No abstract

Conceptus-uterine communication is established during trophoblastic elongation when the conceptus synthesizes and releases estrogen, the maternal recognition signal in the pig. Interleukin-1beta (IL-1beta) is a differentially expressed gene during rapid trophoblastic elongation in the pig. The current investigation determined conceptus and endometrial changes in gene expression for IL-1beta, IL-1 receptor antagonist (IL-1Rant), IL-1 receptor type 1 (IL-1RT1), and IL-1 receptor accessory protein (IL-1RAP) in developing peri- and postimplantation conceptuses as well as uterine endometrium collected from cyclic and pregnant gilts. Conceptus IL-1beta gene expression was enhanced during the period of rapid trophoblastic elongation compared with earlier spherical conceptuses, followed by a dramatic decrease in elongated Day 15 conceptuses. IL-1RT1 and IL-1RAP gene expression was greater in Day 12 and 15 filamentous conceptuses compared with earlier morphologies while IL-1Rant gene expression was unchanged by conceptus development. The uterine lumenal content of IL-1beta increased during the process of trophoblastic elongation on Day 12. Uterine IL-1beta content declined on Day 15, reaching a nadir by Day 18 of pregnancy. IL-1beta gene expression in porcine conceptuses was temporally associated with an increase in endometrial IL-1RT1 and IL-1RAP gene expression in pregnant gilts. Endometrial IL-1beta and IL-1Rant gene expression were lowest during Days 10-15 of the estrous cycle and pregnancy. The temporal expression of IL-1beta during conceptus development and the initiation of conceptus-uterine communication suggests conceptus IL-1beta synthesis plays an important role in porcine conceptus elongation and the establishment of pregnancy in the pig.

The objectives of the present studies were 1) to develop a culture system that has the positive effect of serum on granulosa cell attachment and allows subsequent expression of hormonal effects in serum-free medium and 2) to determine the effect of insulin, epidermal growth factor (EGF), estradiol (E2), and growth hormone (GH) on growth, steroidogenesis, and(or) protein synthesis of bovine granulosa cells. Cells from small (1 to 5 mm) and large (greater than 8 mm) follicles were collected from cattle and cultured for either 4 or 6 d. When cells from small follicles were cultured, insulin (5 micrograms/ml) increased (P less than .05) cell numbers (cells x 10(5)/well) severalfold compared with controls. Alone, EGF (10 ng/ml), FSH (200 ng/ml), LH (200 ng/ml), E2 (2 micrograms/ml), or GH (0 to 1,000 ng/ml) had no effect on cell numbers. However, when included with insulin, 30, 100, and 300 ng/ml of GH increased (P less than .05) granulosa cell numbers on d 4 of culture. Insulin alone increased (P less than .05) progesterone production (ng.10(5) cells-1.24 h-1) by severalfold on d 4, but EGF, FSH, LH, or GH alone had no effect and E2 inhibited progesterone production. In the presence of insulin, FSH and GH (100 ng/ml) increased (P less than .05) progesterone production on d 4 of culture, whereas EGF (10 ng/ml) elicited a decrease (P less than .05) in production. In cells from both sizes of follicles, GH (300 ng/ml) increased synthesis of cellular proteins (greater than 10 kDa). In cells from only large follicles, LH (200 ng/ml) decreased synthesis and secretion of proteins (greater than or equal to 3.5 kDa). These results support the hypothesis that GH may have direct effects on bovine ovarian function.

Secreted phosphoprotein 1 (SPP1, commonly referred to as osteopontin and formerly known as bone sialoprotein 1, early T-lymphocyte activation 1) is an extracellular matrix/adhesion molecule that is upregulated in the pregnant uterus of all mammals examined to date. This study focused on the pig, which has true epitheliochorial placentation and exhibits induction of SPP1 mRNA in luminal epithelium (LE) just before conceptus attachment and in glandular epithelium (GE) after Day 30 of pregnancy. The objective of this study was to determine steroid regulation of SPP1 mRNA and protein in porcine uterine epithelium. To examine the effect of estrogen, cyclic gilts were treated daily (Days 11-14) with 5 mg estradiol benzoate (i.m.) and hysterectomized on Day 15. To evaluate the long-term effect of pseudopregnancy, cyclic gilts were given daily injections (Days 11-15) with steroid as above and hysterectomized on Day 90. In situ hybridization showed high expression of SPP1 mRNA only in LE contiguous with apposing conceptus tissue on Day 15 of pregnancy. In contrast, estrogen injection resulted in moderate but uniform SPP1 mRNA in all LE of Day 15 nonpregnant gilts, with expression maintained through Day 90 of pseudopregnancy. SPP1 mRNA also localized to the GE of Day 90 pseudopregnant gilts, similar to expression in late gestation. Consistent with in situ hybridization results, SPP1 protein localized to the apical surface of LE in all estrogen-treated gilts and in the GE on Day 90 of pseudopregnancy. We conclude that, in pregnant pigs, SPP1 is induced by conceptus estrogen in uterine LE and is regulated in GE in a manner coincident with CL/placental progesterone production.

Conceptus trophectoderm and uterine luminal epithelial cells interact via endocrine, paracrine, and autocrine modulators to mediate pregnancy recognition and implantation. Pig conceptuses not only release estrogens for pregnancy recognition but also secrete interferons during implantation. Because interferon-stimulated genes are increased by interferons secreted for pregnancy recognition in ruminants, we asked whether the interferon-stimulated gene, STAT1, is up-regulated in pig endometrium by conceptus estrogens and/or interferons. STAT1 expression in response to day of pregnancy, estrogen injection, and intrauterine infusion of conceptus secretory proteins in pigs indicated 1) estrogen increases STAT1 in luminal epithelial cells, 2) conceptus secretory proteins that contain interferons increase STAT1 in stroma, 3) STAT1 increases in close proximity to the conceptus, and 4) early estrogen results in conceptus death and no STAT1 in stroma. The interactions of estrogen and interferons to regulate cell-type-specific expression of STAT1 highlight the complex interplay between endometrium and conceptus for pregnancy recognition and implantation.

Published reports indicate that in several mammalian species the oviduct synthesizes and secretes specific glycoproteins which are components of the luminal fluids at the time of ovulation and fertilization. The present study characterized the secretory glycoproteins synthesized by the bovine oviduct at estrus. Oviducts obtained from four crossbred cows in estrus were flushed with saline, and segments of the ampullary and isthmic regions were fixed for immunocytochemical analyses. The remainder of the tissue was subjected to explant culture for 24 h in medium containing either 3H-leucine or 3H-glucosamine. Analysis of culture media by one- and two-dimensional SDS-PAGE followed by fluorography indicated that both the ampullary and isthmic regions synthesized a major class of Mr 97,000 glycoproteins with isoelectric points ranging from 5.5 to 8.1. A polyclonal antibody was generated to the glycoproteins after their isolation by gel filtration followed by electrophoretic separation. Western blot analysis of oviductal culture media indicated that the antisera cross-reacted with a doublet at Mr 97,000 and to a lesser extent with two additional bands at Mr greater than 200,000. Immunoreactive antigens were not identified in serum or in culture media of ovary, uterus, and nonreproductive tract tissues. The Mr 97,000 glycoproteins were present in oviductal flushings obtained from cows in estrus. They were also detected to a lesser degree in oviductal flushings obtained from cows at Days 5, 10, 15, and 18 of the estrous cycle, with the least amount of immunoreactivity being observed in Day 10 samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment of gilts with 5 mg oestradiol benzoate on Day 9.5, 11, 12.5, 14, 15.5 or Days 14-16 resulted in an interoestrous interval of about 30 days. Administration of oestradiol benzoate daily from Days 11 to 15 or two periods of treatment on Days 11 and 14 to 16 resulted in prolonging CL function beyond 60 days from the pre-treatment oestrus. Endometrial secretory response to oestrogen stimulation, based on the ability of oestrogen to release calcium and uterine protein into the lumen appears to occur after Day 10 of the oestrous cycle. The results suggest that maintenance of prolonged CL function appears to require two periods of oestrogen stimulation. The first period occurs on Day 11 when the endometrium has become responsive to oestrogen stimulation followed by a second prolonged increase in oestrogen stimulation after Day 14. These findings accord with the normal patterns of oestrogen released by pig blastocysts during early pregnancy.

The objectives of this study were to develop a rapid method for sex determination for several mammalian species using polymerase chain reaction (PCR) and to use this method to determine whether there is a significant developmental difference in spherical diameter between male and female d-10 or -11 porcine embryos. The PCR system was developed and verified using genomic DNA from pigs of known sex, then it was tested with genomic DNA from several other mammalian species. Sex is determined by amplification of two genes in a single reaction. The presence or absence of a region of the Sry (sex-determining region Y) gene determines sex, and amplification of the Zfy (male) or Zfx (female) genes acts as a positive control for PCR. Sex determination was successful for all animals tested, including pigs, cattle, sheep, goats, llamas, horses, humans, baboons, dogs, cats, rats, and mice. A total of 209 embryos were collected from 21 crossbred gilts on d 10 or 11 of gestation, and their diameters were measured. No significant difference in embryo diameter was detected between male and female embryos, indicating that sexual dimorphism in embryonic growth in pigs does not occur before the period of rapid embryo elongation. The present sexing technique using PCR is rapid (approximately 6 h from receipt of embryos to results), and it may be useful for examining the effects of sex on any trait of interest in early porcine embryos and embryos from several other mammals.