RESUMEN
In teleost, as in other vertebrates, stress affects reproduction. A key component of the stress response is the pituitary secretion of the adrenocorticotropic hormone (ACTH), which binds to the melanocortin 2 receptor (MC2R) in the adrenal glands and activates cortisol biosynthesis. In zebrafish, Mc2r was identified in male and female gonads, while ACTH has been shown to have a physiological role in modulating reproductive activity. In this study, the hypothesis that other melanocortins may also affect how the zebrafish gonadal function is explored, specifically steroid biosynthesis, given the presence of members of the melanocortin signaling system in zebrafish gonads. Using cell culture, expression analysis, and cellular localization of gene expression, our new observations demonstrated that melanocortin receptors, accessory proteins, antagonists, and agonists are expressed in both the ovary and testis of zebrafish (n = 4 each sex). Moreover, melanocortin peptides modulate both basal and gonadotropin-stimulated steroid release from zebrafish gonads (n = 15 for males and n = 50 for females). In situ hybridization in ovaries (n = 3) of zebrafish showed mc1r and mc4r in follicular cells and adjacent to cortical alveoli in the ooplasm of previtellogenic and vitellogenic oocytes. In zebrafish testes (n = 3), mc4r and mc1r were detected exclusively in germ cells, specifically in spermatogonia and spermatocytes. Our results suggest that melanocortins are, directly or indirectly, involved in the endocrine control of vitellogenesis in females, through modulation of estradiol synthesis via autocrine or paracrine actions in zebrafish ovaries. Adult zebrafish testes were sensitive to low doses of ACTH, eliciting testosterone production, which indicates a potential role of this peptide as a paracrine regulator of testicular function.
RESUMEN
Seminal characteristics in teleost fish with an annual reproductive period, such as pacu (Piaractus mesopotamicus), may vary during the breeding season. The sperm formed before the beginning of the spawning period may be stored for a long time, causing damage to the cells. Therefore, re-stripping may be an important way to eliminate the "old" and allow for the collection of "new" spermatozoids. In this study, we analyzed the seminal characteristics of hormonally induced pacu at the beginning, middle and end of the breeding season, and we analyzed samples from re-stripped males (stripped first at the beginning, re-stripped in the middle, and re-stripped again at the end of the season) during two breeding seasons. The sperm density, ionic composition, pH, and osmolality were similar among the groups. The semen volume, seminal plasma protein concentration and incidence of morphologically anomalous sperm increased over time. In addition, some parameters that are associated with good-quality semen decreased, such as sperm motility, viability and DNA integrity. Moreover, we observed a positive association among motility, viability and DNA integrity for sperm with elevated 11-ketotestosterone, but there was no such association for fshb or lhb mRNA levels in the pituitary. The semen that was obtained earlier (at the beginning) or from re-stripped males exhibited better characteristics than the other samples collected. In conclusion, collecting semen from pacu at the end of breeding season should be avoided; it is preferable to strip early and then re-strip later in the season, and this approach may be used for diverse aquaculture purposes.
Asunto(s)
Characiformes/fisiología , Semen/metabolismo , Motilidad Espermática/fisiología , Espermatozoides/metabolismo , Animales , Cruzamiento , Masculino , Concentración Osmolar , Estaciones del Año , Análisis de Semen , Recuento de Espermatozoides , Testosterona/análogos & derivados , Testosterona/metabolismoRESUMEN
The zebrafish has become an important vertebrate model for basic and biomedical research, including the research field of the biology of reproduction. However, very few morphological and stereological data are available regarding zebrafish testis structure and spermatogenesis. In this careful histomorphometric evaluation of the testis, we studied spermatogonial cells using molecular markers, determined the combined duration of meiotic and spermiogenic phases, and examined the formation of the Sertoli cell barrier (tight junctions). We found at least nine spermatogonial generations and propose a morphology-based nomenclature for spermatogonial generations that is compatible with the one used in higher vertebrates. The number of germ cells per cyst increased dramatically (1 to approximately 1360 cells) from undifferentiated spermatogonia type A to early spermatids. The combined duration of meiotic and spermiogenic phases is approximately 6 days, one of the shorter periods among the teleost fish investigated to date. The number of Sertoli cells per cyst increased 9-fold during the maturational cycle of spermatogenic cysts and stabilized in the meiotic phase at a ratio of approximately 100 early spermatids per Sertoli cell (Sertoli cell efficiency). Similarly to mammals, Sertoli cell proliferation ceased in the meiotic phase, coinciding with the formation of tight junctions between Sertoli cells. Hence, the events taking place during puberty in the germinal epithelium of mammals seem to recapitulate the "life history" of each individual spermatogenic cyst in zebrafish.
Asunto(s)
Diferenciación Celular , Espermatogénesis/fisiología , Espermatogonias/fisiología , Pez Cebra/anatomía & histología , Animales , Recuento de Células , Células Germinativas/citología , Células Intersticiales del Testículo/citología , Masculino , Modelos Biológicos , Células de Sertoli/citología , Espermatogonias/citología , Espermatogonias/ultraestructura , Testículo/citología , Testículo/ultraestructura , Uniones Estrechas/fisiología , Uniones Estrechas/ultraestructura , Pez Cebra/crecimiento & desarrolloRESUMEN
Although Kir4.1 channels are the major inwardly rectifying channels in glial cells and are widely accepted to support K+- and glutamate-uptake in the nervous system, the properties of Kir4.1 channels during vital changes of K+ and polyamines remain poorly understood. Therefore, the present study examined the voltage-dependence of K+ conductance with varying physiological and pathophysiological external [K+] and intrapipette spermine ([SP]) concentrations in Müller glial cells and in tsA201 cells expressing recombinant Kir4.1 channels. Two different types of [SP] block were characterized: "fast" and "slow." Fast block was steeply voltage-dependent, with only a low sensitivity to spermine and strong dependence on extracellular potassium concentration, [K+]o. Slow block had a strong voltage sensitivity that begins closer to resting membrane potential and was essentially [K+]o-independent, but with a higher spermine- and [K+]i-sensitivity. Using a modified Woodhull model and fitting i/V curves from whole cell recordings, we have calculated free [SP](in) in Müller glial cells as 0.81 +/- 0.24 mM. This is much higher than has been estimated previously in neurons. Biphasic block properties underlie a significantly varying extent of rectification with [K+] and [SP]. While confirming similar properties of glial Kir and recombinant Kir4.1, the results also suggest mechanisms underlying K+ buffering in glial cells: When [K+]o is rapidly increased, as would occur during neuronal excitation, "fast block" would be relieved, promoting potassium influx to glial cells. Increase in [K+]in would then lead to relief of "slow block," further promoting K+-influx.
Asunto(s)
Neuroglía/fisiología , Neuronas/fisiología , Canales de Potasio de Rectificación Interna/fisiología , Canales de Potasio/fisiología , Retina/fisiología , Animales , Células Cultivadas , Electrofisiología , Potasio/metabolismo , Potasio/farmacología , Canales de Potasio de Rectificación Interna/efectos de los fármacos , Rana pipiens , Ratas , Ratas Long-Evans , Ratas Sprague-Dawley , Proteínas Recombinantes/metabolismo , Retina/citologíaRESUMEN
The retinae and brains of larval and adult amphibians survive long-lasting anoxia; this finding suggests the presence of functional K(ATP) channels. We have previously shown with immunocytochemistry studies that retinal glial (Müller) cells in adult frogs express the K(ATP) channel and receptor proteins, Kir6.1 and SUR1, while retinal neurons display Kir6.2 and SUR2A/B (Skatchkov et al., 2001a: NeuroReport 12:1437-1441; Eaton et al., in press: NeuroReport). Using both immunocytochemistry and electrophysiology, we demonstrate the expression of Kir6.1/SUR1 (K(ATP)) channels in adult frog and tadpole Müller cells. Using conditions favoring the activation of K(ATP) channels (i.e., ATP- and spermine-free cytoplasm-dialyzing solution containing gluconate) in Müller cells isolated from both adult frogs and tadpoles, we demonstrate the following. First, using the patch-clamp technique in whole-cell recordings, tolbutamide, a blocker of K(ATP) channels, blocks nearly 100% of the transient and about 30% of the steady-state inward currents and depolarizes the cell membrane by 5-12 mV. Second, inside-out membrane patches display a single-channel inward current induced by gluconate (40 mM) and blocked by ATP (200 microM) at the cytoplasmic side. The channels apparently show two sublevels (each of approximately 27-32 pS) with a total of 85-pS maximal conductance at -80 mV; the open probability follows a two-exponential mechanism. Thus, functional K(ATP) channels, composed of Kir6.1/SUR1, are present in frog Müller cells and contribute a significant part to the whole-cell K+ inward currents in the absence of ATP. Other inwardly rectifying channels, such as Kir4.1 or Kir2.1, may mediate the remaining currents. K(ATP) channels may help maintain glial cell functions during ATP deficiency.