Mark A. Mirando

The role of essential trace elements in embryonic and fetal development in livestock

This review addresses the concept that essential trace minerals play a vital role in many enzymatic and metabolic pathways that are critical for conceptus development during pregnancy in livestock species. The conceptus relies entirely on the maternal system for a sufficient supply of trace minerals and other nutrients needed for normal development. If this supply is inadequate, growth and/or health of the conceptus can be affected adversely, and many of these effects carry over into the neonatal period. Information, accumulated in our laboratory and presented herein, indicates that zinc, copper and manganese are among the trace minerals that have the greatest impact on reproduction. For example, levels of zinc, copper and manganese were several fold greater in the conceptus than in other reproductive tissues, indicating that the conceptus preferentially accumulates these minerals, an action that may be important for conceptus development, growth and survival. Moreover, some recent results indicate that increasing the biological availability of zinc, copper and manganese, by attachment to short peptide chains (i.e., proteinated trace minerals) can enhance reproductive performance of swine. Mineral concentrations in conceptuses from female pigs consuming proteinated trace minerals were greater than those from females that consumed only inorganic mineral salts. Elucidating the mechanisms whereby conceptus development and survival are enhanced by essential trace minerals may lead to development of specific feeding programs to increase the number and health of offspring at parturition, thereby allowing for further improvements in production efficiency in animal agriculture.

Autocrine/Paracrine Action of Oxytocin in Pig Endometrium

Abstract Luminal epithelial cells of porcine endometrium are unresponsive to oxytocin (OT) in vitro although they express the greatest quantity of OT and receptors for OT in vivo. Therefore, the objective of this study was to determine if oxytocin acted in an autocrine manner on luminal epithelial cells to stimulate prostaglandin (PG)F2α secretion. Treatment of endometrial explants or enriched luminal epithelial cells with OT antagonist L-366,948 decreased (P < 0.05) basal secretion of PGF2α. Oxytocin increased (P < 0.01) PGF2α secretion from luminal epithelial cells that were pretreated with 1:5000 or 1:500 OT antiserum for 3 h to immunoneutralize endogenously secreted OT. However, OT only increased (P < 0.05) PGF2α secretion from glandular epithelial cells when pretreated with 1:500 OT antiserum. Pretreatment with OT antiserum did not alter the ability of OT to induce PGF2α secretion from stromal cells. Medium conditioned by culture of luminal epithelial cells stimulated (P < 0.05) phospholipase C activity in stromal cells, indicative of the presence of bioactive OT. Oxytocin was secreted by luminal epithelial cells and 33% was released from the apical surface. These results indicate that luminal epithelial cells secrete OT that acts in an autocrine and/or paracrine manner in pig endometrium to stimulate PGF2α secretion.

Oxytocin-stimulated phosphoinositide hydrolysis and prostaglandin F2α secretion by luminal epithelial, glandular epithelial and stromal cells from pig endometrium. II. Responses of cyclic, pregnant and pseudopregnant pigs on Days 12 and 1

In pigs, the exact mechanism for the shift in endometrial PGF 2α secretion from an endocrine to an exocrine mode during pregnancy recognition is not known. The objective of this study was to examine whether this shift involved a change in the responsiveness of luminal epithelial, glandular epithelial and stromal cells to 0 or 100 nM oxytocin. Luminal epithelial cells, glandular epithelial cells and stromal cells were isolated from cyclic, pregnant or oestrogen-induced pseudopregnant gilts on Day 12 (Experiment 1) or Day 16 (Experiment 2) post oestrus (oestrus = Day 0). For cells obtained on Day 12, oxytocin stimulated PGF2α secretion by stromal cells (P<0.01) similarly for each reproductive status, whereas oxytocin stimulated PGF 2α secretion from luminal and glandular epithelial cells (P<0.05) from pregnant and pseudopregnant gilts but not from cyclic gilts. For both concentrations of oxytocin, mean PGF2α secretion was less (P<0.05) from stromal cells of pregnant than cyclic gilts. For cells obtained on Day 16, oxytocin stimulated PGF 2α release from stromal cells of cyclic gilts but not from stromal cells of pregnant gilts. Mean PGF 2α secretion also was less (P<0.05) from stromal cells of pregnant gilts than cyclic gilts. Oxytocin tended to stimulate PGF 2α release (P<0.07) from glandular epithelial cells of cyclic but not pregnant or pseudopregnant gilts. Luminal epithelial cells from all reproductive statuses were similarly unresponsive to oxytocin. In conclusion, the increased PGF2α secretory response to oxytocin of luminal and glandular epithelial cells from pregnant gilts on Day 12, combined with the decreased response of stromal cells from pregnant gilts on Days 12 and 16, may contribute, in part, to the shift in endometrial PGF2α secretion from an endocrine to an exocrine direction during early pregnancy in pigs.

Relationship between phosphoinositide hydrolysis and prostaglandin F2α secretion in vitro from endometrium of cyclic pigs on day 15 postestrus

The mechanism for the luteolytic release of prostaglandin (PG)F2 alpha in swine is not known. This study examined the potential role of oxytocin (OT)-induced phosphoinositide (PI) hydrolysis in promoting PGF2 alpha secretion in vitro from the endometrium of cyclic gilts on Day 15 postestrus. In Experiment 1, endometrial PI hydrolysis was increased (P < 0.05) by 100 nM OT and was increased quadratically (P < 0.05) by LiCl, but was not affected by the LiCl x OT interaction (P > 0.30). PI hydrolysis was maximal at 50 mM LiCl and declined at 100 mM LiCl. In Experiment 2, endometrial PI hydrolysis and PGF2 alpha secretion were similarly increased (P < 0.01) by 0, 0.1, 1, 10, and 100 nM OT in a dose-dependent manner. In Experiment 3, the linear increase in PI hydrolysis occurring 0, 3, 5, 10, and 20 min after treatment was greater (P = 0.01) for tissue treated with 100 nM OT than for the tissue treated with 0 nM OT. The quadratic increase (P < 0.05) in PGF2 alpha secretion occurring 0, 3, 5, 10, and 20 min after treatment was greater (P < 0.05) for tissue treated with 100 nM OT than for the tissue treated with 0 nM OT. In Experiment 4, AlF4- (an activator of Gp and phospholipase C) similarly increased (P < 0.01) PI hydrolysis and PGF2 alpha secretion. In Experiment 5, PI hydrolysis (P < 0.01) and PGF2 alpha secretion (P < 0.05) were increased by 100 nM OT but were not stimulated by cholera toxin (an activator of Gs and adenylate cyclase). Cholera toxin also did not enhance PI hydrolysis and PGF2 alpha secretion in response to 0.1 or 100 nM OT. These results are consistent with the hypothesis that OT may induce PI hydrolysis to stimulate the endometrial secretion of PGF2 alpha during corpus luteum regression in swine.

Effect of recombinant alpha interferons on fertility and interestrous interval in sheep.

In Experiment 1, 12 unmated cyclic ewes received twice-daily intrauterine injections on Days 12 to 14 of one of the following treatments: 1) ovine conceptus secretory proteins (oCSP) containing 25 microg of ovine trophoblast protein-1 (oTP-1) as determined by RIA; 2) 25 or 50 microg recombinant human interferon alpha1 (rhlFN); or 3) 1500 microg of serum proteins (oSP) from a Day-16 pregnant ewe (estrus = Day 0) per uterine horn. Ewes receiving oCSP had longer interestrous intervals (27 +/- 2 days; P<0.05) than ewes receiving oSP (17 +/- 2 days). Ewes receiving either dose of rhlFN had an interestrous interval of 16 +/- 2 days which did not differ (P>0.10) from that of oSP-treated ewes. In Experiment 2, 59 normally cycling ewes, mated on Day 0, received twice-daily intramuscular injections of either 2 mg recombinant bovine interferon alpha1 (rblFN) or placebo on Days 12 to 15 post estrus. On Day 16, pregnancy was confirmed by flushing a morphologically normal conceptus from the uterus. Pregnancy rates for rblFN-treated (80%) and placebo-treated (62%) ewes were not different (P>0.10). Uterine flushings and conceptus-conditioned medium were assayed for oTP-1. Total oTP-1 in conceptus-conditioned culture medium was higher (P<0.02) when conceptuses were from placebo-treated (104 +/- 14 microg/conceptus) than from rblFN-treated (56 +/- 12 microg/conceptus) ewes; while total oTP-1 in uterine flushings was similar (P>0.10) for placebo-treated (132 +/- 15 microg/conceptus) and rblFN-treated (147 +/- 17 microg/conceptus) ewes. The interval from mating to subsequent estrus following conceptus removal was 31 +/- 1 and 28 +/- 1 days for pregnant ewes treated with rblFN and placebo, respectively. Interestrous intervals for nonpregnant ewes were longer (P<0.02) for rblFN-treated (27 +/- 3 days) than for placebo-treated (18 +/- 2 days) ewes.

Purification of bovine trophoblast protein-1 complex and quantification of its microheterogeneous variants as affected by culture conditions

The bovine trophoblast protein-1 complex (bTP-1) is a group of glycosylated interferon-alpha 11, molecules secreted by the bovine conceptus that plays a critical role in preventing luteolysis during early pregnancy. In the current studies, secretion of individual variant forms of bTP-1 was quantified under a variety of culture conditions that could affect yields of bTP-1 for preparative-scale production of bTP-1. Additionally, a purification scheme for bTP-1 was developed. Conceptuses from Day 17 produced 13 proteins in the molecular weight and pI range characteristic of bTP-1, with 4-5 isoforms (pI = 5.6-6.6) at each of three molecular weight classes (21, 23.2 and 25.8 kDa). The major forms of bTP-1 were two variants of 23.2 kDa having pIs of 6.2 and 6.6. The relative proportion of bTP-1 variants was generally unaffected by culture conditions. Cultured conceptuses secreted bTP-1 at a sustained rate for 3 days and gaseous environment was without major effect on bTP-1 secretion. Conceptuses from superovulated cows also produced bTP-1 at Day 17 of pregnancy, suggesting that superovulation might be a useful method for increasing total bTP-1 yield per cow. The purification scheme that was developed utilized ultrafiltration with Centricon devices to achieve rapid molecular weight fractionation, desalting and concentration of conceptus secretory proteins prior to purification of bTP-1 using anion-exchange and gel filtration HPLC. The resultant preparation of bTP-1 included 9 variant forms of bTP-1 as well as a slight amount of a 45-kDa contaminant. Purified bTP-1 possessed antiviral activity but the specific activity was apparently reduced when conceptus-conditioned medium used for purification was stored for prolonged periods.

Oxytocin stimulates secretion of prostaglandin F2α from endometrial cells of swine in the presence of progesterone

Oxytocin (OT) stimulates endometrial secretion of prostaglandin (PG) F(2 alpha) during corpus luteum regression in swine but there is differential responsiveness to OT among endometrial cell types. To determine if progesterone influenced responsiveness of luminal epithelial, glandular epithelial, and stromal cells to 100 nM OT during luteolysis in swine, cells were isolated from endometrium of 15 gilts by differential enzymatic digestion and sieve filtration on day 16 postestrus and cultured continuously in the presence of 0, 10 or 100 nM progesterone. For phospholipase C (PLC) activity and PGF(2 alpha) secretion, stromal cells were most responsive to OT (P<0.01) in the absence of progesterone, whereas luminal epithelial cells were unresponsive and glandular epithelial cells displayed an intermediate response to OT (P<0.09). Progesterone enhanced PLC activity linearly in glandular epithelial cells (P<0.05) and influenced it quadratically in stromal cells (P=0.05). The effect of OT and progesterone on PLC activity in luminal epithelial cells was not significant, and progesterone did not increase PLC activity in response to OT in any cell type. Culture in the presence of progesterone, enhanced PGF(2 alpha) secretion in response to OT in luminal epithelial cells (P<0.05) but not in glandular epithelial or stromal cells. Progesterone also increased overall PGF(2 alpha) release from glandular epithelial (P<0.05) and stromal cells (P<0.06) across both levels of OT treatment. These results indicate that progesterone enhanced PGF(2 alpha) secretion from luminal epithelial cells in response to OT and increased basal PGF(2 alpha) release from glandular epithelial and stromal cells.

Oxytocin stimulates prostaglandin F2α secretion from porcine endometrial cells through activation of calcium-dependent protein kinase C☆ ☆

The mechanism for oxytocins (OT) stimulation of PGF2alpha secretion from porcine endometrium is not clear, but is thought to involve mobilization of intracellular Ca2+ and subsequent activation of protein kinase C (PKC). This study determined: (1) if mobilization of inositol trisphosphate-sensitive Ca2+ by thapsigargin or activation of PKC by phorbol 12-myristate 13-acetate (PMA) could stimulate PGF2alpha release from luminal epithelial, glandular epithelial and stromal cells of porcine endometrium and (2) if inhibitors of various PKC isotypes could attenuate the ability of OT, thapsigargin and PMA to stimulate PGF2alpha secretion from these cells. Thapsigargin and PMA each stimulated (P < 0.01) PGF2alpha secretion from all three endometrial cell types examined. However, the effects of thapsigargin and PMA were synergistic (P < 0.05) only in stromal cells. Three protein kinase C inhibitors (i.e. Gö6976, Gö6983 and Ro-31-8220) differentially attenuated (P < 0.05) the ability of OT, thapsigargin and PMA to stimulate PGF2alpha release. These results are consistent with the hypothesis that OT mobilizes Ca2+ to activate a Ca2+-dependent PKC pathway to promote PGF2alpha secretion from porcine endometrial cells. The differing pattern of response to isotype-specific inhibitors of PKC among cell types suggests that distinct PKC isoforms are differentially expressed in luminal epithelial, glandular epithelial and stromal cells.

Angiotensin II increases intracellular calcium concentration in pig endometrial stromal cells through type 1 angiotensin receptors, but does not stimulate phospholipase C activity or prostaglandin F2α secretion

Although the presence of endometrial receptors for angiotensin (Ang) II has been demonstrated, a specific function for AngII in the uterus has not been identified. Cytosolic free Ca2+ concentration [Ca2+]i, phospholipase C (PLC) activity and prostaglandin (PG) F2alpha secretion in response to AngII and oxytocin (OT) were measured in pig endometrial stromal cells collected 16 days after oestrus. Treatment with 100 nM OT or AngII increased (P<0.001) [Ca2+]i in stromal cells similarly (720 +/- 34 v. 690 +/- 33 pM, respectively). Subsequent administration of OT or AngII to the same cells induced smaller [Ca2+]i increases (25% or 35% of the initial responses, respectively) that occurred only if the second exposure to the same agent took place at least 5 min after the first. When administered sequentially, OT and AngII each induced a full response within 1 min of the previous treatment, regardless of which peptide was applied first. Whereas OT increased PLC activity and PGF2alpha secretion in stromal cells (P<0.01), AngII did not increase either PLC activity or PGF2alpha secretion. Type I AngII (AT1) receptors were present on stromal cells, whereas AT2 receptors were absent. Therefore, the effect of AngII in stromal cells was mediated via AT1 receptors. That AngII increased [Ca2+]i in stromal cells, but did not increase PLC or PGF2alpha secretion, indicates that either AngII releases a pool of Ca2+ through a mechanism that is not mediated by PLC and is not involved in PGF2alpha secretion or that a mechanism for PGF2alpha production other than one involving Ca2+ may exist.

Intracellular free calcium in response to oxytocin in pig endometrial cells.

Intracellular free calcium concentration ([Ca2+]i) in response to oxytocin (OT) was studied in stromal, glandular epithelial and luminal epithelial cells obtained from the endometrium of gilts 16 days post-estrus. The amplitude of increased [Ca2+]i in response to 100 nM OT was greatest in stromal cells, intermediate in glandular epithelial cells and not evident in luminal epithelial cells. During continuous OT administration, stromal cells responded initially with a synchronous spike of [Ca2+]i that did not require extracellular Ca2+ and then displayed spontaneous asynchronous [Ca2+]i spikes that required extracellular Ca2+. Each cell possessed its own characteristic response. Increasing concentrations of OT induced an increasing percentage of stromal cells responding, with some cells having nearly equal [Ca2+]i responses at all concentrations and others having graded [Ca2+]i responses as the concentration of OT increased. These results are consistent with the proposed mechanism of OT action in pig endometrium involving activation of phosphoinositide-Ca2+ signaling pathway.