Exogenous human OKSM factors maintain pluripotency gene expression of bovine and porcine iPS-like cells obtained with STEMCCA delivery system.

OBJECTIVES: The use of induced pluripotent stem (iPS) cells as an alternative to embryonic stem cells to produce transgenic animals requires the development of a biotechnological platform for their generation. In this study, different strategies for the generation of bovine and porcine iPS cells were evaluated. Lentiviral vectors were used to deliver human factors OCT4, SOX2, KLF4 and c-MYC (OKSM) into bovine and porcine embryonic fibroblasts and different culture conditions were evaluated. RESULTS: Protocols based on the integrative lentiviral vector STEMCCA produced porcine iPS-like cells more efficiently than in bovine cells. The iPS-like cells generated displayed stem cell features; however, expression of exogenous factors was maintained along at least 12 passages. Since inactivation of the exogenous factors is still a major bottleneck for establishing fully reprogrammed iPS cells, defining culture conditions that support endogenous OKSM expression is critical for the efficient generation of farm animals' iPS cells.

Neuronal degeneration and regeneration induced by axotomy in the olfactory epithelium of Xenopus laevis.

The olfactory epithelium (OE) has the remarkable capability to constantly replace olfactory receptor neurons (ORNs) due to the presence of neural stem cells (NSCs). For this reason, the OE provides an excellent model to study neurogenesis and neuronal differentiation. In the present work, we induced neuronal degeneration in the OE of Xenopus laevis larvae by bilateral axotomy of the olfactory nerves. We found that axotomy induces specific- neuronal death through apoptosis between 24 and 48h post-injury. In concordance, there was a progressive decrease of the mature-ORN marker OMP until it was completely absent 72h post-injury. On the other hand, neurogenesis was evident 48h post-injury by an increase in the number of proliferating basal cells as well as NCAM-180- GAP-43+ immature neurons. Mature ORNs were replenished 21 days post-injury and the olfactory function was partially recovered, indicating that new ORNs were integrated into the olfactory bulb glomeruli. Throughout the regenerative process no changes in the expression pattern of the neurotrophin Brain Derivate Neurotrophic Factor were observed. Taken together, this work provides a sequential analysis of the neurodegenerative and subsequent regenerative processes that take place in the OE following axotomy. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1308-1320, 2017.

Quantitative comparative analysis of the nasal chemosensory organs of anurans during larval development and metamorphosis highlights the relative importance of chemosensory subsystems in the group.

The anuran peripheral olfactory system is composed of a number of subsystems, represented by distinct neuroepithelia. These include the main olfactory epithelium and vomeronasal organ (found in most tetrapods) and three specialized epithelia of anurans: the buccal-exposed olfactory epithelium of larvae, and the olfactory recess and middle chamber epithelium of postmetamorphic animals. To better characterize the developmental changes in these subsystems across the life cycle, morphometric changes of the nasal chemosensory organs during larval development and metamorphosis were analyzed in three different anuran species (Rhinella arenarum, Hypsiboas pulchellus, and Xenopus laevis). We calculated the volume of the nasal chemosensory organs by measuring the neuroepithelial area from serial histological sections at four different stages. In larvae, the vomeronasal organ was relatively reduced in R. arenarum compared with the other two species; the buccal-exposed olfactory epithelium was absent in X. laevis, and best developed in H. pulchellus. In postmetamorphic animals, the olfactory epithelium (air-sensitive organ) was relatively bigger in terrestrial species (R. arenarum and H. pulchellus), whereas the vomeronasal and the middle chamber epithelia (water-sensitive organs) was best developed in X. laevis. A small olfactory recess (likely homologous with the middle chamber epithelium) was found in R. arenarum juveniles, but not in H. pulchellus. These results support the association of the vomeronasal and middle chamber epithelia with aquatic olfaction, as seen by their enhanced development in the secondarily aquatic juveniles of X. laevis. They also support a role for the larval buccal-exposed olfactory epithelium in assessment of oral contents: it was absent in X. laevis, an obligate suspension feeder, while present in the two grazing species. These initial quantitative results give, for the first time, insight into the functional importance of the peripheral olfactory subsystems across the anuran life cycle.

Alterations in the intestine of Patagonian silverside (Odontesthes hatcheri) exposed to microcystin-LR: Changes in the glycosylation pattern of the intestinal wall and inhibition of multidrug resistance proteins efflux activity.

Accumulation and toxicity of cyanobacterial toxins, particularly microcystin-LR (MCLR) have been extensively studied in fish and aquatic invertebrates. However, MCLR excretion mechanisms, which could reduce this toxin's effects, have received little attention. The Patagonian silverside, Odontesthes hatcheri, is an omnivorous-planktivorous edible fish, which has been shown to digest cyanobacterial cells absorbing MCLR and eliminating the toxin within 48h without suffering significant toxic effects. We studied the effects of MCLR on glycoconjugate composition and the possible role of multidrug resistance associated proteins (Abcc) in MCLR export from the cells in O. hatcheri intestine. We treated O. hatcheri with 5µg MCLRg(-1) body mass administered with the food. Twenty four hours later, the intestines of treated and control fish were processed for lectin-histochemistry using concanavalin A (ConA), Triticum vulgaris agglutinin (WGA), and Dolichos biflorus agglutinin (DBA). MCLR affected the distribution of glycoconjugates by augmenting the proportion of ConA-positive at the expense of WGA-positive cells. We studied MCLR effects on the transport of the Abcc-like substrates 2,4-dinitrophenyl-S-glutathione (DNP-SG) and calcein in ex vivo intestine preparations (everted and no-everted sacs and strips). In treated preparations, CDNB together with MCLR (113µg MCLRg(-1) intestine, equivalent to 1.14µmolL(-1) when applied in the bath) or the Abcc inhibitor, MK571 was applied for one hour, during which DNP-SG was measured in the bath every 10min in order to calculate mass-specific DNP-SG transport rate. MCLR significantly inhibited DNP-SG transport (p<0.05), especially in middle intestine (47 and 24%, for luminal and serosal transport, respectively). In middle intestine strips, MCLR and MK571inhibited DNP-SG transport in a concentration dependent fashion (IC50 3.3 and 0.6µmolL(-1), respectively). In middle intestine strips incubated with calcein-AM (0.25µmolL(-1)), calcein efflux was inhibited by MCLR (2.3µmolL(-1)) and MK571 (3µmolL(-1)) by 38 and 27%, respectively (p<0.05). Finally, middle intestine segments were incubated with different concentrations of MCLR applied alone or together with 3µM MK571. After one hour, protein phosphatase 1 (PP1) activity, the main target of MCLR, was measured. 2.5µM MCLR did not produce any significant effect, while the same amount plus MK571 inhibited PP1 activity (p<0.05). This effect was similar to that of 5µM MCLR. Our results suggest that in O. hatcheri enterocytes MCLR is conjugated with GSH via GST and then exported to the intestinal lumen through Abcc-like transporters. This mechanism would protect the cell from MCLR toxicity, limiting toxin transport into the blood, which is probably mediated by basolateral Abccs. From an ecotoxicological point of view, elimination of MCLR through this mechanism would reduce the amount of toxin available for trophic transference.

Overcrowding-mediated stress alters cell proliferation in key neuroendocrine areas during larval development in Rhinella arenarum.

Exposure to adverse environmental conditions can elicit a stress response, which results in an increase in endogenous corticosterone levels. In early life stages, it has been thoroughly demonstrated that amphibian larval growth and development is altered as a consequence of chronic stress by interfering with the metamorphic process, however, the underlying mechanisms involved have only been partially disentangled. We examined the effect of intraspecific competition on corticosterone levels during larval development of the toad Rhinella arenarum and its ultimate effects on cell proliferation in particular brain areas as well as the pituitary gland. While overcrowding altered the number of proliferating cells in the pituitary gland, hypothalamus, and third ventricle of the brain, no differences were observed in areas which are less associated with neuroendocrine processes, such as the first ventricle of the brain. Apoptosis was increased in hypothalamic regions but not in the pituitary. With regards to pituitary cell populations, thyrotrophs but not somatoatrophs and corticotrophs showed a decrease in the cell number in overcrowded larvae. Our study shows that alterations in growth and development, produced by stress, results from an imbalance in the neuroendocrine systems implicated in orchestrating the timing of metamorphosis.

Metformin decreases the incidence of ovarian hyperstimulation syndrome: an experimental study.

BACKGROUND: In assisted reproduction cycles, gonadotropins are administered to obtain a greater number of oocytes. A majority of patients do not have an adverse response; however, approximately 3-6% develop ovarian hyperstimulation syndrome (OHSS). Metformin reduces the risk of OHSS but little is known about the possible effects and mechanisms of action involved. OBJECTIVE: To evaluate whether metformin attenuates some of the ovarian adverse effects caused by OHSS and to study the mechanisms involved. MATERIAL AND METHODS: A rat OHSS model was used to investigate the effects of metformin administration. Ovarian histology and follicle counting were performed in ovarian sections stained with Masson trichrome. Vascular permeability was measured by the release of intravenously injected Evans Blue dye (EB). VEGF levels were measured by commercially immunosorbent assay kit. COX-2 protein expression was evaluated by western blot and NOS levels were analyses by immunohistochemistry. RESULTS: Animals of the OHSS group showed similar physiopathology characteristics to the human syndrome: increased body weight, elevated progesterone and estradiol levels (P<0.001), increased number of corpora lutea (P<0.001), higher ovarian VEGF levels and vascular permeability (P<0.001 and P<0.01); and treatment with metformin prevented this effect (OHSS+M group; P<0.05). The vasoactive factors: COX-2 and NOS were increased in the ovaries of the OHSS group (P<0.05 and P<0.01) and metformin normalized their expression (P<0.05); suggesting that metformin has a role preventing the increased in vascular permeability caused by the syndrome. CONCLUSION: Metformin has a beneficial effect preventing OHSS by reducing the increase in: body weight, circulating progesterone and estradiol and vascular permeability. These effects of metformin are mediated by inhibiting the increased of the vasoactive molecules: VEGF, COX-2 and partially NOS. Molecules that are increased in OHSS and are responsible for a variety of the symptoms related to OHSS.

Effects of bone morphogenic protein 4 (BMP4) and its inhibitor, Noggin, on in vitro maturation and culture of bovine preimplantation embryos.

BACKGROUND: BMP4 is a member of the transforming growth factor beta (TGFbeta) superfamily and Noggin is a potent BMP inhibitor that exerts its function by binding to BMPs preventing interactions with its receptors. The aim of this work was to investigate the role of BMP4 and Noggin, on oocytes in vitro maturation (m experiments) and embryos in vitro development (c experiments) of bovine. METHODS: For m experiments, COCs were collected from slaughterhouse ovaries and in vitro matured in TCM with 100 ng/ml of either BMP4 or Noggin. After 24 h, the nuclear stage of the oocytes was determined by staining with Hoechst 33342. In addition, RT-qPCR was performed on MII oocytes to study the relative concentration of ZAR1, GDF9, BAX, MATER and HSP70 transcripts. Treated oocytes were submitted to parthenogenic activation (PA) or in vitro fertilization (IVF) and cultured in CR2. For c experiments, non-treated matured oocytes were submitted to PA or IVF to generate embryos that were exposed to 100 ng/ml of BMP4 or Noggin in CR2 until day nine of culture. Cleavage, blastocyst and hatching rates, expression pattern of the transcription factor Oct-4 in blastocysts and embryo cell number at day two and nine post-activation or fertilization were evaluated. RESULTS: We found that Noggin, as BMP4, did not affect oocyte nuclear maturation. Noggin supplementation up-regulated the expression of HSP70 and MATER genes in matured oocytes. Moreover, BMP4 during maturation increased the proportion of Oct-4 positive cells in parthenogenic embryos. On the other hand, when Noggin was added to embryo culture medium, developmental rates of parthenogenic and in vitro fertilized embryos were reduced. However, BMP4 addition decreases the development only for in vitro fertilized embryos. BMP4 and Noggin during culture reduced the proportion of Oct-4-expressing cells. CONCLUSIONS: Our results show that BMP4 is implicated in bovine oocytes maturation and embryo development. Moreover, our findings demonstrate, for the first time, that a correct balance of BMP signaling is needed for proper pre-implantation development of bovine embryos.

Daily treatment with α-naphthoflavone enhances follicular growth and ovulation rate in the rat.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and the first protein involved in a variety of physiological and toxicological processes, including those of xenobiotic metabolizing enzymes. AhR has been found in the ovary of many species and seems to mediate the ovarian toxicity of many environmental contaminants, which are AhR ligands. However, the role of AhR in the ovarian function is unknown. Therefore, the aim of this work was to study the action of α-naphthoflavone (αNF), known to be an AhR antagonist, on both follicular growth and ovulation. Immature Sprague-Dawley rats were daily injected intraperitoneally with αNF (0.1-80 mg/kg) or vehicle for 12 days, and primed with gonadotrophins (eCG/hCG) to induce follicular growth and ovulation. Ovaries were obtained 20 h after hCG administration. By means of immunohistochemistry, we found that the numbers of primordial, primary and antral follicles were increased in rats treated with 80 mg/kg αNF and that there were no differences with other doses. Likewise, the ovarian weight and the ovulation rate, measured by both number of oocytes within oviducts and corpora lutea in ovarian sections, were increased when the rats received either 1 or 10mg/kg daily. Although further studies are necessary to know the mechanism of action of αNF, it is possible that the different ovarian processes can be differentially responsive to the presence of different levels of αNF, and that the same or different endogenous AhR ligands can be involved in these ovarian processes in a cell type-dependent manner.

Heterogeneous distribution of G protein alpha subunits in the main olfactory and vomeronasal systems of Rhinella (Bufo) arenarum tadpoles.

We evaluated the presence of G protein subtypes Galpha(o), Galpha(i2), and Galpha(olf) in the main olfactory system (MOS) and accessory or vomeronasal system (VNS) of Rhinella (Bufo) arenarum tadpoles, and here describe the fine structure of the sensory cells in the olfactory epithelium (OE) and vomeronasal organ (VNO). The OE shows olfactory receptor neurons (ORNs) with cilia in the apical surface, and the vomeronasal receptor neurons (VRNs) of the VNO are covered with microvilli. Immunohistochemistry detected the presence of at least two segregated populations of ORNs throughout the OE, coupled to Galpha(olf) and Galpha(o). An antiserum against Galpha(i2) was ineffective in staining the ORNs. In the VNO, Galpha(o) neurons stained strongly but lacked immunoreactivity to any other Galpha subunit in all larval stages analyzed. Western blot analyses and preabsorption experiments confirmed the specificity of the commercial antisera used. The functional significance of the heterogeneous G-protein distribution in R. arenarum tadpoles is not clear, but the study of G- protein distributions in various amphibian species is important, since this vertebrate group played a key role in the evolution of tetrapods. A more complete knowledge of the amphibian MOS and VNS would help to understand the functional organization and evolution of vertebrate chemosensory systems. This work demonstrates, for the first time, the existence of a segregated distribution of G-proteins in the OE of R. arenarum tadpoles.

Amphibian larvae and zinc sulphate: a suitable model to study the role of brain-derived neurotrophic factor (BDNF) in the neuronal turnover of the olfactory epithelium.

The vertebrate olfactory system has fascinated neurobiologists over the last six decades because of its ability to replace its neurons and synaptic connections continuously throughout adult life, under both physiological and pathological conditions. Among the factors that are proposed to be involved in this regenerative potential, brain-derived neurotrophic factor (BDNF) is a candidate for having an important role in the neuronal turnover in the olfactory epithelium (OE) because of its well-documented neurogenic and trophic effects throughout the nervous system. The aim of the present study was to generate a suitable model to study the participation of BDNF in the recovery of the OE after injury in vivo. We developed an experimental design in which the OE of Rhinella arenarum tadpoles could be easily and selectively damaged by immersing the animals in ZnSO(4) solutions of various concentrations for differing time periods. Image analysis of histological sections showed that different combinations of each of these conditions produced statistically different degrees of injury to the olfactory tissue. We also observed that the morphology of the OE was restored within a few days of recovery after ZnSO(4) treatment. Immunohistochemical analysis of BDNF was performed with an antiserum whose specificity was confirmed by Western blotting, and which showed drastic changes in the abundance and distribution pattern of this neurotrophin in the damaged olfactory system. Our results thus suggest that BDNF is involved in the regeneration of the OE of amphibian larvae, and that our approach is suitable for further investigations of this topic.

Inhibition of cyclooxygenase-2 (COX-2) by meloxicam decreases the incidence of ovarian hyperstimulation syndrome in a rat model.

OBJECTIVE: To investigate the effects of selective cyclooxygenase-2 (COX-2) inhibition on the ovarian hyperstimulation syndrome (OHSS) in an experimental model. DESIGN: Controlled laboratory study. SETTING: University-affiliated fertility center. ANIMAL(S): Female Wistar rats. INTERVENTION(S): Female Wistar rats (22 days old) were divided into four groups: group 1 (control group; n = 10) received 0.1 mL of intraperitoneal (IP) saline from days 22-26; group 2 (mild-stimulated group; n = 10) received 10 IU of pregnant mare serum gonadotropin (PMSG) on day 24 and 10 IU of hCG 48 hours later (day 26); group 3 (OHSS group; n = 10) was given 10 IU of PMSG for 4 consecutive days from day 22 and 30 IU hCG on the fifth day to induce OHSS; group 4 was treated the same as group 3, but received 2 muL (15 mg/mL) of meloxicam 2 hours before the PMSG injection for 4 consecutive days, and 2 hours before the hCG injection on the fifth day. All groups were killed on day 26. MAIN OUTCOME MEASURE(S): Number of antral and luteinized follicles, ovarian weight, semiquantitative vascular endothelial growth factor (VEGF) and COX-2 immunohistochemistry. RESULT(S): There were no differences in the ovarian weight between groups 1 and 2. Group 3 showed significantly increased ovarian weight that was suppressed, in group 4, by meloxicam. There was no difference in the number of antral follicles among the four groups. In the mild-stimulated and OHSS groups, the granulosa cells (GC) of preovulatory follicles and the stromal cells showed intense VEGF immunoreactivity. The ovaries from the meloxicam-treated group showed less immunoreactivity than the OHSS group, indicating diminished VEGF expression associated with meloxicam treatment. Group 3 (OHSS group) showed increased COX-2 immunoreactivity that was diminished in the meloxicam-treated group. Meloxicam treatment did not affect the hormone-induced increase in serum E(2) levels seen in OHSS rats. CONCLUSION(S): Our results in a rat model suggest that meloxicam has a beneficial effect on OHSS by reducing the increases in ovarian weight and VEGF expression associated with OHSS. These effects may be mediated by the COX-2 inhibitory capacity of meloxicam.

Immunohistochemical localization of vascular endothelial growth factor and its receptor Flk-1 in the amphibian developing principal and accessory olfactory system.

In the last years several studies have shown that vascular endothelial growth factor (VEGF) is present in neural stem cells and mature neurons from different neural tissues where it may play an important role as a neuroproliferative and/or antiapoptotic factor. The olfactory neuroepithelium has the capability to replace dying neurons with new neurons formed by cell division from stem cells in the basal region of the epithelium. The present study demonstrates, for the first time, that VEGF is present in the olfactory epithelium, nerves and bulbs (both main and accessory) during the development of the toad Bufo arenarum. In this report, we detected VEGF immunoreactivity in mature olfactory neurons from early larval stages until the beginning of the metamorphic climax. VEGF expression decreases dramatically after metamorphosis. VEGF receptor Flk-1 was localized by immunohistochemistry, from premetamorphic larval stages until the climax in the neurons of the olfactory epithelium with a more intense labeling in the basal cell layer. Double-label immunofluorescence studies localized VEGF to the cytoplasm and the nucleus of mature neurons whereas Flk-1 was expressed in cell membranes. Flk-1 was present in neurons of both the main and accessory olfactory bulbs. After the end of metamorphosis, Flk-1 expression was limited to basal cells in the olfactory epithelium and Bowman's glands. The main and accessory olfactory bulbs showed the same pattern of Flk-1 immunostaining before and after the end of metamorphosis. The presence of VEGF and its receptor in the olfactory system suggests that VEGF may play an important role during neural development.

GnRH systems of Cichlasoma dimerus (Perciformes, Cichlidae) revisited: a localization study with antibodies and riboprobes to GnRH-associated peptides.

The distribution of cells that express three prepro-gonadotropin-releasing hormones (GnRH), corresponding to salmon GnRH, sea bream GnRH (sbGnRH), and chicken II GnRH, was studied in the brain and pituitary of the South American cichlid fish, Cichlasoma dimerus. Although the ontogeny and distribution of GnRH neuronal systems have previously been examined immunohistochemically with antibodies and antisera against the various GnRH decapeptides, we have used antisera against various perciform GnRH-associated peptides (GAPs) and riboprobes to various perciform GnRH+GAPs. The results demonstrate that: (1) the GnRH neuronal populations in the forebrain (salmon and sea bream GAPs; sGAP and sbGAP, respectively) show an overlapping pattern along the olfactory bulbs, nucleus olfacto-retinalis, ventral telencephalon, and preoptic area; (2) projections with sGAP are mainly located in the forebrain and contribute to the pituitary innervation, with projections containing chicken GAP II being mainly distributed along the mid and hindbrain and not contributing to pituitary innervation, whereas sbGAP projections are restricted to the ventral forebrain, being the most important molecular form in relation to pituitary innervation; (3) sbGnRH (GnRH I) neurons have an olfactory origin; (4) GAP antibodies and GAP riboprobes are valuable tools for the study of various GnRH systems, by avoiding the cross-reactivity problems that occur when using GnRH antibodies and GnRH riboprobes alone.

Relation between the reproductive status and somatolactin cell activity in the pituitary of pejerrey, Odontesthes bonariensis (Atheriniformes).

The aim of this study was to analyze the relationship between somatolactin (SL) expressing cells and the reproductive status in a multiple spawning fish, the pejerrey Odontesthes bonariensis. Somatolactin cells were identified in adults of both sexes by immunocytochemistry using a heterologous piscine antiserum. The area of the cells that showed immunoreactivity to SL (ir-SL) was compared in specimens with different degrees of reproductive activity as inferred from histological examination of the gonads and calculation of the gonadosomatic index (GSI %). The results showed a significant difference between the area of ir-SL cells of resting/regressing (62.9 +/- 2.1 micron 2) and sexually active/vitellogenic (76.8 +/- 2.3 micron 2) females and a significant positive correlation between the ir-SL cellular area and the GSI % (P < 0.01 in both cases). In males, the correlation between the area of ir-SL cells and the GSI % was not statistically significant. However, in those animals with the highest GSI % values, the ir-SL cells appeared more numerous and showed an increase in the immunostained area when compared to individuals with lower GSI % values. The present in morphological observations are in accordance with biochemical data obtained from other species and support the assumption that SL might be involved in the regulation of reproduction in fish.

Ontogeny and distribution of gonadotropin-releasing hormone (GnRH) neuronal systems in the brain of the cichlid fish Cichlasoma dimerus.

Using immunocytochemistry we have described the distribution and ontogeny of three distinct gonadotropin-releasing hormone (GnRH) neural systems, emphasizing the analysis during the period of sex differentiation in the South American cichlid fish Cichlasoma dimerus. In the forebrain a group of neurones immunoreactive to salmon GnRH that formed clusters in the nucleus olfacto retinalis (NOR), was located at the junction of the olfactory bulb and the telencephalon. These neurones differentiated 3 days after fertilization from the olfactory placodes. GnRH immunoreactive neurones along the olfactory nerves through the rostrobasal olfactory bulb were observed on day 4 and at the NOR on day 10. A group of neurones immunoreactive to chicken GnRH II was seen in the dorsal midbrain tegmentum. They originate from the ventricular ependyma between days 5 and 6. These neurones remained close to blood vessels throughout development. Between days 22 and 30 a group of neurones immunoreactive to seabream GnRH was detected in the anterior basal preoptic area. GnRH innervation of the pituitary was detected after the differentiation of preoptic neurones and in coincidence with gonadal differentiation. We hypothesize that the GnRH neural systems have three distinct embryonic origins. Furthermore, we show that the NOR and the midbrain GnRH neurones might have functions other than gonadal development, whereas the preoptic GnRH neurones in C. dimerus might be associated with gonadal sex differentiation.

Immunocytochemical localization of different cell types in the adenohypophysis of the cichlid fish Cichlasoma dimerus (Heckel, 1840)

The adenohypophysis of the cichlid fish Cichlasoma dimerus was studied using the avidin-biotin-peroxidase method with antisera raised against piscine pituitary hormones and heterologous antisera against mammalian pituitary hormones. Antiserum raised against rabbit ACTH recognized a group of cells bordering the neurohypophysis (NH) in the rostral pars distalis (RPD). Anti-chum salmon prolactin (PRL) identified a compact group of cells in the periphery of the RPD. Gonadotropin II (GTH II), thyrotropin (TSH) and growth hormone (GH)-ir cells were localized in the proximal pars distalis. Ir-GTH II cells were also observed in the dorsal area of the pars intermedia (PI). Ir-GTH I cells could not be identified using anti-chum salmon GTH I, this may be due either to a failure of the antisera to recognize the gonadotropin or to a low expression of the hormone in adults of this species. PAS positive cells from the PI bound specifically with three different antisera raised against somatolactin (SL) of four different fish species. These cells surrounded deep branches of the NH in the PI.

The adenohypophysis of the swamp eel, Synbranchus marmoratus, an immunocytochemical analysis

The adenohypophyseal cell types of the protogynous fish Synbranchus marmoratus were studied by histochemical and immunocytochemical staining with antisera raised against piscine and human pituitary hormones to ascertain their distribution. The prolactin (PRL) cells were distributed in the rostral pars distalis and showed specific binding to antisera to carp and chum salmon prolactin. No reaction was observed with antiserum to human prolactin. The corticotrops showed strong immunoreactivity with anti-human ACTH, these cells bordered the neurohypophysis and islets between PRL cells in the rostral pars distalis. Growth hormone (GH) cells were densely distributed and associated with the neurohypophysis only in pars distalis proximal. They reacted with antisera to piscine GH but not with antisera to human growth hormone. The thyrotrops were scattered in the proximal pars distalis and showed strong immunoreactivity to the human thyrotropin Beta subunit antiserum. Gonadotrops were located in the central area of the proximal pars distalis and in the external border of the pars intermedia. These cells were alcian blue and PAS positive, and reacted with anti-croaker GTH and anti-coho GTH I and GTH II. The PAS positive cells from the pars intermedia bound specifically to anti-chum somatolactin.

Presence and release of immunoreactive atrial natriuretic peptide (ANP) in Bufo arenarum spermatozoa after HCG treatment

Atrial natriuretic peptide (ANP)-like immunoreactivity has been demonstrated in mature spermatozoa of Bufo arenarum. However, after spermiation induced by Gonadotropin Releasing Hormone (GnRH), no ANP immunoreactivity was detected in testicular spermatozoa. Recently, the presence of GnRH and GnRH receptors in amphibian testes has been demonstrated. To clarify if the loss of ANP-like immunoreactivity in spermatozoa is a direct effect of GnRH or pituitary gonadotropins, a study on Bufo arenarum adult males, has been performed. The in vivo treatment with Human Chorionic Gonadotropin (HCG) induced spermiation and loss of ANP-like immunoreactivity. The in vitro treatment with HCG showed the same results. However, in vitro GnRH treatment failed to cause spermiation and loss of ANP-like immunoreactivity. The present results indicate that ANP from mature spermatozoa is regulated via gonadotropic hormones and may be involved in the spermiation process.

Developmental capacity of aged bufo arenarum ovocytes

Se ha estudiado la posibilidad de desarrollo de ovocitos de Bufo arenarum mantenidos en el ovisaco y respectivamente fertilizados a intervalos establecidos, hasta las 24 horas. Los resultados demuestran que en función del envejecimiento disminuye el porcentaje de fertilización y aumenta el número de embriones que se detienen a lo largo de su desarrollo. De los ovocitos con más de 6 horas de permanencia en el ovisaco, se desarrollan larvas que pueden presentar condrodistrofia, espina bífida, nomoftalmia, anoftalmia, etc. No existe relación entre el período de envejecimiento del ovocito y el tipo de anomalía, pero aumenta significativamente el porcentaje de embriones defectuosos. Hay que preguntarse si es conveniente usar, para ensayos experimentales, ovocitos envejecidos. El envejecimiento del ovocito de Bufo arenarum acontece más rápidamente que el señalado en otras especies