# TRENDS IN COMPARATIVE ENDOCRINOLOGY AND NEUROBIOLOGY

EDITED BY: Hubert Vaudry and Olivier Kah PUBLISHED IN: Frontiers in Neuroscience and Frontiers in Endocrinology

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ISSN 1664-8714 ISBN 978-2-88945-399-3 DOI 10.3389/978-2-88945-399-3

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# **TRENDS IN COMPARATIVE ENDOCRINOLOGY AND NEUROBIOLOGY**

Topic Editors: **Hubert Vaudry,** University of Rouen, France **Olivier Kah,** University of Rennes 1, France

White stork (*Ciconia ciconia*) mating in their nest on top of a building in Andalucia (Spain). Comparative endocrinology allows understanding the complex hormonal interactions underlying sexual maturation and behavior in a wide range of animals.

Image: Olivier Kah.

The comparative approach takes advantage of the biological diversity to select the most appropriate model organism to tackle a scientific question. Comparisons between the endocrine and nervous systems accross species have yielded major breakthroughs in endocrinology and neurobiology. For instance: a number of mammalian peptide hormones and neuropeptides have been originally identified in fish or amphibians; studies conducted in a sea slug founded the cellular and molecular basis of learning and memory; observations of neurogenesis in the forebrain of songbirds led to the discovery of adult neurogenesis in the mammalian brain. These examples illustrate the remarkable contribution of the comparative approach for the advancement of neuroendocrinological concepts.

The present e-book is a unique collection of research articles and reviews that provide a representative overview of the latest developments in comparative endocrinology and neurobiology.

**Citation:** Vaudry, H., Kah, O., eds. (2018). Trends in Comparative Endocrinology and Neurobiology. Lausanne: Frontiers Media. doi: 10.3389/978-2-88945-399-3

# Table of Contents

*Axis by Male Odors and Socio-Sexual Interactions*

Claude Fabre-Nys, Keith M. Kendrick and Rex J. Scaramuzzi

*112 Reporter Cell Lines to Evaluate the Selectivity of Chemicals for Human and Zebrafish Estrogen and Peroxysome Proliferator Activated g Receptors*

Marina Grimaldi, Abdelhay Boulahtouf, Vanessa Delfosse, Erwan Thouennon, William Bourguet and Patrick Balaguer

Hans K. H. Ng and Billy K. C. Chow

*153 Evolution of Parathyroid Hormone Receptor Family and Their Ligands in Vertebrate*

Jason S. W. On, Billy K. C. Chow and Leo T. O. Lee

*159 Identification, Functional Characterization, and Pharmacological Profile of a Serotonin Type-2b Receptor in the Medically Important Insect,* **Rhodnius Prolixus**

Jean-Paul V. Paluzzi, Garima Bhatt, Chang-Hui J. Wang, Meet Zandawala, Angela B. Lange and Ian Orchard

Marta C. Romano, Pedro Jiménez, Carolina Miranda-Brito and Ricardo A. Valdez

*188 Bisphenol A and Phthalate Endocrine Disruption of Parental and Social Behaviors*

Cheryl S. Rosenfeld

Katalin Skrapits, Beáta Á. Borsay, László Herczeg, Philippe Ciofi, Zsolt Liposits and Erik Hrabovszky

*227 Divergent Cardio-Ventilatory and Locomotor Effects of Centrally and Peripherally Administered Urotensin II and Urotensin II-Related Peptides in Trout* Gilmer Vanegas, Jérôme Leprince, Frédéric Lancien, Nagi Mimassi, Hubert Vaudry and Jean-Claude Le Mével

# Editorial: trends in Comparative Endocrinology and Neurobiology

*Hubert Vaudry1 \* and Olivier Kah2*

*1Université de Rouen, Mont-Saint-Aignan, France, 2University of Rennes 1, Rennes, France*

Keywords: peptide hormones and neuropeptides, biological rhythms, reproduction, endocrine disrupters, behavior

**Editorial on the Research Topic**

### **Trends in Comparative Endocrinology and Neurobiology**

Neil Campbell once described evolution as the "overarching theme of biology." Indeed, many comparative studies have significantly contributed to major discoveries in biological sciences. This is particularly true in the field of comparative endocrinology and neurobiology. For instance, the concept of neurosecretion has been established from studies conducted in invertebrates and fish (1, 2). Similarly, a number of peptide hormones and neuropeptides [e.g., melanin-concentrating hormone, urocortin, urotensin II (UII), glucagon-like peptide-1, stanniocalcin, etc.] have been identified in non-mammalian vertebrates before being "re-discovered" in mammals including humans (3–12). These breakthroughs bear witness to the power of the comparative approach to the current knowledge in endocrinology and neurobiology. They certainly support the famous assertion by Theodosius Dobzhansky that "Nothing in biology makes sense except in the light of evolution" (13).

This research topic is a compilation of contributions stemming from the 27th Conference of the European Comparative Endocrinologists (CECE2014) held in Rennes, France, that illustrates various facets of current comparative endocrinology and neurobiology investigations.

The neuropeptide allatotropin (AT), which was originally isolated in the central nervous system of the tobacco hornworm, was named after its ability to stimulate juvenile hormone (JH) biosynthesis from the corpora allata. Lismont et al. have characterized the cDNA sequence of AT and its receptor in the desert locust *Schistocerca gregaria*. Expression of the receptor cDNA in a mammalian cell line reveals that AT causes Ca2<sup>+</sup> mobilization and stimulates cAMP production. On locust isolated organs, AT stimulates gut contraction and JH biosynthesis.

Juvenile hormone is an ester of farnesol, a cholesterol precursor found in all eukaryotes. Since, in insects, JH maintains larvae in a juvenile state, De Loof et al. raise the provocative question as to whether farnesol-like endogenous compounds act as anti-aging factors in vertebrates. Indeed, tissue extracts from mammalian organs do display JH activity in insects. In addition, farnesol, its precursor, and/or its metabolites occur in various mouse tissues. Thus, the authors elaborate a hypothesis on the possible roles of these compounds in vertebrates.

A large proportion of regulatory peptides are α-amidated. SALFamides are a family of myorelaxant neuropeptides that occur in echinoderms. Elphick et al. have characterized the SALFamide precursors in representative species of the five echinoderm classes. From these data, they can propose different scenarios regarding the phylogenetic history of the SALFamide neuropeptide family.

The neurohypophysial nonapeptides arginine vasopressin (AVP) and oxytocin are the mammalian representatives of a large family of neuropeptides that appeared very early during evolution. Banerjee et al. have cloned the cDNA encoding the precursors of vasotocin and isotocin, the AVP, and oxytocin counterparts, in two air-breathing catfish. The two precursor genes are expressed both in the brain and the follicular envelope of the ovary and display higher expression during the

*Edited and Reviewed by:*

*Heather K. Caldwell, Kent State University, United States*

*\*Correspondence:*

*Hubert Vaudry<EMAIL_ADDRESS>
### *Specialty section:*

*This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Endocrinology*

*Received: 13 October 2017 Accepted: 16 November 2017 Published: 01 December 2017*

### *Citation:*

*Vaudry H and Kah O (2017) Editorial: Trends in Comparative Endocrinology and Neurobiology. Front. Endocrinol. 8:338. doi: 10.3389/fendo.2017.00338*

reproductive season, suggesting a possible role in the control of ovarian activity.

Three families of G protein-coupled receptors (GPCRs) have been identified in mammals. There is now clear evidence that oligomerization of GPCRs impacts their trafficking, ligand binding, and signaling. While intrafamily oligomer formation has been extensively studied, interfamily oligomerization is less documented. In their review, Ng and Chow describe the different methods applied to study oligomerization of GPCRs and summarize the current knowledge concerning interfamily GPCR heteromerization.

Parathyroid hormone (PTH) is a peptide that plays a crucial role in the regulation of calcium homeostasis. PTH belongs to a family of peptides that includes PTH-related peptide and the tuberoinfundibular peptide of 39 residues (TIP39 also called PTH2). On et al. describe the molecular evolution of these peptides and their receptors in vertebrates. Their review indicates that these peptide–receptor systems appeared early during evolution, possibly in invertebrates.

In mammals, the timing of ovulation is crucial for the success of reproduction: plasma estradiol level acts as an indicator of oocyte maturation while peptidergic neurons within the suprachiasmatic nucleus signal the time of the day and synchronize female fertility with the period of maximal activity and sexual motivation. Simonneaux and Bahougne summarize the literature pertaining to the inhibitory and stimulatory actions of estradiol on the reproductive axis and describe the roles of AVP and vasoactive intestinal peptide in the timing of the luteinizing hormone (LH) surge. They also review the contribution of peripheral clocks within the reproductive organs in the timing of female reproduction. Finally, they discuss the impact of shift work on female fertility.

In photoperiodic mammals, the duration of melatonin secretion during the night regulates seasonal changes of physiological functions. Chakir et al. show that, in the Syrian hamster, nocturnal melatonin exerts chronomodulatory effects on cortisol, leptin, insulin, and glucose daily rhythms. Their study thus provides important information regarding the role of melatonin in the circadian control of energy metabolism.

Besides the pineal gland, melatonin is synthesized by several organs including the retina and gastrointestinal tract. Mukherjee and Maitra review the physiological significance of gut-derived melatonin in vertebrates. While pineal-derived melatonin acts mainly as a neurohormone/neuromodulator, gut melatonin appears to act primarily locally as an autocrine/paracrine factor.

Domestic animals are fruitful models for comparative endocrinologists and neurobiologists. In spring, ewes normally do not ovulate, but exposure to a ram or to ram odor can induce an LH surge and ovulation. This pheromone response, known as the "ram effect," can be used to compact lambing in a herd. Fabre-Nys et al. describe the neuroanatomical pathways and neurochemical mechanisms underlying the ram effect. They also show that the ram effect is a relevant model in which to address more general questions regarding the neuronal and endocrine regulation of the hypothalamic–pituitary–gonadal axis.

Kisspeptin has recently emerged as a major player of the neuroendocrine control of reproduction and puberty. Skrapits et al. review the literature regarding the occurrence of various neurotransmitters and neuropeptides in kisspeptin neurons both in laboratory animals and in humans. Their data reveal that human kisspeptin neurons express far more neuropeptides than those of laboratory species, suggesting that the fine control of fertility differs substantially between primates and non-primates.

Age-related dysfunctions in the endocrine system are usually associated with a decline of cognitive functions and a higher risk of neurodegenerative diseases. Blair et al. review the evidence that changes in gonadal steroids, LH, and even sex hormone-binding globulin can be involved in learning and memory impairment and development of Alzheimer's disease. These data could lead to novel therapeutic strategies for the treatment of neurodegenerative disorders.

Aromatase catalyzes the conversion of testosterone into 17β-estradiol, and the inactivation of 17β-estradiol into catechol estrogens. Charlier et al. summarize the data showing that aromatase activity can be rapidly modified through phosphorylation/dephosphorylation processes. The fine tuning of aromatase phosphorylation in specific brain areas may thus account for the multiple physiological and behavioral effects of estrogens.

The peptide hormones adrenocorticotropin and melanocytestimulating hormones act through five GPCRs collectively called melanocortin receptors. The functional expression of this family of receptors is regulated by two melanocortin receptor accessory proteins, MRAP1 and MRAP2. Jackson et al. elaborate on the impact of MRAP deletions in adrenal deficiency and obesity, as revealed by animal model studies and human mutations.

The neuropeptide UII is a potent vasoconstrictor. UII belongs to a family of peptides that also includes three UIIrelated peptides, i.e., URP, URP1, and URP2. Vanegas et al. have compared the central and peripheral actions of UII, URP1 and URP2 on cardioventilatory and locomotor functions in unanesthetized trout. They show that intracerebroventricular injection of each of the three neuropeptides increases ventilation, blood pressure, heart rate, and locomotion with different potencies, whereas intraarterial administration of only a high dose of UII and URP1 (but not URP2) stimulates ventilation and locomotor activity.

In invertebrates, as in vertebrates, serotonin (5-HT) exerts multiple behavioral and neurophysiological actions. In the insect *Rhodnius proxilus*, the chief Chagas disease vector, 5-HT acts as a diuretic hormone that regulates hemolymph osmolarity after its dramatic feeding bout. Paluzzi et al. have cloned the 5-HT2b receptor cDNA in *R. proxilus* and studied its pharmacological profile on transfected cells and isolated organs.

In mammals, transthyretin (TTR) acts as a thyroxine (T4) binding protein whereas, in sub-mammalian vertebrates, TTR binds preferably the active form of thyroid hormones, triiodothyronine (T3). Richardson reviews the evolution of the TTR gene and describes how subtle changes in the protein structure result in the switch of the ligand from T3 to T4.

The glycoprotein hormone (GPH) family encompasses LH, follicle-stimulating hormone, thyroid-stimulating hormone, and chorionic gonadotropin. Although GPHs are present only in vertebrates, related GPH subunit ancestor genes have been identified in most vertebrates phyla. Cahoreau et al. provide an extensive and critical look at the structure and function of GPHs and their receptors.

5′AMP-activated protein kinase (AMPK) is a protein kinase that is activated by ATP deficiency. AMPK is primarily expressed in the liver and muscle where it acts as an energy sensor. In invertebrates and vertebrates, AMPK is also expressed in the gonads. Bertoldo et al. take a broad comparative view to examine the role of AMPK in the interplay between the regulation of energy homeostasis and reproductive functions.

Steroid hormones influence the fertility, development, and survival of parasites; reciprocally, parasite infection often affects plasma steroid levels in the host. Romano et al. provide evidence that a number of parasites express several steroidogenic enzymes and can synthesize various steroid hormones including ecdysteroids, sex steroids, and corticosteroids.

Endocrine-disrupting chemicals (EDCs) are pharmaceutical or environmental compounds that mimic or impair the action of hormones. EDCs can thus affect a number of physiological and behavioral actions of hormones in animals and humans. Grimaldi et al. compare the effects of EDCs, at the cellular and molecular levels, on human and zebrafish estrogen and peroxisome proliferator-activated γ receptors. They point out that the transcriptional activities of EDCs on human and zebrafish nuclear receptors exhibit marked differences. They conclude that caution

## REFERENCES

should be exercised regarding extrapolation of EDC screening tests in zebrafish models toward hazard assessment for human physiopathology. Rosenfeld focuses on the effect of bisphenol A and phthalate exposure on parental behavior and the impact of developmental exposure to these EDCs on social behavior. They provide convincing evidence that these chemicals alter the organizational and activational programming the brain.

It is our hope that this research topic will become a major set of references for comparative endocrinologists and neurobiologists and will raise the interest of other scientists who are not (yet) involved in this fertile research domain.

## AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.

## ACKNOWLEDGMENTS

We want to thank the authors for their valuable contribution. We are deeply indebted to the dedicated reviewers who helped maintain the articles at the highest standards. We also gratefully acknowledge the excellent secretarial assistance of Mrs. Catherine Beau and the continuous support of the Frontiers staff.

urotensin II-related peptide, and their receptor: from structure to function. *Pharmacol Rev* (2015) 67:214–58. doi:10.1124/pr.114.009480

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Vaudry and Kah. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Molecular cloning, sequencing and tissue expression of vasotocin and isotocin precursor genes from Ostariophysian catfishes: phylogeny and evolutionary considerations in teleosts

### Putul Banerjee<sup>1</sup> , Radha Chaube<sup>2</sup> and Keerikkattil P. Joy <sup>1</sup> \*

### *Edited by:*

Hubert Vaudry, University of Rouen, France

### *Reviewed by:*

Hervé Tostivint, Natural History Museum, France Leo T. O. Lee, The University of Hong Kong, China

### *\*Correspondence:*

Keerikkattil P. Joy, Department of Zoology, Centre of Advanced Study, Faculty of Science, Banaras Hindu University, Varanasi 221005, India<EMAIL_ADDRESS>
### *Specialty section:*

This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Neuroscience

*Received:* 29 November 2014 *Accepted:* 23 April 2015 *Published:* 15 May 2015

### *Citation:*

Banerjee P, Chaube R and Joy KP (2015) Molecular cloning, sequencing and tissue expression of vasotocin and isotocin precursor genes from Ostariophysian catfishes: phylogeny and evolutionary considerations in teleosts. Front. Neurosci. 9:166. doi: 10.3389/fnins.2015.00166 <sup>1</sup> Department of Zoology, Centre of Advanced Study, Banaras Hindu University, Varanasi, India, <sup>2</sup> Zoology Department, Mahila Mahavidhylaya, Banaras Hindu University, Varanasi, India

Basic and neutral neurohypophyseal (NH) nonapeptides have evolved from vasotocin (VT) by a gene duplication at the base of the gnathostome lineage. In teleosts, VT and IT are the basic and neutral peptides, respectively. In the present study, VT and IT precursor genes of Heteropneustes fossilis and Clarias batrachus (Siluriformes, Ostariophysi) were cloned and sequenced. The channel catfish Icatalurus punctatus NH precursor sequences were obtained from EST database. The catfish NH sequences were used along with the available Acanthopterygii and other vertebrate NH precursor sequences to draw phylogenetic inference on the evolutionary history of the teleost NH peptides. Synteny analysis of the NH gene loci in various teleost species was done to complement the phylogenetic analysis. In H. fossilis, the NH transcripts were also sequenced from the ovary. The cloned genes and the deduced precursor proteins showed conserved characteristics of the NH nonapeptide precursors. The genes are expressed in brain and ovary (follicular envelope) of H. fossilis with higher transcript abundance in the brain. The addition of the catfish sequences in the phylogenetic analysis revealed that the VT and IT precursors of the species-rich superorders of teleosts have a distinct phylogenetic history with the Acanthopterygii VT and IT precursors sharing a less evolutionary distance and the Ostariophysi VT and IT having a greater evolutionary distance. The genomic location of VT and IT precursors, and synteny analysis of the NH loci lend support to the phylogenetic inference and suggest a footprint of fish- specific whole genome duplication (3R) and subsequent diploidization in the NH loci. The VT and IT precursor genes are most likely lineage-specific paralogs resulting from differential losses of the 3R NH paralogs in the two superorders. The independent yet consistent retention of VT and IT in the two superorders might be directed by a stringent ligand-receptor selectivity.

Keywords: catfish, cloning, phylogeny, vasotocin-isotocin precursors, synteny analysis

## Introduction

The neurohypophyseal (NH) nonapeptides are an evolutionarily old family which is believed to have originated much before the vertebrates, as one of the first neurotransmitters in the archetype metazoan nervous system (Grimmelikhuijzen et al., 2002).   This has complemented earlier works of characterization of peptide principles of the neurohypophysis by chromatographic methods and has provided useful insights into the distribution of the peptides in different vertebrate species, their precursor structure and evolution. The nonapeptides are synthesized as part of a large precursor molecule that undergo post-translational processing to release the nine amino acid hormone with two half cysteine residues at the first and sixth positions forming a disulphide bridge, and a cysteine- rich protein called neurophysin (Acher, 1996). Neurophysin aids in proper folding of the precursor and its sorting into secretory vesicles (de Bree et al., 2000). In vertebrates, about 15 nonapeptides are known till date. They are classified into two families on the basis of the amino acid at the 8th position, as the basic and neutral peptide families. Each vertebrate possesses at least two peptides, one each from the two families except cyclostomes, where only the basic peptide representative vasotocin (VT) is documented (Gwee et al., 2009). This has led to a widely accepted theory that the VT precursor gene is the ancestor of all vertebrate nonapeptide precursor genes, and has given rise to the two lineages following an event of duplication early in the vertebrate evolution probably at the base of the gnathostome lineage. Evolutionarily too, the two peptide families follow distinct lineages with VT representing the common basic peptide in all nonmammalian vertebrates to be replaced by vasopressin (VP) in mammals, while isotocin (IT) is the neutral peptide in teleosts, mesotocin (MT) in lobefinned fishes and noneutherian tetrapods, and oxytocin (OT) in eutherians. In elasmobranchs, a host of different neutral peptide homologs has been reported which is in contrast with the remarkable stability in the type and numbers of nonapeptides in any other group of Vertebrata (Acher, 1996). Presence of more than two neurohypophyseal peptides has been reported from groups like elasmobranchs, amphibians, and metatherians (Chauvet et al., 1983, 1984, 1985; Rouille et al., 1988, 1989; Parry et al., 2000; Hyodo et al., 2004). In teleosts, VT and IT have been reported as the basic and neutral hormone representatives, respectively. Larhammar et al. (2009) emphasized the need for simplifying the nomenclature with the original names VP for the basic peptides and OT for all neutral peptides.

The two evolutionary lines of basic and neutral nonapeptides across vertebrates represent a contrasting picture in the frequency of substitutions in the 9 amino acid (aa) hormone. In the basic lineage, there have been lesser substitutions than that in the neutral line, as evident from the distribution of the peptides in vertebrates. In fact, the number of cognate receptors for the two peptide families is also contrasting. While the stable basic nonapeptide line has a diverse repertoire of at least five types of cognate receptors (V1A, V1B, V2A, V2B, and V2C), the neutral nonapeptide series has a single type (OTR) in all vertebrate groups (Ocampo Daza et al., 2012; Yagamuchi et al., 2012; Lagman et al., 2013). All types of receptors are present in the early gnathostomes, the cartilaginous fish (Yagamuchi et al., 2012) suggesting that the ligand-receptor selectivity might have been established very early in the vertebrate evolution and it is the co-evolution of the hormone with its cognate receptors which has maintained the two families of basic and neutral nonapeptides, and allowed only a basic amino acid substitution at the 8th position in the basic line and a neutral amino acid substitution at the 8th position in the neutral line. While, the ligand- receptor selectivity might have directed the evolution only in the 9 aa hormone part; the larger neurophysin region is not under this evolutionary pressure. Therefore, to know the evolutionary history of the NH precursors especially in the light of recurrent local and global duplication events occurring throughout the vertebrate history, there is a need to do a phylogenetic study over the entire precursor rather than considering the distribution of peptides in different vertebrate groups.

Teleosts had undergone a third round of whole genome duplication, (3R) (Meyer and Schartl, 1999; Christoffels et al., 2004; Jaillon et al., 2004) in addition to the two vertebratespecific whole genome duplications. After the 3R in teleosts, there was diploidization by the loss of 3R paralogs. These losses of the 3R paralogs were different in various groups of teleosts (Garcia de la serrana et al., 2014). Even in such a scenario, the consistency in the number and type of nonapeptides in teleosts is noteworthy. However, the teleost NH precursors available in the public database are mostly sequences from the speciesrich superorder Acanthopterygii while only few nonapeptide precursor sequences from the other species-rich group, the superorder Ostariophysi are available. The Acanthopterygii and Ostariophysi have undergone independent adaptive radiations (Santini et al., 2009), and are known to have suffered differential losses of 3R paralogs. Therefore, it is pertinent to have sequence information from more species representing all groups in Teleostei, particularly the superorder Ostariophysi to understand the evolution of the NH precursor genes in teleosts.

Previous studies from our laboratory have shown a well-developed and highly organized VT system in the Asian air-breathing catfish Heteropneustes fossilis. VT was characterized in both brain and ovary of the catfish with seasonal changes (Singh and Joy, 2008), and has been shown to be involved in reproductive functions such as steroidogenesis, oocyte maturation, oocyte hydration, ovulation and prostaglandin secretion (Singh and Joy, 2009b, 2010, 2011; Joy and Singh,

2013). IT is less effective in influencing these events. Three VT receptors have been cloned from the catfish V1a1 (accession no.KF434615.1), V1a2 (accession no.KF434616.1), and V2A (accession no.KF434617.1), which display a wide tissue distribution (Rawat et al., 2015). All the three receptors are expressed in the brain and ovary, which are the two major sites of VT production.

The catfish H. fossilis holds an important phylogenetic position as it is believed to be the sole survivor of an old clade that went extinct at the K-T (Cretaceous-Tertiary transition period) boundary (Jansen et al., 2006) when a major extinction event took place due to severe volcanic activity (Deccan traps) in the Asian region. This might have severely polluted and deoxygenated the inland water bodies and only fish with airbreathing apparatus survived. Air-breathing habit also demands a shift in osmoregulation and fluid homeostasis in which apart from other systems, the NH nonapeptides play a key role. In view of the important taxonomic and phylogenetic position of H. fossilis and its air-breathing habit, molecular characterization of the NH peptide genes as a follow on of earlier anatomical and physiological studies on VT has been attempted in the present work. The sequence information was also extended to Clarias batrachus, another air- breathing catfish, and used in the phylogenetic analysis. The catfish NH precursor sequence information from the present study will add to the information pool of teleost nonapeptides from a group other than the superorder Acanthopterygii and allow discussing the phylogeny of the vertebrate NH nonapeptides in the light of 3R and subsequent diploidization in teleosts. A tissue distribution profile for these genes was also carried out since VT was described in the catfish ovary earlier.

## Materials and Methods

## Animal Collection and Acclimation

Adult H. fossilis (40–50 g) were collected from local fish markets in Varanasi. They were maintained in the laboratory for 48 h under natural photoperiod (13.0 L: 11.0 D) and temperature (25 ± 2 ◦C) to overcome stress due to transportation and fed daily with goat liver ad libitum. Adult C. batrachus were obtained in the resting phase for the cloning work. The fish were weighed and sacrificed by decapitation. All experiments were performed in accordance with the guidelines of the Animal Ethics Committee, Banaras Hindu University, Varanasi.

## Chemicals and Reagents

Guanidine thiocyanate—phenol solution (Qiagen), Revert-Aid H Minus First Strand cDNA Synthesis Kit (Fermentas), 2X PCR Master Mix (Fermentas), DNase (Ambion) and veriquest SYBR green qPCR master mix (affymetrix) were purchased through local suppliers. Agarose, Tris base, Glacial acetic acid, EDTA– Na2 and other chemicals were of molecular grade, purchased from E-Merck, India. LB broth, LB Agar, ampicillin, X-Gal and IPTG were purchased from Himedia, India. Hyaluronidase type IV was purchased from Sigma-Aldrich, India. The primers used were synthesized by Integrated DNA Technology, India.

## Cloning of VT and IT genes from *H. fossilis* and *C. batrachus*

For cloning of VT, an approach of first isolating partial VT cDNA using degenerate PCR (polymerase chain reaction) followed by 3 ′ and 5′ Rapid Amplification of cDNA ends (RACE) to obtain the full length VT cDNA sequence was applied. Total RNA from the brain of acclimatized female catfish in the resting phase of the reproductive cycle was prepared by the single-step method of RNA isolation (Chomczynski and Sacchi, 1987) by acid guanidium thiocyanate-phenol-chloroform extraction using Qiazol (Qiagen) as the monophasic lysis buffer. Five µg total RNA was reverse- transcribed using random hexamer primers and Revert Aid M-MuLV reverse transcriptase in a 20µL reaction volume (first strand cDNA synthesis kit, Fermantas) using the manufacturer's protocol. One µL of the resulting cDNA was used to amplify partial VT cDNA in 25µL reaction volume using 2 X PCR master mix (Fermentas) and VT degenerate forward primer and VT degenerate reverse primer in a cycling condition of one cycle of 95◦C, 5 min; then 35 cycles of 95◦C, 30 s; 57◦C, 30 s; 72◦C, 1 min; and a final 7-min elongation at 72◦C. The degenerate primers were designed using the software iCODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primers), in which teleost VT precursor sequences were used as inputs (**Table 1**). The resulting 145 bp partial VT amplicon was purified using Nucleo-pore PCR clean- up gel extraction kit (Genetix) and sequenced taking services of Eurofin genomics, Bangalore, India. For obtaining full length sequence, 3′ RACE was done for which total RNA from the brain was reversetranscribed using oligo dT anchor primer and Revert Aid M-MuLV reverse transcriptase. One µL of the resulting cDNA was amplified using a VT H FP (corresponding to the hormone moiety), designed from the partial sequence and anchor primer to obtain a 700 bp 3′ RACE amplicon, which was cloned and sequenced. This sequence was utilized for designing two genespecific nested reverse primers to do 5′ RACE. Total RNA from the brain was reverse-transcribed using the outer gene specific VT RP and Revert Aid M-MuLV reverse transcriptase. The resulting cDNA was purified using the Nucleo-pore PCR clean- up gel extraction kit (Genetix) to remove residual dNTPs. The purified cDNA was dA tailed using dATP and terminal transferase. The dA tailed cDNA was used for PCR amplification using the nested RP 2 primer and oligo dT anchor primer. The 5′ RACE amplicon was purified, cloned and sequenced. Full length VT cDNA sequence was submitted to the GenBank with the accession no. JX035928.1

For cloning IT precursor cDNA, total RNA from the brain was reverse-transcribed using an oligo dT anchor primer and Revert Aid M-MuLV reverse transcriptase. One µL of the resulting cDNA was PCR amplified using a sense or forward primer IT H FP designed from the IT hormone moiety and anchor primer, following the 3′ RACE protocol. The resulting 700 bp amplicon was sequenced taking services from Eurofin genomics. To get the 5′ end of the precursor cDNA, 5′ RACE was done. Total RNA from the brain was reverse- transcribed using an outer reverse or antisense primer, RP1 (designed over a conserved region of the neurohypophyseal peptide neurophysin) and Revert Aid M-MuLV reverse transcriptase. The resulting cDNA was

### TABLE 1 | List of primers used for cloning and qPCR of vasotocin and isotocin precursors.

purified and dA tailed. The dA tailed cDNA was used for PCR amplification using an overlapping and nested antisense primer RP2 and oligodT anchor primer. The resulting 200 bp 5 ′ RACE amplicon was ligated into pGEMT vector (promega kit), following the manufacturer's protocol and transformed into the E. coli DH5 α competent cells. Transformed cells were plated into LB Agar solid medium containing ampicillin, X- Gal and IPTG to select positive transformants and bluewhite screening was done for recombinant colonies. Plasmids from white colonies (colonies transformed with recombinant plasmids) were extracted by alkaline lysis method and sequenced. Sequence information from the IT precursor containing inserts (as known by in silico translation of the insert sequences) was used to design a sense primer from the 5′ UTR region (IT UTR FP), followed by 3′ RACE with this primer to obtain the entire IT precursor sequence, which was submitted to the GenBank with the accession no. JX669009.1.

Partial sequences of C. batrachus VT and IT precursors were obtained by 3′ RACE with the forward primers VT H FP and IT H FP (the same used for H. fossilis), to obtain a sequence read from the hormone region till the 3′ untranslated region. The primer details are given (**Table 1**).

### Sequencing of VT and IT Transcripts from Ovary

Since the VT and IT transcripts are less abundant in the ovarian follicles, a semi-nested 3′ RACE was done to get a single, sharp amplification of the gene transcripts. Briefly, it included a primary PCR using VT UTR FP, IT UTR FP spanning the 5′ UTR region of VT and IT genes, respectively and anchor primer to amplify 1µL of follicular cDNA (made by reverse transcription of RNA using oligodT anchor primer), followed by a secondary PCR using VT H FP, IT H FP and anchor primer to amplify1µL of the primary PCR product. The amplicons from the secondary PCR was purified and sequenced.

### Data Mining for Phylogenetic Analysis

The NH nonapeptide precursor sequences of species representing most vertebrate classes were tried to be included in the phylogenetic analysis. The sequences were procured from the GenBank. Details of the sequences of the species with accession numbers are given in Supplementary Table 1. For procuring the NH precursors of Ictalurus punctatus, a BLAST search against the Ictalurus EST database (Lu et al., 2010) was done using the H. fossilis VT and IT sequences as queries. The identities of the putative VT and IT precursor mRNAs obtained were confirmed by in silico translation of the sequences. The spotted gar Lepisosteus oculatus, Holostei is a basal actinopterygian species representing a pre 3R genome. The neurohypophyseal nonapeptide precursor sequences of this species were procured by BLAST searches in its Nucleotide database and analyzed.

### Phylogenetic Analysis

Phylogenetic trees were constructed using the Neighbor- Joining (NJ) and Maximum-Likelihood (ML) method. Evolutionary distances were computed using the Poisson correction method for rate of amino acid substitution. All phylogenetic studies were done using the software MEGA 6 (Tamura et al., 2013). In addition to the calculation of evolutionary distance of precursor sequences within and between species, the sequences were grouped according to the taxonomic units for calculation of evolutionary distance between groups (**Table 2**). Keeping in view of the enormous species diversity, the teleost sequences of the two superorders Acanthopterygii and Ostariophysi were independently treated. Multiple sequence alignment used for the tree construction and distance calculation was the same and was done in MEGA6 using Clustal W parameters. Gonnet protein weight matrix with gap opening and gap extension penalties of 10 and 0.2 respectively was used for the alignment. The alignment is provided in a Supplementary data sheet 1.

## Analysis of VT and IT Genomic Location and Synteny Analysis

The nonapeptide gene-containing chromosome blocks of spotted gar and representative teleost species were obtained from the NCBI and Ensembl genome browser. Fugu, medaka, tilapia, and stickleback belong to the teleost superorder Acanthopterygii, and zebrafish and cavefish belong to the superorder Ostariophysi. The selection of the chromosome blocks from Ensembl database was based both on searching the genome assemblies for the nonapeptide genes by named searches as well as BLAST searches using teleost nonapeptides as queries. This is to ensure that nonapeptide loci are obtained even in the species where the genes are not annotated. The chromosome blocks were obtained from the NCBI database mostly by navigating the genomic context of the genes. Blocks obtained from both Ensembl and NCBI were tallied for each species and a consensus assembly of genes on the blocks was arrived at by filling in for the nonannotated genes in one of the databases from information obtained from the other. Apart from the nonapeptide genes, the assemblies were also searched for the genes linked with the nonapeptide genes and the additional chromosomal blocks harboring these genes (paralogous to the linked genes), if distinct from the

### TABLE 2 | Details of the taxonomic groups and the species included in each for the phylogenetic distance calculation between groups.

The accession numbers of the sequences are given in Supplementary Table 1.

nonapeptide loci. Additionally, the loci of the linked genes in human were obtained and used as a reference tetrapod assembly for comparison of conserved synteny.

### Analysis of VT and IT Precursor Gene Expression

A two step qPCR was conducted to show tissue and seasonal expression of VT and IT genes. For tissue expression profile, various tissues viz. brain, gills, liver, muscle, kidney, and gonads were collected. For the ovarian tissue sample, the follicles were collected from the post-vitellogenic ovary and the follicular layer was separated from oocyte using hyaluronidase treatment (Mishra and Joy, 2006). Briefly, the protocol included treatment of a batch of about 500 follicles with 0.3% hyaluronidase (Type IV) for 5 min. The isolated follicular envelope (granulosa and theca cells) and the denuded oocytes were used for the expression studies. For seasonal expression, brain and ovary was collected during the different reproductive phases, resting (November–January), preparatory (February– April), pre-spawning (May–June), spawning (July–August) and post-spawning (September–October). About 100 mg tissues were used for extraction of total RNA by the single-step method of RNA isolation. To remove genomic DNA contamination from the preparation, DNAase (Ambion) treatment (2 units/10µg RNA) was given and subsequently DNAase was heat inactivated at 75◦C in presence of EDTA. RNA purity was checked by A260/A<sup>280</sup> ratio. Samples having a ratio above 1.8 were only considered for reverse transcription. Two µg of the total RNA was reverse-transcribed using random hexamer primers and Revert Aid M-MuLV reverse transcriptase in a 20µL reaction volume (first strand cDNA synthesis kit, Fermantas) using the manufacturer's protocol. The resulting cDNA was diluted 10 times and 1 µL was used in a PCR reaction of 20µL containing veriquest SYBR green 2X master mix and VT/IT FP and IT FP/ RP using manufacturer's protocol in an Applied Biosystem 7500 machine with a thermal condition of 50◦C for 2 min, 95◦C for 10 min, followed by 40 cycles of 95◦C for 15 s and 60◦C for 1 min. The specificity of the PCR product was checked by dissociation curve analysis of the amplicon and was also checked by agarose gel electrophoresis. The relative gene expression in different tissues and across different reproductive seasons was expressed using the comparative CT method with the catfish βactin (accession number FJ409641.2), used as the endogenous control. The resting phase brain cDNA was taken as the calibrator sample for relative quantity calculation (Livak and Schmittgen, 2001). Each reaction was set up in duplicate and the average CT value was taken for calculation. Graphs were plotted with the mean RQ (relative quantity) values (2−11CT) of five fish and represented as mean ± SEM. The results were analyzed by One-Way ANOVA (p < 0.001), followed by Newman-Keuls' test (p < 0.05) for statistical significance.

## Results

## Sequence Analysis of VT and IT Precursors from *H. fossilis*

The isolated full length cDNA of the HfVT precursor is 618 bp long with a cds from 60 to 524 bp and encodes a VT precursor of 155 amino acids (aa; **Figure 1**; Supplementary Figure 1A). The deduced VT precursor codes for a signal peptide (1–20 aa), a 9 aa hormone moiety (21–29 aa), a 3 aa enzymatic processing site (30–32 aa) and a 122 aa neurophysin (34–155 aa). The central part of the neurophysin (40–118 aa) is highly conserved and belongs to the hormone\_5 superfamily (NCBI CDD accession pfam00184) of the neurohypophyseal hormones (NCBI CDD accession smart00003). There are 14 cysteine residues in this region (42, 45, 53, 59, 60, 66, 76, 86, 93, 100, 106, 107, 112, and 118). The C- terminal part of the neurophysin (119–155 aa) is poorly conserved except for the presence of a leucinerich region "LLLRILH" (141–146 aa) (**Figure 1**) that is conserved across all vertebrate groups. All these features conform to the typical features of the neurohypophyseal hormones. There is no putative site for N- linked glycosylation in C-terminal domain corresponding to copeptin, making it akin to other teleost neurohypophyseal hormone precursors, unlike lungfish and tetrapod hormone precursors.

The isolated HfIT precursor cDNA is of 708 bp long with a cds from 56 to 508 bp and encodes the hormone precursor of 151 aa (**Figure 1**; Supplementary Figure 1B). Similar to the VT precursor, the putative IT precursor encodes for a signal peptide (1–20 aa), a hormone moiety of 9 aa (21–29 aa), enzymatic processing signal (30–32 aa) and a 118 aa neurophysin (34–151 aa). Further, the central part of neurophysin (40–118 bp) is highly conserved and belongs to the hormone\_5 superfamily (NCBI CDD accession pfam00184) of the neurohypophyseal hormones (NCBI CDD accession smart 00003) and there are 14 cystine residues in this region (42, 45, 53, 59, 60, 66, 76, 86, 93, 100, 106, 107, 112, 118) (**Figure 1**). The presence of an extended C-terminal part homologous to the C- terminal part of the VT precursors and the mammalian vasopressin-associated copeptin makes it similar to other IT precursor but dissimilar to other neutral peptide precursors like MT and OT precursors and other cartilaginous fish neutral hormone precursors which lack an extended C-terminal region. The C- terminal part of the neurophysin (119–151 aa) is poorly conserved except for the presence of a leucine- rich region "LLKLLHL" (138– 144 aa), which is a signature of the C-terminal region of the neurohypophyseal hormone precursors. There is neither a dibasic cleavage site nor putative site for N- linked glycosylation in this region similar to the VT precursor.

## Sequence Analysis of Other Catfish VT and IT Precursors

The cloned partial sequences of C. batrachus (Cb) VT and IT cDNA extend from the N-terminal neurophysin coding region to the 3′ UTR (Supplementary Figures 1C,D). The putative precursors, like the H. fossilis precursors, have all the essential features of the neurohypophyseal precursor proteins, with a

central conserved neurophysin having 14 cysteine residues. The poorly conserved C- terminal regions have leucine-rich boxes, "LLLRILH" in the VT precursor and "LLKLLHL" in the IT precursor (**Figure 1**).

In silico translation of the clones, recognized as the neurohypophyseal hormone precursors from the EST database of I. punctatus (Ip) by BLAST reveals that they indeed code for VT and IT precursors. The sequence read of the putative VT precursor clone (acc. no. BM495247) starts from 5′ UTR region and ends in the C-terminal part of the neurophysin. On the other hand, the putative IT precursor clone (acc. no. BE213165) sequence extends from the signal peptide region to the neurophysin (Supplementary Figures 1E,F and **Figure 1**).

The 3′ UTR region of the H. fossilis VT gene has a long stretch of 19 CA repeats. The C. batrachus VT gene has only 4 CA repeats in the 3′ UTR at approximately the same position as the H. fossilis VT gene (Supplementary Figures 1A,C).

## Analysis of Spotted Gar Neurohypophyseal Nonapeptides

BLAST search revealed that the spotted gar has three neurohypophyseal nonapeptides, VT as the basic nonapeptide (XM 006626529.2) and two nonapeptides in the neutral family, IT (XM 006626499.1) and a novel peptide (XM 006626523.1, 1–366 nt) with a unique hormone "CFISNCPLG" is present. While the spotted gar IT has an extended C-terminal with a leucine- rich core, the novel nonapeptide has a short C-terminal like all other neutral nonapeptide precursors of vertebrates (**Figure 1**).

## Tissue and Seasonal Expression of VT and IT Precursor Genes

The nonapeptide genes expressed only in the brain and ovary of the catfish (**Figure 2**). In the ovary, the expression was confined to the follicular envelope and not in oocytes. There was no expression in the gill, liver, kidney, testis and muscle. The VT and IT mRNA expressions were significantly higher in the brain than the ovary (**Figure 2**).

The sequence reads of the semi-nested 3′ RACE amplicons of VT and IT in the ovary were partial but good enough to indicate the presence of functional transcripts of the genes in ovarian follicles (not shown). Also, the sequences were similar to the brain transcripts, pointing to the same gene responsible for synthesis of the nonapeptides in both brain and ovary.

VT and IT expressions showed significant seasonal changes both in the brain and ovary (**Figure 3**). In the brain, the VT expression was low in the resting phase and increased in the preparatory, pre-spawning and spawning phases. The expression remained high in the post-spawning phase similar to the preparatory and pre-spawning phases. The expression of IT precursor was similar to that of the VT precursor but it decreased sharply in the post-spawning phase as in the resting phase.

The highest expression was observed in the spawning phase for both the genes (**Figures 3A,B**). In ovary too, the expression of both VT and IT increased in the reproductive phases compared to the resting phase (**Figures 3C,D**). However, while in the spawning phase the expression of the VT precursor was

higher as compared to the preparatory and pre-spawning phases (**Figure 3C**), the IT precursor expression remained equally high in all the reproductive phases (**Figure 3D**).

## Evolutionary History of Teleost NH Peptide Precursor Genes

### Phylogenetic Analysis

The phylogenetic inference on vertebrate NH peptide precursors was drawn from both tree constructions as well as from the evolutionary distance calculation in between groups. Both the NJ and ML trees are similar and only the ML tree is shown in **Figure 4**. The NH sequences of salmonids and catastomids (teleosts) were included in the initial analysis but were removed from the tree as they did not make a definite cluster probably due to polyploidization that led to two copies of VT and IT genes, and gene conversions (Hyodo et al., 1991; Suzuki et al., 1992). Both the phylogenetic tree and evolutionary distance calculation in between the groups (**Figure 5**) show that the NH precursor paralogs of cartilaginous fish, coelacanth and mammals

are phylogenetically closer than their homologs in other groups. The cartilaginous fish VT and neutral hormone precursors share a distance of 0.350, coelacanth VT and MT precursor share a distance of 0.132, and the mammalian VP and OT precursors share a distance of 0.219. The evolutionary distances of the NH precursors of these groups with that of other vertebrate groups are larger (**Figure 5**). Amongst the cartilaginous fish, elephant shark VT and OT precursors share a closer phylogenetic relation, as shown by the ML tree.

The phylogenetic tree with the VT precursor of Agnatha as the outgroup also shows that the teleost VT and IT precursors form a cluster with those of the spotted gar (Holostei) while other gnathostome NH precursors form another cluster, which includes the cartilaginous fish VT and neutral hormone precursors, and coelacanth and lungfish VT and MT precursors. However, the bootstrap support for the node separating the teleost NH precursors and the other gnathostome NH precursors is low.

The catfish NH precursors expectedly segregated into their respective clusters, i.e., the HfVT, CbVT, IpVT and HfIT, CbIT, IpIT, making two distinct clusters. Within these clusters, the CbVT, HfVT and CbIT, HfIT are closer to each other than to IpVT and IpIT, respectively. The spotted gar NH precursors made a common cluster; the two neutral NH peptide precursors (IT precursor and the novel peptide precursor) are evolutionarily closer than the VT precursor. The Acanthoperygii VT and IT precursors are phylogenetically closer than their orthologs in the Ostariophysi. The evolutionary distance in between the groups confirms this. The evolutionary distance between the Acanthopterygii VT and IT precursors is only 0.269 while that between Acanthopterygii VT and Ostariophysi VT precursors is 0.426, and the Acanthopterygii IT and Ostariophysi IT precursors is 0.452. The ostariophysian VT precursor form a cluster having the catfish VT precursors and one of the two VT precursors of cavefish (Astyanax mexicanus), while zebrafish (Danio rerio) VT precursor does not form part of this cluster. The ostariophysian IT precursors make a cluster that includes the catfish and zebrafish IT precursors, and one of the IT precursors of the cavefish. The other cavefish VT and IT precursors, which do not form a part of the ostariophysian clusters, make a separate cluster and share a close phylogenetic relation with each other. Unlike the Acanthopterygii VT and IT precursors, the Ostariophysi VT and IT precursors are phylogenetically distant and share an evolutionary distance of 0.422 (**Figure 5**).

## Linkage and Synteny Analysis of VT and IT in Teleosts and Spotted Gar

Details of the chromosome blocks and linkage groups used in the analysis are summarized in Supplementary Table 2. A schematic representation of conserved synteny in the spotted gar and teleosts is presented in **Figure 6**. In the spotted gar, VT, IT and the novel peptide precursors are present in the same linkage group. But, the VT precursor gene (avp-like) and IT precursor genes (oxt-like) are not arranged in tandem, unlike in human and Xenopus where the nonapeptide genes are present in tandem. However, the oxt-like gene and the novel peptide precursor gene (both in the neutral family) are arranged in tandem. Similarly, in acanthopterygian teleosts (fugu, tilapia, medaka, sticleback), the avp- like (VP- like) and oxt- like genes are in the same linkage group but are not present in tandem.

FIGURE 4 | The phylogenetic tree of vertebrate neurohypophyseal nonapeptides constructed by the Maximum-Likelihood method. The bootstrap consensus tree inferred from 500 replicates is taken to represent the evolutionary history of the taxa analyzed. The analysis involved 67 amino acid sequences. All positions with less than 95% site coverage were eliminated. There were a total of 97 positions in the final dataset. Evolutionary analyses were conducted in MEGA6. References to the sequence and species name are given in Supplementary Table 1.

In the ostariophysian teleosts, two contrasting situations exist. In zebrafish, avp- like and oxt- like are present on two different chromosomes. In contrast, in the cavefish where there are two genes each for VT and IT, one pair is present in the same linkage group like in the acanthopterygian fish. The other avp-like and oxt-like genes are present on different linkage groups so that in the cavefish, there are three linkage groups harboring the nonapeptide genes. The chromosome block in which the two genes are linked shares a conserved synteny with the acathopterygian NH loci and zebrafish avp-like containing linkage group. The other oxt-like gene is present in the paralogon of this block and shares a conserved synteny with the zebrafish oxt-like containing block and the Acanthopterygii paralogon of the NH loci. The genomic context of the other avp-like gene is considerably different from that of the teleost and spotted gar nonapeptide-harboring chromosome blocks or their paralogons. But, on a closer examination it seems to share a conserved synteny with the tetrapod nonapeptide loci as it is present in the same linkage group as the genes fast kd 5 and ddrgk (genes linked with NH genes in tetrapods) but separated from them by 13 genes.

## Discussion

## Catfish VT and IT Precursor Genes and Proteins

In the present study, we cloned a full length cDNA of 618 bp from H. fossilis that coded for a VT precursor of 155 aa. There is no difference in the sequence of the brain and ovarian transcripts and hence the same gene expresses in both the tissues. We could also clone C. batrachus partial VT precursor gene from the Nterminal neurophysin coding region to the 3′ UTR. The catfish VT precursor gene does not show any major departure from the other known teleost sequences. However, a repeat of CA dinucleotide at the 3′ UTR is unique and the number of repeats is varied in different catfishes. In H. fossilis, there are 19 repeats,

vasopressin family peptides have the three domain structure. Only in mammals, a second cleavage apart from the hormoneneurophysin cleavage has been reported, which separates the copeptin from the neurophysin. The copeptin domain possesses

and may have a regulatory role.

N- linked glycosylation sites in mammals, amphibians and lungfish (Hyodo et al., 1997) but unlike in teleosts. The catfish VT copeptin domain does not have a N-linked glycosylation site nor is cleaved. The isolated IT precursor has a multi-domain structure too and, like VT/VP precursors, has an extended Cterminal with a leucine-rich core, while no other vertebrate preprohormone in the neutral line has got an extended C-terminal.

while in C. batrachus, there are only 4 repeats. The putative Ictalurus VT precursor gene from the database do not show any CA repeat. The available sequence read ended with the coding region of the neurophysin and did not extend to the 3′ UTR region so that no CA repeat was observed. It may be inferred that the clone has CA repeats in the 3′ UTR, as it belongs to the microsatellite enriched library. It is very likely that the CA repeats may be a conserved feature of the VT precursor genes of catfishes

The deduced amino acid sequence of the VT precursors shows that, like all other members of neurohypophyseal nonapeptide precursor superfamily, it has a multi- domain structure, each having a separate function. Apart from the signal peptide, the vasopressin family peptides have a three domain structure. The nine amino acid hormone forms the smallest domain, the most biologically active region, and is released with a C-terminal amidation after a three-step enzymatic process. The second domain called neurophysin is joined to the hormone moiety with a GKR, which is the site of the three-step enzymatic process of cleavage of the hormone and amidation. The N-terminal 9 amino acid of the neurophysin is poorly conserved, while the rest is conserved. The third domain is copeptin which is poorly conserved except for the presence of a leucine-rich core (de Bree and Burbach, 1998). The catfish VT precursors like the

gar and teleost NH precursor genes and their paralogons. Human assembly is represented as a reference of tetrapod genomic context of the respective loci. The figure shows the linked situation of the genes in spotted gar (Holostei), acanthopterygian teleosts and one of the loci in cavefish, and unlinked situation in teleosts belonging to Ostariophysi. Conserved syntenic blocks have bars with the same colors. Details of genes and proteins encoded are: ubox5, ring finger protein 37; fast KD5, fast kinase domain containing protein5; lzts3b, leucine zipper, putative tumor suppressor family

## Tissue Expression Profile of Nonapeptides and Functional Implications

In H. fossilis, VT and IT genes are expressed only in the brain and ovary. In a previous study, the distribution of VT has been reported in the preoptico-hypophyseal neurosecretory system of the catfish (Singh and Joy, 2008). Brain VT showed seasonal reductase; smyd1, SET and MYND domain containing protein; fabp1, fatty acid binding protein1; thnsl2, threonine synthase like; avp, arginine vasopressin; oxt, oxytocin; znf367, zinc finger domain protein 367; slc35d2, UDP- N- acetylglucosamine transporter; ccl28, c-c motif chemokine 28; prosapip1, prosap interacting protein 1; dqx, DEAQ box RNA dependent ATPase; znf366, zinc finger protein 366; ptpra, protein phosphatase receptor type A, GnRH2, gonadotropin releasing hormone 2; cdc14b, cell divison cycle 14b; unch, uncharacterized gene.

variation associated with the annual reproductive cycle of the catfish with an increase during the recrudescent phase and a decrease after spawning. The expression of the VT and IT genes shows differences in tissue and seasonal transcript levels. The transcript levels are high in the brain than ovary. In the ovary, the expression was confined to the follicular layer since the denuded

oocytes did not show any signal. In the seasonal study, the VT and IT precursor transcripts in the brain and ovary show significant variations with higher levels during the reproductive phase, suggesting a prominent influence of the reproductive factors on the transcriptional activity of the genes. The expression of both VT and IT precursor genes in the ovary points to local synthesis of the peptides for a paracrine role in ovarian activity. This confirms the earlier findings from our laboratory showing HPLC characterization of both VT and IT, and immunocytochemical localization of VT in the follicular layer (Singh and Joy, 2008). The ovarian VT levels vary with the annual ovarian cycle closely associated with recrudescence and spawning (Singh and Joy, 2008). Moreover, steroid hormones estradiol-17β and 17, 20 β- dihydroxypregnen-3-one, the maturation- inducing steroid, modulate VT secretion as in the brain (Singh and Joy, 2009a, 2011). In vitro studies with ovarian follicles have showed that VT stimulated steroidogenesis, oocyte final maturation, oocyte hydration, ovulation and prostaglandin secretion (Singh and Joy, 2010, 2011; Joy and Singh, 2013). Gwee et al. (2009) have shown VT expression in the ovary of elephant shark. Bobe et al. (2006) have reported VT and IT mRNAs in the preovulatory ovaries of rainbow trout, while the transcripts were not detected in the ovary during the other stages of reproduction. In mammals, there are several reports of the presence of VP and OT in nonneuronal peripheral sites, where they have a paracrine role (Wathes et al., 1983; Clements and Funder, 1986; Sernia et al., 1994; Mechaly et al., 1999). Similarly, in other classes of vertebrates like aves, presence of VT has been reported in the reproductive tissues (Saito and Grossmann, 1999). The peripheral expression of VT and its homolog signifies a phylogenetic pattern and the phenomenon seems to be conserved in all major groups of vertebrates.

## Evolutionary History of Teleost NH Precursors from Phylogenetic Analysis, Genomic Locations and Synteny Analysis

Phylogenetic inference from the present study shows that the precursor paralogs in some vertebrate groups (cartilaginous fish, coelacanth, mammals) are closer than their homologs in other groups. Similarly, studies in salmons (Hyodo et al., 1991; Suzuki et al., 1992) and flounder (Warne et al., 2000) showed that the VT and IT precursors share high sequence identity. Gwee et al. (2008, 2009) also showed that the sequence identity between the respective precursor paralogs is very high in coelacanth and elephant shark. The studies also reported that the high sequence similarity was mostly in the central portion of the neurophysin. Events of gene conversion between the paralogous genes may be responsible for diluting the distance created by the long evolutionary history and make them seem more closely related than what they actually are, typifying "concerted evolution." Concerted evolution is common in duplicated genes, for example, as reported in rRNA genes, the highly conserved histone and ubiquitin gene families, and the heat shock protein gene family (Nei and Rooney, 2005). For the NH precursors, gene conversions seem to encompass the central neurophysin region and, therefore, the high sequence similarity in the central portion. Further evidence in favor of frequent events of gene conversion in between the nonapeptide paralogs is given for the bovine VP and OT precursor mRNA and avian NH precursors, having their central neurophysin not merely similar but identical pointing to a recent event of gene conversion (Ruppert et al., 1984; Levy et al., 1987). Gwee et al. (2008) found a low GC3 content in all the three exons of coelacanth VT and MT precursors as compared to bovine and human VP and OT genes, and attributed the sequence similarity in coelacanth to purifying selection rather than due to gene conversion. In mammals and yeast, gene conversion events are known to be GC-biased so that the GC content increases after gene conversion (Galtier, 2003; Marais, 2003; Noonan et al., 2004; Duret and Galtier, 2009). However, a recent study showed that gene conversions might not always be GC-biased (Assis and Kondrashov, 2012) and in such a scenario one of the evidence in favor of gene conversion can be incompatibility between phylogenetic tree and known duplication history (Mansai and Innan, 2010), as is the case in the present study. The neurophysin is only concerned with precursor processing and axonal transport which is common to all nonapeptides in contrast to the hormone moieties that evolved under the constraints of stringent and differential ligandreceptor selectivity in the two families. Hence, gene conversion events encompassing only the neurophysin might have occurred during evolution. This might also be facilitated by the 3-exon structure of the nonapeptide genes where the central neurophysin is encoded by a separate exon, i.e., the 2nd exon.

Based on the gene structures of salmon VT and IT, Urano and Ando (2011) had proposed a distinct and independent origin of the teleost IT as compared to the neutral hormone precursors of other vertebrates. Evidence in support of it is the presence of an extended C-terminal in IT precursors with a leucine-rich core, like in VT/VP. However, the authors have added that the molecular divergence of VT and IT in teleosts is complicated because of the 3R. The recent addition of the spotted gar sequences in the database and the analysis in the present study has prompted a re-apprisal. The presence of IT with the extended C-terminal in the spotted gar suggests an origin of IT in the actinopterygian lineage before the 3R. The spotted gar has an additional neutral peptide with a short C-terminal, which may be related directly to the evolutionary line of other vertebrate neutral hormone precursors that has originated at the base of the gnathostome lineage. In no teleost studied till date, a neutral peptide with a short C-terminal has been reported. Therefore, it may be hypothesized that the neutral hormone precursor gene with the short C-terminal might have originated very early in vertebrates, as documented in the cartilaginous fish (Hyodo et al., 2004; Gwee et al., 2009) and this early neutral hormone precursor might be the evolutionary forerunner of MT and OT. In teleosts, this neutral hormone precursor gene might have been secondarily lost or needs to be traced in the teleost lineage.

An interesting result that surfaced due to the addition of the catfish nonapeptide precursor sequences in the phylogenetic study is about the differences in the evolutionary distance shared by the precursor paralogs of different groups within teleosts. While the Acanthopterygii VT and IT precursors share a very small evolutionary distance (0.269) between them, the Ostariophysi VT and IT precursors are phylogenetically distant (0.422). The distance between the VT precursors and IT precursors of Acanthopterygii and Ostariophysi is also high (0.426 and 0.452, respectively). Phylogenetic clustering of the teleost NH precursors further supports these results. In the phylogenetic tree, the Acanthopterygii VT and IT precursors clustered together and seemed to have diverged from a common lineage. While the Ostariophysi IT precursors formed a definite cluster that included the catfish IT precursors, one of the IT precursors of the cavefish and the zebrafish IT precursor. The VT precursors of the catfish and one of the cavefish make a cluster which does not include the zebrafish VT precursor. The results from the distance calculation between the groups and the phylogenetic tree suggest that underlying the apparent homogeneity in all teleosts having VT and IT, there might be a difference in the origin of VT and IT precursors in different groups, i.e., these may be lineage-specific paralogs.

The analysis of the genomic loci of the NH hormone genes and the synteny analysis of the loci in different groups of teleosts further support the above assumption. Gwee et al. (2008, 2009) showed that in coelacanth, lamprey, and elephant shark (cartilaginous fish), the NH gene loci has a conserved synteny with the tetrapod NH loci. In fugu, there is a rearrangement. In the present study, the NH gene loci were studied in the spotted gar and teleosts belonging to different groups, which are known to have suffered independent gene losses during the process of diploidization (Garcia de la serrana et al., 2014). Our study indicates that, indeed, the loci and 3R paralogons show the footprints of rearrangements and differential losses of the NH gene paralogs in the different teleost lineages. On examination of the different genes linked with the nonapeptide genes in the spotted gar chromosome segment and the human linkage groups (as a tetrapod reference), it may be inferred that a rearrangement in the position of the genes in the nonapeptide loci of the spotted gar occurred by the fusion of the two blocks of chromosomes after the origin of IT in the actinopterygian lineage, disrupting the tandem arrangement of the genes, before the 3R. After the 3R, in teleosts the two paralogous blocks (3R paralogons) harboring the nonapeptide genes suffered further rearrangement and differential gene losses during the process of diploidization. The rearrangement was common judging from the conserved synteny in both the NH gene loci and its paralogon in all teleosts. However, the gene losses in these loci were differential in the two teleost superorders Acanthopterygii and Ostariophysi. This led to the linked and nonlinked avp-like and oxt-like genes in these different groups. In the acanthopterygian lineage, both the genes might have been lost from the same paralogon maintaining the linked situation of the two genes similar to the spotted gar, i.e., the pre 3R genome. In the zebrafish, avp like and oxt-like genes were lost one each from two paralogons so that a single copy of the NH hormone genes was maintained and they remained unlinked. It is difficult to reconstruct the sequence of events that can explain the present picture of synteny in the NH loci and also the inconsistent phylogenetic relation between the VT and IT precursors in the different groups of teleosts. However, it may be inferred that in the base lineage of acanthopterygians, after one pair of VT and IT precursor genes was lost from one of the 3R paralogon, an event of gene conversion might have occurred between the remaining pair. This might have occurred in the time period between the 3R and the explosive adaptive radiation and could explain the phylogentic relation of the Acanthopterygii VT and IT precursors. The Acanthopterygii VT precursors and Acanthopterygii IT precursors make two distinct clusters but share a very close phylogenetic relation. In the Ostariophysi, the loss of the NH precursor paralog was differential as compared to the Acanthopterygii with VT and IT precursor genes being lost one each from the two 3R paralogons. Rapid speciation event might have occurred after this. Gene conversion might not have occurred in the ostariophysian lineage. This explains the unlinked state of the NH paralogs in the zebrafish and also the distant relation shared by the VT and IT precursors of the Ostariophysi. The situation in the cavefish is difficult to explain. It is likely that the VT precursor gene was lost from one of the 3R paralogons while in the other both genes were retained so that a pair of linked genes remained. An independent event of gene conversion might also have occurred in between the two linked genes explaining the close phylogenetic relation between the two. The evolutionary history of the VT precursor gene of the cavefish which is unlinked can be easily traced phylogenetically as it clusters with the catfish VT precursors. However, the synteny analysis shows that it is present in a unique genomic context which shares a conserved synteny with neither the tetrapod nor the teleost NH loci, making it impossible to arrive at a possible origin of this gene. Genomic information of the NH loci from more teleost species belonging to the Ostariophysi may be helpful in explaining this. Our future direction of work would involve isolating the NH gene loci in the catfish so that the evolutionary history of the VT and IT genes in the Ostariophysi may be inferred more clearly. With the available information from the phylogeny and synteny analysis, it is clear that in the different groups of teleosts the VT and IT precursors are lineage-specific paralogs arising from differential loss of the 3R paralogs in the different superorders. The independent yet consistent retention of VT and IT in the different groups despite the catastrophes of genome duplications, diploidizations and gene conversions might have been directed by stringent ligand- receptor selectivity, established early during the vertebrate evolution.

## Conclusion

The complete VT and IT precursors were cloned from H. fossilis, and partially from C. batrachus. The deduced precursor proteins have the characteristics of the NH precursors. The precursors express in the brain and follicular envelope of the ovary. The expression in both brain and ovary shows seasonal variation with higher expression in the reproductive phases. The phylogenetic analysis of the vertebrate NH precursors and the synteny analysis of the NH gene loci show that the VT and IT precursors are lineage-specific paralogs in the superorders of Teleostei (Acanthopterygii and Ostariophysi).

## Acknowledgments

This study was supported by a Department of Science and Technology, New Delhi Research Grant (No. SA/SO/AS-43/2009) to KPJ and RC. PB is grateful to the Council of Scientific and Industrial Research, New Delhi for Junior and Senior Research Fellowships. We thank the Coordinator of DBT- BHU Interdisciplinary School of life Sciences for providing the qPCR facility.

## References

## Supplementary Material

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fnins. 2015.00166/abstract

linking two neurohypophyseal precursor lineages. Biosci. Rep. 8, 631–636. doi: 10.1007/BF01127675

amphibians through vasotocin precursor processing. Proc. Natl. Acad. Sci. U.S.A. 88, 5272–5275. doi: 10.1073/pnas.86.14.5272

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Banerjee, Chaube and Joy. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# AMPK: a master energy regulator for gonadal function

Michael J. Bertoldo<sup>1</sup> , Melanie Faure<sup>2</sup> , Joëlle Dupont <sup>2</sup> and Pascal Froment <sup>2</sup> \*

<sup>1</sup> Discipline of Obstetrics and Gynaecology, School of Women's and Children's Health, University of New South Wales, Sydney, NSW, Australia, <sup>2</sup> Unité de Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique, UMR85, Nouzilly, France

From C. elegans to mammals (including humans), nutrition and energy metabolism significantly influence reproduction. At the cellular level, some detectors of energy status indicate whether energy reserves are abundant (obesity), or poor (diet restriction). One of these detectors is AMPK (5′ AMP-activated protein kinase), a protein kinase activated by ATP deficiency but also by several natural substances such as polyphenols or synthetic molecules like metformin, used in the treatment of insulin resistance. AMPK is expressed in muscle and liver, but also in the ovary and testis. This review focuses on the main effects of AMPK identified in gonadal cells. We describe the role of AMPK in gonadal steroidogenesis, in proliferation and survival of somatic gonadal cells and in the maturation of oocytes or spermatozoa. We discuss also the role of AMPK in germ and somatic cell interactions within the cumulus-oocyte complex and in the blood testis barrier. Finally, the interface in the gonad between AMPK and modification of metabolism is reported and discussion about the role of AMPK on fertility, in regards to the treatment of infertility associated with insulin resistance (male obesity, polycystic ovary syndrome).

Keywords: AMPK, testis, ovary, germ cells, fertility

## Introduction

The 5′ AMP-activated protein kinase (AMPK) is a serine/threonine heterotrimeric kinase composed of one catalytic α-subunit bound with β- and γ-regulatory subunits. The genes encoding the three subunits of AMPK are highly conserved in eukaryotic species including vertebrates, invertebrates, plants, fungi, and protozoa (Hardie et al., 2003; Ghillebert et al., 2011). Activation of AMPK occurs with the phosphorylation of the α-subunit at Threonine 172. AMPK is sensitive to the AMP to ATP ratio and is activated by an increasing AMP concentration and by the upstream kinases including liver kinase B1 (LKB1) and calcium/calmodulin (CaM) kinase (CaMKK) (Woods et al., 2003; Hawley et al., 2005). It can also be dephosphorylated by phosphatases [Protein kinase phosphatase-1 and -2A (PP2A and PP2C)] (Lu et al., 2010; Joseph et al., 2015). AMPK is activated in pathophysiological situations (exercise, stress), by metabolic hormones (leptin, adiponectin, ghrelin) or pharmacological agents [5-aminoimidazole-4-carboxamide-1-β-D-riboside (AICAR), metformin and thiazolidinediones (TZD)] (Hardie, 2015). It regulates energy homeostasis by maintaining constant intracellular ATP concentrations by stimulation of catabolic pathways and inhibition of anabolic pathways (Hardie, 2015). Several studies have also shown that AMPK is expressed in gonads (Tosca et al., 2005; Dupont et al., 2012; Tartarin et al., 2012a) and could play a key role in the reproductive function in linking the gonadal axis with energy balance. More precisely, AMPK is present in ovarian (granulosa and theca cells, oocytes and corpora lutea) and testicular (Sertoli, leydig and germinal cells) cells in different species [oyster (Guévelou et al., 2013),

### Edited by:

Hubert Vaudry, University of Rouen, France

### Reviewed by:

Luis J. Garcia-Marin, University of Extremadura, Spain Maria Fernanda Riera, Centro de Investigaciones Endocrinologicas-CONICET-GCBA, Argentina

### \*Correspondence:

Pascal Froment, Unité de Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique, UMR85, 37380 Nouzilly, France<EMAIL_ADDRESS>
### Specialty section:

This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Neuroscience

Received: 09 March 2015 Accepted: 19 June 2015 Published: 14 July 2015

### Citation:

Bertoldo MJ, Faure M, Dupont J and Froment P (2015) AMPK: a master energy regulator for gonadal function. Front.  This kinase controls gonad steroidogenesis and germinal cell maturation but also cell proliferation and survival, polarity, formation, and maintenance of cellular junctional complexes, and cytoskeletal dynamics. In this review we report briefly some of the known functions of AMPK in the female and male gonad, and then we describe the potential role of this kinase in the interactions between metabolism and gonadal function. Most of the studies and conclusions are based on animal studies. However, we reported human studies (about granulosa and thecal cells or human embryonic testis) when it was possible.

## Gonadal Steroidogenesis

In female mammals and birds, the role of AMPK has been studied in detail in granulosa cell cultures by using pharmacological agents and adenovirus-mediated expression of dominant negative forms of AMPK (Tosca et al., 2005). AMPK activators inhibit the secretion of progesterone and/or estradiol by granulosa cells in mammals (Tosca et al., 2005, 2007a). In rat and bovine species, this inhibition is associated with a decrease in 3beta-hydroxysteroid dehydrogenase (3β-HSD) mRNA and protein levels and a decrease in MAP kinase (MAPK) extracellular signal-regulated kinases (ERK) 1/2 phosphorylation (Tosca et al., 2005, 2007a, 2010). In rats, AMPK activation induced by metformin does not reduce aromatase expression and estradiol production. However, it decreases progesterone synthesis and the expression of different proteins involved in steroidogenesis [3β-HSD, cytochrome P450 (CYP11A1), steroidogenic acute regulatory protein (StAR)] (Tosca et al., 2006b). Metformin reduces follicle-stimulating hormone (FSH) but not forskolin-stimulated aromatase expression and activity in an AMP-activated protein kinase-independent manner in a human granulosa cell line (Rice et al., 2013). Also in human granulosa cells, metformin decreases androgen synthesis, by directly inhibiting cytochrome P450 17alpha-hydroxylase (Cyp17) activity (La Marca et al., 2000). In contrast, it has been shown that AMPK could improve androgen production by adrenal cells (Hirsch et al., 2012). Even if no studies have shown a role of AMPK in ovarian steroidogenesis in vivo, this has been largely demonstrated in vitro.

A total α1AMPK knock out mouse model has been developed (Tartarin et al., 2012a). The male α1AMPK−/<sup>−</sup> have high levels of testosterone that are not due to adrenal disorders or to glucorticoid resistance but to hyperactive Leydig cells (Tartarin et al., 2012a). Indeed, the Leydig cells of these animals have an increased volume, an altered endoplasmic reticulum area, a high intratesticular cholesterol concentration and a greater expression of proteins involved in steroid production (Tartarin et al., 2012a). These data accord with those described previously in vitro in granulosa cells but also in Leydig cells in response to a modulation of AMPK activity either pharmacologically or genetically. Certainly in MA-10 and MLTC-1 Leydig cells, AMPK activation inhibits cAMP-induced steroidogenesis by repressing the expression of key regulators of steroidogenesis, including the cholesterol carrier, StAR and the nuclear receptor Nr4a1 (Abdou et al., 2014). In the latter study, the authors suggest that some AMPK-sensitive element(s) containing sites for nuclear receptors of NR4A1 are located in the StAR promoter and are required for elevated cAMP dependent activation (Abdou et al., 2014). This suggests that activation of AMPK reduce the activity of NR4A1 and StAR expression. Furthermore, in primary rat Leydig cells, resveratrol, an AMPK agonist impairs human chorionic gonadotropin (hCG)-mediated testosterone production by repressing StAR expression (Svechnikov et al., 2009). In humans, the association of increased steroid production and the inhibition of AMPK could be associated to the Peutz-Jeghers Syndrome (PJS) (Ham et al., 2013). Peutz-Jeghers syndrome is an autosomal-dominant disorder that arises as a consequence of mutations in the serine/threonine kinase 11 (STK11) gene that encodes LKB1. In these PJS patients, excess estrogen and an increase in testicular aromatase expression is associated with a decrease in AMPK phosphorylation in the testis (Ham et al., 2013). Thus, AMPK could be a molecular modulator that inhibits gonadal steroidogenesis to preserve cellular energy homeostasis and prevent excess steroid production.

An important study for human health found that when human and mouse fetal testes were cultured in the presence of metformin, there was a reduction in testosterone secretion and mRNA of key factors which are involved in steroidogenesis (Tartarin et al., 2012b). This was also associated with an increase in lactate production. Furthermore, in vivo administration of metformin during pregnancy reduced the testicular size of fetal and neonatal testes. Although the number of germ cells was not altered by metformin treatment, the number of Sertoli cells was reduced in both fetal and neonatal testes. Interestingly the androgen producing Leydig cell population was only reduced in the fetal period at 16 days post-coitum (Tartarin et al., 2012b). This study presented a potentially harmful effect of metformin treatment on the development of fetal testes (Tartarin et al., 2012b). These effects were likely to result from a metforminstimulated AMPK-mediated reduction in cellular proliferation (Kayampilly and Menon, 2012), indicating that the reduction in steroidogenesis occurred as a result of reduced testicular growth.

## Proliferation and Survival of Somatic Gonadal Cells

Gonadal somatic cells comprise the granulosa, cumulus and theca cells of the ovary, and the Sertoli and Leydig cells of the testis. Proliferation and survival of somatic cells are indispensable for fertility. Indeed, it is well known that proliferating granulosa cells support the progression of follicular growth and oocyte maturation. In males, testis size and sperm production are directly correlated to the total number of adult Sertoli cells. Regulation of proliferation and survival processes involves different hormones including FSH. As AMPK has previously been described as inhibiting proliferation of somatic cells (Tosca et al., 2010; Hardie, 2011; Kayampilly and Menon, 2012; Riera et al., 2012), we will examine the proliferative role of AMPK for these critical cell types.

In C. elegans, AMPK promotes survival and arrests germline development during nutrient stress (Fukuyama et al., 2012). More precisely, AMPKα1 and AMPKα2 (aak-1 and aak-2), the two catalytic α subunits of AMP-activated protein kinase, regulate germline quiescence by suppressing activity of target of rapamycin complex 1 (TORC1) that is involved in cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription. Similarly in rat Sertoli cells, Riera et al. (2012) observed that activation of AMPK induces a decrease in FSH-stimulated Sertoli cell proliferation through a phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mTORC1 mechanisms but also an increase in cyclin dependent kinase inhibitor (CDKI, p19INK4d, p21Cip1, and p27Kip1) expression (Riera et al., 2012). In agreement with these data, Tosca et al. (2010) observed that metformin-induced AMPK activation reduces cell growth, protein synthesis and MAPK ERK1/2 and ribosomal protein S6 kinase (p90rsk) phosphorylation in response to insulin-like growth factor 1 (IGF1) in cultured bovine granulosa cells. Furthermore, Kayampilly and Menon observed that exposure of rat granulosa cells with a pharmacological activator of AMPK increased p27kip expression, an inhibitor of the cell cycle (Kayampilly and Menon, 2009). These latter authors have also observed that activation of AMPK induced by dihydrotestosterone (DHT) treatment decreases granulosa cell mitogenesis and consequently could contribute to ovulatory dysfunction observed in hyperandrogenic states (Kayampilly and Menon, 2012).

## Maturation of Germ Cells

## The Oocyte

The role of AMPK in mammalian oocyte maturation is strikingly species specific. AMPK improves resumption of oocyte meiosis in mice (Chen et al., 2006; Downs and Chen, 2006; Larosa and Downs, 2007; Chen and Downs, 2008) but not in rats (Downs, 2011) and pharmacological activation of AMPK blocks nuclear oocyte maturation in pigs and cattle (Mayes et al., 2007; Tosca et al., 2007b; Santiquet et al., 2014). The oocyte is reliant on the metabolism of lactate and pyruvate from the tricarboxylic acid cycle (TCA cycle) and oxidative phosphorylation for most of its energy stores (Biggers et al., 1967; Leese and Barton, 1984; Roberts et al., 2002). In human granulosa cells, AMPK could be also involved in lactate production which is important for follicular development (Richardson et al., 2009). The cumulus-oocyte complex (COC) also metabolizes glucose via numerous important pathways such as the pentose phosphate pathway and glycolysis. These metabolic pathways are critical for successful oocyte maturation and resumption of meiosis (Downs and Mastropolo, 1994; Downs et al., 1996; Sutton et al., 2003a,b). Cyclic adenosine monophosphate (cAMP), synthesized by adenylate cyclase downstream of the pentose phosphate pathway, is an important negative regulator of meiotic maturation. It is well known that degradation of cAMP by phosphodiestrases triggers resumption of meiosis. The depletion of cAMP results in an increase in the AMP/ATP ratio. As AMP levels rise, AMPK is triggered and activates a number of enzymes involved in energy producing pathways and inhibiting energy consuming pathways (Downs et al., 2002). This makes AMPK critically important for oocyte developmental competence.

In mouse oocytes, the α1AMPK subunit is more abundant than α2AMPK. Immunolocalization of the α1 catalytic subunit of AMPK showed an association with condensed chromatin and the meiotic spindle but not in the spindle poles or midbody. In the absence of α1AMPK specifically in the oocyte, a decrease in mdm2 protein level, a strong negative regulator of p53, leads to an increase in the p53 content and probably induces cell cycle arrest as shown by the few number of oocyte fertilized in IVF or by the lower litter size in comparison to control mice (Bertoldo et al., 2014b, 2015). AMPK activation increases the rate of germinal vesicle breakdown (GVBD), spindle formation and polar body (PB) extrusion whereas the kinase has no effect on peripheral movement of the spindle. The meiosis-inducing actions and localization of AMPK are regulated by microtubule spindle integrity during mouse oocyte maturation (Ya and Downs, 2014). Interestingly, in mice, fatty acid oxidation is required for AMPKinduced maturation in vitro (Vasangkar and Downs, 2013).

The AMPK activator, AICAR, is a potent stimulator of maturation in mouse cumulus cell-enclosed oocytes (CEO) and denuded oocytes (DO), but only marginally stimulatory in rat CEO and ineffective in rat DO (Downs, 2011). AICAR and compound C produced contrasting results on polar body formation in cultured CEO in rat and mouse. Active AMPK was colocalized with chromatin after GVBD in rat and mouse oocytes, but did not appear at the spindle poles in rat oocytes as it did in mouse oocytes. These data highlight significant differences in meiotic regulation between the two species (Downs, 2011).

Contrary to results obtained with mouse oocytes, bovine and porcine oocyte meiosis is inhibited by activators of AMPK which is activated by AMP, the degradation product of cAMP (Bilodeau-Goeseels et al., 2007; Mayes et al., 2007; Bilodeau-Goeseels, 2011). During oocyte in vitro maturation (IVM), Santiquet et al. (2014) cultured porcine oocytes in the presence of AICAR and assessed the oocytes response in reference to oocyte nuclear maturation and cumulus cell expansion. Nuclear maturation was inhibited, however, this effect was only observed in cumulus enclosed oocytes, suggesting that cumulus cells are essential for AICAR's effect on oocyte maturation. In addition, AICAR inhibited cumulus expansion, which normally occurs in response to FSH and/or epidermal growth factor (EGF) during IVM (Harper and Brackett, 1993; Lonergan et al., 1996; Ritter et al., 2015). The results in porcine are supported by those in bovine where supplementation of metformin during embryo in vitro production resulted in AMPK mediated activation of TSC2 (Pikiou et al., 2015) and probably a reduction in TOR complex signaling and protein synthesis inhibition. In bovine COCs, metformin blocks meiotic progression at the germinal vesicle stage, activates AMPK, and inhibits MAPK3/1 phosphorylation in both the oocytes and cumulus cells during in vitro maturation. Moreover, cumulus cells were essential for the effects of metformin on bovine oocyte maturation, whereas MAPK ERK1/2 phosphorylation was not (Tosca et al., 2007b). While the precise targets of AMPK in the COCs are not entirely known, AMPK has been shown to modulate protein synthesis in various cell types (Hormon et al., 2002; Proud, 2004) and proteins involved in communication with somatic cells (see next section).

As observed in the large animal species, AMPK signaling keeps nemertean oocytes from maturing (Stricker et al., 2013). Unlike in mice, where the onset of oocyte maturation (germinal vesicle breakdown, GVBD) is blocked by cAMP and triggered by AMPK, oocytes of the marine nemertean worm Cerebratulus undergo GVBD in response to cAMP elevations and AMPK deactivation (Stricker, 2011). In addition these effects are observed only in the absence of the surrounding somatic cells (Stricker et al., 2010). These results also provide evidence for a novel GVBD-regulating mechanism involving AMPK deactivation by cAMP-mediated S485/491 phosphorylation and further highlight the highly species specific effects of AMPK in regard to oocyte maturation.

 In oyster, it is more highly expressed in male gonad than in female and its expression is more important during the first stage of gametogenesis when germ cells proliferate (Guévelou et al., 2013).

To traverse the female reproductive tract, it is essential that mammalian spermatozoa acquire the functional competence to achieve this objective in order to successfully fertilize the oocyte. These functional markers include motility, capacitation, hyperactivation and the acrosome reaction (Hurtado de Llera et al., 2015). It was recently demonstrated that AMPK protein is highly expressed in ejaculated boar and chicken spermatozoa (Hurtado de Llera et al., 2012a; Nguyen et al., 2014), and in mouse epididymal sperm (Tartarin et al., 2012a; Bertoldo et al., 2014a) and that it localizes in the head of the spermatozoon and in the midpiece of the flagellum (Hurtado de Llera et al., 2013). In the boar, pharmacological inhibition of AMPK lead to a reduction in motility (Hurtado de Llera et al., 2012a) while concomitantly causing changes in mitochondrial membrane potential, sperm plasma membrane fluidity and organization and acrosome integrity (Hurtado de Llera et al., 2013; Martin-Hidago et al., 2013). Similar results have been described in the chicken. These studies highlight the conservation of the AMPK function in sperm activity.

Interestingly, Hurtado de Llera et al. noted that the majority of studies to date that had studied sperm physiology as a function of AMPK had only done so under conditions where AMPK was inhibited (Hurtado de Llera et al., 2012b, 2015) or was genetically silenced (Tartarin et al., 2012a). Consequently they conducted a study to assess sperm physiology while activating AMPK indirectly. They observed under extended periods (24 h) of AMPK activation, boar spermatozoa had reduced motility, acrosome membrane integrity and organization and fluidity of the plasma membrane which was associated with an increase in lipid disorganization (Hurtado de Llera et al., 2015). As these processes are critical under the different environmental conditions experienced by spermatozoa when transiting through the female reproductive tract to accomplish fertilization, it becomes obvious from studies carried out so far that an optimal level of AMPK activation is essential for regulating spermatozoa function (Hurtado de Llera et al., 2015).

Nakada et al. demonstrated that spermatogenesis is intimately linked to mitochondrial respiration (Nakada et al., 2006), and recently Pellicione et al. associated asthenozoospermia with abnormal mitochondrial ultrastructure (Pelliccione et al., 2011). Therefore, it is likely that the motility disturbances observed in our α1AMPK KO model and in the LKB1 KO model that presented with abnormalities in spermatozoa functionality and morphology (motility and head morphology) (Towler et al., 2008; Tartarin et al., 2012a), was directly linked to mitochondrial dysgenesis. Similarly, incubation of sperm from boar with an AMPK inhibitor, compound C, lead to a reduction in motility (Martin-Hidago et al., 2013). Surprisingly, a mouse model inactivated for an oxidative stress sensor protein like glutathione peroxydase 4 (Liang et al., 2009; Schneider et al., 2009) is described with structural abnormalities in spermatozoa analogous to those observed in α1AMPK KO, suggesting that mitochondria dysfunction could affect oxidative stress oxidative status.

Diabetic and infertile men present with a decrease in antioxidant concentrations and an increase in ROS generation in their semen, even before cryopreservation, demonstrating that the sperm from this group are at increased risk of oxidative damage (Lewis et al., 1995; Garcez et al., 2010). Several studies have demonstrated that metformin can reduce the levels of oxidative DNA damage and afford anti-oxidant protection (Attia et al., 2009; Onken and Driscoll, 2010; Martin-Montalvo et al., 2013). Metformin has been shown to guard against diabetesinduced genomic instability in sperm cells and the bone marrow of diabetic rats (Attia et al., 2009), so we can hypothesize that the use of metformin in diabetic patients would have no negative effect in the integrity of spermatozoa. However, we cannot exclude consequences on the paternal genome for oocyte fertilization. For example, treatment of murine sperm with high concentration of metformin increased histone deacetylase activity (Bertoldo et al., 2014a). Curiously, treatment of sperm with known natural activators of AMPK such as resveratrol or a synthetic activator like metformin, present positive effects such as reduction in DNA damage and lipid peroxidation (Bertoldo et al., 2014a). A study in the wood frog (Rana sylvatica), a species with a high freeze tolerance, has revealed that AMPK was more activated in liver and muscle tissue, thus presenting AMPK as a molecule with cryoprotective properties (Rider et al., 2006). Subsequently modification of AMPK has been exploited in the freezing protocols of mammalian semen (Bertoldo et al., 2014a; Cordova et al., 2014). Metformin was used in mouse semen extender (Bertoldo et al., 2014a) and following thawing, spermatozoa showed an improvement in fertilization capability in vitro. This was associated with a reduction in the number of abnormal zygotes following IVF when mouse spermatozoa was frozen in the presence of metformin compared to controls (Bertoldo et al., 2014a). AMPK was also modulated in stallion semen extender where there was an improvement in sperm quality post-thaw (Cordova et al., 2014). As the reports of AMPK

presence in spermatozoa are growing in number, we believe it is reasonable to assume that AMPK activity is likely required for optimal mammalian spermatozoa physiology.

## Germinal and Somatic Cells Interactions

## Cumulus-oocyte Complex

Gap junction communication between cumulus cells and oocytes is crucial for oocyte meiotic maturation and to acquire full developmental competence (Gilchrist et al., 2004; Gilchrist, 2011; Li and Albertini, 2013). Such that maintenance of gap junction communication and delayed meiotic resumption have been shown to increase oocyte developmental competence (Thomas et al., 2004; Gharibi et al., 2013). Gap junction communication between the oocyte and the surrounding cumulus cells is established by the formation of bidirectional channels. The connexins family, which compose gap junctions is involved in oocyte/cumulus cell communication and allows passage of ions and small organic molecules. Loss of connexin 37 and connexin 43 in mouse oocytes or cumulus cells impaired fertility through inhibiting oocyte growth and folliculogenesis (Winterhager and Kidder, 2015). Electron microscopic analysis has shown that junctions between granulosa cells and oocytes are altered or absent as in connexin 37−/<sup>−</sup> mice (Simon et al., 1997). In the mouse, deletion of α1AMPK specifically in the oocyte lead to a reduction in connexin 37 between the oocyte and cumulus cells at the Metaphase II stage which was associated with reduced fertility following IVF, and suggests a reduction in gap junction communication (Bertoldo et al., 2015). Reductions in connexin 26 and connexin 37 expression were also described in a diabetic mouse model, where oocyte quality is poor (Ratchford et al., 2008). In an non-mammalian example, Alesutan et al., demonstrated that active AMPK decreased connexin 26 abundance in the cell membrane in xenopus oocytes (Alesutan et al., 2011), suggesting disparate regulation of gap junction communication by AMPK between species.

Deletion of α1AMPK in oocytes leads to reductions in other proteins associated with intercellular communication within the cumulus oocyte complexes (Bertoldo et al., 2015). These include N-cadeherin and β-catenin (markers for adherens junctions) and occludin (a marker for tight junctions) (Bertoldo et al., 2015). The cumulus-oocyte complex interacts with granulosa cells through adhesion junctions composed of proteins such as E-cadherin and N-cadherin (Rufas et al., 2000; Machell and Farookhi, 2003). Expression of N-cadherin for example, increases throughout maturation, fertilization and early embryogenesis (Ziv et al., 2002), and N-cadherin mediated cell contact is associated with the maintenance of meiotic arrest (Peluso, 2006). Deregulation of these proteins impacts oocyte maturation, fertilization and early embryogenesis (Ziv et al., 2002; Peluso, 2006).

Furthermore in the bovine, it was recently demonstrated that the transzonal processes (TZP) that traverse the zona pellucida transfer RNA molecules from cumulus cells to the oocyte (Macaulay et al., 2014). It was proposed that these TZPs are held in place by adherens like junctions (Macaulay et al., 2014) and are critical for oocyte developmental competence. During repair of lung capillary endothelium α1AMPK promotes the development of intercellular adherens junctions by binding with N-cadherin and contributes to repair (Creighton et al., 2011). This supports the notion that AMPK may have a critical role in oocyte developmental competence by maintaining open oocyte-somatic cell communication channels through at least gap, adherens and tight junctions. Taken together the literature supports the concept that AMPK plays a crucial role in maintaining metabolic and molecular intercellular coupling between the oocyte and its somatic cells and breakdown of this coupling results in reduced oocyte developmental competence.

 Sertoli cells have an important role in the shaping of the spermatid head for example (Kierszenbaum and Tres, 2004). Different transgenic mouse models show that AMPK plays a role in intra-testicular communication. Absence of the upstream AMPK kinase, LKB1 reduced mature spermatozoa production associated with abnormal acrosome morphology and a defect in Sertoli cell polarity and testicular junctional complexes (Towler et al., 2008). Patients with Peutz-Jeghers syndrome present a similar phenotype with alteration of sperm production associated with modifications of tight junctions in the blood-testis-barrier (Ulbright et al., 2007; Chen et al., 2012; Tanwar et al., 2012). The disruption of the α1AMPK gene in the whole murine testis induced altered sperm morphology without presenting abnormalities in Sertoli cell nucleus polarization (Tartarin et al., 2012a). Nonetheless, transmission electron microscopy analyses have shown the presence of some disrupted Sertoli cell/elongated spermatid germ cell junctions (Tartarin et al., 2012a). Interestingly, a similar phenotype in sperm head or midpiece morphology has been already described in mice deleted for adhesion molecules like nectin-2 (Mueller et al., 2003) or Tslc1 (Surace et al., 2006). The fact that absence of the α1AMPK gene lead to a mild phenotype in contrast to LKB1 could be explained by the activation of LKB1 through several other AMPK-related kinases present in the testis such as microtubule-associated protein/microtubule affinity-regulating kinases (MARK2) (Bessone et al., 1999) or SNRK (Jaleel et al., 2005) as a compensatory mechanism. This hypothesis is supported by the decrease in phosphorylation of MARK in testis in LKB1-KO mice (Kojima et al., 2007; Tanwar et al., 2012). The reduction and/or incorrect relocalization of markers of adherens junctions (β-catenin and N-Cadherin) (Kopera et al., 2010) and tight junctions (occludin and ZO-1) (Kopera et al., 2010) in Sertoli cells from LKB1-KO mice suggests a loss of contact with germ cells leading probably to alteration in germ cell shape (Kleymenova et al., 2005). Notably, altered β-catenin expression has previously been described to compromise Sertoli cell function and the maturation of germ cells and lead to infertility (Lee et al., 2005; Tanwar et al., 2010; Kerr et al., 2013).

The use of the AMPK activator (AICAR) can also influence junction complex integrity in rat Sertoli cells as has been described by Galardo et al. (2010). Rat Sertoli cells incubated with AICAR stabilized ZO-1 protein as observed by immunofluorescence (Galardo et al., 2010). The use of EGTA to limit the free calcium concentration in culture medium induced a redistribution of ZO-1 between Sertoli cells. Addition of AICAR or adenosine, in the presence of EGTA permitted the rescue of ZO-1 distribution to normal conditions at the cell membranes (Galardo et al., 2010). As in oocytes, the cAMP/PKA pathway is modified in the absence of α1AMPK in Sertoli cells, raising the question about the interaction between AMPK/cAMP signaling and the functionality of the blood-testis barrier permeability. Indeed, the cAMP signal has been described to be involved for the formation and maturation of male germ cells (Scobey et al., 2001), and can perturb junctions in rat Sertoli cells (Lui and Lee, 2005).

## Interface between AMPK and Modification of Metabolism

Diet restriction is well known to promote longevity and reduce fertility in several species like C. elegans, drosophila melanogaster, birds and mammals. Diet restriction induces a negative energy balance which activates some energy sensors such as AMPK and the sirtuins which promote respiration and energy production by mitochondria. In mice, a deficiency in LKB1 or AMPK in mature Sertoli cells negatively impacts mitochondrial function, and has been associated with loss of quiescence and an activation of cell proliferation (Bertoldo et al., 2013). The association between nutrient availability, mitochondrial function and fertility has been already observed in invertebrates. In C. elegans the germinal stem cells regulate longevity through the TOR pathway (Arrantes-Oliveira et al., 2002), and similarly in drosophila TOR signaling is involved in the regulation of female germinal stem cell proliferation as a function of the availability of nutrients (Drummond-Barbosa and Spradling, 2001; Lafever and Drummond-Barbosa, 2005; Lafever et al., 2010; Shyh-Chang et al., 2013). These results are also observed in Sertoli cells where stimulation with an AMPK activator such as metformin or AICAR has consequences on lactate production and the increase in glucose transport (Galardo et al., 2007). One hypothesis of the action of metformin, is an indirect effect: an inhibition of the respiratory chain in mitochondria leading to an increase in lactate production, and in the AMP: ATP ratio inducing the activation of AMPK. On the other hand, the inactivation of α1AMPK in Sertoli cells, reduces the expression of mitochondrial markers (cytochrome c and PGC1a) and the ATP content, and increases the lactate production (Bertoldo et al., 2013). The increase in lactate, in this case, could be due to a switch in the cell between energy production by respiration to the aerobic glycolysis. Thus, α1AMPK deficiency enhances the Warburg Effect which can be associated with increased cell proliferation in vitro (Faubert et al., 2013). In addition, glycolysis could increase the allocation of glucose carbon into lipids and explain the increase in lipid vesicles. We cannot exclude that a modification in lipid metabolism in Sertoli cells has a consequence on germ cells. Indeed, some studies have described lipid transport from the Sertoli cells to the germ cells (Saether et al., 2003). Moreover, several recent studies using mice deficient in genes related to lipid metabolism, have described that the accumulation of excess lipid droplets in Sertoli cells resulted in impaired spermatogenesis (Coussens et al., 2008). Therefore, a balance of lipid metabolism in Sertoli cells is essential for normalspermatogenesis (Selva et al., 2004).

Ratchford et al. have hypothesized that abnormalities in oocyte metabolism, such as that observed in diabetes, could potentially preprogramme the oocyte for unfavorable outcomes after fertilization (Ratchford et al., 2007). Furthermore, Wang et al. (2009) concluded that maternal diabetes results in numerous oocyte deficiencies. Glucose metabolism is essential for successful oocyte maturation and the recommencement of meiosis (Downs and Mastropolo, 1994). It is well known that mitochondria can influence the developmental competence of the oocyte (Thouas et al., 2004). Certainly mitochondria play a key role in cellular energy generation, the control of cell death (Perez et al., 2000) and the dynamic process of meiosis including DNA reorganization (Wang et al., 2009). In the case of diabetes, mitochondria are abnormally distributed around the spindle or in the oocyte cytoplasm (Wang et al., 2009). Ratchford et al. observed that under hyperglycaemic conditions, phosphorylated ACC, a downstream target of AMPK and phosphorylated AMPK were both decreased in diabetic oocytes, demonstrating decreased AMPK activity (Ratchford et al., 2007). Diabetic oocytes were also metabolically perturbed leading to altered AMPK activity. Interestingly, increasing AMPK with AICAR in these oocytes during the preovulatory phase corrected the metabolic and meiotic perturbations observed. For these crucial activities in oocyte maturation, mitochondrial redistribution, activity or dysfunction have been suggested as markers of oocyte quality and are strongly related to fertilization rates and embryo development (Van Blerkom, 2004; Wang et al., 2009).

During the last decade a variety of natural ligands and synthetic ligands have been shown to activate AMPK including resveratrol (Baur et al., 2006), sulforaphane (Choi et al., 2014), niacin (Thirunavukkarasu et al., 2006), berberine (Brusg et al., 2006), metformin (Zhou et al., 2001), and thiazolidinediones (Fryer et al., 2002). Some of these compounds have nonlinear dose-response characteristics, such as that of hormesis and have the ability to inhibit the mitochondrial complex I at elevated concentrations that mimick diet restriction (Gems and Partridge, 2008). The hypothesis of hormesis lends weight to differences in phenotype associated with differences of metformin concentration. High metformin concentrations (approximately 5 mM) is enough to inhibit the respiratory chain complex 1 in mitochondria leading to an increase in the AMP/ATP ratio (El-Mir et al., 2000; Owen et al., 2000) and different metformin concentrations induce increases in oxidant defenses as well as an extension of lifespan (Onken and Driscoll, 2010; Martin-Montalvo et al., 2013). The difference in species sensitivity has been already observed as mouse tissue is 10 fold less sensitive than human tissue (Tartarin et al., 2012b).

Metformin is a good example for mimicking diet restriction, because in mouse liver, metformin has been shown to induce a similar transcription pattern to diet restriction especially (Dhahbi et al., 2005). However, similar effects have been described in C. elegans where metformin administration increases the

lifespan and produces several diet restriction-like phenotypes such as reduction in fecundity and a decrease in fat storage in animals which are fed ad-libitum (Onken and Driscoll, 2010). In drosophila, metformin exposure for 7 days at 25 and 50 mM concentration increases significantly the number of eggs laid in contrast to untreated controls. But after 14 days of treatment, egglaying in females on 25 mM metformin was similar to controls and at 50 mM of metformin the females laid significantly fewer eggs (Onken and Driscoll, 2010). Interestingly in flies, metformin targets AMPK and inhibits the TOR pathway (Kalender et al., 2010; Slack et al., 2012). From the reports to date, we can conclude that effects on fertility (increases or reductions in the number of egg laid depending the time and concentration of metformin treatment) remains partially understood and controversial (He and Wondisford, 2015).

SIRT1 is widely regarded as a critical regulator of energy homeostasis and is implicated in a wide variety of cellular processes including metabolic diseases, cancer, aging, and reproduction (Bordone and Guarente, 2005; Brooks and Gu, 2009; Haigis and Sinclair, 2010). Furthermore it is known to interact with AMPK (Fulco et al., 2008; Narala et al., 2008; Canto and Auwerx, 2009). We have recently provided evidence in the oocyte that α1AMPK could be involved in chromatin remodeling, because we observed an increase in acetylation of H3 histone in oocytes from α1AMPK knockout oocytes (Bertoldo et al., 2015). This was correlated, as expected with a reduction in histone deacetylase SIRT1 expression in vivo. In vitro Sirt1 has the ability to deacetylate histone substrates in a NAD+-dependent manner (Vaquero et al., 2004b) and hyperacetylation occurs when SIRT1 is knocked down (Vaquero et al., 2004b). Male mice deficient in SIRT1 present with altered germ cell maturation and increased DNA damage in germ cells (Coussens et al., 2008). Together, these data suggest that AMPK can modify oocyte proteins and histone acetylation status. These observations could

## References

be linked to other reports such as those relating to the aorta and heart tissue where a decrease in AMPK and SIRT1 expression is associated with increased H3 acetylation (Bendale et al., 2013). Interestingly, acetylation of histones H3 and H4 appear to be linked to an overexpression of connexin 43 in a prostate cell line (Ogawa et al., 2005; Hernandez et al., 2006), and PGC1α and p53 can modify their accessibility (Vaquero et al., 2004a; Wakeling et al., 2009; Nelson et al., 2012), possibly suggesting some level of control of intercellular communication and apoptosis. Interestingly, inadequate histone deacetylation causes changes in gene expression, which can lead to embryopathy in mice (Akiyama et al., 2006).

## Conclusion

The involvement of AMPK in fertility control is conserved throughout several animal species from the oyster, C. elegans, drosophila, birds and mammals. Its expression is present in different compartments of the ovary and testis and through the different stages of maturation of germ cells, germline stem cells to oocytes and spermatozoa. Apart from its classical functions on metabolism, proliferation and antiinflammatory effects observed in the gonad, AMPK is also able to modulate steroidogenesis, and to impact morphology and normal nuclear maturation of germ cells though interaction of germ cells with their nurse somatic cells. Some mechanisms elucidated are directly linked with mitochondrial function and junctional proteins. Despite the possibility of different sub-unit combinations of AMPK, absence of only α1AMPK leads to moderate failure of fertility in both sexes.

## Funding

This review was financially supported by ANR "Fertinergy grant."

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rat Sertoli cells and disrupts Sertoli cell-gonocyte contact. Biol. Reprod. 73, 482–490. doi: 10.1095/biolreprod.104.037184

to polycystic ovary syndrome. J. Clin. Endocrinol. Metab. 98, E1491–E1500. doi: 10.1210/jc.2013-1865

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Bertoldo, Faure, Dupont and Froment. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Hypothalamic–pituitary–gonadal axis involvement in learning and memory and Alzheimer's disease: more than "just" estrogen

### **Jeffrey A. Blair <sup>1</sup> , Henry McGee<sup>1</sup> , Sabina Bhatta<sup>1</sup> , Russell Palm<sup>2</sup> and Gemma Casadesus <sup>1</sup>\***

<sup>1</sup> Department of Biological Sciences, Kent State University, Kent, OH, USA

<sup>2</sup> University of Toledo School of Medicine, Toledo, OH, USA

### **Edited by:**

Hubert Vaudry, University of Rouen, France

### **Reviewed by:**

Jacques Epelbaum, INSERM, France Vance Trudeau, University of Ottawa, Canada

### **\*Correspondence:**

Gemma Casadesus, Department of Biological Sciences, Kent State University, 256 Cunningham Hall, Kent, OH 44242, USA e-mail<EMAIL_ADDRESS>
Accumulating studies affirm the effects of age-related endocrine dysfunction on cognitive decline and increasing risk of neurodegenerative diseases. It is well known that estrogens can be protective for cognitive function, and more recently androgens and luteinizing hormone have also been shown to modulate learning and memory. Understanding the mechanisms underlying hypothalamic–pituitary–gonadal axis-associated cognitive dysfunction is crucial for therapeutic advancement. Here, we emphasize that reproductive hormones are influential in maintaining neuronal health and enhancing signaling cascades that lead to cognitive impairment.We summarize and critically evaluate age-related changes in the endocrine system, their implications in the development of Alzheimer's disease, and the therapeutic potential of endocrine modulation in the prevention of age-related cognitive decline.

**Keywords: luteinizing hormone, menopause, ovariectomy, estrogen, testosterone, memory, Alzheimer's disease**

## **INTRODUCTION**

As the baby boomer generation continues to age and advances in medicine have resulted in longer life expectancy, it is vital to consider the eminent risk of age-associated neurodegenerative diseases, such as Alzheimer's disease (AD) and their effects on our society. The number of people diagnosed with AD is estimated to be over 13 million by 2050 (1). Furthermore, expense estimates for AD and dementia patient care in the United States alone are over \$180 billion per year, which makes dementia one of the most costly diseases (2). Not only will more individuals continue to be diagnosed and threatened with neurodegenerative disease, but their caregivers will also suffer more physical, mental, and economic burdens.

Senile plaques containing amyloid-β, neurofibrillary tangles comprised of aggregated tau and cell death in the hippocampal formation are the hallmark pathologies of AD (3). Clinically, AD is characterized by prominent deficits in memory and attention leading to deterioration of judgment, language skills, and spatial orientation as the disease progresses (4). While AD pathogenesis has been linked to oxidative stress, inflammation, and neuronal dysfunction, concrete evidence explaining disease pathogenesis is absent, compelling us and others to search beyond the hallmark pathologies for alternative molecular cues underlying AD pathogenesis (5).

Hormones are known to impact central nervous system (CNS) function and stability (6), and gonadal hormones have been extensively studied for their effect on cognition in elderly men and women (7). We believe that age-associated endocrine system dysfunction is a major factor in the onset and progression of neurodegenerative diseases. This is supported by research showing changes in gender and age-dependent reproductive hormones increases the risk of AD, therefore implicating the hypothalamic–pituitary–gonadal (HPG) axis (8–14).

### **HYPOTHALAMIC–PITUITARY–GONADAL AXIS**

The HPG axis is controlled by a negative feedback loop. In the healthy brain, the hypothalamus releases gonadotropin-releasing hormone (GnRH) into the median eminence, and then GnRH is transported via the hypophyseal portal system to the anterior pituitary where it acts on its receptor (GnRHR). Signaling from GnRHR leads to the production and secretion of the gonadotropins, including luteinizing hormone (LH) and folliclestimulating hormone (FSH). Once the gonadotropins are secreted into the blood stream, LH acts on its receptor in the gonads, which in turn stimulates the release of the sex steroids, androgens and estrogens. These sex steroids complete a negative feedback loop by inhibiting the release of GnRH.

Peripheral hormone levels have long been implicated in changes of behavior. Studies of the modulatory effects of hormones on cognition have led to the discovery that hormone receptors are expressed in the CNS (15, 16). Importantly, many of these hormone receptors are present in areas of the brain associated with learning and memory, such as the hippocampus. Crucial to HPG axis dysfunction and its connection to AD is that the rate of hormone synthesis varies throughout one's life. With age, androgen production decreases gradually in men, while there is an abrupt diminution in gonadal secretion of estrogens in women. Besides, the dysregulation of the HPG axis caused by menopause may be due to a diminished ability of estrogens to inhibit the hypothalamus (17, 18). Interestingly, in addition to the negative feedback estrogens have on the HPG axis, estrogens can also produce positive feedback, which is necessary for fertility (19) and is attenuated with age (20). This body of work strongly suggests that cognitive function may rely on the levels of sex steroids and gonadotropins, implicating HPG axis dysfunction as a factor in the strong correlation between aging and dementia.

### **ESTROGENS**

Gender has been associated as an independent risk factor for AD. Females have a higher risk of developing AD as shown by ageadjusted odds ratios regardless whether familial or sporadic AD was acquired (21, 22). The higher prevalence and incidence of AD in women has been linked to the changes in reproductive hormones that occur during menopause. Therefore, women have become a main focus of many aging studies including theWomen's Health Initiative Memory Study (9, 10). Endocrinological evaluations show lower levels of estrogens in women with AD (12, 23), thus encouraging hormone replacement therapy (HRT) to improve cognition and decrease the risk of AD in post-menopausal women (24–27). This theory is based on an abundance of evidence displaying the neuroprotective effects of estrogens observed in healthy cells. Estrogens signal through a multitude of mechanisms to induce axonal sprouting, regeneration, synaptic transmission, and the prevention of cell death (28). Estrogen receptors (ER) can activate production of brain-derived neurotrophic factor (BDNF), which has been shown to protect against ischemic injury *in vitro* and retain cognitive function as measured with passive avoidance in mice *in vivo* (29). Additionally, other*in vitro* studies have shown protective effects of estrogens from excitotoxicity by increasing the apoptosis regulator Bcl-2 (30). Moreover, estrogens have been shown to protect against oxidative stress induced by amyloid-β fibrils alone or in a complex with acetylcholinesterase, making estrogens a target for AD therapeutics (31). This hypothesis is influential in that the only current AD treatment is cholinesterase inhibitors, with which meta-analyses show a modest benefit (32).