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Laura C Schulz and R Michael Roberts

The hormone leptin, which is primarily produced by adipose tissue, is a critical permissive factor for multiple reproductive events in the mouse, including implantation. In the CD1 strain, maternally derived leptin from the oocyte becomes differentially distributed among the blastomeres of pre-implantation embryos to create a polarized pattern, a feature consistent with a model of development in which blastomeres are biased toward a particular fate as early as the two-cell stage. In this study, we have confirmed that embryonic leptin is of maternal origin and re-examined leptin distribution in two distinct strains in which embryos were derived after either normal ovulation or superovulation. A polarized pattern of leptin distribution was found in the majority of both CD1 and CF1 embryos (79.1 and 76.9% respectively) collected following superovulation but was reduced, particularly in CF1 embryos (29.8%; P<0.0001), after natural ovulation. The difference in leptin asymmetries in the CF1 strain arose between ovulation and the first cleavage division and was not affected by removal of the zona pellucida. The presence or absence of leptin polarization was not linked to differences in the ability of embryos to normally develop to blastocyst. In the early blastocyst, leptin was confined subcortically to trophectoderm, but on blastocoel expansion, it was lost from the cells. Throughout development, leptin co-localized with LRP2, a multi-ligand transport protein, and its patterning resembled that noted for the maternal-effect proteins OOEP, NLRP5, and PADI6, suggesting that it is a component of the subcortical maternal complex with as yet unknown significance in pre-implantation development.

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R Michael Roberts, Jonathan A Green and Laura C Schulz

The very apt definition of a placenta is coined by Mossman, namely apposition or fusion of the fetal membranes to the uterine mucosa for physiological exchange. As such, it is a specialized organ whose purpose is to provide continuing support to the developing young. By this definition, placentas have evolved within every vertebrate class other than birds. They have evolved on multiple occasions, often within quite narrow taxonomic groups. As the placenta and the maternal system associate more intimately, such that the conceptus relies extensively on maternal support, the relationship leads to increased conflict that drives adaptive changes on both sides. The story of vertebrate placentation, therefore, is one of convergent evolution at both the macromolecular and molecular levels. In this short review, we first describe the emergence of placental-like structures in nonmammalian vertebrates and then transition to mammals themselves. We close the review by discussing the mechanisms that might have favored diversity and hence evolution of the morphology and physiology of the placentas of eutherian mammals.

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The variation in the number of eggs shed by the two ovaries of mice has been examined by statistical analyses of 697 egg counts and 390 corpora lutea counts, made on mice from a variety of outbred strains, both after natural oestrus and after oestrus induced in adults by pregnant mares' serum (pms) and human chorionic gonadotrophin (HCG). The numbers of eggs or corpora lutea were distributed between sides approximately at random, the variation conforming fairly closely to a binomial distribution. This was true even after superovulation. There was, however, a slight but significant excess of variation between sides over the random amount in the egg counts, particularly after natural ovulation. Corpora lutea counts differed from egg counts in showing a slight but significant reduction of the variation below the random amount. Several possible reasons for these small deviations from a random distribution are discussed.

The correlation between the numbers of eggs shed by the two ovaries was negative after natural ovulation but positive after superovulation. This difference can be fully accounted for by the random distribution between sides together with the differences of mean and variance between natural ovulation and superovulation. The variation of total egg number was proportional to the mean egg number after natural ovulation. The variation after superovulation was much higher than after natural ovulation, even when the difference of mean was taken into account, and the greater variation of total egg number caused the correlation between sides to be positive after superovulation.

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M. T. Zavy, D. C. Sharp, F. W. Bazer, A. Fazleabas, F. Sessions and R. M. Roberts

Summary. Uterine secretions were obtained on Days 4, 8, 12, 14, 16, 18 and 20 of the oestrous cycle and early pregnancy. Acid phosphatase activity was significantly affected by day of the cycle, reaching a maximum at Days 12–14 during the luteal phase and then declining to almost undetectable levels by Day 20. In pregnant animals, activity continued to increase beyond Day 14. Two-dimensional polyacrylamide gel electrophoresis showed that albumin was a major component. However, a number of unique proteins of non-serum origin appeared in mid-cycle but had disappeared by Day 20. One of these was a basic protein indistinguishable in electrophoretic properties from the uterine acid phosphatase of the pig, uteroferrin, which is believed to be involved in iron transport from the uterine endometrial epithelium to the conceptus. These same polypeptides, including the putative uteroferrin, were also present in uterine flushings from pregnant animals until Day 20, and in flushings from ovariectomized mares treated with progesterone but not in those given only oestradiol-17β. Flushings from all ovariectomized animals contained a non-serum, acidic polypeptide (pI 5·3) of molecular weight 70 000. One basic polypeptide (molecular weight ~17 000) appeared by Day 4 of the oestrous cycle and disappeared by Day 16 but was maintained during pregnancy until Day 20. It was absent, however, in flushings from a Day 45 pseudopregnant mare. Like the sow, therefore, the mare possesses a number of proteins associated with cyclic changes in steroid hormones during the oestrous cycle and early pregnancy.

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K. J. McDowell, D. C. Sharp, A. T. Fazleabas and R. M. Roberts

Summary. Conceptuses were obtained from pony mares on each day of pregnancy between Days 12 and 28, and on Days 39, 45, 65 and 100. Endometrium was obtained from mares at Days 12, 14, 16, 18, 39, 45, 65 and 100 of pregnancy, and from non-pregnant mares during anoestrus, during transition into the breeding season, at oestrus, or during dioestrus. Tissues were incubated in vitro for 24 h with l-[3H]leucine. Proteins synthesized and released into the culture medium were analysed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and fluorography.

Conceptuses obtained before Day 14 after ovulation released a characteristic pattern of labelled proteins. These included two groups of apparent isoelectric variants of relative molecular weights (M r) 30 000–40 000 (pI values 4·5–5·5 and 6–7), one group of M r ∼22 000 (pI 6·5–7), and large protein(s) that did not enter the 10% polyacrylamide gel. After Day 14 the array of labelled proteins had changed and resembled that produced by isolated yolk sac at the later stages of pregnancy studied. Included amongst these were several acidic polypeptides with M r 20 000 (pI 5–6).

The endometrial samples released an array of non-dialysable polypeptides into the culture medium. Fluorograms could be assigned to one of three general groups, with endometrium from mares within each group producing similar patterns of labelled proteins. The first group consisted of anoestrous, transitional and ovariectomized mares, and mares at oestrus or Day 1 or Day 18 after ovulation. The second group was comprised of mares at Days 12–16 of dioestrus or Days 12–18 of pregnancy. Mares from Day 39 through 100 of pregnancy made up the third group.

Keywords: embryo; horse; proteins; endometrium; electrophoresis

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K Hardy, C Wright, S Rice, M Tachataki, R Roberts, D Morgan, S Spanos and D Taylor

The advent of human in vitro fertilization (IVF) over 30 years ago has made the oocyte and preimplantation embryo uniquely accessible. This accessibility has given rise to new micromanipulation techniques, such as intracytoplasmic sperm injection for treatment of male infertility, as well as embryo biopsy for preimplantation diagnosis of both genetic disease and aneuploidy, a major cause of early embryo demise and miscarriage. In the UK, average pregnancy rates after IVF and embryo transfer are < 25%, even after transfer of several embryos. Unfortunately, a third of these pregnancies involve multiple gestations. Research is currently focusing on methods to improve IVF success rates while reducing twin and triplet pregnancies and their associated increased morbidity and mortality. One approach is to develop screening methods to identify the most viable embryos, so that transfer of fewer healthy embryos will result in a higher proportion of singleton pregnancies. Screening methods include optimizing culture conditions for prolonged culture and selection of viable blastocysts for transfer, or embryo biopsy and aneuploidy screening. Assisted reproduction is also increasingly important in other branches of medicine: survival rates for cancer sufferers are improving continually and there is now a significant need for approaches to preserve fertility after sterilizing chemo-and radiotherapy treatment. Techniques for cryopreserving male and female gametes or gonadal tissue are being developed, although systems to grow and mature these gametes are in their infancy. Finally, there are also concerns regarding the safety of these new assisted reproductive technologies.

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T. K. Schalue-Francis, P. W. Farin, J. C. Cross, D. Keisler and R. M. Roberts

Summary. In Exp. 1 twice daily i.m. injections of 2 mg recombinant bovine IFN-αI1 (rboIFN-αI1) (N = 24) or placebo (N = 25) were administered to ewes from Day 12 to Day 16 during a normal oestrous cycle. Treatment did not increase (P > 0·10) oestrous cycle length (20·7 ± 1·2 versus 18·5 ± 1·4 days). In Exp. 2, ewes were injected twice daily with 2 mg IFN (N = 34) or placebo (N = 36) from Days 11 to 18 after natural mating. The rboIFN-αI1 significantly (P = 0·05) improved pregnancy rate (79% versus 58%) as determined by a failure of ewes to return to oestrus within 50 days. The number of ewes that lambed was greatest in the rboIFN-αI1-treatment group (71% versus 50%; P = 0·07), and no teratogenic effects were observed in the young born to IFN-treated ewes. The study was repeated a second year with a more fecund group of ewes (Exp. 3). More (P = 0·08) ewes injected with rboIFN-αI1 (58/65) than placebotreated ewes (48/61) were judged pregnant by ultrasound. Again more ewes lambed (55 versus 45) and more lambs were born (98 versus 80) from the rboIFN-αI1-treated group. Combining the data from both studies revealed a significant (P = 0·01) effect of treatment. The amount of antiviral activity in jugular vein blood of ewes injected with rboIFN-αI1 (2 mg) was determined over time in Exp. 4. Activity rose to a maximum (∼450 IRU/ml) within 1–2 h and declined by over 75% in 24 h. Single injections of 1, 2 and 5 mg in buffer or 2 mg emulsified in sesame oil all gave similar profiles of antiviral activity in jugular blood over a 48-h period. In Exp. 5, antiviral activity was measured in uterine vein, ovarian artery and jugular vein serum of untreated pregnant (N = 7) and non-pregnant (N = 11) ewes at Day 15 after mating. Activity was detected in the uterine vein (58 ± 19 IRU/ml) of all pregnant ewes. The observations in Exps 1–5 are consistent with a role for conceptus-derived IFN-α in maternal recognition of pregnancy and suggest that supplemental IFN-α might be useful in improving pregnancy success in sheep.

Keywords: α-interferon; oestrous cycle; pharmacokinetics; pregnancy; sheep

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C Richard Barb, Robert R Kraeling, George B Rampacek and Gary J Hausman

Two experiments (EXP) were conducted in ovariectomized prepubertal gilts to test the hypothesis that neuropeptide Y (NPY) stimulates appetite and modulates LH and GH secretion, and that leptin modifies such acute effects of NPY on feeding behavior and LH and GH secretion. In EXP I, gilts received intracerebroventricular (ICV) injections of 0.9% saline (saline; n=6), or 10 μg (n=7), 50 μg (n=5) or 100 μg (n=7) NPY in saline and blood samples were collected. In EXP II, gilts received ICV injections of S (n=4), or 50 μg leptin (n=4), or 100 μg NPY (n=4) or 100 μg NPY +50 μg leptin (n=4) in saline, and feed intake was measured at 4, 20 and 44 h after feed presentation and blood samples collected. In EXP I, NPY suppressed LH secretion and the 100 μg dose stimulated GH secretion. In EXP II, NPY reversed the inhibitory effect of leptin on feed intake and suppressed LH secretion, but serum GH concentrations were unaffected. These results support the hypothesis that NPY modulates feed intake, and LH and GH secretion and may serve as a neural link between metabolic state and the reproductive and growth axis in the pig.

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F. W. Bazer, J. L. Vallet, R. M. Roberts, D. C. Sharp and W. W. Thatcher

Summary. Conceptuses produce steroids, prostaglandins, proteins and possibly other unidentified agents which may play a role in the establishment and maintenance of pregnancy. A key event in this process is protection of the corpus luteum (CL) from the luteolytic activity of prostaglandin (PG) F-2α of uterine origin. Oestrogens produced by the pig conceptuses between Days 11 and 16 appear to exert an antiluteolytic effect resulting in the sequestering of PGF-2α within the uterine lumen. Failure of the pregnant uterus to release PGF-2α in an endocrine fashion, therefore, allows for maintenance of CL function. Conceptuses of sheep and cattle produce proteins which, when introduced into the uterine lumen of nonpregnant ewes and cows, suppress the ability of oestradiol and oxytocin to stimulate uterine production of PGF-2α. These conceptus secretory proteins appear to exert an antiluteolytic effect by inhibiting uterine production of luteolytic amounts of PGF-2α. The horse conceptus produces both oestrogens and proteins during early pregnancy when uterine production of PGF-2α is suppressed. Co-culture of horse endometrium and conceptus inhibits endometrial production of PGF-2α. Conceptuses of pigs, sheep and cattle undergo elongation to achieve apposition between trophectoderm and endometrium but the horse embryo migrates rapidly and consistently throughout the uterus to achieve endometrial contact.

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Jésus J Muniz, Margaret M Joyce, James D Taylor II, James R Burghardt, Robert C Burghardt and Greg A Johnson

Glycosylation dependent cell adhesion molecule 1 (GlyCAM-1), a mucin component of sheep histotroph produced by glandular epithelium (GE) during early pregnancy, is hypothesized to function in implantation. However, GlyCAM-1 is present in uterine tissues subsequent to implantation suggesting additional functions of this l-selectin-binding ligand. This study focused on uterine GlyCAM-1 expression during placentome development in sheep. Western blot analysis of day 50 pregnant sheep identified 45, 40, and 25 kDa bands in interplacentomal endometrium, 40 and 25 kDa bands in placentomes, and 80 and 40 kDa bands in chorioallantois. The GlyCAM-1 proteins in interplacentomal regions were comparable to those detected in day 15–19 pregnant sheep, however, the 80 kDa form was unique to chorioallantois, and the absence of the 45 kDa GlyCAM-1 in placentomes indicated differences between interplacentomal and placentomal endometrium. Immunofluorescence identified GlyCAM-1 in lumenal epithelium (LE), stromal fibroblasts, and vascular smooth muscle cells. To better define its cellular distribution, GlyCAM-1 was co-localized with either epithelium-specific cytokeratin, smooth muscle-specific alpha-smooth muscle actin (α SMA), or stromal-specific vimentin. In interplacentomal endometrium, GlyCAM-1 co-localized with cytokeratin in LE but not in GE. GlyCAM-1 did not co-localize with α SMA, and was localized in the extracellular matrix of vimentin-positive stroma. In placentomes, GlyCAM-1 did not co-localize with cytokeratin, but did co-localize with α SMA and vimentin. Thus, in contrast to interplacentomal regions, GlyCAM-1 in placentomes was predominantly localized in vasculature rather than epithelial cells. Further, leukocytes expressing L-selectin were localized to the endothelial surface of GlyCAM-1-expressing vessels within placentomes. These data suggest that GlyCAM-1 assumes distinct functions in compartment-specific regions of the sheep uterus.