A proinflammatory response of bovine endometrial epithelial cells to active sperm in vitro.

In the cow, cryopreserved semen is inseminated into the uterus, and most of sperm are removed by backflow and phagocytes. Nevertheless, the mechanism responsible for sperm phagocytosis is unclear. Here, we used cultured bovine uterine epithelial cells (BUECs) to investigate the uterine response to sperm and the mechanism that activates polymorphonuclear neutrophils (PMNs). BUEC monolayers were co-cultured with different numbers of washed sperm obtained from cryopreserved semen (104 , 105 , and 106 sperm/ml) for 3 hr. Sperm dose-dependently up-regulated IL8 (Interleukin 8). Sperm at 106 /ml increased mRNA expression of TNFA (Tumor necrosis factor alpha), IL1B (Interleukin 1B), NFKB2 (Nuclear factor kappa B2), and C3 (Complement factor 3), as well as PGES (Prostaglandin E synthase) expression and PGE2 release. Live sperm, but not dead sperm, attached to BUECs, and dead sperm did not induce an acute inflammatory response. Time-dependent effects were evaluated by co-culture of 106 /ml washed sperm with BUECs for 0, 1, 3, and 6 hr. The number of detached sperm increased gradually toward 6 hr. Maximum mRNA expression of IL8, TNFA, IL1B, and NFKB2 was induced at 3 hr, while C3 continued to increase toward 6 hr. Sperm did not stimulate mRNA expression of anti-inflammatory cytokines TGFB1 (Transforming growth factor beta 1) or IL10 (Interleukin 10). Medium conditioned by sperm co-incubated with BUECs stimulated PMNs phagocytosis of sperm in vitro. Fresh media supplemented with low levels of IL1B, TNFA, and PGE2 up-regulated sperm phagocytosis by PMNs as well. In conclusion, our findings strongly suggest that the active sperm attach to BUECs and trigger uterine local innate immunity with induction of a pro-inflammatory response that enhances sperm phagocytosis by PMNs.

Bovine embryo induces an anti-inflammatory response in uterine epithelial cells and immune cells in vitro: possible involvement of interferon tau as an intermediator.

Recent observations suggest that the bovine uterus starts to react to the early embryo immediately after its arrival from the oviduct. The present study aimed to investigate the effect of the early developing embryo on the immune-related gene profile in bovine uterine epithelial cells (BUECs) in vitro, and to further examine the impact of conditioned media (CM), either from embryo-BUEC co-culture or embryo culture alone, on gene expression in peripheral blood mononuclear cells (PBMCs). First, BUECs were co-cultured with morulae (n = 10) for D5-D9 (D0 = IVF), and gene expression in BUECs was analyzed. Subsequently, PBMCs were cultured in CM from embryo-BUEC co-culture or D5-D9 embryo culture, and gene expression was evaluated. In BUECs, the embryo induced interferon (IFN)-stimulated genes (ISGs: ISG15, OAS1, and MX2), a key factor for IFN-signaling (STAT1), and type-1 IFN receptors (IFNAR1 and IFNAR2), with suppression of NFkB2, NFkBIA and pro-inflammatory cytokines (TNFA and IL1B). The embryo also stimulated PTGES and PGE2 secretion in BUECs. In PBMCs, both CM from embryo-BUEC co-culture and embryo culture alone induced ISGs, STAT1 and TGFB1, while suppressing TNFA and IL17. Similarly, interferon tau (IFNT) at 100 pg/ml suppressed NFkB2, TNFA and IL1B in BUECs, and also stimulated TGFB1 and suppressed TNFA in PBMCs. Our findings suggest that the bovine embryo, in the first four days in the uterus (D5-D9), starts to induce an anti-inflammatory response in epithelial cells and in immune cells. IFNT is likely to act as one of the intermediators for induction of the anti-inflammatory response in the bovine uterus.

Expression of glucocorticoid receptor α and its regulation in the bovine endometrium: possible role in cyclic prostaglandin F2α production.

Cortisol (Cr), the most important glucocorticoid (GC), is well known to suppress uterine prostaglandin F2α (PGF) production. However, the details of the regulatory mechanisms controlling the cyclic changes in endometrial PGF production remain unclear. Here we investigated the expression of the GC receptor (GC-Rα), the actions of cortisol throughout the estrous cycle and the regulatory mechanism of GC-Rα in the bovine endometrium. The levels of GC-Rα protein were greater at the mid-luteal stage (Days 8-12) than at the other stages. Cr more strongly suppressed PGF production at the mid-luteal stage than at the follicular stage. GC-Rα expression was increased by progesterone (P4) but decreased by estradiol-17ß (E2) in cultured endometrial stromal cells. The overall results suggest that ovarian steroid hormones control the cyclic changes in endometrial PGF production by regulating GC-Rα expression in bovine endometrial stromal cells.

PCR detection of Theileria equi and Babesia caballi in apparently healthy horses in Paraguay.

Equine piroplasmosis (EP) is a tick-borne disease caused by Theileria equi and Babesia caballi in equids, including horses. EP has a global distribution and often leads to a significant socioeconomic impact on the equine industry. Infected animals remain as carriers and become a source of infection for tick vectors, thereby posing an immense challenge in the disease management. Therefore, detection of these carriers is crucial to assess the risk of transmission and to implement appropriate control measures in endemic countries. Paraguay is a tropical country where various tick-borne diseases are common among livestock; however, the status of EP remains unknown in this country. Because the tick vectors capable of transmitting T. equi and B. caballi are endemic in Paraguay, we hypothesised that Paraguayan horses are infected with these parasite species. To test our hypothesis, we prepared blood DNA samples from a total of 545 apparently healthy horses in 16 of the 17 departments of Paraguay and analysed them with specific PCR assays to detect T. equi and B. caballi. The PCR results showed that 178 (32.7%) and 8 (1.5%) of the horses were infected with T. equi and B. caballi, respectively. Among the infected horses, two (0.4%) were infected with both parasite species. Our analyses further indicated that the positive rates of T. equi infection did not differ between horse breeds, males and females, or age groups. We also found that haematological parameters were the same between the non-infected animals and animals with single infections. By contrast, the two horses co-infected with T. equi and B. caballi had haemoglobin and haematocrit values lower than the normal ranges. In conclusion, the present study demonstrated that Paraguayan horses are infected with T. equi and B. caballi and that the rate of T. equi infection is higher than that of B. caballi. Our findings highlight the need to add EP to the list of differential diagnoses when anaemic horses are presented to equine clinics in Paraguay.

Roles of cytokines and progesterone in the regulation of the nitric oxide generating system in bovine luteal endothelial cells.

Nitric oxide (NO) produced by luteal endothelial cells (LECs) plays important roles in regulating corpus luteum (CL) function, yet the local mechanism regulating NO generation in bovine CL remains unclear. The purpose of the present study was to elucidate if tumor necrosis factor-α (TNF), interferon γ (IFNG), and/or progesterone (P4) play roles in regulating NO generating system in LECs. Cultured bovine LECs obtained from the CL at the mid-luteal stage (Days 8-12 of the cycle) were treated for 24 hr with TNF (2.9 nM), IFNG (2.5 nM), or P4 (0.032-32 µM). NO production was increased by TNF and IFNG, but decreased by P4 (P < 0.05). TNF and IFNG stimulated the relative steady-state amounts of inducible nitric oxide synthase (iNOS) mRNA and iNOS protein expression (P < 0.05), whereas P4 inhibited relative steady-state amounts of iNOS mRNA and iNOS protein expression (P < 0.05). In contrast, endothelial nitric oxide synthase (eNOS) expression was not affected by any treatment. TNF and IFNG stimulated NOS activity (P < 0.05) and 1400W, a specific inhibitor of iNOS, reduced NO production stimulated by TNF and IFNG in LECs (P < 0.05). Onapristone, a specific P4 receptor antagonist, blocked the inhibitory effect of P4 on NO production in LECs (P < 0.05). The overall findings suggest that TNF and IFNG accelerate luteolysis by increasing NO production via stimulation of iNOS expression and NOS activity in bovine LECs. P4, on the other hand, may act in maintaining CL function by suppressing iNOS expression in bovine LECs.

Roles of prostaglandin F2alpha and hydrogen peroxide in the regulation of Copper/Zinc superoxide dismutase in bovine corpus luteum and luteal endothelial cells.

BACKGROUND: Prostaglandin F2alpha (PGF) induces luteolysis in cow by inducing a rapid reduction in progesterone production (functional luteolysis) followed by tissue degeneration (structural luteolysis). However the mechanisms of action of PGF remain unclear. Reactive oxygen species (ROS) play important roles in regulating the luteolytic action of PGF. The local concentration of ROS is controlled by superoxide dismutase (SOD), the main enzyme involved in the control of intraluteal ROS. Thus SOD seems to be involved in luteolysis process induced by PGF in cow. METHODS: To determine the dynamic relationship between PGF and ROS in bovine corpus luteum (CL) during luteolysis, we determined the time-dependent change of Copper/Zinc SOD (SOD1) in CL tissues after PGF treatment in vivo. We also investigated whether PGF and hydrogen peroxide (H2O2) modulates SOD1 expression and SOD activity in cultured bovine luteal endothelial cells (LECs) in vitro. RESULTS: Following administration of a luteolytic dose of PGF analogue (0 h) to cows at the mid-luteal stage, the expression of SOD1 mRNA and protein, and total SOD activity in CL tissues increased between 0.5 and 2 h, but fell below the initial (0 h) level at 24 h post-treatment. In cultured LECs, the expression of SOD1 mRNA was stimulated by PGF (1-10 microM) and H2O2 (10-100 microM) at 2 h (P<0.05). PGF and H2O2 increased SOD1 protein expression and total SOD activity at 2 h (P<0.05), whereas PGF and H2O2 inhibited SOD1 protein expressions and total SOD activity at 24 h (P<0.05). In addition, H2O2 stimulated PGF biosynthesis at 2 and 24 h in bovine LECs. Overall results indicate that, SOD is regulated by PGF and ROS in bovine LECs. SOD may play a role in controlling intraluteal PGF and ROS action during functional and structural luteolysis in cows.

Seasonal changes in luteal progesterone concentration and mRNA expressions of progesterone synthesis-related proteins in the corpus luteum of mares.

Although circulating progesterone (P4) levels tend to change with the season, little is known about the seasonal changes of P4 synthesis-related proteins in the corpus luteum (CL) of mares. To examine these changes, seventy-four ovaries containing a CL were collected from Anglo-Norman mares at a local abattoir in Kumamoto, Japan (~N32°), five times during one year. The stages of the CLs were classified as early, mid and regressed by macroscopic observation of the CL and follicles. The mid CL, which had the highest P4 concentration, was used to evaluate the seasonal changes in P4 synthesis. The luteal P4 concentration and mRNA expression of luteinizing hormone receptor (LHCGR) were lowest during early winter and highest during late winter. The mRNA expressions of steroidogenic acute regulatory protein (StAR), P450 cholesterol side-chain cleavage enzyme (P450scc) and 3ß-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3ß-HSD) were lowest during early winter and increased during late winter. These results suggest that P4 synthesis in the CL is affected by the seasonal changes in the mRNA expressions of P4 synthesis-related proteins in mares.

Risk factors for equine trypanosomosis and hematological analysis of horses in Paraguay.

Animal trypanosomosis, caused by Trypanozoon trypanosomes (Trypanosoma evansi and T. equiperdum), and Trypanosoma vivax, is endemic to South American countries and has a negative impact on the livestock industry. However, the risk factors for trypanosomosis in Paraguay remain unknown. This study aimed to determine the risk factors for equine trypanosomosis in Paraguay based on a PCR-based molecular survey and individual horse sampling data. In this study, 739 blood samples were collected from horses in 16 departments of Paraguay between August 2019 and November 2020. To elucidate the risk factors for trypanosome infection, the relationship between trypanosome infection status detected by PCR and the location, sex, age, breed of horses, and season of sample collection was analyzed. There were no significant differences in trypanosome prevalence in horses between the eastern and western regions, ages, or breeds of horses in Paraguay. Sex and season were identified as risk factors for trypanosome infection in horses in Paraguay in the current study. These results suggest that the rainy-summer season, when vectors increase in number and their blood-sucking activity, could be the most important risk factor for trypanosome infection in Paraguay horses. Preventive measures and treatments should be developed to address these factors.

First molecular survey of animal trypanosomes in Paraguayan horses.

Despite the epidemic situation of animal trypanosomosis caused by Trypanosoma evansi, Trypanosoma equiperdum and Trypanosoma vivax in South American countries, there are no reports for the prevalence of animal trypanosomes in Paraguay. In this study, 408 blood samples were obtained from apparently healthy horses from sixteen departments of Paraguay, for routine medical check-up from August to September 2019, and a polymerase chain reaction (PCR)-based cross-sectional study was carried out to identify trypanosome prevalence. The prevalence of Trypanozoon (T. evansi and T. equiperdum) and T. vivax was 7.11% (29/408) and 26.23% (107/408), respectively. Mixed infections were detected in 4.90% (20/408) of the samples. Some of the selected trypanosome positive samples were confirmed as T. vivax and T. evansi Type A by sequence analysis of the internal transcribe spacer region and RoTat1.2 variant surface glycoprotein gene, respectively. In conclusion, we found higher prevalence of T. vivax than Trypanozoon in Paraguayan horses. However, the genotypic variation should be verified in further studies.

Is interleukin-1alpha a luteotrophic or luteolytic agent in cattle?

Cytokines are thought to regulate prostaglandin (PG) secretion in the bovine endometrium. However, there is no consensus about the role of interleukin-1alpha (IL1A) on PG secretion. The objective of this study was to examine the influence of IL1A on basal and interferon-tau (IFNT)-regulated PG in vitro secretion, as well its effects on PG secretion, progesterone (P(4)) output, and corpus luteum (CL) in vivo lifespan. Explants of bovine endometrium (days 16-17 of the estrous cycle or early pregnancy) were stimulated with IL1A (10 ng/ml), IFNT (30 ng/ml), or IL1A combined with IFN. IL1A alone strongly stimulated luteotrophic PGE(2) secretion by endometrial tissues of both pregnant and nonpregnant cows. IL1A also stimulated luteolytic PGF(2alpha) output in the late luteal phase. IFNT augmented the stimulatory effect of IL1A on PGE(2) secretion. In an in vivo experiment, saline or IL1A at different doses (0.001-10 microg/per animal) was applied to the uterine lumen on day 16 of the cycle. Only the highest dose of IL1A caused a temporal increase in PGF(2alpha) secretion, while it had no effect on P(4) secretion or CL lifespan. Application of 0.1 and 1 microg IL1A stimulated P(4) and PGE(2) output and prolonged the CL lifespan. Although IL1A may stimulate in vitro luteolytic PGF(2alpha) secretion during the estrous cycle, it only acts as a luteotrophic factor in vivo. IL1A increased luteotrophic PGE(2) and P(4) output, inhibiting spontaneous luteolysis. These luteotrophic effects may result in appropriate luteal development and function in cows during the estrous cycle and early pregnancy.

Effective oxytocin treatment on placental expulsion after foaling in heavy draft mares.

The aim of this study was to establish the effectiveness of administration of oxytocin (OT) on placental expulsion after foaling. Four foaling mares with the placentas retained for up 1 hr after foaling received OT (50 IU) administration at 1 hr intervals before expulsion of the placenta. The changes in the plasma concentrations of OT and the PGF2alpha metabolite (PGFM) were investigated, and the influence of OT administration was considered. The results were as follows. The placenta was expelled after one to three OT administrations in all four mares that received OT. In two mares, which expelled the placenta within 30 min after OT administration, the OT concentration increased and remained high. Expulsion of the placenta was delayed in two mares, and one of these mares, which received three doses of OT beginning 1 hr after foaling, showed only a small increase in the OT concentration after the first administration; the other mare did not receive OT until 3 hr after foaling. The OT concentration was increased before placental expulsion in all the mares, and the PGFM concentration also increased in the two mares with retained placentas. In conclusion, we suggest that intramuscular administration of 50 IU of OT at 1-hr intervals beginning 1 hr after foaling is effective for inducing placental expulsion.

Development of an enzyme immunoassay for urinary pregnanediol-3-glucuronide in a female giant panda (Ailuropoda melanoleuca).

In order to enable monitoring of the reproductive status of the female giant panda after observation of estrus behavior, we developed an enzyme immunoassay (EIA) system for urinary pregnanediol-3-glucuronide (PdG), a progesterone metabolite, using commercial reagents and examined the changes in the urinary concentration of PdG in a female giant panda that showed pseudopregnancy and suspicious pseudopregnancy in 6 consecutive years. The developed EIA system had good reproducibility (intra- and interassay CVs 6.1% and 16.3%, respectively), good parallelism between the standard curve and the dose response curve of serial diluted samples and positive correlation (r=0.836) with the data for PdG in the same samples measured by gas chromatography. Urinary PdG in the female panda showed two phases of increase. The first elevation was observed immediately after estrus with the levels of PdG below 100 ng/Crmg, while the second phase was characterized by a drastic elevation above 100 ng/Crmg until the level began to decrease at the end of pseudopregnancy or suspicious pseudopregnancy. The length of the second phase had wider range than that of the first phase. In the present study, a new EIA assay system for urinary PdG in the female giant panda was developed, and we found that the length of the second phase is unstable in the pseudopregnant and suspicious pseudopregnant giant panda, in contrast with the unstable length of the first phase caused by delayed implantation in the pregnant giant panda.

The role of glucocorticoid in the regulation of prostaglandin biosynthesis in non-pregnant bovine endometrium.

To determine whether glucocorticoids (GCs) play a role in regulating uterine function in cow, the present study examined the expression of mRNA encoding GC receptor (GC-R) alpha, 11beta-hydroxysteroid dehydrogenase (11-HSD) type 1 and type 2, and the activity of 11-HSD1 in bovine endometrial tissue throughout the estrous cycle. We also studied the effects of cortisol on basal, oxytocin (OT)- and tumor necrosis factor-alpha (TNFalpha)-stimulated prostaglandin (PG) production. A quantitative real-time PCR analysis revealed that GC-Ralpha mRNA was expressed more strongly in the mid-luteal stage (days 8-12) than in the other stages. In contrast to GC-Ralpha mRNA expression, 11-HSD1 mRNA expression was greater in the follicular stage than in the other stages, whereas 11-HSD2 mRNA expression was lowest in the follicular stage. The activity of 11-HSD1 was greater in the follicular stage and estrus than in the other stages and was lowest in the mid-luteal stage. Cortisone was dose-dependently converted to cortisol in the cultured endometrial tissue. Although cortisol did not affect either the basal or OT-stimulated production of PGs in the cultured epithelial cells, the production of PGs stimulated by TNFalpha in the stromal cells was suppressed by cortisol (P < 0.05). Cortisol suppressed basal prostaglandin (PG)F2alpha without affecting basal PGE2 production in the stromal cells. The overall results suggest that the level of cortisol is locally regulated in bovine endometrium throughout the estrous cycle by 11-HSD1, and that cortisol could act as a luteoprotective factor by selectively suppressing luteolytic PGF2alpha production in bovine endometrium.

Oxygen concentration is an important factor for modulating progesterone synthesis in bovine corpus luteum.

Oxygen deficiency caused by a decrease in the blood supply is known to induce various responses of cells. Because luteal blood flow has been shown to decrease during luteolysis, a low-oxygen condition seems to be an integral part of the environment during luteolysis. To determine whether a low-oxygen condition is associated with functional luteolysis, we examined the influence of reduced oxygen tension on the luteal progesterone (P4) generating system in cultured bovine midluteal cells. Luteal cells obtained from midcycle corpus luteum (d 8-12) were incubated under different O2 concentrations (20, 10, 5, 3% O2) with or without LH for 24 h. P4 production decreased with decreasing O2 concentration but was significantly stimulated by LH regardless of O2 concentration. After 8 h of culture, both basal and LH-stimulated P4 production was significantly lower under 3% O2 than under 20% O2. Low-oxygen condition also inhibited pregnenolone production. Cytochrome P450 side-chain cleavage enzyme (P450scc) mRNA expression, measured by quantitative PCR, decreased under low-oxygen condition in both non-LH-treated and LH-treated cells. Low-oxygen condition did not affect the expressions of steroidogenic acute regulatory protein mRNA or protein, whereas steroidogenic acute regulatory protein mRNA expression was stimulated by LH during 4 h of culture. Low-oxygen condition also did not affect 3 beta-hydroxysteroid dehydrogenase/Delta 5-Delta 4 isomerase mRNA expression or the activity of the enzyme in the cells, regardless of the incubation period. The overall results indicate that a low-oxygen condition decreases P4 synthesis by attenuating P450scc production and P450scc activity in bovine luteal cells and suggest that oxygen deficiency is an essential condition for the progression of luteolysis in cattle.

Role of intraluteal prostaglandin F(2alpha), progesterone and oxytocin in basal and pulsatile progesterone release from developing bovine corpus luteum.

The present study examined the role of intra-luteal prostaglandin (PG) F(2alpha), progesterone (P4) and oxytocin (OT) on the corpus luteum function by using specific hormone antagonists. Luteal cells from the developing CL (days 5-7 of the estrous cycle) were exposed to P4 antagonist (onapristone, OP, 10(-4)M), OT antagonist (atosiban, AT; 10(-6)M) or indomethacin (INDO; 10(-4)M), for 12h and then stimulated with PGF(2alpha) (10(-8)M) for 4h. Pre-treatment of the cells with OP, AT or INDO resulted in an increase in P4 secretion in response to PGF(2alpha). To examine the temporal effects of P4, OT and PGs on P4 secretion, dispersed luteal cells were pre-exposed to OP, AT or INDO for 1, 2, 4, 6 or 12h. Prostaglandin F(2alpha) stimulated P4 secretion (P<0.05) after 2h of pre-exposition. In the microdyalisis study, the spontaneous release of P4 from developing CL tissue was of pulsatile nature with irregular peaks at 1-2h intervals. Treatment with OP increased the number of P4 peaks (P<0.05), whereas AT and INDO significantly reduced the number of P4 peaks detected (P<0.05). Interestingly, INDO completely blocked the pulsatile nature in the release of P4, but it secretion remained stable throughout the experimental period. These results demonstrate that luteal PGF(2alpha), OT, and P4 are components of an autocrine/paracrine intra-ovarian regulatory system responsible for the episodic (pulsatile) release of P4 from the bovine CL during the early luteal phase.

Phytoestrogens modulate prostaglandin production in bovine endometrium: cell type specificity and intracellular mechanisms.

Prostaglandins (PGs) are known to modulate the proper cyclicity of bovine reproductive organs. The main luteolytic agent in ruminants is PGF2alpha, whereas PGE2 has luteotropic actions. Estradiol 17beta (E2) regulates uterus function by influencing PG synthesis. Phytoestrogens structurally resemble E2 and possess estrogenic activity; therefore, they may mimic the effects of E2 on PG synthesis and influence the reproductive system. Using a cell-culture system of bovine epithelial and stromal cells, we determined cell-specific effects of phytoestrogens (i.e., daidzein, genistein), their metabolites (i.e., equol and para-ethyl-phenol, respectively), and E2 on PGF2alpha and PGE2 synthesis and examined the intracellular mechanisms of their actions. Both PGs produced by stromal and epithelial cells were significantly stimulated by phytoestrogens and their metabolites. However, PGF2alpha synthesis by both kinds of cells was greater stimulated than PGE2 synthesis. Moreover, epithelial cells treated with phytoestrogens synthesized more PGF2alpha than stromal cells, increasing the PGF2alpha to PGE2 ratio. The epithelial and stromal cells were preincubated with an estrogen-receptor (ER) antagonist (i.e., ICI), a translation inhibitor (i.e., actinomycin D), a protein kinase A inhibitor (i.e., staurosporin), and a phospholipase C inhibitor (i.e., U73122) for 0.5 hrs and then stimulated with equol, para-ethyl-phenol, or E2. Although the action of E2 on PGF2alpha synthesis was blocked by all reagents, the stimulatory effect of phytoestrogens was blocked only by ICI and actinomycin D in both cell types. Moreover, in contrast to E2 action, phytoestrogens did not cause intracellular calcium mobilization in either epithelial or stromal cells. Phytoestrogens stimulate both PGF2alpha and PGE2 in both cell types of bovine endometrium via an ER-dependent genomic pathway. However, because phytoestrogens preferentially stimulated PGF2alpha synthesis in epithelial cells of bovine endometrium, they may disrupt uterus function by altering the PGF2alpha to PGE2 ratio.

Lack of Rev7 function results in development of tubulostromal adenomas in mouse ovary.

Rev7 is a subunit of Polζ, one of the translesion DNA synthesis (TLS) polymerases involved in DNA damage repair. We recently found that Rev7 is also essential for germ cell development in mouse. In the present study, we found the development of ovarian tumors in Rev7 mutant mouse, suggesting the involvement of TLS deficiency in the etiology of ovarian tumor. The Rev7 mutant mice showed complete lack of oocytes and follicles in the ovary. The lack of follicles causes a significant increase of gonadotropin level and an increase in the proliferation of ovarian cells. As a result, the weight of the ovaries of Rev7 mutant mice increased with age and they developed tubulostromal adenomas. However, the remarkable overgrowth of ovaries occurred after gonadotropin level decreases at older ages, suggesting gonadotropin-independent progression of the ovarian tumors. In addition, the Rev7 mutant fibroblasts and ovarian cells showed significant accumulation of DNA damage. These findings suggest that not only increased gonadotropin levels but also lack of DNA damage repair function could be responsible for the development of ovarian tumors in the Rev7 mutant mouse.

Endothelin-1 within the corpus luteum during spontaneous luteolysis in the cow: local interaction with prostaglandin F2alpha and angiotensin II.

It is generally accepted that endothelin-1 (ET-1) is involved in the regression of the corpus luteum (CL) in cows, but there are few in vivo data available on the local release of vasoconstrictors, including ET-1. Thus, we aimed to determine in detail the local secretion of ET-1, angiotensin II (Ang II) and prostaglandin F2alpha (PGF2alpha) within the CL during spontaneous luteolysis in the cow. To observe real-time dynamics of the releasing profile of CL-derived factors, a microdialysis system was surgically implanted in the CL on day 15 of the estrous cycle and continuously perfused with Ringer's solution. Local secretion of ET-1, Ang II and PGF2alpha increased immediately after the onset of luteolysis (the time point when progesterone release started to decrease within the CL) and was maintained at high levels. A positive relationship was observed in intra-luteal changes among ET-1, Ang II and PGF2alpha release. This is the first real-time and in vivo evidence that the secretion of ET-1 together with Ang II and PGF2alpha immediately increases within the CL after the onset of spontaneous luteolysis. Consequently, we suggest that the activation of a local positive feedback mechanism among ET-1, Ang II and PGF2alpha might play a functional role in the paracrine modulation of luteolytic cascade and, simultaneously, the elevated ET-1 with Ang II and PGF2alpha should induce a strong vasoconstriction, thereby reducing the blood supplying the CL during spontaneous luteolysis.

Changes in prostaglandin secretion by the regressing bovine corpus luteum.

Secretion of prostaglandins (PGs) by the regressing corpus luteum (CL) was investigated in the cow. Six cows were implanted with microcapillary dialysis membranes of a microdialysis system (MDS) into the CL during Days 8-9 (Day 0 = estrus), and a prostaglandin (PG) F2alpha analogue (Estrumate) was injected intramuscularly (i.m.) to induce luteolysis. Acute increases in intraluteal release of PGF2alpha and PGE2 were observed during the first 4 h, followed by decreases over the next 8 h. Intraluteal release of both PGs gradually increased again during the period 48-72 h. Concentrations of PGF2alpha in ovarian venous plasma (OVP) were 4-13 times higher than those of jugular venous plasma (JVP) (P < 0.001) during the period of the experiment, and increased from 24 h after treatment with Estrumate (P < 0.05). Cyclooxygenase (COX)-2 mRNA expression increased (P < 0.05) at 2 and 24 h after treatment with Estrumate. The results indicated that local release of PGF2alpha and PGE2, and COX-2 mRNA expression were increased by Estrumate in the regressing CL at the later stages of luteolysis. Thus, luteal secretion of PGs may be involved in the local mechanism for structural rather than functional luteolysis.

Effects of LH and PGF2alpha in equine dominant follicles observed by MDS.

Microdialysis System (MDS) is a novel technique used for investigation of molecule secretion between different cell populations. Local hormonal secretion at follicular wall has been still unclear. This MDS study was used to determine progesterone (P4), androstenedione (A4), estradiol-17beta (E2) and Prostaglandin F2alpha (PGF2alpha) release in mare pre-ovulatory follicles. Follicles larger than 30 mm were isolated from the ovary and follicular fluid aspirated for hormone assay. Follicular fluid collected from small, middle and large follicles were analyzed by EIA. The concentrations of P4 and PGF2alpha were similar among the different sizes of follicles. The release of A4 was observed in middle and large follicles. E2 concentration was observed in middle follicles and was higher in large follicles compared with middle follicles. Follicular wall was cut and incubated for MDS and when LH was infused, there was an increase in P4 and A4 release. PGF2alpha release was considerably high after LH infusion compared to the control group. Infusion of PGF2alpha increased P4 and A4 release but there was no change in E2 release. This results suggest that in pre-ovulatory follicles, LH stimulates theca interna cells and also PGF2alpha seemed to have a mediator role to induce steroid hormone production and luteinization of follicular cells. The nature of the mechanisms involved in selection of large follicles is still a perplexing research problem in reproduction.