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In this study, we show that sex hormones (testosterone, estradiol, and progesterone) act as physiological modulators of programmed cell death (PCD) during the frog liver involution observed postvitellogenesis. PCD in parenchymal cells is paralleled by the specific induction of the "tissue" transglutaminase (tTG) gene. tTG protein specifically accumulates in hepatocytes showing the morphological features of apoptosis. The hormone-dependent increase of both PCD and tTG was reproduced in ovariectomized frogs. Treatment of castrated animals with testosterone, estradiol, and progesterone inhibited the induction of both tTG and PCD, thus indicating that in vivo the drop in the circulating sex hormone is the signal favoring the involution phase of the maternal frog liver after mating. Although an affinity-purified polyclonal antibody raised against mammalian transglutaminase reacts in frog liver with a 55- to 60-kDa protein, concomitant with the onset of PCD, tTG cleavage products were detected, suggesting a proteolytic processing of the enzyme protein. These results represent the first evidence indicating that the physiological involution occurring postvitellogenesis of frog liver takes place by programmed cell death and that this, together with the concomitant induction of tTG gene expression, is regulated by sex hormones., (Copyright 1999 Academic Press.)

In male Podarcis s. sicula plasma, a sex steroid binding protein [SSBP(s)] binds testosterone (T) and estradiol-17 beta (E2) with moderate affinity (Kd = 0.23 +/- 0.08 x 10(-8) for 3H-E2, and 0.24 +/- 0.07 x 10(-8) for 3H-T) and high capacity. The SSBP binding affinity is unchanged throughout the sexual cycle, although its capacity is higher in nonreproductive males (winter and postreproductive period). This change may be related to changes in plasma T and E2 levels, and is likely to be involved in mechanisms whereby free steroid is delivered to target organs. SSBP, under isoelectrofocusing, is distributed between pH 5.5-6.5 and pH 7.1-7.5. The concentration of these two forms varies during the annual cycle.

Progesterone (P), 17-OH-progesterone (17-OH-P), androstenedione (A), dehydroepiandrosterone (DHEA), testosterone (T), 5 alpha-dihydrotestosterone (5 alpha-DHT), and 17 beta-estradiol (E2) were measured by RIA in plasma and testes of 114 males of the oviparous lizard Podarcis s. sicula raf, a species that displays annual hibernating cycles. Hormones were determined each month from January until December, except for August. Testosterone peaked at 174.8 ng/ml of plasma after emergence (March), while 5 alpha-DHT and A peaked in April. Plasma DHEA increased during hibernation. During the refractory period there were progressive increases in P and E2 plasma levels. The testicular peak of T, in March, coincided with that observed in plasma. The striking increases in testicular T and A in early July occurred at a time when plasma androgen concentrations were low. 5 alpha-DHT increased in April when spermatogenesis with spermiation occurred and then decreased alongside a second peak of T. There is an apparent separation of plasma and testicular androgen concentrations during the reproductive cycle.

The present investigation was performed to evaluate the effects of mammalian gonadotropin-releasing hormone (mGnRH) on prostaglandin F2 alpha (PGF2 alpha) plasma level in adult male and female water frog, Rana esculenta, during three different periods of the reproductive cycle: recovery period (October), breeding period (May), and postreproductive period (June). Intact, hypophysectomized (HYP), gonadectomized (GON), and hypophysectomized-and-gonadectomized (HYP/GON) animals were injected with 0.6 micrograms of mGnRH and sacrificed 1 hr and 5 hr after peptide administration. Some of each of the groups were sacrificed without having received mGnRH. PGF2 alpha plasma levels were assessed by radioimmunoassay. Hypophysectomy induced a significant increase of PGF2 alpha levels in October and June males. mGnRH induced a significant increase of PGF2 alpha plasma levels only in HYP and HYP/GON frogs. The tissue target of this GnRH action is, at present, unknown, although interrenals could be putative responsive tissues. At present, it is also difficult to assign any physiological role to observed phenomena unless to suppose that the pituitary inhibition is not constant throughout the year. It cannot be excluded that the prostaglandin induction depends on a local paracrine action of GnRH, which could be performed outside any pituitary control.

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.

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.

Estradiol-binding molecules have been found both in cytosol and nuclear extract of hepatocytes of the female green frog Rana esculenta. These molecules show the properties of an estradiol receptor (Re): high and specific affinity for estrogens (2.10-7.10 x 10(-10) M), stability of binding at 0 and 20 degrees, localization in the nucleus, and cytosolic versus nuclear binding shift in estradiol-treated frogs. In hepatocytes, both filled and unfilled Re is detectable in all stages of the R. esculenta annual cycle. It increases during the recovery phase (September to January) when vitellogenetic processes are active in the ovary. These changes are positively correlated with vitellogenin. Only nuclear filled Re, moreover, is positively correlated to level of circulating estradiol. This suggests a direct connection: plasma estradiol level versus nuclear filled Re versus plasma vitellogenin level.

The plasmatic profiles of androgens, estradiol, and progesterone, together with gonads and SSC modifications, have been followed, throughout the post-reproductive period in two Rana esculenta populations, inhabiting, respectively, a mountain pond (Colfiorito) and a sea level lagoon (Lesina). Testosterone and progesterone progressively decrease in the blood until July, while estradiol shows, in both sexes, an increase in the same month. Testosterone depletion accounts for thumb pad atrophy in the male and probably, in both sexes, for the summer interruption of sexual behavior. The estradiol increase could be linked to the induction of vitellogenin synthesis by the liver or, alternatively, could act through negative feed-back on the brain centers involved in GnRF synthesis and therefore it could be responsible for an inhibition of LH release and, in turn, of androgen synthesis/secretion by the gonads. The last function can be framed in the endocrine regulation of the so-called "refractory period" which interrupts the breeding during the summer. The hematic level of progesterone is higher during the ovulation period and this is consistent with the role assigned to the hormone, i.e., the induction of jelly release from oviductal glands. The hormonal trends in the blood of the two frog populations are very similar, although some differences exist in the levels of testosterone and progesterone.