and M. lerani

Molecular, Cellular, of Camerino, 62032

and Animal Camerino,

Biology, Italy

ABSTRACT Prostaglandin F2a (PGFza), prostaglandin E2 (PGE2), progesterone, androgens, and 1‘I&estradiol in vitro release by the abdominal gland of the crested newt, Ttirurus was studied during the prereproductive, reproductive and camifex (Laur.), postreproductive periods. In addition, the in vitro effects of the PGF2a and/or PGE2 on progesterone, androgens and estradiol release by the abdominal gland were evaluated. PGF2a, PGEz and progesterone release was higher during the reproductive period, and in the same period, PGE2 treatment induced a progesterone increase. PGF2o: induced an increase of abdominal gland estradiol release at the end of the reproductive period. These results seemed to confirm the pheromonal role assigned to progesterone, and suggested a PGE2 stimulatory role in inducing progesterone release, even if pheromonal activity of PGF2a and PGE2 cannot be excluded. In addition, PGFza-dependent estradiol increase at the end of reproduction could be interpreted as a mechanism for interruption of the abdominal gland activity. INTRODUCTION Most urodeles of the Trilurus genus rely on pheromones for sexual communication. The abdominal glands of the cloaca1 complex are believed to be responsible for the hedonic-like secretions which the male releases into the water and conveys towards the female, fanning his tail to induce acceptance of the spermatophore (1, 2). The abdominal gland of Ttiturus camifex, well described by Sacerdote (3, 4, 5), is a male secondary sexual character which shows temperature-dependent histological changes. It has been suggested that the abdominal gland of Ttiturus camifex secretes neutral glycoproteins and lipid substances (6); in addition, other authors found that this gland was able to metabolize steroids (7, 8, 9). Preliminary studies (10) suggested that courtship pheromones could be glycoprotein components of the abdominal gland secretion. More recently, Belvedere et al. (11) found that female Ttiturus camifex was completely unresponsive to the glycoproteins secreted by the abdominal gland of the male during courtship, but showed that progesterone was a secretory product of the abdominal gland, and that this steroid was a pheromone for the female. The female goldfish, Carassius auratus, was found to release a mixture of prostaglandins F which can act as a pheromone (12, 13). On the other hand, the implication of prostaglandins in fish sexual attraction was proved by Colombo et al. (14), who found that the administration of prostaglandin F2a(PGF za) and prostaglandin E2 (PGE2) was able to induce the appearance of a pheromonal activity in the urine of male gobiid, Orsinigobius punctatissimus, and PGF2u in the urine of female gobiid, Padogobius martensi. Recently, it was found that PGF2a is involved in the regulation of the reproductive processes and in the modulation of the sex hormone steroidogenesis in the water frog, Rana esculenta (15, 16, 17), and in the crested newt, Ttirurus camjfex ( 18, 19). In this work we studied the in vitro PGF2o, PGE2, progesterone, androgens, and


1992 VOL. 43 NO. 2


PROSTAGLANDINS 178-estmdiol release by the abdominal gland of Triturus camifex during the prereproductive, reproductive and postreproductive periods of the annual sexual cycle. In addition, the in vitro effects of PGFzn and/or PGEz on the steroid release by the abdominal gland were examined in the same periods. MATERIALS


Animals. The reproductive cycle of this TL$UNScamifex population comprises two major phases: during the summer period the animals disappear underground, during the cold months newts are in the pond undertaking reproduction (January to March). Adult male and female crested newts, T~‘~uNscamifex (Laur.), were captured in the Colfiorito pond (Umbria, Italy; 870 m above sea level) during the following months: December (prereproduction), January (reproduction beginning), March (reproduction ending) and April (postreproduction). The animals were transferred to our laboratory aquaria and fed on larvae of Chironomidae ( Tubifex and Dafnia). The aquaria were floored with gravel and furnished with different water plants. The water was kept flowing by slowly running tap water. Water temperature and photoperiod followed the seasonal cvcles. Male courtship observation. In each of the months above reported, after a brief acclimatization (1 week), the male reproductive behavior was observed. The male courtship consisted in four main phases (2), here briefly described: a) approach: the male sniffs the female; b) fan: the male assumes a characteristic posture and slowly beats his tail; c) lashes: the fan becomes more pronounced until the male’s tail begins to strike the female’s head; d) deposition: the male moves in front of the female and after her tail-touch he deposits the spermatophore. In December and April, the males did not show courtship; in January, the males exhibited intense reproductive behavior, and in March one less intense than in January. In vitro exneriment. The method used for the in vitro experiment followed Gobbetti and Zerani (17). For each of the months considered, after courtship observation, 80 males were weighed (8.57 + 0.98 g) and decapitated; the abdominal glands were rapidly removed under dissection microscope following Sacerdote (3, 4), weighed and placed in cold Dulbecco’s modified Eagle medium (DME; Sigma, USA) containing 10 mM Hepes, 1 mg Penicillin G/ml, and 2 mg Streptomycin/ml. Preliminary histological verifications, in according with Sacerdote (4), led us to confirm the validity of the method used for the removal of the abdominal gland. For each month, 16 abdominal glands, randomly chosen, were placed each in one incubation well. Multiwell tissue culture plates (Becton Dickinson, USA) were utilized. Each incubation set of wells was divided into 4 experimental groups (each consisting of 4 wells). The experimental groups were: a) abdominal gland incubated with DME alone; b) abdominal gland incubated with PGF2a (150 ng); c) abdominal gland incubated with PGEz (200 ng); d) abdominal gland incubated with PGFz u plus PGE2 (150 ng PGFza plus 200 ng PGE2). The final volume of each well was 1 ml. Culture plates were wrapped with aluminium foil and incubated in a shaking water bath (19OC), set at 30 revolutions/min. One well of each experimental group was removed, respectively, after 15, 30, 60 or 120 min of incubation. The incubation medium samples were immediately stored at -20°C for later hormone determination; tissues were at once homogenized in amphibian saline (0.64% w/v NaCl solution), and protein content was determined by the Protein Assay method (Bio-Rad, USA). In addition, the experiment was repeated with incubation sets with DME alone (without abdominal gland). Tests on 5 parallel incubation sets were carried out. Preliminary evidence led to



1992 VOL. 43 NO. 2

PROSTAGLANDINS our choosing the incubation conditions and the PGF20, PGE2, or PGF2o plus PGE2 minimum effective dose utilized in the present in vitro study (data not shown). In all the four months, the effects of progesterone on PGFza and PGE2 release by the abdominal gland were studied, but no dose tested was able to increase the PGF2a and PGE2 release (data not shown). mn, PGE2. progesterone , androaens, and 178-estradiol determination. A radioimmunoloaical method (RIA) was used for the measurement of the PGFza content of incubation media, as previously reported ( 15, 17, 18). The RIA method employed for PGFza was used for PGEz determination. The recovery ofadded labelled PGEz was 83.7 + 0.77. The parallelism among the standard curve in buffer, a standard curve in incubation medium (then extracted), and a serial dilution of a single incubation medium sample (extracted) was constant. The progesterone, androgens, and 17D-estradiol content of the abdominal gland incubation media were determined by RIA according to previously reported methods (17,20). The following sensitivities were recorded: PGFzx, 18 pg (intra-assay variability: 5%; inter-assay variability: 8%); PGE2. 19 pg (in&a-assay variability: 6%; inter-assay variability: 10%); progesterone, 8.5 pg (intra-assay variability: 6%; inter-assay variability: 10.5%); androgens, 5.5 pg (intra-assay variability: 6.5%; inter-assay variability: 9%) 17&estmdiol, 7 pg (intra-assay variability: 4%; inter-assay variability: 9%). The PGFza, progesterone, testosterone, and 17B-estradiol antisera were provided by Dr. G.F. Bolelli (CNR-Physiopathology of Reproduction Service, University of Bologna, Italy), the PGE2 antiserum was purchased from Cayman Chemical (USA). Compounds that cross-reacted more than 0.1% with PGF2o antiserum were: PGF 1CI more than 0.0 I % (36.2%) and 6-keto-PGFru (0.2%); compounds that cross-reacted with PGEz antiserum were: 15-keto-PGE2 (9.3%) and PGEI (5.0%). Testosterone was not separated from Sa-dihydrotestosterone and therefore, since the antiserum used is not specific, the data are expressed as androgens. Tritiated PGFza, PGE2, progesterone, testosterone, and 17I3-estradiol were purchased from Amersham testosterone, International (England), non-radioactive PGFzn, PGEz, progesterone, and 17D-estradiol from Sigma. Statistics. Data relative to each hormone were submitted to an analysis of variance (ANOVA) followed by Duncan’s multiple range test (2 1, 22). RESULTS PGFza, PGE2, progesterone, androgens, and estradiol were not detectable in incubation with DME alone (data not shown). PGF2a levels were higher in January with respect to the other months (P

PGF2 alpha, PGE2, and sex steroids from the abdominal gland of the male crested newt Triturus carnifex (Laur.).

Prostaglandin F2 alpha (PGF2 alpha), prostaglandin E2 (PGE2), progesterone, androgens, and 17 beta-estradiol in vitro release by the abdominal gland o...
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