cannabinoid signaling disrupts uterine receptivity for embryo implantation

The mechanisms by which synchronized embryonic development to the blastocyst stage, preparation of the uterus for the receptive state, and reciprocal embryo-uterine interactions for implantation are coordinated are still unclear. We show in this study that preimplantation embryo development became asynchronous in mice that are deficient in brain-type (CB1) and/or spleen-type (CB2) cannabinoid receptor genes. Furthermore, whereas the levels of uterine anandamide (endocannabinoid) and blastocyst CB1 are coordinately down-regulated with the onset of uterine receptivity and blastocyst activation prior to implantation, these levels remained high in the nonreceptive uterus and in dormant blastocysts during delayed implantation and in pregnant, leukemia inhibitory factor (LIF)-deficient mice with implantation failure. These results suggest that a tight regulation of endocannabinoid signaling is important for synchronizing embryo development with uterine receptivity for implantation. Indeed this is consistent with our finding that while an experimentally induced, sustained level of an exogenously administered, natural cannabinoid inhibited implantation in wild-type mice, it failed to do so in CB1(-/-)/CB2(-/-) double mutant mice. The present study is clinically important because of the widely debated medicinal use of cannabinoids and their reported adverse effects on pregnancy.     

Survival of equine embryos transferred to normal and subfertile mares

To test the hypothesis that an abnormal uterine environment was a cause of early embryonic loss in subfertile mares, morphologically normal embryos were transferred to normal mares (n = 20) and subfertile mares (n = 20), and embryo survival rates were compared. Embryos were recovered nonsurgically at Days 7 to 8 postovulation and transferred surgically to normal and subfertile mares that had ovulated on the same day or within 2 d after a donor. Survival of transferred embryos was monitored by ultrasonography of the recipient mare's uterus from Day 9 through Day 28 postovulation. There were no significant differences (P > 0.5) in the embryo survival rates at Day 12 (11 20 vs 9 20 ) or Day 28 (10 20 vs 8 20 ) for normal or subfertile mares, respectively. The uterine environment of subfertile mares was apparently adequate to support the development of transferred embryos from Days 7 or 8 through Day 28 postovulation.     

The position effect in mice on day 14

Previous publications have produced conflicting results concerning the effect and nature of uterine horn position on embryonic weight and development in the uterus bicornis. For this investigation we used 503 embryos from 41 litters of NMRI - mice on gestational day 14. The relative position of each embryo was determined by measuring the distance from the center of each placenta to the cervix in relation to the total length of the uterine horn. After cessation of shrinkage due to fixation the umbilical cord and remaining membranes were removed under the dissecting microscope and the embryos were weighed to the nearest 0.1 mg. Analysis of potential embryonic positions along the uterine horns indicated that 10% at the extreme ovarian and cervical ends were almost devoid of implantations. A mixed nested analysis of variance was used as the appropriate model to test a position on weight effect. Not only was a position effect found but, in contrast to other studies, it is also quantitatively represented. Embryos were lighter both at the ovarian and cervical ends, the mean weight difference being in the range of less than 10% of the average embryo weight.     

Differential effects of p-nitrophenyl-D-xylosides on mouse blastocysts and uterine epithelial cells

A number of studies have implicated glycoconjugates in cell recognition events associated with implantation of mammalian blastocysts into the uterus. We have found that p-nitrophenyl-D-xylosides inhibit mouse embryo attachment and outgrowth on monolayers of uterine epithelial cells when cocultured in vitro. Inhibition of attachment and trophoblast formation by alpha- and beta-xylosides was observed in embryos cultured on tissue culture plastic in serum containing medium or on monolayers of epithelial cells. The biochemical basis for this inhibition has been investigated. Consistent with their accepted mode of action, beta- but not alpha-D-xylosides greatly stimulated glycosaminoglycan chain production by uterine epithelial cells and likewise reduced proteoglycan assembly. In contrast, both alpha- and beta-anomers selectively inhibited embryo attachment and outgrowth without stimulating glycosaminoglycan chain production by embryos. The inhibitory effect of the xylosides on embryos was reversible and did not require concentrations that reduced the rate of protein synthesis. Both alpha- and beta-D-xylosides inhibited the synthesis of proteoglycans including heparan sulfate as well as certain other glycoconjugates by embryos. Collectively, these data indicate that proper assembly of glycoconjugates, including proteoglycans, is required for implantation-related processes, although the inhibition of embryo outgrowth by xylosides may be by an as yet uncharacterized mechanism.     

In vitro development of bovine morulae in bovine serum albumin, normal steer serum and uterine flushings

One hundred fifty-three excellent and good bovine morulae were cultured in Ham's F-10, supplemented with 10 % steer serum, bovine serum albumin, or uterine flushings (64 mg protein/ml) to compare embryo development. Embryos were observed every 12 h in culture. Treatment differences were evaluated by assigning a numerical value of 0 to 4 to each embryo representing the stage of development reached in vitro. The final morphological developmental score of the embryos was comparable for steer serum (2.66) and bovine serum albumin (2.50), but it differed significantly for uterine flushings obtained from ovariectomized, steroid-supplemented cows (< 0.1) or heat-treated uterine flushings (0.07). Since albumin alone was able to support development, it suggests that the albumin component of steer serum may be responsible for the observed development. Uterine fluids were unable to support growth of embryos, suggesting that incompatibility may be due to asynchrony between the early bovine embryo and uterine constituents, or a concentration of uterine components may exacerbate actions of inhibitory substances.     

Placental Type Interactions and Evolutionary Trends of Development of Uterus in Cestodes

In Proteocephalus thymalli and P. torulosus, a contact of the placental type in uterus was shown to be formed at two different levels. At the first level an interaction occurs between outgrowths of uterine epithelium and thin capsule of embryos closely adjacent to uterine wall. The next level is formation of contact between neighboring egg capsules, which allows distributing nutrients among fetuses present in the uterine cavity. Placental interactions in Clestobothrium acheilognathi are limited in time and space. First, a relatively small number of eggs are involved in interaction of the placental type in the uterine sac, while uterine duct is filled with freely lying eggs. Second, the closest contact is observed in eggs with non-sclerosed egg shell. One of the main evolutionary tendencies in cestodes has been shown to be a modification of uterus for formation of close interrelations with embryonic membranes in the course of transition from the extrauterine to the intrauterine type of embryonic development. Uterus in parasites with a polylecital type of the egg is suggested to serve to the greater extent as a reservoir, whereas in cestodes with oligolecital eggs, uterus performs its direct function—supply of developing embryos with nutrients. As a result, modifications of uterine epithelium are formed: from the appearance of the placental type interactions formed repeatedly in phylogenetically distant groups of cestodes to formation of branched outgrowths separating the uterine space into units or disintegration to actively functioning uterine capsules.     

Immunocytochemical analysis of the association of bovine oviduct-specific glycoproteins with early embryos.

The bovine oviductal epithelium synthesizes and secretes a class of oviduct-specific glycoproteins that is present in the luminal fluid when fertilization and early embryonic development occur. The objective of this study was to determine if these characterized glycoproteins become associated with oviductal embryos. Ovarian ova and oviductal embryos were recovered from super-ovulated cows at 72 h after onset of estrus. Eggs were fixed in 3% paraformaldehyde-1% glutaraldehyde and subsequently embedded in Lowicryl K4M. Sections (1 micron) were processed for peroxidase-antiperoxidase immunocytochemistry. Immunolabeling was not detected in any region of ovarian ova. Oviductal embryos, regardless of cleavage stage, exhibited immunoperoxidase staining localized within their zona pellucidae. Sections (100 nm) obtained from a 4- and an 8-cell embryo were also subjected to colloidal gold immunoelectron microscopy to determine conclusively the subcellular distribution of the oviduct-specific glycoproteins. Gold particles were distributed uniformly throughout the width of the zona pellucida. Also, immunoreactivity was observed associated with flocculent material in the perivitelline space and with the vitelline membrane. These results indicate that the bovine oviduct-specific secretory glycoproteins become associated with oviductal embryos. This association may be biologically important to the developing embryo.     

Role of progesterone on peri-implantation stage endometrium-embryo interaction in the primate

Progesterone secretion during the luteal phase influences oviductal and endometrial functions which are essential for embryo viability and implantation in a number of species including primates. Luteal phase estrogen is not essential for progesterone-dependent endometrial receptivity towards implantation and pregnancy in the rhesus monkey and in the human. However, synchronous development of embryo and endometrium is an essential prerequisite for evolutive implantation. Progesterone helps to maintain synchronous development of preimplantation embryo through its action on maternal uterus. The anti-nidatory action of mifepristone, a potent progesterone receptor modulator (PRM) with pronounced antiprogestagenic activity, is known to be associated with desynchronization of endometrium along with repression of glandular secretory differentiation and vascular maturation. Thus, it is likely that early luteal phase administration of mifepristone affects paracrine action of the secretory stage endometrium on the preimplantation stage embryo, and thereby inhibits embryonic development and viability. We shall examine this hypothesis using the rhesus monkey as a primate model.     

Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects of epigenetic alterations originated during preimplantation development on subsequent fetal development and adult health. The use of an effective and consistent embryo transfer technique is crucial to enhance the generation of genetically modified animals and to determine the effect of different treatments on implantation rates and survival to term. Embryos at the blastocyst stage are usually transferred by uterine transfer, performing a puncture in the uterine wall to introduce the embryo manipulation pipette. The orifice performed in the uterus does not close after the pipette has been withdrawn, and the embryos can outflow to the abdominal cavity due to the positive pressure of the uterus. The puncture can also produce a hemorrhage that impairs implantation, blocks the transfer pipette and may affect embryo development, especially when embryos without zona are transferred. Consequently, this technique often results in very variable and overall low embryo survival rates. Avoiding these negative effects, utero-tubal embryo transfer take advantage of the utero-tubal junction as a natural barrier that impedes embryo outflow and avoid the puncture of the uterine wall. Vasectomized males are required for obtaining pseudopregnant recipients. A technique to perform vasectomy is described as a complement to the utero-tubal embryo transfer.