The use of embryo collection techniques in Holstein heifers: A model to study early embryonic death

Twenty-three cyclic Holstein heifers were purchased for use as embryo donors to study the effect of intrauterine exposure to Haemophilus somnus on the number, quality, and viability of embryos produced. Few problems were encountered using standard superovulation and nonsurgical embryo collection techniques on virgin heifers. Based on three or more ovulations, as determined by palpation per rectum of the ovaries at the time of embryo recovery, 28 of 30 heifers responded to the superovulation regimen. Of 29 nonsurgical recoveries, 27 produced one or more embryos. One hundred and seventy-six embryos and ova were collected from heifers synchronized, superovulated, and flushed 7 to 8 d after insemination.     

Some observations on early embryonic death in mares

Early embryonic death (EED) was studied in 354 pregnant mares during two breeding seasons. Pregnant mares from six independent research projects were examined at 15, 20, 25, 30, 35, 40 and 50 days post-ovulation using a real-time ultrasound scanner. Mares that had previously been treated with anabolic steroids tended (P<0.10) toward a higher incidence of EED than untreated mares (21.1% vs 0%). Of the 59 pregnancies obtained from embryo transfer in 1982, 10 had undergone EED by day 50 (16.9%) compared to 10.1% for embryo transfer recipients in 1983. Overall, there was a 13.3% incidence of EED in embryo transfer recipients. This percentage was nearly identical to 13.4% incidence of EED for mares inseminated with fresh semen. In 1982, mares inseminated with frozen-thawed semen tended (P<0.10) to have a higher incidence of EED compared to those inseminated with fresh semen (34.6% vs 15.8%). However, in 1983 there was no difference (P>0.05) in the incidence of EED between mares inseminated with frozen-thawed and fresh semen (16.7% vs 12.5%). Further, combined over both years, there was no difference (P>0.10) in the incidence of EED (24.2% vs 14.4%). There was a 38.9% incidence of EED in infertile mares.Of the 354 pregnant mares, the overall incidence in EED through day 50 postovulation was 17.3%. The majority of EED (77.1%) occurred prior to day 35 post-ovulation. During the period 15 to 35 days post-ovulation, a greater (P<0.05) incidence of EED occurred between days 15 to 20 (26.2%) and 30 to 35 (29.5%) post-ovulation.     

Spontaneous pancreatic islet amyloidosis in 40 baboons

Spontaneous amyloidosis occurs in many nonhuman primate species but remains difficult to diagnose and treat. Nonhuman primates continue to offer promise as animal models in which to study amyloidosis in humans. Amyloidosis was not diagnosed clinically but was found histologically in four male and 36 female baboons. The baboons averaged 18 years of age at death (range, 7-28 years). Clinical signs, if present, were hyperglycemia and cachexia. Blood glucose values were elevated in 12 of 30 baboons with available clinical pathology data. Four baboons had been clinically diagnosed as diabetic and three were treated with insulin. Amyloid was found in the islets of Langerhans of the pancreas in 40 baboons; 35 baboons had amyloid only in the islets of Langerhans. Amyloid was found in nonislet tissue of baboons as follows: five, nonislet pancreas; four, intestine and adrenal; three, kidney; two, prostate and spleen; and one each, lymph node, liver, gall bladder, stomach, tongue, urinary bladder, and salivary gland. Sections of paraffin-embedded tissues were evaluated for amyloid with hematoxylin and eosin (HE) and congo red (CR) staining, and using immunohistochemistry for human islet amyloid polypeptide (IAPP), calcitonin gene-related peptide (CGRP), glucagon, pancreatic polypeptide (PP), somatostatin (SS), and porcine insulin. Islet amyloid was positive with HE in 40 baboons, with CR in 39 baboons, and with IAPP and CGRP in 35 baboons. IAPP and CGRP only stained islet amyloid. PP, SS, glucagon, and porcine insulin did not stain amyloid. Islet amyloidosis in the baboon appears to be difficult to diagnose clinically, age-related, and similar to islet amyloidosis in other species. The baboon may be a good model for the study of islet amyloidosis in humans.     

Ultrasonography for detection of pregnancy and study of embryonic development in heifers

Real-time B-mode ultrasonography was used to evaluate uterine changes associated with the estrous cycle in 22 ovulatory periods in 12 nulliparous heifers. Irregular, nonechogenic (black) areas were seen on the images of uterine horns during the periovulatory period. These nonechogenic areas were presumably due to intraluminal fluids since they coincided with the discharge of clear, viscous mucus preceding ovulation and blood-tinged mucus after ovulation. Eight heifers were bred until five pregnant heifers were obtained for study of the ultrasonic morphology of the conceptus. Ultrasound examinations were done daily to day 50 of pregnancy. Discrete, nonechogenic areas were first visible within the uterus between days 12 and 14, when they were approximately 2 mm in diameter. These discrete nonechogenic structures were identified as the embryonic vesicle, since they were observed only in heifers later confirmed to be pregnant and were always in the uterine horn ipsilateral to the corpus luteum. The presence of an embryo within the embryonic vesicle was confirmed by observing an echogenic (white) area with rhythmic pulsations (heartbeat). The embryonic vesicle gradually increased in length from the day of first observation until day 26 when it extended past the curvature of the horn and began to encroach into the contralateral horn. In all heifers, by day 32 the vesicle extended to the tip of the contralateral horn. The embryo was first visible between days 26 and 29 when the mean length was 10 mm. The embryo increased in length an average of 1.1 mm per day. A heartbeat was detectable in the embryo on the first day observed. In one superovulated heifer, five vesicles were visible in the uterine horns by day 14 and by day 33 seven embryos were observed; two of the seven embryos apparently resorbed by day 43.     

Lysosomal cathepsins in embryonic programmed cell death

During limb development, expression of cathepsin D and B genes prefigure the pattern of interdigital apoptosis including the differences between the chick and the webbed digits of the duck. Expression of cathepsin L is associated with advanced stages of degeneration. Analysis of Gremlin-/- and Dkk-/- mouse mutants and local treatments with BMP proteins reveal that the expression of cathepsin B and D genes is regulated by BMP signaling, a pathway responsible for triggering cell death. Further cathepsin D protein is upregulated in the preapoptotic mesenchyme before being released into the cytosol, and overexpression of cathepsin D induces cell death in embryonic tissues by a mechanism including mitochondrial permeabilization and nuclear translocation of AIF. Combined inhibition of cathepsin and caspases suggests a redundancy in the apoptotic molecular machinery, providing evidence for compensatory activation mechanisms in the cathepsin pathway when caspases are blocked. It is concluded that lysosomal enzymes are functionally implicated in embryonic programmed cell death.     

Spontaneous cyclic embryonic movements in humans and guinea pigs

Motility assessment before birth can be used to evaluate the integrity of the nervous system. Sideways bending (SB) of head and/or rump, the earliest embryonic motility in both humans and guinea pigs, can be visualized sonographically. We know from other species that early embryonic motility is cyclic. This study explores the distribution of SB-to-SB intervals in human and guinea pig embryos before the appearance of more complex movements such as general movements. We hypothesized that the activity in both species is cyclic. We made 15-min sonographic recordings of SBs between 5 weeks and 0 days (5wk0d) and 7wk0d conceptional age (CA) in 18 human embryos of uncomplicated IVF pregnancies (term 38 weeks) and in 20 guinea pig embryos between 3wk4d and 4wk0d CA (term 9 weeks). SB-to-SB interval durations were categorized as long (≥10 s) or short (<10 s) intervals. For human embryos, the median values for long and short intervals were 61 s (range, 10-165 s) and 3 s (range, 1-9 s) respectively; for guinea pigs 38 s (range, 10-288 s) and 5 s (range, 1-9 s), respectively. During development, the duration of long intervals decreased while the number of short intervals increased for both species. The earliest embryonic motility in the human and guinea pig is performed cyclically with distinct developmental milestones. The resemblance of their interval development offers promising possibilities to use the guinea pig as a noninvasive animal model of external influences on motor and neural development.     

Early embryonic death of mice deficient in gamma-adaptin

Intracellular protein transport and sorting by vesicles in the secretory and endocytic pathways requires the formation of a protein coat on the membrane. The heterotetrameric adaptor protein complex 1 (AP-1) promotes the formation of clathrin-coated vesicles at the trans-Golgi network. AP-1 interacts with various sorting signals in the cytoplasmic tails of cargo molecules, thus indicating a function in protein sorting. We generated mutants of the gamma-adaptin subunit of AP-1 in mice to investigate its role in post-Golgi vesicle transport and sorting processes. gamma-Adaptin-deficient embryos develop until day 3.5 post coitus and die during the prenidation period, revealing that AP-1 is essential for viability. In heterozygous mice the amount of AP-1 complexes is reduced to half of controls. Free beta1- or micro1 chains were not detectable, indicating that they are unstable unless they are part of AP-1 complexes. Heterozygous mice weigh less then their wild-type littermates and show impaired T cell development.     

Programmed cell death in the embryonic vertebrate limb

The developing limb bud provides one of the best examples in which programmed cell death exerts major morphogenetic functions. In this work, we revise the distribution and the developmental significance of cell death in the embryonic vertebrate limb and its control by the BMP signalling pathway. In addition, paying special attention to the interdigital apoptotic zones, we review current data concerning the intracellular death machinery implicated in mesodermal limb apoptosis.     

Spontaneous male death during copulation in an orb-weaving spider

Males of some cannibalistic species of spiders and insects appear to sacrifice themselves by allowing the female to eat them, and the adaptive significance of such drastic terminal reproductive investment has recently been demonstrated for a spider. Typically, the female has to kill the male, but it has been suggested that males of some species in the cannibalistic orb-weaving spider genus Argiope may die in copula without female 'collaboration'. Here, we provide the first experimental evidence to our knowledge of programmed sudden death after onset of copulation in males of the spider Argiope aurantia. Our observations reveal that males invariably die during the insertion of their second pedipalp, regardless of whether they mate with newly moulted, defenceless females or with older mature females that often attack them. We determined experimentally that the death of males is triggered immediately upon insertion of the second palp, when males become unresponsive, and heartbeat ceases within minutes of insertion. We discuss the possible adaptive significance of programmed death during copulation, and argue that male death has evolved in a context other than sexual cannibalism.     

Sire effect on early and late embryonic death in French Holstein cattle

We investigated the effect of maternal sire on early pregnancy failure (between D0, day of insemination and D90) in their progeny during the first and second lactations (n=3508) in the Holstein breed. The estimated breeding value (EBV) for cow fertility of 12 bulls (reliability⩾0.95) was used to create the following three groups: low, medium and high EBV (EBV from −0.7 to 1 expressed as genetic standard deviation relative to the mean of the breed). In their daughters (93 to 516 per bull), progesterone measurement was carried out on the day of artificial insemination (AI; D0) to check whether the cows were in the follicular phase and on D18 to 25 to assess non-fertilisation-early embryonic mortality (NF-EEM). Late embryonic mortality (LEM) and early foetal death (FD) were determined by ultrasonography on D45 and D90 and by the return to oestrus after the first AI. Frequencies of NF-EEM, LEM, FD and pregnancy were 33.3%, 11.7%, 1.4% and 48.5% and incidences were 35.1, 19.0, 2.7 and 51.1, respectively. Sire EBV was significantly related to the incidences of pregnancy failure between D0 and D90, fertilisation failure-early embryonic mortality (FF-EEM) and LEM but not to the incidence of FD between D45 and D90 of pregnancy. The relative risk (RR) of FF-EEM was significantly higher (RR=1.2; P<0.05) for the progeny group of low EBV bulls when compared with high EBV bulls. The same effect was observed when comparing LEM of the progeny groups from the low EBV bulls to those from moderate and high EBV bulls (RR, respectively, of 1.3 and 1.4; P<005). The incidence of FF-EEM was significantly higher when cows were inseminated before 80 days postpartum compared with later, and for the extreme values of the difference between milk fat and protein content measured during the first 3 months of lactation. FF-EEM was also significantly related to the year of observation. The incidence of LEM was higher for the highest producing cows and was influenced by interaction between milk yield×lactation rank and milk yield×milk protein content. In conclusion, this study showed large differences in early pregnancy failure between progeny groups and highlights the interest of accurate characterisation of embryonic death in order to identify potential candidate genes for female fertility.     

Spontaneous male death and monogyny in the dark fishing spider

Monogyny (male monogamy) is found in a diverse assemblage of taxa, and recent theoretical work reveals that a male-biased sex ratio can favour the evolution of this relatively rare mating system. We integrate this theoretical framework with field observations and laboratory experiments involving the sexually size dimorphic fishing spider, Dolomedes tenebrosus, to test the prediction that this species exhibits monogyny. Field surveys revealed a male-biased sex ratio, likely resulting from different life-history strategies (early male maturation). Results from mating trials supported our prediction of monogyny as we discovered that males mate with a single female. Unexpectedly, however, we observed that mating results in obligate male death and genital mutilation. Additional field observations of released individuals suggest that males are not limited by their ability to encounter additional females. Controlled laboratory assays demonstrated that males discriminate among virgin and non-virgin female silk cues, consistent with predictions of first-male sperm precedence. In summary, we report a novel case of male self-sacrifice in a species that exhibits female-biased sexual size dimorphism, male-biased sex ratio, genital mutilation and a suggestion of first-male sperm precedence; all of which are consistent with theoretical predictions of the evolution of monogyny.     

Early embryonic death in mice lacking the beta-catenin-binding protein Duplin

The Wnt signaling pathway plays a pivotal role in vertebrate early development and morphogenesis. Duplin (axis duplication inhibitor) interacts with beta-catenin and prevents its binding to Tcf, thereby inhibiting downstream Wnt signaling. Here we show that Duplin is expressed predominantly from early- to mid-stage mouse embryogenesis, and we describe the generation of mice deficient in Duplin. Duplin(-/-) embryos manifest growth retardation from embryonic day 5.5 (E5.5) and developmental arrest accompanied by massive apoptosis at E7.5. The mutant embryos develop into an egg cylinder but do not form a primitive streak or mesoderm. Expression of beta-catenin target genes, including those for T (brachyury), Axin2, and cyclin D1, was not increased in Duplin(-/-) embryos, suggesting that the developmental defect is not simply attributable to upregulation of Wnt signaling caused by the lack of this inhibitor. These results suggest that Duplin plays an indispensable role, likely by a mechanism independent of inhibition of Wnt signaling, in mouse embryonic growth and differentiation at an early developmental stage.     

Rudhira is a cytoplasmic WD40 protein expressed in mouse embryonic stem cells and during embryonic erythropoiesis

We describe a novel murine gene rudhira that is expressed at high levels in embryonic stem cells and is restricted to blood islands and the erythroid lineage during embryonic development. Rudhira is expressed in angiogenic precursors but is excluded from the differentiated endothelium. Rudhira-expressing cells are seen in close proximity to endothelial cells in angiogenic blood vessels. Rudhira encodes a predicted cytoplasmic WD40 protein that is 98% identical to human BCAS3. The gene encoding BCAS3 maps to a breakpoint of hematological neoplasms on human chromosome 17q23, but its expression and function remain to be determined. We demonstrate that mouse Rudhira is a novel marker for analysis of the erythroid lineage.     

Pregnancy rates at Days 2 and 14 and estimated embryonic loss rates prior to day 14 in normal and subfertile mares

Pregnancy rates at Days 2 and 14 postovulation were determined for 15 normal mares and 15 subfertile mares. Embryonic loss rates were estimated by the difference in the Day 2 and Day 14 pregnancy rates. Mares were artificially inseminated with the pooled ejaculates from three stallions, and the embryonic vesicle was detected with ultrasonography at Days 9, 10, 12 and 14. Mares were short-cycled with prostaglandin F(2) alpha (PGF(2alpha)) and rebred to the same stallions, and the Day 2 pregnancy rates were determined by recovery of cleaved ova (embryos) from the surgically excised oviducts. Significantly more (P < 0.01) normal versus subfertile mares were pregnant at Day 14 (12 15 vs 3 15 ). There was no significant difference in the Day 2 pregnancy rate for normal versus subfertile mares (10 14 vs 11 14 ). There were no significant differences (P > 0.5) in the mean number of blastomeres per embryo or in the mean diameter of embryos recovered at Day 2 from normal or subfertile mares. The estimated embryonic loss rate was significantly lower (P < 0.01) for normal verusus subfertile mares (0 10 vs 8 11 ). Fertilization rates were similar for normal and subfertile mares; however, subfertile mares had a higher embryonic loss rate prior to Day 14 postovulation.