Prolonged Effect of Seminal Plasma on Global Gene Expression in Porcine Endometrium.

Seminal plasma (SP) deposited in the porcine uterine tract at the time of mating is known to elicit an initial response that is beneficial for pregnancy outcome. However, whether SP has any long-term effect on alterations in endometrial molecular and cellular processes is not known. In this study, using microarray analyses, differential changes in endometrial transcriptome were evaluated after Day 6 of SP-infusion (6DPI) or Day 6 of pregnancy as compared to corresponding day of estrous cycle. Both, pregnancy and SP induced significant changes in the endometrial transcriptome and most of these changes were specific for a particular group. Functional analysis of differentially expressed genes (DEGs) using Ingenuity Pathway Analysis revealed that inhibition in immune response was affected by both pregnancy and SP infusion. Long-term effects of SP included differential expression of genes involved in inhibition of apoptosis, production of reactive oxygen species and steroid biosynthesis, and activation of processes such as proliferation of connective tissue cells and microvascular endothelial cells. Moreover, interleukin-2 and interferon-γ was identified to be responsible for regulating expression of many DEGs identified on 6DPI. The present study provides evidence for the long-term effects of SP on porcine endometrium that can be beneficial for pregnancy success.

Maternal effect gene expression in porcine metaphase II oocytes and embryos in vitro: effect of epidermal growth factor, interleukin-1ß and leukemia inhibitory factor.

Maternal effect genes (MEG) play a crucial role in early embryogenesis. In vitro culture conditions may affect MEG expression in porcine oocytes and embryos. We investigated whether in vitro culture medium supplementation with epidermal growth factor (EGF), IL-1ß or LIF (leukemia inhibitory factor) affects the mRNA level of ZAR-1 (zygote arrest 1), NPM2 (nucleoplasmin 2) and DPPA3 (developmental associated protein 3) in porcine MII oocytes and embryos. Cumulus-oocyte complexes (COCs) were matured in NCSU-37 medium (control) or in NCSU-37 with EGF 10 ng/ml, IL-1ß 10 ng/ml or LIF 50 ng/ml. After maturation for 44-46 h, MII oocytes were preserved for the analysis of MEG mRNA levels (experiment 1). In experiment 2, COCs were fertilized, and the presumptive zygotes were cultured in the same groups. Then, 2-, 4-, 8-cell embryos, morulae and blastocysts were collected for the analysis of MEG mRNA levels. LIF addition to the maturation medium increased MII oocyte numbers (P < 0.05), while EGF and IL-1ß did not affect oocyte maturation. Medium supplementation with EGF resulted in lower DPPA3 mRNA levels in MII oocytes and in 2- and 4-cell embryos versus control embryos (P < 0.05). LIF treatment increased DPPA3 mRNA levels in morulae and blastocysts (P < 0.05). Culture with EGF and IL-1ß decreased ZAR-1 and NPM2 mRNA levels in 2-cell embryos (P < 0.05). The inclusion of EGF or IL-1ß in the porcine in vitro production system influences ZAR-1, NPM2 and DPPA3 mRNA in MII oocytes and embryos but not beyond the 4-cell stage. LIF stimulates oocyte maturation and affects DPPA3 mRNA in porcine morulae and blastocysts in vitro.

Comparison of the porcine uterine smooth muscle contractility on days 12-14 of the estrous cycle and pregnancy.

BACKGROUND: Uterine contractile activity is very important for many reproductive functions including embryo transport, implantation, gestation and parturition. Abnormal contractility leads to implantation failure, spontaneous miscarriage, preterm birth and many other disorders. The objective of the present study was to assess the effects of acetylcholine (ACh), noradrenaline (NA), oxytocin (OT) and prostaglandins F2α (PGF2α) and E2 (PGE2) on the contraction of uterine strips collected from the horns of cyclic gilts (12-14 days of the estrous cycle-group I) and from pregnant (12-14 days after first insemination gilts in which one of the uterine horn was gravid (group IIa) and the second one was non-gravid (group IIb). Uterine strips consisting of the endometrium with the myometrium and myometrium alone were examined. RESULTS: ACh increased the tension in all groups as compared to the pretreatment period, and the increase was the highest in group IIb; the amplitude decreased in all groups, and the frequency increased mainly in groups I and IIa. NA did not affect the tension in any group, but decreased the amplitude and frequency in group IIb as compared to groups I and IIa. OT caused the highest increase in the tension in group IIb, a decrease in the amplitude and an increase in the frequency of contractions as compared to the pretreatment period. PGF2α induced the highest increase in the tension and amplitude in group IIb, with a decline in the frequency in this group. PGE2 increased the tension and frequency only in group IIb, and caused the greatest eduction in the amplitude in this group. CONCLUSIONS: These results indicate that contractility of the porcine smooth muscle collected from uterine horns with embryos was different from those obtained from the uterine horns without embryos and the horns of cyclic gilts.

Peroxisome proliferator-activated receptors in the regulation of female reproductive functions.

Peroxisome proliferator-activated receptors (PPARs) belong to a ligand-dependent nuclear receptor family. In the past decade, numerous studies have revealed the presence and significance of PPARs in the reproductive system. PPARs are expressed at different levels of hypothalamic-pituitary-gonadal (HPG) axis. They are also present in the uterus as well as in the placenta and embryonic tissues of different species. PPARs significance has been reported during the estrous/menstrual cycle and pregnancy with the gamma isoform studied most frequently. Several studies indicate that PPARs regulate proliferation of ovarian cells, tissue remodeling and steroidogenesis. In the endometrium, PPARs are engaged in the regulation of prostaglandins, steroids and cytokines synthesis. The role of PPARs in the trophoblast differentiation, maturation and invasion as well as in the embryo development has also been demonstrated. In this review, we summarize current findings concerning the role of PPARs in the regulation of reproductive functions at different levels of the HPG axis during various physiological statuses of females. In addition, the role of PPARs in the modulation of uterine functions as well as the placenta and embryo development has also been discussed.

Proteomic analysis of porcine endometrial tissue during peri-implantation period reveals altered protein abundance.

In mammals, successful pregnancy depends upon the readiness of uterus for implantation, followed by correct communication between the endometrium and the developing conceptus. The objective of this study was to elucidate changes in protein abundance associated with progression of estrous cycle and pregnancy from Day 9 to Day 12. We analyzed porcine endometrial tissue lysates by 2D-DIGE. Abundance of several proteins was altered depending upon the pregnancy status of animals. MALDI-TOF/TOF was used to identify a number of these proteins. Endometrial proteins that increased from Day 9 to Day 12 of cycle included annexin A4, beta-actin, apolipoprotein, ceruloplasmin and afamin. Changes in protein abundances associated with conceptus secreted factors, including haptoglobin, prolyl-4-hydroxylase, aldose-reductase and transthyretin, were also observed. Functional analysis revealed that endometrial proteins with altered abundance on Day 12 irrespective of the reproductive status were related to growth and remodeling, acute phase response and free radical scavenging, whereas transport and small molecule biochemistry were the functions activated in the pregnant endometrium as compared to the cyclic endometrium. These data provide information on dynamic physiological processes associated with uterine endometrial function of the cyclic and pregnant endometrium during period of maternal recognition of pregnancy in pigs and may potentially demonstrate a protein profile associated with successful pregnancy. BIOLOGICAL SIGNIFICANCE: In pigs, the fertility rates are generally very high but the early embryonic loss that occurs during the second and third weeks of gestation critically affects the potential litter size. Temporal changes that take place in the uterine environment during the period of early pregnancy in pigs and a cross-talk between the uterus and the embryo play an important role in embryonic survival and successful pregnancy. A better understanding of the molecular changes associated with these processes will pave way for understanding of endometrial functions and help towards increasing embryo survival. In this study, we present a 2D-DIGE based analysis of changes in porcine endometrial proteome that are associated with progression of cycle and progression of pregnancy. The network analysis of the results clearly revealed the pathways that are involved in rendering the endometrium receptive to the presence of embryo and also the changes that are result of molecular communication between the endometrium and the conceptuses. This comprehensive identification of proteomic changes in the porcine endometrium could be a foundation for targeted studies of proteins and pathways potentially involved in abnormal endometrial receptivity, placentation and embryo loss.

The effect of hormonal estrus induction on maternal effect and apoptosis-related genes expression in porcine cumulus-oocyte complexes.

BACKGROUND: The effect of hormonal estrus induction on maternal effect (MATER - maternal antigen that embryo requires, ZAR-1 - zygote arrest 1, and BMP15 - bone morphogenetic protein 15) and apoptosis-related genes expression (BCL-2 and BAX) in porcine cumulus-oocyte complexes (COCs) and selected follicular parameters was investigated in this study. METHODS: Gilts were divided into three groups: (I) with natural estrus; (II) stimulated with PMSG/hCG; and (III) with PMSG/hCG + PGF2alpha. Analysis of maternal effect and apoptosis-related transcripts expression in COCs, and progesterone synthesis pathway genes expression (P450scc and 3betaHSD) in granulosa cells was performed by qPCR. BMP15 protein expression in follicular fluid (FF) was analyzed by western blot. Oocyte nuclear maturation was assessed by aceto-orcein staining. Progesterone (P4) and estradiol (E2) concentrations in FF and serum were measured by ELISA. Data were analyzed with the one-way ANOVA and Bonferroni post-test or Kruskal-Wallis test and Dunns post-test. RESULTS: The highest expression of MATER, ZAR-1, and BMP15 genes was found in COCs recovered from gilts treated with PMSG/hCG when compared to PMSG/hCG + PGF2alpha-stimulated or non-stimulated gilts. Hormonal treatment did not affect the BMP15 protein expression in FF, but increased the expression of genes participating in P4 synthesis in granulosa cells. The higher percentage of immature oocytes was found in PMSG/hCG-treated when compared to the non-stimulated gilts. The expression of BCL-2 and BAX mRNA, and BCL-2/BAX mRNA ratio was significantly higher in COCs derived from PMSG/hCG-treated when compared to PMSG/hCG + PGF2alpha-treated or non-stimulated subjects. The level of P4 in serum was similar in animals from all experimental groups, while its concentration in FF was greater in gilts subjected to PMSG/hCG treatment than in PMSG/hCG + PGF2alpha-stimulated and non-stimulated gilts. The concentration of E2 did not differ in the serum or FF between the control group and the hormonally stimulated groups. CONCLUSIONS: Hormonal induction of estrus affected maternal effect gene transcripts levels in COCs and and oocyte nuclear maturation. The inclusion of PGF2alpha into the stimulation protocol enabled maintaining of physiological concentration of P4 in FF. Additionally, both hormonal treatments seem to be beneficial for apoptosis prevention through increasing BCL-2/BAX transcript ratio.

Effects of seminal plasma and the presence of a conceptus on regulation of lymphocyte-cytokine network in porcine endometrium.

Infusion of seminal plasma in the uterus is known to elicit an instant inflammatory response in the porcine uterus, but whether or not it prepares a uterine immunological response to the presence of conceptuses is not well understood. Seminal plasma induced long-term modulatory effects and conceptus-induced immune changes in leukocyte populations were measured by flow cytometry and mRNAs for various cytokines by quantitative reverse-transcriptase PCR in porcine endometrium collected on Days 6 and 13 from cycling and pregnant animals or from animals given seminal plasma infusions. Seminal plasma infusion induced long-term modulatory effects, resulting in significantly more endometrial FoxP3-positive T-regulatory and T-helper cells 6 days after infusion as compared to cycling and pregnant animals. The number of T-cytotoxic and T-null cells did not change between the studied groups. The early molecular effects of seminal plasma were not observed at 13-days post-infusion, although animals on Day 13 of pregnancy did show significantly more T-cells (of any type investigated). Seminal plasma also showed a delayed effect on cytokine expression, specifically exhibiting a significant increase in interleukin 10 (IL10) and a decrease in granulocyte macrophage colony-stimulating factor (GMCSF) gene expression on Day 13 as compared to Day 6 of cycling or pregnant gilts. The results indicate a delayed regulatory effect of seminal plasma on immune responses in the porcine uterus, which are similar to immune changes generated by implanting conceptuses.

The effect of embryo presence on the expression of peroxisome proliferator activated receptor (PPAR) genes in the porcine reproductive system during periimplantation.

This study was undertaken to determine the effect of the presence of embryos in the uterine horn on peroxisome proliferator activated receptors (PPARs; A, D, G) gene expression in the reproductive tissues of gilts subjected to a surgical procedure. The uterus consisted of one intact horn connected to the uterine corpus and the second horn detached from the uterine corpus but connected with the contiguous ovary. The gilts were hormonally stimulated and divided into two groups: the first group, inseminated (pregnant) and the second group (cyclic), with surgical procedure but not inseminated. The animals of both groups were slaughtered on day 14 of pregnancy or on day 14 of the oestrous cycle, respectively. PPARs mRNA abundance in the endometrium and the corpus luteum (CL) was analysed by quantitative real-time PCR. During pregnancy, PPARA and PPARD µmRNA abundance in the porcine endometrium was significantly higher in the horn containing embryos than in the contralateral horn, where embryos were absent. The endometrial PPARG1 mRNA abundance did not differ between the two horns during pregnancy and the oestrous cycle, but a higher level of the transcript was observed during pregnancy when compared to the oestrous cycle. In the CL, there were no significant differences in PPARA and PPARDµ mRNA abundance between horns in pregnant or cyclic sows. However, there was a significant increase of PPARA and PPARD transcript level in the CL from cyclic compared with pregnant sows. The results of our study suggest that PPARA and PPARD have regulatory functions in early pregnancy, and they indicate that increased levels of endometrial gene expression are correlated with the presence of embryos in the uterine horn. Higher levels of PPARA and PPARD expression in the porcine CL on day 14 of the oestrous cycle than on day 14 of pregnancy suggest that both forms are involved in the regulation of CL functions.

Transcript abundance and apoptosis in day-7 porcine blastocyst cultured with exogenous insulin-like growth factor-I.

Exogenous growth factors may increase the efficiency of embryo development in vitro. The aim of the present study was to examine the effects of insulin-like growth factor (IGF)-I on porcine embryo development. Porcine embryos obtained by in vitro fertilization were cultured for seven days in the presence of IGF-I (50, 100 or 150ng/ml). Subsequently, relative transcript abundance (RA) of IGF-related genes (IGFR1, IGFBP2, and IGFBP3), glucose transporter genes (SLC2A4 and SLC2A8), and apoptosis-related genes (BAX and BCL-XL) was analyzed. No differences were observed in the cleavage rate on day 2 post insemination (pi) and blastocysts rate on day 7pi between IGF-treated and control embryos. IGF-I treatment did not affect RA of IGFR1, IGFBP3, and SLC2A4 genes, but decreased RA of IGFBP2 and SLC2A8 genes. The percentage of TUNEL-positive nuclei in blastocysts did not differ between the experimental groups. However, RA of BAX and BCL-XL genes decreased in response to all IGF-I concentrations, whereas the BCL-XL/BAX RA ratio was enhanced when embryos were cultured in medium containing 150ng/ml of IGF-I. These results indicate that IGF-I did not stimulate in vitro development of porcine embryos through the IGF signaling system, nor did IGF-I stimulate RA of glucose transporter genes. However, IGF-I at the highest dose was able to increase the BCL-XL/BAX transcript expression ratio. This may indicate that the primary role of IGF-I during the first days of embryo development in the pig is associated with anti-apoptotic actions rather than with growth stimulation.

Expression of peroxisome proliferator activated receptor (PPAR) genes in porcine endometrium exposed in vitro to IL-6 and INFγ.

The aim of the present study was to examine the effects of interferon gamma (INFγ) or interleukin 6 (IL-6) on gene expression of PPARs in the porcine endometrium on day 14 of the estrous cycle and pregnancy. Endometrial tissue (200-210 mg), after 18 h of pre-incubation, was incubated for 6 or 12 h in the presence of INFγ (5 or 50 ng/ml) or IL-6 (1 or 10 ng/ml). Gene expression was analyzed by quantitative real time RT-PCR. During the estrous cycle, neither INFγ nor IL-6 affected PPARα and PPARß/δ transcript levels in the endometrium of the cyclic pigs incubated for 6 or 12 hours. The presence of INFγ (5 ng/ml) significantly (p<0.05) increased PPARγ1 gene expression in the tissue incubated for 12 h. During pregnancy, INFγ (50 ng/ml) significantly (p<0.05) enhanced PPARα and PPARß/δ mRNA levels in the endometrium incubated for 6 h, whereas IL-6 (1 or 10 ng/ml) did not change their expression at any incubation time. The effect of both cytokines on PPARγ1 transcript level differed and was dependent on the incubation time. We observed an inhibitory (after 6 h of incubation, p<0.0001) and a stimulatory (after 12 h of incubation, p<0.05) effect of INFγ (5 ng/ml) or IL-6 (10 ng/ml) on PPARγ1 gene expression. The present study indicates that INFγ and IL-6 modulate PPARs gene expression in the porcine endometrium during the estrous cycle and pregnancy. The effect depends on the reproductive status of animals and the length of in vitro incubation of endometrial tissue with the treatments.

Effect of the conceptus on uterine prostaglandin-F2alpha and prostaglandin-E2 release and synthesis during the periimplantation period in the pig.

The present study was conducted to evaluate the effect of the conceptus on uterine prostaglandin-F2alpha (PGF2alpha) and prostaglandin-E2 (PGE2) release and the expression of prostaglandin synthase enzymes during the periimplantation period in the pig. A surgically generated model with conceptuses developing in only one of the uterine horns was created. The highest concentration of PGF2alpha and PGE2 was found in the gravid uterine horn, compared with the non-gravid horn and the intact horn of cyclic gilts. Endometrial concentration of both PGs in pregnant gilts was elevated regardless of the conceptus in the uterine horn, whereas only myometrial PGE2 concentration increased during pregnancy. Expression of prostaglandin-E2 synthase (mPGES-1) mRNA in the endometrium was upregulated during the oestrous cycle, while protein expression presented a similar pattern to that of PGE2 concentration in the uterine flushings. Prostaglandin-F2alpha synthase (PGFS) mRNA and protein expression in the endometrium did not differ between pregnancy and oestrous cycle but PGFS mRNA in the myometrium increased during pregnancy both in the gravid and the non-gravid uterine horns. We suggest a local effect of the conceptus on PG release pathways but also a more systemic effect within the whole uterus with regard to PG synthesis and accumulation in uterine tissues.

Local and systemic effects of embryos on uterine tissues during early pregnancy in pigs.

In the pig, the periimplantation period is critical for successful establishment of pregnancy. We studied the influence of embryos on oxytocin (OT) and progesterone (P(4)) regulated endometrial and myometrial secretion of 1) luteotrophic prostaglandin E(2) (PGE(2)) and 2) luteolytic prostaglandin F(2alpha) and its metabolite (PGFM) on days 12-14 of pregnancy in pigs. We used unilaterally pregnant pigs created by a surgical procedure in which one uterine horn remained intact and the second horn was cut transversely so that part of the horn was detached from the uterine body. The animals were divided into two groups, inseminated gilts (days 12-14 of pregnancy, n=6) and uninseminated cyclic gilts, which were used as controls (days 12-14 of estrous cycle, n=5). Embryos developed only in the patent part of the uterus and not in the occluded horn. The abundance of OTR mRNA was increased in the endometrium and decreased in the myometrium of the gravid uterine horn in the pregnant pigs compared with the non-gravid uterine horn or either uterine horn in the cyclic pigs, indicative of a local effect of the conceptus. The presence of embryos in the uterine horn during the periimplantation period determines endometrial metabolism of PGF(2alpha) and the local response of the endometrium to OT and P(4). OT stimulates PGF(2alpha) secretion and PGFM accumulation in endometrial cultures only from the non-gravid uterine horn and controls PGE(2) secretion from the endometrium and myometrium in the pregnant gilts. The results indicate a more systemic affect of pregnancy on the uterine response to OT and a possibly the local effect of the conceptus in promoting progesterone's inhibition of OT-stimulated PGE(2) secretion and uterine metabolism of PGF(2alpha).

Immune response to lytic peptides conjugated to a betaCG fragment in treated BALB/C mice.

Hecate-betaCG and Phor14-betaCG(ala) are relatively short, amphipathic alpha-helical cationic peptides with the ability to destroy selectively breast, prostate and ovarian cancer cells. Treatment with proteins and peptides frequently initiated antibody formation. Short peptides may minimize the risk of the immune system mobilization after treatment but it is necessary to investigate whether Hecate-betaCG and Phor14-betaCG(ala) induce the immune system to produce antibody and whether they affect the reproductive organs in normal wild-type mice. The results of our experiments showed that specific antibodies, tested by the enzyme-immunoassay, were not detected in the group treated with Hecate-betaCG and Phor14-betaCG(ala). The blood concentrations of both peptides begun to decrease from 60 minutes after injection and after 240 minutes its levels were undetectable. Histopatho-logical examination exhibited degenerative changes in the prostate glands and testes in males and in the ovaries and uteri of females treated with both peptides. In conclusion, our results indicate that both relatively small and rapidly metabolized peptides are not immunogenic and can be used for further investigation as a potential cancer treatment.

Quantitative expression of lysophosphatidic acid receptor 3 gene in porcine endometrium during the periimplantation period and estrous cycle.

Lysophosphatidic acid (LPA) belongs to the group of lipid messengers, which act via lysophosphatidic acid receptor 3 coupled to G-proteins. The participation of LPA3 in reproductive biology was revealed in mice and has not been studied in gilts. The present study was performed to evaluate the gene expression of LPA3 by a quantitative real-time PCR technique in the endometrium during different stages of pregnancy (days 6-30) and corresponding days of the estrous cycle (days 2-20) as well as in periimplantation period in pigs with surgically detached uterine horns. Based on the most conserved segments of human and rodent LPA3 we obtained a product containing 619bp (GenBank: EF137953), which exhibited high homology with human and rodents sequences. The highest transcript level was noted on days 10-12 of gestation in comparison to remaining periods and during pregnancy on days: 6-7, 8-9, 10-12 and 13-14 in comparison with the corresponding days of the estrous cycle. Higher mRNA level was noted in the horn containing embryos compared to the contralateral horn, where embryos did not develop. The results imply the important role of receptor LPA3 during early pregnancy.

Apoptosis inhibition by insulin-like growth factor (IGF)-I during in vitro maturation of bovine oocytes.

Recently, significant progress has been achieved in improving the yield of good quality embryos in vitro. However, efforts are still required to recognize the factors and understand the mechanisms of oocyte maturation, which are essential for subsequent embryo development. The aims of the present study were to determine the frequency of apoptosis in oocytes recovered from slaughterhouse ovaries and to investigate whether insulin-like growth factor (IGF)-I action during oocyte maturation in vitro may withhold apoptosis and improve oocyte quality. Only oocytes of proper morphology with homogenous ooplasm and compact cumulus cells were selected for this study. All oocytes recovered from the slaughterhouse ovaries were divided into two groups. One group of oocytes, chosen for apoptosis detection, was examined immediately after recovery. The other group of oocytes was maturated in vitro. Oocytes were maturated with IGF-I supplementation (100 ng/ml). Oocytes without supplementation were used as a control. Apoptosis in oocytes was determined by positive results of TUNEL assay and active caspase labeling. The percentage of apoptotic oocytes detected by TUNEL fell to zero when the maturation medium was supplemented with IGF-I in comparison to the control matured oocytes (0 vs. 9.87%; P<0.05). However, active caspase labeling was only slightly decreased in the IGF-I matured oocytes compared with the control matured oocytes (1.13 vs. 2.08%; P<0.05). The results indicate that IGF-I may serve as an anti-apoptotic factor during oocyte maturation. We suggest that IGF-I may inhibit apoptosis in oocytes at the stage of caspase activation and may prevent further advancement of oocyte apoptosis.

Scarless skin repair in immunodeficient mice.

Scarring, the end result of the wound healing process in adult mammals, is a problem of significant clinical importance. We observed that athymic nude-nu mice, similar to mammalian fetuses, are able to restore the structure and integrity of injured skin through a process resembling regeneration, where scar formation is absent. Among the postinjured skin tissues collected from athymic nude-nu, wild-type controls (C57BL/6J), severe-combined immunodeficient, Rag (lack of B and T cells), athymic (thymectomized neonates and adult C57BL/6J), and mice treated with an immunosuppressant (cyclosporin A), only athymic nude-nu mice showed: a lack of scar by histological examination (hematoxylin & eosin and Masson's trichrome staining), low levels of collagen (as determined by hydroxyproline content), high levels of hyaluronic acid, a statistically significant increase in elastic modulus for injured samples over unwounded (biomechanical testing) and low levels of the pro-scarring cytokines platelet-derived growth factor-B and transforming growth factor beta1. Additionally, immunohistochemical and Western blot analyses of postinjured tissues as well as flow cytometry analysis of blood samples showed the presence of CD8-positive cells in all studied animals except nude-nu mice. We conclude that scarless skin healing in athymic nude-nu mice provides a new model to study the influence of the immune system on tissue regeneration.

Influence of estrus synchronization of prepubertal gilts on embryo quality.

Synchronization and superovulation are commonly used to obtain large numbers of embryos for experimental and practical purposes. This study compared the number, quality, and in vitro development of embryos recovered from gilts following single or double estrus synchronization and superovulation. Prepubertal gilts from the single synchronization group were injected with 1500 I.U. PMSG and 1000 I.U. hCG 72 h later. The double synchronized group of gilts was treated with 750 I.U. PMSG and 500 I.U. hCG 72 h later. After 17 days, 1500 I.U. PMSG followed by 1000 I.U. hCG was administered. Five days after insemination embryos were recovered and cultured for 6 days. Both single and double hormonal stimulation schedules resulted in recovery of elevated numbers of embryos (28.4 and 23.4 vs. 11.3; p<0.01 and p

Effects of prostaglandin F(2alpha) and nitric oxide on the secretory function of bovine luteal cells.

The objective of the present study was to investigate the influence of prostaglandin F(2alpha) (PGF (2alpha)) and nitric oxide (NO) on production of steroids and PGs by culturing bovine luteal cells obtained from ovaries on days 8-12 of the estrous cycle with a nitric oxide (NO) donor (Spermine NONOate), and a NO synthase inhibitor (N(G)-nitro-L-arginine methyl ester dihydrochloride: L-NAME). When the cells were exposed for 24 h to PGF(2alpha) (10(-7)-10(-5) M), production of progesterone (P(4)) increased significantly at all doses used (P<0.05). Moreover, PGF(2alpha) stimulated PGF(2alpha) production (P<0.01), depressed testosterone (T) production (P<0.05), but did not affect synthesis of prostaglandin E(2) (PGE(2)). Spermine NONOate decreased P(4) production to 66%, 47% and 34% of the control concentration after treatment with 10(-5) M, 10(-4) M and 10(-3) M, respectively, but did not affect T production, and increased PGF(2alpha) synthesis (P<0.05) and PGE(2) (P<0.01) at all doses used. L-NAME increased production of P(4) (P<0.01) but did not affect (P>0.05) secretion of T, PGF(2alpha) and PGE(2). Estradiol-17beta (E(2)) was detectable on the level of sensitivity of assay and was not significantly altered by any treatments. The overall results suggest that PGF(2alpha) and NO produced locally in bovine CL play roles in the regulation of the secretory function of the bovine CL as auto/paracrine factors.