Sex-Based Variation of Gene Expression in the Gonads and Fins of Russian Sturgeon (<i>Acipenser gueldenstaedtii</i>)

Russian sturgeon (Acipenser gueldenstaedtii) is a primitive freshwater fish and a source of black caviar. The genes involved in sexual determination and differentiation are still unknown and there are no molecular markers for sex identification in this species. Studying the variation of the sex-based differences in genomic sequences and in gene expression in the sturgeon may lead to markers of sex in early stages of development and advances in aquaculture, as well as provide novel insights about the evolution of reproduction, sex determination, and sexual differentiation mechanisms in vertebrates. Previous studies by our and other groups have identified differentially expressed genes in the gonads of adult female and male sturgeon. The current study aimed to test whether these genes were also differentially expressed in non-gonadal tissue, namely fins. We measured by qRT-PCR the mRNA levels of 29 known and novel sex-related genes in the gonads and fins of males (4 years old) and females (7 years old;sexual maturation is earlier in males than in females). Six genes (ATP6, IGFRM, LIA1A, S1A, NPL1A, GAPDH and SOX9) showed higher expression in female fins. However, only ATP6 mRNA levels differed in fins of males and females of the same age (4 years old). These findings underscore the impracticality of sex identification based on gene expression in non-gonadal tissue and the need for genetic sex markers in the Russian sturgeon.

Localization of nitric oxide synthase in the developing gonads of amphioxus Branchiostoma belcheri tsingtauense

The localization of nitric oxide synthases （NOS） is dealed with in the developing gonads of amphioxus Branchiostoma belcheri tsingtauense. It was found by NADPH-diaphorase staining that （1） NOS activity was present in the nuclear membranes of germinal vesicles during the entire period ofoocyte development; （2） NOS was localized in both the nuclear membranes and the perinuclear region of cytoplasm in the vitellogenetic oocytes; （3） NOS was relocated in the cortical layer in the mature egg; （4） NOS activity was present in spermatocytes, but not in the spermatogonia in the middle of October; （5） NOS was detected in both spermatozoa and spermatids as well as spermatocytes during the breeding season. This is the first report on the distribution pattern of NOS in the developing gonads in protochordates. These results suggest a role for NOS in the functioning of the nuclear membranes and yolk synthesis during oogenesis and in cell division and differentiation during spermatogenesis.

Identification of global alternative splicing and sex-specific splicing via comparative transcriptome analysis of gonads of Chinese tongue sole(Cynoglossus semilaevis)

Chinese tongue sole(Cynoglossus semilaevis)is an economically important marine fish species with a ZZ/ZW sex determination mechanism,which can be influenced by temperature.Alternative splicing(AS)is an important mechanism regulating the expression of genes related to sex determination and gonadal differentiation,but has rarely been reported in fish.In this study,to explore the molecular regulatory mechanisms of sex determination and gonadal differentiation,we combined isoform and RNA sequencing(Iso-Seq and RNA-Seq)to perform transcriptome profiling of male and female gonads in C.semilaevis.In total,81883 and 32341 full-length transcripts were obtained in males and females,respectively.A total of 8279 AS genes were identified,including 2639 genes showing differential AS(DAS)between males and females.Many intersecting DAS genes and differentially expressed genes(DEGs)were enriched in the meiotic cell cycle pathway,and genes related to gonadal differentiation,such as esrrb and wt1a,were found to have sex-specific isoforms.Thus,this study revealed AS events in the gonadal transcriptomes of male and female C.semilaevis,described the characteristics of active transcription in the testes,and identified candidate genes for studying the regulatory mechanisms of AS during gonadal differentiation.

PATHOLOGICAL CHARACTERISTICS OF GONADS IN NINE PATIENTS WITH TRUE HERMAPHRODITISM

The gonadal histopathology, and its correlation with the clinical features has been investigated in 9 true hermaphroditism patients, aged 5-21 yr. Seven of 9 patients had been raised as females, of which the chromosomal karyotype was 46XX in 5 cases, 46XX/46XY and 46XX/47XXY in 2 cases. Two of 9 pa- tients were raised as males, the chromosomal karyotypes being 46XX and 46XX/46XY. All 9 patients had testicular tissue excised, and biopsies of the conserved ovarian tissue were performed. Ovotestis was the most common form of the abnormal gonads; two of 9 patients had bilateral ovotestes, seven had unilateral ovotestes (5 in right side, 2 in left side). In seven patients with a unilateral ovotestis, 6 had a contralateral ovary and one had a contralateral testis. Microscopically, the ovarian tissue of 11 ovotestes, including 6 biopsies from contealateral ovaris, were normal, with many primordal follicles and a few growing follicles. In two of the patients, aged over 15 years, evidence of ovulation was observed. In comparison, the testicu- lar tissue of the ovotestis and the one left inguinal testis was histologically abnormal, with immature semi- niferous tubules, most of which filled with Sertoli cells only. Three of 9 patients married after surgical treatment. Two of these subsequently conceived and delivered of normal infants by cesarean section.

Histological Observation of Gonads of Male Elk (Elaphurus davidianus)

Elk (Elaphurus davidianus) is a rare species native to China, and much less efforts have been dedicated to gonadal histology of male elk.The microscopic morphology of gonadal tissue of elk was observed by paraffin method. The results showed that the tunica albuginea testis was com-posed of collagenous fibers and elastic fibers, with the thickness of about 1 054 μm, and a large number of blood vessels were distributed in tunica albuginea;the seminiferous tubule was about 203 μm in diameter, with uneven thickness of tubule wall, which was consisted of sustentacular cells and spermatogenic cells;there were interstitial cells and blood vessels between seminiferous tubules. The epididymis contained more than 20 effer-ent ducts and a long curly epididymal duct;the capsule thickness at epididymis head was about 53 μm, and that at epididymis body and tail was400-1 000 μm;the epithelium of efferent duct was pseudostratified columnar epithelial cells;the wall epithelium of epididymal duct was composed of tall columnar cells at proximal lumina and stroma cells at distal lumina;the epithelial basement membrane of efferent duct and epididymal duct contained circular smooth muscle. The wall of spermatic duct was divided into mucosae, tunica muscularis and tunica adventitia. The tunica muscu-laris was well developed, which was smooth muscle fiber.

Odor-Active Volatile Compounds from the Gonads of the Sea Urchin <i>Mesocentrotus nudus</i>in the Wild in Miyagi Prefecture, Tohoku, Japan

Background and Aims: Mesocentrotus nudus is commercially harvested from shallow rocky bottoms in northern Japan. The gonads are targeted as an edible product. The objective of this study was to identify odor-active volatile organic compounds (VOCs) from the gonads of adult M. nudus collected at a fishing ground in Miyagi Prefecture, Tohoku, Japan. Methods and Results: Gas chromatography-mass spectrometry analysis and gas chromatography-sniffing techniques identified 42 compounds categorized as alcohols, aldehydes, aromatic hydrocarbons, esters, halomethanes, hydrocarbons, ketones, sulfur-containing compounds, and nitrogen-containing compounds. GC-sniffing analysis characterized four compounds with preferable odors of sea urchin gonads;limonene, propyl acetate, acetone, dibromochloromethane. On the other hand, the analysis characterized three compounds with unpreferable odors;methyl mercaptane, dimethyl sulfide, and s-methyl thioacetate. Several VOCs from the gonads were derived from seaweeds, terrestrial plants, and fish flesh as food because M. nudus is omnivorous. Conclusion: This is the first study to identify VOCs from edible sea urchin gonads in the wild in Japan. These VOC data comprise a typical standard in order to evaluate a higher quality of sea urchin gonads.

Germ cell development in teleost gonads

Among vertebrate species,fishes are considered to be the most developmentally diverse taxa exhibiting remarkable gonadal plasticity with capacity of sex reversal.Sex determination in teleost fishes is governed via a complex regulatory network involving multiple genetic,environmental(temperature,population density,pH),social interaction/stress factors.Piscine germ-line originates as Primordial germ cells(PGC)from cleavage blastomeres/endoderm and gets specified by maternal inheritance of Vasa,N anos,Tdrds,Dnd,Dazl etc and subsequently migrates to reach the embryonic gonadal primordium.Post-hatching,mitotically active germ cells(Gc)continue to proliferate via two distinct pathways-firstly,stem cell-like mode followed by meiosis-committed cystic division.In Japanese rice fish medaka,Oryzias latipes sex gets determined on the chromosomal basis,where XY becomes a male because of testicular Dmy transcription and XX develops as female owing to Gc restricted Foxl 3 expression.Sex specific germ-line stem cell systems also have been found in adult O.latipes.Intriguingly in teleost fishes,the developmental fate of transplanted Gc do not depend on the sex of the donor rather solely relies upon the sex of the recipient.Furthermore,sexually mature piscine gonads remain developmentally plastic showing extensive responsiveness towards sex steroids.The critical regulation of gonadal differentiation is directed by female specific 17β-estradiol(E2)and bio-active male androgen 11-keto-testosretone(11-kT).Various transcription factors(TFs)e.g.-Dmrt1,DAX1,Ad4BP/SF-1,Foxl 2 regulate the bio-conversion rate of testosterone(T)to either E2 or 11-kT.Similarly,multiple endocrine factors(EFs)e.g.-LH,FSH,E2,11-kT and growth factors(GFs)e.g-AMH,GSDF,IGF-1/3,GDF-9/BMP-15 critically induce Gc differentiation to regulate the gametogenic output.In this article we have precisely discussed the endocrine regulation of Gc development during gonadal maturation of teleosts.

Expression,localization,and a regulated target gene(ccnd1)of miR-202-5p in the Japanese flounder gonads

MiR-202-5p is identified as the maker microRNA(miRNA)of germplasm in fish.However,the role of miR-202-5p in gonadal development is still unclear.Here we investigated the expression patterns of miR-202-5p in Japanese flounder(Paralichthys olivaceus).Real-time PCR displayed that miR-202-5p was specifically expressed in bisexual gonads,and its transcription in the testis was relatively abundant than that in the ovary.Moreover,it was gradually increased in testis from stage II to stage IV.Fluorescent in situ hybridization(FISH)illustrated that miR-202-5p was co-localized with vasa(a marker gene of germ cells)in testis,and it was predominately expressed in spermatogonia and spermatocytes,implying that miR-202-5p might participate in spermatogenesis.Considering the important role of retinoic acid(RA)on regulating meiosis during spermatogenesis,we treated Japanese flounders by injecting all-trans retinoic acid(ATRA).MiR-202-5p in testis was shown to be up-regulated,and ccnd1,a key cell cycle regulator,was found to be down-regulated after ATRA treatment.Furthermore,ccnd1 was demonstrated to be a direct regulatory target of miR-202-5p using Dual-luciferase assay.In the primary testis cell from Japanese flounder,we further confirmed that ccnd1 was down-regulated by miR-202-5p.The results suggest that miR-202-5p exerts a crucial role in gonadal development by inhibiting ccnd1 in Japanese flounder.

Textile dyeing wastewater negatively influences the hematological profile and reproductive health of male Swiss albino mice

Objective:To determine the effects of textile dyeing industrial wastewater on the hematological parameters and reproductive health including histoarchitecture of male gonad(testes)of mice.Methods:Twenty-four Swiss albino mice at 4-weeks old were divided into four groups(n=6 per group).Mice of group 1 supplied with normal drinking water were served as the control group.Mice of group 2,3 and 4 were supplied normal drinking water mixed with textile dyeing wastewater at 5%,10% and 20% concentration,respectively.After completing 24 weeks of treatment,different hematological profile,weight of testes,gonadosomatic index(GSI),sperm concentration and morphology were measured.Moreover,histopathological changes in testes were examined.Results:Hematocrit value and hemoglobin concentrations were decreased in all groups of wastewater-treated mice compared to the control group.Likewise,weight of testes,GSI and sperm concentration were decreased significantly in wastewater-treated mice in comparison to the control group.The percentage of morphologically healthy epididymal sperm was significantly reduced in wastewater-treated mice.Histopathological examination revealed degenerative changes in seminiferous tubules,a smaller number of spermatogenic cells,elongation of seminiferous tubules and degenerative changes of seminiferous tubules in wastewater-treated mice.Conclusions:Textile dyeing wastewater has harmful effects on hematological profile and reproductive health of male mice.

Engineering of ovarian tissue for ovarian dysfunctions:A review

This review explores tissue engineering as a potential solution for reproductive health issues in women caused by genetic or acquired diseases,such as premature ovarian failure or oophorectomy.The loss of ovarian function can lead to infertility,osteoporosis,and cardiovascular disease.Hormone replacement therapy is a common treatment,but it has limitations and risks.The review focuses on two main approaches in tissue engineering:scaffold-based(3D printing,electrospinning,decellularization)and scaffold-free(stem cell transplantation,organoid cultivation).Both approaches show promise in preclinical studies for creating functional ovarian tissue.Challenges include vascularization,innervation,long-term function,and safety.Despite these challenges,tissue engineering offers a potential avenue for restoring fertility and hormone balance in women with ovarian dysfunction.

Immunohistochemical localization of gonadotropin-releasing hormone receptors (GnRHR) in the nervous system,Hatschek's pit and gonads of amphioxus,Branchiostoma belcheri

Using gonadotropin-releasing hormone (GnRH) anti-idiotypic antibodies and APA immunohistochemical method, the immunoreactivity of GnRHR in the nervous system, Hatschek’s pit and gonads of amphioxus has been located. It is found for the first time that the immunoreactivity of GnRHR exists in the nerve cells and fibers in the amphioxus’s brain and nerve tube and the epithelial cells of Hatschek’s pit at the different stages of gonadal development. At the same time, it is also found that GnRHR also exists in the ovary and testis of different developed stages. These findings provide morphological new proof for the informative transfer and regulation between brain and Hatschek’s pit mediation by GnRHR, and for the understanding of the mechanism of action on the reproductive endocrine control axis among brain-Hatschek’s pit-gonads.

Fatty acids in female’s gonads of the Red Sea fish Rhabdosargus sarba during the spawning season

Objective:To determine the fatty acids profiles in female fish,Rhabdosargus sarba(R.sarba)from the Red Sea during the spawning season.Methods:Monthly individual R.sarba were obtained from Bangalah market in Jeddah,Red Sea and transported to the laboratory in ice aquarium.The total length,standard length and weight were measured,fishes were dissected.Ovaries were removed,weighed and 10 mL of concentrated hydrochloric acid were added to 10 g of the ovary in a conical flask and immersed in boiling water until the sample was dissolved and the fat was seen to collect on the surface.The conical was cooled and the fat was extracted by shaking with 30 mL of diethyl ether.The extract was bowled after allowing the layers to separate into a weighed flask.The extraction was repeated three times more and distilled off the solvent then the fat dried at 100℃,cooled and weighed.Then 50 mg of lipid was put in a tube,5 mL of methanolic sulphuric acid was added and 2 mL of benzene,the tube well closed and placed in water bath at 90℃for an hour and a half.After cooling,8 mL water and 5 mL petroleum were added and shaked strongly and the ethereal layer was separated in a dry tube,evaporated to dryness.The fatty acid methyl esters were analyzed by using a Hewlett Packard(HP 6890)chromatography,a split/splitless injector and flame ionization detector.Results:In female R.sarba,a total of 29 fatty acids were detected in ovaries throughout the spawning season.The main fatty acid group in total lipid was saturated fatty acid(SFA,28.9%),followed by 23.5%of polyunsaturated fatty acids(PUFA)and 12.9%of monounsaturated fatty acids(MUFA).The dominant SFA were palmitic and stearic,the major MUFA were palmitoleic and oleic,and the major PUFA were C18:2 and C22:2.During spawning stages,there were no significant differences in total SFA,MUFA and PUFA.The highest value of SFA was in late spawning(36.78%).However,the highest value of MUFA and PUFA was in spawning(16.70%and 24.96%respectively).During spawning season there were significant differences in total SFA between March(late spawning stage)and December(nearly ripe stage),P<0.05.Conclusions:From the present study it has been shown that 29 fatty acids were detected in ovaries of R.sarba throughout the spawning season.The main fatty acid group in total lipid was SFA followed by PUFA and MUFA.The dominant SFA were palmitic and stearic,the major MUFA were palmitoleic and oleic,and the major PUFA were C18:2 and C22:2.The highest value of SFA was in late spawning.However,the highest value of MUFA and PUFA was in spawning.

Characterization and expression analysis of sox3 in medaka gonads

Numerous studies have showed that sox3 is involved in neurogenesis and sex differentiation in vertebrates.However,the accurate expression pattern is still unclear in fish.In this study,medaka sox3 was isolated and its expression patterns were compared with germ cell gene vasa in adult gonads.By reverse transcription-polymerase chain reaction(RT-PCR)analysis,sox3 RNA expression was detected in the brain,eyes and gonads.By chromogenic and fluorescent in situ hybridization,sox3 was present in the oogonia and early stages of oocytes as well as granulosa cells and theca cells in the later stages;in the testis,sox3 was occurred in the Sertoli cells and sperm with a specific signal in the location of chromatid body of sperm;in the embryos,sox3 was expressed in the central nervous.These results suggest that medaka sox3 gene was occurred in somatic and germ cells in adult gonads of both sexes,involved in the process of spermatogenesis,as well as the development of the nervous system.This study provides a precise expression pattern of sox3 and suggests that sox3 may be involved in gonadal and nervous system development of fish.

Sex-biased autophagy as a potential mechanism mediating sex differences in ischemic stroke outcome

Stroke is the second leading cause of death and a major cause of disability worldwide,and biological sex is an important determining factor in stroke incidence and pathology.From childhood through adulthood,men have a higher incidence of stroke compared with women.Abundant research has confirmed the beneficial effects of estrogen in experimental ischemic stroke but genetic factors such as the X-chromosome complement can also play an important role in determining sex differences in stroke.Autophagy is a self-degrading cellular process orchestrated by multiple core proteins,which leads to the engulfment of cytoplasmic material and degradation of cargo after autophagy vesicles fuse with lysosomes or endosomes.The levels and the activity of components of these signaling pathways and of autophagy-related proteins can be altered during ischemic insults.Ischemic stroke activates autophagy,however,whether inhibiting autophagy after stroke is beneficial in the brain is still under a debate.Autophagy is a potential mechanism that may contribute to differences in stroke progression between the sexes.Furthermore,the effects of manipulating autophagy may also differ between the sexes.Mechanisms that regulate autophagy in a sex-dependent manner in ischemic stroke remain unexplored.In this review,we summarize clinical and pre-clinical evidence for sex differences in stroke.We briefly introduce the autophagy process and summarize the effects of gonadal hormones in autophagy in the brain and discuss X-linked genes that could potentially regulate brain autophagy.Finally,we review pre-clinical studies that address the mechanisms that could mediate sex differences in brain autophagy after stroke.

Gonad traits and age structure of the sea urchin Heliocidaris crassispina in central range:a comparison with rangeextenders

The sea urchin Heliocidaris crassispina has northern expanded in geographic range to Toga Bay,Oga Peninsula in the Sea of Japan due to the global warming.To demonstrate the gonad traits and age structure of H.crassispina in the central range and compare the difference with those in northern extended range,we collected 100 H.crassispina individuals from a barren off Shitsumi,Fukui(central range),in August 2018.Their growth,gonad traits(size,development and color),and sex ratio were investigated and compared with those in Toga Bay,in August 2014(extended range).The successive 2012–2016 year classes of H.crassispina indicated successive juvenile recruitment in the central range,as found in the extended range.The individuals at three years old in central range were obviously larger than those in the extended range,showing large test diameter and body weight,possibly due to the higher temperature in central range than that in northern extended range.In addition,a balanced sex ratio in the central range was found,in contrast to the female-skewed sex ratio found in the extended range.Gonadal development was sex-and site-dependent.The testicular development in the central range was not only delayed in comparison to ovarian development,but also less delayed than those in the extended range,which possibly attribute to the higher water temperature in the central range.A significantly lower male gonad index and L*(lightness)value was recorded in central ranges compared to that in extended range,possibly due to the releasement of sperm of H.crassispina in central ranges.

Poultry genetic heritage cryopreservation and reconstruction:advancement and future challenges

Poultry genetics resources,including commercial selected lines,indigenous breeds,and experimental lines,are now being irreversibly lost at an alarming rate due to multiple reasons,which further threats the future livelihood and academic purpose.Collections of germplasm may reduce the risk of catastrophic loss of genetic diversity by guaranteeing that a pool of genetic variability is available to ensure the reintroduction and replenishment of the genetic stocks.The setting up of biobanks for poultry is challenging because the high sensitiveness of spermatozoa to freezing–thawing process,inability to cryopreserve the egg or embryo,coupled with the females being heterogametic sex.The progress in cryobiology and biotechnologies have made possible the extension of the range of germplasm for poultry species available in cryobanks,including semen,primordial germ cells,somatic cells and gonads.In this review,we introduce the state-of-the-art technologies for avian genetic resource conservation and breed reconstruction,and discuss the potential challenges for future study and further extending of these technologies to ongoing and future conservation efforts.

Single-cell RNA sequencing reveals gonadal dynamic changes during sex differentiation in hermaphroditic protogynous orange-spotted grouper(Epinephelus coioides)

DEAR EDITOR,Sex differentiation is a complex process that requires many factors to regulate gonadal proliferation, differentiation,development, and organization. In teleosts, the molecular mechanisms of sex differentiation are diverse and unclear.

The m6A methylation regulates gonadal sex differentiation in chicken embryo

Background:As a ubiquitous reversible epigenetic RNA modification,N6-methyladenosine(m6A)plays crucial regulatory roles in multiple biological pathways.However,its functional mechanisms in sex determination and differentiation during gonadal development of chicken embryos are not clear.Therefore,we established a transcriptome-wide m6A map in the female and male chicken left gonads of embryonic day 7(E7)by methylated RNA immunoprecipitation sequencing(MeRIP-seq)to offer insight into the landscape of m6A methylation and investigate the post-transcriptional modification underlying gonadal differentiation.Results:The chicken embryonic gonadal transcriptome was extensively methylated.We found 15,191 and 16,111 m6A peaks in the female and male left gonads,respectively,which were mainly enriched in the coding sequence(CDS)and stop codon.Among these m6A peaks,we identified that 1013 and 751 were hypermethylated in females and males,respectively.These differential peaks covered 281 and 327 genes,such as BMP2,SMAD2,SOX9 and CYP19A1,which were primarily associated with development,morphogenesis and sex differentiation by functional enrichment.Further analysis revealed that the m6A methylation level was positively correlated with gene expression abundance.Furthermore,we found that YTHDC2 could regulate the expression of sex-related genes,especially HEMGN and SOX9,in male mesonephros/gonad mingle cells,which was verified by in vitro experiments,suggesting a regulatory role of m6A methylation in chicken gonad differentiation.Conclusions:This work provided a comprehensive m6A methylation profile of chicken embryonic gonads and revealed YTHDC2 as a key regulator responsible for sex differentiation.Our results contribute to a better understanding of epigenetic factors involved in chicken sex determination and differentiation and to promoting the future development of sex manipulation in poultry industry.

Characterization of Bacterial Community Associated with Four Organs of the Yesso Scallop (Patinopecten yessoensis) by High-Throughput Sequencing

We used Illumina high-throughput sequencing of PCR-amplified V3-V4 16 S rRNA gene regions to characterize bacterial communities associated with the adductor muscles, gills, gonads and intestines of the Yesso scallop(Patinopecten yessoensis) from waters around Zhangzidao, Dalian, China. Overall, 421,276 optimized reads were classified as 25 described bacterial phyla and 308 genera. Firmicutes, Proteobacteria, Tenericutes, Bacteroidetes, Chlamydiae and Spirochaetae accounted for > 97% of the total reads in the four organs. The bacterial 16 S rDNA sequences assigned to Firmicutes and Proteobacteria were abundant in the adductor muscles, gills and gonads; while reads from Tenericutes were dominant in the intestines, followed by those from Firmicutes, Chlamydiae, Proteobacteria and Bacteroidetes. At the genus level, the dominant genera in the adductor muscles, gills and gonads appeared to be Bacillus, Enterococcus and Lactococcus, whereas Mycoplasma was dominant in the intestines. The relative abundances of Bacillus, Enterococcus, Lactococcus, Alkaliphilus, Raoultella, Paenibacillus and Oceanobacillus were significantly lower in the intestine than in the other three organs. Cluster analysis and principal coordinates analysis of the operational taxonomy units profile revealed significant differences in the bacterial community structure between the intestine and the other three organs. Taken together, these results suggest that scallops have intestine-specific bacterial communities and the adductor muscles, gills and gonads harbor similar communities. The difference in the bacterial community between organs may relate to unique habitats, surroundings, diet and their respective physiological functions.

Seasonal variations in gonad morphology and hypothalamic GnRH-I and GnIH in Eurasian Tree Sparrow,a multi-brooded passerine

The hypothalamic-pituitary-gonadal(HPG)axis ubiquitously regulates seasonal reproduction,following the rhythmicity of a suite of environmental cues.Birds display prominent seasonal variations in gonad size regulated by two hypothalamic peptides,gonadotropin-releasing hormone-1(GnRH-I)and gonadotropin-inhibiting hormone(GnIH).How multi-brooded avian species adjust GnRH-I and GnIH to regulate gonadal morphology seasonally remains largely unknown.Here,we studied the variations in the hypothalamic immunoreactivity(ir)of GnRH-I and GnIH,gonadal proliferation,and apoptosis in a typical multi-brooded species,the Eurasian Tree Sparrow(Passer montanus),across the pre-breeding(PB),first breeding(FB),second breeding(SB),pre-basic molt(PM),and wintering stages(WS).Our results showed that both sexes had higher preoptic area(POA)-GnRH-I-ir but lower paraventricular nucleus(PVN)-GnIH-ir neurons during the breeding stages(FB and SB)relative to other life-history stages,with no significant differences between two broods.The testes and ovaries were significantly larger during the breeding stages.Testicular volume increased during the two broods due to anincreased diameter of seminiferous tubules.Furthermore,there were more testicular apoptotic cells in PB and WS stages than in breeding stages,and in PB stage than in PM stage.Males had higher POA-GnRH-I expression than females during the breeding stages,but both sexes had comparable PVN-GnIH expression throughout the annual cycle.Both sexes of the sparrows may undergo a similar pattern of life-history stage-dependent variation in the hypothalamic GnRH-I,GnIH,and gonadal morphology,except that during breeding stages,males may display higher expression of POA-GnRH-I relative to females.The higher expression of POA-GnRH-I-ir in breeding male sparrows may be critical for male-dependent parental care.