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Spermatogenesis is a complex process that leads to the production of male gametes within the testis through the coordination of mitotic, meiotic and differentiation events, under a deep control of endocrine, paracrine and autocrine modulators along the Hypothalamus-pituitary-gonad (HPG) axis. The kisspeptin system plays a fundamental role along the HPG axis as it is the main positive modulator upstream of the hypothalamic neurons that secrete the Gonadotropin Releasing Hormone (GnRH), the decapeptide that supports pituitary gonadotropins and the production of gonadal sex steroid. Currently, kisspeptins and their receptor, KISS1R, have a recognized activity in the central control of puberty onset, sex maturation, reproduction and sex-steroid feedback mechanisms in both animal models and human. However, kisspeptin signaling has been widely reported in peripheral tissues, particularly in the testis of mammalian and non-mammalian vertebrates, with functions related to Leydig cells physiology and steroid biosynthesis, spermatogenesis progression and spermatozoa functions, but its mandatory role within the testis is still a matter of discussion. This review provides a summary of the main intratesticular effects of kisspeptin in vertebrates, via a comparative approach. Particular emphasis was devoted to data from the anuran amphibian Pelophylax esculentus, the first animal model in which the direct intratesticular activity of kisspeptin was reported., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Kisspeptin, via Gpr54 receptor, regulates puberty onset in most vertebrates. Thus, the direct involvement of kisspeptin activity in testis physiology was investigated in the anuran amphibian, Pelophylax esculentus. In this vertebrate gpr54 mRNA has been localized in both interstitial compartment and spermatogonia (SPG), whereas SPG proliferation requires the cooperation between estradiol and testicular Gonadotropin releasing hormone (Gnrh). In the pre-reproductive period, dose response curve to assess the effects of Kisspeptin-10 (Kp-10) was carried out in vitro (dose range: 10(-9)-10(-6)M; incubation times: 1 and 4h); proliferative activity and germ cell progression were evaluated by expression analysis of proliferating cell nuclear antigen (pcna), estrogen receptor beta (erβ), Gnrh system (gnrh1, gnrh2, gnrhr1, r2, r3) and by the count of empty, mitotic and meiotic tubules. All selected markers were up regulated at 4h Kp-10 incubation. Histological analysis also proved the increase of mitotic activity and the progression of spermatogenesis. Besides Kp-10 modulation of testicular Gnrh system, in vitro treatment with 17β-estradiol (10(-6)M) ± the antagonist ICI182-780 (10(-5)M) revealed gnrh2 and gnrhr3 estrogen dependent expression. In the reproductive period, testes were incubated for 1 and 4h with Kp-10 (10(-7)M) or Kp-10 (10(-7)M)+kisspeptin antagonist [Kp-234 (10(-6)M)]. Results obtained in the pre-reproductive period were confirmed and Kp-234 completely counteracted Kp-10 effects. In conclusion, Kp-10 modulated the expression of pcna, erβ, gnrhs and gnrhrs, inducing the progression of the spermatogenesis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Endocannabinoids - primarily anandamide (AEA) and 2-arachidonoylglycerol (2-AG) - are lipophilic molecules that bind to cannabinoid receptors (CB1 and CB2). They affect neuroendocrine activity inhibiting gonadotropin releasing hormone (GnRH) secretion and testosterone production in rodents, through a molecular mechanism supposed to be hypothalamus dependent. In order to investigate such a role, we choose the seasonal breeder, the anuran amphibian Rana esculenta, an experimental model in which components of the endocannabinoid system have been characterized. In February, at the onset of a new spermatogenetic wave, we carried out in vitro incubations of frog testis with AEA, at 10(-9)M dose. Such a treatment had no effect on the expression of cytochrome P450 17alpha hydroxylase/17,20 lyase (cyp17) nor 3-β-hydroxysteroid dehydrogenase/Δ-5-4 isomerase (3β-HSD), key enzymes of steroidogenesis. To understand whether or not the functionality of the hypothalamus-pituitary axis could be essential to support the role of endocannabinoids in steroidogenesis, frogs were injected with AEA, at 10(-8)M dose. Differently from in vitro experiment, the in vivo administration of AEA reduced the expression of cyp17 and 3β-HSD. Whereas the effect on 3β-HSD was counteracted by SR141716A (Rimonabant) - a selective antagonist of CB1, thus indicating a CB1 dependent modulation - the effect on cyp17 was not, suggesting a possible involvement of receptors other than CB1, probably the type-1 vanilloid receptor (TRPV1), since AEA works as an endocannabinoid and an endovanilloid as well. In conclusion our results indicate that endocannabinoids, via CB1, inhibit the expression of 3β-HSD in frog testis travelling along the hypothalamus-pituitary axis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Recently, we have investigated the endocannabinoid involvement in chromatin remodeling events occurring in male spermatids. Indeed, we have demonstrated that genetic inactivation of the cannabinoid receptor type 1 (Cnr1) negatively influences chromatin remodeling mechanisms, by reducing histone displacement and indices of sperm chromatin quality (chromatin condensation and DNA integrity). Conversely, Cnr1 knock-out (Cnr1(-/-)) male mice, treated with estrogens, replaced histones and rescued chromatin condensation as well as DNA integrity. In the present study, by exploiting Cnr1(+/+), Cnr(+/-) and Cnr1(-/-) epididymal sperm samples, we show that histone retention directly correlates with low values of sperm chromatin quality indices determining sperm nuclear size elongation. Moreover, we demonstrate that estrogens, by promoting histone displacement and chromatin condensation rescue, are able to efficiently reduce the greater nuclear length observed in Cnr1(-/-) sperm. As a consequence of our results, we suggest that nucleus length may be used as a morphological parameter useful to screen out spermatozoa with low chromatin quality., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In the hypothalamus, endocannabinoids affect neuroendocrine activity by means of Gonadotropin-Releasing-Hormone-I (GnRH-I) inhibition. Since most vertebrates, human included, possess at least two GnRH molecular forms, the aim of this work was to investigate the effect of endocannabinoids on GnRH molecular forms other than GnRH-I and on GnRHRs. Thus, we cloned GnRH precursors as well as GnRH receptors (GnRHR-I, GnRHR-II, GnRHR-III) from the diencephalons of the anuran amphibian, Rana esculenta. GnRH-II expression was evaluated in pituitary, whole brain, spinal cord, hindbrain, midbrain and forebrain during the annual sexual cycle. Then, in post-reproductive period (May), GnRH-I, GnRH-II and GnRHRs expression was evaluated by quantitative real time (qPCR) after incubation of diencephalons with the endocannabinoid anandamide (AEA). AEA significantly decreased GnRH-I and GnRH-II expression, up regulated GnRHR-I and GnRHR-II mRNA and it had no effect upon GnRHR-III expression. These effects were counteracted by SR141716A (Rimonabant), a selective antagonist of type I cannabinoid receptor (CB1). In conclusion our results demonstrate a CB1 receptor dependent modulation of GnRH system expression rate (both ligands and receptors) in frog diencephalons. In particular, we show that AEA, besides GnRH-I, also acts on GnRH-II expression., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Many advances have been carried out on the estrogens, GnRH and endocannabinoid system that have impact in the reproductive field. Indeed, estrogens, the generally accepted female hormones, have performed an unsuspected role in male sexual functions thanks to studies on non-mammalian vertebrates. Similarly, these animal models have provided important contributions to the identification of several GnRH ligand and receptor variants and their possible involvement in sexual behavior and gonadal function regulation. Moreover, the use of non-mammalian animal models has contributed to a better comprehension about the endocannabinoid system action in several mammalian reproductive events. We wish to highlight here how non-mammalian vertebrate animal model research contributes to advancements with implications on human health as well as providing a phylogenetic perspective on the evolution of reproductive systems in vertebrates., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Endometriosis is a chronic gynecological disease characterized by the growth of endometrial tissue outside the uterine cavity. Exposure to endocrine disruptors during critical period of development causes long-lasting effects, being the genital system one of the targets. This study describes the effects on female genital system caused by developmental exposure to the endocrine-disrupting chemical bisphenol A (BPA) during pre- and peri-natal development in mice. To this end, timed pregnant Balb-C mice were treated from day 1 of gestation to 7 days after delivery with BPA (100, or 1000 microg/kg/day). After delivery, pups were held for 3 months; then, pelvic organs were analyzed in their entirety and livers of both pups and moms were studied for the presence of BPA. We found in the adipose tissue surrounding the genital tracts of a consistent number of treated animals, endometriosis-like structure with the presence of both glands and stroma and expressing both estrogen receptor and HOXA-10. Moreover, cystic ovaries, adenomatous hyperplasia with cystic endometrial hyperplasia and atypical hyperplasia were significantly more frequent in treated animals respect to the controls. Finally, BPA was found in the livers of exposed moms and female offspring. In conclusion, we describe for the first time an endometriosis-like phenotype in mice, elicited by pre-natal exposition to BPA. This observation may induce to thoroughly reconsider the pathogenesis and treatment of endometriosis, considering the high incidence of endometriosis and the problems caused by associated infertility., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Mouse ubiquitin-specific processing protease (mUBPy) is a deubiquitinating enzyme highly expressed in both brain and testis. In testis, it interacts with the DnaJ protein, MSJ-1; both mUBPy and MSJ-1 are located on the cytoplasmic surface of the developing acrosome and in the centrosomal region during spemiogenesis. Present data show the first appearance in testis of mUbpy mRNA and protein at 10 days post-partum (d.p.p.). In addition, to investigate on a possible role of mUBPy in sperm formation, we took advantage of mutant wr/wr (wobbler) mice characterized by male infertility, which is likely due to the lack of a real, functional acrosome. RT-PCR and Northern blot analyses show that mUbpy is up-regulated in adult wobbler testis. Furthermore, in wild-type testis mUBPy protein is primarily detected by Western blot in the soluble (cytosolic/nuclear) fraction during the first round of spermatogenesis and in the adult. By contrast, mUBPy is primarily detected in membranous/insoluble protein fraction when wobbler phenotype is clearly shown (30 d.p.p.) and in adult wobbler testis. By immunohistochemistry, whereas in wild-type animals mUBPy marks the profile of the acrosomic vesicle in differentiating spermatids, in wobbler mice only a detergent pre-treatment procedure allows to detect mUBPy immunoreactivity, which results in diffuse spotted granules inside the cytoplasm and around the nuclear shape. In conclusion, in wobbler testis expression of mUbpy is up-regulated, while a differential sorting of the protein characterizes wobbler spermatids where acrosome formation is impaired., ((c) 2009 Elsevier Inc. All rights reserved.)

The importance of the endocannabinoid system (ECBS) and its involvement in several physiological processes is still increasing. Since the isolation of the main active compound of Cannabis sativa, Delta(9)-THC, several lines of research have evidenced the basic roles of this signaling system mainly considering its high conservation during evolution. In this chapter the attention is focussed on the involvement of the ECBS in the control of male reproductive aspects at both central and local levels which are both considered from a comparative point of view.

Msj-1 gene encodes a DnaJ protein highly expressed in spermatids and spermatozoa of both rodents and amphibians, possibly involved in vesicle fusion and protein quality control by means of interaction with heat shock proteins. We isolated and characterized the entire murine msj-1 gene and searched for putative msj-1-like genes into the human genome. Furthermore, ultrastructural localization of MSJ-1 was analyzed in mouse germ cells by immunogold electron microscopy. The analysis of murine msj-1 genomic sequence reveals that it is an intron less gene. Putative promoter region was predicted within the 600 bp upstream the transcription start site. In mouse, msj-1 maps on chromosome 1, into an intronic region of UDP glucuronosyl-transferase 1 family cluster. At ultrastructural level, MSJ-1 marks the developing acrosomic vesicle and the sperm centriolar region. A blast search against the human genome database revealed two closed regions (Ha and Hb) on human chromosome 2 having high nucleotide identity with murine msj-1 coding region. Similarly to mouse, in human both regions map into an intronic region of UDP glycosyl-transferase 1 family polypeptide A cluster (ugt1a@). A significant ORF encoding a putative DnaJ protein of 145 aa was predicted from Ha. Finally, expression analysis, conducted by RT-PCR in human sperm cells, demonstrated that Ha mRNA is effectively present in humans; by Western blot, a specific MSJ-1 band of approximately 30kDa was detected in human sperm. Taken together, these data suggest that msj-1 gene might be conserved among vertebrates and might exert fundamental functions in reproduction.

Endogenous and environmental estrogens have been proved to affect male reproduction in vertebrates. Both positive and negative effects in the regulation of the reproductive tract have been described. Since it is well known that amphibians represent a useful model to study several aspects concerning reproductive activity, we have taken advantage of the frog, Rana esculenta, to study the involvement of estrogens in sperm release. We show here that pituitary hormones increased the number of peritubular myoid cells (PMCs) expressing Fra-1 and induced testicular morphological changes related to sperm release. The estrogen antagonist ICI182-780 counteracted the hypophysis driven effects. In vivo and in vitro experiments demonstrated that 17beta-Estradiol acted directly on the testis to switch-on Fra-1 in PMCs. Furthermore, impairment of estrogen activity significantly reduced sperm release mainly affecting the detachment of spermatozoa from Sertoli cells (spermiation). Therefore, estrogens can be considered a new entry in the list of substances involved in spermiation.

Endocannabinoids are endogenous ligands for plasma membrane receptors (CB1 and CB2), belonging to the superfamily of G-protein-coupled receptors. They mimic some of the effects played by D9-tetrahydrocannabinol (THC), the active principle isolated from Cannabis sativa. N-arachidonoylethanolamine (anandamide, AEA) is the main endocannabinoid described to date in the testis and in human seminal plasma. However, the activity of AEA in controlling male reproduction is still poorly understood. In this study we report on physiological activity of endocannabinoids in the male reproductive tract. Using wild type (WT) and CB1 knock out mice (CB1KO) we show that endocannabinoids act in the epididymus. Here, through CB1, they inhibit sperm motility measured as the percentage of motile spermatozoa (SPZ). In particular, while in WT mice, as expected, the percentage of motile SPZ (measured in caput and cauda of epididymus) was significantly lower in the caput as compared with the cauda, in CB1KO mice a strong increase of motile SPZ in the caput was measured.

mUBPy (mouse ubiquitin specific processing protease) is a de-ubiquitinating enzyme expressed in mouse testis and brain. In testis, it interacts with the DnaJ protein MSJ-1 (mouse sperm cell specific DnaJ first homologue), a molecular chaperone expressed in spermatids and spermatozoa. Since MSJ-1 is conserved among vertebrates, to demonstrate an evolutionarily conserved function of UBPy/MSJ-1 system, we assayed mUBPy presence in the anuran amphibian, the frog, Rana esculenta, during the annual sexual cycle. By Western blot we have detected a specific signal of 126kDa in testis and isolated spermatozoa. During the annual sexual cycle, the signal gradually increases as soon as spermatogenesis resumes after the winter stasis. Using immunocytochemistry, we have localized the protein in spermatids and spermatozoa. In conclusion, UBPy/MSJ-1 system is available in R. esculenta testis suggesting a conserved fundamental function in spermatogenesis and sperm formation.

The presence of c-jun like mRNA was assessed in the brain of the frog, Rana esculenta, during the annual sexual cycle. In parallel, Jun protein and GnRH molecular form (mammalian and chicken II also indicated as GnRH1 and GnRH2, respectively) activity was studied in order to establish possible relationships. Northern blot analysis of total RNA reveals the presence of a 2.7 kb c-jun-like mRNA. Western blots, carried out on cytoplasmic and nuclear protein extracts, show the presence of Jun immunoreactive band of 39 kDa in brain and pituitary. Fluctuations of c-jun-like mRNA and Jun immunoreactive protein (cytoplasmic and nuclear) levels in brains during the year indicate relationships among transcription, translation, and nuclear activity. In particular, mRNA levels increase gradually from September until November when Jun protein concentration peaks in cytosolic extracts. Conversely, the nuclear protein reaches highest concentration in July when the cytosolic level shows low values. Immunocytochemical studies confirm the presence of Jun immunoreactivity in both cytoplasmic and nuclear compartments of several brain areas, including those primarily involved in gonadotropin discharge (e.g., anterior preoptic area and preoptic nucleus). GnRH molecular forms and Jun are colocalized in anterior preoptic area and preoptic nucleus. Moreover, during the period characterized by GnRH release, Jun levels strongly decrease in nuclei. Finally, we show that treatments with a GnRH analog (buserelin, Hoechst, Frankfurt) increase Jun levels in brain nuclear extracts.

The mechanisms underlying the complexity of spermatogenesis and spermiogenesis have deeply been studied in recent years. Transgenic animals, gene-targeting techniques, and lower vertebrate animal models have led to the discovery of some of the intratesticular signals involved in germ cell progression. This review wish to give the state of the art about it with particular emphasis on the comparative approach.

This review emphasizes the comparative approach for developing insight into knowledge related to cellular communications occurring in the hypothalamus-pituitary-gonadal axis. Indeed, research on adaptive phenomena leads to evolutionary tracks. Thus, going through recent results, we suggest that pheromonal communication precedes local communication which, in turn, precedes communication via the blood stream. Furthermore, the use of different routes of communication by a certain mediator leads to a conceptual change related to what hormones are. Nevertheless, endocrine communication should leave out of consideration the source (glandular or not) of mediator. Finally, we point out that the use of lower vertebrate animal models is fundamental to understanding general physiological mechanisms. In fact, different anatomical organization permits access to tissues not readily approachable in mammals.

Since proto-oncogenes play a central role in the regulation of cellular growth and differentiation, localization of MYC, FOS, and JUN proteins has been studied in the testis of the lizard, Podarcis s. sicula, during the annual reproductive cycle by immunocytochemistry using antisera against c-myc, c-fos, and c-jun products. MYC was localized in the nuclei of spermatogonia (SPG), I and II spermatocytes (SPC), and spermatids (SPT). Strong immunoreactivity was detected in Sertoli cells just prior to the onset of the early spring spermatogenic wave coinciding with the androgen peak. FOS protein was present in the nuclei of SPG and SPC. In SPG an exclusive nuclear localization was seen during the active spermatogenic period (February-March and September). A perinuclear localization was observed during other months. Immunoreactivity in Sertoli cells was also observed during the periods of active spermatogenesis. JUN protein was localized in the cytoplasm of SPG as well as in I and II SPC and was detected in the nuclei of I and II SPC during April and October when spermatogenic waves occur. These data suggest that proto-oncogene activities have regulatory roles in the spermatogenesis of the lizard., (Copyright 1997 Academic Press. Copyright 1997 Academic Press)

The expression of c-fos and c-jun mRNA has been examined in the testis of a seasonal breeder (the frog, Rana esculenta) during the annual reproductive cycle, using Northern blot analysis along with measurements of plasma levels of estradiol-17 beta and androgens (testosterone + 5 alpha-dihydrotestosterone). A c-fos-like transcript of 1.9 kb was revealed using a 1.1-kb v-fos probe, while three different transcripts of 3.7, 3.4, and 2.7 kb were seen using 1.0-kb human (h)-c-jun fragment. The proto-oncogene-like mRNAs appear during the period of the year associated with the new wave of spermatogenic activity. The levels of fos-like mRNA were highest after the estradiol-17 beta peak, while low levels were concomitant with high androgen concentrations. It is concluded that there is a close correlation between c-fos- and c-jun-like expression and testicular activity in R. esculenta.

Proto-oncogenes are said to influence the regulation of cellular growth and differentiation. Myc, Fos, Jun, and Mos protein localization has been studied by immunocytochemistry in the testis of the frog, Rana esculenta, during the annual reproductive cycle. Oncoproteins have been localized in the primary and secondary (I and II) spermatogonia (SPG). Myc and Mos also appear in I and II spermatocytes (SPC) while Jun appears in II SPC. Myc, Fos, and Jun in SPG translocate in the nucleus during the periods of active spermatogenesis. Myc, Fos, and Jun are also localized in Sertoli cells. Fos is present in interstitial cells during the period characterized by the androgen peak which precedes the sharp increase of estradiol. It is suggested that proto-oncogene activity exerts a regulatory role in steroidogenesis and spermatogenesis.

The distribution of different molecular forms of gonadotropin-releasing hormone (GnRH) in the brain and serum of the spotted dogfish, Scyliorhinus canicula, was investigated by an indirect immunofluorescence method, using antisera against salmon (s-), chicken-II (cII-) and mammalian (m-) GnRHs, and by reverse-phase high-performance liquid chromatography (RP-HPLC) coupled with radioimmunoassays. Five GnRH molecular forms were demonstrated on the basis of the retention time in the RP-HPLC system. The characteristics of four of these GnRH peptides are consistent with those of m-, cII-, dogfish (df-), and sGnRH. The fifth form appears to be novel. Immunoreactive sGnRH structures were confined to the diencephalon; whereas cIIGnRH and mGnRH were found in the telencephalon and diencephalon. cIIGnRH- and dfGnRH-like molecules were detected in the serum. Moreover, a specific, low-affinity GnRH binding protein (GnRH-BP) was found in the serum of the spotted dogfish. The binding of [125I]sGnRHA to the serum GnRH-BP was dependent on incubation time, equilibrium being reached within 1 hr at 4 degrees; binding was rapid and completely reversible. Scatchard analysis yielded a linear plot with a Kd of 7.9 x 10(-7) M. The presence of a GnRH-BP in spotted dogfish serum suggests a probable action of GnRH via the general circulation.

Ethane 1,2-dimethane sulfonate (EDS) destroys Leydig cells in the testis of some rodents (mice excluded), disrupts interstitial and germinal compartments in the frog, Rana esculenta, while it stimulates testicular activity in the teleost, Gobius paganellus. In the Japanese quail the toxin removes mature spermatozoa. There is no information on EDS effects in reptiles. The present study examines the effect of EDS treatment in the lizard Podarcis s. sicula Raf during two different periods of the testicular cycle (winter stasis and breeding season). Animals received a single EDS injection (100 mg/kg body wt) and were sacrificed at 0 and 24 hr and 3, 5, 7, 11, and 28 days after injection. Androgens were measured in plasma and right testes, while left testes were examined histologically. Plasma androgen levels decreased 5-7 days after EDS injection, alongside interstitial tissue destruction and mast cell appearance, with slight but significant increases on Days 11 and 28. Testicular androgen levels did not change. On Day 11 metaphases were present in the interstitial tissue which regenerated on Day 28. Between Days 5 and 7 some pycnotic nuclei of spermatocytes appeared, mitotic activity of spermatogonia was normal, but germ cell stages were disorganized and empty spaces appeared at the boundary of the tubule. These data show that a single EDS injection results in destruction and repopulation of the interstitial cells in a reptile. Moreover, the effects of EDS in the lizard suggest that P. s. sicula Raf testis responds to the toxin in a similar fashion to the rat testis.

The effects of ethane dimethane sulfonate (EDS) on the testes of hypophysectomized frogs (Rana esculenta) were investigated by light and electron microscopy. Initial signs of interstitial cell damage were observed in EDS- and EDS plus pituitary homogenate (PH)-treated animals 5 days after a single injection of EDS (100 mg/Kg body weight). The germinal compartment in these two groups appeared disorganized adjacent to the damaged interstitial tissue only in the EDS-treated animals, and by Day 8, spermatogenesis seemed to be affected in the EDS + PH-treated frogs in which Leydig cells had disappeared in some areas. On Day 28, regeneration of the interstitial tissue was complete and spermatogenesis was restored to normal. These data suggest that, in hypophysectomized frogs, the regeneration of the interstitial compartment is independent of pituitary activity and that the lack of interstitial cells activates the production of local factors responsible for the differentiation and proliferation of new Leydig cells. It is concluded that in addition to gonadotropins, the intratesticular environment is fundamental in the maintenance and regulation of testicular structure and function.

An androgen-binding activity has been identified in nuclear extracts of the testis of the frog, Rana esculenta. A single class of high affinity (Kd = 2.5 +/- 0.6 x 10(-9) M), low-capacity binding sites was found. The binding was specific for androgens; 17 beta-estradiol displaced [3H]testosterone with an ID50 of 0.1 microM. Cytosolic binding activity has a low affinity and a high capacity and lacks specificity. The seasonal fluctuations in binding capacity did not correlate with the androgen peaks in plasma and testes between February and June and in September; periods coinciding with the resumption of spermatogenesis and the development of spermatids, respectively. The present data strongly support androgenic control of intratesticular function in vertebrates generally.

Methionine-enkephalin (Met-Enk) has been detected in the testis of the frog, Rana esculenta, using a reverse-phase high-performance liquid chromatography system coupled with a specific radioimmunoassay. By means of immunocytochemical techniques Met-Enk positive cells have been localized in interstitial and germinal compartments. Particularly, spermatogonia, spermatocytes, and spermatozoa were stained in seminiferous tubules, and numerous interstitial cells showed strong cytoplasmic immunoreactivity in summer animals. Variations in the concentration of Met-Enk immunoreactive material occurred during the annual cycle. Our data show that Met-Enk is present in testes of nonmammalian vertebrate species. These results suggest that autocrine and/or paracrine mechanisms may regulate testicular activity in amphibians.

Steroid concentrations in plasma and follicular tissues (theca plus granulosa layers) were determined by radioimmunoassay in the aplacental viviparous ray, Torpedo marmorata, during various stages of the reproductive cycle. Steroids in the uterine fluid of pregnant animals and in preovulatory atretic follicles were also measured. In the follicular tissue of cyclic animals, levels of progesterone were always lower than those of estradiol-17 beta and androgens (testosterone plus 5 alpha-dihydrotestosterone). Estradiol-17 beta and androgen levels increased as the animals approached the ultimate maturational stage before ovulation. Androgens were not detectable in plasma, while estradiol-17 beta increased dramatically before ovulation. In pregnant animals, only small ovarian follicles (less than 5 mm in diameter) were observed, and these had hormone concentrations that were similar to those of the small follicles of cyclic animals. Progesterone was the only steroid detected in the uterine fluid of pregnant animals. In completely sclerotic atretic follicles of pregnant animals, steroids were not detected. Progesterone was the main hormone in atretic follicles undergoing yolk resorption. This suggests that the latter may contribute to the elevated plasma progesterone concentrations of pregnant animals.

The direct effect of estradiol on testicular androgen biosynthesis was studied in the frog, Rana esculenta, measuring progesterone, 17 alpha-OH-progesterone, androstenedione, and androgens (T + DHT) in supernatants and testes incubated (6 hr, 15 degrees) with estradiol alone (10(-6) M) or in combination with crude pituitary homogenate (1 pituitary equivalent/tube). Estradiol, either alone or in combination with pituitary homogenate, induced decreases of 17 alpha-OH-progesterone, androstenedione, and androgens but was ineffective in modulating progesterone levels. Pituitary homogenate was effective in inducing a significant increase of androstenedione and androgens but was ineffective in modulating both progesterone and 17 alpha-OH-progesterone production. It is concluded that estradiol acts by decreasing the activity of steroidogenic enzymes starting from 17 alpha-hydroxylase, while pituitary homogenate does not affect the 17 alpha-hydroxylase activity, but it acts starting from 17,20-lyase.

Seasonal variations of intratesticular steroid hormones (androgens and estradiol-17 beta) and spermatogenic activity have been studied in the marine teleost fish, Gobius paganellus. In addition, in vivo and in vitro experiments have been carried out in order to investigate the control of androgen production by the testis. While estradiol was never detected, androgens were at low values in autumn and reached maximal levels in spring concomitantly with the highest testis weight and the highest efficiency of the spermatogenic wave. In vitro incubations were carried out using ovine luteinizing hormone (oLH) (400, 4000, and 40,000 micrograms/liter; 20 degrees for 6 and 24 hr). The effective dose 40,000 micrograms/liter was used to induce androgen stimulation in both autumn and spring testes. The responsiveness to oLH was enhanced in spring testis. Estradiol and a gonadotropin-releasing hormone analog GnRHA (HOE766) were ineffective in modulating androgen production either alone (1-1000 nmol/liter) or in concert with oLH during short-term incubations. In intact animals, GnRHA elicited, 3 hr after the injection (10 micrograms), a three-fold increase of intratesticular androgen content. In conclusion, we show that the annual androgen profile in G. paganellus parallels the spermatogenic activity and that the androgen production is not affected in these experimental conditions by putative intratesticular factors (e.g., estradiol-17 beta and GnRH-like substances) which, conversely, are effective in inducing androgen changes in several vertebrate species.

Apart from mice, in rodents ethane dimethane sulfonate (EDS) selectively destroys Leydig cells. This has been indicated as a new method for the study of seminiferous interstitial compartment interaction. No information on the possible destruction and repopulation of Leydig cells exists in lower vertebrates. This study deals with EDS effects in the frog, Rana esculenta. Animals received a single intraperitonial dose (100 mg/kg body wt) and were sacrificed at 0, 12, and 24 hr and 3, 4, 7, 14, and 28 days postinjection. Androgens (testosterone + DHT) were measured in plasma and right testes. Moreover, left testes were fixed and examined for histological observation. Plasma androgen levels were extremely low on Day 4 after EDS treatment and remained unchanged thereafter. In testes, androgen levels decreased on Day 4 but increased to control levels on Day 14. Leydig cells were damaged within 3 days post-treatment and were completely destroyed on Days 4 and 5. Germinal compartment damage appeared only where the adjacent interstitial tissue presented complete destruction. Pale primary spermatogonia (stem cells) were always present. Testes restored to normal on Day 14 and spermatogenesis resumed to the regenerating interstitial tissue. These results show that regenerating testes in R. esculenta retain androgens and that interstitial-germinal compartment communications may have a role in maintaining spermatogenesis.

The possible physiological role of a putative testicular gonadotropin-releasing hormone (GnRH)-like material was studied in the frog, Rana esculenta. We have investigated (a) changes of mitotic index (MI) of primary spermatogonia (SPG) in GnRH agonist (GnRH-Ag)-treated testes in vitro; (b) changes of androgen concentrations in testes of intact frogs treated with a GnRH antagonist (GnRH-Ant); (c) variations of mitotic index of primary SPG in intact GnRH-Ant-injected animals and in testes incubated with GnRH-Ant; (d) changes of MI in hypophysectomized (PDX) animals treated with hypophysis (PD) homogenate, and hCG alone or in combination with GnRH-Ant; and (e) changes of androgen concentrations in plasma PDX frogs treated with hCG and hCG plus GnRH-Ant. Our results indicate that while GnRH-Ag induced accumulation of primary SPG mitosis, GnRH-Ant inhibited androgen production and natural occurring mitosis accumulation. Therefore, GnRH-Ant may counteract an endogenous peptide working into the testis.

The binding of a gonadotrophin-releasing hormone (GnRH) long acting analog (GnRHA), D-Ser (But)6,Pro9-NEt GnRH (HOE 766), to pituitary and testicular extracts and the presence of GnRH-like material in testes and hypothalamuses were measured in the frog, Rana esculenta. Also, the cellular localization of immunoreactive GnRH was investigated in testes by immunohistochemical staining. Furthermore, lyophilized preparations of pituitary crude homogenates from animals caught monthly were tested in vitro for their ability to stimulate androgen production by December testes. Satisfactory results on specific 125I-GnRH binding were difficult to obtain in view of its low binding capacity. Moreover, binding in testicular homogenates was of the same order of magnitude (about 2%) as that found in pituitaries. In a cospecific radioimmunoassay for GnRH nonapeptide, both hypothalamic and testicular extracts gave displacement parallel to the standard curve. Immunoreactive GnRH did not significantly fluctuate in hypothalamuses, while it peaked in testes during December and July. Immunoreactive GnRH was evidenced in June and September testes employing immunohistochemical staining. In particular, the interstitial cells and the Sertoli cells were faintly stained. Testes of December animals stimulated by February pituitaries produced larger quantities of androgens as compared with testes stimulated with hypophyseal preparations from the remaining periods of the year. In conclusion, the present results are consistent with the idea that seasonal changes of the hypothalamus-hypophyseal activity play an important role in regulating the hormonal response in vertebrate testes. Moreover, we report that, in addition to rats, GnRH-like material is present in frog testes and for the first time it has been shown that such putative intratesticular material undergoes seasonal fluctuations in a vertebrate.

The seasonal plasma estradiol-17 beta (E2-17 beta) profile and annual vitellogenin content of liver, fat body, plasma, and ovary were investigated in Rana esculenta. Concomitant with the increase in E2-17 beta, vitellogenin peaked in liver, plasma, and ovary during autumn and winter, while it remained at a relatively high concentration in fat body during spring. In vitro experiments showed that E2-17 beta (10(-9) M) is ineffective in inducing vitellogenin production in fat body, but is effective in inducing vitellogenin production in liver. As fat bodies do not produce the vitellogenin they contain, we suggest that fat bodies are involved in the transfer of vitellogenin to the ovary.

Seasonal plasma and intraovarian estradiol-17 beta (E) and progesterone (P) fluctuations were studied by specific radioimmunoassay in the frog, Rana esculenta. Moreover, incubations of ovine-luteinizing hormone (oLH)-stimulated ovarian pieces at two different temperatures (15 and 24 degrees) have been carried out in order to evaluate the dependence of E and P output on this exogenous factor. Estradiol showed similar changes in plasma and ovaries, while P profile was better evidenced in the gonads since this hormone fluctuated in plasma, giving pulses of difficult interpretation. A shift from E to P production by the ovary near the ovulatory period (February-March) was noted. In vitro experiments were carried out using approximately equal-sized ovarian fragments containing follicles ranging from 0.7 to 1 mm and classified as early vitellogenic. High temperature induced oLH-stimulated P production within 6 h, while E increased after 24 h concomitantly with a P decline. At 15 degrees the stimulatory effect of oLH was achieved only on E output in the incubation medium after 24 h. In conclusion, our results in the frog, R. esculenta, show that E and P intervene at peak values separately during the annual cycle and that the temperature has an important role in the regulation of the steroid hormone-releasing activity.

Seasonal changes in plasma androgens, testicular total protein content, gonosomatic index, and spermatogenic activity were studied in the grey partridge, Perdix perdix. Moreover, testicular androgen output after stimulation with ovine LH (oLH) was tested in vitro during different periods of the sexual cycle. Androgens and the gonosomatic index peaked in April, during which all the spermatogenic stages were observed. Total protein content in the testes was highest in January and March. Gonadal responsiveness to oLH was found to increase in the period April-May in coincidence with the hormone peak in the plasma, while February testes were irresponsive.

Testicular testosterone was determined by radioimmunoassay in the frog (Rana esculenta) kept in total darkness, at a high or a low temperature (24 or 4 degrees C), and treated with a gonadotrophin-releasing hormone analog (GnRHa, HOE 766). Prolonged exposure to dark conditions seemed to inhibit hypotalamic functions. Moreover, it is shown that high temperature interacts positively with GnRHa treatment on testicular testosterone concentration.

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.

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.

The interactions between thyroid hormones and receptors for steroid hormones and prolactin in dorsal skin and caudal fin of Triturus cristatus carnifex were studied during the annual cycle. Thyroidectomy induces an increase of prolactin binding in the dorsal skin and caudal fin in the animals captured in March. In these thyrodectomized animals the androgen receptors became undetectable. Results indicate that in Triturus cristatus carnifex the thyroid induces an increase of androgen receptors and a decrease, that is removed by thyroidectomy, of prolactin receptors.

Seasonal plasma progesterone, androstenedione, estrone, and 17 beta-estradiol concentrations in the female Rana esculenta were determined by radioimmunoassay during the 1979 and 1981 seasons. Plasma levels of these steroids were highest just before the first ovulatory wave in spring and lowest after the breeding season. In the 1979 season (during the 1981 season hormones were not assayed in January, November, and December) progesterone, androstenedione, and estradiol levels showed another peak in November-December. During the breeding months, i.e., late March to late June, progesterone, androstenedione, and estradiol levels showed intermittent ups and downs corresponding roughly to the ovulatory waves. In addition, during the breeding season progesterone and androstenedione levels had a higher average in frogs with "ripe" ovaries than in those with "spent" ovaries. Relationships between seasonal steroid levels and ovarian activity are briefly discussed.

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.

We have studied the annual cycle of estrogen-binding activity in the frog, Rana esculenta, testis to evidence possible fluctuations. The testicular binding for [3H]estradiol-17 beta shows high affinities (cytosolic Kd = 1.94 +/- 0.43 x 10(-9) M; nuclear Kd = 2.72 +/- 1.20 x 10(-9) M) and low capacity (cytosolic: 7.56 +/- 0.66 fmol/mg protein; nuclear: 9.27 +/- 2.5 fmol/mg protein). Nuclear-binding activity appears in spring concomitantly with the estradiol peak in plasma and testes, while, in the remaining periods, binding capacity was extremely low or absent. Present data strongly support an important role played by estrogens as an intratesticular control factor in vertebrates.

The effect of a gonadotropin-releasing hormone analog (GnRHA, HOE766) was studied in hypophysectomized elasmobranch fish Torpedo marmorata and T. ocellata. In addition, estradiol (E2) effects were studied in intact and hypophysectomized (HPX) animals. Plasma androgen concentrations were measured 2 or 6 hr after GnRHA (100 ng or 10 micrograms) or 6 hr after E2 (10(-9) or 10(-6) M) intratesticular injections. Both GnRHA and E2 induced the increase of plasma androgen levels in HPX fish. E2 also enhanced androgen levels in intact animals. It is concluded that GnRH-like substances and E2 may modulate testicular activity in elasmobranch fish.

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.