Anna Gobbetti

Is Corticosterone Involved in the Reproductive Processes of the Male Lizard, Podarcis sicula sicula?

Corticosterone (B) and testosterone (T) plasma levels and the effects of short (1-48 min) and long (6-192 hr) confinement stress during the various phases of the reproductive period of the male lizard, Podarcis sicula sicula, were studied; in addition, the in vitro effects of B on the T secretion by testis and adrenals were evaluated. Plasma B was highest during the mating phase and plasma T was highest during the aggressive phase. Confinement stress caused an increase of B plasma levels within 12 min of capture and a decrease in plasma T within 48 min of capture; B increase and T decrease continued for 48 hr, but, after 192 hr of confinement, the levels of these two steroids became similar to those found a few minutes after capture. The basal release of T by the tests and of B and T by adrenals mirrored the trends seen in the systemic circulation. In in vitro experiments B treatment decreased T by testis and adrenal tissue release in aggressive and mating phases. These data suggest that B could involved in the reproduction of P. s. sicula by acting on T synthesis to reduce aggressive behavior and allow breeding.

EFFECTS OF CAPTIVITY STRESS ON PLASMA STEROID LEVELS IN THE GREEN FROG, RANA ESCULENTA, DURING THE ANNUAL REPRODUCTIVE CYCLE

Abstract 1. 1. Androgen, 17β-estradiol and corticosterone levels were measured by RIA in plasma of Rana esculenta frogs sampled in the field and in the laboratory. 2. 2. Androgen and 17β-estradiol annual cycles of frogs sampled in the field mirrored those of animals sampled in the laboratory 24 hr after capture; on the contrary the absolute values were not similar. 3. 3. 17β-estradiol levels showed a peak 6 hr after capture in the pre-reproduction and reproduction periods. 4. 4. Corticosterone levels increased 72 hr after capture.

Intraluteal regulation of prostaglandin F2α-induced prostaglandin biosynthesis in pseudopregnant rabbits

The objective of the present study was to investigate in rabbit corpora lutea (CL), at both the cellular and molecular level, intraluteal cyclooxygenase (COX)-1, COX-2 and prostaglandin (PG) E2-9-ketoreductase (PGE2-9-K) enzymatic activities as well as in vitro PGE2 and PGF2alpha synthesis following PGF2alpha treatment at either early- (day-4) or mid-luteal (day-9) stage of pseudopregnancy. By immunohistochemistry, positive staining for COX-2 was localized in luteal and endothelial cells of stromal arteries at both the stages. In CL of both stages, basal COX-2 mRNA levels were poorly expressed, but rose (P < 0.01) 4- to 10-fold 1.5-6 h after treatment and then gradually decreased within 24 h. Compared to mid-stage, day-4 CL had lower (P < 0.01) COX-2 and PGE2-9-K basal activities, and PGF2alpha synthesis rate, but higher (P < 0.01) PGE2 production. Independent of luteal stage, PGF2alpha treatment did not affect COX-1 activity. In day-4 CL, PGF2alpha induced an increase (P < 0.01) in both COX-2 activity and PGF2alpha synthesis, whereas that of PGE2 remained unchanged. In day-9 CL, PGF2alpha up-regulated (P < 0.01) both COX-2 and PGE-9-K activities, and PGF2alpha production, but decreased (P < 0.01) PGE2 synthesis. All changes in gene expression and enzymatic activities occurred within 1.5 h after PGF2alpha challenge and were more marked in day-9 CL. Our data suggest that PGF2alpha directs intraluteal PG biosynthesis in mature CL, by affecting the CL biosynthetic machinery to increase the PGF2alpha synthesis in an auto-amplifying manner, with the activation of COX-2 and PGE-9-K; this may partly explain their differentially, age-dependent, luteolytic capacity to exogenous PGF2alpha in rabbits.

Evidence for a Luteotropic Role of Peroxisome Proliferator-Activated Receptor Gamma: Expression and In Vitro Effects on Enzymatic and Hormonal Activities in Corpora Lutea of Pseudopregnant Rabbits

ABSTRACT The expression of peroxisome proliferator-activated receptor gamma (PPARgamma) and its role in corpora lutea (CL) function were studied in pseudopregnant rabbits. Corpora lutea were collected at an early stage (Day 4), midstage (Day 9), and late stage (Day 13) of pseudopregnancy. Immunohistochemistry found evidence for the presence of PPARgamma in the perinuclear cytoplasm and nucleus of all the luteal cells; immunoreactivity decreased from the early to the late stage, with immunonegativity of the nuclei of late stage CL. PPARgamma mRNA transcript was expressed in all the luteal stages with the lowest level in the late stage. In CL cultured in vitro, the PPARgamma agonist (15-deoxy delta12,14 prostaglandin J2 [15d-PGJ2], 200 nM) increased and the antagonist (T0070907, 50 nM) decreased progesterone secretion at early and midluteal stages, whereas 15d-PGJ2 reduced and T0070907 increased PGF2alpha at the same stages. Prostaglandin-endoperoxide synthase 2 (PTGS2) activity was reduced by 15d-PGJ2 and increased by T0070907 in CL of early and midluteal stages. Conversely, 15d-PGJ2 increased and T0070907 reduced 3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity in early and midluteal stage CL. PGE2 in vitro secretion as well as PTGS1 and 20alpha-HSD enzymatic activities were not affected by 15d-PGJ2 and T0070907 in any CL types. These results indicate that PPARgamma plays a luteotropic role in pseudopregnant rabbits, through PTGS2 down-regulation and 3beta-HSD up-regulation, with a consequent PGF2alpha decrease and progesterone increase.

Vitellogenin hormonal control in the green frog, Rana esculenta. Interplay between estradiol and pituitary hormones

1. The effect of estradiol and pituitary hormones on the titre of serum vitellogenin has been studied in Rana esculenta by rocket immunoelectrophoresis. 2. Hepatic synthesis of vitellogenin depends on physiological doses of estradiol. 3. Gonadotrophins enhance the uptake, presumably by acting directly on the oocyte plasma membrane. 4. In addition, our data support direct pituitary intervention on liver synthesis and/or release of vitellogenin. 5. Hormonal response, as evaluated by vitellogenin serum titres, tends to increase from November to July. This could be the expression of a modification, throughout the sexual cycle, of liver sensitivity to the hormones.

Expression of luteal estrogen receptor, interleukin-1, and apoptosis-associated genes after PGF2α administration in rabbits at different stages of pseudopregnancy

The dynamic expression for estrogen receptor subtype-1 (ESR1), interleukin-1beta (IL1B), and apoptosis-associated genes, as well as nitric oxide synthase activity, were examined in corpora lutea (CL) of rabbits after prostaglandin F(2alpha) (PGF(2alpha)) administration on either day 4 or day 9 of pseudopregnancy. By reverse transcriptase polymerase chain reaction, the steady-state level of ESR1 transcript was lower (P < 0.01) and that of anti-apoptotic B-cell CLL/lymphoma 2 (BCL2) -like 1 (BCL2L1) was greater in day 4 (P < 0.01) than in day 9 CL. Western blot analysis revealed that BCL2-associated X protein (BAX) abundance was greater in day 4 (P < 0.01) than in day 9 CL, whereas BCL2L1 protein was undetectable at both luteal stages. After PGF(2alpha), ESR1 transcript decreased (P < 0.01) in day 9 CL, whereas IL1B mRNA showed a transitory increase (P < 0.01) at both stages. The pro-apoptotic tumor protein p53 (TP53) gene had diminished (P < 0.01) on day 4 and on day 9 after a transitory increase (P < 0.01), whereas the BAX/BCL2L1 expression ratio increased (P < 0.01) in day 9 CL 24 h after treatment. Following PGF(2alpha), TP53 protein increased (P < 0.01) at both luteal stages, and BAX decreased (P < 0.01) in day 4 CL but increased (P < 0.01) 24 h later in day 9 CL; BCL2L1 became detectable 6 h later in day 4 CL. Nitric oxide synthase activity temporarily increased (P < 0.01) following PGF(2alpha). These findings suggest that PGF(2alpha) regulates luteolysis by ESR1 mRNA down-regulation and modulation of pro- and anti-apoptotic pathways in CL that have acquired a luteolytic capacity.

Changes in refractoriness of rabbit corpora lutea to a prostaglandin F2 alpha analogue, alfaprostol, during pseudopregnancy

The responsiveness of rabbit corpus luteum to 200 micrograms of the prostaglandin F2 alpha (PGF2 alpha) analogue, alfaprostol, between Days 3 and 9 of pseudopregnancy was assessed by evaluating the decline in plasma progesterone after treatment with PGF2 alpha in 81 New Zealand White (NZW) rabbits. On Days 3-5, functional luteolysis was not observed. On Days 6, 7, and 8 of pseudopregnancy, the number of rabbits responsive to PGF2 alpha, rose from 38% to 71% and 83%, respectively. In the other cases, the effect of the PGF2 alpha analogue was transient as CL recovered in the following 2 or 3 days. By contrast, on Day 9 luteolysis was effective and persistent in all the animals. In rabbits treated on Day 9, progesterone decreased gradually from 10.6 +/- 0.7 within the first 6 h, but fell to 3.6 +/- 1.5 ng/mL (p < 0.01) 12 h after PGF2 alpha and to 0.2 +/- 0.1 ng/mL (p < 0.01) 24 h later.

Seasonal changes in plasma prostaglandin F2α and sex hormones in the male water frog, Rana esculenta

Prostaglandin F2 alpha (PGF2 alpha), progesterone, androgens (testosterone + dihydrotestosterone), and 17 beta-estradiol were measured in the plasma of male frogs, Rana esculenta, by radioimmunoassays. Plasma concentrations of PGF2 alpha were higher from October to December and peaked in March (prereproduction) and in June (postreproduction). Plasma progesterone levels were relatively low but showed an increase from October to December and in June. Plasma androgen titres rapidly increased in early spring, started to fall during the reproductive period (May), and were lowest in July. 17 beta-Estradiol levels peaked in March and in June. The annual profile of the plasma PGF2 alpha levels was positively correlated with those of progesterone and androgens, while it was not correlated to the estradiol plasma pattern, except in March and June. The increase in plasma PGF2 alpha in the autumn may be related to gonadal recovery. The simultaneous increases in PGF2 alpha and 17 beta-estradiol, both in March and June, suggest a PGF2 alpha-dependent estradiol synthesis, a possibility also supported by the increased plasma 17 beta-estradiol previously observed in PGF2 alpha-treated postreproductive females. The effects of captivity and castration on plasma PGF2 alpha concentrations were also studied during the annual cycle. Captivity was associated with a reduced PGF2 alpha titre, while castration did not modify prostaglandin synthesis, which may point to an extragonadal source of plasma PGF2 alpha.

Gonadotropin-releasing hormone stimulates biosynthesis of prostaglandin F2α by the interrenal gland of the water frog, Rana esculenta, in vitro

The present study was carried out to evaluate the in vitro effects of mammalian gonadotropin-releasing hormone (mGnRH) on the production of prostaglandin F2 alpha (PGF2 alpha) and sex steroids (progesterone, androgens, and 17 beta-estradiol) by the interrenal gland of male and female Rana esculenta during three different periods of the sexual annual cycle. In both sexes, mGnRH induced a significant increase in PGF2 alpha in the incubation medium in all examined periods. Progesterone and androgens were undetectable, while 17 beta-estradiol was significantly increased by mGnRH in interrenals incubated during the postreproductive period in both sexes. These results suggest that R. esculenta interrenals could be a GnRH-dependent PGF2 alpha-secreting tissue. In addition, the simultaneous increase in PGF2 alpha and estradiol from postreproductive cultured interrenals support the notion that mGnRH-induced estradiol synthesis is mediated through PGF2 alpha formation. This finding, taken together with other previous studies, strongly suggests that the end of the breeding period in R. esculenta depends on GnRH-induced PGF2 alpha-mediated enhancement of estradiol synthesis in a steroidogenetic organ (probably interrenals).

Prostaglandin F2α in female water frog, Rana esculenta: Plasma levels during the annual cycle and effects of exogenous PGF2α on circulating sex hormones

Prostaglandin F2 alpha (PGF2 alpha) and sex hormones (progesterone, androgens, and estradiol-17 beta) have been determined in the plasma of female water frog, Rana esculenta, utilizing radioimmunoassay methods (RIA). Plasma PGF2 alpha level increases in autumn-winter when recovery processes interest both gonad and oviduct. The PGF2 alpha plasma highest value has been recorded in October (4.52 +/- 0.58 ng/ml) and the lowest in May (1.43 +/- 0.12). The PGF2 alpha annual pattern is positively correlated to that of estradiol (but not to those of progesterone and androgens). Exogenous PGF2 alpha, injected into intact females during the various phases of the annual cycle, induces an increase of ovarian weight in frogs treated in December and February and an increase of plasma estradiol in postreproductive animals (June). Although preliminary, such results propose a putative implication of PGF2 alpha in ovarian recovery processes and in the interruption of reproductive processes in late summer.