A new in vivo model for luteolysis using systemic pulsatile infusions of PGF(2α).

A new in vivo model for studying luteolysis was developed in sheep to provide a convenient method for collecting corpora lutea for molecular, biochemical, and histological analysis during a procedure that mimics natural luteolysis. It was found that the infusion of prostaglandin F(2α) (PGF(2α)) at 20 µg/min/h into the systemic circulation during the mid luteal phase of the cycle allowed sufficient PGF(2α) to escape across the lungs and thus mimic the transient 40% decline in the concentration of progesterone in peripheral plasma seen at the onset of natural luteolysis in sheep. Additional 1h-long systemic infusions of PGF(2α), given at physiological intervals, indicated that two infusions were not sufficient to induce luteolysis. However, an early onset of luteolysis and estrus was induced in one out of three sheep with three infusions, two out of three sheep with four infusions, and three out of three sheep with five infusions. Reducing the duration of each systemic infusion of PGF(2α) from 1h to 30 min failed to induce luteolysis and estrus even after six systemic infusions indicating that, not only are the amplitude and frequency of PGF(2α) pulses essential for luteolysis, but the actual duration of each pulse is also critical. We conclude that a minimum of five systemic pulses of PGF(2α), given in an appropriate amount and at a physiological frequency and duration, are required to mimic luteolysis consistently in all sheep. The five pulse regimen thus provides a new accurate in vivo model for studying molecular mechanisms of luteolysis.

Molecular cloning and characterization of prostaglandin (PG) transporter in ovine endometrium: role for multiple cell signaling pathways in transport of PGF2alpha.

In ruminants, endometrial prostaglandin F(2alpha) (PGF(2alpha)) is the luteolytic hormone. Cellular transport of PGF(2alpha) in the uterine endometrium is critical for regulation of the estrous cycle. Molecular mechanisms responsible for control of PGF(2alpha) transport in endometrium during luteolysis are largely unknown. In the present study, we characterized the prostaglandin transporter (PGT) in ovine endometrium. Ovine PGT cDNA consists of 1935 nucleotides that encode 644 amino acids. In ovine endometria, PGT is highly expressed during the period of luteolysis, between d 14 and 16 of the estrous cycle, in luminal and glandular epithelia. Pharmacological and genomic inhibition of PGT indicates that it is responsible for influx and efflux of PGF(2alpha) in ovine endometrial epithelial cells. Inhibition of PGT during the period of luteolysis prevents the release of oxytocin-induced PGF(2alpha) pulses, and maintains functional corpus luteum and its secretion of progesterone. In ovine endometrial epithelial cells, protein kinase A and protein kinase C pathways are involved in regulating the influx of PGF(2alpha), whereas epidermal growth factor receptor pathways are implicated in regulation of influx and efflux of PGF(2alpha.) The ERK1/2 pathway is associated with efflux of PGF(2alpha), whereas Jun-amino-terminal kinase/stress-activated protein kinase pathways are involved in both efflux and influx of PGF(2alpha.) Phosphatidylinositol 3-kinase pathways are not involved in either influx or efflux of PGF(2alpha) in ovine endometrial epithelial cells. These are the first results to demonstrate a functional role for PGT in regulation of PGF(2alpha) efflux and influx in ovine endometrial cells that influence luteolytic mechanisms in ruminants.

Cyclooxygenase-2 regulates survival, migration, and invasion of human endometriotic cells through multiple mechanisms.

Endometriosis is a debilitating disease characterized by the presence of functional endometrial glandular epithelium and stroma outside the uterine cavity that affects up to 20% of women of child-bearing age. Cyclooxygenase-2 (COX-2), a rate-limiting enzyme in the biosynthesis of prostaglandin E(2) (PGE(2)), is highly expressed in endometriotic tissues and results in increased concentrations of peritoneal PGE(2) in women. In this study, we determined the expression of COX-2 protein in ectopic and eutopic endometria in humans and the role of COX-2 in endometriotic cell survival, migration, and invasion in humans. Our results indicate that COX-2 protein is abundantly expressed in ectopic endometria compared with eutopic endometria. Comparatively, expression of COX-2 protein is higher in eutopic endometria from women with endometriosis compared with women without endometriosis. Inhibition of COX-2 decreases survival, migration, and invasion of endometriotic cells that are associated with decreased production of PGE(2). Cell growth inhibitory effects of COX-2 inhibition/silencing are mediated through nuclear poly (ADP-ribose) polymerase-mediated apoptosis. Cell motility and invasion inhibitory effects of COX-2 inhibition/silencing are mediated through matrix metalloproteinase-2 and -9 activities. Interestingly, effects of COX-2 inhibition is more profound in endometriotic epithelial than in stromal cells. Furthermore, inhibition of COX-2 affects invasion rather than migration of endometriotic epithelial and stromal cells. It is the first evidence showing that inhibition of COX-2 decreases endometriotic epithelial and stromal cell survival, migration, and invasion in humans. Our results support the emerging concept that COX-2/PGE(2) promotes the pathophysiology and pathogenesis of endometriosis in humans.

Expression of prostaglandin transporter in the bovine uterus and fetal membranes during pregnancy.

Uteroplacental prostaglandins (PGs) play pivotal roles in the maintenance and termination of pregnancy in mammals. In the present study, we have characterized the expression of prostaglandin transporter (PGT) in placentome caruncles, intercaruncular tissues, fetal membranes, and utero-ovarian plexus during pregnancy in cattle. Pregnant bovine uteri were collected and classified into six groups covering the entire gestational length. In caruncles and intercaruncular tissues, PGT mRNA (also known as SLC02A1) and PGT protein were highly expressed at the late stage of pregnancy compared to the early and mid stages, whereas the level of expression is constant and low in fetal membranes throughout pregnancy. PGT mRNA and PGT protein were expressed at a constant level in the utero-ovarian plexus both ipsilateral and contralateral to corpus luteum throughout the course of pregnancy. Overall, the relative expression of PGT mRNA and PGT protein were higher in caruncles than in intercaruncular tissue and fetal membranes, whereas no differences were detected between intercaruncular tissues and fetal membranes at any stage of gestation. Immunohistochemistry indicated that PGT was preferentially expressed in caruncular epithelial cells of placentomes and endometrial luminal epithelial and myometrial smooth muscle cells of the intercaruncular regions. The level of PGT expression was comparatively higher in maternal components than in fetal components. In conclusion, differential spatiotemporal tissue-specific expression of PGT in uterine and intrauterine tissues suggests a role for this transporter in the exchange of PGs between the maternal and the fetal compartments, as well as for intrauterine metabolism of PGs during pregnancy.

Effect of interferon-tau on prostaglandin biosynthesis, transport, and signaling at the time of maternal recognition of pregnancy in cattle: evidence of polycrine actions of prostaglandin E2.

Recognition and establishment of pregnancy involve several molecular and cellular interactions among the conceptus, uterus, and corpus luteum (CL). In ruminants, interferon-tau (IFNtau) of embryonic origin is recognized as the pregnancy recognition signal. Endometrial prostaglandin F(2alpha) (PGF(2alpha)) is the luteolysin, whereas PGE(2) is considered a luteoprotective or luteotrophic mediator at the time of establishment of pregnancy. The interplay between IFNtau and endometrial PGs production, transport, and signaling at the time of maternal recognition of pregnancy (MRP) is not well understood. We have studied the expression of enzymes involved in metabolism of PGE(2) and PGF(2alpha), cyclooxygenase-1 (COX-1) and COX-2, PG synthases (PGES and PGFS), PG 15-dehydrogenase, and PG transporter as well as PGE(2) (EP2 and EP3) and PGF(2alpha) receptors. IFNtau influences cell-specific expression of COX-2, PGFS, EP2, and EP3 in endometrium, myometrium, and CL in a spatio-temporal and tissue-specific manner, whereas it does not alter COX-1, PGES, PG 15-dehydrogenase, PG transporter, or PGF(2alpha) receptor expression in any of these tissues. In endometrium, IFNtau decreases PGFS in epithelial cells and increases EP2 in stroma. In myometrium, IFNtau decreases PGFS and increases EP2 in smooth muscle cells. In CL, IFNtau increases PGES and decreases EP3. Together, our results show that IFNtau directly or indirectly increases PGE(2) biosynthesis and EP2-associated signaling in endometrium, myometrium, and CL during MRP. Thus, PGE(2) may play pivotal roles in endometrial receptivity, myometrial quiescence, and luteal maintenance, indicating polycrine (endocrine, exocrine, paracrine, and autocrine) actions of PGE(2) at the time of MRP. Therefore, the establishment of pregnancy may depend not only on inhibition of endometrial PGF(2alpha), but also on increased PGE(2) production in cattle.

Prostaglandin biosynthesis, transport, and signaling in corpus luteum: a basis for autoregulation of luteal function.

The corpus luteum (CL) is a transient ovarian endocrine gland formed from the ovulated follicle. Progesterone is the primary secretory product of CL and is essential for establishment of pregnancy in mammals. In the cyclic female, the life span of CL is characterized by luteal development, maintenance, and regression regulated by complex interactions between luteotrophic and luteolytic mediators. It is universally accepted that prostaglandin (PG) F(2a) is the luteolysin whereas PGE(2) is considered as a luteotropin in most mammals. New emerging concepts emphasize the autocrine and paracrine actions of luteal PGs in CL function. However, there is no report on selective biosynthesis and cellular transport of luteal PGE(2) and PGF(2alpha) in the CL of any species. We have studied the expression of enzymes involved in the metabolism of PGE(2) and PGF(2alpha), cyclooxygenase (COX)-1 and -2, PGE and F synthases, PG 15-dehydrogenase, and PG transporter as well as receptors (EP2, EP3, and FP) throughout the CL life span using a bovine model. COX-1, PGF synthase, and PG 15-dehydrogenase are expressed at constant levels whereas COX-2, PGE synthase, PG transporter, EP2, EP3, and FP are highly modulated during different phases of the CL life span. The PG components are preferentially expressed in large luteal cells. The results indicate that PGE(2) biosynthesis, transport, and signaling cascades are selectively activated during luteal maintenance. By contrast the PGF(2alpha) system is activated during luteal regression. Collectively, our results suggest an integrated role for luteal PGE(2) and PGF(2alpha) in autoregulation of CL function.

Temporal and tissue-specific expression of prostaglandin receptors EP2, EP3, EP4, FP, and cyclooxygenases 1 and 2 in uterus and fetal membranes during bovine pregnancy.

Uteroplacental prostaglandins (PGs) play pivotal roles in maintenance and /or termination of pregnancy in mammals. Regulation of PG biosynthetic and signaling mechanisms in uteroplacental tissues during maintenance of pregnancy is largely unknown. In the present study, we have characterized the expression of PGE2 receptors (EP2, EP3, EP4), PGF2alpha receptor (FP), and cyclooxygenase (COX) types 1 and 2 in placentome caruncle (CAR), intercaruncle, and fetal membrane tissues during pregnancy in cattle. Pregnant bovine uteri were collected and classified into six groups covering the entire gestational length. The levels of expression of EP2, EP3, and FP mRNAs differ depending on tissues and days of gestation (days <50 to >250). EP4 mRNA was undetectable in all the tissues studied. The expression levels of PG receptor mRNAs were as follows: placentome CAR FP>EP2>P3, intercaruncle EP2>EP3> or =FP, and fetal membranes EP3> or =EP2 >>FP. EP2 and EP3 expressions were modulated in uteroplacental tissues, depending on days of pregnancy, whereas FP was uniformly expressed. COX-1 mRNA and protein were constitutively expressed, whereas COX-2 was highly modulated in uteroplacental tissues throughout pregnancy. Immunohistochemistry showed that EP2 and COX-2 proteins were colocalized in most cell types of placentome CAR, endometrium, and myometrium. Our study indicates that EP2 is the primary cAMP-generating PGE2 receptor expressed in uteroplacental tissues during bovine pregnancy. Temporal and tissue-specific expression of PGE2 and PGF2alpha receptors and COX-1 and -2 at the maternal-fetal interface suggests a selective and distinctive role for PGE2 and PGF2alpha in uterine activities during pregnancy in bovine.

Molecular cloning and characterization of bovine prostaglandin E2 receptors EP2 and EP4: expression and regulation in endometrium and myometrium during the estrous cycle and early pregnancy.

Prostaglandins (PGs) play important functions in the reproductive system, and PGE(2) appears necessary for recognition of pregnancy. We have found that PGE(2) is able to increase cAMP generation in the bovine endometrium. There are two PGE(2) receptors (EP), EP2 and EP4, that are coupled to adenylate cyclase to generate cAMP, but these receptors have not been studied in the bovine. We have cloned and characterized bovine EP2 and EP4 receptors and studied their expression in the uterus. The amino acid sequences of bovine EP2 and EP4 possess a high degree (>80%) of identity with the other mammalian homologs. EP2 is expressed in most tissues, and EP4 is expressed only in intestine and testis. EP2 mRNA and protein are expressed in endometrium and myometrium during the estrous cycle, whereas EP4 is undetectable. The Western analysis indicates that EP2 is maximally expressed in both endometrium and myometrium between d 10 and 18 of the estrous cycle. Immunohistochemical localization reveals that EP2 protein is expressed in all cell types of endometrium and myometrium. On d 18, pregnancy up-regulates EP2 protein, primarily in endometrial stroma and myometrial smooth muscle cells. In conclusion, EP2 is the major cAMP-generating PGE(2) receptor expressed and regulated in the bovine uterus during the estrous cycle and early pregnancy.

Testosterone and estradiol differentially regulate thyroid growth in Wistar rats from immature to adult age.

Experimental and clinical data from cancerous thyroid tissue suggest gender as one of the major factors influencing thyroid growth. However, there is not much information on the effect of sex steroids on the normal development and growth of the thyroid. The aim of the present study was to examine the effect of sex steroids on thyroid growth, serum TSH and TSH receptors in the thyroid, at various stages of sexual maturation. Normal Wistar male and female rats from immature to adult age (day 21, 30, 45, 60 and 160 post partum (PP)) were used in the present study. One group of rats was gonadectomized (GDX) (Group II); another 2 groups of GDX rats (Groups III and IV) were treated with replacement doses (i.e. physiological amounts) of either testosterone or estradiol. Thyroid growth indices (thyroid weight, concentration of DNA, mitotic index and numerical density of thyrocytes), serum TSH and concentration of TSH receptors in the thyroid were assessed. It is concluded from the present study that (i) sex steroids influence thyroid growth by interfering with the secretion of TSH and regulating its receptor number in the thyroid (ii) while testosterone facilitates the mitogenic activity of the thyroid gland of rats irrespective of sex and age, estradiol plays a gender-specific stimulatory role on thyroid growth.