Daniel J. Mathew

Interaction of the conceptus and endometrium to establish pregnancy in mammals: role of interleukin 1β.

Implantation and the establishment of pregnancy in mammals involves an intricate interplay of hormones, cytokines, growth factors, proteins, lipids, ions and the extracellular matrix between the uterine epithelium, stroma, immune cells and the conceptus trophectoderm. The divergent nature of implantation in the mouse, human and pig provides not only an interesting contrast in the establishment of pregnancy and early embryonic development but also intriguing similarities with regard to early endometrial-conceptus signaling. An interesting pro-inflammatory cytokine expressed in a number of mammalian species during the period of implantation is interleukin-1β (IL1B). The presence of IL1B might be involved with immunotolerance at the maternal-placental interface and has been proposed as one of the mediators in placental viviparity. The production of IL1B and other proinflammatory cytokines might play a role in establishing pregnancy through modulation of the nuclear factor kappa-B (NFKB) system in a number of species. A model for the regulation of cellular progesterone receptor expression and NFKB activation for endometrial receptivity and conceptus attachment is continuing to evolve and is discussed in the present review.

Activation of the transcription factor, nuclear factor kappa-B, during the estrous cycle and early pregnancy in the pig

Establishment and maintenance of pregnancy in the pig involves intricate communication between the developing conceptuses and the maternal endometrium. This process occurs during trophoblast elongation which is spaciotemporally associated with conceptus synthesis and release of IL1B concomitant with pregnancy-specific endometrial up-regulation of IL-1 receptors, providing the potential for activation of the transcription factor, NFKB. The objective of the current investigation was to determine changes in expression and cellular localization of NFKB and associated factors during the estrous cycle and early pregnancy in the pig. In situ hybridization was used to localize changes in PGR, ESR1, and TNFRSF11A during the peri-implantation period. Quantitative RT-PCR was utilized to demonstrate gene expression changes for NFKB1, RELA, TNFRSF11A, TLR4, NFKBIA and NFKBIB. Transcription factor ELISA demonstrated an overall increase in RELA during the peri-implantation period in both cyclic and pregnant gilts. While the presence of TNFSF11A and TLR4 were both detected, TLR4 expression changes were temporally associated with NFKB expression and activation. Collectively, these data demonstrate that NFKB activation may occur during the period of uterine receptivity in both the cyclic and pregnant endometrium.

Cytokines from the pig conceptus: roles in conceptus development in pigs

Establishment of pregnancy in pigs involves maintaining progesterone secretion from the corpora lutea in addition to regulating a sensitive interplay between the maternal immune system and attachment of the rapidly expanding trophoblast for nutrient absorption. The peri-implantation period of rapid trophoblastic elongation followed by attachment to the maternal uterine endometrium is critical for establishing a sufficient placental-uterine interface for subsequent nutrient transport for fetal survival to term, but is also marked by the required conceptus release of factors involved with stimulating uterine secretion of histotroph and modulation of the maternal immune system. Many endometrial genes activated by the conceptus secretory factors stimulate a tightly controlled proinflammatory response within the uterus. A number of the cytokines released by the elongating conceptuses stimulate inducible transcription factors such as nuclear factor kappa B (NFKB) potentially regulating the maternal uterine proinflammatory and immune response. This review will establish the current knowledge for the role of conceptus cytokine production and release in early development and establishment of pregnancy in the pig.

Uterine Progesterone Receptor Expression, Conceptus Development, and Ovarian Function in Pigs Treated with RU 486 During Early Pregnancy

Establishment of pregnancy in the pig depends on down-regulation of progesterone receptor (PGR) in the uterine luminal and glandular epithelium during the first week after breeding. The present study evaluated the regulation of endometrial PGR by progesterone and the possible role of endometrial tumor necrosis factor (ligand) superfamily member 11 (TNFSF11) and nuclear factor-kappa B (NFKB) activation in PGR expression. Mature, cycling gilts were inseminated (Day 0) and assigned to either untreated control (n = 9) or one of two treatments that employed RU 486 to block progesterone action either before (treatment 1 [T1]) or after (treatment 2 [T2]) the initiation of PGR down-regulation. The T1 gilts were treated with RU 486 (400 mg/day) on Days 3–5 of pregnancy (n = 9), and T2 gilts were treated with RU 486 on Days 6 and 7 of pregnancy (n = 9). Uteri and ovaries were collected on Day 8 or 12 of gestation. The diameter of the conceptuses in T1 gilts was approximately half that in controls by Day 8, and normal conceptuses were not collected from any T1 gilts on Day 12. Endometrial PGR mRNA was more abundant in T1 and T2 gilts compared with control gilts. The PGR-B protein decreased from Day 8 to Day 12 in the luminal epithelium and, to some extent, in superficial glandular epithelium in control and T2 gilts. In T1 gilts, the PGR-B protein remained elevated (i.e., failed to undergo down-regulation) on Day 12. Blocking PGR action early in the cycle (i.e., on or before Day 5), therefore, prevented normal conceptus development, caused elevated PGR mRNA, and prevented the decrease in PGR protein that typically occurs in pigs. We could not confirm a role for NFKB activation in PGR down-regulation, because pigs with extreme differences in PGR and TNFSF11 expression (T1 and controls) had similar NFKB activation on Day 8. Activated NFKB within the luminal epithelium and glandular epithelium (both superficial and deep) was observed in T2 and control pigs on Day 12 when elongating conceptuses (presumably releasing interleukin 1 beta to activate NFKB) were recovered. Gilts treated with RU 486 had greater ovarian follicular growth and greater plasma estradiol concentrations. We conclude that the mechanisms controlling PGR down-regulation are progesterone-dependent and occur between Day 3 and Day 6 of pregnancy. NFKB activation did not appear to have a role in PGR down-regulation within the period that we studied. Blocking progesterone action after Day 6 did not reverse the process of PGR down-regulation, nor did it appear to affect the development of conceptuses collected on Day 12.

Activation of the Transcription Factor Nuclear Factor-Kappa B in Uterine Luminal Epithelial Cells by Interleukin 1 Beta 2: A Novel Interleukin 1 Expressed by the Elongating Pig Conceptus

ABSTRACT Conceptus mortality is greatest in mammals during the peri-implantation period, a time when conceptuses appose and attach to the uterine surface epithelium while releasing proinflammatory molecules. Interleukin 1 beta (IL1B), a master proinflammatory cytokine, is released by the primate, rodent, and pig blastocyst during the peri-implantation period and is believed to be essential for establishment of pregnancy. The gene encoding IL1B has duplicated in the pig, resulting in a novel gene. Preliminary observations indicate that the novel IL1B is specifically expressed by pig conceptuses during the peri-implantation period. To verify this, IL1B was cloned from mRNA isolated from Day 12 pig conceptuses and compared with IL1B cloned from mRNA isolated from pig peripheral blood leukocytes (PBLs). The pig conceptuses, but not the PBLs, expressed a novel IL1B, referred to here as interleukin 1 beta 2 (IL1B2). Porcine endometrium was treated with recombinant porcine interleukin 1 beta 1 (IL1B1), the prototypical cytokine, and IL1B2 proteins. Immunohistochemistry and real-time RT-PCR were used to measure activation of nuclear factor-kappa B (NFKB) and NFKB-regulated transcripts, respectively, within the endometrium. Both IL1B1 and IL1B2 activated NFKB in the uterine luminal epithelium within 4 h. The NFKB activation and related gene expression, however, were lower in endometrium treated with IL1B2, suggesting that the conceptus-derived cytokine may have reduced activity within the uterus. In conclusion, the peri-implantation pig conceptus expresses a novel IL1B that can activate NFKB within the uterine surface epithelium, likely creating a proinflammatory microenvironment during establishment of pregnancy in the pig.

Short communication: Glucose infusion into early postpartum cows defines an upper physiological set point for blood glucose and causes rapid and reversible changes in blood hormones and metabolites

Low blood glucose concentrations after calving are associated with infertility in postpartum dairy cows perhaps because glucose is a master regulator of hormones and metabolites that control reproductive processes. The hypothesis was that low blood glucose postpartum is caused by inadequate glucose entry rate relative to whole-body demand as opposed to the alternative possibility that postpartum cows have a lower regulatory set point for blood glucose. Eight early postpartum (10 to 25 d) dairy cows (5 Holstein and 3 Guernsey) were jugular catheterized. During the first 24 h, cows were infused with physiological saline at 83.3 mL/h. After 24 h, the infusion solution was switched to 50% dextrose that was infused at a rate of 41.7 mL/h (total daily glucose dose=500 g). On d 3 and d 4, the rate of glucose infusion was increased to 83.3 mL/h (daily dose=1,000 g) and 125 mL/h (daily dose=1,500 g), respectively. On d 5, physiological saline was infused at 83.3 mL/h. Blood was sampled hourly through a second jugular catheter (contralateral side) and analyzed for glucose, nonesterified fatty acids, β-hydroxybutyrate, insulin-like growth factor 1, and insulin. Blood glucose concentrations on d 1 (saline infusion) averaged 53.4±1.7 mg/dL. Blood glucose concentrations increased on d 2 when cows were infused with 500 g/d and increased further on d 3 when cows were infused with 1,000g of glucose/d. Increasing the infusion rate to 1,500 g/d on d 4 did not cause a further increase in blood glucose concentrations. Based on a segmented regression analysis, the upper physiological set point for blood glucose was 72.1 mg/dL. Both insulin and insulin-like growth factor 1 concentrations increased in response to glucose infusion and decreased when cows were infused with saline on d 5. Serum nonesterified fatty acids and β-hydroxybutyrate concentrations decreased in response to glucose infusion and rebounded upward on d 5 (saline infusion). In conclusion, early postpartum cows had circulating blood glucose concentrations that were well below the upper set point defined in this study (72.1 mg/dL). Infusing approximately 1,000 g of glucose daily increased blood glucose to the physiological set point and rapidly changed the hormonal and metabolic profile that typifies postpartum cows. The inability of the early postpartum cow to achieve an adequate entry rate for glucose relative to whole-body demand is a possible mechanism that links postpartum physiology and nutrition to reproduction in dairy cows.

Interleukins, interferons, and establishment of pregnancy in pigs

Early pregnancy in mammals requires complex and highly orchestrated cellular and molecular interactions between specialized cells within the endometrium and the conceptus. Proinflammatory cytokines are small signaling proteins released by leukocytes that augment innate and adaptive immune responses. They are also released by the mammalian trophectoderm as the conceptus apposes the uterine surface for implantation. On approximately day 12 of development in pigs, the conceptus undergoes a rapid morphological transformation referred to as elongation while simultaneously releasing estrogens and a novel conceptus form of interleukin-1 beta (IL1β). Following elongation, pig conceptuses express interferon gamma (IFNγ) and, in lesser amounts, interferon delta (IFNδ). Significant IFN signaling takes place within the endometrium between day 14 and 18 of pregnancy as the conceptus intimately associates with the uterine epithelium. Based on studies carried out in pigs and other mammals, the combined spacio-temporal activities of conceptus estrogens, IL1β, and IFN set in motion a series of coordinated events that promote establishment of pregnancy. This is achieved through enhancement of conceptus development, uterine receptivity, maternal-fetal hemotropic exchange, and endometrial leukocyte function. These events require activation of specific signaling pathways within the uterine luminal epithelium, glandular epithelium, and stroma. Here, we review proinflammatory cytokine expression by pig conceptuses and the hypothesized actions of these molecules during establishment of pregnancy.

Rapid conceptus elongation in the pig: An interleukin 1 beta 2 and estrogen regulated phenomenon

Establishment and maintenance of pregnancy in the pig involves activating many physiological, cellular, and molecular signaling pathways between the developing conceptus and hormonally regulated maternal endometrium. Rapid elongation of the pig trophoblast allows for the establishment of sufficient placental surface area for the transport of nutrients to the fetus throughout pregnancy. Estrogens secreted by the conceptus during elongation act on uterine epithelia to induce secretion of uterine factors required for conceptus development and for preventing endocrine secretion of prostaglandin F2α, which would cause luteolysis. Thus, trophoblast expansion within the uterine lumen during early gestation is an essential process for implantation and maintenance of pregnancy in species with an epitheliochorial form of placentation. In the pig, rapid conceptus elongation involves the unique expression of interleukin‐1 beta 2 (IL1B2), which establishes pro‐inflammatory effects that may be tempered by the spatiotemporal secretion of estrogen from the conceptuses. The present review provides current information on pig conceptus remodeling and signaling via estrogen and IL1B2 pathways, as well as endometrial responses to those conceptus factors leading to establishment of pregnancy.

Reproduction in grazing dairy cows treated with 14-day controlled internal drug release for presynchronization before timed artificial insemination compared with artificial insemination after observed estrus.

Progesterone-releasing (controlled internal drug release, CIDR) devices inserted for 14 d are used to presynchronize the estrous cycle for timed artificial insemination (TAI) in beef heifers (14-d CIDR-PGF(2α) program). The objective was to test a similar program in dairy cows by measuring first-service conception rates (FSCR), pregnancy rates after 2 AI, and time to pregnancy compared with a control (AI after observed estrus). Postpartum cows (Holstein, Jersey, or crossbred; n=1,363) from 4 grazing dairy farms were assigned to 1 of 2 programs: 14dCIDR_TAI [CIDR in for 14 d, CIDR out, PGF(2α) injection at 19 d after CIDR removal, GnRH injection 56 h later, and then TAI 16 h later; n=737] or control [AI after observed estrus; reproductive program with PGF(2α) (cycling cows) and CIDR (noncycling cows) to synchronize estrus with the start of the breeding season; n=626]. Body condition was scored (1 to 5; thin to fat) at the start of the trial. The interval from the start of the breeding period (final PGF(2α) injection of either program) to first AI was shorter for 14dCIDR_TAI compared with the control (3.0±0.2 vs. 5.3±0.2 d; mean ± SEM) but 14dCIDR_TAI cows had lesser FSCR than controls (48 vs. 61%). Farm affected FSCR (50, 51, 67, and 58% for farms 1 to 4). The BCS affected FSCR (50, 55, and 62% for BCS=2, 2.5, and 3, respectively). Cows that either calved the year before (carryover) or that calved early in the calving season had greater FSCR than cows that calved later in the calving season (55, 61, and 42%, respectively). The percentage of cows pregnant to AI (first and second inseminations within 31-d breeding season) was similar for 14dCIDR_TAI and control (64 vs. 70%) cows, but farm (64, 62, 80, and 69%) and time of calving (70, 76, and 56%: carryover, early, and late, respectively) affected the percentage. Survival analyses showed an initial advantage for 14dCIDR_TAI (more cows inseminated and more pregnancies achieved early in the breeding season) that was not maintained over time. Conclusions were that the 14dCIDR_TAI program achieved acceptable FSCR (48%) and overall AI pregnancy rates (64%), but did not surpass a control program that used AI after observed estrus (61 and 70%, respectively).