University of Nevada, Reno

Effects of asprosin on steroidogeneses of bovine granulosa cells

A thesis submitted in partial fulfillment of the requirements for the degree of Master of

Science in Animal and Rangeland Science

By

Isadora Maria Batalha

Dr. Luis Fernando Schutz/ Thesis Advisor

December 2020 THE GRADUATE SCHOOL

We recommend that the thesis prepared under our supervision by

ntitled

be accepted in partial fulfillment of the requirements for the degree of i

Abstract

Asprosin is a novel associated with disorders, such as resistance and polycystic ovarian syndrome. Understanding ovarian steroidogenesis is imperative to understand how health and fertility could be managed. Although, asprosin impacts steroidogenesis of ovarian theca cells, its impacts on granulosa cells (GC) remains to be unveiled. Hence, the objective of this study was to investigate asprosin impacts on GC steroidogenesis. Bovine GC from small follicles of were used to investigate cell proliferation, production, and mRNA abundance of steroidogenic enzymes such as steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme

(CYP11A1), and aromatase (CYP19A1). Asprosin enhanced FSH-induced GC production of estradiol, but diminished IGF1-induced estradiol synthesis in comparison to IGF-1 alone. Asprosin decreased GC proliferation induced by IGF-1 alone or in combination with FSH. Asprosin increased CYP11A1 mRNA abundance when combined with FSH- treated or IGF-1-treated GC. Taken together, the present findings indicate that asprosin regulates GC steroidogenesis and proliferation and suggests that asprosin is a promising ovarian steroidogenic and folliculogenesis regulator. Further studies are required to unveil how asprosin affects GC function and its importance to fertility and health.

ii

“It is our choices that show what we truly are, far more than our abilities.” Albus Dumbledore Harry Potter and the Chamber of Secrets

To my mom, for her love and strength examples.

To my sisters, for their academic dedication and friendship.

To my grandparents for their unconditional support.

I dedicate. iii

Acknowledgments

First, I thank God for being so generous and give me the strength to never give up.

It is with immense gratitude that I acknowledge the support and help of my advisor, Dr. Luis Fernando Schutz, who showed me that science can be enjoyable even when difficulties cross your path. He welcomed me and put a lot of effort to make this day happen. He encouraged me and believed in my capacity to overcome challenges. Dr.

Schutz, thank you for the priceless hours of discussion. There will never be enough words, even in Portuguese, to express how thankful I am.

I owe sincere gratitude to Dr. Mozart Fonseca, for all his assistance since my very first day at UNR. I am greatly glad for his patience, insightful discussions and the opportunity to learn from him. Dr. Fonseca, thank you for sharing your expertise and beers.

I want to thank Dr. Bradley Ferguson for his kindness, availability and for inspiring me to be passionate about science. Luckily, I had the opportunity to take a class that took me into a complete “out-of-comfort zone” under his mentoring.

I would also like my appreciation to Dr. Leon Spicer, for all his collaboration and assistance to complete this study. I also would like to acknowledge Excel Maylem, Dr.

Spicer’s student, for her help and support. Thank you both for the patience, kindness and receptivity. iv

I would also like to acknowledge Dr. Tamzen Stringham for her priceless support and Dr. Coretta Patterson for being always available and to believe in my teaching abilities.

I am indebted to faculty and staff of the Department of Animal and Rangeland sciences for the support. Also, I would like to acknowledge Dr. Patricia F. Santos and Dr.

Christie Howard for sharing their equipment and good talks in the hallway.

I would like to express my deepest gratitude to many people that made the path easier during the past years:

- My lab colleagues Evandro Archilia and Camilo Pena Bello for being the best

team I ever had. Thank you for all hard work, countless laughs, and beers.

- My beloved roomie Stefania. You were the best companionship someone

could have had during the easy and hard moments.

- My best “room-memate” Felipe. Your company and care made the days

lighter and easier. Thank you for the insightful discussions, stats lessons, and

being such a good friend.

- The Californian friend I made, Reese. Thank you for taking care of me during

one of the hardest times of my journey. I would have never survived without

ice cream and Netflix.

- My colleagues in Animal Science: Karin, Francine, Lucilaine, Gabriel,

Lucien, Amanda, and Arturo for all your support.

- My friends from Brazil, Dâm, Bombom, Karina, Vivi, Dani, Ju, e Gabi for

being there for me. v

- My Team from Brazil, Beto, Daiani, Renata, and Tião for you hard work to

keep the company running with my mom.

My respectful acknowledgments may also be addressed to the animals. I hope their honorable contribution to science may be a step towards a healthier world.

Finally, I share my greatest gratitude to my family. Mom Elbia, sisters Sophia and Loren, grandparents Maria José e Joaquim, and uncle Heder, thank you! Thank you, million times, for being my strength, for your unconditional love and support.

vi

Contents Abstract ...... i Acknowledgments ...... iii List of Tables ...... viii List of Figures ...... ix INTRODUCTION...... 1 CHAPTER I ...... 4 REVIEW OF LITERATURE ...... 4

1. ASPROSIN AND ITS RECEPTOR ...... 4 1.1. Asprosin ...... 4 1.2. Asprosin receptor ...... 6 2. ASPROSIN IN THE ...... 6 2.1. Role of fibrilin-1 and furin in the ovarian physiology ...... 6 3. ASPROSIN AND POLYCYSTIC OVARIAN SYNDROME ...... 9 4. OVARIAN STEROIDOGENESIS ...... 11 4.1. StAR (Steroidogenic Acute Regulatory Protein)...... 11 4.2. CYP11A1...... 13 4.3. CYP19A1...... 13 5. WHITE ADIPOSE-DERIVED FACTORS AND OVARIAN STEROIDOGENESIS ...... 14 5.1. ...... 14 5.2. ...... 15 5.3. ...... 17 5.4. Visfatin ...... 19 6. BIBLIOGRAPHY ...... 21 CHAPTER II ...... 34 Effects of asprosin on steroidogeneses of bovine granulosa cells ...... 34 Introduction ...... 34 Material and Methods ...... 35 Reagents and ...... 35 Granulosa cell collection and culture ...... 36 Experimental design ...... 37 Radioimmunoassay (RIA) ...... 37 RNA extraction ...... 38 Quantitative real-time reverse transcription polymerase chain reaction (qPCR) ...... 38 Statistics ...... 39 Results ...... 40 vii

Experiment 1: Asprosin effects on estradiol production and proliferation of granulosa cells ...... 40 Experiment 2: Asprosin effects on mRNA relative abundance of enzymes regulating granulosa cells steroidogenesis ...... 41 Discussion ...... 41 References ...... 44

viii

List of Tables

Table 1. Information of primers...... 49

ix

List of Figures

Figure 1. Effect of asprosin on FSH- and IGF1-induced steroidogenesis in granulosa cells (GC). Estradiol production in asprosin-treated GC concomitantly treated with FSH,

IGF1 of both. Means ± SEM without a common letter differ (p <0.05)...... 50

Figure 2. Effects of asprosin (200ng/ml) on granulosa cells (GC) proliferation in presence of FSH and IGF1 or both. Means ± SEM without a common letter (a,b,c,d,e) differ (p<0.05)...... 51

Figure 3. Relative mRNA abundance of CYP11A1 in granulosa cells treated with asprosin, FSH, IGF1, or combined treatments between them. Treatments were applied for

24h. Means ± SEM without common letters (a,b) differ (p <0.05)...... 52

Figure 4. Relative mRNA abundance of CYP19A1 in granulosa cells treated with asprosin, FSH, IGF1, or combined treatments between them. Treatments were applied for

24h. Means ± SEM without common letters (a,b,c) differ (p <0.05)...... 53

Figure 5. Relative mRNA abundance of StAR in granulosa cells treated with asprosin,

FSH, IGF1, or combined treatments between them. Treatments were applied for 24h. No significant effect was observed among treatments...... 54 1

INTRODUCTION

Ovaries are a major player in female endocrine regulation. They are responsible to produce hormones that are important for reproductive function maintenance, such as ovarian folliculogenesis and ovulation (Craig, 2018). Estradiol, the main steroid hormone produced by the ovaries not only regulates reproductive functions, but also regulates and lipid metabolism

(Newell-Fugate, 2017). It is known over the years that ovaries and have a close relationship, in which cytokines produced by white adipose tissues, known as adipokines, have important effects on the reproductive system (Reverchon et al., 2014). Indeed, is an important site for steroidogenesis and a key regulator of health, in which its dysfunction can lead to and polycystic ovarian syndrome (PCOS; Newell-Fugate, 2017).

Hence, the study of interactions between ovarian steroidogenesis and adipokines is critically important to understand how health and fertility can be regulated by steroid hormones.

The reproductive efficiency of cattle is critically important for the cattle industry. For example, as milk productivity of dairy cows increased over the last decades, the fertility of these animals has declined (Lucy, 2001; Walsh et al., 2011). Among several hypotheses proposed to explain the decline in fertility of dairy cows is nutritional management and metabolic disorders

(Walsh et al., 2011). Therefore, the investigation of the impact of hormones associated with nutrition and metabolism, such as adipokines, on regulation of reproduction is important.

According to the Office on Women’s Health in the U.S Department of Health & Human

Services (2019), female infertility in most cases is due to anovulation. The most common cause of anovulatory infertility is PCOS (Prabhakar et al., 2020). Like women, bovine females may have a disrupted secretion of androgens, which is a hormonal pattern also observed in PCOS

(Summers et al., 2014). In addition, humans and bovines share similar patterns of steroid 2

hormone secretion and ovulation (Abedal-Majed & Cupp, 2019; Adams & Pierson, 1995;

Fortune, 1994; Spicer & Echternkamp, 1986). Moreover, ovarian research in humans is challenging because reproductive tissues that have not been subjected to high doses of hormonal treatments are very difficult to obtain from women at a reproductive age (Campbell et al., 2003).

Hence, it is important to understand how ovarian steroidogenesis can interfere in reproductive disorders and the bovine model is well suited for that.

Studies investigating factors associated with nutrition and ovarian steroidogenesis are important for both cattle productivity and human fertility and health. In this way, it is important to study how asprosin, a novel adipokine, can possibly interfere in the process of ovarian steroidogenesis. The objective of the present study is to evaluate how asprosin regulates steroidogenesis in ovarian granulosa cells of cattle.

Bibliography

Abedal-Majed, M. A., & Cupp, A. S. (2019). Livestock animals to study infertility in women. Animal Frontiers, 9(3), 28–33. https://doi.org/10.1093/af/vfz017 Adams, G. P., & Pierson, R. A. (1995). Bovine model for study of ovarian follicular dynamics in humans. Theriogenology, 43(1), 113–120. https://doi.org/10.1016/0093-691X(94)00015-M Campbell, B. K., Souza, C., Gong, J., Webb, R., Kendall, N., Marsters, P., … Baird, D. T. (2003). Domestic ruminants as models for the elucidation of the mechanisms controlling ovarian follicle development in humans. Reproduction (Cambridge, England) Supplement, 61(February 2003), 429–443. https://doi.org/10.1530/biosciprocs.5.032 Craig, Z. R. (2018). Plastic compounds. In Encyclopedia of Reproduction (Second Edi, Vol. 2). https://doi.org/10.1016/B978-0-12-801238-3.64410-0 Fortune, J. E. (1994). Ovarian Follicular Growth and Development in Mammals1. Biology of Reproduction, 50(2), 225–232. https://doi.org/10.1095/biolreprod50.2.225 Lucy, M. C. (2001). ADSA foundation scholar award reproductive loss in high-producing dairy cattle: Where will it end? Journal of Dairy Science, 84(6), 1277–1293. https://doi.org/10.3168/jds.s0022-0302(01)70158-0 Newell-Fugate, A. E. (2017). The role of sex steroids in white adipose tissue adipocyte function. Reproduction, 153(4), R133–R149. https://doi.org/10.1530/REP-16-0417 3

Office on Women's Health, U.S Department of health and Human Services, womentshealth.gov April 01, 2019, Accessed at https://www.womenshealth.gov/a-z- topics/infertility on November 24, 2020 Prabhakar, P., Mahey, R., Gupta, M., Khadgawat, R., Kachhawa, G., Sharma, J. B., … Bhatla, N. (2020). Impact of myoinositol with metformin and myoinositol alone in infertile PCOS women undergoing ovulation induction cycles - randomized controlled trial. Gynecological Endocrinology, 0(0), 1–5. https://doi.org/10.1080/09513590.2020.1810657 Reverchon, M., Ramé, C., Bertoldo, M., & Dupont, J. (2014). Adipokines and the Female Reproductive Tract. International Journal of Endocrinology, 2014(11), 1–10. https://doi.org/10.1155/2014/232454 Spicer, L. J., & Echternkamp, S. E. (1986). Ovarian Follicular Growth, Function and Turnover in Cattle: A Review1. Journal of Animal Science, 62(2), 428–451. https://doi.org/10.2527/jas1986.622428x Summers, A. F., Pohlmeier, W. E., Sargent, K. M., Cole, B. D., Vinton, R. J., Kurz, S. G., … Wood, J. R. (2014). Altered theca and cumulus oocyte complex gene expression, follicular arrest and reduced fertility in cows with dominant follicle follicular fluid androgen excess. PLoS ONE, 9(10), 1–13. https://doi.org/10.1371/journal.pone.0110683 Walsh, S. W., Williams, E. J., & Evans, A. C. O. (2011). A review of the causes of poor fertility in high milk producing dairy cows. Animal Reproduction Science, 123(3–4), 127–138. https://doi.org/10.1016/j.anireprosci.2010.12.001

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CHAPTER I

REVIEW OF LITERATURE

1. Asprosin and its receptor

1.1. Asprosin

Asprosin is a novel protein hormone mainly secreted by the white adipose tissue. It was first identified in 2016 in a study which showed that the Fibrillin 1 gene

(Fbn1) undergoes a 3’ truncating mutation on the last 50 nucleotides of exons 65 and 66, generating a truncated profribillin protein (Romere et al., 2016). Asprosin is the result of a C-terminal cleavage of profribillin protein, which is catalyzed by the enzyme Furin, resulting in a product of 140 amino-acids (Romere et al., 2016). The location of cleavage is unknown, but some authors suggested that it happens between the trans-Golgi network and cell surface (Ritty et al., 1999; Wallis et al., 2003).

Asprosin was first discovered during a study on patients with neonatal progeroid syndrome (NPS; Romere et al., 2016). NPS is a rare genetic condition characterized by extreme leanness, low appetite, and (Hou, 2009), which has been associated with (Bindlish et al., 2015). Although NPS patients kept euglycemia and asprosin has been reported to increase both insulin and glucose levels in mice, NPS patients showed low concentration of Asprosin in their plasma. In addition, when mutated profribillin was overexpressed in wild-types cells, the asprosin production was diminished into the media. It was suggested that the truncated profribillin 5 have a negative effect on asprosin secretion due to the fact that it is a predicted escape from nonsense mediated decay (NMD) mRNA (Romere et al., 2016).

Following its discovery, several studies have aimed to unveil the metabolic effects of asprosin. It is now accepted that this hormone is able to regulate glucose homeostasis by stimulation of hepatic via G protein-cAMP-protein kinase A pathway (Duerrschmid et al., 2017; Romere et al., 2016). Indeed, asprosin has been reported as a key hormone to control diabetes mellitus type II (Elnagar et al., 2018;

Romere et al., 2016; Zhang et al., 2019). Interestingly, besides its gluconeogenic function, asprosin has been shown to regulate appetite. Asprosin follows a circadian cycle in which its levels increase after overnight fasting and decrease right after feeding

(Romere et al., 2016). In fact, it is now established that asprosin’s orexigenic function is made through activation of Agouti-related (AgRP+) neurons by increasing firing frequency and resting membrane potential via Gαs – cAMP – protein kinase A axis. As result, anorexigenic positive neurons ( POMC) are inhibited and appetite is increased (Elnagar et al., 2018).

Some authors also reported that Asprosin is linked to the polycystic ovarian syndrome (PCOS), in which the adipose tissue dysfunction implies metabolic disruptions, such as obesity and insulin resistance (Alan et al., 2019; X. Li et al., 2018). Patients with this condition presented high levels of plasma asprosin compared to control groups. Also, asprosin levels were highly correlated to insulin resistance and free androgen index (Alan et al., 2019). Hence, it is important to understand how asprosin regulates the ovarian physiology.

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1.2. Asprosin receptor

It is currently believed that circulating asprosin binds to the to stimulate gluconeogenesis via cAMP second messenger system to activate protein kinase A (PKA)

(Romere et al., 2016). Indeed, glucose production mediated by asprosin is impaired when g-protein function or cAMP-PKA signaling is blocked (Romere et al.,

2016). Therefore, the receptor for asprosin in the liver is likely to be a G-protein-coupled receptor (GPCR) due to the use of cAMP second messenger system (Li et al., 2019).

The OLFR734 has been described as an asprosin receptor in the hepatocytes to stimulate gluconeogenesis. It is a mouse ortholog of the receptor

OR4M1, a GPCR in the rhodopsin family. Olfactory receptors are generally expressed in olfactory epithelium, in order to detect environmental chemical changes and in the olfactory bulb, in which they are part of the neuronal circuit formation. Additionally,

OLFR734 is also expressed in the testis and liver ( Li et al., 2019). Interestingly, asprosin has a strong binding to olfactory epithelium, olfactory bulb, testis, liver, and , which matches the OLFR734 distribution and identify asprosin as its ligand (Li et al.,

2019).

2. Asprosin in the ovary

2.1. Role of fibrilin-1 and furin in the ovarian physiology

Fibrillin – 1 (FBN1) is a large glycoprotein (320kDa) (Sakai et al., 1986) encoded by the Fbn1 gene on (Kielty et al., 2002; Lee et al., 1991;

Pereira et al., 1993; Zhang et al., 1994). It is produced at first as a propeptide, profibilin-1 7

(350kDa), which suffers a proteolytic action that results in its active form, FBN1

(Milewicz et al., 1995). FBN1 is an important extracellular matrix (ECM) protein produced by stromal cells capable of formation of microfibrils with other proteins

(Sengle et al., 2012; Summers et al., 2009; Wong et al., 2007). It is a multidomain structure in which the second motif contains 8-cysteins domains that are named as TB modules (Pereira et al., 1993). FBN1 is an adult Fibrillin because it remains throughout the fetal ovary development and increases with the years (Bastian et al., 2016; Hatzirodos et al., 2011). In addition, it is a major component of the ECM of ovarian stromal compartments during ovarian development (Hatzirodos et al., 2011).

FBN1 supports the formation of skin, ligaments, blood vessels, and muscles.

In addition, Fbn1 gene is responsible for function of organs, and any perturbations in the stroma can result in impaired organ function, such as pulmonary fibrosis, renal fibrosis, and PCOS (Bastian et al., 2016; Ito et al., 1998; Rock et al., 2011; Z. Wang et al., 2015;

Wong et al., 2007). Also, mutations on this gene is associated to , which is a genetic disorder that affects the connective tissue causing abnormalities in many tissues, such as , blood vessels, bones, and eyes. (Nistala et al., 2010; Ramirez

& Dietz, 2009).

FBN1 expression has been reported in bovine fetal and adult ovaries and in human ovaries (Hatzirodos et al., 2011; Prodoehl et al., 2009). In ovarian follicles of adult bovine ovaries, FBN1 mRNA expression has been detected in granulosa cells

(Hatzirodos et al., 2014), but FBN1 protein has only been detected in theca cells, not in granulosa cells of bovine antral follicles (Prodoehl et al., 2009). On the other hand, it has 8 been reported that FBN1 is present in Call-Exner bodies (Prodoehl et al., 2009), which are round small fluid-fill space between granulosa cells of ovarian follicles (Schwab,

2011; Van Wezel et al., 1999). Although it is not well known how Call-Exner bodies are formed, many of the structural components present in the follicular basal lamina are also found in these structures (Van Wezel et al., 1999). There are two hypotheses used to explain the Call-Exner bodies origin. The most accepted one is that Call-Exner bodies were originated directly from the follicular basal laminal and they may take up thecal

FBN1 during this process (Van Wezel et al., 1999). Indeed, it was shown that FBN1 was not found on follicular basal lamina or the membrane granulosa but was associated with the theca interna (Prodoehl et al., 2009).

The second motif of FBN1 is homologous to the three repeats found in the transforming growth factor β (TGF-β) (Chaudhry et al., 2007; Kielty et al., 2002; Maslen et al., 1991). Not surprisingly, besides the support on strength and elasticity of tissue,

FBN1 regulates bioavailability of TGF-β (Chaudhry et al., 2007). TGF-β (25kDa) is a dimeric polypeptide composed of 112 amino-acids linked by bisulfate bonds (Lyons &

Moses, 1990). It is part of the superfamily that receives the same name and comprises more than 35 different polypeptides, which includes inhibins, anti-Mullerian hormone

(AMH), growth differentiation factors, and bone morphogenic proteins (Chang et al.,

2002). The members of TGF-β regulate steroidogenesis in various species (Mondschein et al., 1988; Roberts & Skinner, 1991; Yeh et al., 1993) and, therefore, FBN1 might regulate the actions of TGF- β on ovarian steroidogenesis via control of its bioavailability. 9

Furin is a calcium dependent serine protease. It is one of the seven protein convertases (PCs) enzymes identified until today (Steiner, 1998; Seidah & Chrétien,

1997). PCs are known to cleave mostly C-terminal to R-R or K-R pairs of basic amino acids (Steiner, 1998). Furin is a membrane associated protein that transits between the

Golgi-apparatus and cell surface (Molloy et al., 1994). TGF- β upregulates furin bioavailability, whereas furin is responsible for the cleavage of TGF- β (Dubois et al.,

1995), profribillin-1 (Lönnqvist et al., 1998), and asprosin (Romere et al., 2016).

Oocyte-specific deletion of furin in mice causes female infertility because it stimulates an early secondary follicle arrest (Meng et al., 2017). A recent study conducted by our group showed that follicle size may influence the furin mRNA abundance in bovine follicles

(Maylem et al., 2020). However, bovine theca cells had higher abundance of furin mRNA than granulosa cells (Maylem et al., 2020).

3. Asprosin and polycystic ovarian syndrome

The polycystic ovary syndrome (PCOS) also known as Stein-Leventhal syndrome (Stein & Leventhal, 1935) is a metabolic and reproductive disorder which affects women of reproductive age worldwide (Azziz et al., 2004; El Hayek et al., 2016;

Kahsar-Miller et al., 2001). This syndrome can be classified by three major criteria: NIH

1990 (El Hayek et al., 2016; Zawadski & Dunaif, 1992), ESHRE/ASRM (Rotterdam)

2003 (Fauser et al., 2004), and AES 2006(Azziz et al., 2006). It is necessary two of the three following characteristics to diagnosis PCOS: polycystic ovaries on ultrasonography, hyperandrogenism, and oligo/amenorrhea (Mathew et al., 2018). 10

The classical phenotype observed on PCOS patients’ ovaries is enlarged ovaries with string-of-pearl morphology, in which the theca cells have hyperplasia that confirms the androgen exposure. In addition, the antral follicles growth is arrested at 5-

8mm (Witchel et al., 2019).

PCOS is a multifaceted disease that presents symptoms of hyperandrogenism, insulin resistance, inhibition of liver production of sex-hormone-binding globulin, ovarian/adrenal androgen secretion, and it has also been described as a genetic disorder

(Azziz et al., 2004; Ehrmann, 2005; Fauser et al., 2004). Previous studies suggested that patients with family history of PCOS were affected by the same condition (Azziz et al.,

2004; Kahsar-Miller et al., 2001) and the identification of PCOS- susceptibility loci support a genetic etiology (Chen et al., 2011). Ovarian dysfunction, such as altered steroidogenesis, and extrinsic ovarian factors, as hyperinsulinemia, support the ovarian hyperandrogenism (Witchel et al., 2019). Moreover, the increase of androgen synthesis is corroborated by the high expression of CYP17A1, enzyme that converts pregnenolone and into (DHEA) and (Gilep et al., 2011), in theca cells isolated from PCOS ovaries (Witchel et al., 2019).

The adipose tissue has attracted attention with the years because of the interest in its reproductive endocrine actions. This tissue excess is highly prevalent in PCOS patients and the present obesity scenario that is observed worldwide is likely that the syndrome diagnosis will increase (Bohler et al., 2010).

The white adipose tissue dysfunction is a contributor factor for insulin resistance in PCOS patients. Previous studies, reported adipose tissue as a storage and 11 metabolic site for a variety of fat-soluble steroids (Azziz, 1989; Pasquali et al., 2006;

Svendsen et al., 2008). According to Li et al.(2018), estradiol and sex hormone binding globulin (SHBG) are highly correlated to asprosin levels in PCOS patients. Furthermore, asprosin has a positive correlation with and a negative correlation with . Therefore, asprosin may be part of the pathway for the development of PCOS via sex hormones interaction besides the obesity and insulin resistance pathways.

Moreover, asprosin is correlated to an inflammatory maker-hs-CRP, which is also present on women with PCOS (Alan et al., 2019). In this way, asprosin might be considered a key player in the development of PCOS via insulin resistance and inflammation pathways

These findings suggest an important role for asprosin on ovarian function.

4. Ovarian Steroidogenesis

The process of production of steroid hormones by specialized cells in specific tissue is called steroidogenesis. Steroid hormones are an important class of terpene-based, small lipid molecules. Steroid hormones can be adrenal glucocorticoids; sex steroids, such as ovarian and placental progesterone and ; and neurosteroids (Stocco,

2001). Knowing the enzymes and signaling involved in steroidogenesis and their intracellular location is vital to understand the regulation of steroid hormones synthesis.

4.1. Follicle-stimulating hormone and its receptor

Follicle-stimulating hormone (FSH) is a dimeric glycoprotein composed by two subunits (α and β), in which the β-subunit confers its specificity and biological 12 activity (Cahoreau et al., 2015; Ciccone & Kaiser, 2009). FSH is a gonadotropin and an integral part of the hypothalamic-pituitary-gonadal axis (George et al., 2011) that regulates steroid hormone synthesis and ovarian folliculogenesis (Fortune, 1994). FSH is synthesized and released by the gland via gonadotropin releasing hormone (GnRH) stimulation (Kaiser et al., 1997). FSH is a major player in ovarian folliculogenesis and it stimulates aromatase activity in the granulosa cells (Fortune,

1994).

FSH acts particularly on granulosa cells via its specific receptor called follicle- stimulating hormone receptor (FSHR; Xu et al., 1995; Yamoto et al., 1992).

FSHR is a G protein-coupled receptor with an extended NH2-terminal extracellular domains with numerous -rich repeats that help ligand specificity (Brown &

Roberson, 2017; George et al., 2011; Schiöth & Lagerström, 2008). The FSH binding triggers several different signaling pathways. The most known signaling event is initiated by adenylyl cyclase, followed by induction of cyclic adenosine monophosphate (cAMP), protein kinase A activation, and protein phosphorylation (Dorrington & Armstrong, 1979;

Heindel et al., 1975). Since FSH acts exclusively via FSHR, mechanisms controlling the receptor expression determine the FSH-induced cell proliferation and hormone production (George et al., 2011).

4.2. StAR (Steroidogenic Acute Regulatory Protein)

Steroidogenic tissues store some steroids and require a response to trophic hormones to start the production of new steroids hormones. These trophic hormones, like adrenocorticotropic hormone (ACTH) for adrenal cortex and (LH)in 13 the case of ovaries, use the pathway signaling of cAMP/PKA signaling to stimulate StAR expression and phosphorylation (Arakane et al., 1997; Stocco et al., 2005).

StAR accommodates cholesterol in a sterol-binding pocket and acts as an activator of mitochondrial cholesterol import rather than a transporter. In this way, it solely acts on the outer membrane of the mitochondria (Jamnongjit & Hammes, 2006).

StAR is present in ovarian theca and granulosa cells (Bao & Garverick, 1998; Kiriakidou et al., 1996) and its expression is regulated by many hormones and growth factors

(Balasubramanian et al., 1997; Ivell et al., 2000; Luo et al., 2011; Mamluk et al., 1999;

Sekar et al., 2000).

4.3. CYP11A1

The primary steroidogenic step is the conversion of cholesterol in pregnenolone by the action of the enzyme CYP11A1. This enzyme is a cytochrome P450 side-chain cleavage enzyme found in the adrenal cortex, ovary, testis, and

(Payne & Hales, 2004). In the ovaries, the expression of CYP11A1 is concentrated in theca and granulosa cells of ovulatory follicles (Oonk et al., 1990), more specifically the inner mitochondrial membrane (Farkash et al., 1986).This is considered the rate-limiting enzyme of the process of steroidogenesis (Hu et al., 2002).

4.4. CYP19A1

CYP19A1 is the gene that encodes the enzyme p450arom commonly known as aromatase. It is an important player in the regulation of the reproductive cycle of females and it is responsible for the conversion of androgens to estrogens (Payne & Hales, 2004).

It was demonstrated that PCOS patients had diminished expression of P450arom and 14 hence low levels of estradiol (Jakimiuk et al., 1998). Aromatase is present in granulosa cells of antral follicles throughout ovarian folliculogenesis and also in luteal cells of preovulatory follicles, located specifically in the endoplasmic reticulum (Stocco, 2012;

Strauss & FitzGerald, 2019). Its expression is responsible to cause changes in the estradiol levels in the blood that modulates changes in the female reproductive tract according to the reproductive cycle stage (Korach et al., 2003). In granulosa cells, FSH is responsible to stimulate the transcriptions of CYP19A1 (Strauss & FitzGerald, 2019).

5. White adipose-derived factors and ovarian steroidogenesis

The adipose tissue is an energy reservoir divided in two lines: brown and white. The white adipose tissue is an endocrine organ responsible to produce cytokines, known as adipokines (Saely et al., 2011). It is an important site for steroid hormone production and metabolism. Steroid hormones can be derived from cholesterol by the action of the enzyme cholesterol side-chain cleavage enzyme or it also may be synthesized through other steroids precursor present in the blood stream (Li et al., 2015).

Moreover, it is known that adipokines, such as leptin, resistin, adiponectin, and visfatin, have important effects on ovarian steroidogenesis (Reverchon et al., 2014).

5.1. Leptin

Leptin (16kDa) is an adipokine discovered in 1995 by Friedman, during a research in which the expression of the obese (ob) gene in the adipose tissue of mutant mouse was identified (Friedman, 2014; Sarkar et al., 2010). Leptin is composed by 167 15 amino acids and an amino-terminal secretory signal sequence of 21 amino acids

(Margetic et al., 2002). It is produced in a circadian rhythm and released in a pulsatile manner (Park & Ahima, 2020).

Leptin has been shown to be a major player on food intake regulation, energy expenditure, and reproductive functions (Barash et al., 1996; Spicer et al., 2000; Weigle et al., 1995). It was identified in mouse, rat, human, monkey, bovine (Leon J Spicer et al.,

2000), and pig (Ruiz-Cortés et al., 2003). Hyperphagia, morbid obesity and insulin resistance are observed when leptin and (or) its receptor (LepRs) are absent

(Pelleymounter et al., 1995).

Leptin also acts in the regulation of ovarian functions such as ovarian steroidogenesis. Folliculogenseis has been affected by leptin through several activities. In porcine, physiological levels of leptin stimulates estradiol production of granulosa cells in vitro whereas excessive leptin inhibits granulosa cells steroidogenesis (Ruiz-Cortés et al.,

2003). In bovine and humans, however, in vitro studies have shown that leptin inhibits steroidogenesis of granulosa cells (Spicer & Francisco, 1997; Greisen et al., 2000; Spicer et al., 2000).

5.2. Resistin Resistin is a pro-inflammatory adipokine discovered in 2001. It is part of a cysteine-rich C-terminal domain protein family called resistin-like molecules (RELMs)

(Steppan et al., 2001). Resistin expression was identified in placenta (Yura et al., 2003), (Sassek et al., 2016), and (Gualillo et al., 2003). According to

Steppan and colleagues (2001), resistin had abundant expression on female gonadal fat 16 and epididymal adipocytes. Moreover, resistin expression was observed in the ovaries of humans (Niles et al., 2012), cows, rats (Maillard et al., 2011) and pigs (Rak-Mardyła et al., 2013).

The production of resistin is associated with different factors. Insulin

(Shojima et al., 2002), hormones (Nogueiras et al., 2003), IGF-1(Chen et al.,

2005), and peroxisome proliferator-activated receptor ɣ ( PPARɣ; Patel et al.,

2003; Steppan et al., 2001) act as negative regulators of resistin. However, resistin expression is positively regulated by Y ( NPY; Yuzuriha et al., 2003) and androgens (Nogueiras et al., 2003).

It is demonstrated that resitin directly affects ovarian follicle function through cell proliferation and steriodogenesis. In rats, resistin stimulates progesterone (P4) production by granulosa cells in vitro (Maillard et al., 2011). In cattle, resistin decreases

17ß-estradiol (E2) and progesterone (P4) production by granulosa cells of small follicles

(Spicer et al., 2010; Maillard et al., 2011), but increases E2 production by granulosa cells of large follicles in vitro (Spicer et al., 2010) In addition, resistin is more efficient on regulating E2 production by granulosa cells rather than by theca cells (Spicer et al.,

2010). In porcine theca cells, resistin had a stimulatory effect on P4, androestenedione, and testosterone production via 3bHSD (Rak-Mardyła et al., 2013). A study conducted on porcine ovarian steriodogenesis concluded that gonadotoropins and steroids increased expression in ovarian follicles. However, IGF-1 steriodogenic pathways were inhibited by resistin and IGF-1 decreased resistin expression in ovarian follicles (Rak et al., 2015). 17

Also, resistin had a positive effect on ovarian follicles expression of CYP17A1 and

17bHSD (Rak-Mardyła et al., 2013; Rak et al., 2015)

The association between resistin and PCOS has been proposed (Lu et al.,

2005; Majuri et al., 2007; Munir et al., 2005; Rak et al., 2017). A study conducted with overweight women with PCOS concluded that circulating levels of resistin was significantly diminish by the insulin sensitizing agent rosiglitazone (Majuri et al., 2007).

Moreover, PCOS patients presented a positive association between serum testosterone and resistin (Munir et al., 2005). Nevertheless, most of the studies did not confirm the association of PCOS and resistin, even though a positive correlation between resistin, IR, and diabetes type II exists.

5.3. Adiponectin

Adiponectin, also known as Acrp 30, AdiopQ, apM1, or GBP28, is a 30kDa protein member of the complement 1q family produced mainly by adipocytes (Kadowaki

& Yamauchi, 2005; Scherer et al., 1995). It is a major regulator of insulin and its deficiency can lead to diabetes type II, PCOS, and others metabolic disorders linked to insulin resistance (Maillard et al., 2010).

Adiponectin has a variety of multimers forms, which can range from low molecular weight (LMW) to high molecular weight (HMW). The HMW is the most abundant when compared to the globular fragment (LMW) (Kadowaki & Yamauchi,

2005). In contrast to leptin, circulating adiponectin concentrations are inversely correlated with adiposity storage in (Campos et al., 2008). HMW form is 18 regulated by post-translational modifications, such as hydroxylation and glycosylation

(Wang et al., 2006).

Adiponectin has three receptors ADIPOR1 and 2 and T-cadherin (Thundyil et al., 2012). ADIPOR1 and ADIPOR2 are specific membrane receptors and their expression is mediated by insulin and sex steroids (Kadowaki & Yamauchi, 2005). These receptors have seven transmembrane domains. However, they are structurally and functionally distinct from G-protein-coupled receptors once intra and extracellular orientations of N-terminal and C-terminal are opposite (Kadowaki & Yamauchi, 2005).

ADIPOR2 is the preferred receptor for HMW isoforms, and it is highly expressed in the liver and endothelial cells (Kadowaki & Yamauchi, 2005). ADIPOR1 and ADIPOR2 are present in human, rat (Chabrolle et al., 2008) , pig (Lord et al., 2002), and bovine ovaries

(Maillard et al., 2010).

The classical pathway used by adiponectin is the AMP-activated protein kinase (Kadowaki et al., 2006), in which the adiponectin isoforms induce glucose uptake and lipid oxidation (Trujillo et al., 2005). Adiponectin also may use the cAMP or protein kinase A (PKA) pathway, phosphoinositol kinase 3/protein kinase B (PKB) or AKT systems on endothelial cells (Campos et al., 2008).Moreover, adiponectin activates the peripheral peroxisome -activated receptor-γ in placental tissues (Lappas et al., 2005). It is now established that APPL1, an adapter protein, interacts with ADIPORs and regulates the cross-talk between insulin and adiponectin transduction pathways (Hosch, et al., 2006; Mao et al., 2006).

Adiponectin affects the reproductive system either indirectly, through the , or directly, through the ovary (Kadowaki & Yamauchi, 2005). 19

Adiponectin has been reported to be present in human and porcine follicular fluid

(Ledoux et al., 2006). Furthermore, adiponectin is produced by theca, granulosa and luteal cells of rat (Chabrolle et al., 2008) and chicken ovaries (Chabrolle et al., 2007).

In granulosa cells, adiponectin stimulates the AMP-activated protein kinase phosphorylation of swine and rat (Chabrolle et al., 2008; Ledoux et al., 2006). In addition, the literature shows that adiponectin can act in granulosa cells of rat, humans, and cattle through the mitogen-activated protein kinase (MAPK) particularly in the phosphorylation of extracellular signal- regulated kinases 1 and 2 (ERK1/2) (Chabrolle et al., 2008; Ledoux et al., 2006; Maillard et al., 2010).

Adiponectin has been shown to affect ovarian steroidogenesis. In bovine, adiponectin inhibits progesterone and androstenedione production when induced by LH and insulin in theca cells (Lagaly et al., 2008). Moreover, adiponectin decreases the expression of LH receptor, CYP11A1 and CYP17A1 mRNA in bovine theca cells, but has no effects on bovine granulosa cells or on insulin-induced proliferation of bovine theca cell (Lagaly et al., 2008). Indeed, ADIPOR2 mRNA is not present in bovine granulosa cells but is abundant in bovine theca cells (Lagaly et al., 2008). In humans, similarly to what happens in cattle, adiponectin did not have effect on granulosa cells proliferation and viability although it increased progesterone and E2 production (Barbe et al., 2019).

5.4. Visfatin

Visfatin (52kDa) is also known as nicotinamide phosphoribosyl transferase

(NAMPT) and pre-B cell colony-enhancing factor (PBEF) (Fukuhara et al., 2005; Samal et., 1994). It is an adipokine hormone produced mainly by the visceral adipose tissue. 20

This hormone is involved in metabolic disease, such as obesity and type II diabetes, and immune disorders (Rongvaux et al., 2008). It also regulates glucose homeostasis, lipid metabolism, inflammation and angiogenesis (Stofkova, 2010).

Visfatin has two molecular forms, extracellular and intracellular. The extracellular form is the cytokine-like, which can act on the and regulate downstream signaling pathways. The intracellular form is a rate-limiting enzyme that acts on the NAD biosynthesis from nicotinamide (Reverchon et al., 2016; Revollo et al.,

2004). Furthermore, it is proposed that NAD biosynthesis mediated by visfatin regulates sirtuin (SIRT1) activity, which is a NAD-dependent deacetylase involved in the pathogenesis of type II diabetes (Revollo et al., 2004).

Visfatin expression is observed in human fetal membranes, amniotic epithelium, and mesenchymal cells during gestation (Ognjanovic & Bryant-Greenwood,

2002). In the ovary, visfatin has been reported to be involved on the development of oocytes competency and old female fertility potential (Choi et al., 2012). Furthermore, visfatin has an increased expression in human granulosa cells when treated with human chorionic gonadotropin (hCG) and E2, hormones that regulate steroidogenesis (Shen et al., 2010).

In chickens, recombinant human visfatin reduces levels of STAR and HSD3B in primary granulosa cells (Diot et al., 2015). This may explain the observed inhibitory effect of recombinant human visfatin on progesterone production (Diot et al., 2015). In the same study, it was observed that recombinant human visfatin reduced MAPK3/1 phosphorylation. In addition, it reduced the IGF-1 induced MAPK3/1 phosphorylation, whereas it did not affect PRKA, AKT1 or MAPK14 (Diot et al., 2015). The MAPK3/1 is 21 an important pathway associated to the regulation of STAR expression and steroidogenesis in granulosa cells.

Visfatin has been suggested to influence many physiological parameters, such as insulin resistance (Shen et al., 2010), which is a condition of PCOS patients (Azziz et al., 2004; Kowalska et al., 2007) Indeed, visfatin is increased in PCOS women (Dahl et al., 2012; Omer et al., 2018; Panidis et al., 2008). It is suggested that visfatin is associated to PCOS by the action on the hypothalamic-pituitary-ovarian axis due to elevated LH levels associated to plasma visfatin (Panidis et al., 2008). In addition, it was found that visfatin serum levels were highly correlated to hyperandrogenic patients and it is known that hyperandrogenism is one of the pathophysiology of PCOS (Kim et al., 2018).

However, the specific role of visfatin on PCOS remains to be unveiled, although studies showed the correlation between it and the syndrome key pathophysiology.

Based on this literature review we hypothesize that asprosin regulates granulosa cells steroidogenesis.

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34

CHAPTER II

Effects of asprosin on steroidogeneses of bovine granulosa cells

Introduction

The ovary is an important endocrine organ, in which ovarian follicular cells produce hormones important to regulate reproduction (Craig, 2018) as well as metabolism and health (Newell-Fugate, 2017). Sex steroid hormones produced by the ovary are known to regulate ovarian folliculogenesis, ovulation, and pregnancy establishment (Craig, 2018). Estradiol, the main hormone produced by ovarian somatic follicular cells, regulates glucose and lipid metabolism and its deficiency has been linked to obesity and metabolic disorders (Jia et al., 2015). Moreover, an impairment in ovarian steroid production may lead to ovarian pathologies, such as polycystic ovarian syndrome

(PCOS; Jamnongjit & Hammes, 2006). Hence, studying the process of ovarian steroidogenesis and factors that can impact ovarian function is critically important to understand how sex steroids can regulate health and fertility.

The bovine is a valuable model to study ovarian physiology. The pattern of ovarian follicular growth is well known in cattle, which facilitates the study of ovarian folliculogenesis in this species (Fortune et al., 2004). In addition, the bovine is a mono- ovulatory species and its processes of ovarian steroidogenesis (Adams & Pierson, 1995) and folliculogenesis (Abedal-Majed & Cupp, 2019; Adams & Pierson, 1995; Fortune,

1994; Spicer & Echternkamp, 1986) are similar to the human. Furthermore, ovarian dysfunctions that disrupt ovarian folliculogenesis are found in both cattle and women 35

(Abedal-Majed & Cupp, 2019) and similarities in the etiology of these dysfunctions exist

(A. F. Summers et al., 2014).

White adipose tissue (WAT) is a key regulator of health and metabolism and its dysfunction has been linked to obesity, polycystic ovarian syndrome (PCOS), and menopause (Newell-Fugate, 2017). Adipokines are a group of cytokines produced by the

WAT that are known to act in endocrine, paracrine. and autocrine ways (Reverchon et al.,

2014). Leptin, adiponectin, resistin, and visfatin are among well-known adipokines that regulate the female reproductive tract (Reverchon et al., 2014). Asprosin is a novel cytokine encoded by the FBN1 gene and synthesized by WAT (Romere et al., 2016).

Interestingly, asprosin has been linked to PCOS (Alan et al., 2019; X. Li et al., 2018) and has been recently reported to affect theca cells androgens production (Maylem et al,

2020). Nevertheless, the role of asprosin on granulosa cell (GC) function remains to be unveiled. Therefore, we hypothesize that asprosin regulates GC steroidogenesis in a mono-ovulatory species such as cattle.

Material and Methods

Reagents and hormones

Reagents used in cell culture were: DMEM/Ham’s F-12 (Genesee Scientific,

San Diego, CA); gentamicin, sodium bicarbonate, DNase I, collagenase, and trypan blue

(MilliporeSigma, St. Louis, MO); fetal bovine serum (FBS; Atlas Biologicals, Fort

Collins, CO); testosterone (Steraloids, Newport, RI); purified ovine follicle-stimulating 36 hormone (FSH; Scripps lab, San Diego, CA); insulin-like growth factor I (IGF-1; R&D

Systems, Minneapolis, MN); recombinant human asprosin (BioLegend, San Diego, CA).

Granulosa cell collection and culture

Ovaries from non-pregnant bovine females were collected at a commercial slaughterhouse that follows humane slaughter practices, according to USDA guidelines.

Following collection, ovaries were washed once with saline 0.9%, once with 0.9% saline plus 70% isopropanol and placed on ice in antibiotic saline (0.9% saline solution with 1% penicillin-streptomycin) to be transported to the lab. Follicular fluid was aspirated from small antral follicles (1-5 mm) and GC were isolated following previously described protocol (Langhout et al.,1991; Spicer et al., 1993) . Briefly, small follicles were classified based on surface diameter (1-5 mm) and then aspirated using needle (20G) and

3 mL syringe (BD, Franklin Lakes, NJ). Follicular fluid of pools of ovaries was centrifuged at 200 x g for 4 min at 4oC and supernatant was discarded. Cells pellets received serum-free medium (1:1 DMEM/Ham’s F12 supplemented with 0.12 mM gentamycin and 23.8 mM sodium bicarbonate) and were once again centrifuged at 200 x g for 4 min at 4oC. Following the second centrifugation, cells were resuspended in serum- free medium supplemented with 1.25 mg/ml collagenase and 0.5 mg/ml DNAse to prevent clumping. Following resuspension, GC viability was assessed using trypan blue exclusion method and averaged 67.7% for experiment 1 and 75% for experiment 2. Cells were seeded on 24-well plates (Corning, Corning, NY) in 1 mL of DMEM/Ham’s F12

o medium containing 10% FBS at 38.5 C in a 5% CO2 atmosphere and medium was 37 changed every 24 h. When cells reached adequate confluence to be treated, they were washed twice with 0.5 mL of serum-free medium and treatments were applied for 24 h.

Experimental design

Experiment 1 was conducted to determine the effects of asprosin on cell number and estradiol production of GC. Cells were treated with asprosin (200ng/mL),

FSH (50ng/mL), and IGF1 (30ng/mL) and their possible combinations. Cells were seeded at a concentration of 2.0 X 105 per well. The dose of FSH and IGF were selected based on previous studies (Spicer et al., 1993; Stewart et al., 1995) and the dose of asprosin was selected based on the higher concentration reported in systemic circulation of women

(Romere et al., 2016; Yuren Wang et al., 2018) and mice (Romere et al., 2016). Cells were cultured for 48 h until reaching 80 to 100% confluence and were then treated for 24 h in serum-free medium followed by collection of medium and cells for radioimmunoassay and cell counting, respectively.

Experiment 2 was conducted to evaluate the expression of the steroidogenic enzymes steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (CYP11A1), and aromatase (CYP19A1) in GC treated with or without Asprosin

(200ng/mL), FSH (50ng/mL), IGF1 (30ng/mL), and their possible combinations. Cells were seeded at an average concentration of 1 x 105 per well and cultured for 24 or 48 h in

10% FBS, depending on the pool of cells. When cells reached 60% confluence, they were treated in serum-free medium with 500ng/mL testosterone for 24h.

Radioimmunoassay (RIA)

38

Estradiol concentrations from cell culture medium were determined by

RIA as previously described (Dentis et al., 2016; Stewart et al., 1996). Intra-assay coefficient of variation for estradiol RIA averaged 5.7%.

Cell counting

GC numbers were determined by the end of experiment 1 using a Coulter counter (Model Z2; Beckman Coulter Inc., Hialeah, FL) as previously described (Spicer et al., 2006). First, 500µL of trypsin (0.25% (w/v) in 0.5 M NaCl) was added to each well for 20min at room temperature. Then, cells were scraped and disrupted via repetitive pipetting, diluted in 0.15 M NaCl, and counted.

RNA extraction

Total RNA from GC was isolated using miRNeasy mini kit (Qiagen,

Hilden Germany) according to the manufacturer’s protocol. Briefly, medium was aspirated from the wells of the cell culture plate and each well received 500µL of Qiazol lysis buffer. Since each treatment was applied in duplicate wells per plate, cells of each two wells were pooled together in lysis buffer in one 1.7 mL microcentrifuge tube for posterior RNA extraction. Samples were stored at -80 oC until RNA extraction. RNA concentration and integrity were determined by NanoDrop One spectrophotometer

(NanoDrop Technologies Inc., Wilmington, DE). Samples were stored at -80oC for posterior analysis.

Quantitative real-time reverse transcription polymerase chain reaction (qPCR)

39

For analysis of relative gene expression, 750 nanograms of total RNA were used to generate single-stranded cDNA by using High-Capacity cDNA Reverse

Transcription Kit (Applied Biosystems, Waltham, MA) following manufacturer’s protocol. Target genes mRNA relative abundance was measured by qPCR with a CFX

Connect Real-Time PCR Detection System (BioRad, Hercules, CA,USA) using a SYBR select Master Mix (Applied Biosystems, Waltham, MA). Primers for StAR, CYP11A1, and CYP19A1 were designed using The Primer Express software v3.0.1 (Applied

Biosystems, Waltham, MA). Primers sequences, amplicon sizes, and annealing temperature are provided in table 1. The protocol used was as follows: 95oC for 10 min (1 cycle), 95oC for 15 seconds followed by 60oC for 1 min (40 cycles). The expression of target genes was normalized to 18S ribosomal RNA (18S) expression levels. The relative abundance of mRNA was calculated using the 2-∆∆Ct method and expressed as fold change in comparison to the negative control group (Livak & Schmittgen, 2001).

Statistics

Data were analyzed via factorial ANOVA with GLM procedures of SAS for Windows (version 9.4, SAS Institute Inc., Cary, NC). If necessary, values were transformed to natural log (x+1) to ensure homogeneity of variance. Data were presented as means of experimental groups ± SEM. Mean differences were assessed using Fisher’s protected least significant differences test (Ott,1977). Differences among treatments were considered significant at p < 0.05 and a trend at 0.05 < p < 0.10. Outliers detected through Grubbs’s test were excluded from analyses. 40

Results

Experiment 1: Asprosin effects on estradiol production and proliferation of

granulosa cells

Asprosin alone did not increase estradiol production in comparison to non- treated control (p=0.781; figure 1). However, asprosin increased estradiol production in

FSH-treated cells in comparison to cells treated with FSH alone (p<0.001). In contrast,

GC treated with IGF-1 produced more estradiol than when asprosin was added together with IGF-1 (p<0.001). Asprosin did not affect estradiol production of cells treated with a combination of FSH and IGF-1 (p=0.0976) and this combination of FSH and IGF-1 resulted in greater estradiol production (p<0.001) than all other treatments irrespective of asprosin (figure .

Numbers of GC were not affected by asprosin alone or FSH alone in comparison to the negative control (p>0.05; figure 2). IGF-1 alone increased the numbers of GC in comparison to negative control or to cells treated with FSH alone (p<0.01). The combination of IGF-1 and asprosin increased the cell numbers in comparison to negative control (p<0.01) but minimized cell proliferation in comparison to cells treated with IGF-

1 alone (p<0.05). The combination of FSH and IGF-1 increased GC numbers in comparison to all other treatments (p<0.01) and this increase in GC numbers was minimized with the addition of asprosin to these treatments, although the combination of asprosin, FSH and IGF-1 still resulted in more GC numbers than negative control, asprosin alone, FSH alone, IGF-1 alone, and asprosin combined with FSH or IGF1

(p<0.01). 41

Experiment 2: Asprosin effects on mRNA relative abundance of enzymes

regulating granulosa cells steroidogenesis

Asprosin did not affect CYP11A1 mRNA abundance of granulosa cells (figure

3) or StAR mRNA abundance (figure 5). However, asprosin alone increased CYP19A1 mRNA abundance in comparison to negative control (figure 4) with no other effects of asprosin observed.

Discussion

Adipokines have been reported to play a role in ovarian steroidogenesis in various species (Reverchon et al., 2014). A recent study reported that asprosin regulates bovine theca cells steroidogenesis and that its receptor has a greater mRNA abundance in

GC than in theca cells (Maylem et al., 2020). Therefore, studies investigating the effects of asprosin on GC are needed. Using bovine as a model, the present study unveils the effects of asprosin on steroidogenesis and proliferation of GC of a mono-ovulatory species, providing a potential link between asprosin and fertility.

The findings on this study demonstrate a positive relationship between FSH and asprosin regarding estradiol production by GC. Asprosin has been reported to increase androstenedione production when combined with LH (Maylem et al., 2020).

Because androgens are an essential substrate for estradiol production by GC (Dorrington et al., 1975), these observations suggest that asprosin works as an important regulator of 42 estradiol synthesis by increasing both the substrate for estradiol production coming from theca cells to GC and the synthesis of estradiol production within GC in cattle.

Considering the role of estradiol in GC survival and differentiation (Hsueh et al., 1984;

Knecht et.al., 2015) as well as in regulation of follicular selection and dominance

(Fortune, 1994), it is possible that asprosin influence ovarian folliculogenesis through its stimulation of estradiol in the presence of FSH. The fact that mRNA abundance of the presumed asprosin receptor, OR4M1, is greater in GC of small follicles than in GC of large follicles (Maylem et al., 2020) suggests that asprosin might be particularly important in regulating early follicular development in mammals, but future research is required to test this hypothesis.

IGF-1 is a known mitogen of GC of several species, including bovine

(Gutiérrez et al., 1997; Spicer & Echternkamp, 1995). In the present study, asprosin reduced the mitogenic effects of IGF-1 on GC. Because GC of dominant follicles have more IGF-1 binding sites than GC of small follicles (Spicer et al., 1994) and concentrations of bioavailable IGF-1 are more abundant in follicular fluid of dominant than subordinate follicles (Fortune et al., 2001; Spicer, 2004), this interaction of asprosin and IGF-1 might be important to regulate mitosis of GC of dominant follicles.

Interestingly, although IGF-1 is a mitogen of GC, studies with mice null for IGF-1 show that IGF-1 is even more important for GC final differentiation than for GC proliferation in vivo (Baker et al., 1996). Because GC proliferation is reduced as GC approaches final differentiation (Robker & Richards, 1998), it is possible that asprosin contributes to final differentiation of GC, but further studies are required to confirm this hypothesis. 43

In order to elucidate which steps of steroidogenic enzyme machinery were affected by asprosin actions, StAR, CYP11A1, and CYP19A1 gene expression were analyzed. StAR is the gene of the enzyme that mediates the transport of cholesterol from the outer to inner mitochondrial membrane (Jamnongjit & Hammes, 2006), where cholesterol side-chain cleavage enzyme is present (Farkash et al., 1986). Cholesterol side- chain cleavage enzyme (CYP11A1) is the key enzyme responsible for the initial rate- limiting step of steroidogenesis in the ovary (Hu et al., 2002) and is responsible for conversion of cholesterol into pregnenolone. Aromatase (CYP19A1) is responsible for the conversion of androgens coming from theca cells to estradiol in GC. In the present study, treatment of GC with asprosin alone increased CYP19A1 mRNA abundance of granulosa cells in comparison to untreated granulosa cells, but no other effect of asprosin was observed when in combination with FSH or IGF-1 on CYP19A1 mRNA abundance.

Moreover, asprosin did not affect mRNA abundance of CYP11A1 and StAR. Future studies assessing protein expression of the steroidogenic enzymes studied herein are required, but it is possible that asprosin is acting through other steroidogenic pathways to influence estradiol production.

Asprosin has been described as a fasting-induced gluconeogenic hormone

(Romere et al., 2016). Since its discovery, many recent studies have been designed to unveil the role of asprosin on health and metabolism. Indeed, excessive levels of asprosin have been linked to obesity and insulin resistance (Elnagar et al., 2018; Lee et al., 2019;

Wong et al., 2019). Excessive circulating asprosin has also been associated with PCOS and altered concentrations of steroid hormones, including excessive androgens and reduced estradiol levels ( Li et al., 2018), but the mechanism behind this association 44 remains to be fully elucidated (Yuan et al., 2020). In the present study, asprosin was found to increase estradiol production by bovine GC when in combination to FSH. It is possible that the role of asprosin on estradiol production is dependable on its concentrations and that excessive asprosin impairs the ovarian steroidogenic process.

Taken together, the findings presented herein indicate that asprosin is a regulator of ovarian estradiol production of cattle and might influence ovarian folliculogenesis. The association of asprosin and ovarian steroidogenesis deserves further attention since estradiol not only regulates fertility, but is known to regulate energy metabolism and influence metabolic diseases (Cooke & Naaz, 2004; D’Eon et al., 2005;

Lundholm et al., 2008; Mauvais-Jarvis et al., 2013).

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Table 1. Information of primers.

ACCESSION GENE SEQUENCES ( 5' - 3') TM(OC) NUMBER FWD: AGAAACGGCTACCACATCCA 18S AF176811.1 57.3 REV:CACCAGACTTGCCCTCCA FWD: GGGTGTGAACACGACATCCA CYP11A1 NM_176644 60 Rev: CGTCGGGCATTCAGAACCTC FWD: CCTGTGCGGGAAAGTACATCA CYP19A1 NM_174305 60 Rev: CAGGTGGAAGCGTCTCAGAA FWD: GCAACACATGCTCTGGTTTTGA STAR NM_174189 60 Rev: GGTGTGTGTTGAAAGCCCCATT 18S, 18S ribosomal; CYP11A1, cytochrome P450 side chain cleavage; CYP19A1, cytochrome P450 aromatase; StAR, steroidogenic acute regulatory protein.

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Figure 1. Effect of asprosin on FSH- and IGF1-induced steroidogenesis in granulosa cells (GC). Estradiol production in asprosin-treated GC concomitantly treated with FSH, IGF1 of both. Means ± SEM without a common letter differ (p <0.05).

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Figure 2. Effects of asprosin (200ng/ml) on granulosa cells (GC) proliferation in presence of FSH and IGF1 or both. Means ± SEM without a common letter (a,b,c,d,e) differ (p<0.05).

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Figure 3. Relative mRNA abundance of CYP11A1 in granulosa cells treated with asprosin, FSH, IGF1, or combined treatments between them. Treatments were applied for 24h. Means ± SEM without common letters (a,b) differ (p <0.05).

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Figure 4. Relative mRNA abundance of CYP19A1 in granulosa cells treated with asprosin, FSH, IGF1, or combined treatments between them. Treatments were applied for 24h. Means ± SEM without common letters (a,b,c) differ (p <0.05).

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Figure 5. Relative mRNA abundance of StAR in granulosa cells treated with asprosin, FSH, IGF1, or combined treatments between them. Treatments were applied for 24h. No significant effect was observed among treatments.