Nodal/Activin signaling drives self-renewal and tumorigenicity of pancreatic cancer stem cells and provides a target for combined drug therapy

Nodal and Activin belong to the TGF-β superfamily and are important regulators of embryonic stem cell fate. Here we investigated whether Nodal and Activin regulate self-renewal of pancreatic cancer stem cells. Nodal and Activin were hardly detectable in more differentiated pancreatic cancer cells, while cancer stem cells and stroma-derived pancreatic stellate cells markedly overexpressed Nodal and Activin, but not TGF-β. Knockdown or pharmacological inhibition of the Nodal/Activin receptor Alk4/7 in cancer stem cells virtually abrogated their self-renewal capacity and in vivo tumorigenicity, and reversed the resistance of orthotopically engrafted cancer stem cells to gemcitabine. However, engrafted primary human pancreatic cancer tissue with a substantial stroma showed no response due to limited drug delivery. The addition of a stroma-targeting hedgehog pathway inhibitor enhanced delivery of the Nodal/Activin inhibitor and translated into long-term, progression-free survival. Therefore, inhibition of the Alk4/7 pathway, if combined with hedgehog pathway inhibition and gemcitabine, provides a therapeutic strategy for targeting cancer stem cells.     

Is the bryophyte soil diaspore bank buffered against nutrient enrichment and grazing exclusion?

Plant and Soil - Soil diaspore banks of bryophytes are poorly known in tundra grasslands, yet can be important for the maintenance of local bryophyte assemblages. We examined the effects of...     

High fertility using artificial insemination during deep anoestrus after induction and synchronisation of ovulatory activity by the "male effect" in lactating goats subjected to treatment with artificial long days and progestagens

The response to the male effect was studied in two Saanen and two Alpine flocks over 5 consecutive years. Adult male and female goats were exposed to artificial long days (16h light and 8h darkness, 16L:8D) in open barns for approximately 3 months (between December 1 and April 15) followed by a natural photoperiod. Goats were treated for 11 days with fluorogestone acetate (FGA) or progesterone (CIDR) immediately before joining. Bucks carrying marking harnesses with adapted aprons joined females 49-63 days after the end of the long-day treatment (between April 30 and June 5) and were left with them for 5 days. In experiment 1 (n=142), FGA- and CIDR-treated goats were inseminated at a time based on the detection of oestrus. Two insemination groups were distinguished by the occurrence of marking over a 48-h period. Earlier (group 1) and later (group 2) buck-marked goats received one single insemination 12-24h or 0-12h after marking, respectively. Unmarked goats were inseminated along with group 2. In experiment 2 (n=344), FGA-treated goats were inseminated 52 and 70 h (52 h:70 h group) or 52 and 75 h (52 h:75 h group) after joining. In experiment 3 (n=285), FGA-treated goats were inseminated 52 h (1-AI group) or 52 and 75 h (2-AI group) after joining. In all experiments, an external control group given the "classical" insemination program was analysed. Over the 5-year period, 92% of the goats exhibited an LH surge during days 1-4 after joining and 98% of them ovulated. Eighty-seven percent of the LH surges detected in milk occurred during the 33-57 h interval after joining, indicating that ovulation took place around 45-69 h. In experiment 1, 96% of the goats were marked 22-70 h after joining. Kidding rate (KR; 78%) was similar between insemination groups and between FGA- and CIDR-treated goats (p>0.05). Most of the goats (95%) were inseminated during the interval between 15h before and up to 4h after ovulation. KR was not affected by the time between detection of marking and insemination or between insemination and ovulation (p>0.05). In experiment 2, KR (75%) was similar in both insemination groups (p>0.05). In experiment 3, KR was higher (p<0.05) in the 1-AI (71%) than the 2-AI group (57%). In all experiments, KR of the control group (68-73%) was similar to that achieved in goats induced to ovulate by the male effect. Prolificity (2.1+/-0.7) was not affected by any of the factors examined (p>0.05). In conclusion, high fertility can be achieved during anoestrus when 1 or 2 inseminations are performed over a 24h period, determined by oestrus or by the introduction of the buck, if light-treated goats receive 11-day FGA or CIDR treatment and are then induced to ovulate by the male effect.     

Cloning and seasonal secretion of the pancreatic lipase-related protein 2 present in goat seminal plasma

The storage of frozen semen for artificial insemination is usually performed in the presence of egg yolk or skimmed milk as protective agents. In goats, the use of skimmed milk extenders requires, however, that most of the seminal plasma is removed before dilution of spermatozoa because it is deleterious for their survival. It has been previously demonstrated that a lipase (BUSgp60) secreted by the accessory bulbourethral gland was responsible for the cellular death of goat spermatozoa, through the lipolysis of residual milk lipids and the release of toxic free fatty acids. This lipase was purified from the whole seminal plasma of goat and was found to display both lipase and phospholipase A activities, this latter activity representing the main phospholipase activity detected in goat seminal plasma. Based on its N-terminal amino acid sequence, identical to that of BUSgP60 purified from bulbourethral gland secretion, and the design of degenerated oligonucleotides, the lipase was cloned from total mRNA isolated from bulbourethral gland. DNA sequencing confirmed it was the goat pancreatic-lipase-related protein 2 (GoPLRP2). The physiological role of GoPLRP2 is still unknown but this enzyme might be associated with the reproductive activity of goats. A significant increase in lipase secretion was observed every year in August and the level of lipase activity in the semen remained high till December, i.e., during the breeding season. A parallel increase in the plasmatic levels of testosterone suggested that GoPLRP2 expression might be regulated by sexual hormones. The lipase activity level measured in goat seminal plasma, which could reach 1000 U/ml during the breeding season, was one of the highest lipase activity measured in natural sources, including gastric and pancreatic juices.     

Highly synchronous and fertile reproductive activity induced by the male effect during deep anoestrus in lactating goats subjected to treatment with artificially long days followed by a natural photoperiod

The response to the male effect was studied in two flocks of Saanen and three of Alpine goats during deep anoestrus in three consecutive years. Males and females were subjected to artificially long days for about 3 months (between December 4 and April 1) followed by a natural photoperiod. Bucks joined goats 42-63 days after the end of the long days treatment (between April 20 and June 3) and fertilisation was ensured by natural mating. In experiment 1 (n=248), female goats were treated or untreated with melatonin at the end of the long days treatment and treated or untreated for 11 days with fluorogestone acetate (FGA) before teasing. The males received melatonin implants. In experiment 2 (n=337), the factor studied was the association or non-association of the 11-day FGA treatment. Neither males nor females received melatonin implants. In experiment 3 (n=180), goats were treated for 11 days with FGA or with natural progesterone (CIDR). Neither males nor females received melatonin implants. In experiment 1, among the non-cycling goats (n=218), 99% ovulated and 81% kidded at 161+/-8 days after joining. Ninety-two percent of FGA-treated goats displayed an LH surge at 65+/-11h after teasing. Melatonin treatment did not affect any parameter but FGA advanced the kidding date. In experiment 2, 94% of the goats ovulated and 87% kidded. A major peak of conception was observed on days 3 and 8 after joining in FGA-treated and untreated goats, respectively. Among the FGA-treated goats, 83% displayed an LH surge. Over all flocks, most of the LH surges occurred over a 24-36 h interval, but the surge was initiated at different times in different flocks (36, 48 or 60 h after joining). FGA treatment did not influence the results, except for advancement of births of about 5 days. Differences among flocks were highly significant. In experiment 3, 94% of the goats displayed the LH surge, 93% ovulated and 68% kidded. Significant differences were found among flocks, but not between the FGA and CIDR groups. Bucks marked 85% of the goats 24-72 h after joining. The time interval between the detection of marked goats and detection of the LH surge depended on the time of marking (r=-0.62; p<0.05). In conclusion, treatment of both males and females goats with artificially long days followed by a natural photoperiod is very effective in inducing highly synchronous and fertile reproductive activity via the male effect in the middle of seasonal anoestrus.     

Reproductive cycle of goats

Goats are spontaneously ovulating, polyoestrous animals. Oestrous cycles in goats are reviewed in this paper with a view to clarifying interactions between cyclical changes in tissues, hormones and behaviour. Reproduction in goats is described as seasonal; the onset and length of the breeding season is dependent on various factors such as latitude, climate, breed, physiological stage, presence of the male, breeding system and specifically photoperiod. In temperate regions, reproduction in goats is described as seasonal with breeding period in the fall and winter and important differences in seasonality between breeds and locations. In tropical regions, goats are considered continuous breeders; however, restricted food availability often causes prolonged anoestrous and anovulatory periods and reduced fertility and prolificacy. Different strategies of breeding management have been developed to meet the supply needs and expectations of consumers, since both meat and milk industries are subjected to growing demands for year-round production. Hormonal treatments, to synchronize oestrus and ovulation in combination with artificial insemination (AI) or natural mating, allow out-of-season breeding and the grouping of the kidding period. Photoperiodic treatments coupled with buck effect now allow hormone-free synchronization of ovulation but fertility results after AI are still behind those of hormonal treatments. The latter techniques are still under study and will help meeting the emerging social demand of reducing the use of hormones for the management of breeding systems.     

Male-induced short oestrous and ovarian cycles in sheep and goats: a working hypothesis

The existence of short ovulatory cycles (5-day duration) after the first male-induced ovulations in anovulatory ewes and goats, associated or not with the appearance of oestrous behaviour, is the origin of the two-peak abnormal distribution of parturitions after the "male effect". We propose here a working hypothesis to explain the presence of these short cycles. The male-effect is efficient during anoestrus, when follicles contain granulosa cells of lower quality than during the breeding season. They generate corpora lutea (CL) with a lower proportion of large luteal cells compared to small cells, which secrete less progesterone, compared to what is observed in the breeding season cycle. This is probably not sufficient to block prostaglandin synthesis in the endometrial cells of the uterus at the time when the responsiveness to prostaglandins of the new-formed CL is initiated and, in parallel, to centrally reduce LH pulsatility. This LH pulsatility stimulates a new wave of follicles secreting oestradiol which, in turn, stimulates prostaglandin synthesis and provokes luteolysis and new ovulation(s). The occurrence of a new follicular wave on days 3-4 of the first male-induced cycle and the initiation of the responsiveness to prostaglandins of the CL from day 3 of the oestrous cycle are probably the key elements which ensure such regularity in the duration of the short cycles. Exogenous progesterone injection suppresses short cycles, probably not by delaying ovulation time, but rather by blocking prostaglandin synthesis, thus impairing luteolysis. The existence, or not, of oestrous behaviour associated to these ovulatory events mainly varies with species: ewes, compared to does, require a more intense endogenous progesterone priming; only ovulations preceded by normal cycles are associated with oestrous behaviour. Thus, the precise and delicate mechanism underlying the existence of short ovulatory and oestrous cycles induced by the male effect appears to be dependent on the various levels of the hypothalamo-pituitary-ovario-uterine axis.