Developmental dynamics of sex reprogramming by high incubation temperatures in a dragon lizard.

BACKGROUND: In some vertebrate species, gene-environment interactions can determine sex, driving bipotential gonads to differentiate into either ovaries or testes. In the central bearded dragon (Pogona vitticeps), the genetic influence of sex chromosomes (ZZ/ZW) can be overridden by high incubation temperatures, causing ZZ male to female sex reversal. Previous research showed ovotestes, a rare gonadal phenotype with traits of both sexes, develop during sex reversal, leading to the hypothesis that sex reversal relies on high temperature feminisation to outcompete the male genetic cue. To test this, we conducted temperature switching experiments at key developmental stages, and analysed the effect on gonadal phenotypes using histology and transcriptomics. RESULTS: We found sexual fate is more strongly influenced by the ZZ genotype than temperature. Any exposure to low temperatures (28 °C) caused testes differentiation, whereas sex reversal required longer exposure to high temperatures. We revealed ovotestes exist along a spectrum of femaleness to male-ness at the transcriptional level. We found inter-individual variation in gene expression changes following temperature switches, suggesting both genetic sensitivity to, and the timing and duration of the temperature cue influences sex reversal. CONCLUSIONS: These findings bring new insights to the mechanisms underlying sex reversal, improving our understanding of thermosensitive sex systems in vertebrates.

Dynamics of epigenetic modifiers and environmentally sensitive proteins in a reptile with temperature induced sex reversal†.

The mechanisms by which sex is determined, and how a sexual phenotype is stably maintained during adulthood, have been the focus of vigorous scientific inquiry. Resources common to the biomedical field (automated staining and imaging platforms) were leveraged to provide the first immunofluorescent data for a reptile species with temperature induced sex reversal. Two four-plex immunofluorescent panels were explored across three sex classes (sex reversed ZZf females, normal ZWf females, and normal ZZm males). One panel was stained for chromatin remodeling genes JARID2 and KDM6B, and methylation marks H3K27me3, and H3K4me3 (Jumonji Panel). The other CaRe panel stained for environmental response genes CIRBP and RelA, and H3K27me3 and H3K4me3. Our study characterized tissue specific expression and cellular localization patterns of these proteins and histone marks, providing new insights to the molecular characteristics of adult gonads in a dragon lizard Pogona vitticeps. The confirmation that mammalian antibodies cross react in P. vitticeps paves the way for experiments that can take advantage of this new immunohistochemical resource to gain a new understanding of the role of these proteins during embryonic development, and most importantly for P. vitticeps, the molecular underpinnings of sex reversal.

Patterns of structural change in gonads of the divine dwarfgoby Eviota epiphanes as they sexually transition.

This study documents changes in gonadal structure for the serial hermaphrodite (or bidirectional sex changer) divine dwarfgoby Eviota epiphanes (family Gobiidae) as individuals transition in both directions. To evaluate transitional gonad morphology, individuals actively producing the same gamete type (oocytes or sperm) were set up into pairs and euthanised over a period of 14 days to get a time series of morphological changes during gonad transformation. Results from this study show that rapid changes in the gonad take place at a structural level as individuals change their reproductive function and gamete production. Changing from oocyte production (o-phase) to sperm production (s-phase) starts with the breakdown of vitellogenic oocytes (i.e., atresia) followed by the appearance and proliferation of spermatogenic tissue which, in most cases, was not previously visible. Changing from sperm production to oocyte production included the cessation of sperm production, a reduction in size and number of seminiferous lobules and the maturation of previtellogenic oocytes already present in the gonads. Experimental fish changed from oocyte production to sperm production more readily than from sperm production to oocyte production. The hypothesis that shifts in sexual function among serially hermaphroditic fish species have a similar cost in either direction is not supported in E. epiphanes.

Changes in reproductive physiology of mangrove rivulus Kryptolebias marmoratus following exposure to environmentally relevant doses of ethinyl oestradiol.

Kryptolebias marmoratus exposed to 4 ng l(-1) of ethinyl oestradiol (EE2) for 30 days experienced significant changes in endogenous 17ß-oestradiol (E2) and 11-ketotestosterone (KT) and qualitative changes in gonad morphology. Both hermaphrodites and males showed a significant decrease in E2, whereas only males exhibited a significant decrease in KT. Exposure to EE2 resulted in a decrease in spermatid and spermatocyte density in males and an increase in the number of early stage oocytes in hermaphrodites.

Novel Targets for the Transcription Factors MEF2 in MA-10 Leydig Cells.

Testosterone production by Leydig cells is a tightly regulated process requiring synchronized expression of several steroidogenic genes by numerous transcription factors. Myocyte enhancer factor 2 (MEF2) are transcription factors recently identified in somatic cells of the male gonad. In other tissues, MEF2 factors are essential regulators of organogenesis and cell differentiation. So far in the testis, MEF2 factors were found to regulate Leydig cell steroidogenesis by controlling Nr4a1 and Star gene expression. To expand our understanding of the role of MEF2 in Leydig cells, we performed microarray analyses of MEF2-depleted MA-10 Leydig cells, and the results were analyzed using Partek and Ingenuity Pathway Analysis software. Several genes were differentially expressed in MEF2-depleted Leydig cells, and 16 were validated by quantitative RT-PCR. A large number of these genes are known to be involved in fertility, gonad morphology, and steroidogenesis. These include Ahr, Bmal1, Cyp1b1, Hsd3b1, Hsd17b7, Map2k1, Nr0b2, Pde8a, Por, Smad4, Star, and Tsc22d3, which were all downregulated in the absence of MEF2. In silico analyses revealed the presence of MEF2-binding sites within the first 2 kb upstream of the transcription start site of the Por, Bmal1, and Nr0b2 promoters, suggesting direct regulation by MEF2. Using transient transfections in MA-10 Leydig cells, small interfering RNA knockdown, and a MEF2-Engrailed dominant negative, we found that MEF2 activates the Por, Bmal1, and Nr0b2 promoters and that this requires an intact MEF2 element. Our results identify novel target genes for MEF2 and define MEF2 as an important regulator of Leydig cell function and male reproduction.

HES-Mediated Repression of Pten in Caenorhabditis elegans.

The hairy/enhancer-of-split (HES) group of transcription factors controls embryonic development, often by acting downstream of the Notch signaling pathway; however, little is known about postembryonic roles of these proteins. In Caenorhabditis elegans, the six proteins that make up the REF-1 family are considered to be HES orthologs that act in both Notch-dependent and Notch-independent pathways to regulate embryonic events. To further our understanding of how the REF-1 family works to coordinate postembryonic cellular events, we performed a functional characterization of the REF-1 family member, HLH-25. We show that, after embryogenesis, hlh-25 expression persists throughout every developmental stage, including dauer, into adulthood. Like animals that carry loss-of-function alleles in genes required for normal cell-cycle progression, the phenotypes of hlh-25 animals include reduced brood size, unfertilized oocytes, and abnormal gonad morphology. Using gene expression microarray, we show that the HLH-25 transcriptional network correlates with the phenotypes of hlh-25 animals and that the C. elegans Pten ortholog, daf-18, is one major hub in the network. Finally, we show that HLH-25 regulates C. elegans lifespan and dauer recovery, which correlates with a role in the transcriptional repression of daf-18 activity. Collectively, these data provide the first genetic evidence that HLH-25 may be a functional ortholog of mammalian HES1, which represses PTEN activity in mice and human cells.

Morphology of gonads, maturity and spawning season of Loricariichthys spixii (Siluriformes, Loricariidae) in a Subtropical Reservoir

The gonad morphology and spawning season of Loricariichthys spixii in Lajes reservoir were described based on 175 males and 613 females. Cells from the spermatogenic lineage were divided in four phases: spermatogonia (primary and secondary), spermatocytes (primary and secondary), spermatids and spermatozoa, and the cells from the ovocitarian lineage were divided in four phases: primary oocytes (O1), previtellogenic oocytes (O2), cortical vesicle oocytes (O3) and yolk globules or vitellogenic (O4). Five gonadal stages were described for the males/females according to oocytes and spermatogenic lineage cells distribution: resting (1); initial maturation (2a); advanced maturation (2b); partially spent/spawned (4a); totally spent/spawned (4b). Spawning was iteroparous, occurring throughout the year. Resting/recover occurred in July/August for the females coinciding with lower temperature and rainfall and decreasing water level. The wide spawning period was the part of the strategy developed to withstand environmental pressure and to get success in this oligotrophic and poorly structured environment.

A morfologia das gônadas e a época de desova de Loricariichthys spixii, no reservatório de Lajes foi descrita baseada em 175 machos e 613 fêmeas, coletados de Janeiro-1996 a Dezembro-1997. Células de linhagem espermatogênica foram divididas em 4 fases: espermatogônias (primárias e secundárias), espermatócitos (primários e secundários), espermátides e espermatozóides, enquanto as células de linhagem ovocitária também foram divididas em 4 fases, baseadas em características do núcleo, ooplasma e folículos dos ovócitos: ovócitos primários (O1), ovócitos previtelogênicos (O2), ovócitos de vesícula cortical (O3) e glóbulos de vitelo ou vitelogênicos (O4). Cinco estádios gonadais foram descritos para machos/fêmeas de acordo com a distribuição das células de linhagem ovocitária / espermatogênicas: repouso (1); maturação inicial (2a); maturação avançada (2b); parcialmente esvaziado/desovado (4a); totalmente esvaziado/desovado (4b). Desova é parcelada, ocorrendo através do ano. O repouso/recuperação ocorreu em Julho/Agosto para fêmeas coincidindo com menores temperaturas e pluviosidades, e diminuições do níveis da água. O amplo período reprodutivo é parte da estratégia desenvolvida por esta espécie para suportar as pressões ambientais (bióticas e abióticas) e obter sucesso neste reservatório oligotrófico e pobremente estruturado.

Intersex females in the red claw crayfish, Cherax quadricarinatus (Decapoda: Parastacidae)

Cherax quadricarinatus is a large freshwater crayfish species Parastacidae) native of north-west Queensland and the Northern Territory of Australia. The species typically exhibits a gonochoristic sexual system, although in cultured populations various types of intersex individuals have been described as functional males. In the present study, the macroscopic morphology and the gonadal histology of one type of intersex are described and discussed. All intersexes having both pairs of genital openings (female and male openings) and lacking both appendix masculinae and red patches were functional females with normal ovaries and oviducts. From a histological point of view, they did not differ from normal females having previtellogenic and/or vitellogenic ovaries according to size.

Cherax quadricarinatus, es un astácido dulceacuícola de gran tamaño de la (familia Parastacidae) originario delnoroeste de Queensland y del norte de Australia. Presenta un sistema sexual gonocórico, aunque en poblaciones decultivo se han descrito varios tipos de individuos intersexos como machos funcionales. En el presente estudio se describe y discute la morfología macroscópica y la histología gonadal de un tipo de intersexos. Todos los intersexos que presentan ambos pares de aberturas genitales (femeninas y masculinas) y carecen de ambos apéndices masculinos y de la mancha roja, fueron hembras funcionales con ovarios y oviductos normales. Desde el punto de vista histológico no difieren de las hembras normales, presentando ovarios previtelogénicos y/o vitelogénicos de acuerdo a su tamaño.