A Deep Learning Model for Accurate Diagnosis of Infection Using Antibody Repertoires.

The adaptive immune receptor repertoire consists of the entire set of an individual's BCRs and TCRs and is believed to contain a record of prior immune responses and the potential for future immunity. Analyses of TCR repertoires via deep learning (DL) methods have successfully diagnosed cancers and infectious diseases, including coronavirus disease 2019. However, few studies have used DL to analyze BCR repertoires. In this study, we collected IgG H chain Ab repertoires from 276 healthy control subjects and 326 patients with various infections. We then extracted a comprehensive feature set consisting of 10 subsets of repertoire-level features and 160 sequence-level features and tested whether these features can distinguish between infected individuals and healthy control subjects. Finally, we developed an ensemble DL model, namely, DL method for infection diagnosis (https://github.com/chenyuan0510/DeepID), and used this model to differentiate between the infected and healthy individuals. Four subsets of repertoire-level features and four sequence-level features were selected because of their excellent predictive performance. The DL method for infection diagnosis outperformed traditional machine learning methods in distinguishing between healthy and infected samples (area under the curve = 0.9883) and achieved a multiclassification accuracy of 0.9104. We also observed differences between the healthy and infected groups in V genes usage, clonal expansion, the complexity of reads within clone, the physical properties in the α region, and the local flexibility of the CDR3 amino acid sequence. Our results suggest that the Ab repertoire is a promising biomarker for the diagnosis of various infections.

Interaction of nuclear ERs and GPER in vitellogenesis in zebrafish.

Estrogens exert their biological functions through the estrogen receptors (ERs). In zebrafish, three nuclear estrogen receptors (nERs) named ERα, ERß1 and ERß2 and one membrane-bound G protein-coupled estrogen receptor (GPER) are identified. Vitellogenin (Vtg) is predominantly expressed in liver and strongly response to the stimulation of estrogen. It has been proposed that all three nERs are functionally involved in vitellogenesis and ERα may act as the major mediator in teleost. However, the role of GPER and its interaction with nERs in this process are not yet defined in teleost species. In the present study, we provide genetic evidence for the functional significance of ERα that the expression of Vtg genes (vtg1, vtg2, vtg3) and their response to estradiol stimulation were significantly decreased in esr1 mutant zebrafish. Activation of ERß1 and ERß2 induced Vtg expression through ERα. Moreover, the involvement of GPER in vitellogenesis and its interaction with nERs in zebrafish were firstly proposed in this work. Activation of GPER induced Vtg genes expression while inhibition of GPER significantly attenuated the estrogenic effect on Vtg. Both treatments altered the expression levels of nERs, suggesting GPER acts interactively with nERs. Collectively, the involvement of both nERs and GPER in regulation of vitellogenesis is demonstrated. ERα is the central factor, acting interactively with ERß1, ERß2 and GPER, and GPER regulates vitellogenesis directly and interactively with nERs.

Fertility Enhancement but Premature Ovarian Failure in esr1-Deficient Female Zebrafish.

It is well established that estrogens regulate female reproduction through estrogen receptors (ERs) in the ovary. However, the precise physiological role of estrogen/ER signaling in reproduction processes remains poorly defined in zebrafish. In this study, we successfully generated an ERα (esr1) mutant line in zebrafish via transcription activator-like effectors nucleases (TALENs). It was found in the mutant females that the fertility was enhanced and the ovarian histology was normal at 90 days post-fertilization (dpf). However, the number of fertile females decreased with age. By 180 dpf, esr1 mutant females were infertile with degenerated ovaries, while the age-matched wild-type females were still fertile. Additionally, few large vitellogenic granules can be found in full grown (FG) follicles at 90 dpf and the expression of vtg genes were down-regulated at both 90 and 180 dpf in esr1 mutant zebrafish. Moreover, steroidogenesis pathway and mTOR signaling pathway were over-activated at 90 dpf, but declined prematurely in esr1 mutant zebrafish by 180 dpf. Collectively, the present study provides evidence that esr1 is fundamental for ovarian maintenance in zebrafish.

Estrogen directly stimulates LHb expression at the pituitary level during puberty in female zebrafish.

The LHb expression is up-regulated during puberty in female zebrafish. However, the molecular mechanism underlying how LHb expression is regulated during puberty remains largely unknown. In this study, we found that the mRNA expression levels of lhb, fshb and cyp19a1b were up-regulated along with the puberty onset in zebrafish. Among the three nuclear estrogen receptors (nERs), the esr2b is the only type whose expression is significantly up-regulated during puberty onset in the pituitary. However, in situ hybridization results revealed that lhb mRNA was colocalized with esr1 and esr2a but not esr2b. Exposure to estradiol (E2) significantly stimulates LHb expression in both wild-type and kiss1-/-;kiss2-/-;gnrh3-/- triple knockout pubertal zebrafish. Moreover, exposure of cultured pituitary cells to E2 increased the LHb expression, indicating that the estrogenic effect on LHb expression could be acted at the pituitary level. Finally, we cloned and analyzed the promoter of lhb by luciferase assay. Our results indicated that the E2 responsive regions of lhb promoter for ERα and ERß2 are identical, suggesting that ERα and ERß2 could bind to the same half ERE region of the promoter of lhb, exhibiting a classical ERE-dependent pathway. In summary, we demonstrate that E2 could directly act on the pituitary level to stimulate LHb transcription during puberty in zebrafish.

Fertility impairment with defective spermatogenesis and steroidogenesis in male zebrafish lacking androgen receptor.

The pivotal role of androgen receptor (AR) in regulating male fertility has attracted much research attention in the past two decades. Previous studies have shown that total AR knockout would lead to incomplete spermatogenesis and lowered serum testosterone levels in mice, resulting in azoospermia and infertility. However, the precise physiological role of ar in controlling fertility of male fish is still poorly understood. In this study, we have established an ar knockout zebrafish line by transcription activator-like effectors nucleases. Homozygous ar mutant male fish with smaller testis size were found to be infertile when tested by natural mating. Intriguingly, a small amount of mature spermatozoa was observed in the ar mutant fish. These mature spermatozoa could fertilize healthy oocytes, albeit with a lower fertilization rate, by in vitro fertilization. Moreover, the expression levels of most steroidogenic genes in the testes were significantly elevated in the ar mutants. In contrast, the levels of estradiol and 11-ketotestosterone (11-KT) were significantly decreased in the ar mutants, indicating that steroidogenesis was defective in the mutants. Furthermore, the protein level of LHß in the serum decreased markedly in the ar mutants when compared with wild-type fish, probably due to the positive feedback from the diminished steroid hormone levels.

Spexin Suppress Food Intake in Zebrafish: Evidence from Gene Knockout Study.

Spexin1 (SPX1) is a newly discovered neuropeptide in vertebrates. Its biological function remains to be elucidated. In this study, we have generated the zebrafish spx1 -/- mutant lines using transcription activator-like effector nucleases. Phenotypes of the spx1 -/- mutant zebrafish were analyzed in order to understand the effects on reproduction and food intake. The reproductive capability is not impaired in spx1 mutant zebrafish. However, we found that the spx1 -/- mutant fish had a higher food intake than the wild type (WT) fish. Real-time PCR revealed that the expression level of agouti-relate protein 1 (AgRP1), a significant appetite stimulant, was significantly higher in spx1 -/- mutant fish after feeding. Intracranial administration of SPX1 could also reduce the mRNA expression of the AgRP1. These data suggest that SPX1 might decrease the food intake by down regulating the expression level of agrp1. Furthermore, spx1 -/- mutant fish exhibited higher glucose, triacylglycerol and cholesterol in the serum than WT fish. However, the hyperphagia did not lead to a higher growth rate or body fat percentage. Taken together, our study suggests that SPX1 may serve as a satiety signal molecular by suppressing the AgRP1 in the brain.

New Insights Into the Role of Estrogens in Male Fertility Based on Findings in Aromatase-Deficient Zebrafish.

It has been demonstrated that estrogens are indispensable for male fertility in mammals. Aromatase (encoded by CYP19) catalyzes the final step of estradiol biosynthesis. However, less is known about the role of aromatase in male fertility in nonmammalian species. Fish aromatase is encoded by two separate genes: the gonad-specific cyp19a1a and the brain-specific cyp19a1b. In a recent study, we used transcription activatorlike effector nucleases to systematically generate cyp19a1a and cyp19a1b mutant lines and a cyp19a1a;cyp19a1b double-mutant line in zebrafish and demonstrated that cyp19a1a was indispensable for sex differentiation. In this study, we focused on male fertility in these aromatase-deficient zebrafish. Our results showed that all aromatase-deficient male fish had normal fertility even at 1 year after fertilization. Interestingly, we observed more spermatozoa in the cyp19a1a and double-mutant males than in the wild-type and cyp19a1b mutant males. The whole-body androgen levels, follicle-stimulating hormone ß and luteinizing hormone ß protein levels in the pituitary, and transcript levels of genes known to be involved in spermatogenesis and steroidogenesis in the testes were significantly higher in the cyp19a1a mutant and aromatase double-mutant males than in the wild-type and cyp19a1b mutant males. These results might explain why more spermatozoa were observed in these fish. Collectively, our findings indicate that estrogens are not needed to achieve and maintain normal fertility in male zebrafish. This finding challenges the traditional view that estrogens are indispensable for male fertility.

Targeted Disruption of Aromatase Reveals Dual Functions of cyp19a1a During Sex Differentiation in Zebrafish.

Aromatase (encoded by the cyp19a1a and cyp19a1b genes) plays a central role in sex differentiation in fish, but its precise roles during sex differentiation are still largely unknown. Here, we systematically generated cyp19a1a and cyp19a1b mutant lines as well as a cyp19a1a;cyp19a1b double mutant line in zebrafish using transcription activatorlike effector nucleases. Our results showed that cyp19a1a mutants and cyp19a1a;cyp19a1b double mutants, but not cyp19a1b mutants, had impaired sex differentiation, and all cyp19a1a mutants and cyp19a1a;cyp19a1b double mutants were males. During sex differentiation, the ovary-like gonads were not observed and the male sex differentiation program was delayed in the cyp19a1a-null fish, and these phenotypes could be partially rescued by 17ß-estradiol treatment. Gene expression analysis indicated that male and female sex differentiation-related genes were significantly decreased in the cyp19a1a mutant. Collectively, our results revealed dual functions of the cyp19a1a gene during sex differentiation: cyp19a1a is not only indispensable for female sex differentiation but also required for male sex differentiation.

Molecular mechanism of feedback regulation of 17ß-estradiol on two kiss genes in the protogynous orange-spotted grouper (Epinephelus coioides).

Kisspeptins are considered critical regulators in the hypothalamic-pituitary-gonadal axis because they can stimulate secretion of Gonadotropin-releasing hormone in mammals, and may also mediate the feedback regulation of sex steroids in the hypothalamus. Two kiss1 paralogues (kiss1 and kiss2) were identified in teleosts, hinting at their increased complexity of signaling for sex-steroid feedback regulation. In the present study, molecular pathways by which 17ß-estradiol (E2 ) exerted feedback regulation on two kiss genes, via three types of estrogen receptors, were investigated in the protogynous orange-spotted grouper (Epinephelus coioides). kiss2 expression in the brain significantly increased in ovariectomized orange-spotted groupers, while E2 replacement in ovariectomized fish reversed these changes to levels in the sham-surgery group; conversely, kiss1 expression did not change. Dual-label in situ hybridization showed that kiss1 and kiss2 neurons express erα, erß1, and erß2, indicating that E2 may directly regulate kiss1 and kiss2. Indeed, E2 treatment of transiently transfected HEK293T cells decreased the activity of both kiss promoters in the presence of erß1 and erß2 rather than erα. Further deletion and site-directed mutagenesis of the kiss promoters indicated that kiss1 is regulated by E2 via an estrogen-responsive element (ERE)-dependent, classical pathway utilized by Erß1, as well as via an Activator protein 1 (Ap1)-dependent, non-classical pathway utilized by Erß2. kiss2 was also differently regulated by E2 through the Creb transcription factor, utilized by Erß1 as well as a half-ERE-dependent, classical pathway utilized by Erß2. Taken together, multiple signaling pathways in orange-spotted grouper are clearly involved in the feedback regulation of E2 on kiss genes via different estrogen receptors.

Genetic Evidence for Multifactorial Control of the Reproductive Axis in Zebrafish.

It is generally believed that kisspeptin and gonadotropin-releasing hormone (GnRH) are required for reproduction in vertebrates. In this study, we generated gnrh3-null zebrafish and found that gnrh3 mutation did not impair gonad development and reproductive capacity. Moreover, zebrafish triple knockout mutant lacking gnrh3 and the 2 kiss1s genes undergo normal puberty and gonad maturation. The expression of follicle-stimulating hormone beta (fshß) and luteinizing hormone beta (lhß) was not significantly altered whereas the expression of neuropeptide Y (npy), tachykinin 3 (tac3), and secretogranin-II (sgII) was significantly increased in the triple knockout mutant, suggesting that compensation mechanisms exist to stimulate the reproductive axis in the absence of kiss and gnrh. Our results challenge the prevailing view that GnRH is indispensable for reproduction across species. These data provide genetic evidence that different mechanisms have evolved for the neuroendocrine control of reproduction between mammals and fish: pulsatile release of GnRH to the portal system is the final gateway to stimulate the reproductive axis in mammals, whereas multiple factors act in parallel with GnRH to stimulate the reproductive axis in certain fish species.

Gene knockout of nuclear progesterone receptor provides insights into the regulation of ovulation by LH signaling in zebrafish.

It is well established that the luteinizing hormone surge triggers ovulation, a dynamic process leading to the release of the mature oocyte from the ovarian follicle. But how this process controlled by LH signaling remains largely unknown in non-mammalian species. In this study, we investigated the roles of nuclear progesterone receptor (npr) in LH-induced ovulation. Our results indicate that the nuclear progesterone receptor serves as an important mediator of LH action on ovulation. This conclusion is based on the following results: (1) the expression level of npr peaks at the full-grown stage of the follicles; (2) the expression of npr is stimulated by LH signaling in vitro and in vivo; and (3) the npr null females are infertile due to ovulation defects. Moreover, we further show that LH signaling could induce ptger4b expression in an npr-dependent manner, and blockage of Ptger4b could also block hCG-induced ovulation. Collectively, our results not only demonstrate that npr serves an indispensable role in mediating the action of LH on ovulation in zebrafish, but also provide insights into the molecular mechanisms of the regulation of ovulation in fish.

The kiss/kissr systems are dispensable for zebrafish reproduction: evidence from gene knockout studies.

The kiss1/gpr54 signaling system is considered to be a critical regulator of reproduction in most vertebrates. However, this presumption has not been tested vigorously in nonmammalian vertebrates. Distinct from mammals, multiple kiss1/gpr54 paralogous genes (kiss/kissr) have been identified in nonmammalian vertebrates, raising the possibility of functional redundancy among these genes. In this study, we have systematically generated the zebrafish kiss1(-/-), kiss2(-/-), and kiss1(-/-);kiss2(-/-) mutant lines as well as the kissr1(-/-), kissr2(-/-), and kissr1(-/-);kissr2(-/-) mutant lines using transcription activator-like effector nucleases. We have demonstrated that spermatogenesis and folliculogenesis as well as reproductive capability are not impaired in all of these 6 mutant lines. Collectively, our results indicate that kiss/kissr signaling is not absolutely required for zebrafish reproduction, suggesting that the kiss/kissr systems play nonessential roles for reproduction in certain nonmammalian vertebrates. These findings also demonstrated that fish and mammals have evolved different strategies for neuroendocrine control of reproduction.