Reproductive biology in the primitive relic Angiosperm Drimys brasiliensis (Winteraceae)

From field observations on Drimys brasiliensis, principally in the Botucatu region of São Paulo State, Brazil, new data on the reproductive biology, the rhythm of growth, and the development of lateral cymose inflorescences, flowers and fruits are presented. Pollination accelerates the rate of flower-development for about 4-6 days. Pollination experiments show that D. brasiliensis is not self-sterile; because of mechanical devices the sticky pollen grains do not normally come into contact with the stigmata unless an animal pollen vector is involved. The pollinators are diurnal Coleoptera, Diptera and Thysanoptera which eat from the pollen, lick from the stigmatic exudates and (in case of the flies) probably also from the staminal glands. Fruit- and seedeaters are birds which seem to be the main dispersal agents. Establishment of new individuals normally is through seedlings, but also by vegetative propagation through plagiotropous branches which may root and separate from the mother plant. The morphological, developmental and reproductive aspects in D. brasiliensis are discussed in a wider context, compared with data from other Magnoliidae, and related to aspects of early Angiosperm evolution. © 1980 Springer-Verlag.

Reproductive biology and pollination mechanisms of Epidendrum secundum (Orchidaceae). Floral variation: a consequence of natural hybridization?

The phenology, flower morphology, pollination mechanism and reproductive biology of Epidendrum secundum were studied in a semi-deciduous forest at the Serra do Japi (SJ), and in the Atlantic rain forest of Picinguaba, both natural reserves in the State of Sao Paulo, southeastern Brazil. E. secundum flowers all year round, with a flowering peak between September and January. This species is either a lithophytic or terrestrial herb in the SJ, whereas, in Picinguaba, it grows mainly in disturbed areas along roadsides. E. secundum is pollinated by several species of diurnal Lepidoptera at both study sites. In Picinguaba, where E. secundum is sympatric with E. fulgens and both share the same pollinators, pollen transference between these two species was recorded. E. secundum is self-compatible but pollinator-dependent. It is inter-compatible with E. fulgens, producing fertile seeds. In contrast to the population of the SJ, in the Picinguaba region, floral morphology is quite variable among plants and some individuals present flowers with characteristics in-between both sympatric species, suggesting that natural hybridization occasionally occurs. The anthropogenic perturbation is probably the cause of the occurrence of E. secundum in the Picinguaba region, enabling its contact with E. fulgens.

Reproductive biology of six Brazilian Myrtaceae: is there a syndrome associated with buzz-pollination?

The floral phenology and reproductive biology of six sympatric arboreal Myrtaceae species were studied in the coastal plain forest (Ubatuba, Brazil, 44 degrees 48`W 23 degrees 22`S), from September 1999 to April 2002. Flowering started in the transition from the driest to the most humid season (Sep/Oct) and lasted until March. The sequence with which the species flowered each year was consistently the same. However, the timing of flowering onset, peak, end, and overlap differed from one year to another. Myrtaceae species were classified as xenogamic according to the pollen:ovule ratios, but two of them seem to present some degree of self-compatibility. Flowers of all species opened at sunrise and lasted for I day. Bombus morio (Apidae: Bombini) was the most common visitor followed by Melipona rufiventris (Apidae: Meliponini). Buzz pollination in Myrtaceae was common at the study area and seems to be related to bees` behaviour and to some aspects of flowers` morphology.

Reproductive biology and seasonal distribution of Mustelus schmitti (Elasmobranchii : Triakidae) in the Rio de la Plata oceanic front, south-western Atlantic

The population structure and reproductive biology of Mustelus schmitti was analysed during autumn (1994, 1995) and spring (1994) in the Rio dc la Plata occanic front. The samples consisted mainly of a adult males and immature females, indicating that nursery areas are Situated elsewhere. In autumn, the highest female densities occurred between 36 degrees S and 35 degrees S, with densities decreasing further south, and high male densities occurred over all latitudes of the study area. In Spring, both Sexes occurred north of latitude 36 degrees 30 degrees S. Males attained Maturity at. 59 cm total length (TL), and females at 72 cmTL Litter size varied From one to ten in spring and two to nine in autumn, With embryos ranging from 9 to 26 cm and 7 to 26 cm TL respcctivcly the mean TL of embryos was significantly higher in spring (22 cm), suggesting that parturition Occurs ill late spring and summer. Litter size was found to Increase with female TL..

Census, reproductive biology, and germination of Astragalus gines-lopezii (Fabaceae), a narrow and endangered endemic species of SW Spain

Astragalus gines-lopezii Talavera, Podlech, Devesa & F.M.Vázquez (Fabaceae) is a threatened endemic species with a distribution restricted to a very small area in Badajoz Province (Extremadura Region, SW Spain) and only 2 populations are known. This species was catalogued in the ?Endangered? category in the 2008 Red List and the 2010 Threatened Spanish Vascular Flora List. Despite its status as an endangered species, at present very little is known about the distribution, census, and reproductive biology of this species. In this study we have carried out an exhaustive census of A. gines-lopezii , and we have evaluated the production of flowers, fruits, and seeds and the existence or not of intra- and interpopulation variability in seed germination. Results have highlighted the high reproductive capacity of this species on the basis of a high production of flowers, fruits, and seeds. Mechanical scarification of seeds was effective for increasing germination. Thus, initial germination (22%?60%) was increased to 97%?99% when seeds were rubbed with sandpapers. A high intra- and interpopulation variability in seed germination was found in this species. A. gines-lopezii produces seeds with different degrees of physical dormancy, varying this grade among different individuals within a population.

Census, reproductive biology, and germination of Astragalus gines-lopezii (Fabaceae),a narrow and endangered endemic species of SW Spain.

Astragalus gines-lopezii Talavera, Podlech, Devesa & F.M.Vazquez (Fabaceae) is a threatened endemic species with a distribution restricted to a very small area in Badajoz Province (Extremadura Region, SW Spain) and only 2 populations are known.This species was catalogued in the "Endangered" category in the 2008 Red List and the 2010 Threatened Spanish Vascular Flora List. Despite its status as an endangered species, at present very little is known about the distribution, census, and reproductive biology of this species. In this study we have carried out anexhaustive census of A. gines-lopezii, and we have evaluated the production of flowers, fruits, and seeds and the existence or not of intra- and interpopulation variability in seed germination. Results have highlighted the high reproductive capacity of this species on the basis of a high production of flowers, fruits, and seeds. Mechanical scarification of seeds was effective for increasing germination. Thus, initial germination (22%-60%) was increased to 97%-99% when seeds were rubbed with sandpapers. A high intra- and interpopulation variability in seed germination was found in this species. A. gines-lopezii produces seeds with different degrees of physical dormancy, varying this grade among different individuals within a population.

Studies of assisted reproduction in the spotted grass frog Limnodynastes tasmaniensis: ovulation, early development and microinjection (ICSI)

Assisted Reproductive Technologies (ART) offer a wide range of techniques that have the potential to augment efforts to conserve and manage endangered amphibians and improve wild and captive population numbers. Gametes and tissues of species nearing endangered or extinct status can be cryopreserved and stored in gene banks, to provide material that can be utilised in the future as ART methods are refined. The Spotted Grass Frog, Limnodynastes tasmaniensis, is an abundant amphibian species in South-Eastern Australia of the family Myobatrachidae, that is suitable for the development of ART systems that can be applied to the threatened and endangered myobatrachid and other amphibian species native to Australia. The aim of this study was to advance the understanding of ovulation, fertilisation and embryo nic development of Lim. tasmaniensis and in vitro manipulations of reproduction and development for use in the development of advanced ART procedures such as intracytoplasmic spermatozoon injection (ICSI), androgenesis and nuclear transfer. Ovulation in amphibians can be induced by protocols utilising natural or synthetic hormones. All protocols tested on Lim. tasmaniensis in this study required two injections and the most effective protocols continued to require a first injection of pituitary extracts to induce ovulation. The second injection was, however, successfully replaced by synthetic chorionic gonadotrophin at a threshold dosage of 100 iu and halved the number of cane toads required to source the pituitaries. A combination of LHRH and Pimozide offered a less effective protocol, that did not require the use of pituitary extracts, and avoided the risk of pathogen transfer associated with unsterilised pituitary extracts. Unfertilised eggs of Lim. tasmaniensis were exposed to media of various osmolalities to determine media effects on eggs and their surrounding jelly layers that might impact on egg viability and fertilisability. Osmolality had no effect upon the egg diameter, however, rapid swelling of the jelly layers occurred within 15 minutes of exposure to various media treatments and plateaued from 30-90 minutes without further expansion. Swelling of the jelly layers was increased in hypotonic media (2.5% SAR, H2O) and minimised in the isotonic media (100% SAR). The optimal conditions for the culture of Lim. tasmaniensis eggs were identified as a holding media of 100% SAR, followed by a medium change to 2.5% SAR at insemination. This sequence of media minimised the rate of swelling of the jelly layers prior to contact with the spermatozoa, and maximised the activation of spermatozoa and eggs throughout fertilisation and embryonic development. Embryos of Lim. tasmaniensis were cultured at four temperatures (13 C, 17 C, 23 C and 29 C), to determine the effect of temperature on cleavage and embryonic development rates. Embryonic development progressed through a sequence of stages that were not altered by changes in temperature. However cleavage rates were affected by changes in temperature as compared with normal embryonic growth at 23 C. Embryonic development was suspended at the lowest temperature (13 C) while embryonic viability was maintained. A moderate decrease in temperature (17 C) slowed cleavage, while the highest temperature (29 C) increased the cleavage rate, but decreased the embryo survival. Rates of embryonic development can be manipulated by changes in temperature and this method can be used to source blastomeres of a specific size/stage at a predetermined age or halt cleavage at specific stages for embryos or embryo derived cells to be included in ART procedures. This study produced the first report of the application of Intracytoplasmic Spermatozoon Injection (ICSI) in an Australian amphibian. Eggs that were activated by microinjection with a single spermatozoon (n=50) formed more deep, but abnormal, cleavage furrows post-injection (18/50, 36%), than surface changes (12/50, 24%). This result is in contrast to eggs injected without a spermatozoon (n=42), where the majority of eggs displayed limited surface changes (36/42, 86%), and few deep, abnormal furrows (3/42, 7%). Three advanced embryos (3/50, 6%) were produced by ICSI that developed to various stages within the culture system. Technical difficulties were encountered that prevented the generation of any metamorphs from ICSI tadpoles. Nevertheless, when these blocks to ICSI are overcome, the ICSI procedure will be both directly useful as an ART procedure in its own right, and the associated refinement of micromanipulation procedures will assist in the development of other ART procedures in Lim. tasmaniensis. A greater understanding of basic reproductive and developmental biology in Lim. tasmaniensis would greatly facilitate refinement of fertilisation by ICSI. Assisted Reproductive Technologies, in conjunction with gene banks may in the future regenerate extinct amphibian species, and assist in the recovery of declining amphibian populations nationally and worldwide.

Reproductive biology of Black Anglerfish (Lophius budegassa) in the northwestern Mediterranean Sea

Gonadal morphology and reproductive biology of the Black Anglerfish (Lophius budegassa) were studied by examining 4410 specimens collected between June 2007 and December 2010 in the northwestern Mediterranean Sea. Ovaries and testes presented traits common among fishes of the order Lophiiformes. Spawning occurred between November and March. Size at first maturity (L50) was 33.4 cm in total length (TL) for males and 48.2 cm TL for females. Black Anglerfish is a total spawner with group-synchronous oocyte development and determinate fecundity. Fecundity values ranged from 87,569 to 398,986 oocytes, and mean potential fecundity was estimated at 78,929 (standard error of the mean [SE] 13,648) oocytes per kilogram of mature female. This study provides the first description of the presence of 2–3 eggs sharing the same chamber and a semicystic type of spermatogenesis for Black Anglerfish. This new information allows for a better understanding of Black Anglerfish reproduction—knowledge that will be useful for the assessment and management of this species.

Occurrence of the Indo-Pacific freshwater prawn Macrobrachium equidens Dana 1852 (Decapoda, Palaemonidae) on the coast of Brazilian Amazonia, with notes on its reproductive biology

O camarão de água doce Macrobrachium equidens, nativo da região do Indo-Pacífico, foi registrada pela primeira vez na costa da Amazônia Brasileira. Esta espécie foi encontrada habitando o mesmo ambiente que duas espécies nativas do gênero Macrobrachium: M. amazonicum e M. acanthurus, e é morfologicamente muito similar à última. A identificação dessa espécie foi confirmada pela análise da sequencia genética do gene mitocondrial Citocromo Oxidase (COI). Uma descrição detalhada das características morfológicas e biologia reprodutiva de M. equidens neste novo ambiente que é apresentada.

Life cycle and reproductive traits of marine podonids (Cladocera, Onychopoda) in a coastal subtropical area

We studied the temporal distribution and reproductive biology of marine podonids during two consecutive years off Ubatuba, southeast coast of Brazil. Podonid specimens and their eggs and embryos were counted, measured and classified into categories. Pseudevadne tergestina was the most abundant species, and was more abundant in surface layers, in warm seasons, when the water column was stratified because of bottom intrusions of the cold and nutrient-rich South Atlantic Central Water (SACW) onto the inner shelf. Evadne spinifera had a similar temporal and vertical distribution, but with lower abundance and frequency. Pleopis schmackeri did not show a clear seasonal distribution, but preferred bottom layers. Pleopis polyphemoides and Podon intermedius occurred in low abundances, and only under SACW influence. Parthenogenetic females were dominant among all podonid species. Gamogenetic females of P. polyphemoides and P. intermedius were observed, but males of neither species occurred. This suggests that in tropical and subtropical regions, P. tergestina, E. spinifera and P. schmackeri reproduce through parthenogenesis during most of the year.

Growth differentiation factor 9 signalling in the ovary

In the ovary, two new members of the large TGF-beta superfamily of growth factors were discovered in the 1990s. The oocyte was shown to express two closely related growth factors that were named growth differentiation factor 9 (GDF-9) and growth differentiation factor 9B (GDF-9B). Both of these proteins are required for normal ovarian follicle development although their individual significance varies between species. GDF-9 and GDF-9B mRNAs are expressed in the human oocytes from the primary follicle stage onwards. This thesis project was aimed to define the signalling mechanisms utilized by the oocyte secreted GDF-9. We used primary cultures of human granulosa luteal cells (hGL) as our cell model, and recombinant adenovirus-mediated gene transfer in manipulating the TGF-b family signalling cascade molecules in these cells. Overexpression of the constitutively active forms of the seven type I receptors, the activin receptor-like kinases 1-7 (ALK1-7), using recombinant adenoviruses caused a specific activation of either the Smad1 or Smad2 pathway proteins depending on the ALK used. Activation of both Smad1 and Smad2 proteins also stimulated the expression of dimeric inhibin B protein in hGL cells. Treatment with recombinant GDF-9 protein induced the specific activation of the Smad2 pathway and stimulated the expression of inhibin betaB subunit mRNA as well as inhibin B protein secretion in our cell model. Recombinant GDF-9 also activated the Smad3-responsive CAGA-luciferase reported construct, and the GDF-9 response in hGL cells was markedly potentiated upon the overexpression of Alk5 by adenoviral gene transduction. Alk5 overexpression also enhanced the GDF-9 induced inhibin B secretion by these cells. Similarly, in a mouse teratocarcinoma cell line P19, GDF-9 could activate the Smad2/3 pathway, and overexpression of ALK5 in COS7 cells rendered them responsive to GDF-9. Furthermore, transfection of rat granulosa cells with small interfering RNA for ALK5 or overexpression of the inhibitory Smad7 resulted in dose-dependent suppression of GDF-9 effects. In conclusion, this thesis shows that both Smad1 and Smad2 pathways are involved in controlling the regulation of inhibin B secretion. Therefore, in addition to endocrine control of inhibin production by the pituitary gonadotropins, also local paracrine factors within in the ovary, like the oocyte-derived growth factors, may contribute to controlling inhibin secretion. This thesis shows as well that like other TGF-beta family ligands, also GDF-9 signalling is mediated by the canonical type I and type II receptors with serine/threonine kinase activity, and the intracellular transcription factors, the Smads. Although GDF-9 binds to the BMP type II receptor, its downstream actions are specifically mediated by the type I receptor, ALK5, and the Smad2 and Smad3 proteins.

In vitro development of islets from human adult pancreatic tissues

Transplantation of isolated islets from cadaver pancreas is a promising possibility for the optimal treatment of type 1 diabetes. The lack of islets is a major problem. Here we have investigated the possibility of generating islets in tissue culture of human pancreatic cells. We first reproduced a previously reported method of in vitro generation of endocrine cells from human adult pancreatic tissue. By tracing the bromodeoxyuridine-labeled cells in differentiated islet buds, we found that the pancreatic progenitor cells represented a subpopulation of cytokeratin 19 (CK19)-positive ductal cells. Serum-free medium and Matrigel overlay were essential for the endocrine differentiation. We then examined the involvement of preexisting islet cells in islet neogenesis. About 6-10% of endocrine cells dedifferentiated and acquired a transitional phenotype by coexpressing CK19. Significant cell proliferation was only observed in CK19-positive cells, but not in chromogranin A-positive endocrine cells. The in vitro-derived human islets were morphologically and functionally immature when compared with normal islets. Their insulin mRNA levels were only 4-5% of that found in fresh human islets, and glucose-stimulated insulin release was 3 times lower than that of control islets. Moreover, some immature endocrine cells coexpressed insulin and glucagon. After transplantation in nude mice, the in vitro-generated islets became mature with one type of hormone per endocrine cell. In addition, we also found that also in both fresh islet transplants many cells coexpressed endocrine markers and ductal marker CK19 as a sign of ductal to endocrine cell transition. Finally, we studied the effects of clinically used immunosuppressive drugs on precursor cell proliferation and differentiation. Mycophenolate mofetil (MMF) severely hampered duct-cell proliferation, and significantly reduced the total DNA content indicating its antiproliferative effect on the precursors. Tacrolimus mainly affected differentiated beta cells by decreasing the insulin content per DNA as well as the proportion of insulin-positive cells. Sirolimus and daclizumab did not show any individual or synergistic side effects suggesting that these drugs are amenable for use in clinical islet transplantation. In summary, we confirm the capacity of endocrine differentiation from progenitors present in the adult human pancreas. The plasticity of differentiated cell types of human pancreas may be a potential mechanism of human pancreas regeneration. Ductal cell differentiation into endocrine cells in transplanted islets may be an important factor in sustaining the long-term function of islet transplants. The immunosuppressive protocol is likely to be an important determinant of long-term clinical islet graft function. Moreover, these results provide new information on the mechanisms of pancreatic islet regeneration and provide the basis for the development of new strategies for the treatment of insulin deficient diabetes mellitus.

不同基因组组合的鱼类抗原基因剂量效应的研究(英文)

本文采用经典免疫遗传学方法研究了鲤鱼不同基因组组合的基因剂量与抗原表达的关系。分别从红鲤（RC）、红鲫（RA）、镜鲤（MC）和鲤鲫二倍体杂种（CA）采血样，制备红细胞悬液。常规方法免疫兔子，使效价高于64，制备抗RC、RA、MC、CA血清。Tab．1为2N、3N、4N鲤鱼来源的红细胞与四种抗血清的凝集实验结果，表明：亲缘关系越近，滴度越高;不过，不同倍性鲤鱼之间，差异不显著。Tab．2为2N、3N、4N鲤鱼来源的红细胞与四种抗血清吸附反应后，与各自靶细胞RC、RA、MC、CA的吸收实验结果，表明：抗血清被

应用RAPD技术对不同基因组组合生物型遗传多态性的研究(英文)

利用ＲＡＰＤ技术对不同基因组合的鱼类进行了基因组指纹图谱构建，在ＤＮＡ水平上对基因组成分进行了分析，探讨了其遗传多态性。ＲＡＰＤ结果发现，在２６个随机引物扩增的产物中，平均每个个体观察到约１４２个ＲＡＰＤ标记，单个引物获得的标记平均为５．４。其中４个引物扩增的图谱可将不同的生物型区分开：Ｓ－２６引物的扩增图谱（Ｆｉｇ．１）可将红鲫（ＲＡ）与其它组合区分开，还可将鲤鲫杂种一倍体（ＣＡ）与鲫鲤杂种三倍体（ＣＡＡ）和人工复合三倍体鲤（ＣＣＡ）区分开；Ｓ－８引物（Ｆｉｇ．２）可区分开红鲤（ＲＣ）和镜鲤（ＭＣ）；Ｓ