Hypothalamic-Pituitary-Gonadal(HPG)Axis and Transcriptional Regulatory Elements Regulate piwil2 Gene Expression During Gametogenesis and Gonadal Development in Japanese Flounder(Paralichthys olivaceus)

The P-element induced wimpy testis(Piwi)proteins,which are associated with PIWI-interacting RNAs(piRNAs),play important roles in meiosis,germ cell division,and germline maintenance.In this study,we identified and characterized the Paralichthys olivaceus piwil2 gene,a constituent factor of the piRNA pathways involved in the biogenesis of reproductive development.The biological analysis indicated that piwil2,which contains PAZ and PIWI domains,was highly conserved between teleosts and tetrapods.The piwil2 distribution profile in different tissues confirmed a sexually dimorphic expression pattern,with a higher expression level in testis.In situ hybridization demonstrated that piwil2 was expressed in the oogonia and oocytes of the ovaries as well as in the Sertoli cells and spermatocytes of the testes.Gene piwil2 showed a maternally inherited expression pattern during embryonic development,and was highly expressed during the early embryonic development.Different luciferase reporters were constructed to determine the transcriptional regulatory mechanisms of piwil2.The piwil2 core promoter region was located at−360 bp to−60 bp.Furthermore,some representative sex hormones,including human chorionic gonadotropin,17α-methyltestosterone,and estradiol-17βhad distinct regulatory effects on piwil2.In a summery,these results indicate that piwil2,regulated by sex hormones and transcriptional elements,has vital functions in the reproductive cycle and gonadal development.

Spindle-E is essential for gametogenesis in the silkworm, Bombyx mori

As a defense mechanism against transposable elements,the PIWI-interacting RNA(piRNA)pathway maintains genomic integrity and ensures proper gametogenesis in gonads.Numerous factors are orchestrated to ensure normal operation of the piRNA pathway.Spindle-E(Spn-E)gene was one of the first genes shown to participate in the piRNA pathway.In this study,we performed functional analysis of Spn-E in the model lepidopteran insect,Bombyx mori.Unlike the germline-specific expression pattern observed in Drosophila and mouse,BmSpn-E was ubiquitously expressed in all tissues tested,and it was highly expressed in gonads.Immunofluorescent staining showed that BmSpn-E was localized in both germ cells and somatic cells in ovary and was expressed in spermatocytes in testis.We used a binary transgenic CRISPR/Cas9 system to construct BmSpn-E mutants.Loss of BmSpn-E expression caused derepression of transposons in gonads.We also found that mutant gonads were much smaller than wild-type gonads and that the number of germ cells was considerably lower in mutant gonads.Quantitative real-time PCR analysis and TUNEL staining revealed that apoptosis was greatly enhanced in mutant gonads.Further,we found that the BmSpn-E mutation impacted gonadal development and gametogenesis at the early larval stage.In summary,our data provided the first evidence that BmSpn-E plays vital roles in gonadal development and gametogenesis in B.mori.

Somatostatin 4 regulates growth and modulates gametogenesis in zebrafish

Somatostatin(SST)plays important roles in growth and development.In teleost fishes six SST encoding genes(sst1 to sst6)have been identified although few studies have addressed their function.Here we aim to determine the function of the teleost specific sst4 in the zebrafish.A CRISPR/Cas9 sst4 zebrafish mutant with loss of function(sst4−/−)was produced which grew significantly faster and was heavier at the onset of gonadal maturation than the wild type(WT).Consistent with their faster growth,liver igf1,igf2a and igf2b expression was significantly upregulated in the sst4−/−fish compared to the WT.Histological examination of the ovaries and testis indicated that sst4−/−fish had slightly delayed testicular gametogenesis compared to the WT.Significantly lower expression of igf3,amh,insl3,hsd17b3,hsd11b2,hsd20b,cyp11b and cyp17 was consistently observed in the sst4−/−testis.In contrast,the ovaries had lower expression of igf1,igf2a and cyp19a1a but increased expression of igf2b and hsd20b.The gonadotrophin beta subunits(fshb and lhb)in the brain were downregulated indicating the brain-pituitary-gonadal axis was downregulated in the sst4−/−fish and suggesting that the steroid production is compromised in the maturing gonads.In addition,analysis of sst1 and sst3 mRNA levels in sst4−/−fish suggests a dosage compensation effect of sst1 in the brain and liver.Altogether,the results from the zebrafish sst4−/−line support the idea that sst4 is involved in the regulation of igf signalling,somatic growth and reproduction since steroidogenesis and gametogenesis at pubertal onset were compromised.

Regulation of Reproduction in Delayed Gametophyte of Saccharina japonica (Laminariales, Phaeophyceae): Effects of Light Intensity, Quality and Photoperiod

Saccharina japonica gametophytes can survive a long period under unfavorable environmental conditions,while they also delay in growth and/or reproduction.Although the reproduction in delayed gametophyte of S.japonica was known to be strongly influenced by light intensity,light quality,and photoperiod,no previous studies have evaluated their interactive effects on gametogenesis.To evaluate these effects,we used an orthogonal experiment to expose delayed gametophytes of S.japonica to different light intensities,light qualities,and photoperiods for 12 days.The results showed that changes in light intensity rather than light quality and photoperiod significantly affected the relative growth rates of the delayed gametophytes.Blue light had the greatest promotion on reproduction rate.The optimal light conditions in the early vegetative growth phase in gametogenesis induction for the delayed gametophytes were at 60–80μmol photons m^(−2) s^(−1) with daylength of 12 or 16 hours under white or blue light.When the delayed gametophytes were maintained in a constant light condition from delayed state to gametogenesis,the beneficial photoperiods for vegetative growth and reproductive rate were both 16L(16 hours of light):8D(8 hours of dark).However,when the delayed S.japonica gametophytes achieve the optimal growth state during the first 6 days and then they were cultured at different light conditions for the following 6 days,the reproduction rate increased as the daylength decreased and attained a peak value in group of 8L:16D photoperiod,indicating that photoperiod adjustment at the transition period is crucial in the gametogenesis induction process of delayed gametophyte of S.japonica.

RebL1 is required for macronuclear structure stability and gametogenesis in Tetrahymena thermophila

Histone modification and nucleosome assembly play important roles in chromatin-related processes.Histone chaperones form different complexes and coordinate histone transportation and assembly.Various histone chaperone complexes have been identified in different organisms.The ciliate protozoa(ciliates)have various chromatin structures and different nuclear morphology.However,histone chaperone components and functions of different subunits remain unclear in ciliates.Tet-rahymema thermophila contains a transcriptionally active macronucleus(MAC)and a transcriptionally inactive micronu-cleus(MIC)which exhibit multiple replication and various chromatin remodeling progresses during vegetative growth and sexual developmental stages.Here,we found histone chaperone RebL1 not only localized evenly in the transcriptionally active MAC but also dynamically changed in the MIC during vegetative growth and sexual developmental stages.REBL1 knockdown inhibited cellular proliferation.The macronuclear morphology became bigger in growing mutants.The abnormal macronuclear structure also occurred in the starvation stage.Furthermore,micronuclear meiosis was disturbed during sexual development,leading to a failure to generate new gametic nuclei.RebL1 potentially interacted with various factors involved in histone-modifying complexes and chromatin remodeling complexes in different developmental stages.REBL1 knockdown affected expression levels of the genes involved in chromatin organization and transcription.Taken together,RebL1 plays a vital role in maintaining macronuclear structure stability and gametogenesis in T.thermophila.

STUDY ON GAMETOPHYTE VEGETATIVE GROWTH OF UNDARIA PINNATIFIDA AND ITS APPLICATIONS

When cultured under certain environmental coriditions (25℃, light intensity 80 μmol/m2 ·s, LD12/12, in enriched seawater medium with 7 × 10-4mol/L NO3,-N, 1.56× 10-4mol/L, PO4-P and supplementsof other elements like Mn, Fe, I, etc.), mate and female gametophytes of U pinnatifida keptgrowing vegetatively and propagated fast at average daily fresh weight increase rate of about 20%. Theempirical formula Gm=Go. 3m was established to estimate the output of vegeative gametophytes. Vigorousvegetative gametophyte cells began to form reproductive structures (oogonium and spermatangium, whenthe tmperature was lower than 25℃ and other environmental factors wrre kept optimal. The sufficientsupply of gametophyte cells provided enough seeds for raising Undaria sporelings on prodiction scale.Controlled cross-breeding experiments using selcted male and female gametophyte clones which increasetheir cell number by mitosis instead of meiosis were also carried out in vitro. Juvenile sporophytes fromthe

Reproductive polyphenism and its advantages in aphids:Switching between sexual and asexual reproduction

Reproductive polyphenism,which allows one genotype to produce sexual and asexual morphs,is an extreme case of phenotypic plasticity and is commonly observed in aphids.Aphids are typical species that switch these reproductive modes,and the pathway orientation is triggered by the environmental conditions(mainly photoperiod and temperature).The typical arm ual life of aphids in eludes a successi on of parthenogenetic gen erations duri ng the spri ng and summer and a single sexual generation in autumn.In this review,we describe how the environmental cues orientate the reproductive mode of aphids from photoperiodic perception to endocrine regulation,and how juvenile hormones may act on the target cells(oocytes)to initiate the gametogenesis and embryogenesis in sexual and asexual reproduction.We also discuss the paradox of sex,especially the advantages of sexual reproduction in aphids.With the recent development of genomic resources in aphids,many potential genes involved in the reproductive polyphenism will enter the public's awareness.In particular,we describe a novel RNAi method in aphids,which may provide a molecular technique for determining the developmental fate and multiple reproductive strategies.

Expression Characteristics of Odf3 in Gonad Are Different Between Chlamys farreri and Vertebrates

Outer dense fibers are specific subcellular components of mammal sperm flagella, apart from the axoneme and its associated proteins. As one of the outer dense fibers components, ODF3 is essential for the formation of mammal sperm flagella. In the present study, we isolated the Odf3 gene, 1033 bp in length, from Zhikong scallop Chlamys farreri, which was named as Cf-Odf3. It was predicted that Cf-Odf3 encodes a protein of 240 amino acids which contains 5 discontinuous Pro-Gly-Pro repeats. The Cf-Odf3 transcripts were detected in both testis and ovary of C. farreri at various stages through q RT-PCR. The Cf-ODF3 protein synthesized by prokaryotes was purified using Ni^(2+)-NTA affinity chromatography and used to produce its polyclonal antibody. The antibody product was effective and specific as was examined by Western blotting. The Cf-Odf3 transcript and Cf-ODF3 protein were located in intragonadal somatic cells(ISCs), spermatogonia and spermatocytes of C. farreri testes, while no transcription of Cf-Odf3 and synthesis of Cf-ODF3 were detected in spermatids and spermatozoa. The location of Odf3 expression is different from that of vertebrates where ODF3 is synthesized specifically in spermatids and sperm flagella. Moreover, the Cf-Odf3 expression was also detected in ISCs, oogonia and oocytes of the scallop ovaries. Our finding revealed a different characteristic of Odf3 expression between scallop and vertebrates, which implied that Cf-Odf3 played a role in the gametogenesis of C. farreri.

Epigenetics in the etiology and management of infertility

BACKGROUND Epigenetic disruptions have been implicated in some cases of infertility and can serve as therapeutic targets.However,the involvement of epigenetics in infertility has not received adequate attention.AIM This study aimed to determine the epigenetic basis of infertility in order to enhance public knowledge.METHODS Relevant articles on the subject were collected from PubMed,RCA,Google Scholar,SpringerLink,and Scopus.The articles were pooled together and duplicates were removed using Endnote software.RESULTS Available information shows that epigenetic mechanisms,mainly DNA methylation,histone modification,and microRNA interference are necessary for normal gametogenesis and embryogenesis.As a result,epigenetic disruptions in genes that control gametogenesis and embryogenesis,such as DDX3X,ADH4,AZF,PLAG1,D1RAS3,CYGB,MEST,JMJD1A,KCNQ1,IGF2,H19,and MTHFR may result in infertility.Aberrant DNA methylation during genomic imprinting and parental epigenetic mark erasures,in particular,may affect the DNA epigenomes of sperm and oocytes,resulting in reproductive abnormalities.in the sperm may also cause reproductive abnormalities.Furthermore,overexpression or repression of certain microRNAs embedded in the ovary,testis,embryo,as well as granulosa cells and oocytes may impair reproduction.Male infertility is characterized by spermatogenesis failure,which includes oligozoospermia,asthenozoospermia,and teratozoospermia,while female infertility is characterized by polycystic ovary syndrome.Some epigenetic modifications can be reversed by deactivating the regulatory enzymes,implying that epigenetic reprogramming could help treat infertility in some cases.For some disorders,epigenetic drugs are available,but none have been formulated for infertility.CONCLUSION Some cases of infertility have an epigenetic etiology and can be treated by reversing the same epigenetic mechanism that caused it.As a result,medical practitioners are urged to come up with epigenetic treatments for infertility that have an epigenetic cause.

What we have learned from transcript profile analyses of male and female gametes in flowering plants

Double fertilization is one of the predominant features of sexual reproduction in flowering plants but, because of the physical inaccessibility of gametes, the essential molecular mechanisms in these processes are largely unknown. Based on the techniques for isolating highly purified gametes from several species and well-developed methods for manipulating RNA from limited quantities of gametes, genome-wide investigations of gamete transcription profiles were recently conducted in flowering plants. In this review, we survey the accumulated knowledge on gamete collection and purification, cDNA library construction, and transcript profile analysis to assess our understanding of the molecular mechanisms of gamete specialization and fertilization.

Development and organisation of gonadal steroidogenesis in bony fishes - A review

Gonadal steroidogenesis is pivotal to synchronize various reproductive stages including sexual development,growth and maturation.In all vertebrates including teleost,steroidogenesis is triggered by the mobilization of cholesterol by steroidogenic acute regulatory protein from outer to inner mitochondrial membrane.Thereafter,the entire process occurs in endoplasmic reticulum or mitochondria wherein various steroidogenic enzyme genes play a crucial role.The onset of steroidogenesis during sexual development in teleost is essentially regulated by differential expression of several transcription as well as steroidogenesis-related factors.More specifically,the role of dmrt,sox9,sox3,other sox forms,ad4bp/sf-1,wt-1,foxl2,ftz-f1,gata4,gsdf,Activator protein-1,fgfs and growth factors and steroidogenic enzymes such as cytochrome P450aromatase(cyp19a1),hydroxysteroid 3β-dehydrogenase(hsd3b),hydroxysteroid 17β-dehydrogenase(hsd17b)have been well characterised.Recently,the role of pax2,THO complex(thoc),pentraxin(ptx)and few signalling molecules like wnt4/5 regulating teleostean steroidogenesis has been reported.In females,cyp19a1 appears to be critical as it converts androgens to estrogens during ovarian differentiation which suggests that estradiol-17βis indispensable for the event.Unlike females,males do not depend on testosterone for testicular determination,yet has a major role along with 11-ketotestosterone in testicular development and growth when compared to early testicular differentiation.In males,onset of sex determining or testis-related genes seems most essential.Considering these,the regulation of steroidogenesis is virtually critical at later stages.In view of this,several steroidogenic motifs pertaining to transcriptional regulation have been analysed in teleost,yet far below than the reports on mammals.In this context,the regulatory influence on HHG axis plays a critical role in teleost.Further,multiple gonadotropin-releasing hormones and duality of gonadotropins play a differential role in gonadal function.As for steroidogenic pathway,the synthesis of sex steroids predominantly usesΔ4,however,Δ5 pathway was also evident.The next aspect is shift in steroidogenesis that usually occurs in the maturing follicles during final oocyte maturation,yet such a mechanism is not clear in males.The review highlights the interactions of steroidogenic enzyme gene regulation in terms of HHG axis and other important transcription factors that are involved in the regulatory pathways as well as the influence of environmental and dietary factors by comparing both sexes to present a holistic view on steroidogenesis onset and organisation during gametogenesis in teleost.

Environmental hypoxia:A threat to the gonadal development and reproduction in bony fishes

Vast stretches of open water bodies are gradually becoming hypoxic as a result of depletion of oxygen levels mainly due to various human anthropogenic activities.This problem of hypoxic stress on the fish population is likely to be exacerbated soon since the aquatic hypoxic environment is continuously spreading over vast areas worldwide.In recent years,various harmful effects of hypoxia to bony fishes have been reported,such as the restriction of energy-consuming metabolic processes,arrest of the growth of ovary and testes that are associated with endocrine disruption,loss of sperm and egg quality,inhibition of fertilization,hatching success,and also the reduction of larval survivability,thereby impairment of overall reproductive and developmental processes in fish.Disruption of the brain-pituitary-gonad axis,and certain enzymes related to steroidogenesis and vitellogenesis in fish have also been reported as the primary targets for an endocrine malfunction during hypoxia.Hypoxia-sensitive downregulation of key genes responsible for controlling sex hormones’synthesis has been documented in certain bony fishes.Further,continuous exposure to hypoxia was reported to induce early expression of pro-apoptotic/tumor suppressor p53 genes,thereby causing immense cell death in hypoxic embryos.However,the cellular responses to long-term hypoxia exposure and the degree of reproductive impairments in bony fishes are still not adequate to figure out the actual underlying mechanisms.The present review intends to highlight the current knowledge about the detrimental impact of chronic/acute hypoxia at different stages of fish reproduction and the associated underlying molecular mechanisms.

Evolutionary dynamics and conserved function of the Tudor domain-containing(TDRD)proteins in teleost fish

Tudor domain-containing(TDRD)proteins,the germline enriched protein family,play essential roles in the process of gametogenesis and genome stability through their interaction with the PIWI-interacting RNA(piRNA)pathway.Several studies have suggested the rapid evolution of the piRNA pathway in teleost lineages with striking reproductive diversity.However,there is still limited information about the function and evolution of Tdrd genes in teleost species.In this study,through genome wide screening,13 Tdrd family genes were identified in economically important aquaculture fish,including spotted sea bass(Lateolabrax maculatus),Asian sea bass(Lates calcarifer),and tongue sole(Cynoglossus semilaevis).With copy number,structure,phylogeny,and synteny analysis,duplication of Tdrd6 and Tdrd7,as well as loss of Stk31 and Tdrd10,were characterized in teleost lineages.Codon based molecular evolution analysis indicated faster evolution of teleost Tdrd genes than that in mammals,potentially associated with the accelerated evolution of the piRNA pathway in teleost lineages.The evolutionary diversity of Tdrd genes was also detected between different teleost lineages.RNA-seq analysis showed that most teleost Tdrd genes were dominantly expressed in gonads,particularly highly expressed in testis,such as Tdrd6,Tdrd7a,Tdrd9,Ecat8,and Tdrd15.The varied expression and evolutionary pattern between the duplicated Tdrd6 and Tdrd7 in teleosts may indicate their functional diversification.All these results suggest a conserved function of teleost Tdrd family in gametogenesis and the piRNA pathway,which could lay a foundation for the evolution of Tdrd genes and be helpful for further deciphering of Tdrd functions in teleosts.