**Preface to "Challenges and New Strategies on Rabbit Breeding"**

Rabbit meat is a healthy source of protein for people: it is low in fat, cholesterol, and sodium and can meet the growing population's requirements in the coming decades. In addition, rabbits are less costly and have less environmental impact than other livestock species. Therefore, this valuable livestock species can potentially help to satisfy the nutritional needs of humans thanks to its sustainable husbandry system. However, intensive rabbit production shows some difficulties that negatively impair the reproduction and lifespan of rabbit females, which ultimately affect productive outcomes; then, there is an increase in the use of antibiotics and decrease in animal welfare that subsequently reduce the farmer's profitability, and negatively prejudices consumer perception of rabbit meat production. In addition, new breeding selection tools and improvement in nutritional strategies should arise to balance animal physiological demands and sustainability. Consequently, rabbit production should adapt to the new green challenges promoting circular bioeconomy, benefiting the rabbits' welfare, and promoting both human and rabbit health in a One Health approach to be a competitive and sustainable production system.

> **Rosa Mar´ıa Garc´ıa-Garc´ıa and Mar´ıa Arias Alvarez ´** *Editors*

### *Review* **Current Knowledge on the Multifactorial Regulation of Corpora Lutea Lifespan: The Rabbit Model**

**Massimo Zerani, Angela Polisca \*, Cristiano Boiti and Margherita Maranesi**

Dipartimento di Medicina veterinaria, Università di Perugia, via San Costanzo 4, 06126 Perugia, Italy; massimo.zerani@unipg.it (M.Z.); boiti.cristiano@gmail.com (C.B.); margherita.maranesi@unipg.it (M.M.) **\*** Correspondence: angela.polisca@unipg.it

**Simple Summary:** Corpora lutea (CL) are temporary endocrine structures that secrete progesterone, which is essential for maintaining a healthy pregnancy. A variety of regulatory factors come into play in modulating the functional lifespan of CL, with luteotropic and luteolytic effects. Many aspects of luteal phase physiology have been clarified, yet many others have not yet been determined, including the molecular and/or cellular mechanisms that maintain the CL from the beginning of luteolysis during early CL development. This paper summarizes our current knowledge of the endocrine and cellular mechanisms involved in multifactorial CL lifespan regulation, using the pseudopregnant rabbit model.

**Abstract:** Our research group studied the biological regulatory mechanisms of the corpora lutea (CL), paying particular attention to the pseudopregnant rabbit model, which has the advantage that the relative luteal age following ovulation is induced by the gonadotrophin-releasing hormone (GnRH). CL are temporary endocrine structures that secrete progesterone, which is essential for maintaining a healthy pregnancy. It is now clear that, besides the classical regulatory mechanism exerted by prostaglandin E2 (luteotropic) and prostaglandin F2α (luteolytic), a considerable number of other effectors assist in the regulation of CL. The aim of this paper is to summarize our current knowledge of the multifactorial mechanisms regulating CL lifespan in rabbits. Given the essential role of CL in reproductive success, a deeper understanding of the regulatory mechanisms will provide us with valuable insights on various reproductive issues that hinder fertility in this and other mammalian species, allowing to overcome the challenges for new and more efficient breeding strategies.

**Keywords:** rabbit; corpus luteum; reproduction

#### **1. Introduction**

Corpora lutea (CL) are temporary endocrine structures that secrete progesterone, which is essential for a healthy pregnancy in most species. In rabbits, the CL develop rapidly following ovulation and reach their maximum size and functional capacity within nine to ten days. This process shows the intense angiogenesis and active granulosa or theca cell luteinization of preovulatory follicles, due to the effects of several local angiogenic growth factors, gonadotropins and other hormones [1,2]. In pregnant rabbits, the CL lifespan lasts for about 30 days [3]; however, if pregnancy does not occur, the lifespan of the CL is much shorter, and luteal regression starts around day 12 and ends 16 days after ovulation when the peripheral plasma progesterone concentrations drop to the baseline values [4,5]. Therefore, the absence of embryonic signals or the end of gestation activates luteolysis, a comprehensive regressive process that leads to total functional and structural CL demise, in which prostaglandin (PG) F2 α (PGF2α) plays a central role [6].

Many regulatory factors, including cytokines, growth factors, prostaglandin E2 (PGE2) and PGF2α released by different CL cell types, including endothelial and local immune cells and fibroblasts, as well as progesterone and 17β-estradiol released by luteal and follicular cells and hormones, control the functional lifespan of the CL, with luteotrophic

**Citation:** Zerani, M.; Polisca, A.; Boiti, C.; Maranesi, M. Current Knowledge on the Multifactorial Regulation of Corpora Lutea Lifespan: The Rabbit Model. *Animals* **2021**, *11*, 296. https://doi.org/ 10.3390/ani11020296

Academic Editor: Rosa María García-García and Maria Arias Alvarez Received: 30 November 2020 Accepted: 21 January 2021 Published: 25 January 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and luteolytic effects [7]. However, the overall balance between these contrasting actions varies considerably with the age of the CL and/or in the presence/absence of an embryo [8]. Many facets of luteal physiology have been clarified, but others are still poorly understood, including the molecular and/or cellular mechanisms that protect the CL from luteolysis from the early luteal phase. Moreover, the mechanisms that are induced by the administration of exogenous PGF2α have been extensively investigated in rabbits [9–11] in order to evaluate PG paracrine and/or autocrine functions and other possible regulators that switch on (luteotropic)/off (luteolyic) progesterone production by the CL at a specific stage of its life cycle. However, there are few data on the mechanisms that protect the developing CL from functional luteolysis in the early luteal phase, which starts on day six of a pseudopregnancy, when the luteal cells acquire the ability to respond to the luteolytic effects of exogenous PGF2α (luteolytic capacity) [9]. Luteolysis is a key event in reproduction for spontaneously ovulating species, as well as for rabbits, whose mating activity triggers a neuroendocrine reflex, which, combined with GnRH or exogenous human chorionic gonadotropin (hCG) exogenous administration, induces ovulation [12,13].

This paper provides a summary of our current knowledge on the endocrine and cellular mechanisms of multifactorial CL lifespan regulation, acquired using the pseudopregnant rabbit model, which was able to determine the relative luteal phase following GnRH-induced ovulation. Most of the mechanisms described in this review were observed during our studies on the progressive age-dependent response of the CL to PGF2α conducted over a 20-year period [9]. A better understanding of these mechanisms may provide us with valuable insights in the challenge to find more efficient breeding strategies for rabbits, as well as for other species.

#### **2. Prostaglandins**

Prostaglandins (PGs) play a key regulatory role in CL function and the lifespan: PGF2α is the main luteolytic agent produced by the uterine endometrium of numerous mammals, including rabbits, but not by primates [14–16], while PGE2 plays a crucial luteoprotective role, with luteotrophic and/or antiluteolytic effects [6]. In some species, PGF2α and PGE2 are produced by the CL [17–21].

An essential step in PG biosynthesis is the cyclooxygenase (COX) 1 (COX1) and/or COX2 enzymatic conversion of arachidonic acid (AA)—produced by phospholipase A2 (PLA2) activity—into PGH2 [22–24]. This latter PG is then transformed into four structurally active PGs (PGE2, PGF2α, PGD2 and PGI2) by specific PG synthases [25]. PGF2α biosynthesis is particular, since three specific ketoreductases catalyze this PG from PGH2, PGD2 or PGE2, respectively [26]. PGE2-9-ketoreductase (PGE2-9-K) is present in the rabbit ovary [27] and CL [28]. This ketoreductase also converts progesterone into its inactive metabolite through its 20α-hydroxysteroid dehydrogenase (HSD) catalytic activity.

We previously reported [21] that, in rabbits, intra-luteal PGF2α activates luteolysis with an auto-amplification loop: during the mid- and late-luteal phases, it activates COX2 and PGE2-9-K; the former converts AA into PGH2, which is then transformed into PGF2α and PGE2, while the latter is converted into PGF2α through PGE2-9-K activation. Moreover, this enzyme significantly reduces PGF2α-induced progesterone through its 20α-hydroxysteroid dehydrogenase (20α-HSD) activity that converts progesterone into 20α-OH-progesterone. Late-luteal phase PGE2 production plays another essential role: PGE2-9-K enzymatic activity make this PG the main source of PGF2α synthesis.

Arosh et al. [29] suggested that CL PG biosynthesis is mainly directed toward PGE2 production rather than PGF2α. In fact, PGH2 conversion into PGE2 (PGE synthase) is 150-fold higher than that of PGH2 into PGF2α (PGF synthase) [30]. These results [29,30], combined with our data [21,31], allow us to hypothesize [31] that rabbit CL in the early and mid-luteal phases use the same cellular enzymatic pathways (PLA2/AA/COX2/PGH2/PGE synthase/PGE2) to produce an initial PGE2 amount, while the final luteal production of PGE2 (early CL) or PGF2α (mid-CL) is regulated by PGE2-9-K inactivation or activation, respectively (Figure 1, upper, functional luteolysis).

**Figure 1.** Schematic model reporting the functional (upper) and structural (lower) luteolytic pathways induced by prostaglandin F2 α (PGF2α) in rabbit mid-corpora lutea (CL) (day 9 of pseudopregnancy). Since prostaglandin E2 (PGE2)-9-K and 20α-hydroxysteroid dehydrogenase (HSD) represent two different activities of a single enzyme, they are joined. Figure from the study by Maranesi et al. 2010 [31]. For acronyms, see the list of abbreviations in the text.

Several studies have investigated the possible factors involved in PGF2α-induced luteolytic capacity during the mid-luteal phase [7,9,32–36]. Interleukin 1 (IL1), with other cytokines that are normally present in rabbit luteal cells [32,33], are locally involved in the CL function control leading to apoptosis as proinflammatory mediators [34]. Moreover, locally acting hormones and pro- and antiapoptotic intra-luteal factors may interact dynamically. 17β-Estradiol is one of the main luteotropic effectors, since its absence leads to luteolysis through apoptosis activation [7]. Nitric oxide synthase (NOS) and its product nitric oxide (NO) are also known to have pro- and antiapoptotic properties that modulate various intracellular pathways—in particular B-cell CLL/lymphoma 2 (BCL2)-like 1 (BCL2L1) and tumor protein p53 (TP53) proteins [35]. In rabbits, NOS luteal inhibition favors apoptosis [36].

Our study [31] on the key protein-encoding genes involved in apoptotic mechanism control revealed that PGF2α induces luteolysis in luteal cells with an acquired luteolytic capacity through the upregulation of luteal IL1B and TP53 gene transcripts and the downregulation of the estrogen receptor 1 (ESR1) and BCL2L1 receptors. This PGF2α-induced CL regression seems to be the result of two distinct mechanisms: the steroidogenic pathway, by ESR1 downregulation, and the apoptotic pathway, by the dynamic changes of the TP53 and BCL2L1 proteins and gene transcripts (Figure 1, lower, structural luteolysis). Finally, aglepristone (RU534), an antiprogestinic, increases progesterone release in rabbit mid- and

late-CL, whereas this antiprogestinic reduces PGF2α and enhances PGE2 only during the late-luteal stage [37].

#### **3. Nitric Oxide**

Nitric oxide is a potent vasodilator factor involved in several biological processes, such as neurotransmissions and cytotoxicity, under both physiological and pathological conditions [38,39]. NO is produced by the enzymatic action of NOS, which converts L-arginine into NO and L-citrulline. There are three forms of NOS: two constitutive Ca2+-dependent forms neuronal NOS (nNOS) and endothelial NOS (eNOS) and an inducible Ca2+-independent form (iNOS) [38,40]. With the exception of neuronal and endothelial cells, constitutive eNOS and nNOS are normally expressed in various cell types and produce low levels of NO. Contrastingly, the inducible form only produces large quantities of NO when the expression is activated [38,40]. NOS is present in both ovarian stroma and follicular granulosa cells of several mammalian species, including rabbit ovaries, where it regulates steroidogenesis [17,41–44]. The NO/NOS system present in rabbit, rat and mare ovaries is also involved in ovulation [43–49]. All of these studies suggest that NO regulates the key mechanisms of ovarian physiology.

In rabbits, NO has a direct antisteroidogenic effect at the luteal level. Numerous in vivo and in vitro experiments have found that NO and NOS are the main targets of PGF2α and effectors of PGF2α-induced luteolysis in competent CL [10,11,17,18,33,50]. Ovarian NO is known to be a mediator of the luteolytic action induced by PGF2α in rabbits and other mammalian species [17,51–55]. Ovarian NO might also control the CL lifespan by regulating 17β-estradiol and progesterone concentrations. However, in contrast to earlier findings in rat and human in vitro cultured CL [41,56], NO did not affect the total androgens and 17β-estradiol production in rabbit CL [17]. Contrastingly, in rabbit CL cultured in vitro, the NO donor, sodium nitroprusside, greatly reduced progesterone secretion in all luteal developmental stages [17]. Luteal NOS activity decreases between the early-to mid-luteal phases with elevated steroidogenesis levels [17,57], which increase again in late-CL when the progesterone levels drop and natural luteolysis initiates [5,57].

#### **4. Leptin**

Leptin is a cytokine secreted mainly by adipocytes and encoded by the obese gene [58]. Leptin regulates the hypothalamic centers of satiety and energy metabolism through the modulation of various neurotransmitters [59,60].

The leptin receptor (ObR) has six isoforms (a–f) resulting from mRNA splice variants [61,62]. ObRa–d and ObRf have identical extracellular and transmembrane domains [62,63]. A long intracellular domain of ObRb activates the Janus kinase (JAK)/signal transducer with the subsequent signal transducer and activator of transcription (STAT) phosphorylation [64]. Contrastingly, the short intracellular domain of ObRa, ObRc,d and ObRf activates the mitogen-activated protein kinase (MAPK) pathway [61,65].

Several studies have found that various key mammalian reproductive processes are modulated by leptin [66], including steroidogenesis [67,68], ovulation [69,70], pregnancy [71,72] and menstrual cycles [73,74]. Moreover, leptin is the crucial link between adipose tissue and the reproductive system, since it provides information on whether energy reserves are adequate for normal reproductive function [75].

Leptin receptors are present in several tissues of the hypothalamic–pituitary–gonadal (HPG) axis and in pituitary [76], granulosa, theca and interstitial ovary cells [77]. Various studies have reported that leptin directly inhibits steroidogenesis in intracellular signaling pathways in theca, granulosa and luteinized granulosa cells of rodents, bovines and primates [67,68,77–79].

Our studies on rabbit CL [80] show that leptin affects progesterone and PGF2α release with different intracellular signaling pathways through different receptors (long ObR and short ObR). More specifically, leptin inhibits progesterone release through the MAPK

cascade (short ObR) and stimulates PGF2α release through the JAK pathway (long ObR) (Figure 2).

**Figure 2.** Schematic representation of the leptin mechanisms regulating progesterone release in rabbit mid-CL. For acronyms, see the list of abbreviations in the text.

#### **5. Gonadotropin-Releasing Hormone (GnRH)**

Gonadotropin-Releasing Hormone (GnRH) is a hypothalamic-releasing decapeptide and a key regulator of the mammalian reproductive system. GnRH regulatory action on the reproductive functions is exerted largely via luteinizing hormone (LH) and folliclestimulating hormone (FSH) secretion, which also affect steroidogenesis and germ cell development [81]. Although the hypothalamus and pituitary gland are the main GnRH synthesis and action sites, several studies have reported an extra-hypothalamic presence of GnRH and its cognate receptor (GnRHR) in numerous peripheral tissues, including reproductive organs such as the gonads, prostate, uterine tube, placenta and mammary glands [82]. Previous studies have highlighted that GnRH regulates the ovarian steroid hormones [82]. In rabbit CL, GnRH administration was found to be associated with CL regression with decreased levels of serum progesterone [83]. Contrastingly, no GnRH effects were observed on ovarian tissue steroid production by other authors [84].

The studies conducted in our laboratory [85] highlighted that the autocrine, paracrine and/or endocrine roles of GnRH type I (GnRH-I) directly diminished the progesterone secretion in rabbit CL that had acquired luteolytic competence (Figure 3): GnRH-I acts via GnRHR-I by activating phospholipase C (PLC) and stimulating the inositol trisphosphate (IP3) and diacylglycerol (DAG) pathways. Through the activation of protein kinase C (PKC), these two intracellular messengers stimulate COX2 activity and PGF2α release. This PG induces (via paracrine, autocrine and/or intracrine mechanisms) an increase in NOS activity and NO levels [11], which downregulates the progesterone levels [18,31] (Figure 1, upper, functional luteolysis).

**Figure 3.** Schematic representation of the post-receptorial mechanism of GnRH-I regulating the progesterone release in rabbit CL. The other possible protein kinase C (PKC) targets are represented by hatched lines. Figure from the study by Zerani et al. 2010 [85]. For acronyms, see the list of abbreviations in the text.

#### **6. Endothelin 1**

Endothelin 1 (ET1), a 21-amino acid peptide, is a potent vasoconstrictor secreted by vascular endothelial cells [86,87]. Many tissues other than the vascular endothelium are known to express ET1, including follicular granulosa cells [88–92].

In rabbit CL, ET1 receptors are expressed in the vascular compartments and luteal cells, thus evidencing that the ET1 system is related to ovarian blood flow and steroid hormone production [91,92]. Moreover, ET1-induced luteolysis in rabbits on day nine of the pseudopregnancies was prevented by administering captopril, the angiotensinconverting enzyme inhibitor (ACE). It is important to note that PGF2α-induced luteolysis was not influenced by captopril. These findings indicate that the cascade mechanism triggered by PGF2α does not require the renin–angiotensin system for inducing luteolysis in rabbits [92], which is in good agreement with the data obtained for cows [93]. Strict cooperation between endothelin and NO is required for endothelial cell migration and angiogenesis [94]. ET1 was found to stimulate endothelial NOS under different physiopathological conditions [95], while NO/NOS is a recognized system involved in both PGF2α [11] and ET1 [96]-induced luteal regression.

#### **7. Adrenocorticotropic Hormone**

Adrenocorticotropic hormone (ACTH) is a major component of the hypothalamic– pituitary–adrenal (HPA) axis, which is synthesized and secreted by the anterior pituitary gland in response to stress. This response is activated by the hypothalamic corticotropinreleasing hormone (CRH), which stimulates pituitary ACTH release, with subsequent glucocorticoid secretion from the adrenal glands.

There is strong evidence that female reproduction can be impaired by stress [97]. In fact, CRH, ACTH and glucocorticoid negatively affect fertility by targeting the hypothalamic GnRH neurons [98], as well as pituitary LH and/or FSH production and

sex steroid synthesis by ovarian follicles and CL. However, the mechanisms by which hormones released during stress may inhibit reproductive mechanisms have yet to be clarified; however, any direct action of ACTH on ovarian functions requires the activation of melanocortin receptor 2 (MC2R) [99], while any indirect action requires glucocorticoid receptor (GR) activation.

The presence of ACTH and glucocorticoid receptors in the luteal cells of rabbit CL [100] supports the hypothesis that ACTH affects ovarian functions both directly and indirectly. During the early and mid-luteal phases (days four and nine of the pseudopregnancies), ACTH increased the in vitro progesterone and PGE2 releases but reduced the PGF2α release. Contrastingly, ACTH increased the in vivo plasmatic cortisol levels within four hours, while the progesterone levels dropped 24 h later and for the following 48 h. Daily injections of ACTH did not affect the progesterone profile following ovulation. Taken together, these findings indicate that ACTH directly induces the upregulation of luteal progesterone synthesis through MC2R (Figure 4), while it indirectly blocks CL functions through the cortisol/GR system.

**Figure 4.** Schematic diagram of the adrenocorticotropic hormone (ACTH), kisspeptins (KiSS) and peroxisome proliferator-activated receptor (PPAR) mechanisms modulating progesterone release in early rabbit CL. The effectors that could directly modulate the KiSS/KiSSR (receptor) system at the CL level are represented by blue lines. For acronyms, see the list of abbreviations in the text.

#### **8. Immunity Mediators**

It is now widely accepted that luteolysis is an event mediated by immune effectors in rabbits and other species, as demonstrated by the presence of immune cells during spontaneous luteal regression [32]. Luteal immune cells are key modulators of CL activity, affecting the luteal, endothelial and stromal cells through several cytokines, including IL1, tumor necrosis factor (TNF)α, monocyte chemoattractant protein-1 (MCP1) and interleukin 2 (IL2) [33,101,102]. In rabbits, during spontaneous luteolysis, the expression levels of MCP1 and IL1β increased on day 15 of the pseudopregnancies [33]. These findings show the greater influx of macrophages and immune cells observed during luteal regression [103]. The IL2 transcript increases earlier (day 13 of the pseudopregnancies) than the other cytokines [33]; in fact, T lymphocytes were detected in rabbit CL before the macrophages [103].

The IL-1 cytokine is present in the ovaries of various species, including rabbits [104,105]. IL1β has various effects on the ovaries [106]: it inhibits progesterone production, increases PG synthesis and PGF2 receptor expression, it inhibits COX2 mRNA degradation [107], enhances NO production and induces the activation of constitutive and inducible NOS [108].

Our studies report [21] that injecting pseudopregnant rabbits with PGF2α markedly upregulated COX2 and IL1β mRNA expression and increased PGF2α release and COX2 activity only in CL with acquired luteolytic capacity [31]. These data suggest that IL1β enhances intra-luteal PGF2α synthesis by upregulating the luteal function of COX2 and NOS, thus promoting functional regression in luteal cells that have achieved luteolytic capacity.

#### **9. Peroxisome Proliferator-Activated Receptor**

The peroxisome proliferator-activated receptors (PPARs) include a family of three (a, d and c) nuclear receptor/transcription factors, which regulate steroidogenesis, angiogenesis, tissue remodeling, cell cycle and apoptosis [109], which are all essential processes for normal ovarian function [110]. All three PPARs have been detected in the ovaries of numerous species [111], including rats [110,112], mice [113], pigs [114], sheeps [115], cows [116–118], rabbits [119] and humans [120,121].

Komar [110] reported that PPARc activation affected the progesterone synthesis in ovarian cells. In particular, an endogenous activator of PPARc 15d-PGJ2 inhibited both the basal and gonadotropin-induced production of progesterone in human granulosa cells [122], while 15d-PGJ2 and ciglitazone, a synthetic PPARc activator, increased progesterone production by granulosa cells in equine chorionic gonadotropin (eCG)-primed immature rats [123]. PPARc activation by 15d-PGJ2, ciglitazone or another synthetic activator, troglitazone, also increased progesterone release by porcine theca and bovine luteal cells [114,124]. Taken together, these findings indicate that the cell type, stage of cell differentiation, stage of the ovarian cycle and/or animal species influence the effects of PPARc on progesterone production [110].

Our study [125,126] suggests that PPARc may play a luteotropic role in rabbit CL through a mechanism that upregulates 3β-hydroxysteroid dehydrogenase (3β-HSD) and increases progesterone while it downregulates PGF2α and its correlated enzyme COX2 [21] (Figure 4). Moreover, the significant decrease in PPARc in the luteal cell nucleus during the late-luteal stage supports the aforementioned mechanism, thus suggesting that this reduction may be required for luteolysis to take place.

#### **10. Dopamine**

The catecholamine dopamine (DA) is a neurotransmitter widely distributed in the brain and in various peripheral organs of numerous species [127]. DA exerts its physiological actions by binding to specific receptors (DR). In mammals, there are five dopamine receptor subtypes, which are grouped into the D1R-like and D2R-like receptor superfamilies [127,128].

D1R-like receptors stimulate the production of the second messenger cyclic adenosine monophosphate (cAMP); contrastingly, D2R-like receptors inhibit cAMP synthesis, which

decreases the protein kinase A (PKA) activity [128]. In mammals, dopamine receptors are widely expressed in many organs and tissues, including the reproductive system [128]. D1R has been detected in the luteal cells of humans [129,130], horses [131], rats [132], cows [118] and rabbits [133], suggesting that DA might be directly involved in the physiological pathways regulating the CL function.

Our studies [133] provide evidence that CL produce DA and that the DA/D1R-D3R system regulates the CL lifespan by exerting either luteotrophic or luteolytic actions depending on the luteal stage. In fact, the DA/D1R-D3R system stimulated PGE2 and progesterone synthesis by early CL, while it increased PGF2α production and decreased progesterone production by mid- and late-CL (Figure 5).

A multi-synaptic neural pathway connects the ovaries to the central nervous system in mammals [134]. Moreover, the ovarian interstitial stroma is composed of many different cell types, including neuron-like or neuroendocrine cells [135]. These data suggest that extrinsic and intrinsic neurons are another paracrine source of DA that can bind its cognate receptors D1R and D3R in the CL, thus supporting the hypothesis that the DA/DR system plays a physiological role in regulating the CL lifespan and functions.

#### **11. Kisspeptin**

The hypothalamic neuropeptide kisspeptins (KiSS) are greatly involved in mammalian reproduction. In fact, they regulate the synthesis and production of GnRH that are required to initiate puberty and sustain normal reproductive function [136].

KiSS and its receptor KiSS1R are expressed in various ovarian structures, including the CL of several mammalian species [137–139], supporting the hypothesis that these neuropeptides can regulate the CL lifespan by modulating the steroidogenic enzymes controlling progesterone synthesis. Moreover, Laoharatchatathanin et al. [140] suggested that KiSS is involved in the luteinization of rat granulosa cells.

Based on data obtained in our laboratory [141], we hypothesize that, besides the wellknown hypothalamic mechanism, the KiSS/KiSS1R system may also directly control the rabbit CL lifespan via local mechanisms. In fact, KiSS was found to exert a luteotrophic action by increasing luteal progesterone synthesis, likely through autocrine and/or paracrine mechanisms that simultaneously reduce PGF2α production and increase PGE2 production by blocking COX2 activity (Figure 4). The lack of KiSS1R expression in late-CL suggests

that the functional activity of the KiSS/KiSS1R system is mainly regulated by the gene and/or protein expression of the receptor.

Interestingly, there is sufficient evidence to suggest that the hypothalamic KiSS-1 gene expression is regulated by several factors, including melatonin, gonadal steroids and leptin, which convey environmental cues and reproductive and metabolic conditions, respectively [142,143]. The theory that these factors could modulate the luteal KiSS/KiSS1R system cannot be ruled out (Figure 4).

#### **12. Nerve Growth Factor**

The nerve growth factor (NGF), together with brain-derived growth factor and other neurotrophins, belong to the neurotrophin family [144]. These neurotrophins maintain normal physiological functions in the central and peripheral nervous systems, including neural development, differentiation and synaptic plasticity [145,146]. NGF and its receptors neurotrophic receptor tyrosine kinase 1 (NTRK1) and nerve growth factor receptor (NGFR) have been found in rabbit ovaries [147,148]. In particular, our studies [149] have evidenced that NGF from seminal plasma supports the neuroendocrine ovulatory reflex induced by mating and/or vaginal stimulation through a novel mechanism exerted on the uterus and/or cervix.

Although there is sufficient experimental evidence suggesting that seminal plasma NGF is able to induce ovulation in rabbits [147], its potential role in regulating the CL lifespan has not yet been thoroughly explored. To date, we only know that NGF and its cognate receptor NTRK1 are expressed in rabbit CL at various stages of a pseudopregnancy [149]. Contrastingly, using purified NGF obtained from seminal plasma, Silva et al. [150,151] observed that, in llamas, CL increased vascularization, upregulated cytochrome P450, family 11, subfamily A, member 1/P450 side chain cleavage and steroidogenic acute regulatory protein transcripts and increased progesterone secretion. All of these findings support the hypothesis that NGF positively affects CL development. Tribulo et al. [152] and Stewart et al. [153] obtained similar results in heifers; however, no luteotrophic effect was observed in alpaca CL using recombinant human NGF [154,155].

#### **13. Conclusions**

In conclusion, it is now well-documented that the progressive acquisition of luteolytic competence by rabbit CL is not only due to their increased sensitivity to PGF2 induced by the upregulation of PGF2α and its receptors and to the decrease of the luteotropic factors (E2, PGE2 and ACTH), but it is also caused by several antisteroidogenic factors. These include, among others, GnRH, ET1 and leptin, which influence the inflammatory, vascular and apoptotic processes involved in the luteolytic process through interaction with PGF2α and the NO/NOS system. During PGF2α-induced CL regression with luteolytic competence, all these factors concomitantly induce the upregulation of NOS, COX2 and PGE2-9-K activities and gene transcripts for ETI, COX2, IL1B and TP53, as well as the downregulation of several other transcripts, including ESR1 and BCLXL. Therefore, the luteolytic effect of PGF2α is the result of its influence on distinct processes involving the regulation of vasoactive peptides, steroidogenic pathways and apoptotic pathways. However, despite the increased knowledge on the physiology of rabbit CL, it is recommended that further research should be undertaken in the near future by a younger generation of researchers who will be able to apply these new discoveries in the challenge for new rabbit breeding strategies.

**Author Contributions:** Conceptualization, C.B., M.M. and M.Z.; infographic, M.Z.; writing—original draft preparation, M.M. and A.P. and writing—review and editing, C.B. and M.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was partially funded by the BC Red-water (Perugia, Italy), PFZM Kitchenbrown (Matelica, Italy) and MMZM Bighead (Settevalli, PG, Italy) trusts.

**Acknowledgments:** In memory of Francesco Parillo (1964–2018), friend and colleague, who substantially contributed to the results of our research group.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**



#### **References**


### *Review* **Strategies for Highly Efficient Rabbit Sperm Cryopreservation**

**Kazutoshi Nishijima 1,2,3,\*, Shuji Kitajima 4, Fumikazu Matsuhisa 4, Manabu Niimi 5, Chen-chi Wang <sup>6</sup> and Jianglin Fan 5,7,\***


**Simple Summary:** The rabbit is a valuable animal for both the economy and biomedical sciences. Therefore, the preservation of many rabbit strains is vitally important. So far, sperm cryopreservation is one of the most efficient ways to preserve rabbit strains because it is easy to collect ejaculate repeatedly from a single male and perform artificial insemination to multiple females. Although this method is widely used, there are still some concerns regarding the cooling, freezing and thawing process of sperms, which markedly affects the quality of preserved sperms. In this article, we will review the progress made during the past years in terms of cryopreservation of rabbit sperms and discuss those factors that would possibly influence sperm damage including freezing extender, cryoprotectant, supplements, and procedures.

**Abstract:** The rabbit is a valuable animal for both the economy and biomedical sciences. Sperm cryopreservation is one of the most efficient ways to preserve rabbit strains because it is easy to collect ejaculate repeatedly from a single male and inseminate artificially into multiple females. During the cooling, freezing and thawing process of sperms, the plasma membrane, cytoplasm and genome structures could be damaged by osmotic stress, cold shock, intracellular ice crystal formation, and excessive production of reactive oxygen species. In this review, we will discuss the progress made during the past years regarding efforts to minimize the cell damage in rabbit sperms, including freezing extender, cryoprotectants, supplements, and procedures.

**Keywords:** rabbit; sperm quality; cryopreservation; animal model; assisted reproductive technology

### **1. Introduction**

Rabbits have been indispensable for human life because they are not only valuable for agriculture but also for biomedical research. Rabbits are widely used as a source of meat, hair and fur, and it is estimated that each year, around 300 million rabbits (and hares) are used in the world [1]. Because of their tame characters, rabbits are also raised as a pet. In addition, rabbits are the most-used animals for antibody production for biomedical research. Furthermore, rabbits are similar to humans in terms of cardiovascular physiology and lipid metabolism, and they play an important role in studying human diseases such

**Citation:** Nishijima, K.; Kitajima, S.; Matsuhisa, F.; Niimi, M.; Wang, C.-c.; Fan, J. Strategies for Highly Efficient Rabbit Sperm Cryopreservation. *Animals* **2021**, *11*, 1220. https:// doi.org/10.3390/ani11051220

Academic Editor: Rosa María García-García

Received: 15 March 2021 Accepted: 21 April 2021 Published: 23 April 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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as atherosclerosis and hypercholesterolemia [2,3]. Along with the development of genetic engineering, a number of gene-modified rabbits have been established as experimental models of human diseases. In addition to transgenic rabbits produced with the conventional pronuclear microinjection technique, knockout rabbits have been established using CRISPR/Cas9 genome editing technology [4]. These established genetically modified rabbits are rare and valuable and thus it is vitally important to breed and maintain rabbit strains for different purposes and preserve them as bio-resources [5].

There are two major ways to preserve rabbit strains. The common way to maintain a rabbit colony is carried out simply by repeat breeding. However, several difficulties with this method exist including space and cost. In particular, rabbit shows severe inbreeding depression [6–9], thus a considerable number of rabbits are required to keep a colony. For laboratory rabbits, they are usually housed in strictly controlled conditions in terms of temperature, humidity, illumination and microbiological examinations. Furthermore, living animals have a risk of annihilation or escape in the case of a disaster or accident.

The second method of maintaining the rabbit colony is the cryopreservation of gametes. Cryopreservation of gametes requires less space and cost than animal breeding. It is generally believed that properly cryopreserved zygotes and gametes can be preserved semi-eternally in a liquid nitrogen tank to keep their fertile and developmental ability. In the case of employing ovum or embryo preservation, ova or embryos are generally obtained with oviduct–uterus dissection from sacrificed females, and a skillful surgical operation is required for the embryo transfer. In contrast, ejected semen can be collected repeatedly without sacrificing males (Figure 1) and artificially inseminated into females can be conducted without specific skills, and thus, sperm cryopreservation would be the first choice for preservation of rabbit strains even though sperms preserved can bring paternal hereditary information only, and immediate offspring is always heterozygosity. However, when concerned with one specific transgene, homozygotes can be obtained in the second generation. Even in the case of livestock animals concerning pedigree related with polygenetic factors, the inbreeding can be avoided by mating live females and cryopreserved sperm with a generation gap.

As mentioned above, the successful preservation of rabbits depends on the efficiency and reliability of procedures in sperm cryopreservation. It is known that the process of sperm cryopreservation, including cooling, freezing and thawing, leads to cellular damage on membrane, cytoplasm and genome structures [10,11] caused by osmotic stress, cold shock, intracellular ice crystal formation, and excessive production of reactive oxygen species (ROS) [12] (Figure 2). During the cooling process, the sperm membrane is injured by cold shock which can be diminished by cooling rate [13] or materials stabilizing the membrane including egg yolk or skim milk [14]. The addition of cryoprotectants causes osmotic and toxic stress, which increases due to prolonged exposure during slow cooling [15]. In following freezing process, major problem is ice crystal formation which grow larger by recrystallization and injures cell [16]. The freezing rate and cryoprotectants application should be considered to diminish the problem. Recrystallization occurs during the thawing process because of entry through the recrystallization temperature zone. Since sperms suffer oxidative stress throughout the cryopreservation process [12], supplementation of antioxidants is considerable for the improvement of sperm quality.

Enormous efforts have been exerted to minimize these detrimental effects, and increase the efficiency and reliability of sperm cryopreservation in the rabbit. In this review, we will discuss recent findings and perspectives including extenders, cryoprotectants, supplements and procedures.

**Figure 1.** Schematic illustration of the essential process of sperm cryopreservation. (**A**) Semen is collected with an artificial vagina; (**B**) semen is diluted with a freezing extender; (**C**) sperm solution is cooled at slow rate (with a programming incubator); (**D**) sperm solution packed in freezing straws are frozen in vapor of liquid nitrogen; (**E**) vitrification is another option for sperm freezing; (**F**) sperms are cryopreserved in liquid nitrogen, and (**G**) sperms are thawed by immersion in a warm bath.

**Figure 2.** Schematic diagram of temperature changes during sperm cryopreservation process and associated problems.

#### **2. Effects of Extender and Cryoprotectants**

The sperm freezing extenders are commonly composed of a buffer (commonly Tris buffer for rabbit sperms), salt(s) and cryoprotectant(s) to avoid cell damage caused by inadequate pH, osmolality and cryogenic injury [13]. As a component in the freezing extender, egg yolk provides optimal results in rabbit sperm cryopreservation [17,18]. Nevertheless, the egg yolk contains both beneficial and detrimental components for sperms [19,20]. Additionally, there is a sanitary concern in using fresh biotic materials, like the egg yolk, which is subjected to quarantine inspection in the case of import/export. To avoid these risks, lecithin, also known as phosphatidylcholine, a component of egg yolk, is often used to prevent cold shock in sperm cryopreservation [21,22]. It is reported that non-animal originated soybean lecithin with minimal sanitary risks can be used as a substitute for egg yolk based on the motility and fertility of the frozen–thawed rabbit sperm [23]. Skim milk, another substitute, contains advantageous components in the freezing extender in various animal species [24–30] including rabbits [31]. However, using skim milk in the sperm freezing extender is less common than egg yolk in the rabbit.

The major problem in sperm cryopreservation is the mechanical invasion of sperm cells by ice crystals generated during the freezing process, which decreases the viability of sperm after thawing [32]. Dehydration of cellular water and percolation and immersion of cryoprotectants into the cell are usually performed to avoid the generation of the ice crystals. Both permeable (such as glycerol, dimethyl sulfoxide (DMSO), ethylene glycol, and amides) and non-permeable (saccharides, lipoprotein and Ficoll) are used as cryoprotectants. The permeant agent binds to intracellular free water leading to suppression of the generation of ice crystals. On the other hand, the non-permeant agents surrounding the cell increase extracellular osmotic pressure and enhance dehydration of the cell [33–35].

Lots of studies have been conducted regarding concentrations and combination effects of the cryoprotectants on rabbit sperm cryopreservation as encyclopedically reviewed by Mocé and Vicente [14]. Though glycerol and DMSO are the most common permeable cryoprotectant, it was suggested that glycerol is not suitable for rabbit sperm cryopreservation [14] possibly because of its low water permeability and high activation energy [36]. Additionally, it is known that high concentrations of DMSO show adverse effects on sperm quality in terms of motility and acrosome integrity [37,38]. Amides, namely lactamide and acetamide, are another candidate as a permeable cryoprotectant for rabbit sperm cryopreservation [39,40] and provide better results than glycerol or DMSO [41,42].

Lactose, sucrose, maltose, raffinose, treharose, and dextrans are used as non-permeable cryoprotectants in sperm cryopreservation, and have been shown to have an effect of stabilizing the plasma membrane during freeze–thaw process by interacting with membrane phospholipids [33–35] in addition to increasing the osmotic pressure.

#### **3. Effects of Freezing and Thawing Procedure**

Two principal techniques, slow freezing and vitrification, were employed for sperm freezing (Figure 1). During the freezing process of cells, extracellular water is frozen more quickly than intracellular water [42]. Generation of the ice corresponds to the decrement of water, which produces an imbalance of cellular osmotic pressure and intracellular water moving out of the cell. In slow freezing, generation of the intracellular ice crystal is suppressed since the cellular dehydration progresses slowly during a longer time of freezing. Concomitantly, concentrations of solute are deleterious to the cells and exposure time will be increased within a slow freezing procedure [42]. On the other hand, in vitrification, the cells are immediately cooled to −196 ◦C in liquid nitrogen and exposure time to high concentrated cryoprotectant is minimized. A countermeasure, including the application of high concentrated cryoprotectans, to suppress the ice crystal generation is required since dehydration is not always sufficient in vitrification.

Vitrification procedure has been explored in various mammalian sperm including humans [10,43]. It is known that sperms with larger heads are more susceptible to cold shock [44], which reduces sperm survivability [45]. The head of rabbit sperms is relatively

large [32] and no efficient procedure in vitrification has been established. Rosato and Iaffaldano [46] compared the frozen–thawed sperm survival and fertility with/without various cryoprotectants between slow freezing and vitrification, and showed that the outcome from vitrification was far inferior to slow freezing. It is known that cells with a high water permeability show better tolerance for rapid freezing than those with a low water permeability [47], and rabbit sperm shows a low water permeability [36]. Therefore, conventional slow freezing is the prime choice for sperm cryopreservation at the current moment.

In the slow freezing procedure, sperms are cooled down to 5 ◦C before freezing to avoid the cold shock [45]. In the case of rabbits, Mocé et al. [48] reported that slow cooling to 5 ◦C improved neither fertility rate nor prolificacy in relation to cryopreservation. On the other hand, Maeda et al. [49] revealed that the viability of frozen–thawed rabbit sperm cooled at −0.1 or −0.2 ◦C/min (slower) were higher than at −0.8 ◦C/min. These data indicate that a faster cooling rate negatively affects sperm viability and does not improve reproductive performance after freezing and thawing. Obtaining more viable sperm results in efficiency improvement of cryopreservation, since the possibility of successful fertilization is increased. Additionally, in rabbits, time held at 5 ◦C affects the quality and reproductive performance of sperm. It was reported that longer holding time (90 min) at 5 ◦C increases the quality of frozen–thawed rabbit sperm and their fertilizing ability [50], and conversely, and shortened holding time (10 min) decreases it [51].

The ice crystal formation during the freezing process damage cells as described above and freezing protocol is also one of the concerns. In rabbit sperms, it was revealed that sperms frozen at slow (−15 ◦C/min) and fast (−60 ◦C/min) rates were lower in the quality and fertilizing ability than those frozen at medium (−40 ◦C/min) rate and in static liquid nitrogen vapor [52].

The thawing rate of frozen sperms is also known to affect the quality of sperms. Though frozen sperms are usually thawed at temperatures close to body temperature, it is generally recognized that high thawing rates provide better results [32,53]. It is possible that low thawing rates enhance recrystallization, a phenomenon that relatively many small ice crystals aggregate and form fewer larger ice crystals, which causes more severe damage to the cells [16]. Though thawing temperatures over 60 ◦C were adopted in some cases [54–57], exposing duration to a high temperature must be strictly controlled. Mocé et al. [58] compared the fertility rate and prolificacy of frozen rabbit sperm between thawed at 50 ◦C and 70 ◦C for 10–12 s, and concluded that thawing at 50 ◦C provided better results. In contrast, Chen and Foote [59] reported that the mortality of sperm thawed at 25 ◦C for 1 min was superior to those thawed at 45 ◦C for 30 s or 65 ◦C for 7 s following mechanical seeding at −6 ◦C. Therefore, appropriate thawing temperature can be affected by other conditions including cooling or freezing procedures, and further studies are demanded.

#### **4. Cryopreservation Device**

The choice of the freezing devices affects the quality of frozen–thawed sperm. Glass vials [31], plastic ampoules, polyvinylchloride tubing, [60] and pellet [61] addition to straws, which are widely employed in recent years, have been utilized for rabbit sperm cryopreservation. Thermal conductance to sperm depends on the shape, size and material of the device, which influences cooling, freezing and thawing rates. It was shown that rapid warming had a more dominant effect on survival than rapid cooling in mouse oocyte [62], and rapidly warming to the critical temperature range (−70 ◦C to −35 ◦C) at which intracellular ice is likely to form by recrystallization would improve cryopreservation efficiency of cell [63]. In the case of rabbit sperms, though the rapid freezing method (vitrification) has not been commonly utilized, the development of a new method and device for rapid thawing could lead to a great improvement of sperm cryopreservation efficiency.

#### **5. Effects of Supplements**

As mentioned above, lots of studies have been performed to address components of the sperm freezing extenders; however, results were not always satisfying. Some additional supplements can improve the efficiency in sperm cryopreservation (Table 1).

The antioxidant is one of the most expected supplements for sperm cryopreservation by eliminating the excessive production of ROS [12]. Zhu and colleague investigated the effects of supplementation of amin E analogue [64], cysteine [65], glutamine [66], trehalose [67] and melatonin [68], and revealed that supplementation of these antioxidative agents in Tris-citrate-glucose extender decreased ROS levels and improved the quality of frozen–thawed rabbit sperms. Fadl et al. confirmed 1.0 mM melatonin improved the motility, viability, membrane and acrosome integrities, and DNA integrity of frozen– thawed rabbit sperms in different extender (INRA-82) [69] Additionally, curcumin and curcumin nanoparticles were confirmed to improve the post-thawed quality of rabbit sperms via redox signaling and reduce the apoptosis process [70]. On the other hand, Maya-Soriano et al. reported that supplementation of bovine serum albumin, retinol and retinyl in the sperm freezing extender has no beneficial effect on the viability, mortality, progressivity, and acrosome and morphological integrity of frozen–thawed rabbit sperms at the concentrations they tested [71]. In this sense, other researchers have revealed the importance of the antioxidant concentration to achieve beneficial effects on rabbit sperm quality after thawing. Thus, while 4 mM of glutathione (GSH) improved [72] the viability, mortality, progressivity and acrosome integrity of frozen–thawed rabbit sperms, 0.5 mM of GSH did not provide similar results [73]. More studies are necessary for the adjustment of the freezing media in this species.

There are also some supplements with controversy surrounding their effects on rabbit sperm cryopreservation. Bovine serum albumin (BSA) is known to have a dual effect to protect sperm from osmotic stress by increasing membrane resistance [12] and oxidative stress by trapping free radicals [74]. Therefore, supplementation of BSA has been shown to improve the quality of frozen–thawed sperms in some species [75–78]. However, in rabbits, controversial results regarding the usage of BSA have been reported. Thus, while Maya-Soriano et al. [71] did not find any beneficial effect on sperm viability, mortality, progressivity, and acrosome and morphological integrity, Rosato and Iaffaldano [46] showed that frozen–thawed rabbit sperm display better mortality and DNA integrity when BSA is combined with sucrose or trehalose. These facts suggest that concentration and/or combination with other contents of the extender are important for exerting the beneficial effects of BSA. Moreover, gelatin is known to have protective effects including reduction of the sperm sedimentation and maintenance of the pH homogeneity in cooled semen [79] which has been confirmed in rabbits as well [80,81]. However, Cortell et al. [82] reported that gelatin addition did not improve the motility and viability nor fertility and prolificacy of frozen–thawed sperms in the rabbit. In later years, it was reported adding 2% gelatin enhanced the freezability and fertility of frozen–thawed rabbit sperms [83].

Antifreeze proteins (AFPs) are known to stabilize cell membranes and inhibit ice crystal growth and ice recrystallization [84,85]. With such functions, supplementation of AFP type III in the sperm freezing extender can improve the quality of frozen–thawed rabbit sperms [86]. However, AFPs are not easy to extract from natural resources such as fungi, bacteria, plants, insects and fish that are adapted to cold environments [87] and AFPs derived from other organisms can be detrimental to sperms or inseminated female animals. Tekin and Da¸skın [88] utilized polyvinyl alcohol (PVA) instead of AFPs for ice recrystallization inhibition and showed that supplementation enhances motility, viability, acrosome integrity and mitochondrial activity in frozen–thawed rabbit sperms.


**Table 1.** Effects of supplements on quality of thawed rabbit sperm frozen by slow freezing method.

\*: Minitüb, Tiefenbach, Germany. Concetrations of glycerol when supplemented with a: 0.001, b: 0.01, c: 0.1, d: 1, e: 2% of polyvinl alchool.

By stabilization of cell membrane, supplementation of a silk protein, sericin [89] and cholesterol-loaded cyclodextrin [90] improved motility and quality of frozen–thawed rabbit sperm. However, since these supplements are known to inhibit acrosome reaction [89,91], further studies on enhancing its fertility are required for practical use.

#### **6. Another Preservation Strategy**

The freeze-drying technique is an alternative technology for the long-term preservation of sperms [92]. Liu et al. [93] showed that freeze-dried rabbit sperms maintain the ability for full-term development in spite of immobilization, membrane breaking, and tail fragmentation. It is important to store freeze-dried sperms at low temperatures for stable, long-term preservation, and properly stored freeze-dried sperms maintain their fertility for years [92]. It is necessary to operate intracytoplasmic sperm injection (ICSI) [94] and embryo transfer, which require a skillful technique and particular device for using the freeze-dried sperms. One of the advantages of sperm preservation in rabbits is the applicability of artificial insemination as mentioned above, and preservation of freeze-dried sperms would be just a spare option in rabbit sperm preservation.

#### **7. Conclusions and Perspectives**

As stated above, numerous studies on improving rabbit sperm cryopreservation have been conducted from various aspects including freezing procedure, type, concentration and combination of cryoprotectants. In spite of this, a standard procedure for rabbit sperm cryopreservation has not been well established due to various and irreproducible results from each study.

One of the reasons for the irreproducibility in rabbit sperm cryopreservation may be derived from the differences in sperm conditions. It is known that freezabilities of rabbit sperms differ among individuals [95] or breeds [96]. However, rabbit breeds used in some reproductive studies have not strictly and well defined rather than other livestock animals such as cattle, horses or pigs, and some authors even did not provide enough information about the rabbit breed examined. There are several reports which indicate associations between the sperm freezability and abundance of particular components in seminal plasma including proteins and fatty acids in some species [97–102]. The individual and breed difference in sperm freezability can be explained by such seminal plasma traits. It would be possible to improve the sperm freezability and resolve the individual or breed differences by complemental supplementations according to the seminal plasma trait. In the rabbit, it was revealed that genotype, i.e., breed, affects the abundance of some seminal plasma proteins [103], which are associated with sperm quality [104]. Furthermore, there is no information about the association between sperm freezability and seminal plasma traits in rabbits, and further studies are needed. Again, lack of information about the rabbit breed can disturb the improvement of rabbit sperm cryopreservation efficiency.

Another possible reason for the irreproducibility is disunified evaluation criteria of the quality of the sperms among the reports. Some studies report both the quality of the frozen–thawed sperms and their fertility and prolificacy, and others do only one of them. Examination of fertility and prolificacy involves artificial insemination procedures which affect the results of the study. On the other hand, fertility and prolificacy cannot be estimated by the sperm quality alone, even though obtaining more motile sperms is generally advantageous for efficient reproduction [105–107]. Additionally, the rate of rapid and progressive motile sperm would be important for successful artificial insemination, since the inseminated sperm need to reach the ova via a long reproductive tract [108–110].

In any case, to achieve a consensus on the efficient method for rabbit sperm cryopreservation, extensive investigations are required under unified evaluation criteria and conditions except for factors like extenders, cryoprotectants and procedures to be examined. On the other hand, it seemed that the supplements like antioxidants generally just add their effects without interfering with other components in the extender. Therefore, most of the supplements exert expected effects on the quality of frozen–thawed rabbit sperm even in various conditions including the extender (Table 1). The dose and combination of the supplements can be a key subject for highly efficient rabbit sperm cryopreservation in future studies.

As Dr. Robert G. Edwards was awarded The Nobel Prize in Physiology or Medicine for the development of in vitro fertilization (IVF) in 2010, assisted reproductive technology (ART) including IVF is an indispensable medical procedure in this modern age. The rabbit is also known as a prime reproductive model for human health, because of (1) exact staging of early embryonic developmental and maternal pregnancy stages, (2) large-sized blastocysts amenable to micromanipulation, (3) cell-lineage-specific analyses, (4) gastrulation stages representative of mammalian development, and (5) placental morphology and function similar to the human [111]. Therefore, the development of reproductive technology in rabbits, which leads to the improvement of medical procedures in humans, is very important and desirable.

**Author Contributions:** Writing—original draft preparation, K.N.; review and editing, J.F. and M.N.; supervision, S.K., F.M. and C.-c.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported in part by JSPS KAKENHI (JP17K08783 and JP15H04718), the National Natural Science Foundation of China (No. 81941001 and 81770457) and the JSPS-CAS Bilateral Joint Research Program (JPJSBP 120187204).

**Acknowledgments:** The authors express their sincere respect and thanks to the emeritus Teruo Watanabe, Saga University and Masatoshi Morimoto of Fukuoka Jo Gakuin Nursing University for their valuable advice in preparing the article.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Modulation of Cathepsin S (***CTSS***) Regulates the Secretion of Progesterone and Estradiol, Proliferation, and Apoptosis of Ovarian Granulosa Cells in Rabbits**

**Guohua Song 1,†, Yixuan Jiang 1,†, Yaling Wang 1, Mingkun Song 1, Xuanmin Niu 2, Huifen Xu 1,\* and Ming Li 1,\***


**Simple Summary:** In goat and sheep, *CTSS* is reported to be important for the development and maturation of oocytes by regulating cell proliferation and apoptosis. The purpose of this study was to investigate the role of *CTSS* in regulating cell apoptosis and hormone secretion in rabbit granulosa cells. Our results suggested that the *CTSS* gene can promote the proliferation of granulosa cells and reduce its apoptosis in vitro, while overexpression of *CTSS* promoted the secretion of progesterone and estrogen in rabbit granulosa cells. Therefore, manipulation of *CTSS* may improve development of oocytes, and thus provide an approach for better manipulation of rabbit reproductive performance.

**Abstract:** Cathepsin S (*CTSS*) is a member of cysteine protease family. Although many studies have demonstrated the vital role of *CTSS* in many physiological and pathological processes including tumor growth, angiogenesis and metastasis, the function of *CTSS* in the development of rabbit granulosa cells (GCS) remains unknown. To address this question, we isolated rabbit GCS and explored the regulatory function of the *CTSS* gene in cell proliferation and apoptosis. *CTSS* overexpression significantly promoted the secretion of progesterone (P4) and estrogen (E2) by increasing the expression of *STAR* and *CYP19A1* (*p* < 0.05). We also found that overexpression of *CTSS* increased GCS proliferation by up-regulating the expression of proliferation related gene (*PCNA*) and anti-apoptotic gene (*BCL2*). Cell apoptosis was markedly decreased by *CTSS* activation (*p* < 0.05). In contrast, *CTSS* knockdown significantly decreased the secretion of P4 and E2 and the proliferation of rabbit GCS, while increasing the apoptosis of rabbit GCS. Taken together, our results highlight the important role of *CTSS* in regulating hormone secretion, cell proliferation, and apoptosis in rabbit GCS. These results might provide a basis for better understanding the molecular mechanism of rabbit reproduction.

**Keywords:** CTSS; granulosa cells; proliferation; apoptosis; hormone secretion

#### **1. Introduction**

Rabbits are multi-purpose domestic animals, which can be used as pet animals or biomedical model animals for scientific research, but in people's lives, rabbits are mainly used for meat and fur production [1–3]. Rabbit meat has high nutritional value and is widely accepted by many people for its low content in fat, cholesterol, and high content in protein [4,5]. However, as the demand for rabbit meat increases rapidly, global rabbit meat production is still relatively low [6]. Accelerating the reproductive performance is an important strategy to increase rabbit meat production, thus it is vital to investigate the regulatory mechanism of factors affecting rabbit reproduction.

Ovarian granulosa cells (GCS) are an important component of follicles and play a vital role in follicle growth and development. Each follicle contains an oocyte, numerous

**Citation:** Song, G.; Jiang, Y.; Wang, Y.; Song, M.; Niu, X.; Xu, H.; Li, M. Modulation of Cathepsin S (*CTSS*) Regulates the Secretion of Progesterone and Estradiol, Proliferation, and Apoptosis of Ovarian Granulosa Cells in Rabbits. *Animals* **2021**, *11*, 1770. https:// doi.org/10.3390/ani11061770

Academic Editors: Rosa María García-García and Maria Arias Alvarez

Received: 28 May 2021 Accepted: 10 June 2021 Published: 13 June 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

GCS and theca cells [7]. GCS deliver regulatory signals to oocytes through zone projections (TZPs), and also provide nutrition and metabolite for oocyte [8]. Many studies reported that apoptosis of GCS affect cellular connection between GCS and oocytes [9]. It causes ovarian follicular atresia [10], which occurs in more than 99% of developmental follicles in mammals [11,12]. Another important role of GCS is the secretion of progesterone and estradiol, both of which play an important role in animal breeding and reproduction. It was reported that estradiol can promote the expression of FSH receptors in GCS [13], and promote the formation of rat antral follicles [14]. In bovine, low concentration of progesterone enhanced the follicular development, and the diameter of follicular increased [15,16]. Moreover, after follicle ovulation, GCS differentiated into luteal granulosa cells to form the corpus luteum. The main function of corpus luteum is to secrete progesterone to maintain pregnancy. Therefore, both GCS progesterone secretion disorder and GCS apoptosis will result in a decrease in rabbit reproduction performance.

Cathepsin S (*CTSS*), which is located on chromosome 13 in rabbits, is one member of 11 cysteine proteases, and plays an important role in extracellular matrix degradation and remodeling, antigen presentation, inflammation, and angiogenesis [17]. In malignant tumors, *CTSS* induces tumor proliferation, invasion and metastasis through various mechanisms [18–20]. It was reported that *CTSS* might be a potential predictor of chemotherapy response because up-regulation of *CTSS* is associated with tumor progression and poor prognosis [21]. Studies in vitro also showed that *CTSS* promoted adipocyte differentiation by degrading fibrinolytic proteins [22]. Studies have also found that the polymorphisms of the *CTSS* gene are associated with obesity-related traits [23], and *CTSS* circulating levels are associated with triglycerides synthesis and accumulation [24,25]. *CTSS* can also regulate blood sugar by reducing glucose output [26].

The regulatory function of *CTSS* in cell proliferation has been widely investigated, while its function in the development and maturation of rabbits' oocytes has not been reported. Our hypothesis was that *CTSS* plays an important role in follicle development and ovulation through regulating cell proliferation and hormone secretion. To verify this hypothesis, the present study aimed to investigate the functional role of the *CTSS* gene in cell proliferation and apoptosis, as well as progesterone and estradiol secretion in rabbit GCS. These results provide evidence with which to further uncover the regulatory mechanism which mediated the ovulation process and reproductive performance of rabbits.

#### **2. Materials and Methods**

#### *2.1. Ethic Statement*

The present study was designed and performed according to the guidelines of Institutional Animals Care and Use Committee College (IACUC) of College of Animal Science and Technology of Henan Agricultural University, China (Permit Number: 11-0085; Data: 06-2011).

#### *2.2. Tissue Sample Collection*

In this experiment, three female New Zealand white rabbits (180 days old) were selected from the animal house of Henan Agricultural University. The rabbits were anesthetized and slaughtered for ovary collection. The collected ovaries were placed in PBS (containing 1% penicillin/streptomycin, 37 ◦C) for rabbit granulosa cells isolation.

#### *2.3. Isolation and Culture of Rabbits GCS*

The rabbits' GCS were isolated and cultured according to the previously published papers with few modifications. Briefly, the collected ovaries were washed three times with PBS (phosphate buffer saline) supplemented with 1% penicillin/streptomycin at 37 ◦C. Follicles were needled with a 1 mL syringe in a basal medium supplemented with DMEM/F12 medium (Gibco, MD, USA), 15% FBS (Gibco, CA, USA), and 1% penicillin/streptomycin (Hyclone, Logan, UT, USA) [27]. The GCS were centrifuged (5 min, 1000 rpm/min) and incubated with basal medium at 37 ◦C in 5% CO2; cell medium was changed every 24 h [28]. The GCS isolated from the 3 rabbits were isolated individually and pooled together

for experimental treatments. Before experiments, GCS were seeded in 6-well plate at a density of 3 × 105 cells per cm2. After 24 h incubation (approximately 90% confluence), GCS were treated with siRNA and recombinant adenovirus for 24 h. All treatments were performed with 3 independent biological replicates.

#### *2.4. Immunostaining*

FSHR (follicle stimulating hormone receptor) protein is usually used as a marker protein to identify GCS. The cells were seeded on culture plates plated with cell-climbing slices. After reaching 70% confluence, cells were fixed with 4% paraformaldehyde and then permeated with 0.1% Triton X-100. For immunohistochemical staining, the cells were incubated with anti-FSHR (MAB65591, 1: 200, R&D Systems, Minneapolis, MN, USA) for 1 h at 37 ◦C, and then incubated with goat anti-rabbit IgG (bs-0296G-FITC, 1:400, Bioss, Beijing, China) at 37 ◦C for 45 min. Finally, the cells were taken out and mounted with DAPI (4 ,6-Diamidino-2-phenylindole, Dihydrochloride) medium (Solarbio, Beijing, China). The cells were then observed and imaged under a fluorescence microscope (Nikon Eclipse C1, Tokyo, Japan).

#### *2.5. HE (Hematoxylin-Eosin) Staining*

The morphology examination of GCS was measured by HE staining. GCS were seeded in 6-well culture plates plated with cell-climbing slices. Then, the cells were fixed with 95% ethanol for 20 min and stained with hematoxylin dye solution for 3 min. After dehydration with gradient alcohol, the cells were dyed with eosin dye solution for 5 min. Finally, the cells were mounted with neutral gum and observed by microscope (Nikon Eclipse E100, Tokyo, Japan).

#### *2.6. SiRNA (Small Interfering RNA) Interference*

GCS were seeded into 6-well plates and transfected with siRNA-*CTSS* and siRNA-NC by using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The specific siRNA sequence targeting *CTSS* was designed and synthesized by GenePharma (Shanghai, China). siRNA-*CTSS*, sense: 5 -GGAAGAAAGCCUACG GCAATT-3 ; antisense:5 -UUGCCGUAGGCUUUCUUCCTT-3 . siRNA-NC, sense: 5 - UUCUCCGAACGUGUCACGUTT-3 , antisense: 5 -ACGUGACACGUUCGGAGAATT-3 .

#### *2.7. Recombinant Adenoviruses Generation*

The production of recombinant adenovirus has been previously described [29]. In short, *CTSS* was amplified and subcloned into the shuttle vector pAdTrack-CMV. Then the linearized product of pAdTrack-CMV-*CTSS* was transformed into Escherichia coli BJ5183 competent cells containing the backbone vector pAdEasy-1 to obtain the positive recombinants of pAd-*CTSS*. Then, linearized pAd-*CTSS* fragment was transfected into 293 cells for adenovirus packaging and amplification. GCS were infected with Ad-*CTSS* at MOI (multiplicity of infection) value of 40.

#### *2.8. RNA Extraction and RT-qPCR*

Total RNA of rabbit GCS were extracted by using Trizol reagent (Invitrogen, CA, USA) according to the instructions of the manufacturer. Total RNA was quantified with a Nanodrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized by using Prime Script RT reagent Kit (Takara, Tokyo, Japan) following the instructions. The SYBR Premix Ex Taq II kit (Takara, Tokyo, Japan) was used for RT-qPCR. Primers used for RT-qPCR were listed in Table 1. *β-actin* was selected as reference gene.


**Table 1.** Name, accession number, sequences, amplicon length of primer pairs of RT-qPCR.

Annealing temperature for all primers listed in this table is 60◦C: F, forward primer; R, reverse primer. *PCNA*, proliferating cell nuclear antigen; *STAR1*, steroidogenic acute regulatory protein 1; *CYP19A1*, cytochrome P450 family 19 subfamily A member 1; *CYP11A1*, cytochrome P450 family 11 subfamily 11 subfamily A member 1; *BCL2*, B-cell lymphoma-2.

#### *2.9. Measurement of Progesterone and Estradiol Secretion*

After 48 h of treatment with Ad-*CTSS* and siRNA-*CTSS*, cell culture medium was collected for determination of progesterone and estradiol concentration. Secretion levels of progesterone and estradiol were evaluated with enzyme linked immunosorbent assay (ELISA) according to the instructions of the manufacturer. The kits were all obtained from Nanjing Ruixin Biology (Quanzhou, China). The absorbance was measured using a microplate reader (Bio-Rad) at 450 nm.

#### *2.10. Cell Viability Assay*

GCS were seeded onto 96-well plates at 1 × 104/well and treated with recombinant adenovirus and siRNA for 0 h, 12 h, 24 h, 36 h and 48 h, separately. Cell proliferation was detected with Cell Counting Kit-8 (CCK8) (US Everbright® Inc., Nanjing, China). According to the instructions of the manufacturer, the cells were added to 10 μL of CCK8 solution and incubated at 37 ◦C for 2 h. Then, the optical density (OD) value at a wavelength of 450 nm was detected by a microplate reader (Bio-Rad, Hercules, CA, USA).

#### *2.11. Cell Apoptosis Analysis*

After treatment with recombinant adenovirus and siRNA for 48 h, GCS apoptosis was detected by Annexin V-Alexa Fluor 647/7-AAD Apoptosis Kit (4Abiotech., Beijing, China). According to the instructions of the manufacturer, GCS were digested with trypsin (without EDTA) and resuspended with cold PBS, and then mixed with 5 μL Annexin V/Alexa Fluor 647 at room temperature for 5 min. Finally, the cells were treated with 10 μL 7-AAD and 400 μL PBS. The apoptosis rate of the GCS was measured with a CytoFLEX flow cytometer (Beckman CytoFlex, CA, USA).

#### *2.12. Statistical Analysis*

All data analyses were conducted using SPSS 22.0 software (SPSS, Chicago, IL, USA). All the treatments were performed in three independent biological replicates and three

technical replicates. Statistical analyses were performed with one-way ANOVA to compare the effects of *CTSS* overexpression and knockdown relative to negative control. The fixed effect was treatment and the random effect was replicated in the statistic model. Relative mRNA expression of RT-qPCR was calculated by using 2−ΔΔCt method, where Ct is the cycle threshold. *p* value < 0.05 was indicative of a statistically significant difference, and data are presented as means ± SE.

#### **3. Results**

#### *3.1. Isolation and Identification of Rabbit GCS*

Rabbit GCS were identified by HE staining and immunofluorescence by using FSHR antibody, our HE staining results showed that the nucleus of GCS is blue and the cytoplasm is red, and the pseudopodia between the cells was closely connected (Figure 1A). As shown in Figure 1B, cell nuclei were stained with DAPI and presented to be blue; FSHR protein was green and located in the cytoplasm. The positive rate of FSHR was 99%, indicating that the isolated GCS were 99%.

**Figure 1.** Isolation and identification of rabbit GCS: (**A**): GCS were stained with Hematoxylin and eosin (H&E) staining, (**a**–**c**): Original magnification, 40×, 100×, 200×. (**B**): The identification of rabbit GCS was measured by immunofluorescence staining with fluorescein isothiocyanate (FITC)-goat anti-rabbit IgG. (**d**): Cell nucleus were stained by DAPI (blue), (**e**): FSHR were stained with fluorescein isothiocyanate (FITC)-goat anti-rabbit IgG (green), (**f**): Merge of (**d**) and (**e**). (Scale bar = 100 μm).

#### *3.2. Efficiency of CTSS Overexpression and SiRNA Interference*

RT-qPCR was performed to determine the efficiency of Ad-*CTSS* and siRNA-*CTSS* in rabbit GCS. Our results showed that *CTSS* expression was remarkably increased by Ad-*CTSS* compared with Ad-GFP (Figure 2A, *p* < 0.05), and its expression was significantly decreased by siRNA-*CTSS* relative to siRNA-NC (Figure 2B, *p* < 0.05).

**Figure 2.** The efficiency of Ad-*CTSS* and siRNA-*CTSS* in rabbit GCS: (**A**): mRNA expression changes of *CTSS* after infection with Ad-GFP. (**B**): mRNA expression changes of *CTSS* after transfection with siRNA-*CTSS*. Values are presented as means ± SEM for three biological replicates. \*\* *p* < 0.01.

#### *3.3. CTSS Promotes the Secretion of Progesterone and Estradiol in Rabbit GCS*

To explore the role of *CTSS* on rabbit reproduction, we measured the effects of *CTSS* overexpression and knockdown on hormone secretion in rabbit GCS. As shown in Figure 3A,D, genes responsible for steroid hormone synthesis (Steroidogenic acute regulatory protein, *STAR*) and estrogen synthesis (Cytochrome p450 family 19 subfamily A member 1, *CYP19A1*) were remarkably increased and decreased by *CTSS* overexpression and knockdown, respectively (*p* < 0.05). Consistently, levels of progesterone and estradiol in cell medium were significantly increased by *CTSS* overexpression (Figure 3B,C). While *CTSS* knockdown only decreased progesterone secretion (Figure 3E, *p* < 0.05), estradiol secretion was not changed by siRNA-*CTSS* (Figure 3F, *p* > 0.05).

**Figure 3.** *CTSS* overexpression and interference on progesterone and estradiol secretion in rabbit GCS: Effects of *CTSS* overexpression and knockdown on (**A**,**D**) mRNA expression changes of genes related to progesterone and estradiol secretion; (**B**,**E**) progesterone secretion and (**C**,**F**) estradiol secretion. Values are presented as means ± SEM for 3 biological replicates. \* *p* < 0.05.

#### *3.4. CTSS Promotes Cell Proliferation Activity in Rabbit GCS*

After treatment with Ad-*CTSS* and siRNA-*CTSS* for 0 h, 12 h, 24 h, 36 h and 48 h, the proliferation activity of GCS was measured. As shown in Figure 4A, genes responsible for cell proliferation (*PCNA*, proliferating cell nuclear antigen) were significantly increased by *CTSS* overexpression (*p* < 0.05), and cell proliferation activity of the Ad-*CTSS* infected group was remarkably higher than the Ad-GFP infected group (Figure 4B, *p* < 0.05). *CTSS* knockdown decreased *Notch2* expression and cell proliferation activity in rabbit GCS (Figure 4C,D, *p* < 0.05).

**Figure 4.** *CTSS* overexpression and interference on cell proliferation in rabbit GCS: Effects of *CTSS* overexpression and knockdown on (**A**,**C**): mRNA expression changes of cell proliferation related genes and (**B**,**D**): cell proliferation activity. Values are presented as means ± SEM for 3 biological replicates. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001.

#### *3.5. Activation of CTSS Deceased Cell Apoptosis in Rabbit GCS*

As shown in Figure 5A, compared with the Ad-GFP infected group, mRNA level of *BCL2* (one of the main anti-apoptotic gene) was significantly increased by *CTSS* overexpression (*p* < 0.05). The other important pro-apoptotic gene, *BAX,* was decreased significantly by *CTSS* knockdown (Figure 5E, *p* < 0.05). Flow cytometry analysis revealed that *CTSS* overexpression inhibited the apoptosis of rabbit GCS significantly (Figure 5B–D, *p* < 0.01). As shown in Figure 5F–H, *CTSS* knockdown led to a remarkable increase in apoptotic cells in rabbit GCS (*p* < 0.05).

**Figure 5.** Effect of *CTSS* overexpression and interference on cell apoptosis in rabbit GCS: Relative mRNA expression changes of marker genes related to cell apoptosis after *CTSS* overexpression (**A**) and knockdown (**E**). Cell apoptosis rate was measured by using flow cytometry after *CTSS* overexpression (**B**–**D**) and knockdown (**F**–**H**). \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001.

#### **4. Discussions**

Reproductive performance is an important index for animal husbandry. GCS play an important role in oocyte development and affect female breeding performance. A number of studies have shown that small molecules including Vitamin D3 [30], FSH [31], Bisphenol A [32] and genes including *CITED4* [33], *CXCL12* [34], and *STAT4* [35] regulate cell proliferation, apoptosis and hormone secretion in mammals, thus affecting estrus of female animals and oocyte maturation. However, as one species of the high fertility husbandry animals, the effect of *CTSS* gene on rabbit reproduction has been rarely studied.

Cathepsin S (*CTSS*), one kind of lysosomal protease, is a major participant in proteolysis [36], and participates in many physiological processes. Studies have shown that *CTSS* gene played an important role in lipid metabolism in tumor tissue. To further investigate the role of *CTSS* in rabbit reproduction, we isolated rabbit GCS. GCS exist in the follicle and play an important role in the development of the follicle, as it can provide nutrients to oocytes during follicular development [37]. Studies have reported many methods for in vitro isolation and culture of GCS in pigs [38], cattle [39], and mice [40]. Isolation of rabbit GCS in the present study was performed according to the published methods with little modifications [41], providing material for further study of the complex mechanism of rabbit reproduction.

Progesterone and estradiol are mainly secreted by GCS and play an important role in the breeding process of mammals. They can promote estrus of female rabbits and early fetal pregnancy. To investigate whether *CTSS* affects progesterone and estradiol secretion in GCS, we overexpressed and interfered the expression of *CTSS* gene in rabbit GCS. Our results demonstrated that *CTSS* gene can regulate the secretion of progesterone hormone, and that this effect might be mediated by *STAR* and *CYP19A1*. As summarized in Figure 6, activation of *CTSS* resulted in up-regulation of progesterone hormone-related genes (*STAR* and *CYP19A1*) and promoted the secretion of progesterone. In mammals, progesterone plays a key role in follicular development and early fetal pregnancy [40]. Progesterone promotes follicular development, and low levels of progesterone increase follicular diameter [16]. However, the insufficiency of progesterone secretion will cause the fetus to be unable to survive or, even worse, will result in the abortion phenomenon [42]. In this experiment, our results demonstrated that *CTSS* play an important role in regulating the secretion of progesterone and estradiol in GCS.

**Figure 6.** Molecular mechanism of *CTSS* regulating cell apoptosis and progesterone secretion in rabbit GCS.

By providing nutrition and metabolite to oocytes, GCS play an important role in oocyte development. GCS also transmit signals and information for oocytes, and make oocytes mature. Apoptosis of GCS affects follicle development and leads to follicular atresia. In this study, we found that *CTSS* gene could affect the proliferation and apoptosis of GCS, and that the overexpression of *CTSS* gene in GCS increased the expression of proliferation gene (*PCNA*) and anti-apoptotic gene (*BCL2*), and promoted the proliferation of GCS while decreasing its apoptosis (Figure 6). Consistently, *CTSS* knockdown decreased the proliferation rate while increasing the apoptosis of rabbit GCS. These findings are in

accordance with recent research reporting that *CTSS* interference inhibited the proliferation while inducing the apoptosis in mhCC97-H cells [43,44], and recent studies have also shown that *CTSS* plays a crucial role in the invasion and apoptosis of cancer cells [45,46]. Taken together, our results have demonstrated that *CTSS* can promote the proliferation while reducing the apoptosis of rabbit GCS.

#### **5. Conclusions**

In summary, our data suggest that *CTSS* regulates the proliferation and apoptosis of granulosa cells by regulating the expression of key genes involved in these processes. In addition, *CTSS* is also involved in the process of progesterone and estradiol secretion in rabbit GCS. Overall, the present study provided evidence that *CTSS* may play an important role in regulating follicular development by regulating cell apoptosis and hormone secretion in rabbits. However, whether manipulation of *CTSS* expression in vivo can improve the reproductive performance of rabbits needs further research.

**Author Contributions:** Conceptualization, H.X., M.L. and G.S.; funding acquisition, M.L.; supervision, X.N.; formal analysis, Y.W.; validation, Y.J. and M.S.; writing—original draft, G.S. and Y.J.; writing—review and editing, H.X. and M.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** The research was jointly supported by the National Key Research and Development Program of China (2018YFD0502203) and the Special Fund for Henan Agriculture Research System (S2013-08-G01).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of Declaration of Helsinki, and approved by the Ethics Committee of College of Animal Science and Technology of Henan Agricultural University, China (Permit Number: 11-0085; Data: 06-2011).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no competing financial interest.

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