BMC Molecular andCell Biology

Zareifard et al.
BMC Molecular and Cell Biology(2023) 24:21
https://doi.org/10.1186/s12860-023-00482-5

Open Access

RESEARCH ARTICLE

Janus Kinase 3 phosphorylation and the JAK/STAT pathway are positively modulated
by follicle‑stimulating hormone (FSH) in bovine
granulosa cells
Amir Zareifard1,2, Francis Beaudry2,3 and Kalidou Ndiaye1,2*   

Abstract
Janus kinase 3 (JAK3) is a member of the JAK family of tyrosine kinase proteins involved in cytokine receptor-mediatedintracellular signal transduction through the JAK/STAT signaling pathway. JAK3 was previously shown as differentially expressed in granulosa cells (GC) of bovine pre-ovulatory follicles suggesting that JAK3 could modulate GC
function and activation/inhibition of downstream targets. We used JANEX-1, a JAK3 inhibitor, and FSH treatments
and analyzed proliferation markers, steroidogenic enzymes and phosphorylation of target proteins including STAT3,
CDKN1B/p27Kip1 and MAPK8IP3/JIP3. Cultured GC were treated with or without FSH in the presence or not of JANEX1. Expression of steroidogenic enzyme CYP11A1, but not CYP19A1, was upregulated in GC treated with FSH andboth were significantly decreased when JAK3 was inhibited. Proliferation markers CCND2 and PCNA were reduced in
JANEX-1-treated GC and upregulated by FSH. Western blots analyses showed that JANEX-1 treatment reduced pSTAT3
amounts while JAK3 overexpression increased pSTAT3. Similarly, FSH treatment increased pSTAT3 even in JANEX-1treated GC. UHPLC-MS/MS analyses revealed phosphorylation of specific amino acid residues within JAK3 as well as
CDKN1B and MAPK8IP3 suggesting possible activation or inhibition post-FSH or JANEX-1 treatments. We show that
FSH activates JAK3 in GC, which could phosphorylate target proteins and likely modulate other signaling pathways
involving CDKN1B and MAPK8IP3, therefore controlling GC proliferation and steroidogenic activity.
Keywords Janus Kinase (JAK), Granulosa Cells, STAT3, CDKN1B/p27Kip1, MAPK8IP3/JIP3, Ovary, Proliferation,
Steroidogenesis, JAK/STAT​, UHPLC-MS/MS*Correspondence:
Kalidou Ndiaye
k.ndiaye@umontreal.ca
1Centre de Recherche en Reproduction Et Fertilité, Département
de Biomédecine Vétérinaire, Faculté de Médecine Vétérinaire, CRRF,
Université de Montréal, Saint‑Hyacinthe, Québec, Canada
2
Département de Biomédecine Vétérinaire, Faculté de Médecine
Vétérinaire, Université de Montréal, Saint‑Hyacinthe, Québec 3200,
Canada3
Centre de Recherche Sur Le Cerveau Et L’apprentissage (CIRCA),
Université de Montréal, Montréal, Québec, Canada

Background
The ovary is responsible for producing oocytes through a
precise regulation by gonadotropins (follicle-stimulating
hormone (FSH) and luteinizing hormone (LH)) and by
steroid hormones (estrogen and progesterone) within theovarian follicles [1–4]. In early stages of folliculogenesis,
it is known that the ovarian follicles develop independently of gonadotropins while ovary-derived paracrine
factors, such as cytokines and other growth factors play
crucial roles in these early stages [5]. Although the beginning of folliculogenesis is gonadotropin-independent,
receptors of gonadotropins are present in follicles prior© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​
mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Zareifard et al. BMC Molecular and Cell Biology(2023) 24:21

to antrum formation advancing the importance of gonadotropins in early stages of follicular development [6].
As follicles progress into the antral stage, gonadotropins, especially FSH, become crucial for follicle survivaland growth [6]. Granulosa and theca cells are two typesof somatic cells that provide a suitable environment forthe development of the oocyte within the follicle. Thesecells are responsive to gonadotropins and steroid hormones, which affect follicular development and ovulation for the release of a mature oocyte for subsequentfertilization [7–11]. Granulosa cells in particular are an
important component of the follicle as they contribute tosteroid hormone synthesis [12], oocyte maturation [13],and corpus luteum formation after ovulation [14]. The
control of granulosa cells proliferation and function istherefore complex and depends on the precise regulation
and activation or inhibition of specific genes and signaling pathways. FSH binds to its receptors on granulosa
cells and initiates a series of molecular responses to regulate the expression of specific genes that are required forcell proliferation and growth as well as cumulus expansion and differentiation. While the initiation of folliculardevelopment is under the control of FSH, the release of
the oocyte from the ovulatory follicle is under the influence of the LH surge (see [3] and [4] for reviews). However, the molecular mechanisms and underlying signaling
pathways to these critical biological processes are still not
fully investigated.
Previous in vivo gene expression analysis using
bovine granulosa cells identified several candidate
genes differentially regulated in the ovarian follicles[15–17]. Of interest, Janus kinase 3 (JAK3) was shown
to be differentially expressed in granulosa cells of preovulatory follicles and downregulated in bovine ovulatory follicles by LH or human Chorionic Gonadotropin(hCG) injection [17] suggesting a potential role of JAK3
in regulating ovarian follicular development. JAK3 is a
non-receptor tyrosine kinase that belongs to the JAK
family proteins along with JAK1, JAK2 and TYK2 [18].
JAK proteins have seven JAK homology (JH) domains,
namely the 4.1, ezrin, radixin and moesin (FERM), Src
Homology 2 (SH2), pseudokinase and kinase domains[19, 20]. The FERM region at the N-terminus mediates attachment to cytokine receptors, while the SH2
domain provides a docking site for downstream proteins such as signal transducers and activator of transcription (STAT) proteins [21]. The kinase region
bearing the activation loop at the carboxyl terminus
and responsible for the tyrosine phosphorylation is
located next to a pseudokinase region, which lacks any
demonstrated kinase activity [20]. Within the JAK/STAT pathway and upon activation, JAK3 recruits andphosphorylates downstream effectors including STAT

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proteins, which leads to the regulation of target genes
and modulation of cell proliferation, function and survival [22]. This well-defined pathway is yet to be fully
investigated in reproductive cells including ovarian
granulosa cells. Concurrently, additional JAK3 targets
that can be affected by JAK3 activation in granulosa
cells might include Cyclin-dependent kinase inhibitor 1B (CDKN1B also known as ­p27Kip1) and Mitogen-activated protein kinase 8 interacting protein 3(MAPK8IP3) also known as JNK-interacting protein 3(JIP3) previously identified as JAK3 binding partners[17]. CDKN1B binds to and prevents the activation of
cyclin E-CDK2 or cyclin D-CDK4 complexes, and thus
controls the cell cycle progression at G1 [23]. CDKN1B
is therefore often referred to as a cell cycle inhibitor
protein since its major function is to stop or slow down
the cell division cycle when it is not phosphorylated.
As for MAPK8IP3, it was shown to interact with andregulate the activity of numerous protein kinases of thec-Jun N-terminal kinase (JNK) signaling pathway andthus functions as a scaffold protein in signal transduction [24]. The JNK pathway is one of the major signaling cascades of the mitogen-activated protein kinase(MAPK) signaling pathway and regulates a number ofcellular processes including proliferation, embryonicdevelopment and apoptosis [25].
Overall, the available data regarding JAK3 regulation in reproductive cells point to a crucial role incontrolling granulosa cells activity and proliferation
during follicular development prior to the LH surge
and subsequent ovulation and luteinization processes. Previous studies have shown the importanceof JAK signaling in the formation of primordial follicles as well as germline cyst breakdown as inhibition of JAK3 decreased pre-granulosa cell formation
through the downregulation of NOTCH2 signaling[26]. Other studies have shown stronger abundanceof STAT3 phosphorylation in granulosa cells of smallfollicles after follicle deviation as related to granulosa
cells death and follicular atresia [27]. However, within
the selected follicle, the exact role of JAK3 in the regulation of granulosa cells activity and phosphorylationof target proteins still remains poorly defined. We
hypothesized that JAK3 could modulate granulosa cells
proliferation and steroidogenic activity as well as activation or inhibition of downstream targets. It is wellestablished that phosphorylation on serine, threonine
and tyrosine residues is an extremely important modulator of protein function as these modifications canbe critical in the activation or inactivation of proteinsof interest [28]. Therefore, the present study was conducted to unveil the function and mechanism of actionof JAK3 in granulosa cells during ovarian follicularZareifard et al. BMC Molecular and Cell Biology(2023) 24:21

development. The study was conducted using inhibition and overexpression strategies as well as stimulation of granulosa cells to elucidate the regulation andphosphorylation sites within target proteins. We showthat JAK3 influences granulosa cells proliferation, steroidogenesis activity and function through phosphorylation of target proteins, activation of the JAK/STAT
pathway and likely modulation of other signaling pathways involving CDKN1B and MAP8IP3 in responseto stimulations such as FSH. This study provides evidence that significantly deepen our molecular understanding of JAK3 activity and role in the regulation ofa proper ovarian follicular development and mammalian folliculogenesis.

Results
Expression and regulation of JAK family members in GC
during follicular development

Previously, we reported that JAK3 was differentiallyexpressed in GC of preovulatory follicles and downregulated in ovulatory follicles in vivo by luteinizing
hormone (LH) or following human Chorionic Gonadotropin (hCG) injection [15, 17]. To generate an overall
analysis of the regulation of JAK family members inbovine granulosa cells, total RNA was extracted fromsmall follicles (SF), dominant follicles at day 5 of the
estrous cycle (DF), ovulatory follicles obtained 24 hpost-hCG injection (OF) and corpus luteum (CL) at
day 5 of the estrous cycle in order to analyze JAK1,
JAK2, JAK3 and TYK2 expression. Gene expression
analyses by RT-qPCR revealed the strongest expression of JAK3 in SF and DF and its significant downregulation in OF and CL (Fig. 1a; p ≤ 0.001), confirming
previously reported data from our laboratory [17]. In
addition, JAK1 was induced by hCG in OF as compared
to SF, DF and CL (Fig. 1b; p ≤ 0.001). JAK1 expression
was also stronger in DF as compared to SF (p ≤ 0.05).
JAK2 expression was substantially present in the CL ascompared to small, dominant and ovulatory follicles(Fig. 1c; p ≤ 0.0001) while among the different groupsof follicles, JAK2 expression was stronger in OF as
compared to SF and DF (Fig. 1c; p ≤ 0.05). As for TYK2
expression, it was stronger in DF and OF compared to
the SF and CL samples (Fig. 1d; p ≤ 0.05).
These results suggest that JAK members might playdifferent functions in the ovary and modulate different
downstream targets in GC and therefore might not present functional redundancy. Specifically, JAK3 might beinvolved in GC proliferation and follicular development
while other JAK members might play significant roles in
the ovulation or luteinization processes or maintenanceof a functional corpus luteum.

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JAK3 inhibition reduces expression of proliferation
markers while FSH stimulates proliferation markers
in cultured granulosa cells

JAK3 function in GC was analyzed using JANEX-1(JNX), a specific JAK3 pharmacological inhibitor. RTqPCR analyses were used to measure proliferation
markers cyclin D2 (CNND2), a cell cycle activator andproliferating cell nuclear antigen (PCNA) expression.
The results showed that inhibition of JAK3 led to significant decrease in GC proliferation demonstrated
by a decrease in both CCND2 (Fig. 2a; p ≤ 0.0001) and
PCNA (Fig. 2b; p ≤ 0.0001) expression as compared tothe control group. FSH significantly increased both
CNND2 (Fig. 2a; p ≤ 0.01) and PCNA (Fig. 2b; p ≤ 0.05)
expression as compared to the control and following
JNX treatment. PCNA was used to assess and confirm
GC proliferation since it is expressed in the nuclei ofcells during the DNA synthesis phase of the cell cycle,
which could be an indication of cell proliferation in
GC. Moreover, expression of CCND2 was also analysed using the same in vitro samples as another proliferation marker, which functions as a regulator of
cyclin-dependent kinases required for cell cycle G1/S
transition. Negative impacts of JAK3 inhibition on
expression of CCND2 and PCNA in JNX-treated GC
suggest that JAK3 plays a central role in the regulationof ovarian granulosa cells proliferation and function.
We also showed that FSH stimulation of cell proliferation following JNX inhibitory effects resulted in
an increase in proliferation markers expression in GC(Fig. 2a and b) likely through activation of the JAK/STAT pathway. While we couldn’t detect a stimulatory
effect of FSH on JAK3 expression at the mRNA level,
FSH clearly tended to increase JAK3 expression following JNX treatment, likely as a consequence of FSH
stimulation of cell proliferation following JNX inhibitory effects resulting in an increase in JAK3 expressionin GC (Fig. 2c; p ≤ 0.01).
JAK3 inhibition modulates granulosa cells steroidogenic
activity

To verify the effects of JAK3 inhibition on the steroidogenic activity of GC in vitro, expression of CYP11A1
and CYP19A1 enzymes were analysed as steroidogenesis markers and important regulators of the development and function of mammalian ovaries. RT-qPCR
analyses showed that expression of both CYP11A1 andCYP19A1 was significantly decreased when JAK3 wasinhibited with JNX as compared to the control (Fig. 3aand b; p ≤ 0.0001). Conversely, FSH stimulated thesteroidogenic activity of treated GC and significantly
increased the expression level of CYP11A1 (Fig. 3a;

Zareifard et al. BMC Molecular and Cell Biology(2023) 24:21

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Fig. 1 Regulation of JAK family members during follicular development. mRNA expression and regulation of JAK family members were analyzed in
GC of bovine follicles at different stages using RT-qPCR. Total RNA was extracted from GC of small follicles (SF; N = 3), dominant follicles (DF; N = 4),
ovulatory follicles (OF; N = 4), and corpus luteum at day 5 (CL; N = 4) as described in materials and methods and target genes were analyzed (JAK1,
JAK2, JAK3, TYK2) relative to RPL19 as reference gene. JAK3 mRNA expression was strongest in DF and SF and was significantly decreased in OF
and CL (p ≤ 0.05) while other JAK members were differentially expressed in different stages of follicular development and CL. This result suggests
a different regulation of JAK family in granulosa cells at different stages of follicular development and CL. RT-qPCR data are presented as geneabundance using the ­2−ΔΔCt method. *, p ≤ 0.05, **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001 (ANOVA, Tukey–Kramer multiple comparison), ns Not
significant

p ≤ 0.001), but not CYP19A1 (Fig. 3b), as compared tothe control. Although CYP19A1 expression was not significantly increased by FSH treatment alone, the results

demonstrated that CYP19A1 expression was significantly increased in cells treated with FSH following JNX
treatment (Fig. 3b; P ≤ 0.05), a result that mirrors JAK3

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Fig. 2 Regulation of JAK3 mRNA expression in vitro. Total RNA was extracted from cultured GC treated with or without FSH or JNX and analyzed by
RT-PCR for the expression of proliferation markers Cyclin-D2 (CCND2) and Proliferating cell nuclear antigen (PCNA) as well as JAK3 relative to RPL19
as reference gene. FSH stimulated both CCND2 (a) and PCNA (b) mRNA expression levels in GC while JANEX-1 significantly decreased both of theseproliferation markers after 24 h. In addition, addition of FSH following JNX treatment significantly increased expression of both CCND2 and PCNA
as compared to JNX. RT-qPCR analysis showed that JAK3 mRNA expression in GC (c) significantly decreased after JNX treatment as compared tothe control and FSH-treated cells. FSH treatment alone did not stimulate JAK3 expression as compared to control group; however, FSH treatment
significantly increased JAK3 expression following JNX treatment. CTRL, control; FSH, Follicle-stimulating hormone; JNX, JANEX-1 RT-qPCR data
are presented as gene abundance using the ­2−ΔΔCt method. *, p ≤ 0.05, **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001 (ANOVA, Tukey–Kramer multiple
comparison); ns Not significant

regulation in Fig. 2c. These data confirm a positive effectof FSH on GC and suggest a potential role of JAK3 inregulating the expression of steroidogenic enzymes andits involvement in follicular development and differentiation as its inhibition is associated with negative effects
on GC steroidogenic activity.

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Fig. 3 Effect of JAK3 inhibition on steroidogenesis markers (CYP11A1 and CYP19A1) in granulosa cells. Total RNA was extracted from cultured GC
treated with or without FSH or JANEX-1 and analyzed by RT-PCR for the expression of steroidogenic enzymes cytochrome P­ 450 family 11 subfamily
A member 1 (CYP11A1) and cytochrome P450 family 19 subfamily A member 1 (CYP19A1) normalized against the house keeping gene RPL19 asreference. The results show that FSH significantly upregulated CYP11A1 in GC (a); however, CYP19A1 (b) did change following FSH treatment. In
addition, JAK3 inhibition with JNX resulted in a significant decrease of both CYP11A1 and CYP19A1. FSH treatment following JANEX-1 significantly
increased CYP19A1 expression but not CYP11A1. CTRL, control; FSH, Follicle-stimulating hormone; JNX, JANEX-1. RT-qPCR data are presented asgene abundance relative to RPL19 using the ­2−ΔΔCt method. *, p ≤ 0.05, **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001 (ANOVA, Tukey–Kramer multiple
comparison), ns Not significant

JAK3 inhibition negatively affects STAT3 phosphorylation
while JAK3 overexpression and FSH positively affect STAT3
phosphorylation in granulosa cells

To further analyse the possible regulatory influence of
JAK3 on downstream effectors in GC, total extracted
protein of in vitro samples was subjected to immunoblotting analyses. Relative abundance of STAT3 phosphorylation (pSTAT3) was quantified and presented as
an indication of either STAT3 activation or inhibition
within the JAK/STAT signaling pathway following overexpression or inhibition of JAK3 in GC using the pQE
system or JNX treatment, respectively. Phosphorylation of STAT3 was also analysed and quantified following FSH treatment for 4 h. We previously showed that at
8 h post JNX treatment, a significant decrease in pSTAT3
was observed as compared to the control while overexpression of JAK3 significantly increased pSTAT3 levels
as compared to JNX group and back to the level of thecontrol group [17] (Fig. 4a; p ≤ 0.05). However, JNX treatment for 24 h significantly decreased pSTAT3, whether
in JAK3-overexpresed cells or not (Fig. 4a; p ≤ 0.05, red
bar vs black bar) meaning that JAK3 overexpression was
not enough to reverse JNX effects after 24 h of treatment
as compared to 8 h.

In addition, in the current study, phosphorylation of
STAT3 was analysed and quantified following FSH treatment alone, or in combination with JNX treatment.
FSH administration for 4 h increased pSTAT3 to thesame level as JAK3 overexpression compared to JNXtreated GC (Fig. 4b and c; p ≤ 0.0001). Treatment with
FSH also significantly increased pSTAT3 amounts ascompared to JNX-treated cells even after 24 h as compared to JAK3 overexpression alone post-JNX treatment(Fig. 4b; p ≤ 0.0001, blue bar vs. black bar). This result
suggests a specific effect on STAT3 phosphorylation levels via JAK3 modulation. These data support a role of
FSH in positively modulating JAK/STAT signaling pathway by increasing pSTAT3 amounts in treated cells. The
results also confirm that STAT3 phosphorylation levels
are affected by JAK3 manipulation and suggest a central
role of JAK3 in regulating the transmission of initiatedsignals through a functional JAK/STAT signaling pathway in GC, which would contribute to GC proliferationand follicular development. Overall, JNX negative effects
on the JAK/STAT pathway were less drastic in the presence of FSH although JNX tended to decrease pSTAT3 in
FSH-treated/JAK3-overexpressed granulosa cells (Fig. 4b;
p = 0.0535).

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Fig. 4 Effects of JAK3 inhibition or overexpression and FSH treatment on STAT3 phosphorylation in granulosa cells. a To analyse the effects of JAK3
manipulations on downstream effectors in GC, Western blot analyses were performed and revealed phosphorylation status of STAT3 following
different treatments. JAK3 was overexpressed in GC using the pQE system (+ JAK3) with or without JNX treatment for 8 or 24 h to inhibit JAK3
phosphorylation. Detected bands and their relative abundances were quantified and phosphorylation status of STAT3 (pSTAT3) was analysed.
A significant increase in pSTAT3 amounts was observed when JAK3 was overexpressed. JNX treatment for 8 h resulted in a significant decrease
in pSTAT3. Overexpression of JAK3 following 8 h JNX treatment restored pSTAT3 amounts to the control levels. However, overexpression of JAK3
was not able to restore or increase pSTAT3 within GC after 24 h of JNX treatment, which significantly decreased pSTAT3 as compared to thecontrol group and JAK3-overexpressed cells (red box). b and c Further experiments allowed to evaluate the effects of FSH administration on thephosphorylation status of STAT3 in cultured GC. Fold changes of pSTAT3 over to total STAT3 were compared following treatments with JNX (24 h),
FSH (4 h) or JAK3 overexpression. JNX significantly reduced phosphorylation of STAT3 after 24 h of treatment, while overexpression of JAK3 without
JNX treatment significantly increased the amount of pSTAT3 within transfected GC similar to the result in A. Addition of FSH significantly increased
pSTAT3 compared to the JNX group. Moreover, combination of JAK3 overexpression with FSH treatment increased pSTAT3 but not to the samelevels as either FSH alone or JAK3 overexpression alone. Addition of FSH in the presence of JNX weakened the negative effect of JNX on pSTAT3 as
compared to JAK3 overexpression in the presence of JNX (blue bar). CTRL, control; FSH, Follicle-stimulating hormone; JNX, JANEX-1; + JAK3, JAK3
overexpression. Different letters denote significant differences among samples (ANOVA, Tukey–Kramer multiple comparison)

JAK3 inhibition differentially impacts binding partners
CDKN1B & MAPK8IP3

UHPLC-MS/MS analyses revealed relative total peptide abundances as well as modifications at the aminoacid levels including phosphorylation status of JAK3 andbinding partners CDKN1B/p27Kip1 and MAPK8IP3/JIP3.
Total abundances of these target proteins were analysedin GC treated with FSH or JNX as compared to a control group. Analysis and quantification of JAK3 protein
revealed a substantial increase in total abundance following FSH treatment as compared to control and JNX
treatment (Fig. 5a and b), which suggests an increase in
JAK3 phosphorylation and activation of JAK3 signaling

pathway in FSH-treated GC. In contrast, JNX treatment for 24 h notably decreased the abundance of JAK3
protein in treated granulosa cells as compared to thecontrol (Fig. 5a; p = 0.094) and FSH treatment (Fig. 5a;p ≤ 0.05). Furthermore, peptides from JAK3 protein wererecovered with different modifications including phosphorylation in specific amino acid residues. Quantification analysis of these recovered peptides revealed theirabundances following treatments with FSH and JNX.
Although not all JAK3 phosphorylated peptides showedsignificant changes in their abundances following FSH or
JNX treatments when compared to the control or to eachother, these phosphorylated fragments could indicate(See figure on next page.)
Fig. 5 Effects of FSH and JANEX-1 treatments on JAK3 total abundance and peptides modifications in granulosa cells. HPLC/MS–MS was used toanalyze and quantify total abundances of various recovered peptides for JAK3 in GC samples following treatments with FSH or JNX. a JAK3 totalabundance slightly increased following FSH treatment but not significantly while JNX treatment significantly decreased JAK3 abundance. b Ratioof fold change in JAK3 abundance in FSH-treated cells over the control and in JNX-treated cells over the control are compared and showed JAK3
as significantly more abundant following FSH treatment (p = 0.05). c-f Peptides abundance and phosphorylation at specific amino acid residues.
The abundances of various JAK3 phosphorylated peptides were quantified and evaluated following FSH or JNX treatments. Peptide numbers andpositions in the JAK3 protein sequence are shown. Phosphorylated residues are shown in red. CTRL, control; FSH, Follicle-stimulating hormone; JNX,JANEX-1. *, p ≤ 0.05 (ANOVA, Tukey–Kramer multiple comparison), ns Not significant

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Fig. 5 (See legend on previous page.)(2023) 24:21

Page 8 of 19

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Fig. 6 Effects of FSH and JANEX-1 treatments on CDKN1B total abundance and peptides modifications in granulosa cells. HPLC/MS–MS wasused to analyze and quantify total abundances of various recovered peptides for CDKN1B in GC samples following treatments with FSH or JNX.
a CDKN1B total abundance tended to decrease following FSH treatment (P = 0.075) while CDKN1B was significantly more abundant in JNX-treatedcells. b Ratio of fold change in CDKN1B abundance in FSH-treated cells over the control and in JNX-treated cells over the control are comparedand showed CDKN1B more abundant following JNX treatment. c-d Peptides abundance and phosphorylation at specific amino acid residues.
The abundances of two CDKN1B phosphorylated peptides were quantified and evaluated following FSH or JNX treatments. Peptide numbers andpositions in the CDKN1B protein sequence are shown. Phosphorylated residues are shown in red. CTRL, control; FSH, Follicle-stimulating hormone;
JNX, JANEX-1. *, p ≤ 0.05; **, p ≤ 0.01 (ANOVA, Tukey–Kramer multiple comparison), ns Not significant

phosphorylation status of JAK3 leading to its activationand contribution to the subsequent molecular mechanisms. More specifically, the abundance of peptide #10.2(amino acids 391–403 within JAK3 sequence) was not
significantly affected following FSH or JNX treatment
but exhibited phosphorylation on ­S394 and ­S398 residues
and a relative decrease following JNX and (Fig. 5c). Similarly, peptide #23 (amino acids 871–887 within JAK3

Y886
sequence) exhibited phosphorylation on ­S880 and ­residues but there were no changes in its abundance following FSH or JNX treatment (Fig. 5d). Conversely, otherphosphorylated JAK3 fragments were showing significant
changes in their abundances and phosphorylation status
following FSH treatment as compared to the control and
JNX treatment, suggesting a more stringent regulationof these JAK3 regions. Of interest, peptide #20 (amino

Zareifard et al. BMC Molecular and Cell Biology(2023) 24:21

acids 735–748 within JAK3 sequence) was phosphorylated at Y
­ 738 and was significantly less abundant following
JNX treatment as compared to FSH and control (Fig. 5e;p ≤ 0.05). Similarly, peptide 24 (amino acids 877–895
within JAK3 sequence) phosphorylated at ­S4 tended to beless abundant in JNX-treated cells as compared to FSHtreated cells and control (Fig. 5f ).
In addition, total protein abundances of CDKN1B and
MAPK8IP3 were analysed in the same sets of in vitro
samples than JAK3. The results revealed that total

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abundance of CDKN1B, which negatively affects cell
cycle progression, is considerably decreased with FSH
treatment (Fig. 6a and b; p = 0.075) and significantly
increased post-JNX treatment (Fig. 6a and b; p ≤ 0.05),
while MAPK8IP3 total abundance did not change significantly with FSH treatment but decreased significantly
in JNX-treated cells as compared to control and FSHtreated cells (Fig. 7a and b; p ≤ 0.05). Further analyses ofrecovered peptides of CDKN1B and MAPK8IP3 revealed
modifications at specific amino acid residues related

Fig. 7 Effects of FSH and JANEX-1 treatments on MAPK8IP3 total abundance and peptides modifications in granulosa cells. HPLC/MS–MS wasused to analyze and quantify total abundances of various recovered peptides for MAPK8IP3 in GC samples following treatments with FSH or JNX.
a MAPK8IP3 total abundance did not change following FSH treatment but JNX treatment significantly decreased MAPK8IP3 abundance. b Ratioof fold change in MAPK8IP3 abundance in FSH-treated cells over the control and in JNX-treated cells over the control are compared and showed
MAPK8IP3 as significantly more abundant following FSH treatment. c-d Peptides abundance and phosphorylation at specific amino acid residues.
The abundances of two MAPK8IP3 phosphorylated peptides were quantified and evaluated following FSH or JNX treatments. Peptide numbers andpositions in the MAPK8IP3 protein sequence are shown. Phosphorylated residues are shown in red. CTRL, control; FSH, Follicle-stimulating hormone;
JNX, JANEX-1. *, p ≤ 0.05; **, p ≤ 0.01 (ANOVA, Tukey–Kramer multiple comparison); ns, not significant

Zareifard et al. BMC Molecular and Cell Biology(2023) 24:21

to treatments with FSH or JNX. Abundance of peptide#1 (amino acids 6–15 within CDKN1B sequence) was
increased following JNX treatment as compared to control and FSH treatment (Fig. 6c; p ≤ 0.05) but no phosphorylation was noted in any residue of this peptide
suggesting a possible active CDKN1B protein in these
JAK3-inhibited cells. However, peptide #7 (amino acids
190–198 within CDKN1B sequence) was phosphorylated
at ­T9 and was significantly increased in FSH-treated cells
as compared to JNX-treated cells (Fig. 6d; p ≤ 0.05) suggesting an inactivation of CDKN1B via this specific residue in the presence of FSH, which correlates with FSH
positive effect on JAK3 activation and cell proliferation.
As for peptides recovered for MAPK8IP3, peptides #9(amino acids 201–219 within MAPK8IP3 sequence) and#34 (amino acids 595–605 within MAPK8IP3 sequence)
exhibited phosphorylation modifications, respectively atresidues ­S17 (peptide #9) and S­ 1 and T/S4/8 (peptide #34)
and both peptides were significantly decreased following JNX treatment (Fig. 7c and d; p ≤ 0.05) but did notchange following FSH as compared to control (Fig. 7c).
These data suggest that FSH stimulates JAK3 phosphorylation, which leads to the activation of the JAK/STAT
signaling pathway through increased STAT3 phosphorylation as one of the downstream effectors, while likely
inducing MAPK8IP3 activation and CDKN1B inactivation through phosphorylation of specific amino acid residues leading to cell cycle progression and granulosa cells
proliferation.
Conserved modifications for JAK3 and CDKN1B suggest
activation and inactivation status in granulosa cells

Amino acid sequences of JAK3 and CDKN1B frombovine and human species were aligned using proteinalignment tools (Clustal: Multiple Sequence Alignment)in order to analyse the similarities in phosphorylationsites and conserved areas between these two species.
The analysis of recovered peptides from bovine samples
revealed modifications at specific locations that wereassociated with JAK3 activation and CDKN1B inactivation in human [29–31] and are conserved in the bovine
species. As shown in the section above, these modifications were observed in this study following treatmentswith FSH or JNX and could be indicative of the JAK/STAT pathway activation or inactivation of CDKN1B
in GC. Amino acid sequences alignment for JAK3
showed residues previously shown to be phosphorylated in human, which were retrieved in this study using
bovine samples (Fig. 8a; peptide 29 and related peptides).
Modifications include phosphorylation of Tyrosine residues at 980–981 (­ Y980−981) in JAK3 following FSH treatment, which could be an indication of JAK3 activation.
As shown in Fig. 5, peptides in different regions of JAK3

Page 11 of 19

were detected and were differentially regulated following
the various treatments suggesting a potential phosphorylation pattern in different regions of JAK3 as an indication of JAK3 activity modulation.
In addition, we detected phosphorylation modifications for a specific region of CDKN1B, which is related
to CDKN1B inactivation (Fig. 8b; peptide 7 shown). In
contrast to JAK3 phosphorylation, which activate theprotein, phosphorylation of Threonine (T) residues at
positions 187 and 198 of CDKN1B are an indication of
CDKN1B inactivation, which results in cell cycle progression and cellular growth. As shown in Fig. 6, CDKN1B
total abundance decreased following FSH treatment,
while JAK3 inhibition following JNX treatment resulted
in an increase in total CDKN1B abundance. These data
provide evidence that FSH stimulates JAK3 phosphorylation and activity and suppresses CDKN1B activity, whichallows GC proliferation as demonstrated by the increasein proliferation markers.

Discussion
The JAK/STAT signaling pathway begins with extracellular binding of members of a family of structurally
related cytokines, interleukins, interferons, colonystimulating factors, and some hormones to their corresponding structurally related transmembrane receptors
as previously described [32, 33]. This enables transactivation of receptor-bound JAKs that catalyze tyrosine
phosphorylation of receptors and STATs, resulting in
the formation of homodimers and/or heterodimers that
accumulate in the nucleus and regulate gene transcription. JAK/STAT signaling pathways are major players
in controlling cellular proliferation and differentiation
processes. From this study, we show that FSH-induced
phosphorylation of JAK3 (revealed via UHPLC-MS/MS) and overexpression of JAK3 seem to directly affectprotein abundance and phosphorylation of downstream target STAT3. STAT proteins and in particular
STAT3 are involved in transducing regulatory signals
initiated following activation of JAK3 within the JAK/STAT signaling pathway to regulate the expression of
specific genes associated with proliferation, migrationand survival [26, 34, 35]. Upon activation of JAK3, thenon-phosphorylated STAT3 proteins are recruited andphosphorylated, then dimerize and translocate into the
nucleus where they bind to specific regions within the
DNA and regulate expression of specific target genes.
In contrast, inactivation of JAK3 protein in GC following JNX treatment significantly decreased STAT3 phosphorylation, which could alter the JAK/STAT signaling
pathway in these cells. This is in accordance with previous studies showing downregulation of pSTAT3 following inhibition of JAK3 [36]. The positive effect of FSH

Zareifard et al. BMC Molecular and Cell Biology(2023) 24:21

Page 12 of 19

Fig. 8 Recovered peptides for JAK3 and CDKN1B from HPLC/MS/MS analysis. a Peptides sequences of JAK3 from human and bovine species
were aligned to evaluate similarities and conserved modifications associated with JAK3 activation. Peptides positions within the JAK3 sequence
are indicated and phosphorylated residues are shown in blue. In particular, phosphorylation of the two Tyrosine (Y) residues at locations 980–981(peptide #29.1 in the figure) of JAK3 amino acid sequence in human were retrieved in this study. Similarly, different peptides with residues known
to be phosphorylated in the human sequence were also found in this study meaning that these amino acid residues were also phosphorylated
in bovine granulosa cells following JAK3 activation with FSH. b For CDKN1B, the two highlighted Threonine (T) residues at locations 187 and 198
within peptide #7 are an indication of phosphorylation status of CDKN1B and are related to CDKN1B inactivation. Phosphorylated of this peptide
was more abundant following FSH treatment (and JAK3 activation) as shown in Fig. 6d, while JNX treatment (and JAK3 inhibition) resulted in the
reduction in CDKN1B phosphorylationin JAK3 activation and STAT3 phosphorylation supports the activation of JAK/STAT pathway by FSH aswell as the potential role of JAK3 activation in regulating GC proliferation, steroidogenic activity and survival
through the JAK/STAT signaling and altering downstream targets activity. We have shown that FSH stimulates proliferation-associated genes CCND2 and PCNAand differentiation-associated genes CYP11A1 and, toa lesser degree, CYP19A1, which encode for enzymes
involved in steroidogenic activity of granulosa cells[37–40]. These genes are involved in different stages offollicular development and growth and are associatedwith several intracellular signaling pathways including
the JAK/STAT signaling pathway.

It has been reported that type II cytokine receptors
are associated with JAK3 phosphorylation leading to
the recruitment and phosphorylation of target proteinsrequired for cell proliferation and survival [26, 34]. JAK3
was shown to be involved in mediating signals initiatedby cytokine signaling through coupling with the common γ chain of receptors for interleukins (IL)2, IL4,
IL7, IL9, IL15 and IL21 and subsequently to play a critical role in the development, proliferation and differentiation of immune cells [33, 41, 42]. JAK3 is specifically
responsive to these cytokines since it only binds to thecommon γ chain. Interleukins, which are key mediatorsof immune responses, may affect mechanisms crucial
for granulosa cells function including their maturation

Zareifard et al. BMC Molecular and Cell Biology(2023) 24:21

and differentiation depending on the presence or not of
FSH and thus may play a regulatory role in reproductive function [43]. It was shown that less differentiatedgranulosa cells from small follicles are more responsive
to cytokines than are highly differentiated granulosa cells
within large follicles [44]. Similarly, small follicles are also
more responsive to FSH than large follicles while JAK3
was shown to be differentially expressed in small andgrowing dominant follicles [15, 17]. FSH affects follicular growth, maturation, dominant follicle selection as wellas estradiol production [45]. On the other hand, growth
factors and their receptors may affect the signaling network that is commonly activated by FSH receptors,
which eventually may activate several signaling pathways
including ERK1/2, the phosphatidylinositol-4,5-bisphosphate 3-kinases (PI3K)/protein kinase B (AKT) [46, 47]
and also the JAK/STAT pathway as suggested in thisstudy. UHPLC-MS/MS data showed that FSH induced
JAK3 phosphorylation at specific residues, which areassociated with JAK3 activation and related to increased
cell proliferation as previously described [48, 49]. Theseobservations imply a synergically-induced effect of FSH
and cytokines in granulosa cells that initiate a cascade ofactions leading to JAK3 autophosphorylation and activation and recruitment of downstream target proteins.
A more accurate determination of the number andfunction of FSH‐regulated genes as well as LH-regulated
genes have been reported over the years [15, 16, 50–52]
and demonstrate the importance of functional studies
during the later stages of follicular development to better coordinate the ovarian activity. In this regard, we
have shown previously the suppression of JAK3 in vivo
by endogenous LH or hCG injection in bovine species
and identified JAK3 binding partners in GC using theyeast two-hybrid method [15, 17]. We hypothesized thenthat, in contrast to LH downregulatory effects on JAK3
expression, FSH could induce JAK3 expression/activation, which in turn would regulate GC function by activating the JAK/STAT signaling pathway and modulatesignaling pathways associated with JAK3 binding partners such as CDKN1B, which negatively affects cells
proliferation if not phosphorylated, and MAPK8IP3 tomaintain GC proliferation and follicular development.
The data reported here support this hypothesis sinceinhibition of JAK3 led to a decrease in GC proliferation
shown by a reduction in cell proliferation markers as well
as disturbing the steroidogenic activity of GC. In contrast, JAK3 phosphorylation by FSH led to an increase in
CDKN1B phosphorylation at specific target amino acids,
while JAK3 inhibition resulted in a decrease in MAPK8IP3 abundance and phosphorylation.
CDKN1B ­(p27Kip1) is a cyclin-dependent kinase
inhibitor that binds to and prevents the activation ofPage 13 of 19

cyclin E-CDK2 or cyclin D-CDK4 complexes, and thus
controls the cell cycle progression at G1 [53, 54]. The
degradation of CDKN1B, which is triggered by its CDKdependent phosphorylation and subsequent ubiquitination, is required for the cellular transition from thequiescence to the proliferative state [55]. Therefore,
CDKN1B acts either as an inhibitor or an activator of
cyclin type D-CDK4 complexes depending on its phosphorylation state and/or stoichiometry. The phosphorylation of CDKN1B occurs on serine, threonine andtyrosine residues. Phosphorylation on S
­ 10 is the majorsite of phosphorylation in resting cells and takes placeat the G(0)-G(1) phase leading to protein stability [56].
Phosphorylation on other sites is potentiated by mitogens or growth factors and in certain cancer cell lines[57, 58], meaning that phosphorylated CDKN1B found in
the cytoplasm is inactive. Consistent with these observations, we showed in this study CDKN1B phosphorylation
following FSH treatment and subsequent JAK3 activation. This observation is concomitant with increased
JAK3 total abundance and phosphorylation level suggesting that CDKN1B phosphorylation at specific residues
is necessary following FSH addition in order to allow
progression of the cell cycle and GC proliferation. Basedon these reports, and in light of our current findings, it
may be possible that CDKN1B binding to JAK3 leads to
its phosphorylation and inactivation in granulosa cells ofsmall and growing dominant follicles, thus allowing follicular development.
Additionally, we previously reported the strongest
expression of CDKN1B in the corpus luteum [17] suggesting a role for CDKN1B in establishing the non-proliferative state, which is required for differentiation or
for proper functioning of the differentiated luteal. Ourfindings are therefore consistent with previous results
showing that the luteinization process is associated with
up-regulation of CDKN1B that accumulated during initial phases of luteinization and remained elevated until
termination of the luteal function [59]. It was also demonstrated that inhibition of JAK3/STAT3 signaling significantly decreased viability of colon cancer cells due toapoptosis and cell-cycle arrest through down-regulationof cell cycle genes including cyclin D2 and up-regulationof CDKN1B [36]. Our data show similar findings following JAK3 inhibition with decreased expression of cellproliferation markers cyclin D2 and PCNA, decreased
STAT3 phosphorylation and increased CDKN1B protein abundance, confirming the biological importance of
JAK3 and the JAK/STAT pathway in the ovarian function.
Moreover, our findings confirmed that downstream targets of the JAK/STAT signaling that are involved in cellcycle regulation, including cyclin D2 and CDKN1B, also
play a major role in the regulation of GC proliferation.

Zareifard et al. BMC Molecular and Cell Biology(2023) 24:21

Conversely, MAPK8IP3 (JNK-interacting protein 3;
JIP3) is a mitogen activated tyrosine kinase (MAPK) that
contributes to the C-Jun signalling pathway. The different classes of MAPKs play important roles in various
cellular processes including cell proliferation, differentiation and apoptosis. JNKs are activated by diverse stimuli
including DNA damage or inflammatory cytokines [60].
MAPK activation may be facilitated by the formation of
signaling modules, and it has been well established that
scaffold proteins play important roles by interacting with
MAPKs and their upstream kinases [61]. MAPK8IP3 hasbeen reported as a scaffold protein within the JNK signaling cascade [62]. We show here that MAPK8IP3 exhibited increased phosphorylation of residues associatedwith cell proliferation. Although FSH did not increase
the overall total MAPK8IP3 abundance, JAK3 inhibition
by JNX resulted in a notable decrease in total abundanceof MAPK8IP3. This insight might show a direct effect of
JAK3 inhibition on the activation of MAPK8IP3, therefore affecting other associated signaling pathways in GC.
The data from this study show that FSH regulates
expression of genes involved in GC proliferation and follicular development. Most importantly, these findings
implicate FSH in modulating JAK3 phosphorylation and
the JAK/STAT signaling pathway likely through phosphorylation of STAT3 proteins. The results also demonstratea crucial role of JAK3 in GC proliferation and modulation of downstream targets CDKN1B and MAPK8IP3 in
response to a hormonal signal (FSH) leading to follicular
growth and development. Although the exact mechanisms by which FSH affects JAK3 and regulates STAT3
and the downstream targets were not fully elucidated,
this study serves as a basis for studies targeting GC regulation during late stages of follicular development and in
JAK3‐inhibited GC to identify pathways and downstream
targets affected by FSH‐induced genes and more specifically JAK3.

Methods
Chemicals and Products

Rabbit antibodies against STAT3 (Cat. # ab32500) andphospho STAT3 (Cat. # ab32143) were from Abcam.
Anti-β-actin antibodies (Cat. # SC-47778) used as loading reference was from Santa Cruz Biotechnology. Follicle-stimulating hormone (FSH; Cat. # F4021-10UG)
were purchased from Sigma while the pharmacologicalinhibitor JANEX-1 was purchased from Santa Cruz Biotechnology (SC-205354, LOT # B1011). Complete protease inhibitors were purchased from Roche Diagnostics(Laval, QC, Canada). M-PER Mammalian Protein Extraction Reagent (Cat. # 78,503) was obtained from Thermo
Fisher.

Page 14 of 19

Experimental animal models

Animal Ethics Committee of the Faculty of Veterinary
Medicine of the University of Montreal reviewed andapproved all the experimental protocol as the cows werecared for in accordance with the Canadian Council on
Animal Care guidelines [63] and according to ARRIVE
guidelines. Cows from the Faculty of Veterinary Medicineof the University of Montreal were used for these in vivo
experiments and the animals were returned to the Facultyat the end of the experiments. A total of eight cows wereused. Experimental animal model and sample preparation including groups being compared and experimental
unit have been previously described [16]. The expression
and regulation of all JAK family members was analyzed
during different stages of follicular development and ovulation using an in vivo model previously described [15,17]. Granulosa cells were collected from a dominant follicle group (DF, n = 4 cows) and an hCG-induced ovulatory follicle group (OF, n = 4 cows) obtained from ovaries
of synchronized cows [64]. DF samples were obtained
from the ovaries bearing the dominant follicle on day 5of the estrous cycle (day 0 = day of estrus) as previously
reported [15]. Follicles were dissected in order to isolate
granulosa cells (GC). The OF samples were obtained following an injection of 25 mg of ­PGF2α (Lutalyse) on day 7of the estrous cycle to induce luteolysis, thereby promoting the development of the dominant follicle of the firstfollicular wave into a preovulatory follicle. An ovulatory
dose of hCG (3000 IU, iv; APL, Ayerst Lab, Montréal,
QC) was injected 36 h after the induction of luteolysis,
and the ovary bearing the hCG-induced OF was collected
24 h post-hCG injection for GC isolation. Samples werestored at -80 °C until further analyses. Furthermore, GC
were collected from 2 to 4 mm small antral follicles (SF;n = 3 groups of 20 small follicles each) obtained fromslaughterhouse ovaries, and a total of three pools of 20 SF
were prepared. Corpora lutea (CL; n = 3 cows) were collected at day 5 of the estrous cycle from the same cows
used for DF sampling and stored at -80 °C. These samples are referred to as in vivo samples and were used tovalidate JAK family members regulation during folliculardevelopment.
Functional analyses

For in vitro experiments, GC were aspirated from small
to medium size follicles (< 8 mm in diameter) of slaughterhouse ovaries and put into culture in DMEM/F12
medium. GC were cultured in 12-well plates (n = 4 independent experiments with duplicate wells for each treatment) for RNA and protein extraction. Cells were seeded
at a density of 0.1 × ­106 cells/well. The complete DMEM/
F12 culture medium was supplemented with L-glutamineZareifard et al. BMC Molecular and Cell Biology(2023) 24:21(2 mM), sodium bicarbonate (0.084%), bovine serum
albumin (BSA; 0.1%), HEPES (20 mM), sodium selenite(4 ng/mL), transferrin (5 μg/mL), non-essential aminoacids (1 mM), androstenedione (100 nM), penicillin(100 IU) and streptomycin (0.1 mg/mL) as previously
published [65, 66]. For FSH treatment, cells were incubated in complete culture medium without serum or
insulin for 2 days, then in complete medium containing
FSH (1 ng/mL) for 4 days. In JAK3 inhibition experiments, granulosa cells were cultured and treated with
100 μM of JANEX-1. This concentration was based on
previously published data [17]. JANEX-1 treatment waseither followed by FSH treatment (1 ng/mL) or not. Cells
for all experiments were incubated at 37 °C in a humidified 5% C
­ O2 atmosphere for a total of 6 days with media
changed every 48 h. Effects of FSH and JANEX-1 treatments on GC were assessed by analyzing expressionof JAK3 by RT-qPCR and JAK3 downstream targets
STAT3, CDKN1B and MAPK8IP3 phosphorylation status by western blot or HPLC/MS–MS. Cell proliferation
markers cyclin D2 (CCND2) along with proliferating cell
nuclear antigen (PCNA) were also evaluated by RT-qPCR
analyses. To further analyze GC steroidogenic activity, steroidogenic genes CYP19A1 ­(P450 aromatase) andCYP11A1 ­(P450 cholesterol side-chain cleavage) were
measured by RT-qPCR.
For overexpression experiments, the JAK3 open reading frame was amplified by PCR using the Advantage 2polymerase mix (Takara Bio) with JAK3-ORF primers(Table 1) amplifying the entire 3372 bp open reading
frame. The JAK3 PCR fragment was purified and cloned
into the pQE-Tri System His-Strep2 vector (Qiagen). The
final pQE-JAK3 construct was used to transfect GC using
the Xfect transfection kit (Takara Bio) according to the
manufacturer’s protocol. The effects of JAK3 overexpression were assessed by measuring phosphorylation levelsof STAT3.

Page 15 of 19

Proteomic analysis: Cell extracts and UHPLC‑MS/MS

Protein pellets from control cells, FSH- and JANEX1treated cells were dissolved in 100 µL of 50 mM TRIS–
HCl buffer (pH 8), and the solution was mixed with a
Disruptor Genie at maximum speed (2,800 rpm) for
15 min and sonicated to improve the protein dissolution yield. Proteins samples were denatured by heating
at 120 °C for 10 min using a heated reaction block andallowed to cool for 15 min. Proteins were reduced with
20 mM dithiothreitol (DTT), and the reaction was performed at 90 °C for 15 min. Then, proteins were alkylatedwith 40 mM iodoacetamide (IAA) protected from light atroom temperature for 30 min. Then, 5 µg of proteomicgrade trypsin was added, and the reaction was performed
at 37 °C for 24 h. Protein digestion was quenched by adding 10 µL of a 1% trifluoroacetic acid (TFA) solution.
Samples were centrifuged at 12,000 g for 10 min, and100 µL of the supernatant was transferred into injection vials for analysis using a Thermo Scientific Vanquish
FLEX UHPLC system (San Jose, CA, USA). Chromatography was performed using gradient elution along witha Thermo Biobasic C18 microbore column (150 × 1 mm)with a particle size of 5 μm. The initial mobile phase conditions consisted of acetonitrile and water (both fortified
with 0.1% formic acid) at a ratio of 5:95. From 0 to 3 min,the ratio was maintained at 5:95. From 3 to 123 min, alinear gradient was applied up to a ratio of 40:60, which
was maintained for 3 min. The mobile phase composition ratio was then reverted to the initial conditions, andthe column was allowed to re-equilibrate for 30 min. The
flow rate was fixed at 50 µL/min, and 5 µL of each sample
was injected. A Thermo Scientific Q Exactive Plus Orbitrap Mass Spectrometer (San Jose, CA, USA) was interfaced with the UHPLC system using a pneumatic-assisted
heated electrospray ion source. Nitrogen was used as thesheath and auxiliary gases, which were set at 15 and 5
arbitrary units, respectively. The auxiliary gas was heated

Table 1 Primers used in the expression analyses of Bos taurus genes by RT qPCR
Gene names

Primer sequences (5’-3’)a

Accession #AS (bp)

JAK1

Fwd: GCC​C TG​TGT​T TT​GGT​ATG​C T; Rv: ATC​TGG​ACA​GTT​CGG​TGG​AC

XM_024989564.1

239

JAK2

Fwd: AAC​GCT​GAG​GGG​GAT​TAT​C T; Rv: ATG​GTT​GGG​TGG​ATA​CCA​GA

XM_005209981.4

156

JAK3

Fwd: GGG​AGA​TCC​AGA​TCC​TCA​AAG; Rv: GCA​GAT​C TG​TGA​GGC​GTA​GAG​

XM_010806603.3

194

TYK2

Fwd: TCC​TGG​AGA​TCT​GCT​TCG​AT; Rv: TTC​TGG​GGC​TGT​AGC​TGA​GT

NM_001113764.1

208

PCNA

Fwd: AAG​CCA​C TC​CAC​TGT​C TC​C TA; Rv: TTA​AGT​GTG​TGC​TGG​CAT​C TC​

NM_001034494

207

CCND2

Fwd: GGG​CAA​GTT​GAA​ATG​GAA​CCT; Rv: TGG​CAA​ACT​TGA​AGT​CAG​TGG​

NM_001076372

155

CYP19A1

Fwd: GAC​CAT​C TG​TGC​TGA​T TC​CAT; Rv: TGG​T TT​GAG​AAG​GAG​AGC​T TG​

NM_174305.1

211

CYP11A1

Fwd: ATC​ATT​CAC​CCT​GAA​GAC​GTG; Rv: GCT​GAC​GAA​GTC​C TG​AGA​CAC​

NM_176644.2

240

RPL19

Fwd: GAC​CAA​TGA​AAT​CGC​CAA​TGC; Rv: ACC​TAT​ACC​CAT​ATG​CCT​GCC​

NM_001040516

154

AS Amplicon size (base pairs), Fwd Forward primer, Rv Reverse primeraAll primers were designed and validated by the authors. Each primer was used at a final concentration of 600 nM

Zareifard et al. BMC Molecular and Cell Biology(2023) 24:21

Page 16 of 19

to 300 °C. The heated ESI probe was set to 4000 V, andthe ion transfer tube temperature was set to 300 °C. Mass
spectrometry (MS) detection was performed in positive
ion mode operating in TOP-10 data dependent acquisition (DDA) mode. A DDA cycle entailed one ­MS1 survey scan (m/z 400–1500) acquired at 70,000 resolution(FWHM) and precursor ions meeting the user-defined
criteria for charge state (i.e., z = 2, 3 or 4), monoisotopic
precursor intensity was selected for ­MS2 acquisition
2(dynamic acquisition of M
­ S -based TOP-10 most intenseions with a minimum 2 × ­104 intensity threshold). Precursor ions were isolated using the quadrupole (1.5 Da isolation width), activated by HCD (28 NCE) and fragment
ions were detected in the ORBITRAP at a resolution of
17,500 (FWHM). Data were processed using Thermo
Proteome Discoverer (version 2.4) in conjunction withSEQUEST using default settings unless otherwise specified. The identification of peptides and proteins withSEQUEST was performed based on the reference protein sequences proteome extracted from UniProt (JAK3:
E1BEL4_BOVIN, CDKN1B: A6QLS3_BOVIN, MAPK8IP3: A0A3Q1LQI8_BOVIN) as FASTA sequences.
Parameters were set as follows: M­ S1 tolerance of 10 ppm;
2
­MS mass tolerance of 0.02 Da for Orbitrap detection;
enzyme specificity was set as trypsin with two missedcleavages allowed; carbamidomethylation of cysteine wasset as a fixed modification; and oxidation of methionine
as well as phosphorylation of serine, threonine and tyrosine were set as a variable modification. The minimum
peptide length was set to six amino acids. Data were further analyzed with a target-decoy database to filter incorrect peptide and protein identifications. For protein or
peptide quantification and comparative analysis, we used
the peak integration feature of Proteome Discoverer 2.4
software.

experiments were performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) following the
manufacturer’s protocol. RT-qPCR data were analyzed
using the Livak method ­(2−ΔΔct) [69] with RPL19 used as
reference gene [70].

mRNA Expression analysis

Data are presented as mean ± SEM from three or more
independent experiments, unless otherwise specified in
the text. Values for JAK3 and other target genes mRNA
were normalized to the reference gene RPL19. Homogeneity of variance between groups was verified by
O’Brien and Brown-Forsythe tests. Corrected values ofgene specific mRNA levels were compared between follicular or CL groups using one‐way analysis of variance(ANOVA). When ANOVA indicated a significant difference (p ≤ 0.05), the Tukey‐Kramer test was used for multiple comparison of individual means among SF, DF, OF,
and CL and for in vitro experiments, whereas the Dunnett test (p ≤ 0.05) was used to compare different samples from UHPLC-MS/MS. For Western blot analyses,
cells were grown to appropriate confluence and treatedas described. Protein bands were quantified using ImageJsoftware (NIH), corrected with beta-actin as loading

Expression and regulation of JAK3 and other JAK family members (JAK1, JAK2 and TYK2) mRNA were analyzed by RT-qPCR during follicular development using
in vivo samples and specific primers (Table 1). Total
RNA was extracted from bovine GC collected from follicles at different developmental stages (SF, DF, OF) andCL as described above and previously published [67].
Total RNA were also extracted from in vitro samples ofcultured GC and reverse transcription reactions were
performed using the SMART PCR cDNA synthesis technology (Takara Bio.) according to the manufacturer’s
procedure and as previously published [68]. In vitro
samples were used to analyze the expression of JAK3
binding partners (CDKN1B and MAPK8IP3), proliferation markers (PCNA and CCND2) as well as steroidogenesis markers (CYP19A1 and CYP11A1). RT-qPCR

Cell extracts and immunoblotting analysis

Cultured GCs were collected and homogenized with the
M-PER mammalian protein extraction reagent (Thermo
Fisher) supplemented with complete protease inhibitors.
Total proteins from the different treatment groups wereextracted and cell debris were removed by centrifugation (14,000 × g for 5 min at 4 °C) and the supernatants
were collected and stored at -80 °C until further analyses.
Total protein concentrations were determined using the
Bradford method [71] (Bio-Rad Protein Assay, Bio-Rad
Lab, Mississauga, ON, Canada) and immunoblotting was
performed as described previously [72]. Protein samples (50 µg) were resolved by one-dimensional denaturing 12% Novex Tris–glycine gels (Invitrogen, Burlington,
ON, Canada) and transferred onto polyvinylidene difluoride membranes (PVDF; GE Healthcare Life Sciences).
Membranes were incubated with specific first antibodies against STAT3 at a concentration of 0.069 μg/mL
and phospho-STAT3 (pSTAT3) at a concentration 1 μg/
mL to verify the effects of JAK3 inhibition or activationon the JAK/STAT pathway. Membranes were also incubated with anti-beta actin (ACTB) as reference protein.
Immunoreactive proteins were visualized by incubation
with appropriate horseradish peroxidase-linked secondary antibodies (1:10,000 dilution) followed by incubation
with the chemiluminescence system (Thermo Scientific)
according to the manufacturer’s protocol and revelation
was done using the ChemiDoc XRS + system (Bio-Rad).
Experimental design and statistical rationale

Zareifard et al. BMC Molecular and Cell Biology(2023) 24:21

reference and compared using ANOVA. For proteomics
analyses, data sets were further analyzed with a targetdecoy database to filter incorrect peptide and protein
identifications. For protein or peptide quantificationand comparative analysis, we used the peak integration
feature of Proteome Discoverer 2.4 software. Statisticalanalyses were performed using GraphPad PRISM version
9 for macOS.
Abbreviations
FSH	Follicle-stimulating hormone
LH	Luteinizing hormone
hCG	Human Chorionic Gonadotropin
JAK	Janus kinase
TYK	Tyrosine kinase
JH	JAK homology
FERM	4.1, Ezrin, radixin and meosin
SH2	Src homology 2
STAT​	Signal transducers and activators of transcription
CDKN1B	Cyclin-dependent kinase inhibitor 1B
MAPK8IP3	Mitogen-activated protein kinase 8 interacting protein 3GC	Granulosa cells
DF	Dominant follicles
SF	Small follicles
OF	Ovulatory follicles
CL	Corpus luteum
CCND2	Cyclin D2
PCNA	Proliferation cell nuclear antigen
CYP19A1	Cytochrome P450 family 19 subfamily A member 1
CYP11A1	Cytochrome P450 family 11 subfamily A member 1
UHPLC-MS/MS	Ultra-high performance liquid chromatography-tandem
mass spectrometer
Acknowledgements
The authors thank Dr. Jacques G. Lussier for his suggestions and critical comments throughout the progress of this project.
Authors’ contributions
Investigation, A.Z. and K.N.; Visualization, A.Z. and K.N.; Validation, A.Z. and K.N.;
Formal analysis, A.Z. and K.N.; Proteomics analyses, F.B., A.Z. and K.N.; WritingOriginal draft preparation, A.Z.; Methodology, A.Z., F.B. and K.N.; Conceptualization, K.N.; Resources, K.N. and F.B.; Writing-Review and Editing, A.Z., F.B.
and K.N.; Supervision, K.N.; Project Administration, K.N.; Funding Acquisition,
K.N and F.B. All authors have read and agreed to the published version of themanuscript.
Funding
This study was supported by a Discovery Grant from the Natural Sciences andEngineering Research Council of Canada (NSERC) (RGPIN#04516) to K. Ndiaye.
Proteomics equipment was funded by the Canadian Foundation for Innovation (CFI) and the Fonds de Recherche du Québec (FRQ), the Government ofQuebec (F. Beaudry CFI John R. Evans Leaders grant no. 36706). The funders
had no role in study design, data collection, and analysis, decision to publish,
or preparation of the manuscript.
Availability of data and materials
All data pertinent to this study are contained in the article. Proteomics raw
data are available upon request (k.ndiaye@umontreal.ca).

Declarations
Ethics approval and consent to participate
Animal Ethics Committee of the Faculty of Veterinary Medicine of the University of Montreal reviewed and approved all the experimental protocol as thecows were cared for in accordance with the Canadian Council on Animal Careguidelines [63] and data are reported in accordance with ARRIVE guidelines.

Page 17 of 19

Consent for publication
Not applicable.
Competing interests
The authors declare that there is no conflict of interests.
Received: 10 September 2022 Accepted: 9 May 2023

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