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L-Proline (proline) in amniotic fluid was markedly increased during pregnancy in both pigs and sheep. However, in vivo data to support a beneficial effect of proline on fetal survival are not available. In this study, pregnant C57BL/6J mice were fed a purified diet supplemented with or without 0.50% proline from embryonic day 0.5 (E0.5) to E12.5 or term. Results indicated that dietary supplementation with proline to gestating mice enhanced fetal survival, reproductive performance, the concentrations of proline, arginine, aspartic acid, and tryptophan in plasma and amniotic fluid, while decreasing the concentrations of ammonia and urea in plasma and amniotic fluid. Placental mRNA levels for amino acid transporters, including Slc36a4, Slc38a2, Slc38a4, Slc6a14, and Na+/K+ ATPase subunit-1α (Atp1a1), fatty acid transporter Slc27a4, and glucose transporters Slc2a1 and Slc2a3, were augmented in proline-supplemented mice, compared with the control group. Histological analysis showed that proline supplementation enhanced labyrinth zone in the placenta of mice at E12.5, mRNA levels for Vegf, Vegfr, Nos2, and Nos3, compared with the controls. Western blot analysis showed that proline supplementation increased protein abundances of phosphorylated (p)-mTORC1, p-ribosomal protein S6 kinase (p70S6K), and p-eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), as well as the protein level of GCN2 (a negative regulator of mTORC1 signaling). Collectively, our results indicate a novel functional role of proline in improving placental development and fetal survival by enhancing placental nutrient transport, angiogenesis, and protein synthesis.

Masanaga Muto, Yoshitaka Fujihara, Tomohiro Tobita, Daiji Kiyozumi, Masahito Ikawa, Lentiviral Vector-Mediated Complementation Restored Fetal Viability but Not Placental Hyperplasia in Plac1-Deficient Mice, Biology of Reproduction, Volume 94, Issue 1, 1 January 2016, 6, 1–9, https://doi.org/10.1095/biolreprod.115.133454, The X-linked Plac1 gene is maternally expressed in trophoblast cells during placentation, and its disruption causes placental hyperplasia and intrauterine growth restriction. In contrast, Plac1 is also reported to be one of the upregulated genes in the hyperplastic placenta generated by nuclear transfer. However, the effect of overexpressed Plac1 on placental formation and function remained unaddressed. We complemented the Plac1 knockout placental dysfunction by lentiviral vector-mediated, placenta-specific Plac1 transgene expression. Whereas fetal development and the morphology of maternal blood sinuses in the labyrinth zone improved, placental hyperplasia remained, with an expanded the junctional zone that migrated and encroached into the labyrinth zone. Further experiments revealed that wild-type placenta with transgenically expressed Plac1 resulted in placental hyperplasia without the encroaching of the junctional zone. Our findings suggest that Plac1 is involved in trophoblast cell proliferation, differentiation, and migration. Its proper expression is required for normal placentation and fetal development.

The X-linked Plac1 gene is maternally expressed in trophoblast cells during placentation, and its disruption causes placental hyperplasia and intrauterine growth restriction. In contrast, Plac1 is also reported to be one of the upregulated genes in the hyperplastic placenta generated by nuclear transfer. However, the effect of overexpressed Plac1 on placental formation and function remained unaddressed. We complemented the Plac1 knockout placental dysfunction by lentiviral vector-mediated, placenta-specific Plac1 transgene expression. Whereas fetal development and the morphology of maternal blood sinuses in the labyrinth zone improved, placental hyperplasia remained, with an expanded the junctional zone that migrated and encroached into the labyrinth zone. Further experiments revealed that wild-type placenta with transgenically expressed Plac1 resulted in placental hyperplasia without the encroaching of the junctional zone. Our findings suggest that Plac1 is involved in trophoblast cell proliferation, differentiation, and migration. Its proper expression is required for normal placentation and fetal development.

The cloning of cattle by somatic cell nuclear transfer (NT) is associated with a high incidence of abnormal placentation, excessive fluid accumulation in the fetal sacs (hydrops syndrome), and fetal overgrowth. Fetal and placental development was investigated at Day 50, during placentome formation; at Day 100, when placentation was completed; and at Day 150, when the hydrops syndrome frequently develops. The NT fetuses were compared with contemporary half-siblings generated from in vitro-produced embryos or by artificial insemination (AI). Fetal cotyledon formation and vascularization of the chorioallantoic membranes was initiated normally in NT conceptuses, but fewer cotyledons successfully formed placentomes. By Day 100, the mean number of placentomes was significantly lower in surviving NT fetuses. Only those with normal placentome numbers were represented in surviving NT pregnancies at Day 150. The mean total caruncle tissue weight of the placentomes was significantly higher in the surviving NT groups at Days 100 and 150, irrespective of the placentome numbers, indicating that increased NT placental weight was caused by excessive uterine tissue growth. By Day 100, NT fetuses exhibited growth deregulation, and those that survived to Day 150 were 17% heavier than contemporary AI controls. Placentome, liver, and kidney overgrowth accompanied the hydrops syndrome at Day 150. The NT fetal overgrowth was not a consequence of in vitro embryo culture and showed no correlation with placental overgrowth. However, in vitro culture and incomplete reprogramming of the donor genome are epigenetic effects that may override genetic traits and contribute to the greater variability in placental and fetal development in the NT group compared with AI half-siblings.

Leukemia inhibitory factor (LIF) enhances in vitro murine preimplantation development in a time- and dose-dependent fashion. Knockout experiments have demonstrated that endometrial LIF is essential for in vivo murine implantation. We assessed the impact of LIF and an anti-LIF polyclonal antibody (pab) on in vivo development and developed a novel and successful nonsurgical method of embryo transfer for this species, a transcervical blastocyst transfer technique. The objectives of this study were to evaluate the effects of LIF and the anti-LIF pab on 1) implantation, resorption, pregnancy, and viability rates and 2) the overall structural and skeletal development. Two-cell embryos were recovered from superovulated mated donors, cultured to the expanded blastocyst stage, and transferred transcervically into pseudopregnant recipients. Exposure to 5000 U/ml LIF resulted in significant increases in implantation, pregnancy, and viability rates compared with controls. A similar dose of pab produced overall inhibitory effects with a significant decrease in implantation rate. Paradoxically, lower pab doses resulted in significantly increased viability rates. Exposure to LIF had no effect on fetoplacental development. However, pab treatments had variable but significant negative effects on placental length, ossification of the exoccipital bone, and vertebral space width compared with controls. Exposure of murine blastocysts to LIF at the time of transcervical transfer resulted in pronounced positive effects on implantation and pregnancy rates without affecting fetal development. A similar pab dose dramatically reduced implantation and pregnancy rates; at high and low doses, pab produced deleterious effects on placental and skeletal development.

The production of cloned animals is, at present, an inefficient process. This study focused on the fetal losses that occur between Days 30-90 of gestation. Fetal and placental characteristics were studied from Days 30-90 of gestation using transrectal ultrasonography, maternal pregnancy specific protein b (PSPb) levels, and postslaughter collection of fetal tissue. Pregnancy rates at Day 30 were similar for recipient cows carrying nuclear transfer (NT) and control embryos (45% [54/120] vs. 58% [11/19]), although multiple NT embryos were often transferred into recipients. From Days 30-90, 82% of NT fetuses died, whereas all control pregnancies remained viable. Crown-rump (CR) length was less in those fetuses that were destined to die before Day 90, but no significant difference was found between the CR lengths of NT and control fetuses that survived to Day 90. Maternal PSPb levels at Days 30 and 50 of gestation were not predictive of fetal survival to Day 90. The placentas of six cloned and four control (in vivo or in vitro fertilized) bovine pregnancies were compared between Days 35 and 60 of gestation. Two cloned placentas showed rudimentary development, as indicated by flat, cuboidal trophoblastic epithelium and reduced vascularization, whereas two others possessed a reduced number of barely discernable cotyledonary areas. The remaining two cloned placentas were similar to the controls, although one contained hemorrhagic cotyledons. Poor viability of cloned fetuses during Days 35-60 was associated with either rudimentary or marginal chorioallantoic development. Our findings suggest that future research should focus on factors that promote placental and vascular growth and on fetomaternal interactions that promote placental attachment and villous formation.

Previous studies have demonstrated that during parturition the physiological ripening that permits dilation of the cervical canal is due to a widespread collagenolysis that follows a heavy polymorphonuclear leukocyte invasion of the uterine cervix. The aim of this study was to investigate whether there is any association between the ovarian steroid hormones involved in rat parturition and this phenomenon. Pregnant or pseudopregnant rats were ovariectomized (OVX) at Day 9 and then given a hormonal treatment (sufficient to maintain fetal viability) until Day 23. Cervical biopsies, taken from animals killed intrapartum or 1 h before expected parturition, were studied for eosinophilic infiltration and collagen birefringence. Intrapartum or sham-OVX pregnant rats showed a massive eosinophilic infiltration and a widespread collagenolysis as indicated by a loss of collagen birefringence. Ovariectomized pregnant rats treated with estrogen plus progesterone or with progesterone alone showed neither infiltration nor collagenolysis. In rats OVX during pseudopregnancy, estrogen given alone induced a significant infiltration of eosinophils in the cervical stroma ; however, treatment with the combination of estrogen and progesterone was not able to promote eosinophilic infiltration. Collagenolysis was absent in all pseudopregnant animals. These results show that estrogen induced a cervical eosinophilic infiltration in rats but that when progesterone was added to the estrogen treatment the infiltration was not present ; in addition, none of the steroid hormones assessed were responsible for the collagenolysis found in the cervical tissue at term.

The importance of placental circulation is exemplified by the correlation of placental size and blood flow with fetal weight and survival during normal and compromised human pregnancies in such conditions as preeclampsia and intrauterine growth restriction (IUGR). Using noninvasive magnetic resonance imaging, we evaluated the role of PKBalpha/AKT1, a major mediator of angiogenesis, on placental vascular function. PKBalpha/AKT1 deficiency reduced maternal blood volume fraction without affecting the integrity of the fetomaternal blood barrier. In addition to angiogenesis, PKBalpha/AKT1 regulates additional processes related to survival and growth. In accordance with reports in adult mice, we demonstrated a role for PKBalpha/AKT1 in regulating chondrocyte organization in fetal long bones. Using tetraploid complementation experiments with PKBalpha/AKT1-expressing placentas, we found that although placental PKBalpha/AKT1 restored fetal survival, fetal PKBalpha/AKT1 regulated fetal size, because tetraploid complementation did not prevent intrauterine growth retardation. Histological examination of rescued fetuses showed reduced liver blood vessel and renal glomeruli capillary density in PKBalpha/Akt1 null fetuses, both of which were restored by tetraploid complementation. However, bone development was still impaired in tetraploid-rescued PKBalpha/Akt1 null fetuses. Although PKBalpha/AKT1-expressing placentas restored chondrocyte cell number in the hypertrophic layer of humeri, fetal PKBalpha/AKT1 was found to be necessary for chondrocyte columnar organization. Remarkably, a dose-dependent phenotype was exhibited for PKBalpha/AKT1 when examining PKBalpha/Akt1 heterozygous fetuses as well as those complemented by tetraploid placentas. The differential role of PKBalpha/AKT1 on mouse fetal survival and growth may shed light on its roles in human IUGR.

The nutrient requirements and metabolic pathways used by the developing embryo transition from predominantly pyruvate during early cleavage stages to glucose at the blastocyst; however, the complexities involved in the regulation of metabolism at different developmental stages are not clear. The aims of this study were to examine the role of the malate-aspartate shuttle (MAS) in nutrient metabolism pathways in the developing mouse blastocyst and the consequences of impaired metabolism on embryo viability and fetal and placental growth. Eight-cell-stage mouse embryos were cultured in the presence of the MAS inhibitor amino-oxyacetate, with or without pyruvate as an energy substrate in the media. When the MAS was inhibited, the rate of glycolysis and lactate production was significantly elevated and glucose uptake reduced, relative to control cultured embryos in the presence of pyruvate. Despite these changes in embryo metabolism, this did not influence development to the blastocyst stage, but it did reduce the number of inner cell mass and trophectoderm cells. When these embryos were transferred to psuedopregnant females, inhibition of the MAS significantly reduced the proportion of embryos that implanted and developed into fetuses on Day 18 of pregnancy. Finally, fetal growth was reduced while placental weight was maintained, leading to a decreased fetal:placental weight ratio relative to control embryos. These results suggest that impaired metabolism of glucose in the blastocyst via the MAS alters the ability of the embryos to implant and form a pregnancy and leads to reduced fetal weight, likely via altered placental development and function.

The presence of ammonium in culture medium has a detrimental effect on embryo physiology and biochemistry; however, the stage at which the embryo is most sensitive to this effect is unknown. The aim of this study was to determine the exact stage at which the embryo is most vulnerable to ammonium by exposing the preimplantation embryo to 300 muM ammonium either at the precompaction stage (between the zygote and two-cell or the two-cell to eight-cell) or at the postcompaction stage (between the eight-cell and blastocyst). This study determined that exposure of embryos to ammonium at the precompaction stage from either the zygote to two-cell stage or from the two-cell to the eight-cell stage did not affect the rate of development to the blastocyst stage; however, the resultant blastocysts had decreased cell numbers and inner cell mass cells. Furthermore, these blastocysts had increased levels of cellular apoptosis and perturbed levels of Slc2a3 expression and glucose uptake. Transfer of these blastocysts revealed that, while implantation was not affected, the number of fetuses was reduced by culture with ammonium at the precompaction stage and fetal development was delayed, as observed by reduced crown-rump length and maturity. In contrast, the later stage embryo was more resistant to the negative effects of ammonium, with only Slc2a3 expression and fetal maturity affected. This raises the possibility that the later stage embryo is more able to protect itself from in vitro-derived stress and that the majority of in vitro-induced damage to mouse embryos is inflicted at the early stages of development.

The cloning of cattle by somatic cell nuclear transfer (NT) is associated with a high incidence of abnormal placentation, excessive fluid accumulation in the fetal sacs (hydrops syndrome), and fetal overgrowth. Fetal and placental development was investigated at Day 50, during placentome formation; at Day 100, when placentation was completed; and at Day 150, when the hydrops syndrome frequently develops. The NT fetuses were compared with contemporary half-siblings generated from in vitro-produced embryos or by artificial insemination (AI). Fetal cotyledon formation and vascularization of the chorioallantoic membranes was initiated normally in NT conceptuses, but fewer cotyledons successfully formed placentomes. By Day 100, the mean number of placentomes was significantly lower in surviving NT fetuses. Only those with normal placentome numbers were represented in surviving NT pregnancies at Day 150. The mean total caruncle tissue weight of the placentomes was significantly higher in the surviving NT groups at Days 100 and 150, irrespective of the placentome numbers, indicating that increased NT placental weight was caused by excessive uterine tissue growth. By Day 100, NT fetuses exhibited growth deregulation, and those that survived to Day 150 were 17% heavier than contemporary AI controls. Placentome, liver, and kidney overgrowth accompanied the hydrops syndrome at Day 150. The NT fetal overgrowth was not a consequence of in vitro embryo culture and showed no correlation with placental overgrowth. However, in vitro culture and incomplete reprogramming of the donor genome are epigenetic effects that may override genetic traits and contribute to the greater variability in placental and fetal development in the NT group compared with AI half-siblings.

To gain a better understanding of global methylation differences associated with development of nuclear transfer (NT)-generated cattle, we analyzed the genome-wide methylation status of spontaneously aborted cloned fetuses, cloned fetuses, and adult clones that were derived from transgenic and nontransgenic cumulus, genital ridge, and body cell lines. Cloned fetuses were recovered from ongoing normal pregnancies and were morphologically normal. Fetuses generated by artificial insemination (AI) were used as controls. In vitro fertilization (IVF) fetuses were compared with AI controls to assess effects of in vitro culture on the 5-methylcytosine content of fetal genomes. All of the fetuses were female. Skin biopsies were obtained from cloned and AI-generated adult cows. All of the adult clones were phenotypically normal and lactating and had no history of health or reproductive disorders. Genome-wide cytosine methylation levels were monitored by reverse-phase HPLC, and results indicated reduced levels of methylated cytosine in NT-generated fetuses. In contrast, no differences were observed between adult, lactating clones and similarly aged lactating cows produced by AI. These data imply that survivability of cloned cattle may be closely related to the global DNA methylation status. This is the first report to indicate that global methylation losses may contribute to the developmental failure of cloned bovine fetuses.

Cryopreservation of sheep embryos with ethylene glycol as a protectant appears to be more effective than glycerol, particularly at the morula stage, as has been demonstrated on the basis of in vitro and in vivo development rates after thawing. In this study we compare the ultrastructure of fresh morulae, thawed morulae, and blastocysts cryopreserved with either ethylene glycol or glycerol at the electron microscopic level, to look for cellular damage that could be responsible for proven differences in embryo survival after transfer. Embryos cryopreserved with glycerol showed unequal degrees of conservation even among blastomeres within a single embryo. In morulae, inner blastomeres were completely damaged, whereas external ones appeared to be intact. Both morulae and blastocysts cryopreserved with ethylene glycol showed a higher uniformity in blastomere conservation than embryos with glycerol. The most remarkable features in this experimental group were the presence of desmosomes following tight junctions between blastomeres and the presence of many microvilli on the outer surface of external blastomeres. These characteristics are similar in fresh embryos of the control group. Our results show that ethylene glycol protects membrane and cytoplasmic structures of embryonic cells from cryoinjury much better than glycerol. In vivo survival of embryos confirmed the ultrastructural observations. A limited permeability of glycerol would explain the observed ultrastructural differences in blastomere integrity, which depends on blastomere location and the differences between morulae and blastocysts. We conclude that the low reproductive yield after cryopreservation using glycerol can be attributed to the lack of protection of inner cells.

The production of cloned animals is, at present, an inefficient process. This study focused on the fetal losses that occur between Days 30-90 of gestation. Fetal and placental characteristics were studied from Days 30-90 of gestation using transrectal ultrasonography, maternal pregnancy specific protein b (PSPb) levels, and postslaughter collection of fetal tissue. Pregnancy rates at Day 30 were similar for recipient cows carrying nuclear transfer (NT) and control embryos (45% [54/120] vs. 58% [11/19]), although multiple NT embryos were often transferred into recipients. From Days 30-90, 82% of NT fetuses died, whereas all control pregnancies remained viable. Crown-rump (CR) length was less in those fetuses that were destined to die before Day 90, but no significant difference was found between the CR lengths of NT and control fetuses that survived to Day 90. Maternal PSPb levels at Days 30 and 50 of gestation were not predictive of fetal survival to Day 90. The placentas of six cloned and four control (in vivo or in vitro fertilized) bovine pregnancies were compared between Days 35 and 60 of gestation. Two cloned placentas showed rudimentary development, as indicated by flat, cuboidal trophoblastic epithelium and reduced vascularization, whereas two others possessed a reduced number of barely discernable cotyledonary areas. The remaining two cloned placentas were similar to the controls, although one contained hemorrhagic cotyledons. Poor viability of cloned fetuses during Days 35-60 was associated with either rudimentary or marginal chorioallantoic development. Our findings suggest that future research should focus on factors that promote placental and vascular growth and on fetomaternal interactions that promote placental attachment and villous formation.

After compaction, the preimplantation mouse embryo switches to a glucose-based metabolism, whereas for the 2- to 4-cell stage embryo, glucose can be inhibitory. In this study, we investigated the adaptability of preimplantation embryos to different periods of glucose starvation by culturing in vitro fertilized (IVF) and in vivo-fertilized 1-cell OF1 mouse embryos. Blastocysts obtained from exposure to glucose starvation for different periods of time were examined for the number of cells in the trophectoderm and inner cell mass, and for glycolytic activity and viability. A high percentage of blastocysts was obtained when 1-cell embryos fertilized in vitro or in vivo were cultured in M16 until the 2-cell stage, were transferred to M16 without glucose (M16-G) until the 4- or 8-cell stage, and then were transferred to fresh M16-G. When in vivo-fertilized 1-cell embryos were cultured to the 2-cell stage and then left in M16, less than 5% formed blastocysts compared to 26% of those transferred into M16-G. Blastocysts obtained when in vivo-fertilized 1-cell embryos were left in M16-G after the 2-cell stage, however, showed a significantly elevated glycolytic activity compared to those transferred to fresh M16 or M16-G medium at the 4- or 8-cell stage. Interestingly, even though these embryos displayed elevated glycolytic activity, they did not exhibit differences in the numbers of inner cell mass and trophectoderm cells or in viability compared to embryos cultured according to other protocols. Blastocysts from all cultured protocols had a significantly lower total cell number and a lower trophectoderm, but not inner cell mass, cell number compared to blastocysts developed in vivo. This study documents the metabolic adaptability of the preimplantation embryo by highlighting its ability to proceed with development and retain viability when challenged with glucose starvation at different periods.

The objectives of the present experiment were to compare survival after transfer of bovine embryos produced in vivo with those produced in vitro and to examine the physical characteristics of fetuses produced from these transfers. Embryos produced in vivo (Holstein x Angus) were recovered from uterine flushings of superovulated heifers 7 days after first artificial insemination, and embryos produced in vitro (Holstein x beef breeds) were collected 7 days after insemination. Embryos were paired by source (in vivo, in vitro), stage (compact morula, blastocyst), and quality grade (excellent = 1, good = 2), and transferred nonsurgically to recipient heifers on Day 7 (+/- 1 day) of the estrous cycle. Pregnancy status was monitored by determination of serum progesterone concentrations, ultrasonography, and palpation through 7 mo of gestation, at which time fetuses were recovered. In comparison with grade 1 embryos produced in vivo, the risk of embryonic death after transfer was similar for grade 2 embryos produced in vivo (p = 0.56) and for grade 1 embryos produced in vitro (p = 0.88). By contrast, grade 2 embryos produced in vitro were at greater (p = 0.04) risk of embryonic death. Embryo loss was associated (p = 0.01) with increased serum concentrations of progesterone in recipients at the time of transfer. At 7 mo of gestation, fetuses from embryos produced in vitro were heavier (p = 0.02) than fetuses from embryos produced in vivo and had skeletal measurements that were disproportionate (por = 0.04) to body weight.

In vivo embryogenesis, embryo migration, and survival were studied in the domestic cat. Queens were naturally mated (three times daily) on the second and third days of behavioral estrus and, if ovulation occurred, ovariohysterectomized at 64 (n = 8), 76 (n = 11), 100 (n = 8), 124 (n = 7), 148 (n = 6), and 480 h (n = 8) after first copulation. Of 52 cats mated, 48 (92.3%) ovulated (as evidenced by the presence of ovarian CL), and of these, 38 (79.2%) either produced good-quality embryos or had implantation sites. From the remaining cats, only unfertilized oocytes (n = 5), degenerating embryos (n = 4), or no oocytes/embryos (n = 1) were recovered. Embryos at 64, 76, 100, and 124 h after the first copulation typically were 1 to 4 cells (17 of 20; 85.0%), 5 to 8 cells (18 of 28; 64.3%), 9 to 16 cells (14 of 24; 58.3%), and morulae (15 of 21; 71.4%), respectively; all were within the oviducts. At 148 h, embryos primarily were compact morulae or early blastocysts (15 of 18; 83.3%), and all were within the uterus. For the preimplantation groups, the overall recovery of embryos plus oocytes per CL was 80.6%, and the mean (+/- SEM) numbers of CL and embryos were 64 h, 4.8 +/- 0.3, 3.1 +/- 0.8; 76 h, 4.7 +/- 0.3, 3.9 +/- 0.6; 100 h, 5.8 +/- 0.5, 3.3 +/- 0.8; 124 h, 4.4 +/- 0.5, 4.0 +/- 0.6; and 148 h, 6.5 +/- 1.1, 3.7 +/- 0.7, respectively. Cats in the 480-h group produced a mean of 5.6 +/- 0.5 CL and 3.9 +/- 0.5 implantation sites. In six of eight cats in this group, there was a disparity between CL number on a given ovary and number of implantation sites in the ipsilateral horn, supporting the concept of transuterine embryo migration. In summary, results indicated that 1) more than 90% of cats ovulated following this multiple mating regimen, but approximately 21% of these failed to produce any fertilized or viable embryos; 2) embryo developmental rate in vivo was biphasic, with a rapid cleavage rate to the 5- to 8-cell stage followed by a slower cleavage rate to the morula stage; 3) cat embryos entered the uterus as compact morulae or early blastocysts approximately 5.5 days after the first copulation; and 4) on the basis of implantation/CL ratio, approximately 30% of all ovulated cat oocytes underwent either fertilization failure or preimplantation embryonic mortality.

In this study we examined the role of glucose, and the use of fructose as a replacement, in embryo culture medium. Three embryo culture media were used: a routine embryo culture medium (M16), M16 without glucose (M16-G) and M16-G supplemented with fructose (M16F-G). Their effect on fertilization, rate of cleavage, and embryo viability were examined in both an outbred (OF1) and an inbred (C57Bl) mouse strain. Of the three media, only M16 was found to support fertilization. In vitro-fertilized embryos from OF1 oocytes and C57Bl oocytes and OF1 sperm were placed in the different media at the early 2-cell stage. In 65% of OF1 embryos cultured in M16, development was blocked at the 2-cell stage, whereas in M16-G and M16F-G embryos, only 22% and 32%, respectively, were blocked. M16F-G medium also produced morulae and blastocysts with higher cell numbers than M16-G. In vitro-fertilized C57Bl 2-cell embryos cultured in M16 displayed retarded cleavage to the 4-cell stage compared to embryos cultured in M16-G and M16F-G. In contrast, the morulae and blastocyst cell numbers were significantly lower in M16-G compared to M16 and M16F-G. The viability of morulae and blastocysts obtained from OF1 and C57Bl embryos cultured in M16-G and M16F-G was lower compared to control C57Bl morulae and blastocysts cultured in M16. The results show that although morphologically normal embryos could be obtained in M16-G and M16F-G, inherent anomalies existed that limited viability.

This study was conducted to determine whether the tolerance of embryos to the stress of freezing and thawing can be modified by including in the incubation medium stimulators or inhibitors of membrane lipid peroxidation. Mouse zygotes were cultured in medium M16, supplemented or not with FeSO4, apotransferrin, and/or ascorbate. In each culture supplement, 8-cell embryos were randomly allocated to an untreated (nonfrozen) control group, or treatment by freezing using slow or ultra-rapid cooling. In the slow-frozen group, FeSO4 (49.3%, 66 of 134), decreased embryo survival, whereas apotransferrin (82.7%, 110 of 133) and ascorbate (86.4%, 114 of 132), increased significantly the percentage of intact embryos recovered after thawing compared to those cultured in the basic medium M16 (66.9%, 99 of 148). Apotransferrin and ascorbate also increased significantly the percentage of blastocysts on Day 5 (79.7%, 106 of 133, and 89.4%, 118 of 132 vs. 62.2%, 92 of 148, respectively). Ascorbate, in addition, increased significantly the percentage of implantations compared to those in the basic medium M16 (84.5%, 60 of 71 vs. 61.1%, 37 of 56). Changes in the ultra-rapidly frozen group were not so evident. However, a significant fetal wastage after implantation was observed when embryos were cultured without additives and then ultra-rapidly frozen (31.7%, 13 of 41 vs. 7.0%, 3 of 43, in the nonfrozen control group). Fetal weight was similar between culture conditions, but it was significantly lower in slow-frozen and ultra-rapidly frozen embryos than in the nonfrozen control group.(ABSTRACT TRUNCATED AT 250 WORDS)

A study was conducted to determine whether embryos recovered from first-estrous (pubertal) and second-estrous gilts differed in survival when transferred to first- or third-estrous recipients. Embryos were recovered surgically from first- and second-estrous donors 48-72 h postmating and 6-10 normal embryos/zygotes (1-4 cells) were transferred to oviducts (3-5 embryos/ampulla) of nonmated synchronous first- (n = 40) or third- (n = 15) estrous recipients. Blood samples were collected from the jugular vein of recipient gilts on Days 3, 12, and 30 of gestation and the sera were analyzed for progesterone and free (unconjugated) estrogens by use of radioimmunoassays. Recipient gilts were subsequently slaughtered between Days 30 and 40 to assess embryonic losses. Mean number of ovulations was lower among first-estrous vs. third-estrous recipients (8.9 +/- 0.7 vs. 11.4 +/- 0.7; p0.05). Percentage of recipients that maintained pregnancy was similar between first- and third-estrous gilts (67.5 vs. 60.0%) and recovery of total conceptuses (normal and degenerating) resulting from transfer of one-cell- and cleavage-stage embryos did not differ among first- vs. third-estrous gilts (76.1 vs. 78.2%). Similarly, percentage of viable fetuses in first-estrous gilts that were pregnant from transfer of one-cell- and cleavage-stage embryos was not different from that of third-estrous gilts (69.3 vs. 75.6%). Percentages of total conceptuses and viable fetuses in first- and third-estrous gilts that were recipients of cleavage-stage embryos only also did not differ (p0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Embryos were collected from ewes on Day 6 after estrus (Day 0 = estrus), placed in M2 culture medium, and assigned to 1 of 4 treatment groups. Some embryos were transferred to recipient ewes on Day 6 of their estrous cycle either in pairs (group 1) or singularly (group 2) within 3 h of collection. The remaining embryos were individually cultured for 48 h in an atmosphere of 5% CO2 in humidified air in either synthetic oviduct fluid (SOF) medium (group 3) or SOF containing 1,000 U/ml of recombinant human leukemia inhibitory factor (hLIF) (SOF + hLIF: group 4). These embryos were then transferred to recipient ewes on Day 8 of their estrous cycle. The addition of hLIF to culture medium significantly improved the development of the embryos compared with control embryos prior to transfer (blastocysts hatching from the zona pellucida: group 3 = 16% vs. group 4 = 64%, p less than 0.05; those degenerative: group 3 = 27% vs. group 4 = 9%, p less than 0.05) and the subsequent pregnancy rates of the recipient ewes, receiving a single embryo, at Day 70 of pregnancy (group 3 = 16% vs. group 4 = 50%, p less than 0.05). The pregnancy rate of ewes given embryos cultured for 48 h in SOF + hLIF prior to transfer (50%; group 4) was similar to the group 2 ewes receiving a single embryo soon after collection (52%), but the pregnancy rate for both groups was significantly lower than that for the group 1 ewes receiving two embryos soon after collection (89%: 53% twins, 36% singles; p less than 0.05).

Anti-inflammatory drugs were given to pregnant rabbits during the 24-h period prior to the increase in the uterine vascular permeability which occurs on Day 7. Their effect on the vascular response was monitored by quantifying the concentration of extravascular Evans blue dye. The increase in vascular permeability normally seen on Day 7 was inhibited by either indomethacin or a combination of H1-(mepyramine) and H2-(cimetidine) receptor antagonists. When given alone, neither cimetidine nor mepyramine was as effective as the combination in reducing vascular permeability. Prostaglandins and histamine may be acting together since simultaneous administration of lower doses of indomethacin and the antihistamines reduced vascular permeability below that observed following administration of either class of anti-inflammatory drugs alone. In a second experiment, anti-inflammatory drugs were administered during the peri-implantation period (Days 6-8) and their effect on the weights of maternal and fetal tissues, and on fetal viability were evaluated on Day 14 of pregnancy. Indomethacin had a more deleterious effect on both parameters than did the combination of histamine receptor antagonists. Results from these experiments suggest that both prostaglandins and histamine may participate in the uterine vascular response, whereas the overall process of implantation appears to be more dependent upon the synthesis of prostaglandins than the action of histamine.

In vitro evidence suggests that fibrinolytic activity is involved in the implantation process. In vivo experiments were performed in rats to determine if fetal development can be prevented by epsilon amino caproic acid (EACA), a fibrinolytic inhibitor. Timed pregnant rats were laparotomized on various days of pregnancy, and EACA or control injections were made directly into a uterine horn. Animals were autopsied on Day 15 of pregnancy when implantation and fetal viability rates were determined. Intraluminal EACA did not affect implantation rate, but fetal viability was adversely affected when 3 mg EACA were injected on Day 6. This effect was doserelated and was partially reversed by simultaneous injection of plasmin. No effect was observed on Day 3, 7, or 8. The effect on Days 4 and 5 could not be tested because sham injections alone caused a high resorption rate. Subcutaneous injections, as high as 60 mg daily from Days 3 to 10, did not affect fetal viability. The experiments support the hypothesis that fibrinolytic activity around the time of implantation is important for maintenance of pregnancy.

The pregnant rat has been used as a model for the testing of drugs applied directly into the uterus to determine their effects on implantation and fetal viability. Because of current interest in developing new contraceptive agents, it appeared pertinent to define the experimental model more precisely. Timed pregnant rats were laparotomized under light ether anesthesia. A sterile 25 gauge needle attached to a 1 ml tuberculin syringe was inserted into the lumen of 1 uterine horn at the cervical end. Sterile saline (0.1 ml, 0.9%) was injected into the horn on 1 occasion between Days 3-9 of pregnancy (day sperm found in vagina = Day 1). Rats were autopsied on Day 15. Percent implantation (number of sites/number of corpora lutea) was decreased when 0.1 ml saline was injected on Day 4, but not affected by injection on any other day. Fetal viability (number of viable fetuses/number of sites) was decreased by the injection of 0.1 ml saline on Days 4, 5, 6 or 7; the greatest effect was observed with injection on Day 6. On this day complete resorption occurred in the injected horn of 26 of 28 rats. No effect was observed in untreated contralateral horns. Complete fetal resorption could be effected by injection on Day 6 of 0.1 ml saline into both horns. Decreasing the volume of saline injected on Day 6 resulted in a decrease in the number of resorbed sites. Day’6 sham controls (needle inserted but nothing injected) showed normal fetal viability. Uterine horns injected on Day 6, when observed histologically on Day 9, contained implanted blastocysts. However, undifferentiated cells were seen in the blastocyst cavity along with maternal blood cells, typical of a resorbing blastocyst. These data illustrate that intrauterine normal saline may greatly affect fetal viability depending on the volume and the day of injection; thus defining some limitations of the model as used for antifertility drug testing. The results also describe an animal model with which physiologic correlates of the resorption process can be studied

The production of live young during successive pregnancies was investigated in female CF-1 house mice (Mus musculus) identified at cesarean delivery as having developed in utero between 2 male fetuses (2M females) or not next to a male fetus (0M females). 2M female mice have previously been found to be exposed to higher concentrations of testosterone than 0M females during fetal life, presumably as a result of the transport of steroids between contiguous fetuses. 0M and 2M females were paired with stud males. The males were removed prior to delivery of a litter and replaced by other males when the litter was weaned. This process was repeated until: 1) a female did not become pregnant within 2.5 mo or 2) two successive litters were produced in which all of the pups were dead. In Experiment 1 females were first mated when 25 days old, and 2M females ceased producing litters containing live pups at a younger age and after fewer litters than did 0M females; however, many females were terminated from the study as a result of producing 2 successive litters of dead young rather than failing to become pregnant during a 2.5-mo period. There was a gradual decline in the number of live young produced by 0M females as a function of age and parity, but 2M females abruptly ceased producing any live young after producing a litter of normal size. For the last live litter, there were thus significantly fewer live young produced by 0M females than by 2M females. None of these differences were observed in Experiment 2, in which 0M and 2M females were mated for the first time beginning at 7 mo of age. The 2M females in this experiment ceased producing live young at a significantly older age than did the 2M females first mated at puberty. In contrast, there was no effect of age at initial mating on the age at which 0M females ceased producing live young. This finding suggests that exposure of 2M females to elevated titers of testosterone during fetal life results in a reduction in reproductive life span if they first become pregnant during the pubertal period.