Findings in a fox rescued from hounds.
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Animal Welfare↗
Biomedical subjects
W R Allen
Publications and source records attributed to W R Allen.
Hybridizing Old and New World camelids: Camelus dromedarius x Lama guanicoe.
Thirty female dromedary camels were inseminated on a total of 50 occasions with 2-4 ml of fresh guanaco semen diluted with an equal volume of commercially available camel semen extender. Similarly, nine female guanacos were inseminated on 34 occasions with 4-6 ml of fresh, diluted camel semen. Only two of the dromedary females conceived; one aborted a female foetus on day 260 of gestation and the other gave birth to a stillborn female calf on day 365. Six conceptions occurred in the female guanacos. Two of these conceptuses, diagnosed by ultrasound, were resorbed between days 25 and 40 of gestation, one female foetus was aborted on day 291, another female foetus was aborted on day 302, and one female calf was stillborn on day 365 of gestation. The sixth foetus, a male, was born prematurely but alive after a 328-day gestation. It had a phenotypic appearance intermediate between that of a camel and a guanaco and its hybrid parentage was confirmed by the DNA fingerprinting of eight llama microsatellites. To our knowledge, this is the first viable hybrid ever to be produced between Old World and New World camelids, which have been reproductively isolated from one another for at least 11 million years. The preponderance of female hybrids is in accordance with Haldane's law. Histological examination of their ovaries revealed a failure of meiosis, with only an occasional abnormal oocyte surrounded by follicle cells. Although the diploid chromosone number of camels and guanacos is the same (2n = 74), sufficient genetic change has taken place to make the pairing of homologous chromosomes no longer possible.
Animals↗
Factors controlling epidermal growth factor (EGF) gene expression in the endometrium of the mare.
Previous studies showed a dramatic increase in EGF gene expression in the endometrial glands of pregnant mares around day 40 after ovulation. To investigate how the steroid hormones of pregnancy might regulate this expression, in situ hybridization was used to monitor the levels of EGF mRNA in endometrial biopsies obtained from seasonally anoestrous or ovariectomised mares given exogenous progesterone and oestrogen, alone or in combination, for up to 46 days. Biopsies were also taken from mares during the non-pregnant cycle, during normal pregnancies and pregnancies compromised by endometrial pathology (endometriosis) or because of incompatible extraspecific embryo transfers (donkey-in-horse pregnancies). Only a few samples showed weak EGF expression during the late luteal phase of the oestrous cycle. During normal pregnancy, the previously observed dramatic increase of expression after day 40 of gestation was confirmed. Although aged mares suffering from endometriosis and mares carrying an extraspecific donkey conceptus showed the same increase of EGF mRNA in normal glands, this was virtually absent from gland cross-sections compromised due to inflammatory or fibrotic changes. Administration of various doses and combinations of progesterone and oestrogen for < 35 days yielded negative or only weakly positive hybridization results, whereas progesterone alone for > or = 40 days upregulated EGF expression strongly irrespective of additional treatment with oestrogen. This is the first experimental evidence that EGF expression in the endometrium can be induced by progesterone alone. The requirement for prolonged progesterone priming is of considerable interest in the context of the unusually late stage of gestation at which placental attachment commences in equids.
Animals↗
Cell proliferation patterns in the equine endometrium throughout the non-pregnant reproductive cycle.
Immunohistochemical detection of the proliferation marker Ki-67 antigen was used to monitor mitotic activity in the endometrium of mares. The monoclonal antibody MIB1 was validated for use on equine tissues by demonstrating its reaction with activated peripheral blood lymphocytes, and endometrial biopsies were recovered from 26 non-pregnant mares at selected stages during the reproductive cycle. The proportion of positively stained nuclei was counted in five random areas on each histological section to determine the percentage and type of proliferating cells. Multiplication rates in the types of cell found in the superficial strata, comprising the luminal epithelium, the epithelium of the gland necks and the stromal cells of the stratum compactum, were greatest during oestrus, presumably under the influence of oestrogens secreted by the growing ovarian follicles. In contrast, the mitotic activity in the cells of the deeper secretory portions of the endometrial glands was restricted to a brief phase between day 3 and day 7 of dioestrus, most likely as a delayed response to the decreasing oestrogen concentrations after ovulation. Some of the degenerate glands in subfertile mares did not follow this pattern of increased epithelial proliferation at that stage. After day 7 of dioestrus, the proliferation rates of cells in the endometrium decreased to basal values and remained low for as long as progesterone concentrations remained evaluated, even during prolonged dioestrus. The technique enabled characterization of normal cell proliferation patterns in the endometrium of mares and it will be a useful tool in the future for monitoring the endometrial responses of reproductively healthy and subfertile mares.
Analysis of Variance↗
Oxytocin administration prolongs luteal function in cyclic mares.
Recent evidence indicates that, in mares, as in the domestic ruminants, oxytocin and its endometrial receptor play important roles in stimulating the pulsatile releases of prostaglandin F2 alpha from the endometrium that effect luteolysis. In the present experiment, continuous administration of oxytocin by subcutaneous minipump to five mares during days 8-20 after ovulation abolished luteolysis in four of them, while all four of the control mares infused similarly with saline underwent luteolysis at the expected time. When oxytocin administration began on day 10, instead of on day 8, after ovulation luteolysis occurred rapidly in two of the five treated mares, indicating that the development of oxytocin responsiveness begins on or about day 10 of dioestrus in cyclic mares.
Animals↗
Cell proliferation patterns during development of the equine placenta.
Placentation involves considerable growth and reorganization of both maternal and fetal tissues. In this investigation, immunohistochemical localization of the proliferation marker Ki-67 antigen was used to monitor cell division during placentation in mares. Endometrial biopsies were obtained from eight mares between day 14 and day 26 of pregnancy and from eight anoestrous mares that had been treated with various combinations of progesterone and oestrogen. Samples of endometrium and fetal membranes were obtained from 19 mares carrying normal horse conceptuses between day 30 and day 250 of gestation and from three failing extraspecific donkey-in-horse pregnancies. Proliferation in the superficial strata of the endometrium was increased by day 18 of gestation and this effect could be mimicked by supplementing with oestradiol benzoate during the last 6 days of a prolonged period (18-36 days) of progesterone administration. Fetal chorionic girdle cells were proliferating vigorously at days 30-32 of gestation, but stopped dividing after they invaded the endometrium, while the trophoblast cells of the allantochorion showed an increase in mitotic activity after day 38. The luminal epithelium of the endometrium started to proliferate only after the primary villi of the true epitheliochorial placenta had been formed, and during days 58-70 this effect was seen only in the pregnant horn in which placentation was further advanced. During the second half of gestation, most of the mitotic activity was confined to the periphery of the microcotyledons which were still growing. In the donkey-in-horse pregnancies, proliferation rates of the maternal and fetal epithelial at day 70 of gestation were markedly reduced in areas of heavy endometrial lymphocyte infiltration and poor placentation. These results provide a basis for further studies on factors that influence invasive and non-invasive placentation.
Animals↗
Transfer of a uterine lipocalin from the endometrium of the mare to the developing equine conceptus.
One of the major, progesterone-dependent proteins secreted into the uterine lumen of the mare is a 19-kDa lipocalin (P19). It associates strongly with the embryonic capsule that envelops the young horse conceptus in early gestation, suggesting that it may be involved in sustaining early development. However, it was not known whether the protein was transported through the capsule and/or trophoblast layer and into the yolk sac cavity. To address this question, polyclonal antisera were raised against a C-terminal peptide (based on the deduced amino acid sequence of P19) and a recombinant-derived P19 fusion protein. The antiserum raised against the C-terminal peptide recognized P19 on Western blots of denatured uterine secretions (subjected to SDS-PAGE), but it did not bind to the protein in tissue sections. However, the antiserum raised against the recombinant-derived fusion protein recognized P19 both on Western blots and in histological sections. Western blot analysis of tissues and fluids collected from early-pregnant mares demonstrated significant quantities of P19 in the endometrium and uterine secretions and in the embryonic capsule, the chorion, and the yolk sac fluid, showing that the protein is transferred through to the developing embryo. Concentrations of immunoreactive P19 declined during gestation so that, by Day 30, it had virtually disappeared from both maternal and fetal tissues and fluids. Immunohistochemical staining of endometrial biopsies collected during early pregnancy localized P19 to the glandular and luminal epithelia and to the lumina of the endometrial glands. The capsule and the trophoblast layer of the chorion from early (Days 16-17) horse conceptuses also stained positively with localization of P19 to the apical surface of the trophoblast cells. There was no detectable staining either in or on the embryonic disc. The presence of P19 in both the trophoblast layer and the yolk sac fluid suggests that P19 passes into the yolk sac fluid via trophoblast cells.
Animals↗
Expression of epidermal growth factor and its receptor in equine placental tissues.
Northern blot and in situ hybridization techniques have demonstrated a marked increase in mRNA encoding epidermal growth factor (EGF) in the endometrium of mares, coincident with the start of interdigitation between the allantochorion and endometrium during placentation. In the present study, the unusually high EGF expression in the epithelium of the endometrial glands was shown to be maintained until at least day 250 of gestation (term = 320-340 days) in mares carrying normal horse conceptuses. However, in mares carrying failing donkey-in-horse pregnancies created by embryo transfer, EGF expression was severely retarded in those areas of the endometrium that were heavily infiltrated with lymphocytes and which showed a failure of placental development. Specific receptors for EGF were also detected in tissue homogenates from pregnant mares using 125I-labelled human EGF. Binding was high in the fetal membranes (allantochorion), both before implantation (days 30-34) and in the fully developed placenta (days 150-250), and was equivalent to the level of binding to homogenates of adult liver and kidney. Binding was much reduced in endometrial homogenates before implantation and from non-pregnant mares but increased after implantation to reach values equivalent to those exhibited by the fetal membranes. Scatchard analysis of displacement curves indicated a single class of high-affinity binding sites in the fetal membranes and pregnant endometrium sampled at day 150 of pregnancy and chemical cross-linking of the receptor-125I-labelled EGF complexes in fetal membranes revealed two radiolabelled bands of 170 kDa and 150 kDa. A large excess of insulin-like growth factor I (IGF-I) failed to displace any labelled EGF from the tissue homogenates. The marked and sustained upregulation of endometrial EGF expression during pregnancy in mares, and the presence of EGF receptors in the fetal allantochorion and maternal endometrium, suggest a possible role for EGF in the marked growth of these two tissues during placentation in equids.
Allantois↗
Endometrial oxytocin receptor and uterine prostaglandin secretion in mares during the oestrous cycle and early pregnancy.
Circulating concentrations of 13,14-dihydro-15-ketoprostaglandin F2 alpha (PGFM) were measured before and after administration of oxytocin and after endometrial biopsy, with or without uterine flushing performed per vaginam, on days 10, 14 and 18 after ovulation in nine pregnant and nine cyclic mares. Concentrations of oxytocin receptor were measured in endometrial biopsy samples. Neither pregnancy status nor time after ovulation affected basal PGFM concentrations. PGFM concentrations were increased after oxytocin administration on each of the days studied in cyclic mares; on day 14 the mean response was 4.5 times higher than the mean response on days 10 and 18. In contrast, during pregnancy, responses to oxytocin administration occurred only on days 10 and 18. Marked increases in PGFM concentrations in response to endometrial biopsy occurred only on day 14 in cyclic mares and on day 18 in pregnant mares. Mean concentrations of oxytocin receptor were between 200 and 300 fmol mg-1 protein on day 10 in both pregnant and cyclic mares; in cyclic mares oxytocin receptor concentrations were increased approximately threefold on day 14 compared with days 10 and 18, but no such increase was evident during pregnancy. Total amounts of PGFM secreted after oxytocin treatment correlated with endometrial oxytocin receptor concentrations in cyclic (P < 0.001) but not in pregnant (P > 0.5) mares, and the same was true for PGFM release induced by endometrial biopsy (cyclic: P = 0.0025; pregnant: P > 0.5). The data support the hypothesis that endometrial concentrations of oxytocin receptor determine uterine prostaglandin F2 alpha secretion in cyclic mares and that endometrial oxytocin receptor concentrations are reduced in early pregnancy by a product of the conceptus. The increase in response of the pregnant uterus to oxytocin treatment or biopsy-flushing between days 14 and 18 was not due to an increase in the concentration of oxytocin receptors but presumably reflected increased receptor sensitivity.
Analysis of Variance↗
Control of luteolysis in the one-humped camel (Camelus dromedarius).
Blood plasma concentrations of 13,14-dihydro-15-keto PGF2 alpha (PGFM) were measured in groups of mature non-pregnant and pregnant camels to study PGF2 alpha release patterns around the time of luteolysis and the timing of the signal for pregnancy recognition. Injection of each of four camels with 10 and 50 mg of PGF2 alpha showed clearly that five times the dose of exogenous hormone produced five times the amount of PGFM in peripheral plasma, thereby indicating that, as in other animal species, PGFM is the principal metabolite of PGF2 alpha in the camel. Serial sampling of three non-pregnant camels on each of days 8, 10 and 12, and three pregnant camels on day 10, after ovulation for 8 h showed a significant (P < 0.05) rise in mean plasma PGFM concentrations only on day 10 in the non-pregnant, but not the pregnant, animals. A single intravenous injection of 20, 50 or 100 iu oxytocin given to three groups of three non-pregnant camels on day 10 after ovulation did not increase their basal serum PGFM concentrations. However, daily treatment of six non-pregnant camels between days 6 and 15 (n = 3) or 20 (n = 3) after ovulation with 1-2 g of the prostaglandin synthetase inhibitor, meclofenamic acid, inhibited PGF2 alpha release and thereby resulted in continued progesterone secretion throughout the period of meclofenamic acid administration. These results showed that, as in other large domestic animal species, release of PGF2 alpha from, presumably, the endometrium controls luteolysis in the dromedary camel. Furthermore, reduction in the amount of PGF2 alpha released is associated with luteal maintenance and the embryonic signal for maternal recognition of pregnancy must be transmitted before day 10 after ovulation if luteostasis is to be achieved. However, the results also indicate that, in contrast to ruminants, the release of endometrial PGF2 alpha in the non-pregnant camel may not be controlled by the release of oxytocin.
Animals↗
Detection of equine X chromosome abnormalities in equids using a horse X whole chromosome paint probe (WCPP).
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Animals↗
Maternal response to paternal trophoblast antigens.
PROBLEM: What is the function of the immunoglobulin (Ig) G antibody bound to trophoblast in normal pregnancy, and what is the antigen? METHOD: IgG was acid eluted from term human placental microvesicles and reacted with the antigen, R80K, left on the vesicles. The eluted antibody was used to detect the antigen on monocytes, lymphocytes, and lymphoblastoid cell lines. The eluted antibody is highly polymorphic, but monoclonal antibodies (mAbs) were made against conserved regions of the molecule. These also reacted with the murine equivalent of the human R80K and were used in inhibition studies of natural killer (NK) cell killing and the mouse abortion models, CBA x DBA2 F1 resorption in CBA females, the endotoxin-induced resorption model, and a sonic stress-induced murine resorption model. RESULTS: All 600 syncytiotrophoblast microvesicle preparations of human term placenta had IgG antibody bound, elutable at pH 3.0. The eluted antibody reacted with about 15% of unrelated human placentae. In horses mares make detectable antibody early in pregnancy, at about the time of implantation. The IgG antibody was bound to an 80-kDa protein (R80K) also detected on B lymphocytes and monocytes. In HLA homozygous lymphoblastoid B cell lines, which reacted with one or more eluted antibodies, had a pattern of cytotoxicity independent of HLA Class I; and as a single 80-kDa peptide chain, R80K did not resemble HLA molecules. Genetic studies in horses show that of the two paternal allotypes of R80K detectable by placental alloantibodies, only one, usually the grandpaternal one, is present in all the placentae of a sibship. Two of 26 eluted human antibodies had affinity for K562 and inhibited killing by human peripheral blood NK cells. One mAb, BA11, against a conserved site on R80K inhibited killing of K562, and also reacted with the murine R80K homologue. BA11 inhibited murine NK cell killing and virtually completely inhibited three NK cell-dependent mouse resorption models. CONCLUSION: R80K protein is a target molecule for NK cell activity expressed on all placentae. It has a polymorphic alloantigenic determinant completely covered with maternal antibody in all successful term pregnancies. In murine NK cell-dependent models of abortion, a mAb against a monomorphic determinant present in human and murine R80K prevents abortion very effectively. It seems that the R80K molecule must be covered with antibody to prevent NK attacks on trophoblast.
Animals↗
A 19 kDa protein secreted by the endometrium of the mare is a novel member of the lipocalin family.
Large quantities of an unusual 19 kDa protein (p19) are secreted into the lumen of the uterus of the mare (Equus caballus) during the oestrous cycle and early pregnancy. p19 associates strongly with the acellular capsule that surrounds the young horse conceptus and is believed to be important in maintaining pregnancy. Here we report the complete cDNA sequence encoding p19, its expression patterns in horse tissues and a Southern blot analysis of the gene in horse DNA. The predicted amino acid sequence of the p19 cDNA demonstrated a signal peptide of 18 residues and a mature protein of 162 residues, giving a predicted molecular mass of 18.8 kDa for the secreted protein. Analysis of the predicted amino acid sequence showed that p19 belongs to the lipocalin family of proteins, which are classified on the basis of three conserved amino acid sequence motifs and an eight-stranded anti-parallel beta-barrel conformation. Overall, p19 was most similar to the mouse major urinary protein (MUP) lipocalins (30-34% identity). Secondary structural predictions and structural modelling using the MUP 1 crystal structure coordinates gave convincing evidence that the three-dimensional structure of p19 closely resembles that of the lipocalins. However, p19 has several unusual substitutions in one of the three conserved lipocalin motifs and therefore seems to be a novel member of the family. Lipocalins perform many different functions but most bind small hydrophobic molecules and the majority act as transport proteins. The function of p19 might therefore be as a carrier of a maternal factor needed to sustain the developing embryo during pregnancy, or it could be incorporated into the embryonic capsule and perform some other function. Northern blot analysis demonstrated that expression of p19 is confined to the endometrial lining of the mare's uterus, and hybridization studies in situ showed that the mRNA for p19 is localized to the glandular and luminal epithelia of the endometrium. A Southern blot analysis of horse DNA indicated a single gene for p19 that seems to be at least 4.5 kb in size.
Amino Acid Sequence↗
Recovery rate and quality of embryos from mares inseminated after ovulation.
The aim of this study was to evaluate the quality of embryos and their recovery rate from mares inseminated at different intervals after ovulation. Finnhorse and warmblood mares were inseminated with fresh semen 8 to 16 h, 16 to 24 h, or 24 to 32 h after ovulation. Control mares were inseminated before ovulation. Sixty-seven embryo flushings were performed between Days 7 and 9 after ovulation/insemination. Thirteen mares were not flushed, but their uteri were scanned for pregnancy on Days 14 to 16. Embryo recovery rates decreased as time from ovulation to insemination increased, although embryo quality remained normal as evaluated by morphological criteria and mitotic index. However, postovulatory insemination in this trial appeared to delay embryo development, since the embryos recovered from mares inseminated after ovulation were appreciably smaller and at an earlier stage of development than control embryos recovered from mares inseminated prior to ovulation. Part of this delay in embryo development in the postovulation group could be due to the time needed for sperm capacitation. In addition, as the time from ovulation to insemination increased, embryo development might have been further delayed by defects in the aging oocyte.
Journal Article↗
Implantation and early placentation in the one-humped camel (Camelus dromedarius).
The ovaries and uteri were removed from four pregnant camels on days 14, 25, 35 and 56 after ovulation. The day 14 and 25 uteri were perfuse-fixed with 3 per cent glutaraldehyde: 3 per cent paraformaldehyde whereas the day 35 and 56 specimens were opened dorsally for photography before biopsies of allantochorion attached to endometrium were taken and fixed in either 3 per cent glutaraldehyde: 3 per cent paraformaldehyde or Bouin's fluid. Samples of each uterus were then processed and sectioned for light and transmission electron microscopy. By day 14 the majority of the trophoblast had become closely apposed to the luminal epithelium of the endometrium to form the start of an epitheliochorial placenta with microvillar interdigitation initiated in some places. By day 25 a well-developed microvillar junction had formed between the fetal and maternal tissues. The fetus was situated in the middle of the left uterine horn in the day 35 and 56 specimens and, histologically, large multinucleate giant trophoblast cells had developed at frequent but irregular intervals along the, otherwise single-cell, trophoblast. These giant cells were often situated over the mouth of an endometrial gland but their actual function in pregnancy is not yet known.
Animals↗
Patterns of hormone secretion throughout pregnancy in the one-humped camel (Camelus dromedarius).
Peripheral serum samples were collected from 8 pregnant dromedary camels and hormone secretion patterns were examined at specific time intervals. Mean serum progesterone concentrations began to rise 3-4 days after ovulation and remained reasonably constant at 3-5 ng mL-1 for the first 90-100 days of gestation. Concentrations then showed a definite fall, but thereafter remained constant again at 2-4 ng mL-1 throughout the rest of pregnancy. In contrast, serum oestrogen concentrations showed pronounced fluctuations during the first 100 days of gestation. Mean oestradiol-17 beta concentrations increased at around Day 50 to about 100 pg mL-1 and then remained relatively constant from Day 90 to Day 300. Mean oestrone sulfate concentrations, however, showed two definite peaks in early gestation, each reaching about 10 ng mL-1, with the first peak occurring around Day 25 and the second peak around Day 75. Oestrogen production then remained fairly constant until around Day 300, after which concentrations of both oestrone sulfate and free oestradiol-17 beta rose steeply over the next 80 days to reach mean peak values of 46 ng ML-1 and 518.7 pg mL-1, respectively, at the time of parturition. Concentrations of 13,14 dihydro-15-keto prostaglandin F2 alpha (PGFM) remained low and reasonably steady at 100-200 pg mL-1 during the first 320 days of pregnancy; thereafter, PGFM concentrations rose steeply over the next 50 days, before an explosive further increase to a peak of 1900 +/- 141 pg mL-1 mean +/- sem on the day of calving. These results suggest that, as in the cow, a major change in steroid synthetic capability and/or enzyme content of the placenta may occur at around 80% (Day 300) of gestation in the pregnant camel.
Animals↗
Female pseudohermaphroditism due to congenital adrenal hyperplasia complicated by adenocarcinoma of the prostate and clear cell carcinoma of the endometrium.
The presence of prostatic glandular tissue in female pseudohermaphrodites has previously been documented. However, prostatic neoplasia in this clinical setting has not been reported. A karyotypic female with congenital adrenal hyperplasia due to 21-hydroxylase deficiency developed a prostatic adenocarcinoma associated with elevated serum prostatic specific antigen levels and osteoblastic skeletal metastases. This demonstrates that this tissue in pseudohermaphrodites can become malignant. In addition, the patient subsequently developed clear cell carcinoma of the endometrium, possibly related to radiation therapy for the prostatic adenocarcinoma. This demonstrates that female pseudohermaphrodites may be at risk not only for malignancies seen in genotypic females but also prostate cancer.
Adenocarcinoma↗
Anomalous inheritance of a paternally derived trophoblast antigen.
PROBLEM: Recurrent spontaneous abortion occurs in 1 in 500 random matings and usually results in abortion of all pregnancies. If absence of antibody to a paternally derived antigen caused abortion, the woman would be expected to make antibody to the other paternal antigen and abort only half her pregnancies. METHODS: Microvesicles were prepared from equine placentae. Acid-eluted IgG antibody was eluted from the polymorphic R80K antigen and used to type the residual R80K antigen on vesicles or on peripheral blood leucocytes. RESULTS: In several equine sibships all the half-sibs had the same paternal R80K alloantigen. In the extended horse family descended from the stallion Nearco, three allotypes were found. The allele present was usually the grandpaternal one, but exceptions are seen. Whichever allele is transmitted all the progeny have the same alloantigen (probability of this occurring by chance = 2(-45)). CONCLUSION: Because only one paternal allotype is present in all progeny, lack of antibody to the R80K antigen would result in loss of all pregnancies, not one half.
Alleles↗