Lysophosphatidic acid expression in theca cells depends on the type of bovine ovarian follicle.

Lysophosphatidic acid (LPA) exerts various actions on the mammalian reproductive system. In cows, LPA stimulates the synthesis and secretion of luteotropic factors in the ovary, which affects the growth and development of ovarian follicles. The role of LPA in granulosa cells, oocyte and oocyte-cumulus complex (COC) has previously been investigated; but its role in the theca layer, which is an important structural and functional component of the ovarian follicle, is still unclear. The goal of this study was to investigate the expression of LPA in theca cells originating from different bovine ovarian follicle types. Theca cells were separated from healthy, transitional and atretic ovarian follicles, based on intrafollicular estradiol: progesterone ratios. LPA concentration in the follicular fluid (FF) in different follicle types was measured, and expression of the enzymes responsible for LPA synthesis (autotaxin [AX], phospholipase A2 [PLA2]) and receptors for LPA (LPAR1-4) were determined. The obtained results confirmed the follicle-type dependent presence of LPA in the FF of the bovine ovarian follicles. The highest concentration of LPA was detected in follicles classified as healthy and dominant. LPAR1-4, PLA2 and AX expression in theca cells in all of the types of follicles examined were detected at mRNA and protein level. These results suggest that theca cells can be a source of LPA synthesis other than granulosa cells and COCs, as well as the target for its action in the bovine ovarian follicle, with PLA2 and LPAR4 playing major roles in LPA synthesis and action.

Lysophosphatidic Acid Synthesis and its Receptors' Expression in the Bovine Oviduct During the Oestrous Cycle.

Lysophosphatidic acid (LPA) is a naturally occurring simple phospholipid which in the bovine reproductive system can be produced in the endometrium, corpus luteum, ovarian follicle and embryo. In this study, we examined the possibility that LPA receptors are expressed, and LPA synthesized, in the bovine oviduct. We found that the concentration of LPA was highest in infundibulum in the follicular phase of the oestrous cycle and was relatively high during the early-luteal phase in all examined parts of the oviduct. We also documented that LPA synthesis engages both available pathways for LPA production. The autotaxin (ATX) protein expression was significantly higher in the infundibulum compared to the isthmus during the follicular phase of the oestrous cycle. During the early-luteal phase of the oestrous cycle, ATX and phospholipase A2 (PLA2) protein expression was highest in ampulla, although the expression of LPARs was not as dynamic as LPA concentration in the oviduct tissue, and we presume that in the bovine oviduct, the most abundantly expressed receptor is LPAR2. In conclusion, our results indicate that the bovine oviduct is a site of LPA synthesis and a target for LPA action in the bovine reproductive tract. We documented that LPAR2 is the most abundantly expressed in the bovine oviduct. We hypothesize that in the bovine oviduct, LPA may be involved in the transport of gametes, fertilization and cellular signalling between the oviduct and cumulus-oocyte complex.

Studies on lysophosphatidic acid action during in vitro preimplantation embryo development.

Assisted reproductive technologies, including in vitro embryo production (IVP), have been successfully used in animal reproduction to optimize breeding strategies for improved production and health in animal husbandry. Despite the progress in IVP techniques over the years, further improvements in in vitro embryo culture systems are required for the enhancement of oocyte and embryo developmental competence. One of the most important issues associated with IVP procedures is the optimization of the in vitro culture of oocytes and embryos. Studies in different species of animals and in humans have identified important roles for receptor-mediated lysophosphatidic acid (LPA) signaling in multiple aspects of human and animal reproductive tract function. The data on LPA signaling in the ovary and uterus suggest that LPA can directly contribute to embryo-maternal interactions via its influence on early embryo development beginning from the influence of the ovarian environment on the oocyte to the influence of the uterine environment on the preimplantation embryo. This review discusses the current status of LPA as a potential supplement in oocyte maturation, fertilization, and embryo culture media and current views on the potential involvement of the LPA signaling pathway in early embryo development.

Lipopolysaccharides, cytokines, and nitric oxide affect secretion of prostaglandins and leukotrienes by bovine mammary gland epithelial cells.

The aims of this study were to determine the effects of lipopolysaccharides (LPS), tumor necrosis factor (TNF), interleukin 1 alpha (IL-1α), nitric oxide donor (NONOate), or the combination of TNF + IL-1α + NONOate on the following: (i) secretion of prostaglandin (PG)-F(2α), PGE(2), leukotriene (LT)-B(4), and LTC(4) by epithelial cells of the teat cavity and lactiferous sinus of bovine mammary gland; (ii) messenger RNA (mRNA) transcription of enzymes responsible for arachidonic acid (AA) metabolism (prostaglandin-endoperoxide synthase 2 [PTGS2], prostaglandin E synthase [PTGES], prostaglandin F synthase [PGFS], and arachidonate 5-lipooxygenase [ALOX5]); and (iii) proliferation of the cells. The cells were stimulated for 24 h. Prostaglandins and LT were measured by enzyme immunoassay, mRNA transcription of enzymes was determined by real-time reverse transcription polymerase chain reaction, and the cell viability was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. All factors increased PG secretion, but the highest stimulation was observed after TNF and IL-1α (P < 0.001). Tumor necrosis factor, NONOate, and TNF + IL-1α + NONOate increased LTB(4) production (P < 0.01), whereas LTC(4) was increased by LPS, TNF, and IL-1α (P < 0.01). Lipopolysaccharides, TNF, IL-1α, and the reagents combination increased PTGS2, PTGES, and PGFS mRNA transcription (P < 0.01), whereas ALOX5 mRNA transcription was increased only by TNF (P < 0.001). Lipopolysaccharides, TNF, IL-1α, NONOate, and the combination of reagents increased the cell number (P < 0.001). Mediators of acute-clinical Escherichia coli mastitis locally modulate PG and LT secretion by the epithelial cells of the teat cavity and lactiferous sinus, which might be a useful first line of defense for the bovine mammary gland. Moreover, the modulation of PG and LT secretion and the changing ratio of luteotropic (PGE(2), LTB(4)) to luteolytic (PGF(2α), LTC(4)) metabolites may contribute to disorders in reproductive functions.

Acute changes in the concentrations of prostaglandin F2α (PGF) and cortisol in uterine and ovarian venous blood during PGF-induced luteolysis in cows.

Prostaglandin F2α (PGF) is considered to be the main luteolysin in cattle. We have previously demonstrated that cortisol (Cr) suppresses PGF production in non-pregnant bovine endometrium. This study was carried out to test whether exogenous PGF increases ovarian and/or uterine PGF production and to determine the temporal relationship between PGF and Cr in ovarian and uterine circulations during PGF-induced luteolysis in cows. Catheters were inserted into the ovarian vein (OV), uterine vein (UV) and jugular vein (JV) of 10 cows on Day 9 of the oestrous cycle (Ovulation = Day 0) for frequent blood collection. On Day 10, the cows were divided randomly into two groups and treated with a luteolytic dose of a PGF analogue (cloprostenol) or saline solution. Blood samples were collected at -0.25, 0, 0.25, 0.5, 1 and 2 h and then at 2-h intervals until 12 h after treatment (0 h). The basal concentrations of PGF and Cr in OV and UV plasma were not significantly different. Injection of a PGF analogue induced more than twofold increases in the levels of PGF between 0.25 and 1 h in UV plasma, but not in OV plasma. PGF increased (p < 0.05) the concentrations of Cr in OV, UV and JV plasma between 0.5 and 1 h. The Cr levels in OV, UV and JV plasma were similar. The PGF levels in UV plasma decreased after Cr reached its highest levels. The overall results suggest that the uterus rather than the ovary increases PGF production in response to PGF injection. Based on the temporal changes of PGF and Cr in the ovarian and uterine circulations, Cr may act to reduce uterine PGF production in non-pregnant cows in vivo.

Experimentally induced mastitis and metritis modulate soy bean derived isoflavone biotransformation in diary cows.

The present study compared the changes in isoflavone (daidzein and genistein) and their metabolite (equol and para-ethyl-phenol) concentrations in the blood plasma of cows with induced mastitis and metritis after feeding with soy bean. Sixteen cows were divided into four groups: control for mastitis group, cows with induced mastitis group, control for metritis group, and cows with induced metritis group. All cows were fed a single dose of 2.5 kg of soy bean and then blood samples were taken from the jugular vein for 8 h at predetermined intervals. The concentrations of soy bean-derived isoflavones and their active metabolites were measured in the blood plasma on HPLC system. ß-Glucuronidase activity in the blood plasma of cows was measured by fluorometric method. In the blood plasma of cows with induced mastitis and metritis, we found considerably higher concentrations and time-dependent increase in isoflavone metabolites (equol and para-ethyl-phenol) with reference to cyclic cows (P < 0.05). Moreover, we noticed significant decrease of genistein in the blood plasma of the cows with induced metritis compared with control cows (P < 0.05). In addition, in the blood plasma of the cows with induced metritis, we found an increase in ß-glucuronidase activity compared with control cows (P < 0.05). In conclusion, health status of the females influenced the concentrations of isoflavone metabolites in the blood plasma of the cows. Experimentally induced mastitis and metritis increased isoflavone absorption, biotransformation and metabolism. Therefore, we suggest that cows with induced mastitis and metritis are more exposed to active isoflavone metabolite actions than healthy cows.

Ovarian steroids modulate tumor necrosis factor-α and nitric oxide-regulated prostaglandin secretion by cultured bovine oviductal epithelial cells.

Ovarian steroids assure an optimum environment for the final maturation of oocytes, gamete transport, fertilization, and early embryonic development. The aim of experiment 1 was to examine the influence of ovarian steroids on tumor necrosis factor-α (TNF-α)- or nitric oxide (NO)-regulated prostaglandin (PG), and nitrite/nitrate (NO2/NO3) secretion by cultured bovine oviductal epithelial cells (BOECs). BOECs were pretreated with 17ß-estradiol (E2; 10⁻9 M) and/or progesterone (P4; 10⁻7 M) for 24 h. For the next 24 h, BOECs were treated with TNF-α (10 ng/mL) or spermine nitric oxide complex (NONOate; 10⁻5 M). Prostaglandin F(2α) and PGE2 secretion was measured in medium by ELISA. The pretreatment of cells with P4 (progesterone), E2 (17 ß-estradiol), or E2/P4 augmented TNF-α-induced PGF(2α) and PGE2 secretion (P < 0.01). The pretreatment of cells with E2 or E2/P4 increased NONOate-induced PGF(2α) and PGE2 secretion (P < 0.01). TNF-α induced NO2/NO3 production by BOECs. The pretreatment of cells with E2 augmented only TNF-α-induced NO2/NO3 production (P < 0.05). The aim of experiment 2 was to examine the influence of TNF-α, NO, and ovarian steroids on the protein content of enzymes specifically involved in PG and NO production, PG synthases, and NO synthases (NOSs). BOECs were treated with TNF-α (10 ng/mL) or NONOate (10⁻5 M). TNF-α increased the protein content of PGG/H synthase, PGF synthase, and PGE synthase (P < 0.05) and endothelial and inducible NOSs (P < 0.05). Nitric oxide increased the protein content of PGF synthase, PGE synthase, endothelial NOS, and inducible NOS (P < 0.05). These results show possible linkage between TNF-α and NO, modulated by ovarian steroids, in the regulation of PG synthesis by BOECs that may be important for triggering the process of oviductal contractions.

Nitric oxide stimulates progesterone and prostaglandin E2 secretion as well as angiogenic activity in the equine corpus luteum.

Cytokines and nitric oxide (NO) are potential mediators of luteal development and maintenance, angiogenesis, and blood flow. The aim of this study was to evaluate (i) the localization and protein expression of endothelial and inducible nitric oxide synthases (eNOS and iNOS) in equine corpora lutea (CL) throughout the luteal phase and (ii) the effect of a nitric oxide donor (spermine NONOate, NONOate) on the production of progesterone (P4) and prostaglandin (PG) E(2) and factor(s) that stimulate endothelial cell proliferation using equine luteal explants. Luteal tissue was classified as corpora hemorrhagica (CH; n = 5), midluteal phase CL (mid-CL; n = 5) or late luteal phase CL (late CL; n = 5). Both eNOS and iNOS were localized in large luteal cells and endothelial cells throughout the luteal phase. The expression of eNOS was the lowest in mid-CL (P < 0.05) and the highest in late CL (P < 0.05). However, no change was found for iNOS expression. Luteal explants were cultured with no hormone added or with NONOate (10(-5) M), tumor necrosis factor-α (TNFα; 10 ng/mL; positive control), or equine LH (100 ng/mL; positive control). Conditioned media by luteal tissues were assayed for P4 and PGE(2) and for their ability to stimulate proliferation of bovine aortic endothelial cells (BAEC). All treatments stimulated release of P4 in CH, but not in mid-CL. TNFα and NONOate treatments also increased PGE(2) levels and BAEC proliferation in CH (P < 0.05). However, in mid-CL, no changes were observed, regardless of the treatments used. These data suggest that NO and TNFα stimulate equine CH secretory functions and the production of angiogenic factor(s). Furthermore, in mares, NO may play a role in CL growth during early luteal development, when vascular development is more intense.

Effects of exogenous tumour necrosis factor-alpha on the secretory function of the bovine reproductive tract depend on tumour necrosis factor-alpha concentrations.

The aim of study was to correlate tumour necrosis factor-alpha (TNF) infused doses used with the TNF concentrations achieved and with the secretory function of both the ovary and the uterus in cows. We evaluated the concentrations of progesterone (P4), prostaglandin (PG)F(2alpha), PGE(2) nitric oxide (NO) and TNF in the jugular vein and vena cava caudalis as parameters of exogenous TNF action on the female reproductive tract. Aortae abdominalis of cows (n = 18) were infused with saline or two doses of TNF (luteolytic--1 microg or luteotrophic--10 microg). In the peripheral blood, 1 microg TNF concentrations achieved within the range of 30-45 pg/ml, and 10 microg TNF provoked a sharp increase in achieved concentrations at a range of 250-450 pg/mL). The TNF concentrations achieved in vena cava caudalis were five to six times higher than that in peripheral blood (p < 0.001). One microgram TNF increased PGF(2alpha) and NO (p < 0.001) and decreased P4 (p < 0.05). The higher TNF dose stimulated P4 and PGE(2) (p < 0.01). TNF infusion at luteolytic dose achieved its concentrations at the physiological range previously observed in cows. Luteotrophic TNF dose achieved the concentrations in vena cava caudalis that are much higher than physiological level and were previously noted in pathological circumstances (i.e. mastitis, metritis).

Assessment of selected quality parameters of epididymal cat (Felis catus s. domestica, L. 1758) sperm using flow cytometry method and computer assisted sperm analyser.

The improvement of biotechnical methods connected with fast and precise semen quality assessment and its utilization in assisted reproductive techniques is an urgent necessity in felids. The aim of this study was to evaluate some quality parameters (i.e. the viability and share of cells with intact plasma membrane) of epididymal sperm of cats using the flow cytometry method and computer-assisted sperm analysis (CASA) examination. The material consisted of epididymal spermatozoa flushed from 22 pairs of epididymes after routine neutering procedures obtained from domestic cats aged between 8 and 36 months. The epididymes were cut and incubated with an extender without egg yolk. The samples were assessed for sperm viability (Live/Dead Sperm Viability Kit), percentage of subtle membrane changes (Apoptosis Detection Kit) and motility using FACScalibur flow cytometer and assisted sperm analyser HTM IVOS version 12.2. The flow cytometry method revealed 71.3% and 84.4% of live sperm using Live/Dead Sperm Viability Kit and Apoptosis Detection Kit respectively. The population of early-apoptotic and late-apoptotic sperm were 0.8% and 1.1% respectively. The CASA examination found 51.5% of motile sperm. However, the motility examination under light microscope revealed 69.5% of motile sperm. The data revealed an indistinctive per cent of apoptotic cells and 18.9% and 15.6% of dead cells using Live/Dead Sperm Viability Kit and Apoptosis Detection Kit, respectively, which indicate that the sperm obtained after flushing the epididymis possess potential properties for further assisted reproduction techniques.

Luteolytic effect of prostaglandin F 2 alpha on bovine corpus luteum depends on cell composition and contact.

Prostaglandin F(2 alpha) (PGF(2 alpha)) is a main luteolytic factor in vivo; however, its direct luteolytic influence on steroidogenic cells of bovine corpus luteum (CL) is controversial and not fully understood. The aim of the study was to clarify PGF(2 alpha) action on bovine CL in different in vivo and in vitro conditions and to examine whether the contact among all main types of CL cells is necessary for luteolytic PGF(2 alpha) action. In experiment 1, the bovine CL (day 15 of the oestrous cycle) was perfused using in vivo microdialysis system with dinoprost (an analogue of PGF(2 alpha)) for 0.5 h. Dinoprost caused a short-time increase in progesterone (P4), whose concentration decreased thereafter (at 6-, 10-, 12- and 24-h after treatment). In experiment 2, the direct effect of PGF(2 alpha) on P4 accumulation in CL steroidogenic cells cultured in monolayer (day 15 of the cycle) was determined. PGF(2 alpha) after 24 h of incubation increased P4 accumulation in steroidogenic CL cells. In experiment 3 steroidogenic, endothelial CL and immune cells (day 15 of the cycle) were incubated with PGF(2 alpha) in cocultures for 24 h in glass tubes and the levels of P4, stable metabolites of nitric oxide (NO) and leukotriene (LT) C(4) were determined. Although PGF(2 alpha) treatment increased P4 secretion in homogeneous steroidogenic CL cell culture, the decrease in P4 secretion in cocultures of all types of CL cells was observed. The secretion of NO and LTC(4) increased after the treatment of PGF(2 alpha) both in pure cultures of CL cells and in cocultures. The interactions between endothelial and immune cells with steroidogenic CL cells are needed for luteolytic PGF(2 alpha) action within the bovine CL. Our results indicate that the cell coculture model, including the main types of CL cells, is the most approximate to study PGF(2 alpha) role in vitro.

Expression of enzymes of cyclooxygenase pathway and secretion of prostaglandin E2 and F2alpha by porcine myometrium during luteolysis and early pregnancy.

UNLABELLED: Past studies of uterine prostaglandin (PGs) and pig reproduction have focused on endometrial rather than myometrial PGs. This study documents the synthesis and secretion of myometrial prostaglandins (PGs) in pigs and the involvement of oxytocin (OT) in these processes. Cyclooxygenase-2 (COX-2) expression was similar in myometrial explants from cyclic and pregnant pigs (days 14-16) and OT (10(-7) M) in vitro significantly increased COX-2 protein regardless of reproductive state. Basal expression of prostaglandin E2 synthase (PGES) was higher during pregnancy than during luteolysis. Conversely, prostaglandin F synthase (PGFS) was highest during luteolysis and lower in myometrium from gravid animals. OT had no influence on the expression of PGES and PGFS. In another tissue culture experiment, myometrial slices produced more PGE2 than PGF2alpha regardless of reproductive state of the female. OT stimulated PGE2 production in myometrium harvested during luteolysis and increased PGF2alpha production in all tissues examined. Progesterone (P4; 10(-5) M) blocked stimulatory effect of OT on myometrial PG release. Myometrial OTr mRNA was higher (P=0.03) during luteolysis than during pregnancy. IN CONCLUSION: (1) oxytocin increases myometrial COX-2 expression, but does not influence the expression of terminal enzymes of PGs synthesis (PGES and PGFS); (2) porcine myometrium preferentially produces PGs during early pregnancy and secretes more PGE2 than PGF2alpha; (3) myometrial OT and OTr support secretion of PGs from myometrium during luteolysis.

Phytoestrogen metabolites are much more active than phytoestrogens themselves in increasing prostaglandin F(2alpha) synthesis via prostaglanin F(2alpha) synthase-like 2 stimulation in bovine endometrium.

Phytoestrogens have recently been suggested to be the cause of infertility by stimulating luteolytic prostaglandin (PG) F(2alpha) secretion from endometrium in cattle. The purpose of this study was to examine the enzymatic and molecular mechanisms involved in the preferential induction of PGF(2alpha) synthesis by phytoestrogens, and whether phytoestrogens influence endometrial cell viability. Cultured bovine endometrial epithelial and stromal cells were exposed to phytoestrogens (daidzein and genistein) and their metabolites (equol and p-ethyl phenol) for 24h. Prostaglandin F(2alpha) and PGE2 were stimulated by phytoestrogens in both stromal and epithelial cells, with a preference for PGF(2alpha) synthesis in epithelial cells (P<0.001). Although RT-PCR and Western Blot analyses did not reveal the influence of phytoestrogens on either gene expression or protein level of cyclooxygenase-2 (COX-2) and PGE2 synthase (PGES) in stromal and epithelial cells (P>0.05), the stimulative effects of equol and p-ethyl phenol on PGF(2alpha) synthase-like 2 (PGFSL2) gene expression and protein level were observed only in epithelial cells (P<0.05). The same compounds did not affect PGFSL2 gene expression and protein in stromal cells (P>0.05). Exposure to phytoestrogens and their metabolites decreased cell viability in both stromal and epithelial cells. Stromal cell viability decreased to 50% of the control and was more evident than that in epithelial cells (P<0.001). The overall results suggest that infertility in cattle, caused by phytoestrogen-dependent preferential stimulation of luteolytic PGF(2alpha) synthesis, is caused by increasing PGFSL2 in epithelial cells, and by decreasing stromal cell viability, which are the main source of luteotropic PGE2 production.