SIK2 and SIK3 Differentially Regulate Mouse Granulosa Cell Response to Exogenous Gonadotropins In Vivo

Abstract

Salt-inducible kinases (SIKs), a family of serine/threonine kinases, were found to be critical determinants of female fertility. SIK2 silencing results in increased ovulatory response to gonadotropins. In contrast, SIK3 knockout results in infertility, gonadotropin insensitivity, and ovaries devoid of antral and preovulatory follicles. This study hypothesizes that SIK2 and SIK3 differentially regulate follicle growth and fertility via contrasting actions in the granulosa cells (GCs), the somatic cells of the follicle. Therefore, SIK2 or SIK3 GC-specific knockdown (SIK2^GCKD and SIK3^GCKD, respectively) mice were generated by crossing SIK floxed mice with Cyp19a1pII-Cre mice. Fertility studies revealed that pup accumulation over 6 months and the average litter size of SIK2^GCKD mice were similar to controls, although in SIK3^GCKD mice were significantly lower compared to controls. Compared to controls, gonadotropin stimulation of prepubertal SIK2^GCKD mice resulted in significantly higher serum estradiol levels, whereas SIK3^GCKD mice produced significantly less estradiol. Cyp11a1, Cyp19a1, and StAR were significantly increased in the GCs of gonadotropin-stimulated SIK2^GCKD mice. However, Cyp11a1 and StAR remained significantly lower than controls in SIK3^GCKD mice. Interestingly, Cyp19a1 stimulation in SIK3^GCKD was not statistically different compared to controls. Superovulation resulted in SIK2^GCKD mice ovulating significantly more oocytes, whereas SIK3^GCKD mice ovulated significantly fewer oocytes than controls. Remarkably, SIK3^GCKD superovulated ovaries contained significantly more preantral follicles than controls. SIK3^GCKD ovaries contained significantly more apoptotic cells and fewer proliferating cells than controls. These data point to the differential regulation of GC function and follicle development by SIK2 and SIK3 and supports the therapeutic potential of targeting these kinases for treating infertility or developing new contraceptives.

According to the World Health Organization, infertility is estimated to affect 1 in 6 adults worldwide (1). Of the cases of infertility, 25% are attributed to ovulatory dysfunction (2). Successful ovulation requires the proper, highly coordinated development of the ovarian follicle, consisting of the oocyte and surrounding steroidogenic somatic cells called granulosa cells (GCs) and theca cells.

The master regulators of the latter half of follicle development and ovulation are the gonadotropins: follicle-stimulating hormone (FSH) and luteinizing hormone (LH) (3). FSH is produced and released from the pituitary and binds the FSH receptor (FSHR), whose expression in the ovary is restricted to the GCs (4, 5). FSHR is a Gs-coupled protein receptor and thus triggers downstream signaling cascades, including the canonical cAMP/PKA/CREB (cyclic adenosine monophosphate/protein kinase A/cyclic AMP response element-binding protein) pathway (6). FSHR activation stimulates GC proliferation and steroidogenic gene expression needed to produce estradiol and progesterone (7). However, the mechanisms governing normal follicle development and the perturbations in these pathways that lead to infertility are incompletely understood.

Recently discovered players in the regulation of fertility in the ovary include the salt-inducible kinases (SIKs). The SIKs, consisting of SIK1, SIK2, and SIK3, are a family of AMPK-related serine-threonine kinases with well-established roles as crucial regulators of cAMP-, PKA-, and CREB-mediated signaling pathways (8). Our group was the first to demonstrate the critical roles of SIK2 and SIK3 in female fertility (9). We demonstrated that global SIK2 knockout (SIK2^KO) mice have an enhanced ovulatory response to a superovulation protocol, ovulating approximately 3 times as many oocytes as wild-type controls. In contrast, SIK3^KO mice are infertile, do not ovulate in response to exogenous gonadotropin stimulation, and lack large antral follicles in their ovaries. Furthermore, we showed that broad inhibition of the SIKs with small molecule inhibitors in rat and human GCs and targeted knockdown of SIK2 in rat GCs potentiates FSH-induced transcription of steroidogenic genes (9). Mechanistically, the potentiation of FSH-induced gene expression in rat GCs caused by SIK inhibition is likely through modulation of the cellular localization of CREB-regulated transcriptional coactivator 2 (CRTC2) (10), a well-characterized target of SIK phosphorylation.

Because global knockout mice revealed that SIK2 and SIK3 are involved in follicle development and fertility and that SIK inhibition in vitro enhances GC steroidogenesis (9, 10), we hypothesized that SIK2 and SIK3 differentially affect GC function and, therefore, also ovulation and female fertility. To test this hypothesis, we developed SIK2 and SIK3 GC-specific knockdown mice, characterized their reproductive phenotypes, and determined the effect of loss of SIK2 or SIK3 on steroidogenesis, proliferation, and apoptosis in GCs in vivo. Further understanding the roles of SIK2 and SIK3 in the ovary will provide mechanistic insight into the regulation of ovarian fertility and may shed light on the potential of the SIKs to be targeted for treating infertility or developing new contraceptive strategies.

Methods

Animals

Animals were treated following the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All protocols were approved by the University of Illinois at Chicago Animal Care Committee. Mice were housed at the University of Illinois at Chicago Biologic Resources Laboratory under constant temperature, humidity, and light (14 hours light/10 hours dark) with free access to food and water. All transgenic mice were on a C57BL/6J background. Mice containing LoxP sites flanking exon 5 of the SIK2 or SIK3 genes (SIK2^flox/flox and SIK3^flox/flox) were obtained from Marc Foretz (11) and Hiroshi Takemori (12). Global genomic knockout of SIK2 alleles was generated by crossing SIK2^flox/flox mice with transgenic Zp3-Cre mice (Jackson Laboratory, Bar Harbor, ME). SIK2^flox/– mice were generated by crossing SIK2^flox/flox with SIK2^flox/− mice. SIK2^flox/− or SIK3 floxed mice were bred with mice expressing Cre recombinase driven by the CYP19A1 (aromatase) proximal promoter II (Cyp19a1pII-Cre). The CYP19A1 proximal promoter II is active exclusively in the GCs in the ovary in adult mice in follicles transitioning from the preantral to the preovulatory stage (13). However, it is low or undetectable in GCs of primordial, primary, and secondary follicles, theca cells, and oocytes. Cyp19a1pII-cre mice were obtained from Joanne Richards (13). Therefore, the genotype of SIK2^GCKD mice is SIK2^flox/−;Cyp19a1pII-Cre, and that of SIK3^GCKD mice is SIK3^flox/flox;Cyp19a1pII-Cre. Littermates were used as controls (flox/+ or flox/flox SIK2 or SIK3 without Cre) to the corresponding experimental genotypes.

Fertility Studies

Control and experimental female mice (30-65 days old) were continuously paired with male mouse breeders for 6 months. Breeding cages were checked weekly, and the litters produced by each breeding pair were counted for the number of pups and recorded. The total number of pups was determined by counting live and dead pups. Two female breeders were housed in each cage; thus, visibly pregnant females were noted weekly and moved to another cage to ensure accurate records of which female delivered the pups. The number of pups accumulated by the end of each month of breeding was calculated for each female breeder, and then the mean number of pups accumulated by the end of each month was calculated for each genotype. The mean litter size of each genotype was calculated. Pups produced from these experiments were genotyped and either used for further experimentation or breeding or were euthanized.

Granulosa Cell Isolation

Prepubertal (D21-30) mice were stimulated with 5 IU equine chorionic gonadotropin (eCG; Calbiochem, Cat. No. 367222, San Diego, CA) via intraperitoneal injection. Forty-eight hours later, mice were sacrificed, and ovaries were harvested and placed in ice-cold phosphate-buffered saline (PBS). Ovaries were cleaned of surrounding tissue and then placed in 1 mL of fresh ice-cold PBS. Antral follicles, identified under a dissection scope as visibly large, translucent spheres > 200 µm in diameter concentrated along the periphery of the ovary, were gently pressed for 1 to 2 minutes using two 25-gauge needle syringes to release GCs. The residual ovaries were discarded, and the PBS containing GCs was filtered through a 40-µm cell strainer (Greiner, Monroe, NC) to remove oocytes. After that, the cells were centrifuged at 4 °C for 3 minutes at 3000 rpm, and then the PBS was aspirated from the cell pellet. The cell pellets were stored at −80 °C until total RNA or protein isolation.

RNA Isolation and Quantification

Total RNA was isolated from GCs using TRIzol (Invitrogen, Carlsbad, CA) following manufacturer's instructions. The cDNA was made by reverse transcribing 0.5 µg total RNA using anchored oligo-dT primers (Integrated DNA Technologies, Coralville, IA) and Moloney Murine Leukemia Virus reverse transcriptase (Genscript, Piscataway, NJ). To measure gene expression, intron-spanning primers (Table 1) were designed to amplify each gene of interest, and then the copy number was determined using a standard curve consisting of a serial dilution of the PCR product for each gene of interest. For measuring Sik2 and Sik3, intron-spanning primers were designed such that either the forward or reverse primer was specific to exon 5 of the gene, which is removed upon recombination by Cre recombinase. Gene expression was normalized to the copy number of ribosomal protein L19 (Rpl19).

Table 1.

Primers used to quantify gene expression

Gene of interest	Primer sequences
Rpl19	Forward: 5′-GTA TCA CAG CCT GTA CCT GA-3′ Reverse: 5′-GGA AGC TTT ATT TCT TGG TC-3′
Cyp19a1	Forward: 5′-ATT GCA GCC CCT GAC ACC AT-3′ Reverse: 5′-TGG CGA TGT ACT TCC CAG CA-3′
Cyp11a1	Forward: 5′-GAT GTT CCA CAC CAG TGT CCC-3′ Reverse: 5′-AGG GTA CTG GCT GAA GTC TCG C-3′
StAR	Forward: 5′-TTT TGG GGA GAT GCC GGA GC-3′ Reverse: 5′-GCG AAC TCT ATC TGG GTC TGC G-3′
Sik1	Forward: 5′-ACC ACC CAA ATA TCA TCA A-3′ Reverse: 5′-GAC CCT CGT ACT CCT TCC-3′
Sik2	Forward: 5′-CTT TAA AAC TGG TGA ACT GC-3′ Reverse: 5′-TAC AGG AAC CTC TAT GAG CA-3′
Sik3	Forward: 5′-CAG CAA CCT CTT CAC TCC-3′ Reverse: 5′-CTT TCC TCC TTC AGT TGC-3′
Fshr	Forward: 5′-GTG CAT TCA ACG GAA CCC AGC -3′ Reverse: 5′-CGC CTC CAG TTT GCA AAG GC-3′
Lhr	Forward: 5′-TGT AAC ACA GGC ATC CGG ACC-3′ Reverse: 5′-ACT CCA GCG AGA TTA GCG TCG-3′

Immunoblotting

Protein was isolated from GCs using RIPA buffer (Sigma, St. Louis, MO) containing Halt Protease Inhibitor Cocktail and Halt Phosphatase Inhibitor Cocktail (Thermo Scientific, Waltham, MA). Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA). Thereafter, 10 µg of protein from each sample was denatured and separated using Bis-Tris polyacrylamide (12%) gel electrophoresis. Proteins were transferred to an Immobilon-P PVDF Membrane (Millipore, Burlington, MA). The membrane was blocked for 1 hour at room temperature using 5% milk in Tris-buffered saline, 0.1% Tween 20 (TBST), then incubated overnight in primary antibody diluted in 1% bovine serum albumin (BSA) in TBST. The primary antibodies used were SIK2 (1:1000, Cell Signaling Technology, Cat. No. 6919, Danvers, MA, RRID: AB_10830063), SIK3 (1:1000, Cell Signaling Technology, Cat. No. 39477S, Danvers, MA, RRID: AB_3251492), and GAPDH (1:10 000, ProteinTech, Cat. No. 60004-1-Ig, Rosemont, IL, RRID: AB_2107436). Membranes were washed with TBST and then subjected to a 1-hour incubation at room temperature with secondary antibodies diluted 1:5000 in 5% milk in TBST. The secondary antibodies used were horseradish peroxidase-conjugated goat anti-mouse IgG (Jackson Immunoresearch, Cat. No. 115-035-003, West Grove, PA, RRID: AB_10015289) and goat anti-rabbit IgG (Jackson Immunoresearch, Cat. No. 111-035-003, West Grove, PA, RRID: AB_2313567). Membranes were washed with TBST, then proteins were visualized using SuperSignal West Pico PLUS or SuperSignal West Femto Maximum Sensitivity Substrate enhanced chemiluminescent (ECL) substrate (Thermo Scientific, Waltham, MA) and imaged on a Chemidoc MP Imaging System (Bio-Rad, Hercules, CA).

Superovulation

Prepubertal (D21-28) mice were stimulated with 5 IU equine chorionic gonadotropin (eCG) (Calbiochem, Cat. No. 367222, San Diego, CA) via intraperitoneal (IP) injection. Forty-eight hours later, mice were stimulated with 5 IU human chorionic gonadotropin (hCG) (Calbiochem, San Diego, CA) via IP injection. Seventeen hours later, mice were sacrificed, and the intact ovaries and oviducts were harvested. Cumulus-oocyte complexes were released from the ampulla and were counted using a dissection scope.

Measuring Oocytes Ovulated in Unstimulated Mice

Reproductive-age mice (6-8 weeks old) were given daily vaginal lavages at 3:00 Pm to track their estrus cycles. Vaginal lavage was performed using 10 uL PBS to wash the vaginal opening 10 to 20 times. The lavage sample was smeared on a microscope slide and fully dried. Thereafter, slides were submerged in 0.1% crystal violet (Sigma, Cat. No. C0775, St. Louis, MO) for 1 minute, then washed with dH2O for 1 minute and allowed to dry. Using brightfield microscopy at 10×, cytology was assessed to determine the estrus cycle stage based on established criteria (14). When mice were determined to be in the proestrus stage in the afternoon, it was anticipated that the mice would begin and complete ovulation by 11:00 Am the following day (15). At 11:00 Am the following morning, mice were sacrificed, and the intact ovaries and oviducts were harvested. Cumulus-oocyte complexes were released from the ampulla and were counted using a dissection scope for visualization.

Immunohistochemistry

Ovaries were fixed overnight at 4 °C in modified Davidson's fixative and stored in 75% ethanol at 4 °C before paraffin embedding. Sections of 5 µm width were deparaffinized and rehydrated. Antigen retrieval was performed by gently boiling slides in 1 mM EDTA, 0.5% Tween 20 (pH 8.0) for 3 minutes in a microwave, allowing to cool, then repeating for an additional 3 minutes. Sections were blocked for 1 hour at room temperature in 5% BSA in permeabilization buffer (0.2% w/v gelatin, 0.2% v/v Triton in PBS) and then incubated overnight at 4 °C in primary antibody diluted in 1% BSA in permeabilization buffer. The primary antibodies used were Cleaved Caspase-3 (1:500, Cell Signaling Technology, Cat. No. 9661S, Danvers, MA, RRID: AB_2341188), and proliferating cell nuclear antigen (PCNA) (1:2000, ProteinTech, Cat. No. 60097-1-Ig, Rosemont, IL, RRID: AB_2883063). Slides were washed in 0.1% Tween 20 in PBS (PBST) and then incubated in secondary antibody diluted 1:400 in 1% BSA in permeabilization buffer. The secondary antibodies used were horseradish peroxidase-conjugated goat anti-mouse IgG (Jackson Immunoresearch, Cat. No. 115-035-003, West Grove, PA, RRID: AB_10015289) and goat anti-rabbit IgG (Jackson Immunoresearch, Cat. No. 111-035-003, West Grove, PA, RRID: AB_2313567). Slides were washed in PBST and then exposed to DAB substrate (Vector, Cat. No. SK-4100, Newark, CA) for 2 minutes. Sections were counterstained with hematoxylin from the Hematoxylin and Eosin (H&E) Stain Kit (Vector, Cat. No. H-3502, Newark, CA). Sections were dehydrated, cleared, and mounted using Cytoseal (Epredia, Portsmouth, NH).

Quantifying Proliferation and Apoptosis

Ovary sections stained via immunohistochemistry for cleaved caspase 3 and PCNA were imaged with a 10× objective on a Nikon Eclipse Ti2E Inverted Microscope (Nikon, Tokyo, Japan) using identical brightfield settings. Images of whole ovary sections were opened in ImageJ software, version 1.54f (National Institutes of Health, USA). Using the Colour Deconvolution function, images were separated into brown and blue channels, representing DAB and hematoxylin, respectively. Next, using the Threshold function, the maximum threshold was set to select only the dark brown DAB-positive cells for cleaved caspase-3 and only the distinct brown nuclei for PCNA, and this threshold number was kept constant between images. The ovary was selected using the Freehand selections tool, and then the DAB-positive pixels and area of the ovary were quantified using the Measure function. The sum of the values of the pixels within the selection (RawIntDen) was divided by the area of the ovary and compared between genotypes. One middle section was analyzed per ovary.

H&E and Follicle Counts

Serial 5 µm sections were deparaffinized, rehydrated, and then stained using the Hematoxylin and Eosin Stain Kit (Vector, Cat. No. H-3502, Newark, CA) following manufacturer's instructions. Every tenth section was visualized using a 10× objective on a brightfield microscope, and follicles were counted. Only follicles with the oocyte nucleus visible in the section were counted. Follicles were classified as “2 layer secondary” if the oocyte was surrounded 2 layers of GCs, “3 to 5 layer secondary” if the oocyte was surrounded by 3 to 5 layers of GCs, “large preantral” if the oocyte was surrounded by more than 5 layers of GCs without any antrum formation, as “early antral” if there were several small cavities scattered throughout the granulosa cell layers, as “antral” if there was one fluid-filled antrum but no cumulus stalk, and as “preovulatory” if there was one large antrum and a well-formed cumulus stalk, as previously described (16). Ten sections from one ovary per animal were counted. Follicle numbers for each animal were multiplied by 10 to account for counting one-tenth of the total sections in the ovary. To correct for the size of the ovary, follicle numbers were then normalized to the relative number of sections per ovary. Corpora lutea, characterized by cells with a hypertrophied, lighter-stained appearance, were counted in 2 middle sections at least 200 µm apart per ovary. The mean number of follicles per follicle type was compared between genotypes.

Estradiol Measurement

The eCG-stimulated prepubertal animals were anesthetized, and blood was collected via cardiac puncture using a 25-gauge needle. Blood was incubated at room temperature for 30 to 60 minutes and then centrifuged at 4 °C for 10 minutes at 1.5 RCF. Thereafter, serum was removed and stored at −80 °C. Undiluted serum was subjected to an estradiol ELISA (DRG International, Springfield, NJ, RRID: AB_2924716) in accordance with the manufacturer's instructions.

Statistics

Data were analyzed using Prism 6 (GraphPad, Boston, MA). Differences between 2 groups were determined by Student t test. Differences between multiple groups were determined by one-way analysis of variance (ANOVA) with Tukey post hoc test. To compare pups accumulated over time between genotypes, significant differences were determined by repeated measures ANOVA. To compare follicle counts of each follicle type between genotypes, significant differences were determined by two-way ANOVA with Bonferroni post hoc test. Data are represented as mean ± standard error of the mean (SEM). Significant differences were recognized at P < .05.

Results

To evaluate the reproductive effects of SIK2 or SIK3 loss in GCs, we developed SIK2 and SIK3 GC-specific knockdown mouse models (referred to hereon as SIK2^GCKD and SIK3^GCKD, respectively) using the Cre-loxP system. Mice carrying floxed exon 5 of the SIK2 or SIK3 genes were previously described (11, 12). Floxed mice were crossed with transgenic mice containing the Cre recombinase gene downstream of the Cyp19a1 proximal promoter II (Cyp19a1pII-Cre), which is active specifically in the GCs of follicles between the preantral and preovulatory stages (13). Because the Cyp19a1 pII promoter is activated by the gonadotropins, the experimental design utilized here was to stimulate prepubertal mice with equine chorionic gonadotropin (eCG) for 48 hours to stimulate follicle growth as well as Cre recombinase expression.

To validate the SIK2^GCKD and SIK3^GCKD mouse models, ovaries were harvested from eCG-stimulated mice, and GCs were isolated from antral follicles to quantify SIK mRNA and protein levels (Fig. 1). SIK2^GCKD GCs had significantly less Sik2 expression than controls, with no significant change in Sik3 expression (Fig. 1A). Further, SIK2^GCKD GCs showed little to no SIK2 protein expression compared to wild-types (Fig. 1B). Unexpectedly, SIK2^+/− GCs showed a visible reduction in SIK2 protein expression, suggesting possible haploinsufficiency of SIK2. SIK3^GCKD GCs had significantly decreased expression of Sik3 compared to controls, whereas there was no significant change in Sik2 expression (Fig. 1C). This corresponded with a visible reduction in SIK3 protein expression compared to controls (Fig. 1D). Surprisingly, SIK2^GCKD GCs showed a nonsignificant trend toward increased Sik1 (Fig. 1A), and SIK3^GCKD GCs had significantly increased Sik1 expression (Fig. 1C). These data show that a significant knockdown of Sik2 or Sik3 mRNA and protein levels was achieved in the GCs of gonadotropin-stimulated follicles.

Figure 1.

Measurement of SIK knockdown in SIK2^GCKD and SIK3^GCKD GCs. Prepubertal (21-30 days old) mice were stimulated with 5 IU eCG for 48 hours, then GCs were harvested, and total RNA and protein were isolated. (A, C) Expression of Sik1, Sik2, and Sik3 was measured relative to Rpl19 by quantitative qPCR in wild-type control (“C”) vs SIK2^GCKD (A) or SIK3^GCKD (C) GCs (n = 3). (B) Protein levels of SIK2 and GAPDH in wild-type (WT), SIK2^+/−, SIK2^GCKD, and SIK2^KO GCs (n = 3). (D) Protein levels of SIK3 and GAPDH in WT and SIK3^GCKD GCs (n = 3). Values are displayed as the mean ± SEM. Statistical significance was determined by Student t test. *P < .05; **P < .01 vs controls.

To determine the effects of the loss of SIK2 and SIK3 in GCs on fertility, reproductive-age females were bred for 6 months, and their litters were tracked (Fig. 2). SIK2^GCKD animals had a similar litter size compared to wild-type controls, while SIK3^GCKD animals showed a significant decrease in litter size (Fig. 2A). Similarly, SIK2^GCKD animals accumulated a similar number of pups over 6 months compared to controls, whereas SIK3^GCKD breeders accumulated significantly fewer pups (Fig. 2B), likely due to significantly smaller litter sizes (Fig. 2A). Thus, this suggests that SIK2^GCKD did not affect normal fertility, whereas SIK3^GCKD resulted in subfertility.

Figure 2.

Determining the effect of the loss of SIK2 and SIK3 in GCs on female fertility. Reproductive-age (30-65 days old) female mice were continuously paired with a male breeder for 6 months, and the litters were tracked. (A) The number of pups per litter was recorded for each litter and compared between litters from wild-type controls (WT, n = 26), SIK2^GCKD (n = 28), and SIK3^GCKD (n = 17) female breeders. Error bars represent the mean ± SEM. Statistical significance was determined by one-way ANOVA. (B) The total number of pups accumulated by the end of each month of continuous breeding was calculated for WT (n = 7), SIK2^GCKD (n = 5), and SIK3^GCKD (n = 5) female breeders. Values are displayed as the mean ± SEM. Statistical significance was determined by repeated measures ANOVA. *P < .05; **P < .01; ***P < .001 vs WT.

One of the main functions of ovarian follicles is to produce estradiol to support female fertility. To investigate whether estradiol production is altered in the GC knockdowns, sera from eCG-stimulated animals were collected, and estradiol concentration was measured (Fig. 3). SIK2^GCKD sera contained significantly more estradiol than controls (Fig. 3A). In contrast, SIK3^GCKD animals had significantly less serum estradiol (Fig. 3B), suggesting a possible mechanism by which the loss of SIK3 in GCs resulted in subfertility (Fig. 2).

Figure 3.

Loss of SIK2 and SIK3 in GCs differentially regulates serum estradiol. Prepubertal (21-30 days old) mice were stimulated with 5 IU eCG for 48 hours, and then serum was collected to measure estradiol concentration via ELISA in wild-type (Control, n = 8) vs SIK2^GCKD (n = 6) (A) and SIK3^GCKD (n = 4) (B) mice. Error bars represent the mean ± SEM. Statistical significance was determined by Student t test. **P < .01; ***P < .001 vs Control.

The differential effect of SIK2 or SIK3 knockdown in fertility and estradiol levels led to the hypothesis that SIK2 and SIK3 differentially regulate the expression of steroidogenic enzymes involved in the production of estradiol, including steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc, encoded by Cyp11a1), and aromatase (encoded by Cyp19a1). Thus, gene expression was measured in the GCs of eCG-stimulated animals (Fig. 4). In concordance with the serum estradiol data (Fig. 3), SIK2^GCKD showed significantly increased steroidogenic gene expression of Star, Cyp11a1, and Cyp19a1 (Fig. 4A), whereas SIK3^GCKD showed significantly decreased gene expression of Star and Cyp11a1 (Fig. 4B). Strikingly, the knockdown of SIK3 only tended to decrease Cyp19a1 expression, suggesting that SIK3 may indirectly regulate estradiol production (Fig. 4B). The data indicate that SIK2 may directly regulate estradiol production in GCs via steroidogenic gene expression. In contrast, although SIK3 knockdown also decreased the expression of steroidogenic genes, it appears that this effect is indirect, at least at the level of estradiol production.

Figure 4.

SIK2 and SIK3 differentially regulate steroidogenic gene expression in GCs. Prepubertal (21-30 days old) mice were stimulated with 5 IU eCG for 48 hours, then GCs were isolated and total RNA was harvested. Expression of Star, Cyp11a1, and Cyp19a1 was measured relative to Rpl19 by quantitative qPCR in wild-type controls (C) vs SIK2^GCKD (A) or SIK3^GCKD (B) GCs (n = 3). Values are displayed as the mean ± SEM. Statistical significance was determined by Student t test. *P < .05; **P < .01 vs controls.

Estradiol is essential for promoting follicle growth to the preovulatory stage. Therefore, it was hypothesized that the alterations in estradiol seen in the SIK2^GCKD and SIK3^GCKD animals impact follicle development and ovulation. Hence, we next subjected mice to a superovulation protocol, and then the number of oocytes ovulated was counted in the ampulla of the oviduct (Fig. 5A). SIK2^GCKD mice ovulated significantly more oocytes than controls (Fig. 5A), recapitulating the ovulatory response of SIK2 global knockout (SIK2^KO) mice (9). Interestingly, SIK2^KO mice ovulate a similar number of oocytes as wild-type animals under physiologic conditions (unpublished observation), suggesting that this hyper-fertile response in SIK2^GCKD and SIK2^KO mice may only be seen with supraphysiologic levels of gonadotropins.

Figure 5.

SIK2 and SIK3 in GCs differentially regulate ovulatory response to superovulation. (A) Prepubertal (21-30 days old) mice were stimulated with 5 IU eCG for 48 hours followed by 5 IU hCG for 17 hours to stimulate ovulation. Thereafter, oocytes were counted in the ampulla of each oviduct. The total number of oocytes ovulated by each animal is shown for wild-type controls (Control), SIK2^GCKD, and SIK3^GCKD animals (n = 3). Error bars represent the mean ± SEM. Statistical significance was determined by one-way ANOVA. (B) Representative H&E-stained ovary sections from prepubertal WT, SIK2^GCKD, and SIK3^GCKD mice (n = 3) treated with eCG for 48 hours. Ovaries were imaged with 20× objective, scale bar = 700 µm. *P < .05; ***P < .001 vs controls.

In contrast, SIK3^GCKD mice ovulated significantly fewer oocytes than controls in response to superovulation (Fig. 5A), in concordance with the significantly smaller litter sizes of SIK3^GCKD animals (Fig. 2A). Histological analysis of the ovaries from eCG-stimulated animals revealed that SIK2^GCKD ovaries mainly consisted of large, antral follicles with few preantral follicles, whereas SIK3^GCKD ovaries appeared to have few antral follicles and more secondary, preantral, and early antral follicles than wild-type and SIK2^GCKD ovaries (Fig. 5B).

To more quantitatively determine how the knockdown of SIK3 in GCs affected follicle development, resulting in a decrease in the number of ovulated oocytes, we evaluated and counted the follicles at different stages of development in superovulated ovaries (Fig. 6). This experiment revealed that SIK3^GCKD ovaries contained significantly more preantral follicles and significantly fewer corpora lutea than controls (Fig. 6). The preantral stage precedes the gonadotropin-dependent development of the fluid-filled antrum. Therefore, SIK3 expression in the GCs is important for the preantral-to-antral transition.

Figure 6.

Loss of SIK3 in GCs disrupts folliculogenesis. Prepubertal (21-30 days old) mice were stimulated with 5 IU eCG for 48 hours followed by 5 IU hCG for 17 hours. Thereafter, ovaries were fixed, serially sectioned, and stained with H&E, then follicles were counted by follicle stage. The total number of 2-layer secondary, 3- to 5-layer secondary, large preantral, early antral, antral, preovulatory follicles, and corpora lutea from one ovary per animal was calculated for controls and SIK3^GCKD mice (n = 3). Values are displayed as the mean ± SEM. Statistical significance was determined by two-way ANOVA for follicles and by Student t test for corpora lutea. *P < .05 vs controls.

As SIK2^GCKD and SIK3^GCKD mice showed alterations in folliculogenesis, which may be due to influences on GC proliferation or follicle atresia, we next investigated whether the loss of SIK2 and SIK3 in GCs affects proliferation and follicle survival. Ovaries from eCG-stimulated animals were subjected to immunohistochemistry to measure the expression of proliferating cell nuclear antigen (PCNA), a marker of proliferation, and cleaved caspase-3, a marker of apoptosis (Fig. 7). Confirming results presented in Fig. 5, histology examination suggests that SIK2^GCKD ovaries appear to have more antral follicles than controls, while SIK3^GCKD ovaries appear to have fewer antral follicles and more secondary and preantral follicles than controls (Fig. 7A and 7C). Proliferation and apoptosis quantification revealed that SIK3^GCKD ovaries had significantly more cleaved caspase 3-positive (Fig. 7A and 7B) and fewer PCNA-positive (Fig. 7C and 7D) GCs than controls. The cleaved caspase-3 staining was predominantly observed in the GCs of preantral and early antral follicles (Fig. 7A). There was no significant difference in cleaved caspase-3 between controls and SIK2^GCKD mice (Fig. 7B). Thus, SIK3 may regulate both the follicle growth and survival necessary to grow past the preantral stage.

Figure 7.

SIK2 and SIK3 in GCs differentially affect markers of proliferation and apoptosis. Prepubertal (21-30 days old) mice were stimulated with 5 IU eCG for 48 hours, then ovaries were harvested, fixed, and used for immunohistochemistry studies to detect cleaved caspase-3 (apoptosis marker) (A) and PCNA (proliferation marker) (C). Brown coloring represents positive staining against blue hematoxylin counterstain. The quantification results are displayed in B and D (n = 4-6) as the sum of cleaved caspase-3 or PCNA positive pixels divided by the area of the ovary section. Representative 10× images of wild-type control (Control), SIK2^GCKD, and SIK3^GCKD ovaries are displayed. The area inside the white boxes is displayed at a higher magnification below each image. Values are displayed as the mean ± SEM. Statistical significance was determined by one-way ANOVA with Tukey post hoc test. *P < .05, **P < .01.

Finally, to determine whether SIK2 and SIK3 may influence follicle development by regulating the gonadotropin receptors, the FSH receptor, and the LH receptor, we measured Fshr and Lhr gene expression in eCG-stimulated GCs (Fig. 8). Interestingly, there was significantly more Lhr in GCs from SIK2^GCKD than in GCs from SIK3^GCKD (Fig. 8B) and a nonsignificant trend toward increased Fshr expression in SIK2^GCKD GCs (Fig. 8A). Thus, the loss of SIK2 may promote more ovulation by upregulating the expression of the LH receptor.

Figure 8.

Effects of the loss of SIK2 or SIK3 on FSH receptor and LH receptor gene expression. Prepubertal (21-30 days old) mice were stimulated with 5 IU eCG for 48 hours, then GCs were isolated and total RNA was harvested. Expression of Fshr (A) and Lhr (B) was measured relative to Rpl19 by quantitative qPCR in wild-type controls (WT) vs SIK2^GCKD or SIK3^GCKD GCs (n = 5-6). Values are displayed as the mean ± SEM. Statistical significance was determined by one-way ANOVA with Tukey post hoc test. *P < .05.

Discussion

The GCs are the somatic cells of the ovarian follicle that proliferate, participate in steroidogenesis, and nurture the oocyte. In particular, during the latter half of folliculogenesis, GC function is mainly controlled by FSH, which triggers signaling pathways downstream of the FSHR and promotes GC proliferation, differentiation, and survival, all essential for antral follicle growth. In this work, SIK2 and SIK3 showed differential regulation of steroidogenesis (Figs. 3 and 4)—a hallmark of GC terminal differentiation—and apoptosis (Fig. 7). Further, SIK2 and SIK3 promoted and inhibited follicle growth, respectively; eCG-stimulated SIK2^GCKD animals ovulated significantly more oocytes (Fig. 5A), while SIK3^GCKD ovulated fewer oocytes (Fig. 5A) and had ovaries with more follicles stalled at the preantral stage (Fig. 6). Thus, SIK2 and SIK3 mediate the effects of FSH in GCs in vivo in opposite ways.

Interestingly, although the loss of SIK2 and SIK3 showed differential effects in mouse GCs, multiple lines of evidence in various areas of physiology suggest redundancy between the SIK isoforms. Sleep regulation studies using mice that contain a mutated conserved PKA phosphorylation site in SIK1, 2, or 3 all show increased non-rapid eye movement (NREM) sleep delta density, a marker of sleep need (17, 18). In osteoclastogenesis, knocking down either SIK2 or SIK3 in vitro reduces osteoclast differentiation (19). Mice with SIK1/3 or SIK2/3 chondrocyte-specific double knockdown show chondrocyte hypertrophy that is delayed further than the knockout of SIK3 alone (20). It was also shown that in T cells, the loss of SIK2 and SIK3 results in a greater reduction in T cells vs the loss found with either one (21). This redundancy has also been suggested due to SIK mutations being infrequent in human cancers, combined with the observation that all SIKs share tumor suppressor functions (22). Our findings suggest that ovarian GCs represent a unique system in which SIK2 and SIK3 control different cellular functions. Although the molecular mechanisms involved remain to be determined, our previous in vitro reports suggest that SIK2 inhibits steroidogenesis, particularly the expression of Cyp19a1, a finding we confirm here in vivo (9). In contrast, SIK3 knockdown did not affect Cyp19a1 expression significantly, suggesting that SIK2 and SIK3 differentially regulate this gene. In speculating on how SIK3 may be affecting granulosa cell function, it is known that SIK3, but not SIK1 or 2, regulates glucose and lipid metabolism in the liver (23), and thus it may be possible that SIK3 regulates metabolism in the GCs. However, further studies are required.

In considering compensation between the SIK isoforms, it was unexpected to find that Sik1 was significantly upregulated in eCG-stimulated SIK3^GCKD GCs (Fig. 1). This was surprising considering that SIK1 global knockout (SIK1^KO) mice have normal fertility and ovulate a similar number of oocytes in response to superovulation compared to wild-type controls. It may be possible that SIK1 is also important for normal fertility in GCs and that these effects are masked in SIK1^KO mice by SIK3 compensation. Similarly, if SIK1 promotes fertility, then the upregulation of Sik1 in SIK3 knockdown GCs (Fig. 1) may be one possible explanation as to why the SIK3^GCKD animals were not completely infertile. Experiments to determine whether forced expression of SIK1 can rescue SIK3 knockdown are warranted. Further, experiments in fertility should investigate the reproductive phenotypes of SIK1/2 and SIK1/3 double knockouts to understand better the contributions of SIK isoforms and possible compensatory mechanisms.

We hypothesized that the loss of SIK3 in the GCs would recapitulate the infertile, gonadotropin-resistant phenotype seen in SIK3 global knockout mice (9). However, the SIK3^GCKD animals in this study produced litters, though they accumulated significantly fewer pups than WT controls over 6 months (Fig. 2). Outside of possible compensation by SIK1, there are 3 other possibilities for why the SIK3^GCKD animals were subfertile but not infertile. First, the SIK3^GCKD GCs showed a significant knockdown but not a knockout of SIK3 (Fig. 1). Thus, it is possible that low but not absent SIK3 in the GCs may preserve fertility. Second, SIK3 may have essential roles in the GCs prior to or during differentiation. Our experimental approach utilized the Cyp19a1 proximal promoter II to drive Cre recombinase expression, thus requiring gonadotropin stimulation for Cre recombinase expression and thereby initiating differentiation (13). Third, SIK3 may have important fertility regulatory actions in the theca cells and the oocyte. Previously, our group showed that SIK3 is the most abundant SIK expressed in the theca cells and that broad SIK inhibition using the small molecule inhibitor HG-9-91-01 regulates androgen synthesis in theca cells (24). In the oocyte, the critical activator of SIK kinase activity, liver kinase B1 (LKB1, also known as STK11), has been shown to regulate primordial follicle activation; oocyte-specific knockout of LKB1 in a mouse model promotes premature ovarian failure (25). Thus, it is plausible that SIK3 may be involved in theca cell and oocyte function, although further work is required to determine whether SIK3 regulates fertility via actions in the other follicle components. Regardless, illuminating the follicle-attenuating role of SIK3 in the GCs supports the potential of targeting the SIK3 pathway as a nonhormonal strategy for contraception.

The specific mechanisms by which the SIKs act in GCs require further elucidation. The canonical pathway downstream of the Gs-coupled protein receptor FSHR is the cAMP-PKA-CREB pathway, in which CREB is the transcription factor promoting the expression of steroidogenic enzymes. One family of targets of SIK phosphorylation is the CREB-regulated transcriptional coactivators (CRTCs). Interestingly, our group recently showed that SIK inhibition and FSH stimulation in rat GCs in vitro promote nuclear localization of CRTC2 (10). Moreover, we showed that SIK inhibition does not affect PKA or CREB phosphorylation, suggesting that the SIKs act downstream of CREB to affect CREB-mediated transcription (10). Thus, CRTC2 may be regulated by SIKs in GCs in vitro and in vivo. In our SIK2^GCKD mice, we saw increased serum estradiol and steroidogenic gene expression (Figs. 3 and 4), consistent with our previous work in rat and human GCs in vitro (9, 10). Therefore, steroidogenesis may be regulated via an FSH-SIK2-CRTC2 pathway that augments CREB activity in the nucleus. However, further experimentation is required to establish this pathway in the ovary.

In addition to promoting steroidogenesis, the SIK2^GCKD mice ovulated significantly more oocytes than wild-type controls, recapitulating the hyper-response to superovulation seen in the SIK2 global knockout (SIK2^KO) mice (9) and demonstrating that SIK2 actions in the GCs mediate the effects observed in the global knockout. Interestingly, compared to wild-type controls, the SIK2^GCKD mice produced a similar number of pups over 6 months, and both SIK2^KO and SIK2^GCKD mice had similar litter sizes (9). We also determined that SIK2^KO mice ovulate a similar number of oocytes without stimulation compared to wild-type animals (unpublished observation). Thus, this suggests that other mechanisms under physiological conditions may compensate for the loss of SIK2 but that this compensation may be overwhelmed by supraphysiologic doses of gonadotropins. As discussed above, SIK1 may partially compensate the lack of SIK2. These combined observations highlight SIK2 as a potential target for improving egg retrieval yield during controlled ovarian stimulation, an essential part of the in vitro fertilization process.

Although our previous and current work demonstrated that SIK inhibition and the loss of SIK2 in GCs and theca cells in vitro and in vivo promote steroidogenesis (9, 10, 24), SIK3^GCKD mice showed significantly decreased levels of serum estradiol and significantly decreased steroidogenic gene expression (Figs. 3 and 4). Further, the SIK3^GCKD ovaries showed follicle growth defects and increased atretic follicles (Figs. 6 and 7), suggesting that the lack of SIK3 may indirectly affect steroidogenesis by promoting GC death. This finding also suggests that it is unlikely that SIK3 signals via the CRTCs as these factors appear to be crucial for normal steroidogenesis. Another well-established target of the SIKs is the class IIa histone deacetylases (HDACs), which seem to mediate SIK3 effects on circadian rhythm, metabolism, and skeletal development (26-29). It may be possible that SIK3 actions in GCs are mediated by class IIa HDACs. Other important signal transducers downstream of the FSHR are ERK1/2, which may mediate anti-apoptotic, proliferative, and steroidogenic effects, and Akt, which is also anti-apoptotic in GCs (30). However, we have demonstrated that SIK activity inhibition in GCs does not affect FSH stimulation of AKT (10). Interestingly, SIK inhibition decreases FBS-induced Akt phosphorylation in vascular smooth muscle cells, and this effect was attributed to SIK3 (31). Thus, the evidence does not suggest that ERK1/2 or AKT may mediate the effect of SIK3, although this remains to be determined.

Finally, it is worth considering how the loss of SIK2 or SIK3 in GCs may affect the reproductive lifespan. SIK3 global knockout animals show nearly empty ovaries that are visibly devoid of follicles by 1 year of age, indicating diminished ovarian reserve (9). We showed that SIK3^GCKD ovaries had follicle growth defects (Fig. 6) and a greater percentage of apoptosis-positive cells (Fig. 7), indicative of more atretic follicles. As fewer follicles progress to the antral stages in the absence of SIK3 in the GCs, factors secreted by larger follicles that protect the ovarian reserve may be reduced, leading to increased recruitment of primordial follicles into the growing follicle pool and consequently to a premature decrease of the ovarian reserve. In regard to SIK2, aged SIK2 global knockout ovaries have not yet been studied. Because SIK2^GCKD animals produce a similar number of pups per litter over 6 months compared to wild-type controls (Fig. 2), it is unclear whether SIK2^GCKD animals have more growing follicles under physiologic conditions that may lead to premature depletion of the ovarian reserve. The characterization of aged SIK2^GCKD and SIK3^GCKD ovaries is necessary to begin to understand the role of SIK2 and SIK3 in ovarian aging.

In conclusion, we showed that SIK2 and SIK3 play opposite in vivo roles in regulating GC function, follicle development, and—in the case of SIK3—fertility. Together, our findings contribute to a better understanding of the in vivo role of SIKs in female fertility. Further, this work hopes to inform future studies investigating the role of the SIKs in reproductive pathophysiology as well as the potential of the SIKs as therapeutic targets for treating infertility and developing new contraceptives. However, based on our findings, highly specific SIK2 or SIK3 inhibitors must be developed to take advantage of their possible therapeutic applications.

Abbreviations

ANOVA

analysis of variance

BSA

bovine serum albumin

cAMP

cyclic adenosine monophosphate

CREB

cyclic AMP response element-binding protein

CRTC2

CREB-regulated transcriptional coactivator 2

eCG

equine chorionic gonadotropin

FSH

follicle-stimulating hormone

FSHR

follicle-stimulating hormone receptor

GC

granulosa cell

hCG

human chorionic gonadotropin

H&E

hematoxylin and eosin

LH

luteinizing hormone

PBS

phosphate-buffered saline

PCNA

proliferating cell nuclear antigen

PKA

protein kinase A

SEM

standard error of the mean

SIK

salt-inducible kinase

SIK2^GCKD

SIK2 granulosa cell knockdown

SIK2^KO

SIK2 global knockout

StAR

steroidogenic acute regulatory protein

TBST

Tris-buffered saline with 0.1% Tween-20

Funding

The authors thank the National Institutes of Health (NIH) for the financial support grant R01HD097202 to C.S.

Author Contributions

Conceptualization: E.H., M.A., and C.S.; Methodology: E.H., M.H., O.L., R.F., and M.A.; Formal analysis and investigation: E.H. and C.S.; Resources: M.F., N.T., H.T.; Draft preparation: E.H.; Review and editing: E.H., M.H., O.L., R.F., M.A., M.F., N.T., H.T., and C.S.; Project administration and funding acquisition: C.S.

Disclosures

The authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

Data Availability

Original data generated and analyzed during this study are included in this published article or in the data repositories listed in References.