Effect of administration of estradiol and progesterone,and bacterial contamination,on endometrial morphology of acyclic mares

Acyclic mares (free of uterine infection and with typical acyclic endometria on biopsy) received daily intramuscular injections in oil of either 1.5 mg estradiol benzoate (n - 4) or progesterone (n = 5). Estradiol and progesterone treatment induced biopsy changes consistent with estrus or diestrus respectively (Kenney, 1978). Subsequently, seven mares with typical acyclic endometria were given ten daily intra-uterine infusions of from 400 to 10 000 pg estradiol-17β. All mares developed Streptococcus zooepidemicus infections which masked any endometrial changes which might have been induced by estradiol infusion.     

Timed artificial insemination plus heat II: gonadorelin injection in cows with low estrus expression scores increased pregnancy in progesterone/estradiol-based protocol

The use of tail chalk and estrus/heat expression scores (HEATSC) evaluation is instrumental in identifying cows with greater estrus expression and greater artificial insemination pregnancy rates (P/AI) in cows submitted to timed artificial insemination (TAI), and cows with low or no estrus expression present lower P/AI. It was intended in this study to improve the pregnancy rates in TAI for Bos indicus beef cows, and gonadotrophin-releasing hormone (GnRH) injection was hypothesized to increase pregnancy rates in a TAI program for cows submitted to progesterone–estradiol-based protocols with low or no estrus expression, evaluated by HEATSC. Cows (n= 2284) received a progesterone device and 2 mg estradiol benzoate, after 8 days the device was removed and 1 mg estradiol cypionate, 150 μg of d-cloprostenol and 300 IU equine chorionic gonadotropin was administered. All cows were marked with chalk and HEATSC evaluated (scales 1 to 3) at TAI performed on day 10. Animals with HEATSC1 and HEATSC2 (n= 937) received 100 μg de gonadorelin (GNRH group; n= 470), or 1 ml saline (Control group; n= 467), and cows with HEATSC3 (named HEAT group; n= 1347) received no additional treatment. The larger dominant follicle, evaluated on day 8and at TAI (day 10), was greater in HEAT group (P= 0.0145 and P <0.001, respectively). Corpus luteum (CL) area and progesterone concentration was evaluated on day 17, and CL area was larger in HEAT group, intermediary in Control and lower in GnRH group (Control= 2.68 cm², GnRH= 2.37 cm², HEAT group= 3.07 cm², P <0.001). Greater progesterone concentrations were found in HEAT group than in Control and GnRH groups (Control= 4.74 ng/ml, GnRH= 4.29 ng/ml, HEAT group= 6.08 ng/ml, P<0.001). There was a difference in ovulation rate, greater in HEAT group than GnRH and Control groups (Control= 72.5%; GnRH= 81.25%; HEAT group= 90.71%; P= 0.0024). Artificial insemination pregnancy rates was greater in HEAT group (57.09% (769/1347) than in Control and GNRH groups, with positive effect of GnRH injection at the time of TAI in P/AI (Control= 36.18% (169/467), GnRH= 45.95% (216/470); P<0.0001). In conclusion, GnRH application in cows with low HEATSC (1 and 2) is a simple strategy, requiring no changes in TAI management to increase pregnancy rates in postpartum beef cows submitted to progesterone–estradiol-based TAI protocols, without reaching, however, the pregnancy rates of cows that demonstrate high estrus expression at the TAI.     

Regulation of the 24h body temperature rhythm of women in luteal phase: role of gonadal steroids and prostaglandins

An investigation into whether the rise in the 24h body temperature rhythm observed in the luteal menstrual phase is antagonized by the administration of prostaglandin synthesis inhibitors has been made. Intravaginal body temperature was monitored continuously for 24h, once in the follicular and twice in the luteal phase. In the luteal phase, women were studied both without and with the simultaneous administration of a prostaglandin synthesis inhibitor (lysine acetylsalicylate; 1.8 g every 6 h orally). The progesterone/estradiol ratio (measured at 17:00h each day) was related to mesor (r = 0.825; P < 0.001), acrophase (r = 0.682; P < 0.02), and amplitude (r = -0.731; P < 0.001) of the 24h body temperature rhythm. Luteal phase elevation of the progesterone/estradiol ratio was associated with a 0.32 +/- 0.07 degrees C increase in mesor (P < 0.01), a 0.11 +/- 0.02 degrees C decrease in amplitude (P < 0.001), and a 34.8 +/- 11.6 min delay in acrophase (P < 0.03) of the 24h body temperature rhythm. Prostaglandin synthesis inhibitors did not counteract these modifications. The present data shows that the modifications of the circadian parameters of the 24h body temperature rhythm observed during the luteal phase of the menstrual cycle are strictly related to modifications of the progesterone/estradiol ratio, and presumably independent of prostaglandin synthesis.     

The effect of exogenous progesterone on plasma LH and milk progesterone concentrations in multiple-suckling post-partum cows

Fourteen Friesian cows each suckling four calves were treated for a 7-day period (a) between days 20–40 post partum with progesterone-releasing intravaginal devices (PRID) containing 2% progesterone (Group 1; n = 5), (b) between days 51–264 post partum with PRIDS containing 2% progesterone (Group 2; n = 6) and (c) between days 29–214 days post partum with PRIDS containing 0% progesterone (Group 3; n = 3). Mean plasma LH concentrations decreased during PRID treatment in Group 2 cows only and pre-ovulatory LH surges were observed in $\text{5}\text{6}$ of these cows between 38 and 84 h after coil removal. All Group 2 cows underwent at least one ovarian cycle following PRID removal. No pre-ovulatory LH surges were observed in either Group 1 or Group 3 cows and only one cow (Group 3) underwent an ovarian cycle after treatment. It is suggested that there is an increase in pituitary responsiveness to the feedback effects of progesterone during the post-partum period.     

Temporal aspects of ovarian follicular growth and steroidogenesis following exogenous follicle-stimulating hormone in Angus heifers

Ultrasonography and endocrine assay techniques were used to monitor structural and hormonal alterations made by the ovary in response to the biological actions of pituitary-derived follicle-stimulating hormone (FSH-P). Angus heifers (n = 36) were allotted to receive injections (twice per day) of either FSH-P (up to a total of 28 mg over a maximum of 4 days beginning on Day 10 of a synchronized estrous cycle) or saline in order to quantify temporal relationships among follicle growth and steroid hormone profiles. Transrectal ultrasonography was utilized at 12-h intervals to monitor and record follicle growth. Plasma was collected every 12 h for the first 48 h of the experiment and then every 6 h for the remainder of the experiment. At 48 and 60 h after the onset of treatments, prostaglandin F2α (PGF2α; 25 mg) was administered (i.m.). FSH-treated heifers (n = 6 at each time) were terminated at 24, 48, 72 and 96 h following the onset of treatment. Saline-treated heifers were terminated at 24 and 96 h (n = 6 at each time). After ovaries were obtained, follicular number and size were recorded and follicular fluid (FF) was collected. Plasma concentration of progesterone (P) and estradiol (E2) and FF concentration of P, E2, estrone, testosterone and androstenedione were determined by radioimmunoassays. Plasma concentration of E2 increased (P < 0.05) within 36 h of initiation of FSH treatment. Plasma P decreased (P < 0.0001) by 12 h post-PGF2α. Ultrasonographic examination revealed a significant decrease in the number of small follicles by 48 h, whereas the number of medium follicles increased (P < 0.05) by 60 h after the initiation of FSH treatment. The number of large follicles (LF ≥ 10 mm diameter) increased (P < 0.01) over the course of the experiment. The total number of ovarian follicles (TF) 24 h after the start of FSH treatment was correlated (r = 0.99; P < 0.0001) with the number of small follicles (SF ≤ 5 mm). At 72 h after the onset of FSH treatment, the number of medium follicles (i.e. 6–9 mm) was correlated with TF (r = 0.97; P < 0.0001). Estradiol was the predominant FF steroid. Follicular fluid E2 was greatest in follicles at 72 h after FSH treatment. Follicular fluid E2 and plasma E2 were positively correlated (r = 0.66; P < 0.001). Follicular aromatase activity was estimated by evaluating the ratio of FF estrogens (E) to androgens (A). Elevated aromatase activity (E:A ratio > 1.0) was detected in 196 of 206 follicles. The estrogen to progesterone ratio was used as an estimate of follicle viability. Eighty-five percent of the follicles were estimated to be viable (E:P ratio > 1.0). The peak E:A ratio in LF preceded by 24 h the peak concentration in FF E2 and plasma E2. In MF and SF the E:A ratio increased by 72 h. Enhancement of ovarian follicular growth (i.e. increased number and size of follicles; increased steroidogenesis) by exogenous, pituitary-derived FSH is characterized by (1) increased activity of aromatase, and (2) accumulation of FF E2, events which temporally preceded the increase in plasma concentration of E2. These observations will aid efforts to incorporate recombinant bovine FSH and somatotropin in an effort to develop more predictable superstimulation and ovulation induction protocols.     

Effects of two estradiol esters (benzoate and cypionate) on the induction of synchronized ovulations in Bos indicus cows submitted to a timed artificial insemination protocol

The effects of estradiol benzoate (EB) and estradiol cypionate (EC) on induction of ovulation after a synchronized LH surge and on fertility of Bos indicus females submitted to timed AI (TAI) were evaluated. In Experiment 1, ovariectomized Nelore heifers were used to evaluate the effect of EB (n = 5) and EC (n = 5) on the circulating LH profile. The LH surge timing (19.6 and 50.5 h; P = 0.001), magnitude (20.5 and 9.4 ng/mL; P = 0.005), duration (8.6 and 16.5 h; P = 0.001), and area under the LH curve (158.6 and 339.4 ng/mL; P = 0.01) differed between the EB and EC treatments, respectively. In Experiment 2 (follicular responses; n = 60) and 3 (pregnancy per AI; P/AI; n = 953) suckled Bos indicus beef cows submitted to an estradiol/progesterone-based synchronization protocol were assigned to receive one of two treatments to induce synchronized ovulation: 1 mg of EB im 24 h after progesterone (P4) device removal or 1 mg of EC im at P4 device removal. There was no difference (P > 0.05) between EB and EC treatments on follicular responses (maximum diameter of the ovulatory follicle, 13.1 vs. 13.9 mm; interval from progesterone device removal to ovulation, 70.2 vs. 68.5 h; and ovulation rate, 77.8 vs. 82.8%, respectively). In addition, P/AI was similar (P < 0.22) between the cows treated with EB (57.5%; 277/482) and EC (61.8%; 291/471). In conclusion, despite pharmacologic differences, both esters of estradiol administered either at P4 device removal (EC) or 24 h later (EB) were effective in inducing an LH surge which resulted in synchronized ovulations and similar P/AI in suckled Bos indicus beef cows submitted to TAI.     

Gestational and hormonal regulation of human placental lipoprotein lipase

The fetal demand for FFA increases as gestation proceeds, and LPL represents one potential mechanism for increasing placental lipid transport. We examined LPL activity and protein expression in first trimester and term human placenta. The LPL activity was 3-fold higher in term (n = 7; P < 0.05) compared with first trimester (n = 6) placentas. The LPL expression appeared lower in microvillous membrane from first trimester (n = 2) compared with term (n = 2) placentas. We incubated isolated placental villous fragments with a variety of effectors [GW 1929, estradiol, insulin, cortisol, epinephrine, insulin-like growth factor-1 (IGF-1), and tumor necrosis factor-alpha] for 1, 3, and 24 h to investigate potential regulatory mechanisms. Decreased LPL activity was observed after 24 h of incubation with estradiol (1 micro g/ml), insulin, cortisol, and IGF-1 (n = 12; P < 0.05). We observed an increase in LPL activity after 3 h of incubation with estradiol (20 ng/ml) or hyperglycemic medium plus insulin (n = 7; P < 0.05). To conclude, we suggest that the gestational increase in placental LPL activity represents an important mechanism to enhance placental FFA transport in late pregnancy. Hormonal regulation of placental LPL activity by insulin, cortisol, IGF-1, and estradiol may be involved in gestational changes and in alterations in LPL activity in pregnancies complicated by altered fetal growth.     

Anandamide targets aromatase: A breakthrough on human decidualization

In each menstrual cycle endometrial stromal cells (hESC) proliferate and differentiate into specialized decidual cells, a process termed decidualization, which regulates endometrial receptivity. Decidualization is mainly controlled by sex ovarian hormones, estradiol (E₂) and progesterone. E₂ plays an important role in the expression of the progesterone receptor and promotes the endometrial stromal cells differentiation. Our group previously reported that anandamide (AEA) impairs decidualization through cannabinoid receptor 1 (CB1). In this study, we hypothesized whether AEA inhibitory effect on cell decidualization could be mediated through interaction with aromatase and consequent interference in estradiol production/signaling. We used an immortalized human endometrial stromal cell line (St-T1b) and human decidual fibroblasts (HdF) derived from human term placenta. In cells exposed to a differentiation stimulus, AEA-treatment prevents the increase of the expression of CYP19A1 gene encoding aromatase, E₂ levels and of estradiol receptor expression, that are observed in differentiating cells. Regarding CYP19A1 mRNA levels, the effect was partially reverted by a CB1 receptor antagonist and by a COX2 inhibitor. In addition, we report that AEA presents anti-aromatase activity in placental microsomes, the nature of the inhibition being the uncommon mixed type as revealed by the kinetic studies. Structural analysis of the AEA-Aromatase complexes determined that AEA may bind to the active site pocket of the enzyme. In overall we report that AEA inhibits aromatase activity and may affect E₂ signaling crucial for the decidualization process, indicating that a deregulation of the endocannabinoid system may be implicated in endometrial dysfunction and in fertility/infertility disorders.     

Long-term hormonal promotion overcomes genetic resistance to mammary cancer

It is well known that ovarian steroids estradiol and progesterone play a vital role in the development of mammary cancer. Here, using the genetically highly resistant Copenhagen rats we demonstrate that sustained exogenous treatment with estradiol and progesterone overcomes genetic resistance to mammary cancer. It has been demonstrated that Copenhagen rats develop preneoplastic lesions upon exposure to carcinogens. However, these preneoplastic lesions fail to progress to ductal carcinomas in situ or overt mammary carcinomas. The preneoplastic lesions eventually decrease in number and are absent by 60 days post-carcinogen treatment. In the present study, we exposed 7-week-old female Copenhagen rats to N-methyl-N-nitrosourea (MNU; 50 mg/kg BW). Immediately after MNU treatment the rats were divided into the following groups: (1) control; (2) 30 mg estradiol 17β; (3) 30 mg progesterone; and (4) 30 mg estradiol 17β plus 30 mg progesterone. All hormone treatments were administered via individual silastic pellets for a period of 9 months post-carcinogen treatment. The control animals displayed a low incidence of mammary cancer (10%). Hormone treatments produced significantly higher incidences of mammary cancer, with estradiol at 50%, progesterone at 65% and estradiol plus progesterone at 90%. Hormone treatment sustained the growth of the lesions induced by MNU by increasing expression of Areg, Bcl-2, Ccnd-1 and Vegf genes, while decreasing expression of Bad, Bax, Casp 3, 8, 9 and p53 genes. Furthermore, hormone treatment increased CCND-1 and PARP proteins levels. The data clearly demonstrates that hormonal environment supports mammary cancer progression by increasing cell proliferation, and angiogenesis while inhibiting apoptosis.     

Influence of perioestrous suprabasal progesterone levels on cycle length,oestrous behaviour and ovulation in heifers

In order to induce suprabasal plasma concentrations of progesterone after luteolysis and to determine their effect on oestrous behaviour and ovulation, heifers subcutaneously received silicone implants containing 2.5 (n = 4), 5 (n = 4), 6 (n = 3), 7.5 (n = 3) or 10 (n = 4) g of progesterone, or an empty implant (controls, n = 5) between days 8 and 25 of the cycle (ovulation designated Day 0). Growth of dominant follicles and time of ovulation were determined by ultrasound, and signs of oestrus were recorded and scored. Blood was collected at 2–4 h intervals from Days 15 to 27 and assayed for progesterone concentration. In all heifers, plasma concentrations of progesterone sharply decreased during Days 16–18. Control heifers had their lowest progesterone levels on Days 20.5 and 21, standing oestrus on Day 19.5 ± 0.4 (mean ± SEM), and ovulated on Day 20.7 ± 0.4. A similar pattern was observed in heifers treated with 2.5 and 5 g progesterone. Heifers treated with 6, 7.5 and 10 g of progesterone showed an extended (P < 0.05) interovulatory interval. Onset of prooestrus and time of maximum expression of signs of oestrus were not significantly different from those in controls. However, there was an absence of standing oestrus in most of the cases, signs of oestrus lasted longer (P < 0.05) and were weaker in intensity when doses increased. In these groups, the lowest progesterone concentrations were attained shortly after implant removal. Some heifers treated with 6 and 7.5 g of progesterone had standing oestrus and post oestrous bleeding as seen in the controls but ovulation occurred from Days 24.5 to 27. When plasma progesterone concentrations were over 1 nmol 1⁻¹, disturbed oestrus and delayed ovulation occurred. The extended period of prooestrus and oestrus and delayed ovulation were similar to that described in cases of repeat breeding. It is suggested that suprabasal plasma concentrations of progesterone, after luteolysis, may lead to asynchrony between onset of oestrus and ovulation and consequently be a cause of repeat breeding in cattle.     

Ovarian surface epithelium receptors during pregnancy and estrus cycle of rats with emphasis on steroids and gonadotropin fluctuation

The present study is designed to demonstrate the ovarian surface epithelial cells’ (OSE) estrogen receptor α (ERα) and progesterone receptor (PR) during pregnancy and estrous cycle in rat. Moreover, determination of the levels of plasma progesterone, estradiol, FSH and LH was also made. The levels of plasma progesterone, estradiol, FSH and LH concentrations were determined on days 7 (n = 5), 14 (n = 5), and 21 (n = 5) of pregnancy in three groups of rats and during the estrous cycle (n = 5) using an ELISA kit. Immunohistochemical method for PR and ERα expressions was also made on the ovary. During pregnancy, FSH and LH remained low except at term when LH levels began to increase from 16 ng/ml to 47 ng/ml. Progesterone levels significantly exceeded estradiol values in all pregnant rats with a peak value of 202 ng/ml on day 14. Elevated progesterone levels were associated negatively with LH and estradiol levels during pregnancy. The levels of estradiol surged significantly on day 21. Immunohistochemistry of the ovary showed low levels of OSE cells staining positive for ERα expression. ERα positive cells were absent on day 7 and 14 of pregnancy, only day 21 recorded a very low percentage of immunostaining (0.5%) within the nuclei of OSE cells. On the contrary, immunostaining of PR was not observed within the nuclei of OSE cells in all groups of study. In conclusion, these results may suggest that the progesterone effect during pregnancy seems to be overriding the positive effect of estrogens on OSE cells. High progesterone levels may have a direct negative effect on gonadotropin production and thereby it might inhibit events leading to both follicular development and OSE proliferation. Understanding the factors affecting OSE proliferation may help elucidating the mechanism(s) of assisted diseases such as ovarian cancer.Keyword: OSE pregnancy rat steroid receptors gonadotropins     

Short-term oral progesterone administration antagonizes the effect of transdermal estradiol on endothelium-dependent vasodilation in young healthy women

Very few studies have explored the cardiovascular effects of progesterone in premenopausal women. This study aimed to examine the short-term effects of oral progesterone alone, transdermal estrogen alone, and progesterone and estrogen combined on flow-mediated dilation (FMD) in healthy reproductive-aged women. We suppressed endogenous estrogens and progesterone in 17 premenopausal women for 10-12 days using a gonadotropin-releasing hormone antagonist. On day 4 (hormone suppression condition), subjects were tested (n = 17) and were then supplemented with either 200 mg micronized progesterone (n = 8) orally or 0.1 mg estradiol (n = 9) transdermally per day. On day 7 (progesterone-first or estradiol-first condition), subjects were tested and began supplementation with both hormones (n = 17) and were tested again on day 10 (combined hormone condition). FMD of the brachial artery was assessed using B-mode arterial ultrasound, combined with synchronized Doppler analysis. As a result, significant differences in FMD were observed between hormone suppression (7.85 ± 1.06%) and estrogen-first conditions (10.14 ± 1.40%; P < 0.05). The estradiol-induced increase was abolished when oral progesterone was also supplemented (6.27 ± 0.96%). In contrast, we observed a trend toward a decrease in FMD with unopposed progesterone administration, but no statistically significant differences were found between the progesterone-first (6.66 ± 1.23%), hormone suppression (7.80 ± 1.23%), and combined hormone conditions (7.40 ± 1.29%). In conclusion, these data suggest that short-term oral micronized progesterone administration antagonizes the beneficial effect of transdermal estradiol on FMD.     

Shearing during late pregnancy increases size at birth but does not alter placental endocrine responses in sheep

Shearing during the latter half of pregnancy is a common practice to improve flock health and productivity. Previous studies have demonstrated that shearing pregnant ewes at mid or late pregnancy is associated with an increase in lamb birth weight. In the present study, we used singleton Polypay × Dorset pregnant sheep, to investigate the potential roles of placental function and changes in maternal metabolism in underlying this increased birth weight response. Two groups were randomly established and blocked at enrollment by animal BW, body condition score and subcutaneous adipose tissue depth. The groups were shorn (SH; n = 18) or not (C; n = 20) at gestational day (GD) 107 ± 1 (mean ± SEM). Weekly maternal plasma samples were collected between shearing and birth, but only six samples were assayed for progesterone, pregnancy-associated glycoproteins (PAG1), glucose and non-esterified fatty acids (NEFAs). At birth, sex, birth weight, and newborn body mass index (BMI) were recorded. Maternal BW during mid- to late-pregnancy was similar between groups. Shearing resulted in increased lamb birth weight and BMI (P < 0.05) regardless of fetal sex but did not affect the maternal concentration of PAG1 or progesterone from GDs 100 to 142. After shearing (GD100) and up to lambing, shorn females had higher circulating glucose concentrations (P < 0.05), but not NEFA, compared to the control group. Maternal circulating PAG1, progesterone, glucose or NEFA concentration across pregnancy did not differ according to lamb sex. Across pregnancy, birth weight was positively associated with PAG1 (P < 0.001), but not with progesterone concentrations. In conclusion, weight and BMI at birth were higher in both sexes upon shearing in singleton pregnancies. Despite PAG1 being associated with birth weight, late-pregnancy shearing did not alter the placental endocrine response. Whether other placental factors are altered upon shearing and may influence the increase in birth weight and BMI remain to be investigated.     

Dietary l-arginine supplementation enhances placental growth and reproductive performance in sows

Suboptimal embryonic/fetal survival and growth remains a significant problem in mammals. Using a swine model, we tested the hypothesis that dietary l-arginine supplementation during gestation may improve pregnancy outcomes through enhancing placental growth and modulating hormonal secretions. Gestating pigs (Yorkshire?×?Landrace, n?=?108) were assigned randomly into two groups based on parity and body weight, representing dietary supplementation with 1.0% l-arginine–HCl or 1.7% l-alanine (isonitrogenous control) between days 22 and 114 of gestation. Blood samples were obtained from the ear vein on days 22, 40, 70 and 90 of gestation. On days 40, 70 and 90 of gestation, concentrations of estradiol in plasma were higher (P?<?0.05) in arginine-supplemented than in control sows. Moreover, arginine supplementation increased (P?<?0.05) the concentrations of arginine, proline and ornithine in plasma, but concentrations of urea or progesterone in plasma did not differ between the two groups of sows. Compared with the control, arginine supplementation increased (P?<?0.05) the total number of piglets by 1.31 per litter, the number of live-born piglets by 1.10 per litter, the litter birth weight for all piglets by 1.36?kg, and the litter birth weight for live-born piglets by 1.70?kg. Furthermore, arginine supplementation enhanced (P?<?0.05) placental weight by 16.2%. The weaning-to-estrus interval of sows was not affected by arginine supplementation during gestation. These results indicate that dietary arginine supplementation beneficially enhances placental growth and the reproductive performance of sows.     

Distribution and diversity of cytotypes in Dianthus broteri as evidenced by genome size variations

Background and Aims

Studying the spatial distribution of cytotypes and genome size in plants can provide valuable information about the evolution of polyploid complexes. Here, the spatial distribution of cytological races and the amount of DNA in Dianthus broteri, an Iberian carnation with several ploidy levels, is investigated.

Methods

Sample chromosome counts and flow cytometry (using propidium iodide) were used to determine overall genome size (2C value) and ploidy level in 244 individuals of 25 populations. Both fresh and dried samples were investigated. Differences in 2C and 1Cx values among ploidy levels within biogeographical provinces were tested using ANOVA. Geographical correlations of genome size were also explored.

Key Results

Extensive variation in chromosomes numbers (2n = 2x = 30, 2n = 4x = 60, 2n = 6x = 90 and 2n = 12x =180) was detected, and the dodecaploid cytotype is reported for the first time in this genus. As regards cytotype distribution, six populations were diploid, 11 were tetraploid, three were hexaploid and five were dodecaploid. Except for one diploid population containing some triploid plants (2n = 45), the remaining populations showed a single cytotype. Diploids appeared in two disjunct areas (south-east and south-west), and so did tetraploids (although with a considerably wider geographic range). Dehydrated leaf samples provided reliable measurements of DNA content. Genome size varied significantly among some cytotypes, and also extensively within diploid (up to 1·17-fold) and tetraploid (1·22-fold) populations. Nevertheless, variations were not straightforwardly congruent with ecology and geographical distribution.

Conclusions

Dianthus broteri shows the highest diversity of cytotypes known to date in the genus Dianthus. Moreover, some cytotypes present remarkable internal genome size variation. The evolution of the complex is discussed in terms of autopolyploidy, with primary and secondary contact zones.     

Microsatellite and flow cytometry analysis to help understand the origin of Dioscorea alata polyploids

Background and Aims Dioscorea alata

is a polyploid species with a ploidy level ranging from diploid (2n = 2x = 40) to tetraploid (2n = 4x = 80). Ploidy increase is correlated with better agronomic performance. The lack of knowledge about the origin of D. alata spontaneous polyploids (triploids and tetraploids) limits the efficiency of polyploid breeding. The objective of the present study was to use flow cytometry and microsatellite markers to understand the origin of D. alata polyploids.

Methods

Different progeny generated by intracytotype crosses (2x × 2x) and intercytotype crosses (2x × 4x and 3x × 2x) were analysed in order to understand endosperm incompatibility phenomena and gamete origins via the heterozygosity rate transmitted to progeny.

Results

This work shows that in a 2x × 2x cross, triploids with viable seeds are obtained only via a phenomenon of diploid female non-gametic reduction. The study of the transmission of heterozygosity made it possible to exclude polyspermy and polyembryony as the mechanisms at the origin of triploids. The fact that no seedlings were obtained by a 3x × 2x cross made it possible to confirm the sterility of triploid females. Flow cytometry analyses carried out on the endosperm of seeds resulting from 2x × 4x crosses revealed endosperm incompatibility phenomena.

Conclusions

The major conclusion is that the polyploids of D. alata would have appeared through the formation of unreduced gametes. The triploid pool would have been built and diversified through the formation of 2n gametes in diploid females as the result of the non-viability of seeds resulting from the formation of 2n sperm and of the non-viability of intercytotype crosses. The tetraploids would have appeared through bilateral sexual polyploidization via the union of two unreduced gametes due to the sterility of triploids.     

Regulation of human placental progesterone synthesis in vitro by naturally occurring steroids

A regulatory model of human placental progesterone synthesis is based on studies with isolated placental enzymes. Steroids causing a dose-dependent inhibition are listed in the standing order of their inhibitory potency (I50 (microM)/Ki value (microM)/type of inhibition: c = competitive and nc = non competitive). Cholesterol side chain cleavage enzyme (mitochondria): Mainly regulated by hydroxylated cholesterol derivates. No inhibition was observed by cholesterylesters and by other naturally occurring steroids tested. 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase (mitochondria): 6 beta-hydroxyprogesterone (nc), dehydroepiandrosterone (0.32/0.82/c), 20 alpha-dihydroprogesterone (0.38/-/nc), progesterone (0.46/-), estrone (0.56/0.1/c), estradiol (0.1/0.8/c), 17 alpha-hydroxyprogesterone (2.1/-/nc), 17 alpha-hydroxypregnenolone (0.4/-/c), dehydroepiandrosterone sulfate (2.5/-/c), cortisone (5.0/-), cortisol (100/-). 20 alpha-hydroxysteroid dehydrogenase (cytoplasmic): estrone (0.26/0.7/c), estradiol (0.28/0.9/c), pregnenolone (4.4/9.2/c), 5 alpha-pregnan-3 beta-ol-20-one (4.6/-/nc), estriol (5.1/11.5/c); dehydroepiandrosterone (7.2/14.0/c), 5 alpha-dihydrotestosterone (26.0/-/nc), progesterone (33.0/48.0/c), dehydroepiandrosterone sulfate (50.0/23.0/nc), and testosterone (59.0/63.0/c). An autoregulatory mechanism of placental progesterone synthesis is postulated which is in good agreement with data published by others proving that placental progesterone synthesis is independent of the endocrine organs of the mother and the fetus.     

Maximum likelihood inference implies a high, not a low, ancestral haploid chromosome number in Araceae, with a critique of the bias introduced by 'x'

Background and Aims

For 84 years, botanists have relied on calculating the highest common factor for series of haploid chromosome numbers to arrive at a so-called basic number, x. This was done without consistent (reproducible) reference to species relationships and frequencies of different numbers in a clade. Likelihood models that treat polyploidy, chromosome fusion and fission as events with particular probabilities now allow reconstruction of ancestral chromosome numbers in an explicit framework. We have used a modelling approach to reconstruct chromosome number change in the large monocot family Araceae and to test earlier hypotheses about basic numbers in the family.

Methods

Using a maximum likelihood approach and chromosome counts for 26 % of the 3300 species of Araceae and representative numbers for each of the other 13 families of Alismatales, polyploidization events and single chromosome changes were inferred on a genus-level phylogenetic tree for 113 of the 117 genera of Araceae.

Key Results

The previously inferred basic numbers x = 14 and x = 7 are rejected. Instead, maximum likelihood optimization revealed an ancestral haploid chromosome number of n = 16, Bayesian inference of n = 18. Chromosome fusion (loss) is the predominant inferred event, whereas polyploidization events occurred less frequently and mainly towards the tips of the tree.

Conclusions

The bias towards low basic numbers (x) introduced by the algebraic approach to inferring chromosome number changes, prevalent among botanists, may have contributed to an unrealistic picture of ancestral chromosome numbers in many plant clades. The availability of robust quantitative methods for reconstructing ancestral chromosome numbers on molecular phylogenetic trees (with or without branch length information), with confidence statistics, makes the calculation of x an obsolete approach, at least when applied to large clades.     

The effect of cloprostenol in non-pregnant and pregnant Norwegian semi-domestic reindeer (Rangifer tarandus tarandus L)

Two non-pregnant and five pregnant 1.5-year-old semi-domestic reindeer were used to study the effect of cloprostenol given during the luteal phase of the oestrous cycle, or at two stages of pregnancy (early December, n = 3, and mid-January, n = 2). Blood samples were collected at 3-hourly intervals from immediately prior to treatment until 6–8 days after treatment, after which blood samples were collected every second day. Prior to each blood collection, the animals were observed for signs of oestrus. Plasma progesterone, oestradiol-17β, luteinizing hormone (LH) and 15-ketodihydro-PGF_2α were analyzed to characterize variations in ovarian function.Treatment with cloprostenol resulted in an immediate and rapid decrease in plasma progesterone concentrations in all treated animals. The fall in plasma progesterone was associated with increase in 15-ketodihydro-PGF_2α. Oestrus, indicated by standing behaviour, was observed on three out of four occasions in the two non-pregnant animals. The average duration of standing behaviour (oestrus) was 27 h (range: 24–30 h).Of the pregnant females that were treated with cloprostenol in the beginning of December (n = 3), two aborted between 72 and 96 h after treatment. One of these developed pyometra after abortion. In one female the foetus died 2 days after treatment, but was retained within the uterus until slaughter 2.5 months later. One of the two females that were treated in mid-January aborted between 62–65 h after injection. The other female retained a live foetus until slaughter in February. There were few endocrinological differences between animals that aborted and those that did not, though aborting animals had lower progesterone concentrations for a longer period of time after treatment.It was concluded that cloprostenol can be used during the luteal phase of the oestrous cycle to induce luteolysis and oestrus. When given during pregnancy, cloprostenol can induce abortion, though undesired side effects make it inappropriate for practical use.