Correlation of plasma progesterone concentrations to ovarian H-type lipase activity during pseudopregnancy in the rat

Conditions for extraction and assay of hepatic type (H-type) lipase from rat ovaries were studied. An alkaline buffer with protease inhibitors and detergents gave the most efficient extraction. The specificity of the assays was ascertained using antiserum to H-type lipase from heparin perfusates of rat livers. H-type lipase activity was determined in ovarian compartments during pseudopregnancy (1-13 days) as well as during the ensuing period of luteal regression (Day 17). The activity was low in the luteal compartment immediately after ovulation, increased 6-fold to a maximum between Day 5 and Day 8 and then decreased again. This is similar to previously known changes in blood flow. There was a significant correlation between luteal H-type lipase activity and plasma progesterone regardless of luteal age. In contrast, neither the activity in the remainder of the ovary nor the activity in plasma changed during the luteal phase or correlated to plasma progesterone. Injection of heparin at the height of the luteal cycle (Day 8) caused a pronounced decrease in luteal lipase and in plasma progesterone. These studies suggest that the H-type lipase activity has an important role in luteal steroidogenesis, probably to facilitate uptake of cholesterol from lipoproteins.     

Lipid peroxidation at various estradiol concentrations in human circulation during ovarian stimulation with exogenous gonadotropins.

The aim of our study was to investigate the correlation between serum malondialdehyde levels and serum estradiol concentrations in healthy human female subjects. Nine hundred and fifty-five blood samples, from infertile women undergoing controlled ovarian hyperstimulation treatment with recombinant follicle-stimulating hormone, were collected for estradiol and malondialdehyde measurements. Five groups were formed according to serum estradiol levels: Group I (< 50 pg/ml), group II (50 - 299 pg/ml), group III (300-999 pg/ml), group IV (1000-1999 pg/ml) and group V (> or = 2000 pg/ml). One-way analysis of variance was used for comparisons. Mean malondialdehyde concentrations were 1.74 +/- 0.24 mmol/ml (group I), 1.53 +/- 0.20 mmol/ml (group II), 1.69 +/- 0.24 mmol/ml (group III), 1.77 +/- 0.21 mmol/ml (group IV) and 1.86 +/- 0.20 mmol/ml (group V), respectively. Mean serum malondialdehyde level at physiological estradiol concentrations (50-199 pg/ml, group II) was significantly (p < 0.01) lower than the mean malondialdehyde levels in other groups. Mean malondialdehyde concentrations among the remaining groups did not significantly differ. Our findings suggest that in vivo lipid peroxidation might be increased when circulating estradiol concentrations are below (< 50 pg/ml) or above (> 300 pg/ml) the physiological limits. High blood estradiol levels in human female subjects during ovarian stimulation with exogenous gonadotropins could be associated with increased serum malondialdehyde concentrations.     

Ovarian dynamics, serum estradiol and progesterone concentrations during the interovulatory interval in goats

Ovarian changes determined by daily transrectal ultrasonic scanning, and its correlation with serum progesterone (P4) and estradiol (E2) concentrations were studied in seven cyclic Saanen goats. Estrous cycles were synchronized with 2 injections of a PGF2 alpha analogue 9 d apart. All follicles > or = 2 mm in diameter and CL were measured each day. One goat showed a longer interestrous interval, associated with development of a cystic-luteinized structure. The mean interovulatory interval for the other 6 goats was 20.8 +/- 0.4 d. The incidence of goats with 4, 3, and 2 follicular waves was 3, 1 and 2 respectively; follicular waves emerged on Days 0.5 +/- 0.6, 7.2 +/- 0.7, 10.7 +/- 0.5 and 13.7 +/- 0.8 for Wave 1, 2, 3 and the Ovulatory wave, respectively. The largest follicle of Wave 2 was smaller (4.9 +/- 0.1 mm) than the largest follicles of Wave 3 (6.2 +/- 0.1 mm; P < or = 0.01) and of the Ovulatory wave (7.0 +/- 0.5 mm; P < or = 0.01), and tended to be smaller than the largest follicle of Wave 1 (6.3 +/- 0.6 mm; P < or = 0.09). Interval between emergence of Wave 1 and Wave 2 was longer than interval between emergence of Wave 2 and Wave 3 (7.3 +/- 0.9 d vs 4.0 +/- 0.4 d; P < or = 0.01), and between Wave 3 and the Ovulatory wave (3.8 +/- 1.1 d; P < or = 0.05). Two days before ovulation, the diameter of the ovulatory follicle was larger (P < or = 0.01) than the first subordinate follicle. Serum E2 concentrations increased from the day of ovulation (2.7 +/- 0.3 pg/mL) to Day 2 (7.6 +/- 0.9 pg/mL; P < or = 0.01), associated with the early-mid growing phase of the largest follicle of Wave 1, and then decreased to basal levels on Day 5 (P < or = 0.01) and peaked again (16.5 +/- 2.4 pg/mL) 2 d before ovulation. The CL were detected ultrasonically on Day 3 post ovulation and attained a mean maximum diameter of 13.5 +/- 0.8 mm between Days 8 and 14. The following characteristics were observed: 1) ovarian follicular development in goats is wave-like; 2) increased P4 concentrations may be promoting follicular wave turnover; 3) it is suggested that the presence of follicular dominance and the production of E2 are different among waves. While in Wave 1 and in the Ovulatory wave, follicular dominance is present and production of E2 is consistent, no changes in serum E2 concentrations were found in other stages of the interovulatory interval. In the intervening waves, no indicators of follicular dominance could be firmly documented.     

Changes of progesterone content of rat uterine flushings in relation to serum concentrations of progesterone during the oestrous cycle.

Uterine fluid was collected from four-day cyclic rats at each stage of the oestrous cycle and assayed for progesterone and protein content. Progesterone was determined by radioimmunoassay either after ethanol (or 2.5% NaOH) denaturation of proteins from uterine flushings ('total' progesterone) or without protein denaturation ('ether-extractable' progesterone). The amount of 'ether-extractable' progesterone in the lumen was constant from metoestrus to pro-oestrus (340 pg per uterus) but lower in oestrus (200 pg per uterus). However, 'total' progesterone content of uterine fluid was subject to cyclic variations and was highest in dioestrus (890 pg per uterus) and lowest in oestrus (350 pg per uterus), in contrast to serum progesterone which is lowest in dioestrus and highest in oestrus. Protein content of uterine flushings peaked to 780 micrograms per uterus in pro-oestrus then fell to about 140 micrograms per uterus until the end of the oestrous cycle. Changes in protein content of the lumen were followed by qualitative variations since the mean amount of 'bound' progesterone ('total' progesterone minus 'ether-extractable' progesterone) released per milligram of denatured lumen protein rose from 1.8 pmol in pro-oestrus to 18.2 pmol in dioestrus. The changes of luminal 'bound' progesterone during the oestrous cycle suggest that progesterone binding to luminal proteins could be an important modulator of progesterone action in rat uterus. Moreover, the variations in progesterone content of the lumen, irrespective of serum progesterone concentrations, are consistent with the hypothesis that progesterone synthesis occurs in the uterus.     

Effects of adrenalectomy on serum concentrations of progesterone during the luteal phase of sheep

An experiment was conducted to test the hypothesis that the adrenal gland influences luteal activity in sheep. Twelve Finnish Landrace x Southdown ewes were either bilaterally adrenalectomized (n = 6) or sham adrenalectomized (n = 6) during the breeding season. At approximately 37 and 47 d after surgery, all ewes received intramuscular injections of cloprostenol to synchronize estrus. Blood samples were taken via jugular venipuncture at 48-h intervals between 1 and 19 d after the last cloprostenol treatment. Serum concentrations of progesterone were determined in each of these samples. Analysis of variance showed that concentrations of progesterone during the luteal phase were lower (P<0.05) in adrenalectomized ewes than in sham-operated controls, but that patterns of progesterone were similar for both groups of sheep. Based on these results, we conclude that the adrenal gland does not appear to be necessary for initiation of luteal regression in ewes.     

Cytokine profile of human septic shock serum inducing cardiomyocyte contractile dysfunction

This study was designed to measure nitrite/nitrate and cytokine levels of serum obtained from septic shock patients and to describe potential depressant effects of human septic serum on rat cardiomyocytes. Serum was prepared from 10 non-septic patients and 10 patients with documented septic shock. Adult rat ventricular myocytes were exposed to 20 % serum in the medium. Cardiomyocyte contractility was assessed by measuring shortening fraction and shortening velocity. Serum levels of nitrite/nitrate, a marker of nitric oxide final metabolites, and cytokines (tumor necrosis factor (TNF)-alpha, interleukin (IL) 1beta, 6, 10, 8 and 12p70) were measured. Compared with serum from non-septic patients, serum of septic shock patients induced rapid reduction of the extent and velocity of shortening in isolated cardiomyocytes. Nitrite/nitrate, TNF-alpha, IL-1beta and IL-12p70 concentrations of tested serum for cardiomyocyte studies were not increased in septic serum compared with controls. In contrast, septic serum that induced a depression of in vitro contractility, had increased levels of IL-6, IL-8 and IL-10. We can conclude that the depression of in vitro contractility induced by septic serum is not directly dependent on elevated levels of nitric oxide metabolites, TNF-alpha or IL-1beta. Our results support the view that other cytokines, including IL-6, IL-8 and IL-10, are potent circulating mediators of myocardial depression in cardiomyocytes.     

Puberty and serum concentrations of ovarian steroids during prepuberal period in Friesian heifers artificially infected with Fasciola hepatica

An experiment was conducted to study the effect of fascioliasis on puberty in 13 Friesian heifers divided into a control group (n = 7) and an infected group (n = 6). At 4 m.o. of age each infected heifer received 600 metacercariae of F. hepatica. Blood samples were taken twice a week, at which time permanent heat mount detectors were checked, from 8 m.o. of age until 2 estruses were detected in all heifers. Serum concentrations of progesterone (P4) and estradiol-17 beta (E2) were assayed by ELISA and RIA, respectively. There was a significant delay in the onset of puberty in infected heifers compared with controls. All animals reached puberty at similar body weights but at different ages. First estrus was delayed by 39 d in infected compared with control animals. Serum concentrations of E2 were significantly higher in the infected group than the control group, while P4 concentrations were lower. Fascioliasis alters the serum concentration of E2, which in turn results in an abnormally low concentration of P4, probably due to underdeveloped or absent corpora lutea.     

Changes in LH pulse frequency and serum progesterone concentrations during the transition to breeding season in ewes

To characterize the changes in LH pulse frequency during the transition to breeding season. LH pulse patterns and serum progesterone profiles were determined in 8 intact ewes from mid-anoestrus to the early breeding season. Overall, 8 increases in LH pulse frequency were observed and these were restricted to 5 ewes. Of the 8 increases, 7 occurred during the 4 weeks before the first cycle, 5 of them within 1 week after a pulse frequency typical of anoestrus (0-2 per 8 h). Six of them occurred less than 1 week before either a full-length luteal phase (n = 2) or a 1-3-day increment in progesterone (n = 4). Seven of these brief progesterone increases were observed in 6 ewes, 5 of them immediately preceding the first full-length luteal phase. These results are consistent with the hypothesis that the seasonal decrease in response to oestradiol negative feedback at the beginning of the breeding season causes an increase in GnRH, and thereby LH pulse frequency. In addition, they demonstrate that the first increase in tonic LH secretion occurs in less than 1 week and, in most ewes, initiates either the first full-length cycle or a transient increase in progesterone, the latter occurring more often.     

Adiponectin concentrations increase during acute FFA elevation in humans treated with rosiglitazone.

The adipocytokine adiponectin is released by adipocytes upon activation of the peroxisome proliferator-activated receptor gamma (PPAR gamma). PPAR gamma has binding sites for thiazolidinediones and free fatty acids (FFAs). To evaluate if adiponectin serum concentrations are synergistically regulated by FFAs and thiazolidinediones IN VIVO plasma FFAs were acutely elevated in healthy subjects pre-treated with rosiglitazone or placebo. Sixteen healthy male subjects (23-37 years) were included in this double-blind, randomized, placebo-controlled parallel-group study. Rosiglitazone 8 mg or placebo was administered daily for 21 days. On the last day plasma FFA concentrations were increased by an intravenous triglyceride/heparin infusion. Blood for determination of adiponectin, C-reactive protein (CRP), leptin, resistin, FFAs, glucose, and insulin was drawn at baseline and on day 21 before and after 5 hours of triglyceride/heparin infusion. Adiponectin concentrations increased and FFA levels decreased in subjects receiving rosiglitazone (all p<0.05 VS. baseline). Lipid infusion significantly increased FFA plasma concentrations, with an attenuated elevation in rosiglitazone-treated subjects. However, adiponectin concentrations were only increased in subjects on rosiglitazone (p=0.018 VS. before lipid infusion), but not in controls. Leptin increased during lipid infusion in subjects receiving placebo but not in those on rosiglitazone. CRP and resistin were not affected by rosiglitazone or FFAs. The acute increase in circulating adiponectin concentrations during acutely elevated FFA depends on PPAR gamma activation in healthy subjects.     

Ratios of serum concentrations of testosterone and progesterone from yearling bulls with small testes

Thirty crossbred bulls, 12 to 13 mo of age, were used to examine the relationship of testosterone and progesterone concentrations and testosterone: progesterone ratio to measurements of testicular function. Bulls were allotted to 1 of 2 groups based on scrotal circumferences (SC) as follows: the Small SC (n=20) group had scrotal circumference less than 28 cm while the Large SC (n=10) group had scrotal circumference greater than 28 cm. All bulls were administered GnRH (100 mug, im), and blood was obtained immediately prior to injection (t=0), 30 min after injection (t=30) and 2 to 3 h after injection (t=150). Serum was assayed for concentrations of testosterone and progesterone. Semen was evaluated for the percentage of morphologically normal spermatozoa. Testicular parenchyma was sectioned and stained, and 300 cross sections per testis of seminiferous tubules were examined under a light microscope and classified as either active (spermatocytes and spermatids present) or inactive (no spermatocytes or spermatids present). Although progesterone concentrations varied widely (range: 21 pg/ml to 1070 pg/ml), repeated measurements from individual bulls were highly correlated (r(2)=0.74) and did not change significantly (P > 0.1) in response to GnRH treatment. Small SC bulls had a higher percentage of inactive seminiferous tubules (P < 0.001) and a lower percentage morphologically normal spermatozoa (P < 0.001) than Large SC bulls, but no differences in testosterone or progesterone concentrations or in the ratio of testosterone: progesterone were detected. Mean serum testosterone concentration increased (P < 0.0001) by 30 min after GnRH treatment and continued to increase (P < 0.0001) through t=150 but did not differ (p > 0.1) between groups. Normal testosterone secretion in response to GnRH injection suggested that no biochemical lesions in the testosterone production pathway were present in bulls with very small scrotal circumference.     

Acute reduction in serum progesterone concentrations after feed intake in dairy cows

This study tested the hypothesis that high feed consumption will acutely decrease circulating progesterone concentrations. In the first experiment, a Latin Square design was used to test whether feeding pattern would alter circulating progesterone in pregnant lactating Holstein cows (n = 12). Feed was removed for 12h before the experiment and cows were then either fed 100% of the total mixed ration (TMR), 50% of TMR every 12h, 25% of TMR every 6h, or left unfed for an additional 12h. Blood samples were taken every hour for 24h. Provision of 100 or 50% of TMR decreased circulating progesterone by 1h after feeding and progesterone remained depressed until 8-9h after feeding. Feeding 25% of TMR did not reduce circulating progesterone concentrations. Experiment 2 used a crossover design to measure the effect of acute feeding on circulating progesterone and LH concentrations during delivery of a constant amount of exogenous progesterone (Eazi-Breed CIDRs) in lactating Holstein cows (n = 8) and nonpregnant dry Holstein cows (n = 6). Blood samples were taken every 15min for 8h. There was no change in serum progesterone during the 8h treatment period in unfed cows; however, feeding decreased (P<0.05) circulating progesterone between 2 and 6h after feeding. In lactating cows, feeding increased mean LH (P<0.05). There were more LH pulses (P = 0.01) in lactating than nonlactating cows. Thus, acute feeding reduced circulating progesterone in pregnant lactating cows apparently due to an increase in progesterone metabolism. Interestingly, feeding multiple smaller meals eliminated the acute effect of feeding on circulating progesterone.     

Time-course relationships between serum LH, serum progesterone and urinary preganediol concentrations in normal women

A specific and sensitive gas chromatographic method was used to investigate the concentration of pregnanediol glucuronide in urine in relation to the time of ovulation. Serum LH and progesterone concentrations in the same subjects were used as evidence for the occurrence of ovulation. The urinary concentration of pregnanediol glucuronide in 24-hour collections and in overnight specimens increased 2-fold or more from the day of the midcycle LH peak to the time of predicted ovulation (24-48 hour after the LH peak) in parallel with the rise in serum progesterone concentration.     

Alterations in follicular fluid estradiol, progesterone and insulin concentrations during ovarian acyclicity in water buffalo (Bubalus bubalis)

Ovarian acyclicity is one of the most important causes of infertility in water buffalo. Recent studies have indicated alterations in the composition of follicular fluid during the condition. The aim of this study was to determine the changes in follicular fluid concentrations of estradiol, progesterone and insulin during ovarian acyclicity in water buffalo. Ovaries were collected from 50 acyclic and 95 cyclic (control) buffaloes and follicular fluid was aspirated from small (5.0-6.9 mm), medium (7.0-9.9 mm) and large (≥10.0 mm) sized follicles. Estradiol concentration was lower (P<0.0001) in acyclic (1.4 ± 0.09 ng/ml) than in cyclic (3.3 ± 0.18 ng/ml) buffaloes. Regardless of the ovarian cyclic status, there was an increase (P<0.01) in estradiol concentration with the increase in follicle size; the mean concentrations were 2.4 ± 0.16 ng/ml, 2.8 ± 0.29 ng/ml and 3.5 ± 0.41 ng/ml in small, medium and large follicles, respectively. A higher (P<0.001) progesterone concentration was recorded in acyclic (24.3 ± 2.61 ng/ml) compared to the cyclic (7.6 ± 0.79 ng/ml) group. Furthermore, acyclic buffaloes had a lower (P<0.05) concentration of insulin in the follicular fluid than that of cyclic buffaloes (15.2 ± 1.55 μIU/ml versus 25.9 ± 2.78 μIU/ml, respectively). In conclusion, acyclic buffaloes have lower concentrations of estradiol and insulin concurrent with higher concentrations of progesterone in the follicular fluid. These hormonal changes in the follicular microenvironment are possibly a manifestation of the disturbances in the normal follicular development leading to anovulation and anestrus in acyclic buffaloes.     

The role of progesterone elevation in IVF

Elevation of progesterone during the late follicular phase of stimulated in-vitro fertilization cycles is a frequent event, which negatively impacts the outcome. Over the years evidence has demonstrated a direct relationship between late-follicular elevated progesterone and endometrial receptivity. In this regard, elective cryopreservation of all good quality embryos and transfer in a subsequent frozen/thawed cycle is the most common strategy adopted by clinicians in case of elevated progesterone. Nonetheless, recent evidence suggests that elective cryopreservation might not entirely resolve the reduced pregnancy outcomes associated with the elevation of progesterone, considering that the increase may affect not only implantation, but also embryo quality.     

Detection of pregnancy by radioimmunoassay of a novel pregnancy-specific protein in serum of cows and a profile of serum concentrations during gestation

The development of a double antibody radioimmunoassay for a bovine pregnancy-specific protein (pregnancy-specific protein B; PSPB) is presented. By means of this assay, PSPB could be measured in serum of pregnant cows. Five dairy cows were bled throughout gestation to measure serum levels of PSPB. Serum concentrations (means +/- SE) exceeded 1 ng/ml by 30 days postbreeding and increased gradually through three months (9 +/- 0.6 ng/ml), six months (35 +/- 6 ng/ml), and nine months (150 +/- 75 ng/ml) of gestation. Maximum levels of PSPB (542 +/- 144 ng/ml) were reached two days before parturition and then steadily declined to less than 78 ng/ml by 21 days postpartum. In 21 cows bled daily from 15 through 30 days postbreeding, PSPB could be measured in a few cows before and in most cows by 24 days after breeding. In a commercial herd of 102 beef cows, the assay could detect pregnancy earlier and more accurately than the routine method of rectal palpation. This radioimmunoassay measures a unique antigen that, for the first time, provides a serological method for detecting pregnancy in cows.     

Effect of treatment with progesterone and oestradiol when starting treatment with an intravaginal progesterone releasing insert on ovarian follicular development and hormonal concentrations in Holstein cows

Ovarian follicular development and concentrations of gonadotrophin and steroid hormones were studied in non-lactating Holstein cows following administration of progesterone (P(4)) or oestradiol benzoate (ODB) at the start of treatment with an intravaginal progesterone releasing insert (IVP(4)) in a 2 by 2 factorial experiment. Cows were treated at random stages of the oestrous cycle with an IVP(4) device (Day 0) and either no other treatment (n=8), 200 mg of P(4) IM (n=9), 2.0 mg of ODB IM (n=8) or both P(4) and ODB (n=9). Seven days later devices were removed and PGF(2alpha) was administered. Twenty-four hours later 1.0mg of ODB was administered IM. Oestrus was detected in 97.1% and ovulation in 64.7% (effect of treatment, P=0.41) of cows within 96 h of removing inserts. In the cows that ovulated, day of emergence of the ovulatory follicle was delayed (P<0.01) and more precise (P<0.05) in cows treated with ODB compared to the cows treated with P(4). Interval from wave emergence to ovulation and the diameter of the ovulatory follicle was less (P<0.05) in cows treated with ODB compared to cows treated with P(4). Combined treatment with P(4) and ODB at the time of starting treatment with an IVP(4) device did not significantly change the pattern of ovarian follicular development compared to treatment with ODB alone. Concentrations of LH and FSH in plasma were less in cows treated with ODB between Days 0 and 4 (P<0.05) while treatment with P(4) increased concentrations of FSH in plasma between Days 0 and 4 (P<0.05). When anovulatory cows were compared to ovulatory cows, diameters of follicles (P<0.001) and growth rate of follicles (P<0.01) were less in anovulatory cows between Days 7 and 9, while concentrations of FSH in plasma were greater (P<0.01), concentrations of LH similar (P>0.90) and concentrations of oestradiol were less (P=0.01) in the anovulatory cows between Days 4 and 10. Our findings support a hypothesis that ovarian follicular development following administration of P(4) or ODB at the start of treatment with an IVP(4) device differs. Anovulatory oestrus may have been associated with reduced maturity and/or later emergence of ovarian follicles.