Sergio Minucci

Cell Biology of the Harderian Gland

The harderian gland is an orbital gland of the majority of land vertebrates. It is the only orbital gland in anuran amphibians since the lacrimal gland develops later during phylogenesis in some reptilian species. Perhaps because it is not found in man, little interest was paid to this gland until about four decades ago. In recent years, however, the scientific community has shown new interest in analyzing the ontogenetic and morphofunctional aspects of the harderian gland, particularly in rodents, which are the preferred experimental model for physiologists and pathologists. One of the main characteristics of the gland is the extreme variety not only in its morphology, but also in its biochemical properties. This most likely reflects the versatility of functions related to different adaptations of the species considered. The complexity of the harderian gland is further shown in its control by many exogenous and endogenous factors, which vary from species to species. The information gained so far points to the following functions for the gland: (1) lubrication of the eye and nictitating membrane, (2) a site of immune response, particularly in birds, (3) a source of pheromones, (4) a source of saliva in some chelonians, (5) osmoregulation in some reptiles, (6) photoreception in rodents, (7) thermoregulation in some rodents, and (8) a source of growth factors.

Stimulatory effect of a GnRH agonist (buserelin) in in vitro and in vivo testosterone production by the frog (Rana esculenta) testis

The summary testicular effects of an agonistic analogue of gonadotropin-releasing hormone (buserelin, GnRHa) have been studied in vitro and in vivo in the frog, Rana esculenta. During 3 h incubation GnRHa (8 X 10(-7) M) potentiated pituitary factors in stimulating testosterone production by minced testes in vitro. After 6 h of incubation 8 X 10(-7) M GnRHa stimulated maximal testosterone output. Testes of 10-day hypophysectomized animals did not show any GnRHa effect in vitro. In vivo, a direct effect of GnRHa on testicular testosterone production was demonstrated in hypophysectomized animals, although this effect was temperature-dependent, requiring the frog to be maintained at a high temperature (24 degrees C). No effect of GnRHa was detectable in frogs kept at a low temperature (4 degrees C).

Plasma and testicular estradiol and plasma androgen profile in the male frog Rana esculenta during the annual cycle.

Seasonal plasma and testicular estradiol levels were measured in the male frogs, Rana esculenta, by radioimmunoassay. In plasma samples a simultaneous measurement of androgens was carried out in order to investigate a possible relationship between androgens and estradiol-17 beta. Concomitantly with the estradiol-17 beta peak in plasma and testes during the April-May period, plasma androgens sharply decreased.

Intratesticular feedback mechanisms in the regulation of steroid profiles in the frog, Rana esculenta.

Testosterone (T), 5 alpha-dihydrotestosterone (DHT), estradiol-17 beta (E), and progesterone (P) were measured in the plasma of the frog, Rana esculenta, during the annual cycle. Moreover, in vitro experiments were carried out in order to investigate the local regulation of steroidogenesis. Testosterone and DHT showed high values during autumn and early spring and had a T/DHT ratio which increased during summer, while E peaked in midspring, remaining at detectable values thereafter. Progesterone increased in autumn, winter, and spring. In vitro incubations of minced testes showed that E, stimulated by pituitary factors, inhibited androgen synthesis while T did not. Our results indicate that paracrine and/or autocrine mechanisms operate in the frog testis to regulate annual steroid profiles.

The amphibian testis as model to study germ cell progression during spermatogenesis.

Testicular morphology of vertebrate testis indicates requirement of local control. In urodeles, the testis is organized in lobes of increasing maturity throughout the cephalocaudal axis. The anuran testis is organized in tubules. Spermatogenesis occurs in cysts composed by Sertoli cells enveloping germ cells at synchronous stages. Moreover, in numerous species germ cell progression lasts a year which defines the sexual cycle. Due to the above quoted features, research on factors regulating germ cell progression in amphibians may reach greater insight as compared with mammalian animal models. In particular, studies on endocrine and paracrine/autocrine factors involved in the regulation of germ cell functions reveal that fos activation and a J protein, previously specifically found in mouse testis, exert an important role in spermatogonial proliferation and maturation of post-meiotic stages, respectively.

Molecular forms of immunoreactive gonadotropin-releasing hormone in hypothalamus and testis of the frog, Rana esculenta.

The hypothalamus and the testis of the frog, Rana esculenta, contain gonadotropin-releasing hormone (Gn-RH)-like peptides which are recognized by an antiserum raised against mammalian Gn-RH. Two molecular forms which coelute with synthetic chicken II and salmon Gn-RH from reverse-phase HPLC were distinguished in the hypothalamus. A single peak coeluting with synthetic chicken II Gn-RH was present in the testis.

GPR30 is overexpressed in post-puberal testicular germ cell tumors

GPR30 is a 7-transmembrane G protein-coupled estrogen receptor that functions alongside traditional estrogen receptors to regulate cellular responses to 17β-estradiol and environmental estrogens. In this study, we have evaluated by immunohistochemical analysis GPR30 expression in post-puberal testicular germ cell tumours (30 seminomas, 5 teratomas, 12 embryonal carcinomas, and 20 intratubular germ cell tumors). The GPR30 protein expression was detected at high level in all intratubular germ cell tumours, seminomas, and embryonal carcinomas, whereas in teratomas the expression was low. The immunohistochemical data were further confirmed by Western blot analysis. GPR30 protein expression has also been analyzed in GC1 and TCam-2 cell lines, respectively derived from immortalized type B murine spermatogonia and human seminoma. Our results indicate that GPR30 could be a potential therapeutic target; the design of a specific GPR30 inhibitors could be a useful molecular target to block neoplastic germ cells with a high proliferative rate for the treatment of TGCTs. See commentary: Role of GPR30 in testicular germ cell tumors: A potential new anticancer target

Regulation of androgen production by frog (Rana esculenta) testis: An in vitro study on the effects exerted by estradiol, 5α-dihydrotestosterone, testosterone, melatonin, and serotonin

The possible role of estradiol-17 beta (E2), testosterone (T), 5 alpha-dihydrotestosterone (DHT), melatonin, and serotonin on the regulation of androgen (A) production by the frog, Rana esculenta, testes was studied in vitro. E2 (10(-6) M) inhibited A production whether alone or in combination with oLH (20 micrograms) after 6 hr incubation. After 24 hr incubation. A production was reduced by E2 concentration of around 10(-6) and 10(-9) M. Melatonin and serotonin did not induce any change whichever experimental condition was used. Preincubation for 6 hr with 10(-6) M T or DHT enhanced the oLH-stimulated A production after 18 hr incubation. These data suggest that steroids may regulate their intratesticular levels without passing into the blood stream.

IN SITU CHARACTERIZATION OF MAST CELLS IN THE FROG RANA ESCULENTA

Abstract The number, distribution, and ultrastructural characteristics of mast cells were assessed in the tongue, heart, and kidney of the frog Rana esculenta. The density of tongue mast cells (253±45 mast cells/mm2) was significantly higher than that of the heart (5.3±0.4/mm2) and kidney (15.3±1.4 /mm2). A striking feature of this study was the remarkable association of frog mast cells to nerves. The ultrastructural study of the mast cell/nerve association demonstrated that mast cells were closely apposed to or even embedded in nerves. Mast cells were also physically associated with melanocytes even in the heart. Mast cells were Alcian blue+/safranin+ in the tongue and in the peritoneum, whereas in the heart and in the kidney they were Alcian blue–/safranin+. The mast cells in the lamina propria of the gastrointestinal tract were Alcian blue+/safranin–. The cytoplasm of frog mast cells was packed with numerous heterogeneous, membrane-bound granules. The ultrastructure of these cytoplasmic granules was unique, being totally unlike any other previously described granules in other animal species as well as in man. The histamine content/frog mast cell (≈0.1 pg/cell) was approximately 30 times lower than that of human mast cells isolated from different tissues (≈3 pg/cell). A monoclonal anti-histamine antibody was used to confirm the ultrastructural localization of histamine in secretory granules in frog mast cells.

A gonadotropin releasing hormone analog induces spermiation in intact and hypophysectomized frogs,Rana esculenta

The sperm-releasing activity of a gonadotropin releasing hormone (GnRH) agonist, Buserelin (GnRH*) and hypophysis homogenate (PD) preparations was studied in intact and hypophysectomized (PDX) frogs,Rana esculenta. In addition, human chorion gonadotropin (hCG) was tested in PDX animals, and GnRH antagonist (GnRHA) treatments were carried out in intact and PDX animals, in combination with the hormonal injections. GnRH* or PD treatments were able to elicit spermiation in intact and PDX animals. While GnRH*, injected 24 h later, was again effective in inducing spermiation in intact animals, this was not the case in PDX frogs. GnRHA counteracted GnRH* effects in intact frogs. Moreover, in PDX animals GnRHA injections counteracted the spermreleasing activity induced by hCG or GnRH*, but failed to inhibit sperm-releasing activity induced by PD homogenate.