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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.

Frog, Rana esculenta, pituitary and testis gonadotropin-releasing hormone (GnRH) receptors were characterized by using 125I-chicken IIGnRH (cIIGnRH) as radiolabeled ligand. At 4 C equilibrium binding of 125I-cIIGnRH to pituitary homogenates was achieved after 90 min of incubation; binding of 125I-cIIGnRH to testis membrane fractions reached its maximum at 60 min of incubation. Binding of the radioligand was a function of tissue concentration, with a positive correlation over the range 0.5-2 tissue equivalents per tube. One pituitary and one testis per tube were used as standard experimental condition. Incubation of the pituitary homogenate with increasing concentrations of 125I-cIIGnRH indicated saturable binding at radioligand concentrations of 1 nM and above while for the testis membrane preparation saturation was achieved using 5 nM 125I-cIIGnRH. The binding of 125I-cIIGnRH was found to be reversible after addition of the cold analog and the displacement curves could be resolved into one linear component for both tissues. Scatchard analysis suggested the presence of one class of binding sites for both pituitary and testis (Pituitary: Kd = 1.25 +/- 0.14 nM and Bmax = 8.55 +/- 2.72 fmol/mg protein; testis: Kd = 2.23 +/- 0.89 nM and Bmax = 26.48 +/- 7.39 fmol/mg protein). Buserelin displaced the labeled 125I-cIIGnRH with a lower IC50 as compared with cIIGnRH cold standard, while Arg-vasopressin (AVP) was completely ineffective, confirming the specificity of binding.

The inner horizontal cell (IHC) of the retina of Rana pipiens was studied by light and electron microscopy of Golgi stained or horseradish peroxidase injected cells. Responses of IHCs were recorded with intracellular electrodes. Both axon and dendritic terminals of the IHC make synaptic contact with all classes of receptor. The terminals occur as lateral or medial processes at synaptic ribbons. Reciprocal invaginations of receptor into horizontal cell process are common. Different classes of receptor are contacted in about the proportions with which they occur in the retina. No tendency for contacts of one type of receptor to occur on a particular part of the cell was found. The IHC generates L-type S-potentials. Both rod and cone input is evident in the waveform of the response, its spectral sensitivity, and the effect on it of adaptation.

The effects of a GnRH analog (GnRHA), D-Ser-t-Bu6,desGly-NH2(10) (HOE 766) on spermatogenesis were analyzed in the frog, Rana esculenta. Intact animals caught at two different periods of the year (January and March) were treated with HOE 766 (GnRHA, 45 ng/g BW) at low (4 +/- 1 C) and high (22 +/- 2 C) temperatures. Hypophysectomized frogs were used also and, in addition to GnRHA, these animals were treated with crude pars distalis homogenate. In vitro incubations were carried out at 15 C, for 0, 6, and 24 h with the addition of 1 microgram GnRHA. Half of each testis was used as the untreated control. Histological sections of the testes were analyzed for the evaluation of the mitotic index of the primary spermatogonia. Intact March animals had mitotic indices higher than January animals. GnRHA treatment elicited an increase of the mitotic index in both intact and hypophysectomized animals. High temperature potentiated the GnRHA effect while low temperature favored pars distalis treatment. In conclusion, the present results are consistent with the fact that in the frog, R. esculenta, the magnitude of spermatogonial proliferation is temperature dependent, and for the first time it is shown that GnRH-like substances have direct stimulatory effect on the mitotic activity of the primary spermatogonia in a vertebrate.

Seasonal plasma testosterone and androstenedione levels were determined by radioimmunoassay in the Gentile di Puglia ram. Androstenedione remains steady throughout the years, while testosterone shows seasonal changes. Testosterone is at high levels in winter, spring and summer and drops to a low concentration in autumn.

Ile-de-France ewes had high plasma progesterone concentrations during early summer-late winter. Gentile-di-Puglia ewes had high progesterone values during the winter-spring-summer period but during autumn progesterone values were very low and oestrous behaviour was not displayed. The comparison with Ile-de-France ewes indicates that a phase shift occurs in the annual ovarian activity in ewes of the Gentile-di-Puglia breed.

The profiles of estradiol (E2), progesterone (P), and androstenedione (A), have been studied for the first time in cyclic Gentile di Puglia ewes. Estradiol peaks at estrus whereas progesterone levels are high during the luteal phase. Androstenedione does not show meaningful cyclic fluctuation. All hormones examined show also a pulsatile pattern when plasma was collected hourly.

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.

The outer horizontal cell (OHC) of the frog Rana pipiens was studied by light and electron microscopy of Golgi-stained and horseradish peroxidase-injected cells. Responses of OHCs were recorded with intracellular electrodes. The OHCs are probably axonless cells. Their dendritic terminals are lateral processes at synaptic ribbons and are involved frequently in reciprocal invaginating contacts with receptors, ie, contacts characterized by invagination of receptor membrane deeply into the horizontal cell process. Two classes of OHC were distinguished on the basis of size and receptor contacts. Small OHCs (dendritic area about 5,000 micron2) contact all classes of receptor. These cells were not successfully penetrated with microelectrodes. Giant OHCs (dendritic area about 30,000 micron2) apparently contact only blue-sensitive rods and red-sensitive cones. They generate chromaticity or C-type responses, hyperpolarizing to short and depolarizing to long wavelength light.

We measured the plasma concentrations of luteinizing hormone (LH), oestradiol (E2), progesterone (P), 17 alpha-hydroxy progesterone (17P), androstenediona (A) and testosterone (T) at oestrus and during the oestrous cycle for four consecutive cycles in a group of 15 normal sows. The results show that at oestrus the peak LH value was preceded, 24 hours earlier, by an E2 peak, and indicate that the LH rise begins when E2 concentrations reach their highest value. During diestrus, concentrations of LH and E2 were constantly low, P and 17P were characterized by lowest concentrations during the oestrous period which showed significant (p less than 0.001), progressive increases from the second day after the LH ovulatory peak, to reach their highest values after 8-14 days. The 17P decrease in proestrus precedes that of P.T and A concentrations showed a significant (p less than 0.001) increase 2 days before the LH ovulatory peak; high plasma concentrations of both androgens were maintained until the LH peak occurred. Measurements taken in consecutive cycles in the same animals showed a high reproducibility of the hormone concentrations examined, which showed similar patterns and values in each of the cycles studied. This high reproducibility suggests that these hormones have an important physiological role and may affect oestrous behaviour.

The effects of a GnRH antagonist (GnRHA) on GnRH agonist (GnRH*)-induced androgen production and spermatogonial multiplication were studied in the frog, Rana esculenta, in vivo and in vitro. Intact and hypophysectomized (PDX) animals were kept at 22 +/- 2 C and treated with GnRH (45 ng/g BW) and GnRH* plus 1X and 10X concentrations of GnRHA on alternate days for 2 weeks. Androgen concentration in GnRH* plus GnRHA-treated animals decreased in the testis by about 50% with the 10X dose whereas the increase obtained in GnRH*-treated PDX group was completely abolished with the 1X dose. Histological sections were evaluated with respect of the mitotic index (MI) of the primary spermatogonia. Both GnRHA-treated intact and PDX frogs showed a dose-dependent MI decrease which reached 59% and 57% of control, respectively. In vitro incubations were carried out on testis halves at 15 C for 0, 2, 4, 6, and 8 h with the addition of 1 microgram GnRH* and 1 microgram GnRH* plus 1 or 10 micrograms GnRHA. The stimulatory effect of GnRH* and the inhibitory effect of GnRHA were apparent within 2 h. The basal mitogenic activity was affected by antagonist treatment and the inhibitory effect on the MI was evident within 2-4 h in the 10X-treated groups or within 6-8 h in the 1X treated groups. Since GnRH* and GnRHA bind to the same receptor these data strongly indicate that the effects of putative GnRH-like materials in the frog, Rana esculenta, are mediated throughout stereospecific recognition sites in both pituitary and testis.

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.

Ultrafine glass micropipettes can be easily beveled in a jet stream of grinding compound suspended in saline. The beveling is gradual and continuous, highly reliable, and can be accomplished with common laboratory apparatus. The beveled electrodes are comparable in performance to those prepared with expensive commercial bevelers.