Abstract
Glycogen synthase kinase 3 alpha (GSK3α) has emerged as a prospective target to develop male contraceptives. Global or testis-specific loss of GSK3α, but not GSK3β, results in male infertility. Previous studies demonstrated that the GSK3α-selective inhibitor BRD0705 induces reversible infertility in heterozygous mice. Here, we show that BRD0705 induces similar infertility in wild-type male mice. Fertility outcomes remain largely unaltered following superovulation in female mice mated with BRD0705-treated males. Further, no evidence of foetal resorption was observed in these females. Sperm count in treated mice was not altered; however, morphological defects appeared during their passage through the epididymis. The GSK3α inhibitor affected pathways related to the enzyme’s catalytic activity in epididymal sperm. Phosphoproteomic analysis of sperm from inhibitor-treated mice showed that phosphorylation of over 50 proteins involved in motility, metabolism, and fertility, among others, was significantly altered. Among the highly hypophosphorylated proteins, four are likely substrates of GSK3 (viz., AKAP4, NT5C1B, SPATA18, and TRA2B). Analysis of the seminiferous tubules of BRD0705-treated mice showed normal spermatogenesis, suggesting that the drug did not affect developing spermatogenic cells. However, in vitro exposure of post-meiotic germ cells demonstrated their susceptibility to this inhibitor. BRD0705, therefore, shows only a non-genomic effect at the post-translational level. These findings support GSK3α inhibition via BRD0705 as a non-hormonal, on-demand, and reversible male contraceptive strategy.
Keywords: glycogen synthase kinase 3 alpha, sperm maturation, male contraceptive, BRD0705, spermatogenesis, phosphoproteomics
Graphical abstract
Graphical Abstract.
Selective inhibition of glycogen synthase kinase 3 alpha (GSK3α) by BRD0705 impairs epididymal sperm maturation, reducing fertility while preserving spermatogenesis, demonstrating reversible, non-hormonal male contraception. Created in BioRender. Singh, A. (2026) https://BioRender.com/e2tqunw
Introduction
The global population is expected to reach 9.73 billion by the year 2064, with most growth occurring in developing countries (Vollset et al., 2020). Unintended pregnancies remain a major global health and socioeconomic challenge, highlighting the need for novel and effective contraceptive strategies. Male contraception remains limited to barrier methods, such as condoms, and permanent surgical procedures, such as vasectomy, placing a greater contraceptive burden on women (Thirumalai and Amory, 2021). Both these current methods have limitations in efficacy, reversibility, and acceptance (James et al., 2020).
Sperm maturation is a unique process for normal male fertility in mammals: an essential part of this maturation takes place during their passage through the epididymis (Kabi et al., 2025). Here, the sperm gain motility essential for successful fertilization. Among the key regulators of this maturation is the signalling enzyme paralogue glycogen synthase kinase 3 alpha (GSK3α) (Koch et al., 2015). Targeting epididymal sperm maturation offers several advantages, including a faster onset of action and better reversibility than contraceptives that affect spermatogenesis. Since spermatogenesis is a longer process, any contraception affecting this stage can take longer to show effects.
GSK3 is a multifunctional serine/threonine kinase involved in a wide range of cellular processes such as insulin signalling, cell survival, apoptosis, embryonic development, and Wnt/β-catenin signalling (Doble and Woodgett, 2003; Forde and Dale, 2007; Amaral et al., 2023). Its two paralogues, GSK3α and GSK3β, are encoded by two separate genes. They share ∼98% sequence similarity in their catalytic domains, differing at their N- and C-termini. Unlike most kinases, GSK3 is constitutively active under basal conditions (Fang et al., 2000). Its activity is regulated by phosphorylation at serine residues (Ser21 in GSK3α and Ser9 in GSK3β) that leads to inhibition. In contrast, autophosphorylation of their tyrosine residues (Tyr279 in GSK3α and Tyr216 in GSK3β) allows for catalytic activity (Bhattacharjee et al., 2015). The two paralogues are largely redundant in many tissues, except in the testis, where there is a paralogue-specific requirement for GSK3α in sperm function and male fertility (Bhattacharjee et al., 2018; Dey et al., 2018). Pan inhibitors of GSK3 (which inhibit both GSK3α and GSK3β) have been used for the last three decades. However, a loss of activity of both protein functions would disrupt their other necessary physiologic functions in male germ cells and adversely affect the functioning of other cell types. Recently, paralogue-selective inhibitors were developed by Dr Wagner and their team at the Broad Institute, exploiting the aspartic/glutamic acid difference in the ATP-binding sites of GSK3α and GSK3β (Wagner et al., 2018).
In our previous study, Gsk3a+/− mice were used as they are phenotypically similar to the wild-type male mice (Kabi et al., 2025). This allowed assessment of GSK3α inhibition without the effects of complete gene knockout. This made it suitable for initial confirmation of GSK3α inhibition by BRD0705, the paralogue-specific inhibitor for GSK3α. We demonstrated that a 1-week pharmacological inhibition of GSK3α in heterozygous Gsk3a+/− male mice leads to reversible infertility. This was characterized by impaired sperm motility as recorded via Computer-Assisted Semen Analysis (software version 1.17.2 HT CASA II; Hamilton Thorne Biosciences, Beverly, MA, USA), reduced ATP levels, and loss of tyrosine phosphorylation at the GSK3α auto-activation site. Fertility of the treated mice was restored following cessation of treatment. Together, these results show that GSK3α is a ‘male fertility kinase’ and provide proof-of-concept that its selective inhibition is a non-hormonal option for a safe and reversible male contraception.
Although the previous study provided key insights, it also raised new questions about the role of GSK3α in male fertility, particularly its potential secondary effects on female reproductive health. In this follow-up article, heterozygous (Gsk3a+/–) mice were paired with superovulated females to ensure oestrous synchrony and that all females were at the same stage before mating setup. Second, one rationale for utilizing the Gsk3a+/– model in our prior research was that a single copy of Gsk3a produces only 50% of the protein, which, while enough for sustaining fertility, is more amenable to inhibition by BRD0705 due to its reduced quantity. Therefore, the fertility tests in the present investigation were conducted in wild-type mice (Gsk3a+/+) to verify whether comparable results were observed to ensure translatability in humans. Furthermore, this paper investigates whether sperm from BRD0705-treated males impact embryo implantation or induce embryo resorption in the female reproductive tract. Assessment of implantation sites provides a clear distinction between any pre- and post-fertilization defects. This would confirm that BRD0705 has a contraceptive action that is male-specific and is non-toxic to the female reproductive system. We also provide evidence that the effect of BRD0705 during in vivo treatment was restricted to its effect on sperm during their passage through the epididymis. Finally, understanding the phosphoproteomic consequences attributable to GSK3α signalling in sperm sheds light on the essential role of this kinase in regulating motility and fertilization. We provide clarity on the possible downstream pathways affected by the inhibition of GSK3α by BRD0705. This insight is essential for the possible use of GSK3α as a male contraceptive target. Together, these experiments provide an in-depth assessment of the physiological, cellular, and molecular consequences of GSK3α inhibition in the male reproductive system.
Materials and methods
Ethics statement
All experimental procedures were performed with C57BL/6J wild-type (Gsk3a+/+) and heterozygous (Gsk3a+/−) male mice. For fertility experiments, C57BL/6J (Gsk3a+/+) female mice were also utilized. Mice were housed at the Central Animal Research Facility, Manipal Academy of Higher Education. Handling was preapproved by the Institutional Animal Ethics Committee (IAEC) of Kasturba Medical College, Manipal, India (Approval #IAEC/KMC/43/2023). The wild-type (Gsk3a+/+) and heterozygous (Gsk3a+/−) male mice were derived from the Gsk3a global knockout mouse line, which was generated at InStem, Bangalore, Karnataka, India, by the method described earlier (Choudhari et al., 2026). The animals were kept under controlled conditions at 23 ± 2°C, with a 12-h light/dark cycle, and were provided with a continuous supply of food and water.
Formulation of BRD0705
The average body weight of 8- to 10-week-old C57BL/6J male mice was around 25 g. The intraperitoneal injections of BRD0705 were given at 20 mg/kg body weight at a 200 µl dosage volume per mouse. This contained 10% of the DMSO (Dimethyl sulfoxide)-solubilized BRD0705 dissolved in the vehicle corn oil. All control male mice received a vehicle consisting of only 10% DMSO in corn oil. In brief, 30 mg of BRD0705 was solubilized in DMSO, and 10 aliquots of 100 µl were stored at −20°C. This dose corresponds to 20 mg/kg of BRD0705, considering the average mouse weight of ∼25 g. The solution was thoroughly mixed before use to ensure even distribution of the drug.
In vivo fertility experiments
BRD0705 treatment in Gsk3a+/− male mice mated with superovulated females
The heterozygous (Gsk3a+/−) mice were obtained by mating either Gsk3a+/+ or Gsk3a+/− males with Gsk3a+/− or Gsk3a−/− females, and their genotypes were confirmed by PCR using tissue DNA taken by ear punches. Ten fertile Gsk3a+/− male mice, aged ∼3 months, were divided equally into treatment and control groups. The treatment group was given the BRD0705 inhibitor (20 mg/kg) intraperitoneally (i.p.) for 10 days. The control group received a dose of 10% DMSO in corn oil administered i.p. for 10 days. After the course of the injections, each male mouse was allowed to mate with two wild-type (Gsk3a+/+) females each. Each Gsk3a+/+ female was superovulated prior to the mating setup using PMSG (Pregnant Mare’s Serum Gonadotropin; 100 IU; ProSpec, East Brunswick, NJ, USA; Cat. No. # HOR-272), followed by HCG (50 IU; Bharat Serums and Vaccines, Maharashtra, India), 46 h after. Any pregnant females during this course were separated. Litter size and day of birth, post-conception, were recorded. The study was terminated 45 days after the mating setup.
BRD0705 treatment in Gsk3a+/− male mice—sperm parameters
Six Gsk3a+/− male mice were divided into control and treatment groups and were administered i.p. 20 mg/kg body weight of BRD0705 for 10 days. The control group received a dose of 10% DMSO in corn oil. The testis, cauda epididymis, and vas deferens from each mouse were collected in separate tubes. The spermatozoa were squeezed out from the vas deferens, and the cauda epididymis was nicked. Spermatozoa were allowed to swim out in the Human Tubal Fluid (HTF: NaCl: 101.6 mM, KCl: 4.69 mM, MgSO4: 0.20 mM, KH2PO4: 0.37 mM, CaCl2: 2.04 mM, NaHCO3: 25 mM, Glucose: 2.78 mM, Na-Pyruvate: 0.33 mM, Na-Lactate: 21.4 mM, BSA: 5 mg/ml; adjusted to pH 7.4) media-containing petri dish and incubated at 37°C for 20 min. After removing tissue debris, the sperm suspension from each sample was mixed and pipetted into fresh tubes; 10 μl of each sample was diluted in 90 μl of ultrapure water, and sperm count and morphology imaging were performed immediately. Sperm motility was analysed with a computer-assisted sperm motility analyser (software version 1.17.2 HT CASA II; Hamilton Thorne Biosciences). Five microlitres of diluted sperm was loaded into a 100-lm Leja chamber slide, prewarmed to 37°C. For each chamber, five random fields were analysed as described earlier (Dey et al., 2020). The CASA settings for analysis were as follows: 90 frames at 60 frames/s, minimum contrast was set to 30, default cell-size of 13 pixels, minimum cell size at 4 pixels, static cell intensity was 60, high size gate of 2.26, low size gate of 0.17, low and high intensity gate of 0.35 and 1.84, respectively, minimum VAP (average path velocity: VAP is the average velocity of a sperm cell measured along its smoothed or mean trajectory, expressed in µm/s) of 50 μm/s and a VSL (straight-line velocity: VSL is the average velocity of a sperm cell measured as the straight-line distance between its initial and final positions over time, expressed in µm/s) cut off of μm/s.
BRD0705 treatment in Gsk3a+/− male mice—phosphoproteomic analysis
Four Gsk3a+/− mice, ∼3 months old, were divided into treatment and control groups. The cauda epididymis and vas deferens were collected. The caudal part was nicked, and sperm were squeezed out into separate Petri dishes containing HTF medium (pH 7.4) without NaHCO3 and CaCl2. The dishes were incubated at 37°C for 15 min to allow the sperm to swim out. The sperm suspension was centrifuged at 700× g for 5 min at 4°C. The pellet was subjected to cell lysis. To each sample, 150 μl of lysis buffer [20 mM HEPES with 8 M Urea supplemented with 1× Protease Inhibitor Cocktail (Roche, Basel, Switzerland; Cat. No. # 11836170001) and 1× PhosSTOP (Roche; Cat. No. # 04906837001)] was added. The samples were vortexed for even mixing and sonicated under the following conditions: 10 s, three times, at 40% amplitude with a 10-s pause between runs. They were then centrifuged at 16 000 × g for 10 min at room temperature. Supernatants were transferred to fresh tubes and subjected to BCA (Bicinchoninic acid) using the Pierce BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA; Cat No.# 23225). After protein estimation, the samples were stored at −80°C for 6 h and then lyophilized for further processing at the Cleveland Clinic, Cleveland, OH, USA.
For phosphoproteomic profiling of mouse sperm, lyophilized samples were first resuspended in urea lysis buffer [20 mM HEPES, 9 M urea, 1× HALT protease/phosphatase inhibitor, pH 8.0 (Thermo Fisher Scientific, Cat No.#78442)]. Protein concentrations were measured using the Pierce BCA assay. Two pooled samples underwent in-solution digestion: proteins were reduced with DTT, alkylated with IAA, and digested overnight with trypsin (Promega, Madison, WI, USA; Cat No. # V5113). Digestion was stopped with trifluoroacetic acid (0.5–1% final), and peptides were desalted using Sep-Pak® C18 cartridges. From each digest, 400 μg of peptide material was dried and subjected to Fe-NTA-based phosphopeptide enrichment (Thermo Fisher Scientific; Cat No. # A32992). Enriched peptides were then dried, reconstituted in 25 μl of 0.1% formic acid, and clarified using a 0.22 μm Ultrafree-MC centrifugal filter prior to LC-MS/MS (Liquid Chromatography-Tandem Mass Spectrometry) analysis.
Mass spectrometric analyses were performed on a Thermo Fisher Scientific Exploris 480 coupled to a Vanquish Neo UHPLC system. Peptides were separated on a 50 cm × 75 μm EasySpray PepMap NEO C18 column (2 μm, 100 Å) and eluted with an acetonitrile/0.1% formic acid gradient at 0.3 μl/min. Five microliters of each sample were injected. Gene Ontology (GO) for phosphoproteomic analysis was performed using SR Plot (Tang et al., 2023).
BRD0705 treatment in Gsk3a+/+ male mice to assess long-term contraceptive reversal
Eighteen Gsk3a+/+ male mice, aged around 3 months, were divided equally into treatment and control groups. After the course of 10-day injections, each male mouse was allowed to mate with two Gsk3a+/+ females. Pregnancy was monitored daily, and any birth was recorded. The pups were removed from the cages at birth, and the breeding pair was retained. After the 45-day time point, five male mice were randomly selected from each group for extended monitoring. Breeding and fertility were monitored by recording litter size and date of birth for 90 days to check the withdrawal of BRD0705 from the male reproductive system.
BRD0705 treatment in Gsk3a+/+ male mice–embryo count and foetal resorption
Ten Gsk3a+/+ mice, aged 3 months, were divided into treatment and control groups. To optimize natural mating conditions without superovulation and to exclude female-related fertility confounders, males were separated into individual cages 3 days prior to the start of pairing. One day before the final injection, males were briefly removed from their cages, and a single Gsk3a+/+ female was introduced into each of the same cages. This exposure ensured that females were housed in cages previously inhabited by males. This allows pheromonal cues to potentially induce oestrous cycling. After the final treatment (Day 10), each male was reintroduced into its original cage and paired with that female for natural mating. On Day 12, post-mating, which is around the midpoint of the typical female mouse’s gestation period, the females from both groups were sacrificed for embryo count and to check any signs of foetal resorption in the uterus.
BRD0705 treatment in Gsk3a+/+ male mice–gene expression and histology
The testes from three control and three BRD0705-treated wild-type mice were collected in separate tubes. Testes analysed for histology were preserved in 10% neutral buffered formalin, and H&E slides were prepared. The remaining testis, still attached to the caput epididymis, was collected in Krebs–Ringer Buffer (KRB), without collagenase. A precise nick was made in the region between the caput epididymis and the testis connective region to expose the rete testis. Gentle pressure was applied to expel the spermatozoa. This sperm suspension was then collected and passed through a 70 μm cell strainer to remove any tissue debris and large cell aggregates. The rete testicular sperm was collected and subjected to homogenization in RNA Isoplus (Takara, Shiga, Japan; Cat. No. # 9108). To each sample, chloroform was added and incubated at RT for 7 min, followed by centrifugation at 4°C for 15 min at 12 000 × g. The aqueous layer was collected, and an equal amount of isopropyl alcohol was added. The samples were centrifuged at 4°C for 10 min at 12 000 × g. The pellets were then broken, and RNA was precipitated in 75% ethanol twice with centrifugation at 4°C for 5 min at 12 000 × g. After air drying, the pellet was dissolved in Diethyl pyrocarbonate H2O. After quantification of the isolated RNA, 200 ng of RNA was used to prepare cDNA using the Takara PrimeScript RT Reagent kit [Perfect Real Time] (Cat #RR037A). Quantitative PCR was performed to check for the basal level expressions of the following genes: Actb (Forward primer: 5′-CATTGCTGACAGGATGCAGAAGG-3′/Reverse primer: 5′-TGCTGGAAGGTGGACAGTGAGG-3′), Gapdh (Forward primer: 5′-CCCAGCTTAGGTTCATCAGG-3′/Reverse primer: 5′-TGATGGCAACAATCTCCACT-3′), Prm1 (Forward primer: 5′-ATGGCCAGATACCGATGCTG-3′/Reverse primer: 5′-CTAGTATTTTTTACACCTTATGG-3′), Sycp2 (Forward primer: 5′-GACACTGAAACCGAATGTGGA-3′/Reverse primer: 5′-TGTGGGTCTTGGTTGTCCTTT-3′), and Akap4 (Forward primer: 5′-CAGAGGCGTGTGCAAGGTAGAT-3′/Reverse primer: 5′-AACTCGCCTTCTGACCTGGAAC-3′).
BRD0705 treatment in Gsk3a+/+ male mice—HPLC analysis
BRD0705 standards were readily soluble in a 70:30 acetonitrile:water (ACN:H2O) mixture. The collected tissues: liver, testis, and epididymis from two BRD0705-treated and control mice for each day were minced and extracted in the same 70:30 ACN:H2O solvent. This was followed by centrifugation at 16 000 × g for 15 min at 4°C. The supernatants were transferred to a fresh tube and filtered using a 0.22 μm filter. Extracts were adjusted to 200 mg/ml and analysed on an Agilent 1290 Infinity II LC system. The column used was InfinityLab Poroshell 120 Aq-C18 2.1 × 150 mm, 2.7 µm (Agilent, Santa Clara, CA, USA; Cat No. 693775-742); 5 µl of each sample was run at 0.4 ml/min using a water (A)/acetonitrile (B) gradient (30:70) with UV monitoring at 330 nm. Blank-subtracted chromatograms were compared to BRD0705 standards and untreated controls. To quantify BRD0705 levels across tissues, HPLC measurements were normalized to protein content. Protein concentration of each tissue extract was determined using the BCA assay. Based on the measured protein concentration, the amount of protein present in the injected sample volume (5 µl) was calculated. BRD0705 concentrations obtained from HPLC peak area analysis were then normalized to a standard protein input of 100 µg by applying a scaling factor from the protein content of each sample. This allowed expression of BRD0705 levels as ng/100 µg of protein, enabling direct comparison across tissues.
In vitro BRD0705 treatment of sperm and spermatids
Gsk3a+/+ male mice were sacrificed, and the testes, caput, and caudal epididymis were collected. The testis was collected in KRB, and the caput and cauda were collected in HTF media without NaHCO3 and CaCl2. Male mouse testes were collected in KRB buffer and washed in DPBS (Dulbecco’s Phosphate Buffered Saline) containing 1% antibiotics. A nick was made near the rete testis, and the blood was allowed to drain out. The testis was detunicated, and the seminiferous tubules were kept for digestion in a Petri dish containing 0.5 mg/ml collagenase II in KRB at 32°C for 1 h. The tissue was then digested using an enzyme mix (Trypsin-EDTA, 1× KRB, DNase I, 2.5 mg/ml Hyaluronidase) and incubated at 32°C for 16 min; 100 µl of this was collected as the input. Using a 70 µm mesh, this input was strained, followed by filtration through a 40 µm tip filter. The observed cells were microscopically confirmed as spermatids. These spermatids were divided into two groups: control and treatment and were cultured in DMEM/F-12 media for 24 h in a 37°C CO2 incubator. This was repeated for 6 h of incubation. The treatment group was incubated with 40 μM of BRD0705. Isolation of sperm from the caput and cauda epididymis was performed as mentioned earlier. The sperm from each was pooled and divided into control and treatment groups. The treated sperm received 40 μM of BRD0705 and were incubated for 2 h at 37°C in a CO2 incubator. In vitro treatment of caudal spermatozoa was repeated at different concentrations of BRD0705 as well as at different time points.
Protein extraction was performed using RIPA lysis buffer (Tris-HCl: 50 mM pH 7.4, NaCl, 150 mM, 0.25% deoxycholic acid, 1% NP-40, and 1 mM EDTA, supplemented with 10 mM benzamidine HCl, 1 mM PMSF, 1 mM TPCK, and 1 mM activated sodium orthovanadate). Total protein concentration was estimated using the BCA assay. The same concentration of protein from all groups was loaded into SDS-PAGE. This was followed by wet transfer onto a PVDF (Polyvinylidene fluoride) membrane (porosity: 0.45 μm). The membrane was blocked with 5% bovine serum albumin. The blot was incubated with a GSK3alpha/beta phosphotyrosine antibody, 1:1000 dilution (Invitrogen, Waltham, MA, USA; Cat. No. # 702230) overnight, followed by a rabbit secondary antibody at 1:6000 dilution. The blot was developed and visualized using enhanced chemiluminescence. The blot was reprobed with a GSK3alpha/beta monoclonal antibody (Invitrogen; Cat. No. # 44-610) overnight.
Statistical analysis
Statistical analyses were conducted utilizing GraphPad Prism 9.0 (GraphPad Prism version 9.0; GraphPad Software, Boston, MA, USA; https://www.graphpad.com/) through unpaired Student’s t-tests; significance levels were determined based on a minimum P-value < 0.05 in all instances. Data are presented as mean ± SD. The number of biological replicates and the statistical significance for each experiment are provided in the figure legends.
Results
BRD0705 treatment in Gsk3a+/− males delays parturition and reduces litter size in superovulated females mated with them
Wild-type females were mated with control and BRD0705-treated male mice post 10 days of injections. The females mated with the control group males gave birth from Days 18 to 30 post-mating setup. The females that were mated with the BRD0705-treated males gave birth from Days 23 to 37 post-mating (Fig. 1A). The females mated with the control mice gave birth on an average of 23 days post-mating, and the females mated with the BRD0705-treated mice gave birth 33 days post-mating, leaving a 10-day delay between the two groups (Fig. 1A).
Figure 1.
BRD0705 treatment in Gsk3a+/− males delays delivery and reduces litter size in mated females. (A) Delivery dates of females mated with control and BRD0705-treated Gsk3a+/− male mice show an ∼10-day delay in the treatment group (n = 5 males in each group, n = 10 females in each group; two females were mated with each male; unpaired t-test, ****P = 0.000002). (B) Twenty-three days post-mating, following the usual gestation period, 7 of the 10 female mice mated with the control group have given birth. No births are seen from females mated with BRD0705-treated males (unpaired t-test, ***P = 0.0005). (C) Forty-five days post-mating, females mated with the treatment mice had given birth, with a mean litter size decreased by half; (unpaired t-test, *P = 0.0128). In all cases, data have been represented as mean ± SD.
After 23 days post-mating, seven females mated with the control male mice had given birth to a healthy litter size. None of the females mated with the BRD0705-treated male mice had given birth (Fig. 1B). Following 45 days post-mating, a total of 9 females from the control group had given birth to an average litter count of 10 pups. At 45 days post-mating, eight females from the treatment group had given birth to an average litter count of five pups, indicating a gradual recovery in fertility (Fig. 1C). Two females from the treatment group did not give birth (Fig. 1C) and neither showed any signs of implantation in the horn of the uterus (data not shown).
While most analyses included eight or more mice per group, some experiments were conducted with five mice per group because statistically significant differences were observed at this sample size. In accordance with the ethical principle of reducing animal use, additional animals were not included once sufficient statistical power was achieved. Nevertheless, larger cohorts and extended fertility recovery studies have been performed in wild-type mice to further strengthen the assessment of fertility restoration following BRD0705 treatment, as described in this article.
Sperm count and morphology
To investigate the basis of BRD0705-mediated suppression of male fertility in Gsk3a+/− male mice, we examined sperm morphology, count, and motility. Sperm collected from the cauda epididymis of the BRD0705-treated Gsk3a+/− males showed more morphological abnormalities than those from controls. The defects observed in the treated group included several distinct categories, each representing a disruption of a specific aspect of sperm maturation or function (Fig. 2A and B). Head defects included small, amorphous, and bent morphologies. Defects in the midpiece region were also observed, including coiling and bending in the centre region, and midpieces with breaks or cuts. Many of the spermatozoa also presented tails with coiling throughout the midpiece and principal piece, indicating a more pronounced morphological abnormality. In addition, BRD0705-treated males produced sperm with elongated and thinning tails, coiled tails, and multiple simultaneous abnormalities.
Figure 2.
Sperm morphology, count, and motility in control vs in vivo BRD0705-treated Gsk3a+/− males. (A) The morphology of in vivo control Gsk3a+/− sperm shows normal morphology with intact head, one midpiece, and principal piece (n = 3 in control group). (B) The morphology of in vivo BRD0705-treated Gsk3a+/− sperms shows various types of morphological defects (indicated by white arrows) (n = 3 in treatment group). The percentage of each abnormal morphology type is higher in vivo BRD0705-treated sperm than in control. Data analysis of sperm morphology was performed from three biological replicates (control: 157 sperm analysed in total, treatment: 151 sperm analysed in total). Unpaired t-test: long and thinning tail ***P = 0.0005, coiled tail *P = 0.0280, head defects *P = 0.0135, midpiece defects **P = 0.0060, and multiple defects **P = 0.0053. (C) Sperm count between control and treated mice remains the same, suggesting that the inhibition of GSK3α is significant in epididymal maturation (n = 4 in each group; unpaired t-test: ns P = 0.9459). (D) A decrease in sperm kinematic parameters is seen in sperm of male mice treated with BRD0705 (VCL, VAP, and VSL was analysed using the CASA software (n = 5 for each group); ALH was calculated manually by observing individual sperm from control and treatment (n = 12 in each group). Unpaired t-test: VSL ****P = 0.000067, VCL ***P = 0.0002, ALH ****P = 0.000001, VAP ***P = 0.0001). In all cases, data have been represented as mean ± SD. VCL, curvilinear velocity; VAP, average path velocity; VSL, straight-line velocity; ALH, amplitude of lateral head displacement.
Despite these pronounced morphological alterations, caudal sperm counts did not differ between control and treated groups, indicating that BRD0705 does not reduce sperm production (Fig. 2C).
Treatment with BRD0705 significantly reduced multiple sperm motility parameters (Fig 2D). VSL decreased in the BRD0705-treated group (∼33 µm/s vs ∼55 µm/s in the control). Similarly, curvilinear velocity (VCL) was significantly reduced following treatment (∼135 µm/s vs ∼178 µm/s in controls). The amplitude of lateral head displacement (ALH) was also drastically lower in the treated group (∼10 µm) compared to controls (∼15 µm), indicating reduced lateral head movement during sperm progression. In addition, VAP was also significantly decreased in BRD0705-treated samples (∼63 µm/s) relative to controls (∼82 µm/s). Overall, these findings demonstrate that BRD0705 treatment impairs sperm motility, resulting in slower, less vigorous movement.
Phosphoproteomic analysis
Phosphoproteomic analysis was performed to identify downstream substrates and signalling pathways regulated by GSK3α, which are critical for sperm motility, structure, and fertilization. As GSK3α is a serine/threonine kinase, its inhibition is expected to alter phosphorylation patterns across multiple direct protein substrates and other kinases or phosphatases regulated by it. From an initial identification of 2721 peptides, including 2021 phosphopeptides, 1634 differentially phosphorylated peptides were chosen for further analysis (Fig. 3A). The differential phosphorylation analysis was performed using stringent criteria: a P-value < 0.05 and a fold change >2 (either hypo- or hyperphosphorylation) between treated and control samples. Applying these filters, we obtained a refined list of phosphoproteins showing significant regulation in response to BRD0705 (Fig. 3B). We then manually selected unique proteins that are testis- and/or sperm-specific in function or origin, or have previously been observed to play a role in the testis or sperm of the male reproductive system, as per current scientific literature. This shortened list had 28 proteins that were significantly hypophosphorylated and 50 proteins that were hyperphosphorylated. GO Biological Process enrichment analysis of the differentially expressed proteins revealed significant enrichment of pathways associated with ciliary and flagellar motility, as well as germ cell development and differentiation. These findings indicate that the altered proteins are predominantly involved in the development of mature spermatozoa and in regulating sperm tail structure and motility. The cellular component suggests that the differentially phosphorylated proteins are primarily localized to sperm flagellar and motility-related structures such as the fibrous sheath and 9 + 2 axoneme arrangement, supporting a role in sperm movement (Fig. 3C).
Figure 3.
Method of manual selection and identification of the most differentially phosphorylated proteins. (A) This figure shows the method of selection of the most affected/hypophosphorylated proteins having sperm/testis functions. (B) The heat map shows over 50 differentially phosphorylated proteins affected the most by the inhibition of GSK3α via BRD0705. (C) Gene ontology of 28 hypophosphorylated and 50 hyperphosphorylated proteins that were manually selected based on their function in the testis and spermatozoa (C1, C2, C3, T1, T2, T3 represent the technical replicates of n = 2 pooled samples of each group).
To further analyse the significance of the 28 hypophosphorylated proteins, we set three criteria: (i) if the protein was a possible direct substrate of GSK3α or Protein Kinase A (PKA), (ii) if any of these proteins are testis-/sperm-specific in origin and function, and (iii) if they have already been studied for their knockout effect in male fertility. Sixteen such hypophosphorylated proteins were observed to be testis/sperm specific, of which four proteins (AKAP4, NT5C1B, SPATA18, and TRA2B) contained the canonical pre-phosphorylated GSK3α substrate recognition motifs (S/T-X-X-X-pS/pT), suggesting that they are direct downstream targets of GSK3α inhibition by BRD0705 (Table 1—denoted by #). Additionally, two proteins, RGS22 and TMEM190, were also hypophosphorylated and contained PKA consensus phosphorylation motifs (R/K-R/K-X-S/T). This suggests that a few hypophosphorylated proteins were affected through reduced PKA activity (Table 1—denoted by ^). Interestingly, the knockout phenotype of most of these proteins had previously been reported to be indispensable for male fertility. Therefore, the loss of GSK3α activity may have led to the decreased function of such proteins that are heavily involved in regulating motility, fertilization, and overall development of the spermatozoa. We also analysed proteins hyperphosphorylated in BRD0705-treated sperm (Table 2). Twenty of the hyperphosphorylated proteins were testis/sperm specific, and the majority of them already had studies describing their knockout phenotype in male fertility. The manually chosen testis-/sperm-specific (16 hypophosphorylated and 20 hyperphosphorylated) proteins were categorized into three main groups: proteins associated with germ-cell structural formation (Fig. 4A), those associated with sperm motility (Fig. 4B), and those that had functions in fertilization (Fig. 4C).
Table 1.
List of hypophosphorylated proteins having known functions in sperm and testis and their localization in the male reproductive system.
| Protein name | Sequence | Localization | Importance for male fertility as per scientific literature |
|---|---|---|---|
| #TRA2B | HSHS99PMST103RRRHVGN | Spermatocytes | Loss of Tra2b leads to azoospermia due to cell death during the meiotic prophase stages of spermatogenesis (Dalgliesh et al., 2025) |
| #+NT5C1B | SPST71GAPS75VDEPRSR | Connecting piece/midpiece | Mice with deleted Nt5c1b exhibited normal fertility (Song et al., 2020) |
| #SPATA18 | NRRSEALKS196DHRS200TK | Midpiece | Spata18 knockout male mice are fertile in vivo, but their sperm are impaired in vitro due to diminished sperm motility (Panahi et al., 2022) |
| #+AKAP4 | SAVS267KIAS271EMAHEAV | Principal piece | Loss of Akap4 leads to abnormal sperm morphology, motility and infertility (Fang et al., 2019) |
| ^RGS22 | QKR1153K1154AS1156DTEEDKAGK | Spermatids | – |
| ^TMEM190 | HR118K119CS121KLSKTVSSL | Inner acrosomal membrane | Mice with deleted Tmem190 exhibited normal fertility (Nishimura et al., 2011) |
| TMEM232 | LFFPSLDVAPPKT538PVEVDLPR | Spermatids | Deletion of Tmem232 in mice causes male-specific infertility (He et al., 2023) |
| OAZ3 | SRPS12LYSLSYIK | Spermatids | Loss of Oaz3 in male mice results in infertility (Tokuhiro et al., 2009) |
| SPATA32 | HHRS149PQEK | Spermatids | Homozygous Spata32-KO male mice suffer from severe subfertility (Lee et al., 2012) |
| TEX29 | S 104 PTPELIPEPIPE | Acrosome/midpiece | Mice with deleted Tex29 exhibited normal fertility (Chen et al., 2026) |
| CABYR | AVEPKTKIS339IESLK | Spermatids/principal piece | Cabyr-knockout male mice showed subfertility with a defect in sperm motility as well as a significant disorganization in the fibrous sheath (Young et al., 2016) |
| LDHC | GEALDLLHGSLFLS73TPK | Principal piece | The targeted disruption of Ldhc severely impaired fertility in male mice. Ldhc−/− sperm did not acquire hyperactivated motility, characteristic of capacitation (Odet et al., 2008) |
| AKAP3 | TPCERPIT644PPAPK | Principal piece/acrosome | Male mice lacking Akap3 are infertile, with abnormal fibrous sheath structure (Xu et al., 2020) |
| FSIP2 | EKAS5837KETETK | Principal piece/acrosomal head | – |
| PDHA2 | YHGHS294MSDPGISYR | Principal piece/acrosomal head | Pdha2 knockout results in male infertility in mice (Wang et al., 2025a) |
| HK1 | IDKYLY83AMR | Principal piece | Depletion of Hk1s leads to infertility in male mice and reduces sperm glycolytic pathway activity (Tian et al., 2024) |
The table lists the hypophosphorylated proteins that exhibit the GSK3α (#) (S/T-X-X-X-pS/pT) or PKA (R-R/K-X-S/T), (^) consensus motif for substrate binding. Bold and underlined letters are the sites identified as affected by GSK3α inhibition. Only bold letters signify the pre-phosphorylated sites indicating that the identified protein may be a possible direct substrate of GSK3α or PKA. Knockout data for these proteins regarding their effect on male fertility are also provided. Plus (+) represents the proteins that have an identified serine/threonine site that has been previously reported as a phosphosite.
Table 2.
List of hyperphosphorylated proteins that are testis/sperm specific according to origin or function and their effect on male fertility when knocked out.
| Protein name | Localization | Importance for male fertility as per scientific literature |
|---|---|---|
| CCDC105 | Entire length of tail | Mice with deleted Ccdc105 exhibited normal fertility (Nguyen et al., 2024) |
| TSSK1B | Spermatids/midpiece | Targeted deletion of Tssk1 results in infertility due to haploinsufficiency (Xu et al., 2008) |
| TEX43 | Spermatids/entire length of tail | Tex43-KO mice showed reduced sperm count but normal testicular architecture, sperm motility, and fertility (Chen et al., 2026) |
| ACTL7A | Head | Actl7a deficiency led to malformed formation of sperm acrosomes, male infertility, and fertilization failure during IVF (Zhou et al., 2022) |
| FSCB | Principal piece | Mice with deleted Fscb exhibited normal fertility (Sun et al., 2020) |
| HNRNPK | Spermatocytes/spermatids | Deletion of Hnrnpk disturbed the expression of genes involved in male reproductive development, among which the meiosis genes were significantly affected, and Hnrnpk cKO spermatocytes failed to complete the meiotic prophase (Xu et al., 2022) |
| ODF2 | Midpiece/principal piece | Male mice were infertile due to haploinsufficiency caused by the heterozygous deletion of the Odf2 (Ito et al., 2019) |
| CFAP91 | Entire length of tail | Cfap91 knockout mice exhibit impaired sperm flagellum formation and male infertility (Wang et al., 2025b) |
| ODAD4 | Entire length of tail | Ttc25 (Odad4) knockout mice presented with laterality defects (Wallmeier et al., 2016) |
| AKAP1 | Midpiece | Mice with deleted Akap1 exhibited normal fertility (Newhall et al., 2006) |
| PRKAR2A | Midpiece | Mice with deleted Prkar2a exhibited normal fertility (Burton et al., 1999) |
| IZUMO1 | Head | The acrosome-reacted sperm of Izumo1 knockout mice are completely unable to adhere to or fuse with the oocyte plasma membrane (oolemma) (Matsumura et al., 2021) |
| AKAP3 | Spermatids/principal piece | Lack of AKAP3 causes mislocalization of sperm proteins and displacement of PKA subunits in mature sperm (Xu et al., 2020) |
| ROPN1 | Spermatids/principal piece | Ropn1 KO male mice are subfertile (Fiedler et al., 2013) |
| SPACA1 | Anterior acrosome | Spaca1-null sperm exhibit a round-headed morphology, a clinical trait mimicking human globozoospermia (Fujihara et al., 2012) |
| DNAH3 | Spermatids/entire length of tail | Dnah3 knockout male mice are infertile, showing a severe reduction in sperm movement with abnormal mitochondrial structure (Wang et al., 2024) |
| NKAPL | Spermatocytes/spermatids | Depletion of Nkapl causes male infertility in mice by blocking the meiotic exit and downregulating haploid genes (Kang et al., 2025) |
| SPAG8 | Outer acrosomal membrane/midpiece | Mice with deleted Spag8 exhibited normal fertility (Yamamoto et al., 2024) |
| CCDC136 | Acrosome | Mice with deleted Ccdc136 were infertile, due to a severe defect in disrupting acrosome formation (Geng et al., 2016) |
| SPESP1 | Equatorial segment of acrosome | Spesp1 knockout sperm showed a lower fusing ability compared with the wild-type sperm (Fujihara et al., 2010) |
| SLC26A8 | Spermatids/equatorial segment of acrosome | Tat1 null males were sterile due to a complete lack of sperm motility and reduced sperm fertilization potential (Touré et al., 2007) |
Figure 4.
Thirty-six hypophosphorylated and hyperphosphorylated proteins that are testis/sperm specific or are possible substrates of GSK3α or Protein Kinase A (PKA), categorized according to their function. (A) Proteins associated with germ-cell structural development: hyperphosphorylated proteins: TSSK1B (**P = 0.002677), TEX43 (*P = 0.021849), FSCB (***P = 0.000124), HNRNPK (*P = 0.03897), ODAD4 (**P = 0.009793), and SPAG8 (***P = 0.000633); hypophosphorylated proteins: TRA2B (**P = 0.0051), TMEM232 (*P = 0.0312), OAZ3 (**P = 0.0019), SPATA32 (*P = 0.0113), RGS22 (**P = 0.0089), DNALI1 (**P = 0.0017), DNAJC5B (*P = 0.0349), and TEX29 (*P = 0.0117). (B) Proteins associated with sperm motility: hyperphosphorylated proteins: CCDC105 (****P = 0.000004), ODF2 (***P = 0.000271), CFAP91 (****P = 0.000029), AKAP1 (***P = 0.000264), PRKAR2A (****P = 0.000017), ROPN1 (****P = 0.00003), and DNAH3 (****P = 0.000004); hypophosphorylated proteins: SPATA18 (****P = 0.002751), NT5C1B (***P = 0.0008), AKAP4 (*P = 0.0101), FSIP2 (*P = 0.0482), and PDHA2 (**P = 0.0035). (C) Proteins associated with fertilization: hyperphosphorylated proteins: ACTL7A (****P = 0.000055), IZUMO1 (**P = 0.004731), SPACA1 (*P = 0.003894), NKAPL (*P = 0.000105), CCDC136 (**P = 0.00473), SPESP1 (***P = 0.000632), and SLC26A8 (*P = 0.011435); hypophosphorylated proteins: TMEM190 (*P = 0.0307), CBYR (****P = 0.0022), LDHC (*P = 0.0319), and HK1 (**P = 0.0016). For all three graphs, the box-and-whiskers plot for each protein shows the median line in the colour of the box, and the mean is represented by a black line.
We also performed STRING pathway analysis of the 50 manually selected hyperphosphorylated proteins with a function in the testis/sperm. It was observed that these proteins are regulated by multiple kinases, making it difficult to identify a specific kinase regulated by GSK3α. We were unable to establish a direct link between these hyperphosphorylated proteins and GSK3α (Supplementary Fig. S1). Future studies would be needed to identify how GSK3α affects their phosphorylation. Alternatively, it is possible that hyperphosphorylation could be the net result of altered kinase and phosphatase activity. Other hypophosphorylated and hyperphosphorylated proteins that followed the initial criteria of the P-value and fold change cut-off have also been summarized. Many of these proteins also have roles in male fertility established via their knockout (Supplementary Table S1).
In our previous paper (Kabi et al., 2025), we performed a western blot analysis to check the protein expression levels of phosphorylated Hexokinase 1 (HK1) in sperm treated with BRD0705. Further, we observed a significant reduction in HK1 phosphorylation in sperm from the treatment group. It was also previously observed in Gsk3α−/− male mice that the loss of GSK3α impaired sperm hexokinase activity, resulting in low ATP levels (Dey et al., 2018). The reduction of HK1, therefore, is associated with inhibition of GSK3α. In the phosphoproteomic analysis, we also observed that the sperm-specific HK1 is significantly hypophosphorylated in BRD0705-treated sperm, as previously validated (Fig. 4C).
Reversal of fertility in BRD0705-treated wild-type mice
Heterozygous mice provided a sensitized genetic background to detect pharmacological effects; however, validation in wild-type animals is essential to establish translational relevance in humans who have two functional copies of GSK3A. Hence, we examined whether wild-type male mice exhibit the same contraceptive effect as seen in Gsk3a+/− mice. The treated wild-type male mice showed a marked reduction in the total number of offspring (66 pups), especially in the early weeks post-mating. Control mice displayed a steeper cumulative increase in pup numbers (103), while the treated group also exhibited a delayed and reduced rise in the number of offspring (Fig. 5A, left plot). The litter size was also decreased between the control and treatment groups in both the litter sets (Fig. 5A, right plot). At 26 days post-mating, none of the females paired with BRD0705-treated males produced litters. This indicates a potent suppression of fertility immediately following BRD0705 treatment (Fig. 5B, upper left plot). By 45 days post-mating, although litter production in the treated group increased slightly, it remained significantly lower than in the control group (Fig. 5B, upper right plot). By 68 days post-mating, most of the females in the control group had given birth to their second litter (Fig. 5B, lower left plot). There was evidence of a gradual recovery in fertility in the males in the treated group; however, they still sired significantly fewer litters compared to the controls. By 90 days post-mating, there is no significant difference in litter size between control and treated groups, suggesting a substantial recovery of fertility as BRD0705 may be cleared from the male reproductive system (Fig. 5B, lower right plot). Spermatogenesis in mice is completed in ∼34 days. The fertility recovery observed at later time points (68 and 90 days) reflects the passage and epididymal maturation of freshly produced spermatozoa.
Figure 5.
BRD0705 treatment transiently suppresses fertility in males, with gradual recovery following treatment withdrawal. (A) Cumulative number of pups born to control (blue) and BRD0705-treated (brown) mice days post mating (Litter 1: n = 9 male mice in each group, n = 18 females in each group; 2 females per male. Litter 2: n = 5 male mice in each group, n = 5 females; 1 female per male). BRD0705-treated mice showed a reduced total litter count compared to control over a 90-day period, indicating potential effects of BRD0705 treatment on reproductive output. (B) Litter counts from control and BRD0705-treated mice were compared at 26, 45, 68, and 90 days post-mating. Significant reductions in litter count were observed in the BRD0705-treated group post-mating. A gradual recovery in the number of pregnant females can be seen over 90 days, indicating a recovery in fertility (unpaired t-test: 26 days post-mating **P = 0.0015, 45 days post-mating **P = 0.0045, 68 days post-mating *P = 0.0192, 90 days post-mating ns P = 0.3463). In all cases, data have been represented as mean ± SD.
Embryo count and evaluation of foetal resorption
We have established that administering BRD0705 in male mice leads to infertility, but to rule out the possibility of any adverse side effects in the female reproductive tract, we performed a mid-term sacrifice to assess female cofounders and embryo physiology. There was no significant difference in the body weight of male mice before and after BRD0705 treatment. Body weights remained similar in both groups, indicating that BRD0705 treatment did not affect overall health or normal body weight in the male mice (Fig. 6A). Body weights of the females were recorded before mating set-up, as well as at the time of the mid-term sacrifice, to assess weight loss due to the indirect effect of BRD0705 from the male sperm.
Figure 6.
BRD0705 treatment in Gsk3a+/+ males leads to reduced mid-term foetus count without signs of embryonic resorption in mated females. (A) Body weight of male mice pre and post BRD0705 treatment shows no significant difference or overt toxicity caused by the treatment (n = 5; unpaired t-test ns P = 0.1983). (B) An increase in body weight of females mated with control male mice before mating and at mid-term can be attributed to pregnancy (n = 5 males in each group, n = 5 females in each group; one female per male; unpaired t-test ns P = 0.5536). (C) Body weight of females mated with BRD0705-treated male mice before mating and at mid-term remains unchanged (unpaired t-test *P = 0.0174). (D) Twelve days post-mating setup, females from both groups were sacrificed to check the mid-term foetus count and resorption of the embryo. After 12 days, 4 females mated with control males gave an average of 10 foetuses with no sign of resorption. Females mated with treatment males did not show signs of pregnancy/resorption of the embryo. (E) Graphical representation of foetus count between females mated with control and BRD0705 males (unpaired t-test *P = 0.0479). In all cases, data have been represented as mean ± SD.
The increase in weight in females mated with the control group can be attributed to pregnancy (Fig. 6B). There was no significant change in body weight during these two periods in the females mated with the BRD0705-treated male mice, indicating no adverse effect (Fig. 6C). Uterine morphology was assessed at mid-gestation period at Day 12 post-mating (Fig. 6D). Females mated with control males (C1–C5) showed normal implantation sites with multiple developing foetuses. In contrast, females mated with BRD0705-treated males (T1–T5) displayed markedly reduced implantation sites or complete absence of pregnancies, with no evidence of resorption. Quantification of foetal counts further supported this observation (Fig. 6E), with females mated to control males exhibiting normal litter sizes, whereas those paired with BRD0705-treated males showed a significant reduction in the number of foetuses. In the control group, females displayed normal pregnancy outcomes, with clear implantation sites and developing foetuses visible along the uterine horns. In contrast, females paired with BRD0705-treated males did not exhibit signs of resorption within the uterine horns; however, several females showed an absence of implantation sites altogether. This indicates that embryo resorption did not occur, and the rate of successful pregnancy was significantly reduced in the treatment group.
Gene expression and histology
To determine whether GSK3α inhibition affects gene expression at the very end of spermatogenesis before the onset of epididymal maturation, we performed qPCR analysis using RNA isolated from rete testis sperm. There were no expression changes in key marker genes involved in spermatogenesis, head shaping, and tail assembly (Fig. 7A). There was also no difference in the housekeeping genes between the two groups, validating that BRD0705 administration was not interfering with any other foundational genes. Consistently, histological H&E analysis of the testis seminiferous tubules revealed normal organization. All stages of spermatogenesis are present and comparable across control and treated mice (Fig. 7B).
Figure 7.
BRD0705 treatment in Gsk3a+/+ males shows no change in gene expression of key structural proteins and histology. (A) Gene expression remains insignificant between the control and treated mice for genes that are often linked to head and tail development in normal functioning sperm (n = 3; unpaired t-test Actb ns P = 0.9188, Gapdh ns P = 0.0814, Akap4 ns P = 0.4036) (n = 4; unpaired t-test Prm1 ns P = 0.7845, Sycp2 ns P = 0.2269). (B) No changes are seen in the seminiferous tubules, and structural integrity is maintained (n = 3). Histology images were taken at 20×. The individual tubules, enlarged for visual depiction, have been zoomed in by a factor of 1.54. In all cases, data have been represented as mean ± SD.
Determination of BRD0705 levels in testis and epididymis in treated mice
To determine whether BRD0705 was present in reproductive tissues following the in vivo treatment with BRD0705, chromatograms from treated and control mice were compared with the freshly prepared BRD0705 standards (Supplementary Fig. S2A). The retention time of the standards was ∼2.7 min. After 1 day of injections, the liver showed the highest concentration of BRD0705 and very low levels in the testis and epididymis. After 1 day of injection, a higher concentration in the liver is expected, as the liver is the primary site of drug uptake and metabolism. In comparison, the testis and epididymis show lower levels, likely due to limited initial penetration into reproductive tissues and the presence of protective barriers such as the blood–testis barrier (Supplementary Fig. S2B).
Post 5 days of injections, in the treated animals, a small but distinct peak corresponding to BRD0705 was detected in the liver at the expected retention time, indicating a small accumulation of the compound in hepatic tissue (Supplementary Fig. S2C). In contrast, the testis extract from treated mice displayed a very strong and sharply defined peak at around 2.5 min. This demonstrates the accumulation of BRD0705 in the testis either due to systemic circulation or local absorption through the testicular vasculature. Both the liver and testis peaks shifted to ∼2.5 min. This indicates that BRD0705 was being metabolized within these tissues. Further, epididymal extracts from treated males did not exhibit any strong peak at the BRD0705 retention time. This suggests that the compound either does not penetrate the epididymis or is present at levels below the detection threshold. The concentration of BRD0705 was calculated by normalizing to 100 μg of protein per tissue (Supplementary Fig. S2D). None of the control liver, testis, or epididymis extracts showed any signal at this retention time. This confirms that no natural tissue compounds appear at the same retention time as the inhibitor.
In vitro analysis of BRD0705 in sperm and spermatids
Western blot analysis was performed to assess the effect of BRD0705 on the auto-activating Tyr279/216 phosphorylation status of GSK3α and GSK3β in various late developmental stages. Spermatids cultured with 40 μM BRD0705 for 24 h showed a marked decrease in the phosphorylation of GSK3α (∼51 kDa) compared to DMSO-treated controls. This decrease is not observed in spermatids incubated with BRD0705 for 6 h. In contrast, when only the whole protein levels of GSK3α and GSK3β were examined, no difference was observed in their expression upon the inhibitor treatments (Fig. 8-I). Further, this decrease in tyrosine phosphorylation of GSK3α is also observed in rete testicular sperm (Fig. 8-II). These results indicate that BRD0705 treatment reduces Tyr279 phosphorylation of GSK3α without affecting its total protein level.
Figure 8.
Differential regulation of Glycogen Synthase Kinase 3 alpha (GSK3α) tyrosine phosphorylation by BRD0705 during spermiogenesis and epididymal sperm maturation. (I) Isolated spermatids were incubated for 24 and 6 h in vitro with DMSO (control) and 40 µM of BRD0705. Following protein extraction, proteins were run on an SDS-PAGE followed by western blotting. The blot was blocked with 5% bovine serum albumin. A GSK3a/b phosphotyrosine antibody was used for incubation overnight, followed by a rabbit secondary antibody. The blots were developed and visualized using an enhanced chemiluminescence. (II) Rete testicular sperm was isolated from the testis and was incubated in vitro with DMSO (control) and 40 µM of BRD0705. Following protein extraction, the proteins were run on an SDS-PAGE gel and then subjected to western blot analysis. (III) Sperm was isolated from the caput epididymis and was incubated in vitro with DMSO (control) and 40 µM of BRD0705. Following protein extraction, the proteins were run on an SDS-PAGE gel and then subjected to western blot analysis. (IV) Sperm was isolated from the caudal epididymis and was incubated in vitro with DMSO (control) and 40 µM of BRD0705. Following protein extraction, the proteins were run on an SDS-PAGE gel and then subjected to western blot analysis. For all blots, n = 3, a representative image is shown here.
However, the same decrease in GSK3α tyrosine phosphorylation does not occur in the immature spermatozoa of the caput and fully mature sperm cells from the cauda epididymis (Fig. 8-III/8-IV). Caput and caudal spermatozoa, when incubated with BRD0705 in HTF medium for 24 h, lyse and are viable only up to 6 h. The uncropped version(s) of the western blots have been provided in Supplementary Fig. S3.
Discussion
Mating with BRD0705-treated Gsk3a+/− males resulted in a significant reduction in fertility, with fewer females giving birth and smaller litter sizes compared to controls. Additionally, some females showed no signs of implantation. These findings demonstrate that BRD0705-mediated inhibition of GSK3α in male mice results in a pronounced effect on fertility outcomes. This aligns with our previously published data, showing that the use of paralogue-selective GSK3α inhibition, such as with BRD0705, can transiently impair male fertility (Kabi et al., 2025). Contraceptive effect may be induced during BRD0705 treatment, with fertility recovering upon inhibitor withdrawal. The same has been implicated by earlier studies (Bhattacharjee et al., 2018; Dey et al., 2019). The use of PMSG and HCG ensured that each female was in an optimal, consistent physiological state for ovulation and fertilization. By standardizing female reproductive synchronization, any differences observed in pregnancy rates, litter size, or delivery timing could be attributed solely to the functional capacity of the sperm from BRD0705-treated males.
Further, BRD0705 treatment in Gsk3a+/− male mice impaired sperm quality, resulting in increased morphological abnormalities across the head, midpiece, and tail regions. Head abnormalities typically reflect impaired chromatin condensation or acrosomal shaping, both of which are essential for oocyte recognition and fertilization. Defects in the midpiece region indicate disturbances in tubular organization or energy regulation, which are critical for sustained motility and flexibility. In our previous paper (Kabi et al., 2025), we observed that in vivo-treated spermatozoa from the cauda epididymis showed around 50% more abnormal morphologies than the control. This result, therefore, corroborates that finding and highlights the other morphological defects these sperm exhibit. Such morphological defects have also been observed in caudal sperm from Gsk3a knockout mice: ∼30% of the spermatozoa showed bent heads at the head-connecting piece junction, and similar percentages of sperm cells exhibited bent midpieces (hairpin bend at mid- and principal-piece junction) as well; and overall, morphologically normal sperm dropped close to only 40% mark (Bhattacharjee et al., 2018). All of these aberrations are associated with compromised flagellar beating, reduced forward progression, and impaired overall motility.
The significant reduction in the sperm kinetic parameters provides further evidence for the loss of fertility in BRD0705-treated male mice. Parameters, such as VSL, VCL, ALH, and VAP, are indicators of sperm quality and fertilizing potential, as they reflect the ability of spermatozoa to travel through the female reproductive tract. In particular, a significant decrease in ALH indicates reduced lateral oscillation of the sperm head, reduced flagellar beating amplitude, and reduced force generation, all of which are important for sperm hyperactivation (Sansegundo et al., 2022). Lower ALH values may therefore reflect weakened tail activity and compromised sperm propulsion.
Despite clear morphological and motility defects, BRD0705 treatment in these Gsk3a+/− male mice did not affect sperm count, indicating that sperm production or spermatogenesis remained unchanged. This confirms that BRD0705 does not interfere with those transcriptional pathways that govern germ cell development. Instead, sperm obtained from BRD0705-treated males showed reduced motility and increased morphological abnormalities, implicating defects in sperm function rather than formation. This suggests that GSK3α inhibition by BRD0705 does not interfere with the production of sperm in terms of quantity but may work on the post-translational level, especially at the phosphoproteomic level, to disturb essential pathways that lead to altered sperm functional parameters.
Given the pronounced defects in sperm morphology and motility despite unchanged sperm counts, we performed phosphoproteomic analysis to identify key fertility-related proteins and signalling pathways affected by BRD0705 treatment. There were found to be 1634 differentially phosphorylated peptides between the sperm extracts from control and BRD0705-treated Gsk3a+/− male mice. Twenty-eight hypophosphorylated and 50 hyperphosphorylated proteins were identified that had a role in the testis and/or spermatozoa. Their GO analysis showed that most of these differentially phosphorylated proteins were associated with germ cell development, motility, and structure. The enrichment of sperm flagellum and cilium-associated cellular components is consistent with the observed reduction in sperm motility, suggesting that dysregulation of proteins localized to the axoneme, fibrous sheath, and other flagellar substructures may underlie the motility and morphological defects observed in the BRD0705-treated male mice.
Among the 28 hypophosphorylated proteins manually selected for their established roles in the testis or sperm, 15 have been demonstrated to be essential for male fertility, as their gene knockouts result in compromised spermatogenesis or suboptimal sperm motility and morphology. The altered phosphorylation of these proteins resulting from BRD0705 may modify their physiological roles. Interestingly, four proteins may be direct downstream targets of GSK3α (AKAP4, NT5C1B, SPATA18, and TRA2B). Further, the specific serine/threonine residue hypophosphorylated was examined using PhosphositePlus, and it was found that both AKAP4 (Ser267) and NT5C1B (Thr71) were already reported to be phosphorylated at this site (Ficarro et al., 2003; Hoffert et al., 2006). This further suggests that these sites could be the actual motifs through which GSK3α binds to these two proteins and further emphasises that the inhibition of GSK3α via BRD0705 has directly affected the optimal functioning of AKAP4 and NT5C1B.
AKAP4 is a scaffold protein essential for the structure and function of the fibrous sheath (Nixon et al., 2019). AKAP4 localizes the phosphorylation of target proteins that are usually involved in regulating glycolytic enzymes. This signal transduction role is essential for ATP production and flagellar movement. It has been observed earlier that the loss of Akap4 results in a reduction in flagellar length and the loss of proteins such as glyceraldehyde-3-phosphate dehydrogenase (GAPDHS), which contributes to male infertility (Brown et al., 2003). The role of NT5C1B in sperm is not fully known. However, this protein may be involved in energy charge regulation and is known to be both serine/threonine- and tyrosine-phosphorylated. SPATA18 plays a crucial role in sperm mitochondrial quality control and is linked to sperm motility. It has been observed that sperm motility is directly associated with SPATA18 expression; lower SPATA18 levels are associated with asthenozoospermia (Panahi et al., 2022). A decrease in SPATA18 expression impairs the capacity of sperm mitochondria to maintain energy production, resulting in decreased motility and contributing to impaired fertility (Bornstein et al., 2011).
It has previously been shown that GSK3 phosphorylates TRA2B at 11 sites, allowing TRA2B to regulate alternative splicing (Liu and Klein, 2018). It was also identified by Dey et al. (2020) that SLC16A7 (MCT2) and Basigin (BSG), among other proteins, exhibited altered phosphorylation in calcineurin-deficient sperm, and they proposed them as potential downstream targets of the calcineurin–GSK3α signalling pathway. Given the established interaction between Basigin and MCT2 in spermatozoa, these findings suggest that GSK3α may regulate sperm energy metabolism and function through modulation of the Basigin–MCT2 complex. Thus, it can be inferred that the inhibition of GSKα via BRD0705 has directly or indirectly led to a disruption of these key fertility regulatory proteins, resulting in infertility.
Interestingly, it has been found that GSK3α phosphorylates and inhibits phosphodiesterase PDE4, which is expected to lead to increased levels of cAMP (Dey et al., 2018). Higher concentrations of cAMP cause the release of PKA active subunits from inhibitory subunits. This could lead to more widespread phosphorylation of target proteins involved in fertility. Therefore, the inhibition of GSK3α may reduce PDE4 phosphorylation and relieve inhibition on PDE4, leading to degradation of cAMP molecules and decreased PKA activity. Two of the hypophosphorylated proteins, RGS22 and TMEM190, contain PKA motif-binding sites, suggesting that they may be potential PKA substrates. RGS22 and TMEM190 both have functional importance in spermatozoa. RGS22 was shown to interact directly with G-protein α subunits GNA12, GNA13, and GNA11, and it may help mediate the translocation of GNA13 from the cytoplasm to the nucleus during the elongation and maturation of spermatids (Hu et al., 2008). TMEM190 is a testis-specific, small transmembrane protein found in the inner acrosomal membrane of mouse sperm. It co-localizes with IZUMO1 (a crucial protein for sperm–egg fusion), both before and after the acrosome reaction (Nishimura et al., 2011). How increased or decreased PKA phosphorylation affects sperm function remains to be determined.
The long-term fertility assessment showed that the contraceptive effect induced by BRD0705 was reversible following withdrawal of treatment. At 26 days post-mating, treated males failed to sire litters, indicating a near-complete suppression of fertility. Although litter size remained significantly reduced at 45 days post-mating, partial recovery was evident, suggesting that fertility restoration had begun following inhibitor withdrawal. By 90 days post-mating, litter sizes in the treatment group were no longer significantly different from those of controls, indicating substantial recovery of reproductive capacity. Since sperm present in the epididymis during BRD0705 treatment may have undergone impaired maturation, reduced fertility persisted for some time even after BRD0705 withdrawal. The gradual recovery of fertility likely occurred as these affected sperm were replaced by newly produced sperm that developed and matured without exposure to the inhibitor. These findings suggest that BRD0705 mainly affects epididymal sperm maturation and the development of normal sperm motility, rather than causing permanent damage to spermatogenesis.
It was also important to perform the long-term fertility study in wild-type mice to reflect the physiological condition in humans, who possess two functional copies of GSK3α. Demonstrating similar, reversible effects in wild-type animals strengthens the translational relevance of BRD0705 as a potential contraceptive, ensuring that its efficiency is not limited.
In men, short-term abstinence (one day) is associated with improved sperm motility and reduced DNA damage (Blackwell and Zaneveld, 1992). The epididymis is less clearly divided and has a smaller storage region, so sperm pass through it more quickly (Jonge et al., 2004). Consequently, some andrologists question the importance of epididymal sperm maturation itself in humans. Although longer abstinence periods (more than 3–4 days) result in higher semen volume and sperm concentration, they also result in poorer sperm quality (Sullivan and Mieusset, 2016). There is reduced sperm motility, lower vitality, diminished mitochondrial function, and increased DNA fragmentation. This suggests that prolonged storage of sperm in the epididymis may be harmful (Comar et al., 2017). In mice, the epididymis is clearly divided into distinct regions, providing a long pathway for sperm maturation and storage and serving as an indispensable transitional stage towards gaining competence to fertilize eggs (James et al., 2020).
It is therefore important to consider such species-specific differences between rodents and humans. While these variations may impact the efficacy of pharmacological interventions on sperm maturation and function across different species, it is important to recognize that GSK3α inhibition occurs within the testis, with its effects becoming evident during epididymal maturation. Interestingly, Bhattacharjee et al. (2015) showed that the morphological abnormality of sperm cells in Gsk3a knockout mice is acquired during their transit from the rete testis to the caput, and its effects extend only during epididymal maturation. However, further studies in humans will be required to evaluate the extent to which these findings translate to human physiology.
Importantly, targeting epididymal sperm maturation as a potential contraceptive target may minimize the risk of genetic damage to the germ cells, as it avoids direct interference with the spermatogenesis process. Processes such as DNA replication, recombination, and chromatin packaging and maintenance of optimal transcription levels during spermatogenesis are essential, and disruptions in them can lead to permanent defects. Hence, its potential efficacy as a male contraceptive in humans should be largely indisputable.
Recently, several non-hormonal small-molecule strategies have been explored as male contraceptives, targeting spermatogenesis, sperm function, or epididymal signalling. Retinoic acid pathway inhibitors, such as WIN 18,446, effectively suppress spermatogenesis and have been trialled in men (Paik et al., 2014). Though they performed as contraceptives, many males faced severe side reactions with alcohol consumption due to off-target effects on the ALDH2 enzyme (Chen et al., 2018). Several ion-channel and transporter regulators have shown contraceptive potential, such as CatSper/SLO3 pathway modulators (e.g. RU1968, nifedipine) and CFTR/Cl− blockers (e.g. AF-2785, CFTRinh-172, DPC, DIDS). While these molecules affect sperm motility or capacitation, whether they can be used in humans remains unknown (Yunaini and Ari Pujianto, 2023). S-α-chlorohydrin, a GAPDHS inhibitor, disrupts spermatogenesis and sperm function by blocking the cAMP–PKA pathway in spermatozoa, but concerns about toxicity have limited its development as a clinical contraceptive. Compounds like CDB-4022 act on Sertoli cells to disrupt the blood–testis barrier, causing temporary infertility at low doses but possibly leading to testicular damage and toxicity at higher doses (Hild et al., 2001).
Soluble adenylyl cyclase (sAC/ADCY10), essential for sperm capacitation, has been targeted by compounds such as catechol oestrogens, KH7, and the allosteric inhibitor LRE1 (Ramos-Espiritu et al., 2016). These clearly demonstrated that sAC inhibition blocks capacitation, motility, and acrosome reaction in mouse and human sperm. However, sAC inhibitors like KH7 are not highly selective for sperm sAC and can inhibit sAC activity in other tissues. Because sAC is expressed in multiple organs, sAC inhibition can raise the risk of off-target effects (Ramos-Espiritu et al., 2016b; Balbach et al., 2020). Moreover, sAC is the only class of cytosolic form of adenylyl cyclase, whereas the other nine types are all transmembrane in structure (Li et al., 2024). Due to this non-redundancy of sAC, it may not be an ideal target for contraceptive development. Targeting it for contraception (e.g. male non-hormonal options) risks side effects in non-reproductive tissues where sAC regulates pH, metabolism, and other processes, making it less ideal. Small molecules, such as adjudin, an indazole derivative, have been shown to impair chloride-dependent signalling in sperm. This results in decreased sperm hyperactivation, acrosome reaction, and lower intracellular cAMP levels (Mok et al., 2011; Li et al., 2013). While these studies show the pharmacological disruption of sperm function, many such small-molecule candidates lack in vivo validation and may have off-target effects. For example, TSSK2 has recently been shown to be targeted by novel chemically synthesized molecules in vitro (Holdaway et al., 2026).
Compared to the aforementioned non-hormonal male contraceptive molecules, BRD0705 offers more advantages. Many small-molecule approaches mainly act on capacitation or fertilization-associated pathways in ejaculated or activated sperm. This often requires the continuous presence of drugs and is associated with the female reproductive tract. In contrast, BRD0705 acts during the late stages of spermatogenesis and epididymal maturation, a critical developmental phase of spermatozoa. Importantly, BRD0705 exhibits paralogue specificity and selectively inhibits GSK3α, making it a safer option. The functional effect of BRD0705 is mediated at the phospho-proteomic level. The restoration of fertility following drug withdrawal further highlights its reversibility. The in vivo fertility assays, wild-type validation, stage-specific in vitro studies, and phosphoproteomic pathway analysis provide deeper insight that is not present in previous studies.
Further, BRD0705 treatment in Gsk3a+/+ males leads to compromised reproductive outcomes, likely by reducing successful fertilization, without involvement of any female factors involved in foetal development. This led to delayed or a lack of pregnancies in the females mated with treated males. No overt signs of systemic toxicity were observed in male mice treated with BRD0705. Body weights remained comparable between control and treated groups throughout the treatment period, and no abnormal behavioural changes were noted. Similarly, females mated with treated males did not exhibit any observable behavioural abnormalities or change in body weight post-mating set-up with BRD0705-treated male mice. Importantly, there was no evidence that BRD0705 exerted a direct adverse effect on the female reproductive tract, ensuring its safety in female reproductive physiology and the foetus. No signs of foetal resorption were observed. As observed in earlier results, BRD0705 does not interfere with transcriptional pathways governing germ cell development; instead, it alters sperm function and motility at the proteomic level. Gene expression analysis also revealed the same, where there were no expression changes in key marker genes involved in spermatogenesis. BRD0705 also does not impair spermatogenic progression or testicular architecture. This observation can be advantageous as a contraceptive that controls sperm function without causing genetic or structural changes in the testis is considered safer.
HPLC was performed to detect and quantify BRD0705 in different tissues, thereby confirming its distribution and accumulation following in vivo treatment. At Day 1 post-injection, BRD0705 was predominantly detected in the liver, with minimal levels in the testis and epididymis. At Day 5, BRD0705 accumulated strongly in the testis, showed low levels in the liver, and was undetectable in the epididymis. These findings align with our in vitro results, indicating that the developmental stages of sperm occurring within the testis are affected by the inhibition of GSK3α via BRD0705. Due to membranous modifications to maturing sperm during their transit in the epididymis, spermatozoa are impenetrable by BRD0705. The absence of BRD0705 in the epididymis suggests that the defects seen in caudal sperm are not caused by drug exposure within the epididymis itself. Instead, these defects may begin slightly earlier, during the late stages of testicular sperm development (spermiogenesis), when the testis is exposed to BRD0705. Alteration of sperm development during spermiogenesis manifests during its transit in the epididymis, where the spermatozoa mature and gain fertilization ability and motility. In the epididymis, spermatozoa with inhibited GSK3α exhibit decreased motility, abnormal morphology, and have significantly hypophosphorylated key fertility proteins. This may be because phosphorylation changes established during spermiogenesis are carried forward as sperm transit through the epididymis. Since mature sperm have very limited transcriptional and translational activity, their function is largely regulated by post-translational modifications such as phosphorylation. These altered phosphorylation patterns may carry on or be further modified during epididymal transit, contributing to impaired sperm function. A thorough pharmacokinetics and pharmacodynamics analysis may be undertaken in the future for BRD0705 distribution in male reproductive organs.
BRD0705 specifically inhibits GSK3α activity through its tyrosine autoactivation site without affecting the overall protein abundance in spermatids and rete testicular spermatozoa. The absence of any change in total GSK3α/β protein levels indicates that the protein is not degraded and its presence is not altered at the genomic level. Because spermatids and rete testicular sperm are the developmental stages at which structural refinement, head shaping, and flagellar assembly begin, disruption of GSK3α signalling at this point would be expected to show morphological defects without altering sperm production or testicular gene expression. Thus, the absence of detectable effects in caput and cauda sperm shows limited epididymal permeability to BRD0705, as also confirmed by the HPLC analysis.
The morphological abnormalities observed in vivo can be best explained by the early interference of BRD0705 in spermatids and rete testicular sperm as they initiate the maturation process. This highlights the stage-specific role and importance of GSK3α during the final phases of spermiogenesis and the phenotypic manifestation of infertility during its transit through the epididymis.
While this study and our previous paper (Kabi et al., 2025) provide detailed insights into morphological and molecular alterations in sperm following GSK3α inhibition, additional functional assays such as capacitation status, acrosome reaction, and fertilization competence would provide further mechanistic insight into how GSK3α inhibition affects sperm function and represent an important direction for future studies.
Conclusion
This study establishes GSK3α as a critical regulator of sperm maturation and male fertility. We demonstrate that the selective inhibition of GSK3α by BRD0705 produces a reversible, non-hormonal, and male-specific contraception. Using mating assays in both Gsk3α heterozygous and wild-type males, BRD0705 treatment consistently resulted in delayed pregnancies, reduced litter sizes, and complete absence of implantation in many females. These findings confirm that the contraceptive effect is due to fertilization failure and not post-implantation complications.
Despite normal sperm counts and unchanged expression of genes, males treated with BRD0705 produced spermatozoa with marked morphological abnormalities and impaired motility, indicating that GSK3α inhibition disrupts sperm function, rather than sperm production. Phosphoproteomic analysis showed four hypophosphorylated proteins to be possible direct substrates of GSK3α and two to be possible direct substrates of PKA (Fig. 9). In vitro assays revealed that BRD0705 effectively suppresses GSK3α tyrosine autophosphorylation in spermatids and rete testicular sperm, but not in mature epididymal spermatozoa. Complementing these findings, HPLC analysis revealed the accumulation of BRD0705 in the testis but not in the epididymis, indicating that the inhibitor acts during spermiogenesis or early maturation.
Figure 9.
Graphical depiction of the importance of identified hypophosphorylated proteins in male fertility. (A) Diagrammatic representation of hypophosphorylated proteins affected by direct GSK3α inhibition by BRD0705. (B) Diagrammatic representation of hypophosphorylated proteins affected indirectly through the suppression of PKA due to the inhibition of GSK3α. Certain elements were created in Biorender, Singh, A. (2026) https://BioRender.com/dokilso.
Importantly, long-term mating studies demonstrated a gradual recovery of fertility after withdrawal of BRD0705. Together, these results demonstrate that BRD0705 induces a potent yet reversible contraceptive effect by disrupting phosphorylation-dependent regulatory pathways.
Overall, this work provides strong mechanistic evidence supporting GSK3α as a promising target for non-hormonal male contraception. We highlight BRD0705 as a paralogue-selective inhibitor, offering reversibility and safety characteristics for future translational development.
Supplementary Material
Author Biography
Dr Souvik Dey: Dr Souvik Dey, PhD, is a Senior Assistant Professor in the Department of Biotherapeutics Research at the Manipal Academy of Higher Education, India. His research focuses on the molecular regulation of spermatogenesis, sperm maturation, and male fertility, with particular emphasis on GSK3α signalling and RNA epitranscriptomics using transgenic mouse models. His laboratory develops innovative diagnostic tools and non-hormonal therapeutic strategies for male contraceptive development. Supported by multiple competitive grants from the Department of Biotechnology, Anusandhan National Research Foundation, and the Indian Council of Medical Research, his work integrates biochemistry, molecular biology, and translational reproductive medicine to advance male reproductive health.
Contributor Information
Aditi Khamamkar, Department of Biotherapeutics Research, Manipal Academy of Higher Education, Manipal, Karnataka, India.
Rounak Roy, Department of Biotherapeutics Research, Manipal Academy of Higher Education, Manipal, Karnataka, India.
Dhanu Anneyplar Shivakumar, Department of Biotherapeutics Research, Manipal Academy of Higher Education, Manipal, Karnataka, India.
Neha Choudhari, Department of Biotherapeutics Research, Manipal Academy of Higher Education, Manipal, Karnataka, India.
Rahul Dutta, Department of Reproductive Science, Kasturba Medical College of Manipal, Manipal Academy of Higher Education, Manipal, Karnataka, India.
Michel Weïwer, Centre for the Development of Therapeutics, Broad Institute, Cambridge, MA, USA.
Satish Kumar Adiga, Department of Reproductive Science, Kasturba Medical College of Manipal, Manipal Academy of Higher Education, Manipal, Karnataka, India.
Srinivasan Vijayaraghavan, Department of Biological Sciences, Kent State University, Kent, OH, USA.
Souvik Dey, Department of Biotherapeutics Research, Manipal Academy of Higher Education, Manipal, Karnataka, India.
Supplementary data
Supplementary data are available at Molecular Human Reproduction online.
Data availability
The phosphoproteomic data have been deposited in the PRIDE database under the project accession ID PXD079474.
Authors’ roles
Experiments and investigation: AK, RR, DAS, and NC. Writing—original draft preparation: AK. Writing—review and editing: SD and SV. Inputs and suggestions: RD, SKA, and MW. Supervision: SD.
Funding
This research was made possible by funding from the Department of Biotechnology, Ministry of Science and Technology, Grant Number: BT/RLF/Re-entry/08/2019, and the Indian Council of Medical Research (ICMR), Government of India, Grant Number: IIRPSG-2024-01-06557, and the National Institutes of Health (NIH) [HD107333 01 (SV)].
Disclosures
The authors declare no conflicts of interest.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The phosphoproteomic data have been deposited in the PRIDE database under the project accession ID PXD079474.