Souvik Dey

Idiopathic male infertility is a rising global concern characterised by ejaculatory defects, absence or low sperm count with abnormal morphology, and poor sperm motility. Deciphering the aetiology of male infertility requires a fundamental understanding of multiple spermatogenic events, including sperm maturation. Glycogen synthase kinase 3 paralog‐α (GSK3α) plays a critical role in sperm maturation, specifically during epididymal motility, capacitation and hyperactivation. Methylation modulates mRNA transport, stability, turnover, and translational efficiency to meet cellular requirements. The mRNA demethylase, fat mass and obesity‐associated protein (FTO), is a target for phosphorylation by GSK3α, suggesting the potential role of this enzyme in male fertility. This study focuses on the high‐affinity spatiotemporal interaction between FTO and GSK3α to delineate the post‐transcriptional modifications in the murine testis. Expression of Gsk3a and Fto increases temporally starting with day 18–20 postpartum testis, coinciding with meiosis I and gradually peaks by day 25–34 with the formation of spermatids and completion of spermatogenesis. Co‐immunoprecipitation of GSK3α and GSK3β with FTO using respective antibodies and super‐resolution microscopy shows a preferential interaction of GSK3α with FTO. Moreover, Gsk3a knockout mice showed significantly low m6A levels in testis, presumably due to enhanced FTO activity. In silico protein–protein docking and molecular dynamics analysis demonstrated an energetically favourable, consensus phosphorylation motif‐dependent high‐affinity interaction between FTO and GSK3α, further validating our observation. A specific missense mutation (Cys326 > Ser) permitted an additional GSK3‐phosphorylation site in FTO, leading to teratozoospermia in a patient. Collectively, this study affirms GSK3α as a spatiotemporal regulator of FTO function inside the mammalian testis.

Background: N6-methyladenosine profiles of mRNA transcripts regulate their translocation from the nucleus to the cytosol, stability, and translational efficiency; hence, they have been implicated in gene expression and disease progression. The m ⁶ A-methylation is widely associated with various cancers and neurological, cardiovascular, and developmental disorders, which demand early diagnosis. A robust m ⁶ A-motif prediction is necessary to enable us to identify the regulatory nucleic acid sequences that determine mRNA fate in normal and diseased conditions. Methods and Results: We have developed a transcript-aware computational pipeline, termed m ⁶ A Functional Index in Transcription (m ⁶ A-FINDiT), that can identify potential m ⁶ A sites on mRNA transcripts, considering molecular intricacies associated with their secondary structure. This tool can separately identify m ⁶ A motifs within the coding sequences as well as in non-translatable regions, i.e., 5’UTR and 3’UTR, of mRNA transcripts. Parallelly, another technique was developed that quantifies specific m ⁶ A methylation motifs through a probe-based ELISA process, MAQ-G. This second method successfully validated the N⁶-methyladenosine motifs predicted by the initially developed motif-finder program. Conclusion: This integrated m ⁶ A-FINDiT and MAQ-G, coupled with a real-time qPCR assay, could correlate the methylation profiles of N6-methyladenosine motifs with the expression and stability contours of a gene. To establish the physiological implications of these techniques, we chose three tumour-suppressor genes, viz., IRF8 , RB1 , and TP53 mRNA transcripts, which may undergo m ⁶ A methylation at certain DRACH motifs. The m ⁶ A-FINDiT pipeline could successfully predict the specific m ⁶ A motifs, and the MAQ-G confirmed the methylation profile of the latter. These duo techniques hold potential for use in clinical settings for early cancer detection.

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.

Germ cells are pivotal for the continuation of biological species. The metazoan germline develops from primordial germ cells (PGCs) that undergo multiple rounds of mitotic divisions. The PGCs are specified by either maternal inheritance of asymmetrically polarized cytoplasmic mRNAs/proteins (found in roundworms, flies, fishes, frogs, and fowl) or via direct induction of epiblast cells from adjacent extraembryonic ectoderm in mammals. In all vertebrates, PGCs remain uncommitted to meiosis and migrate to colonize the developing gonadal ridge before sex determination. Multiple RNA‐binding proteins (e.g., Vasa, Dnd, Dazl, etc.) play crucial roles in PGC identity, expansion, survival, and migration. Postsex determination in mouse embryos, Gata4, expressing nascent gonads, induces Dazl expression in newly arriving germ cells that supports retinoic acid–mediated induction of meiotic onset. This article briefly discusses the developmental events regulating the PGC specification and commitment in metazoans. We also highlight the recent progress towards the in vitro generation of functional PGC‐like cells in rodents and humans.

Background and objectives: Epididymal transit renders key competence to mammalian spermatozoa for fertilizing eggs. Generally, the two paralogs of glycogen synthase kinase 3, GSK3α and GSK3β, functionally overlap except in testis and sperm. We showed that GSK3α is essential for epididymal sperm maturation and fertilization. Male infertility is the only phenotype of mice with a global or testis‐specific knockout (KO) of Gsk3α . Their sperm maturation is impaired, and sperm cannot fertilize eggs in vitro and in vivo. This suggests that GSK3α is a “male fertility kinase” in mammals and that GSK3α‐selective inhibitor is a potential male contraceptive. Materials and methods: A set of eight heterozygous Gsk3α(±) male mice received daily intraperitoneal injections of BRD0705, an isoform‐selective GSK3α inhibitor, at 20 mg/kg body weight for 1 week. Five vehicle‐treated and BRD0705‐treated mice were tested for in vivo fertility and the remaining mice were sacrificed; their caudal spermatozoa were examined for motility and biochemical properties. Results: The treated mice did not sire any pups while the control group sired 46 pups with a normal gestation period of 19–23 days. Continued fertility testing up to 6 weeks post‐treatment, showed that the treated mice regained fertility siring 56 pups, with 76 in the control group. Sperm motility was impaired, its abnormal morphology increased during epididymal transit, Adenosine triphosphate (ATP) levels were low, and tyrosine‐phosphorylation of hexokinase was absent: these phenotypes imitated those observed in Gsk3α KO mice. Tyrosine ²⁷⁹ ‐phosphorylation of GSK3α was reduced in sperm from the treated mice showing that the GSK3α activity was inhibited. The altered sperm phenotypes returned to normal following recovery of fertility. Conclusions: Complete infertility resulted after 1 week of BRD0705‐treatment and fertility recovered after cessation of the treatment. Work is ongoing to determine the minimum dose and treatment time and the testing of new compounds with increased selectivity and inhibitory activity against GSK3α.

In brief: Protein phosphatase 1 catalytic subunit gamma isoform 2 (PP1γ2) is a unique phosphatase expressed only in mammalian testes and sperm cells. The PP1γ2 isoform is indispensable for sperm motility and fertility and cannot be replaced by the PP1γ1 isoform for these functions. The serine–threonine phosphatase has four paralogs – PP1α, PP1β, PP1γ1 and PP1γ2 – encoded by three genes, Ppp1ca , Ppp1cb and Ppp1cc . Protein phosphatase PP1γ2, one of two isoforms of the gene Ppp1cc , is expressed in spermatogenic cells in the testes and sperm, while PP1γ1 is found in somatic cells. The two PP1γ isoforms, formed by alternate splicing that occurs only in mammals, are identical except at their C-termini. Global or testis-specific knockout of Ppp1cc in mice results in male infertility due to disrupted spermiation and mid-to-late spermiogenesis. Transgenic expression of PP1γ2, driven by a testis-specific promoter in differentiating spermatogenic cells, rescues spermatogenesis and fertility in the Ppp1cc -null mice. Why PP1γ2 is essential and present only in mammalian sperm is a mystery. We have generated a knock-in mouse where the Ppp1cc gene is edited to express only PP1γ1. Spermatogenesis was normal in knock-in mice. Testis-expressed PP1γ1 in the knock-in mice and PP1γ2 in the wild-type mice were incorporated in equal amounts into sperm. Sperm bearing PP1γ1 have reduced flagellar beat amplitude and motility, and male mice were severely sub-fertile. Although in the wild-type mice, PP1γ2 is present in both the head and tail, in the knock-in mice, PP1γ1 is absent in sperm heads, leading to an altered intra-sperm protein phosphatase landscape. Phosphoproteomic analysis of sperm proteins suggested a plausible molecular basis for compromised PP1γ1 functions: it identified GSK3α, a known substrate of PP1, to be dysregulated in knock-in sperm. This study provides a preliminary explanation for the isoform-specific requirement of PP1γ2 for male fertility.

Alpha-fetoprotein (AFP) belongs to the albuminoid protein family and is considered as the fetal analog of serum albumin. This plasma protein is initially synthesized in the fetal liver and yolk sac and shows a maximum peak near the end of the first trimester. Later, concentrations begin to decline prenatally and drop precipitously after birth. This protein has three key ligand-binding pockets for interactions with various biomolecules. It contains multiple phosphorylation and acetylation sites for the regulation of physiological and pathophysiological states. High serum AFP titer is an established biomarker for yolk sac, embryonal and hepatocellular carcinoma. The present review critically analyzes the chemical nature, receptors, clinical implications, and therapeutic aspects of AFP, underpinning the development of different types of cancer.

Leydig cells (Lc) reside in the interstitial compartment of the testis and are the target of Luteinising hormone (LH) for Testosterone (T) production, thus critically regulates male fertility. Classical histological studies have identified two morphologically different populations of Lc during testicular development [fetal (FLc) and adult (ALc)]. Recent progress inex vivocell/organ culture, genome-wide analysis, genetically manipulated mouse models, lineage tracing, and single-cell RNA-seq experiments have revealed the diverse cellular origins with differential transcriptomic and distinct steroidogenic outputs of these populations. FLc originates from both coelomic epithelium and notch-active Nestin-positive perivascular cells located at the gonad–mesonephros borders, and get specified as Nr5a1 (previously known as Ad4BP/SF-1) expressing cells by embryonic age (E) 12.5 days in fetal mouse testes. These cells produce androstenedione (precursor of T, due to lack of HSD17β3 enzyme) and play critical a role in initial virilization and patterning of the male external genitalia. However, in neonatal testis, FLc undergoes massive regression/dedifferentiation and gradually gets replaced by T-producing ALc. Very recent studies suggest a small fraction (5-20%) of FLc still persists in adult testis. Both Nestin-positive perivascular cells and FLc are considered to be the progenitor populations for ALc. This minireview article summarizes the current understanding of Lc development in fetal and adult testes highlighting their common or diverse cellular (progenitor/stem) origins with respective functional significance in both rodents and primates. (227 words)