RESUMEN
Brushtail possums exhibit a distinct preovulatory pattern of prolactin (Prl) secretion suggesting that Prl is involved in normal reproductive function. In some mammals, Prl is essential for corpus luteum (CL) function and/or modulation of steroidal effects on hypothalamic-pituitary activity. The aim of this study was to test the effects of biologically active recombinant possum Prl (recPosPrl) on both pituitary gland and CL function in possums. To confirm biological activity, administration of recPosPrl-N2C1 (10 µg) resulted in an 18-fold stimulation (P<0.05) of progesterone (P(4)) production by possum granulosa cells in vitro. Based on these findings, minipumps containing either recPosPrl-N2C1 (n=10) or saline (n=8) were inserted into lactating female possums. The expression levels of pituitary-derived PRL, LHB, FSHB and GNRHR and CL-derived LHR mRNA were quantified. Following a resumption of reproductive activity, no differences in ovulation incidence or plasma Prl concentrations were observed. Plasma Prl levels were less variable (P<0.001) in Prl-treated possums, confirming a self-regulatory role for Prl in this species. There was a marked down-regulation (P<0.001) of FSHB mRNA at the mid-luteal stage in Prl-treated possums, whereas mean PRL, LHB, GNRHR and LHR mRNA expression levels were not different between experimental groups. Plasma P(4) concentrations were not different (P=0.05) in Prl-treated possums, although tended to be higher in the peri-ovulatory and early-luteal phase. We conclude in the brushtail possum that Prl is self-regulated via a short-feedback loop common to all mammals studied and is able to modulate FSHB expression probably at the level of the hypothalamus and/or pituitary gland.
Asunto(s)
Hormona Folículo Estimulante/genética , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Sistema Hipotálamo-Hipofisario/metabolismo , Hipófisis/metabolismo , Prolactina/farmacología , Trichosurus/metabolismo , Animales , Cuerpo Lúteo/efectos de los fármacos , Cuerpo Lúteo/metabolismo , Femenino , Hormona Liberadora de Gonadotropina/genética , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Hormona Luteinizante de Subunidad beta/genética , Hipófisis/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Progesterona/genética , Radioinmunoensayo , Receptores de HL/genética , Trichosurus/genéticaRESUMEN
In eutherian mammals, the gonadotrophins (LH and FSH) are synthesized and stored in gonadotroph cells under the regulation of multiple mechanisms including GnRH. Very little is known about the regulation of gonadotrophin secretion and storage in pituitary glands of marsupials. This study revealed, using quantitative PCR and heterologous RIA techniques, that LHB mRNA expression levels remained constant over the oestrous cycle, regardless of the presence of a preovulatory LH surge, which is characteristic of a hormone secreted under regulation. Our sampling regime was unable to detect pulses of LH during the follicular phase, although GNRHR mRNA levels had increased at this time. Pulses of LH were, however, detected in the luteal phase of cycling females, in anoestrus females and in males. There was a positive correlation between gene expression of FSHB and plasma levels of FSH at different stages of the oestrous cycle and no pulses of FSH were detected at any time; all characteristics of a hormone secreted via the constitutive pathway. Using in situ hybridisation and immunohistochemistry methods, we determined that mRNA expression of LHB and FSHB, and protein storage of gonadotrophins exhibited a similar pattern of localisation within the pituitary gland. Additionally, sexual dimorphism of gonadotroph populations was evident. In summary, these findings are similar to that reported in eutherians and considering that marsupial evolution diverged from eutherians over 100 million years ago suggests that the regulation of gonadotrophins is highly conserved indeed.
Asunto(s)
Evolución Biológica , Hormona Folículo Estimulante de Subunidad beta/genética , Hormona Luteinizante de Subunidad beta/genética , Hipófisis/metabolismo , Receptores LHRH/genética , Trichosurus/metabolismo , Animales , Femenino , Hormona Folículo Estimulante de Subunidad beta/análisis , Fase Folicular , Expresión Génica , Hormona Liberadora de Gonadotropina/metabolismo , Inmunohistoquímica , Fase Luteínica , Hormona Luteinizante de Subunidad beta/análisis , Hipófisis/química , ARN Mensajero/análisis , Radioinmunoensayo/métodos , Receptores LHRH/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
Prolactin (Prl) has been implicated in reproduction in many mammalian species and is illustrated by the distinctive patterns of secretion during the breeding season, the oestrous cycle and lactation. The recent development of a homologous RIA for measuring the circulating Prl concentrations in brushtail possums has facilitated the reliable measurement of Prl in plasma during different physiological states in this species for the first time. Determination of Prl concentrations during lactation involved the collection of weekly blood samples from eight female possums from the time of parturition through either one or two consecutive lactational cycles. Prl was at baseline levels during early lactation (weeks 0-14 post-partum), and then increased markedly to maximum concentrations at weeks 19-21 before returning to nadir levels at a time coincident with the weaning of pouch young (weeks 23-27). The profile of Prl secretion over the oestrous cycle and in particular at the time of the preovulatory LH surge was obtained from 14 possums during the reproductive cycle, in which preovulatory follicle development and ovulation were monitored by laparoscopy. There was no distinct daily pattern of Prl secretion during the oestrous cycle; however, in 3/4 possums in which a typical preovulatory LH surge was measured, a biphasic preovulatory Prl surge was also observed. The preovulatory Prl surge commenced 2-6 h prior to, and had returned to baseline close to the onset of, the preovulatory LH surge, and a second surge of Prl occurred concomitantly with the delayed preovulatory FSH surge. Seasonality of Prl levels was established from weekly blood samples collected from six barren female possums, and concentrations of Prl were lower during the breeding season compared to the non-breeding season. Additionally, a circadian pattern of Prl secretion was evident in both female and male possums, with Prl levels higher in the morning compared to the afternoon. In conclusion, interpretation of endogenous secretory patterns suggests that Prl may be important during late lactation and at impending ovulation, but the involvement of the circannual rhythm of Prl in the regulation of seasonality in the brushtail possum remains to be determined.
Asunto(s)
Ritmo Circadiano , Estro/sangre , Preñez/sangre , Prolactina/sangre , Estaciones del Año , Trichosurus/fisiología , Animales , Femenino , Hormona Folículo Estimulante/sangre , Lactancia/sangre , Hormona Luteinizante/sangre , Masculino , Embarazo , Radioinmunoensayo/métodos , Trichosurus/sangreRESUMEN
We report the production of recombinant possum prolactin (posPrl), and its use in the development and validation of a highly specific homologous radioimmunoassay for the measurement of prolactin (Prl) in brushtail possums. This enabled the subsequent investigation of some basic mechanisms involved in the regulation of Prl secretion in this species. Recombinant posPrl spanning the entire coding region was expressed in Escherichia coli, resulting in a 199 amino acid protein with a molecular weight approximately 23 kDa. The potency of posPrl was 45.3 +/- 4.8% that of ovine Prl in a radioreceptor assay using possum mammary gland receptors and induced a 3.4 +/- 0.8-fold increase in progesterone secretion in primary possum granulosa cells. Antiserum (G27) was raised against recombinant posPrl and was highly specific for possum Prl (approximately 30% binding at 1:60,000 final dilution), and exhibited negligible cross-reactivity (<0.0001%) with possum growth hormone. Serial dilutions of pituitary gland extracts, and plasma samples from male and female possums gave parallel inhibition curves to recombinant posPrl standards in the assay. Biological validation of the RIA included treating possums with drugs known to alter Prl secretion in other mammals. In seasonally anoestrous female possums, administration of 20 microg thyrotropin-releasing hormone (TRH) resulted in a 15-fold increase (P < 0.01) in plasma Prl concentrations. In mid-late lactating female possums, a bolus of cabergoline (dopamine agonist; 75 microg) reduced (P < 0.05) plasma Prl levels to baseline for 24 h, while repeated administration (6 x 75 microg at 12 h intervals) suppressed (P < 0.01) plasma Prl concentrations until 24h after the last injection. Prolonged inhibition of Prl levels subsequently caused marked (P < 0.01) attenuation in rate of bodyweight increase of pouch young. The amplitude of the Prl surge in response to a bolus of TRH (15 microg) was 5-fold lower in cabergoline-treated, compared to control mid-late lactating possums. In conclusion, we report the development and validation of a robust and sensitive RIA for measuring Prl concentrations in the plasma of brushtail possums.
Asunto(s)
Zarigüeyas/fisiología , Prolactina/fisiología , Radioinmunoensayo/veterinaria , Animales , Bioensayo/veterinaria , Western Blotting/veterinaria , Cabergolina , Agonistas de Dopamina/farmacología , Ergolinas/farmacología , Femenino , Células de la Granulosa , Masculino , Nueva Zelanda , Zarigüeyas/metabolismo , Progesterona/análisis , Prolactina/análisis , Prolactina/química , Prolactina/genética , ARN/química , ARN/genética , Radioinmunoensayo/métodos , Ensayo de Unión Radioligante/veterinaria , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Hormona Liberadora de Tirotropina/farmacologíaRESUMEN
The purpose of this study was to determine the occurrence of and the regulatory mechanisms involved in priming of the pituitary to GnRH before the preovulatory LH surge in sheep. Experiment 1: Forty-two ewes had progestagen devices removed after 14 days and were assigned to luteal (Lut) or follicular (Foll) groups. Fifteen days later, blood sampling was initiated either immediately or 36 h after induced luteolysis in groups Lut and Foll, respectively. After 4 h, ewes were administered either saline (n = 5) or 250 ng (n = 8) or 10 microg (n = 8) of GnRH. Five ewes per treatment group were killed 1 h later, while remaining animals were blood sampled for a further 7 h. Experiment 2: Eighteen ewes were allocated to Lut and Foll groups (described above). Blood samples were collected from 2 h before GnRH (10 microg) treatment until 7 h after. Despite up-regulated GnRH-R mRNA levels in Foll ewes, pituitary content and plasma levels of LH and LHbeta mRNA levels were similar between groups. Mean FSHbeta mRNA and plasma FSH levels were elevated in Lut ewes but declined after GnRH treatment. Inversely, plasma estradiol and inhibin-A concentrations were higher in Foll ewes and declined after GnRH treatment. Fewer LH(+ve)/secretogranin II(-ve) (SgII(-ve)) granules were present in gonadotropes of Foll ewes, coincident with increased basal LH levels. Fewer smaller sized granules were present after GnRH treatment. In conclusion, there was no evidence of self-priming before onset of the preovulatory LH surge. Constitutive release of LH(+ve)/SgII(-ve) granules may maintain basal LH levels while smaller sized, presumably mature granules may be preferentially released after GnRH stimulation.
Asunto(s)
Fase Folicular/metabolismo , Hormona Liberadora de Gonadotropina/farmacología , Hormona Luteinizante/metabolismo , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , Animales , Estradiol/sangre , Femenino , Hormona Folículo Estimulante/sangre , Hormona Folículo Estimulante/metabolismo , Inhibinas/sangre , Fase Luteínica , Hormona Luteinizante/sangre , Ovario/anatomía & histología , Progesterona/sangre , ARN Mensajero/metabolismo , Distribución Aleatoria , Ovinos , Factores de TiempoRESUMEN
While the role of oestradiol and progesterone in the control of GnRH pulsatile secretion and generation of the preovulatory GnRH surge to induce release of the LH surge has been fully investigated, less attention has been given to changes in the pituitary gland that may sensitize gonadotrophs to switch from pulsatile release to surge release of LH, in particular. Furthermore, in the follicular phase while pulsatile secretion of LH is maximal, FSH secretion is reduced, yet both hormones are produced by the same gonadotrophs. The mechanisms whereby this differential release can occur are still unclear. The main regulator of FSH secretion is through the negative feedback effects of oestradiol and inhibin, which directly affect FSHbeta mRNA content and subsequent synthesis of FSH. FSH is then released predominantly via a constitutive pathway and the amount released is closely related to the rate of synthesis. In contrast, while basal LH secretion occurs via a constitutive pathway, the principal release of LH through pulsatile secretion is through the regulated pathway with GnRH stimulating the release of pre-synthesized LH contained in storage granules without significant changes in LHbeta mRNA. Secretogranin II (SgII) is associated with LH in these electron-dense storage granules and LH-SgII granules appear to be the principal form of granule released in response to GnRH through the regulated pathway. At the time of the preovulatory LH surge, granule movement to the gonadotrope cell membrane abutting a capillary, polarization, appears to play an important part in the priming mechanism for release of LH during the preovulatory LH surge in response to the GnRH surge. As there appears to be limited or no gonadotroph cell division in the adult pituitary gland, each gonadotroph passes through this synthesis and secretion pathway repeatedly through successive oestrous cycles. Packaging of LH and FSH into different secretory granules within the same cell is thus pivotal for the differential secretion of these gonadotrophins.
Asunto(s)
Estro/metabolismo , Hormona Liberadora de Gonadotropina/fisiología , Gonadotropinas Hipofisarias/metabolismo , Ovulación/metabolismo , Hipófisis/metabolismo , Animales , Estradiol/metabolismo , Retroalimentación Fisiológica , Femenino , Hormona Folículo Estimulante/genética , Hormona Folículo Estimulante/metabolismo , Expresión Génica , Gonadotropinas Hipofisarias/genética , Humanos , Inhibinas/metabolismo , Hormona Luteinizante/genética , Hormona Luteinizante/metabolismoRESUMEN
The granin proteins secretogranin II (SgII) and chromogranin A (CgA) are commonly found associated with LH and/or FSH within specialised secretory granules in gonadotroph cells, and it is possible that they play an important role in the differential secretion of the gonadotrophins. In this study we have examined the regulation of the biosynthesis and secretion of SgII and CgA, in relation to LH secretion, in the LbetaT2 mouse pituitary gonadotroph cell line. Three experiments were carried out to investigate the effects of oestradiol (E2) and dexamethasone (Dex) in the presence and absence of GnRH (experiment 1), differing GnRH concentrations (experiment 2) and alterations in GnRH pulse frequency (experiment 3). In experiment 1, exposure to E2, Dex or E2+Dex, either with or without GnRH treatment, resulted in increased LH secretion. Steroids alone had no effect on LHbeta mRNA levels, but in the presence of GnRH LHbeta mRNA levels were increased in Dex- and E2+Dex-treated cells. GnRH receptor (GnRH-R) mRNA levels were up-regulated by Dex and E2+Dex, but were unaffected by GnRH. There were no steroid-induced changes in SgII or CgA mRNA, but increased levels of CgA mRNA were observed after GnRH treatment in cells cultured in the presence of Dex. In experiment 2, increasing concentrations of GnRH resulted in increases in LH secretion that were inversely dose-dependent. No changes in LHbeta, GnRH-R or SgII mRNA levels were observed, but there were dose-dependent increases in CgA mRNA levels. In experiment 3, GnRH was given as either 1 pulse/day or 4 pulses/day for 3 days. Both pulse regimes resulted in increased LH, SgII and CgA secretion compared with controls during the first 15 min pulse on day 3. Exposure to GnRH at 4 pulses/day increased LH and SgII secretion compared with controls during all 4 pulses, but secretion of both proteins was reduced during pulses 2-4 compared with pulse 1. CgA secretion also increased due to GnRH in pulse 1, but was decreased by GnRH treatment during pulse 2, and unchanged by GnRH during pulses 3 and 4. Total daily secretion of LH and SgII from cells given 1 pulse/day of GnRH increased compared with controls on all three treatment days, while total CgA secretion increased in response to GnRH on days 2 and 3 only. Intracellular levels of SgII, but not LH, decreased after GnRH treatment. In contrast, intracellular CgA was increased, but only after 4 pulses/day of GnRH. Levels of LHbeta, but not SgII, mRNA were increased by both pulse regimes, while CgA mRNA levels increased after 1 pulse/day of GnRH. These results indicate that there is a close correlation between the GnRH-stimulated release of LH and SgII from LbetaT2 cells, suggesting that SgII may have an influential role in the regulated secretion of LH, possibly by inducing LH aggregation to facilitate trafficking into secretory granules. CgA secretion does not appear to be closely associated with that of LH, but CgA expression does appear to be regulated by GnRH, which may indicate involvement in the control of LH secretion, possibly by influencing the proportion of LH in the different types of secretory granules.
Asunto(s)
Cromograninas/biosíntesis , Hormona Liberadora de Gonadotropina/farmacología , Hormona Luteinizante/metabolismo , Adenohipófisis/metabolismo , Biosíntesis de Proteínas , Esteroides/farmacología , Análisis de Varianza , Animales , Northern Blotting/métodos , Línea Celular , Cromogranina A , Cromograninas/metabolismo , Dexametasona/farmacología , Esquema de Medicación , Estradiol/farmacología , Hormona Luteinizante/genética , Hormona Luteinizante de Subunidad beta/análisis , Hormona Luteinizante de Subunidad beta/genética , Hormona Luteinizante de Subunidad beta/metabolismo , Ratones , Proteínas/metabolismo , ARN Mensajero/análisis , Radioinmunoensayo/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estimulación QuímicaRESUMEN
Associations between granins (secretogranin II (SgII) and chromogranin A and B (CgA and CgB)) and gonadotrophins (LH and FSH) have been reported in rodents and they may interact to facilitate differential storage and secretion of LH and FSH. This study investigated the relationship between granins and gonadotrophins in sheep at different stages of the oestrous cycle. Thirty-four cycling ewes had their oestrous cycles synchronised, and were divided into late luteal (LL; n=5) and early (EF; n=4), mid (n=3) and late (LF; n=11) follicular stages, and 24-53 h (n=5), 80-100 h (n=3) and 120-144 h (n=3) after the preovulatory LH surge (PS). LHbeta mRNA levels were low in LF ewes (when plasma levels and pulse frequency of LH were high) but had increased by 80-100 h PS. In contrast, FSHbeta mRNA levels decreased during the follicular phase and plasma FSH concentrations followed a similar pattern, to peak at 24-53 h PS due to low plasma oestradiol levels. While alpha-gonadotrophin subunit (alpha-GSU), SgII and CgA mRNA levels did not change, CgB mRNA levels were elevated in EF ewes and had declined in ewes around the surge. Four distinctly sized mRNA transcripts ( approximately 1.3, 2.0, 2.8 and 3.2 kb) were observed for CgA mRNA, while a double band was observed for LHbeta mRNA that was subsequently reduced to a single band after 3'-poly(A) tail truncation. The long and short LHbeta transcripts were prevalent in follicular and luteal ewes respectively. Numbers of LH(+ve)/FSH(-ve) granules stored within gonadotrophs were not different in LL and LF ewes (even though proportions of LH(+ve) granules were higher in LF ewes), but were reduced at 24-53 h PS. The majority of LH(+ve) granules also contained SgII, although few CgA(+ve) granules were found. Granule partitioning was evident whereby FSH and CgA were located near the periphery, and LH and SgII throughout the matrix. In conclusion, increases in both storage of LH(+ve) granules and secretion of LH in LF ewes despite constant LHbeta mRNA levels was facilitated, at least in part, by improved LHbeta mRNA transcript stability. Fewer LH(+ve)/FSH(-ve) granules were in storage after the PS, which was mirrored by a reduction in LH pulsatile release. Surprisingly, in view of results in rodents indicating significant changes, SgII and CgA mRNA levels did not change over the oestrous cycle in sheep. Conversely, CgB mRNA levels decreased around the time of PS. These novel results illustrate major differences in granin-gonadotrophin interactions between sheep and rodents.
Asunto(s)
Cromograninas/análisis , Ciclo Estral/fisiología , Gonadotropinas Hipofisarias/análisis , Adenohipófisis/química , Ovinos/fisiología , Análisis de Varianza , Animales , Northern Blotting , Cromogranina A , Cromograninas/genética , Femenino , Hormona Folículo Estimulante/análisis , Hormona Folículo Estimulante/sangre , Hormona Folículo Estimulante/genética , Hormona Folículo Estimulante de Subunidad beta , Hormona Luteinizante/análisis , Hormona Luteinizante/sangre , Hormona Luteinizante/genética , Microscopía Inmunoelectrónica , Proteínas/análisis , Proteínas/genética , ARN Mensajero/análisisRESUMEN
Intracellular associations indicate that granins may play a role in the regulatory mechanisms involved in differential secretion of gonadotrophins. The effect of GnRH on mRNA expression, storage and secretory patterns of granins and gonadotrophins was investigated in male mice. GnRH antiserum (G/A) was injected into mice in the treatment group (n = 15) at 12 h intervals for 2 days and a subset (n = 9) was killed. Buserelin (G/A + B) was administered to the remaining mice (n = 6), which were killed 2 h later; control mice (n = 6) were killed at the onset of the study. LHb mRNA content was lower in G/A and G/A + B mice compared with controls, whereas plasma LH concentrations were higher in G/A + B mice. FSHbeta mRNA content did not change, whereas plasma FSH concentrations were lower in G/A mice compared with controls, and higher in G/A + B mice compared with both G/A and control mice. Secretogranin II (SgII) and CgA mRNA contents were not different between experimental groups. There were more granules per gonadotroph in G/A mice, and considerably fewer after Buserelin treatment. Immunogold labelling of gonadotrophs revealed the presence of LH(+ve)/SgII(+ve) and LH(+ve)/SgII(-ve) granules, and negligible numbers of LH(-ve)/SgII(+ve) granules. Both the numbers of LH(+ve)/SgII(+ve) granules and overall granule antigenicity for SgII were higher in G/A mice compared with controls and G/A + B mice. In contrast, there were fewer LH(+ve)/SgII(-ve) granules per gonadotroph in G/A mice compared with controls. In conclusion, absence of GnRH input to the pituitary gland resulted in preferential storage of SgII and subsequently increased intragranular co-aggregation with LH. Administration of Buserelin to G/A mice resulted in the apparent release of LH(+ve)/SgII(+ve) granules that was reflected by an increase in plasma LH concentrations, indicating that these granules were in the regulated secretory pathway. In contrast, secretion of LH(+ve)/SgII(-ve) granules did not appear to be influenced by the actions of Buserelin and, therefore, may have been destined for constitutive release, possibly to maintain basal plasma LH concentrations.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Hormona Liberadora de Gonadotropina/fisiología , Hormona Luteinizante/metabolismo , Adenohipófisis/metabolismo , Proteínas/metabolismo , Animales , Buserelina/farmacología , Cromograninas , Gránulos Citoplasmáticos/efectos de los fármacos , Hormona Folículo Estimulante/sangre , Hormona Folículo Estimulante/genética , Gonadotropinas/biosíntesis , Gonadotropinas/genética , Hormona Luteinizante/sangre , Masculino , Ratones , Microscopía Confocal , Microscopía Electrónica , Neuropéptidos/metabolismo , Adenohipófisis/efectos de los fármacos , Adenohipófisis/ultraestructura , ARN Mensajero/genéticaRESUMEN
The pattern of replenishment of LH secretory granule stores in sheep pituitary gonadotrophs, after an induced LH surge, was determined by immunogold localisation at the ultrastructural level by electron microscopy. Twenty-four Welsh Mountain ewes were initially synchronised with progestagen devices for 14 days before luteolysis was induced by a prostaglandin F(2 alpha) analogue, cloprostenol. A further 24 h later, a preovulatory LH surge was induced by intravenous injection of a GnRH agonist, buserelin. Animals were divided into four groups (n=6) and blood sampled at 2 h intervals from 4 h prior, to 18 h after, buserelin administration and then at infrequent intervals (1 to 8 h) thereafter until death. Pulse profiles of LH were also obtained by an additional collection of blood samples within a 6 h window directly preceding death. Groups of animals were killed at 24, 48, 72 or 96 h after buserelin treatment. Pituitaries were dissected and processed for transmission electron microscopy and frozen for later molecular biological analysis. A characteristic preovulatory surge of LH was observed in all animals. The cytoplasm of gonadotrophs, in animals killed 24 h after buserelin treatment, was completely empty of secretory granules. This was associated with diminutive pituitary LH content, low pituitary GnRH binding levels and an almost complete absence (one pulse in one animal) of LH pulsatile secretion. Despite the lack of apparent secretory activity, clusters of exposed LH beta label present within the cytoplasm at this time and constant LHbeta mRNA expression levels irrespective of tissue collection time, suggest that the cell is actively synthesising LHbeta. The formation of sparse numbers of small LH beta immuno-labelled electron-dense secretory granules was apparent at 48 h after buserelin treatment, and replenishment of LH beta immuno-labelled granule stores continued until total granule numbers had increased two-fold (P<0.01) by 96 h post-treatment. Affiliated with granule replenishment was a significant increase in pituitary LH content (P<0.01), pituitary GnRH binding levels (P<0.01) and the restoration of LH pulsatile secretion. Despite the replenishment of granule stores with time, cytoplasmic area did not vary. These results suggest that restoration of pulsatile LH secretion after a preovulatory LH surge is related to replenishment of LH beta secretory granule stores and an increase in GnRH binding levels.
Asunto(s)
Fase Folicular/metabolismo , Hormona Luteinizante/biosíntesis , Hipófisis/metabolismo , Vesículas Secretoras/metabolismo , Ovinos/metabolismo , Análisis de Varianza , Animales , Buserelina/farmacología , Femenino , Fármacos para la Fertilidad Femenina/farmacología , Hormona Liberadora de Gonadotropina/metabolismo , Hormona Luteinizante/sangre , Hormona Luteinizante/genética , Microscopía Inmunoelectrónica , Hipófisis/efectos de los fármacos , Hipófisis/ultraestructura , Unión Proteica , Tasa de Secreción , Vesículas Secretoras/ultraestructura , Factores de TiempoRESUMEN
The common brushtail possum (Trichosurus vulpecula) is a pest of considerable economic importance in New Zealand. Attempts to develop methods of suppressing reproduction in this species are currently hampered by the lack of reliable methods to synchronise oestrus and ovulation in this species. The objective of this study was to compare antral follicle populations in anoestrous and cyclic brushtail possums and to assess the efficacy of exogenous FSH to induce follicle development in anoestrous animals. Ovaries were recovered from anoestrous possums after administration of either exogenous FSH (1.0 mg/injection) or the saline vehicle alone (0.5 ml/injection) at 12-h intervals for 3 days (n = 6/group), and from cyclic animals (n = 6) that were euthanised in mid-follicular phase (5 days after removal of their pouch young). All antral follicles > or =1.0 mm in diameter were dissected free of extraneous tissue, incubated in vitro to measure oestradiol production, and then processed for histological assessment of health status. Mean weight of ovaries and vaginal cul-de-sac tissues were both significantly greater (P<0.001) in FSH-treated anoestrous females (24.2+/-5.1 mg and 6.50+/-1.34 g, respectively), but did not differ significantly between saline-treated anoestrous possums (12.4+/-3.0 mg and 1.31+/-0.27 g) and cyclic animals (13.5+/-1.6 mg and 2.62+/-0.95 g). Mean uterine weights in both cyclic (889+/-161 mg) and FSH-treated (1098+/-184 mg) animals were significantly heavier(P<0.001) than those of anoestrous possums (414+/-61 mg). The mean number of follicles (> or =1.0-mm diameter) present was significantly greater (P<0.001) in FSH-treated, than in cyclic and anoestrous possums (38.0+/-4.4, 23.2+/-3.2 and 10.7+/-3.4 follicles/animal, respectively). Cyclic animals had significantly more (P<0.01) follicles than anoestrous possums. The proportion of follicles that were classified as healthy, was significantly lower (P<0.01) in cyclic possums(38%) than in anoestrous (69%) and FSH-treated (88%) animals. The mean diameter of the largest healthy follicle present was 2.5+/-0.41, 2.1+/-0.08, and 3.1+/-0.15 mm for cyclic, anoestrous and FSH-treated animals, respectively. None of the follicles harvested from saline-treated anoestrous possums produced measurable levels of oestradiol in vitro, whereas 7% and 59% of those from cyclic and FSH-treated animals did so. In summary, cyclic possums had more antral follicles present than anoestrous animals, but a lower percentage of these follicles were healthy. Less than 10% of healthy follicles from cyclic possums, and none of those from anoestrous animals, were capable of producing oestradiol when incubated in vitro. Treatment with ovine FSH promoted follicle development in anoestrous possums, to significantly increase the number of follicles present, the proportion that were healthy and the percentage capable of producing oestradiol.
Asunto(s)
Estro/fisiología , Hormona Folículo Estimulante/farmacología , Zarigüeyas/fisiología , Folículo Ovárico/fisiología , Animales , Estradiol/análisis , Femenino , Hormona Folículo Estimulante/fisiología , Laparoscopía/veterinaria , Folículo Ovárico/citología , Folículo Ovárico/efectos de los fármacos , Ovario/fisiología , Radioinmunoensayo/veterinaria , Distribución Aleatoria , Útero/fisiología , Vagina/fisiologíaRESUMEN
Changes in plasma concentrations of follicle-stimulating hormone (FSH) and luteinizing hormone (LH), and their relationship to antral follicle development and ovulation, were determined in female brushtail possums (Trichosurus vulpecula) in experiments in which pouch young were removed (RPY) from lactating females to promote ovarian activity. In Experiment 1 (n = 8), the development of preovulatory follicles and ovulation was monitored by laparoscopy. In Experiment 2 (n = 15) estrus and mating were monitored by cytology of urine. Ovulation occurred in 4/8 (Experiment 1) and 9/16 (Experiment 2) possums, and in these animals, plasma FSH concentrations fell progressively over the period of preovulatory follicle development and returned to pretreatment levels after ovulation. With the exception of samples taken at the time of the preovulatory gonadotropin surge, mean plasma LH levels remained basal. In those possums that failed to ovulate, plasma FSH concentrations were elevated while plasma LH concentrations were low; these patterns remained unchanged throughout the sampling period. It was not possible to distinguish between animals that would ovulate and those that would not ovulate after RPY on the basis of gonadotropin profiles at the time of RPY. A further group of possums (Experiment 3, n = 10) were blood-sampled at hourly intervals for 48 h to characterize preovulatory gonadotropin surges, using laparoscopy to monitor preovulatory follicular development and predict ovulation. A preovulatory LH surge (max. conc. 10.2-43.5 ng/ml, duration 7-9 h) was recorded in 4 animals, with a coincident preovulatory FSH surge (max. conc. 1.4-21.4 ng/ml, duration 3-11 h) observed in 3 of these possums. The patterns of gonadotropin secretion in the cycling brushtail possum conform to those reported for eutherians that ovulate spontaneously and appear to be regulated by similar mechanisms.
Asunto(s)
Hormona Folículo Estimulante/sangre , Hormona Luteinizante/sangre , Zarigüeyas/sangre , Animales , Células Epiteliales , Estro/fisiología , Femenino , Folículo Ovárico/fisiología , Ovulación , Progesterona/sangre , Orina/citologíaRESUMEN
The aim of this study was to describe and quantify the changes that occur in cul-de-sac tissue, in particular to epithelial cells and their constituents, at specific stages of the estrous cycle in the brushtail possum. Stereological techniques were used to quantify changes in cul-de-sac epithelial cells collected at four stages of the estrous cycle; the time of removal of pouch young (RPY; n = 5), of initial follicle development (n = 5), of preovulatory follicle formation (n = 5), of midluteal stage (n = 4), and again at RPY (n = 5) after completion of the experiment to examine for any effects due to season or time. Tissue was weighed and processed for light microscopy, transmission electron microscopy, and stereological analysis. Cul-de-sac epithelial cell volume increased approximately 17-fold at the time of preovulatory follicle formation compared with that at the time of RPY, before declining (approximately four-fold greater than at RPY) during the midluteal phase. Epithelial cell volume enlargement was correlated strongly with the size of the preovulatory follicle present, and maximum size was coincidental with the formation of extracellular spaces and projection of cell processes between lateral cell membranes. Maximum cell volume was associated with an approximate 25-fold and six-fold increase in cytoplasmic and nuclear volume, respectively. Enlargement of the epithelial cells coincided with an increase in cytoplasmic organelle numbers, microvilli prominence, and accumulation of secretory vesicles. In the possum, the cul-de-sac epithelial cell undergoes phenomenal remodelling during the estrous cycle to accommodate an approximate 17-fold increase in volume. This increase in cell volume is coincident with morphological changes characteristic of secretory activity and appears to be under estrogen regulation.
Asunto(s)
Células Epiteliales/citología , Estro , Vagina/citología , Animales , Tamaño de la Célula , Gránulos Citoplasmáticos/ultraestructura , Células Epiteliales/ultraestructura , Femenino , Marsupiales , Microscopía Electrónica , Microvellosidades/ultraestructura , Orgánulos/ultraestructura , Factores de TiempoRESUMEN
BACKGROUND: Although routine interdental cleaning is important and recommended by dental professionals, compliance has been relatively low. To aid in improving compliance, an electrically powered device has been developed. METHODS: This six-month randomized, single-blinded, parallel-group study was conducted to compare the long-term efficacy and safety of a new interdental cleaning device (Braun Oral-B Interclean, model ID2) with those of an ADA-approved waxed dental floss in healthy adults. RESULTS: The authors found no statistically significant difference between the two products with respect to the gingival index or gingival bleeding index after three or six months of use. A one-time product use, at the six-month examination, confirmed the equivalency of the two methods with respect to removal of dental plaque. The oral soft-tissue status of both groups of subjects also remained comparable throughout the study. CONCLUSION: Use of the interdental cleaning device and dental floss resulted in comparable benefits with respect to gingival health and plaque removal. CLINICAL IMPLICATIONS: Although it was not shown to be an improvement over dental floss, the cleaning device was comparable in every respect. Since it can be used with one hand and does not require as much dexterity as floss, the device warrants consideration by those who lack the motivation or are unable to use dental floss.
Asunto(s)
Dispositivos para el Autocuidado Bucal , Adulto , Análisis de Varianza , Placa Dental/prevención & control , Electricidad , Diseño de Equipo , Seguridad de Equipos , Femenino , Hemorragia Gingival/prevención & control , Gingivitis/prevención & control , Humanos , Masculino , Motivación , Destreza Motora , Índice Periodontal , Reproducibilidad de los Resultados , Método Simple CiegoRESUMEN
PURPOSE: To compare the efficacy on plaque, gingivitis and calculus of an oscillating/rotating power toothbrush (Braun Oral-B Ultra Plaque Remover-D9) and a high frequency toothbrush (sonicare). MATERIALS AND METHODS: A 6-week, randomized crossover study was carried out. A total of 62 healthy adult subjects completed the study and were assessed for plaque control, gingival condition and calculus control. At the end of the study, subjects completed a questionnaire which assessed their preferences for the two devices. RESULTS: Both toothbrushes were found to be safe and effective, but a significantly lower gingival index (P = 0.002) and a lower calculus index (P = 0.022) was found in the D9 group. Assessment of patient preferences revealed that a significant majority of subjects in the study (65%) preferred the D9 to the high frequency toothbrush (27%). It is concluded that the D9 may offer greater potential for the maintenance of good gingival condition than the high frequency toothbrush, and that the greater preference for the D9 may be important with respect to long-term compliance. The implications of these findings are discussed.
Asunto(s)
Placa Dental/prevención & control , Gingivitis/prevención & control , Cepillado Dental/instrumentación , Adulto , Análisis de Varianza , Estudios Cruzados , Índice de Placa Dental , Diseño de Equipo , Femenino , Humanos , Masculino , Índice Periodontal , Método Simple Ciego , Encuestas y CuestionariosRESUMEN
Studies using transgenic mice that overexpress ciliary neurotrophic factor (CNTF), direct injection of CNTF into brain parenchyma, and ectopic expression of CNTF by an adenoviral vector have demonstrated that CNTF activates astrocytes. Paradoxically, studies to date have failed to show an effect of CNTF on the expression of GFAP by cultured astrocytes. Therefore, the goal of this study was to use nuclear hypertrophy and GFAP expression as indices of glial activation to compare the responsiveness of forebrain type 1 and type 2 astrocytes to CNTF. As reported by others, CNTF did not increase GFAP in type 1 astrocytes; however, it rapidly increased their nuclear size by 20%. Nuclear hypertrophy was apparent within 4 h after CNTF exposure and persisted for at least 48 h. In contrast, type 2 astrocyte GFAP increased 2-fold over the course of 48 h of CNTF treatment. During this same treatment period type 2 astroglial nuclei enlarged by 25%. We conclude that CNTF stimulates both type 1 and type 2 astrocytes directly. Together with our in vivo studies (Levison et al., 1996: Exp. Neurol. 141: 256), these data support the concept that CNTF is responsible for many of the progressive astroglial changes that appear after CNS injury and disease.
Asunto(s)
Astrocitos/efectos de los fármacos , Núcleo Celular/patología , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas del Tejido Nervioso/farmacología , Animales , Animales Recién Nacidos , Astrocitos/patología , Núcleo Celular/efectos de los fármacos , Células Cultivadas , Factor Neurotrófico Ciliar , Hipertrofia , Factores de Crecimiento Nervioso/farmacología , Prosencéfalo/citología , RatasRESUMEN
The traditional method for inducing and synchronising oestrus in the brushtail possum (Trichosurus vulpecula) is by removal of their suckling pouch young (RPY). However, our studies have recently shown that, in addition to wide variation between animals in the time of ovulation after RPY, a proportion of animals failed to ovulate. Evidence from several mammalian species indicates that the presence of males can stimulate ovarian activity and synchronise oestrus in females. The aim of this study was to determine the effect of the male on the oestrous cycle of the female brushtail possum after RPY. A total of 67 adult female brushtail possums were treated as three replicates. In order to observe the day of preovulatory follicle emergence and ovulation, animals underwent laparoscopic examination at 1-4 day intervals over a period from 0-21 days after RPY. The first replicate (N = 18, May/June 1995) involved only animals kept in isolation from males, whereas the two remaining replicates compared ovarian responses between animals kept with ( N = 10, July 1995; N = 14, June 1996) or in isolation from ( N = 10, July 1995; N = 15, June 1996) males. The incidence of ovulation after RPY was significantly higher in females that were housed with males than in those kept in isolation from males (100%, 92.8% vs. 50.0%, 66.7%, 14.3%; P < 0.001). Every animal that ovulated, had previously had a preovulatory follicle present at the site where the corpus luteum formed. Conversely, none of the animals that failed to ovulate, developed a preovulatory follicle during the period of the study. The range of mean day of preovulatory follicle emergence (6.00-6.86 days), of ovulation (11.80-12.20 days) and the synchrony of ovulation between animals (range 8-17 days) after RPY, were not significantly affected by the presence of males. This study demonstrates for the first time, that the presence of males significantly increases the incidence of ovulation after RPY in the brushtail possum. However neither the timing of reproductive events nor the synchrony of ovulation were affected by presence of the male.
Asunto(s)
Zarigüeyas/fisiología , Ovulación/fisiología , Animales , Sincronización del Estro , Femenino , Masculino , Inducción de la Ovulación , Reproducción , Estaciones del Año , DesteteRESUMEN
The common method for synchronizing oestrus in brushtail possums is by removal of their pouch young (RPY). However, there is little information on the ovarian response to this treatment, the timing and incidence of ovulation is poorly defined, and methods of identifying oestrus are unreliable. In this study, the development of preovulatory follicles, ovulation and reproductive tract changes following RPY were monitored by repeated laparoscopic observation. A total of 120 adult female possums underwent laparoscopy at intervals of 1-4 days over the period from 0 to 21 days after RPY. Tissue was collected from a further 30 animals for correlative histology of ovarian structures, and to quantify changes in reproductive tract organs. Only 80 of 120 animals ovulated, and the time of ovulation ranged from 7 to 18 days following RPY. In most animals, enlargement of vaginal cul-de-sac and uterine tissue occurred within 10 days. Correlative histology supported the macroscopic classification of ovarian structures, and healthy and atretic follicles could be identified by laparoscopy. Vaginal smears and plasma progesterone concentrations verified the occurrence of ovulation as observed by laparoscopy. A 'presumptive' preovulatory follicle, first identifiable approximately 5 days before ovulation, was recorded in all animals that ovulated and in none that failed to ovulate. Changes to its surface morphology indicated impending ovulation. This study has enabled the day of ovulation to be identified accurately for the first time in this species. It has also shown that there is wide variation in follicle development, lack of synchrony in the time of ovulation in the brushtail possum, and that some animals fail to ovulate following RPY.
Asunto(s)
Marsupiales/fisiología , Folículo Ovárico/fisiología , Ovulación/fisiología , Animales , Sincronización del Estro , Femenino , Laparoscopía , Marsupiales/anatomía & histología , Progesterona/sangre , Factores de Tiempo , Útero/anatomía & histología , Vagina/anatomía & histología , Frotis VaginalRESUMEN
Graft inclusion and vessel reattachment to openings made in the graft were employed in the treatment of 605 patients with thoracoabdominal aortic aneurysms. These patients were divided into four groups on the basis of the extent of aneurysm. Group I consisted of those patients with involvement of most of the descending thoracic and upper abdominal aorta; group II involved most of the descending thoracic aorta and most or all of the abdominal aorta; group III involved the distal descending thoracic aorta and varying segments of abdominal aorta; and group IV involved most or all of the abdominal aorta including the segment from which the visceral vessels arose. The cause of aneurysm formation was medial degenerative disease in 80%, and dissection in 17%; other causes were responsible in the remaining 3%. The median age was 65 years and associated diseases including aneurysms involving other segments, atherosclerotic occlusive disease, heart disease, chronic obstructive pulmonary disease (COPD), hypertension, and renal insufficiency were frequent. The aneurysm was symptomatic in 70% of cases and rupture had occurred in 4% of cases. There were 54 (8.9%) early (30-day) deaths and 151 late deaths; 400 (66%) patients were still alive 3 months to 20 years after operation, including 60% at 5 years. Statistically significant pre- and intraoperative variables by univariate analysis that were predictive of increased risk of early death were advancing age, associated diseases that included COPD, renal artery occlusive disease, atherosclerotic heart disease, renal insufficiency, and long aortic clamp time. Three of these (age, clamp time, and the presence of COPD) retained significance by multivariate analysis. Variables predictive of risk of late death were age, dissection, extent of aneurysm, rupture, heart disease, cerebrovascular disease, COPD, hypertension, and poor renal function. Age, rupture, renal dysfunction, extent of aneurysm, and dissection retained their significance by multivariate analysis. Variables predictive of neurologic disturbances of the lower extremities included rupture, reattachment of intercostal and lumbar arteries, clamp time, dissection, extent and age. Rupture, reattachment of vessels, dissection, and extent of aneurysm retained significance by multivariate analysis. Thus, the risk of this complication was greatest in patients with extensive lesions (group II) with aortic dissection. The greatest risk of renal failure after operation that required dialysis was in patients who had impaired renal function before operation. Methods employed did not prevent these complications.