Ovarian transplantation causes a loss of follicular reserve, requiring new strategies to optimize post-transplant follicular growth. This study investigated N-(2-hydroxy-3-piperidin-1-ylpropoxy)pyridine-3-carboximidamide; dihydrochloride (BGP-15), a mitochondrial modulator, as a follicular growth stimulator and a collagen-depositing inhibitor, thereby improving graft quality. In vitro, neonatal mouse ovaries were treated with different concentrations of BGP-15. In vivo, adult mice undergoing ovarian autotransplantation received 10 mg/kg of BGP-15. Follicular quantification, gene expression by qRT-PCR (Sirt1, Sirt3, Nampt, Foxo3a, KitL, Pten, Amh), collagen deposition by Picrosirius assay, neoangiogenesis, macrophage infiltration, biometric parameters, and the estrous cycle were evaluated. In vitro, 0.001 µM BGP-15 induced follicular growth. reducing the percentage of primordial follicles (p < 0.0001) and upregulating Sirt1 (p < 0.0001), Sirt3 (p = 0.026), Nampt (p = 0.002), Pten (p = 0.012), and downregulating Amh (p = 0.031). In vivo, BGP-15 treatment in transplanted mice resulted in an earlier resumption of the estrous cycle (p = 0.0458). After transplantation, BGP-15 improved follicular outcome on day 22, increasing healthy follicles (transitional p = 0.0206; primary p = 0.0125; secondary p = 0.0428) and reducing atretic follicles (p = 0.0381). Treated animals exhibited decreased macrophage numbers (p < 0.0001) and collagen deposition (ovary: p = 0.0465; capsule: p = 0.0344), and increased blood vessel density (p < 0.0001). BGP-15 improves follicular growth and survival in ovarian grafts, coinciding with mitochondrial gene dysregulation and reduced collagen deposition. These findings suggest BGP-15 as a promising candidate for improving follicular outcomes after ovarian transplantation.

Pyroptosis, a proinflammatory form of programmed cell death mediated by the NLRP3 inflammasome, has recently been implicated in Sertoli cell inflammatory injury. Crocin, a water-soluble carotenoid glycoside derived from Crocus sativus L. and Gardenia jasminoides Ellis, exhibits anti-inflammatory, antioxidant, and anti-apoptotic properties, yet its role in Sertoli cell pyroptosis remains unclear. Here, we investigated the pharmacological effects and mechanisms of crocin in regulating TM4 Sertoli cell viability and pyroptosis. Network pharmacology based on oligoasthenospermia (OA)-related candidate targets, together with molecular docking, identified HSP90AA1 as a candidate molecular target associated with crocin-mediated regulation of Sertoli cell pyroptosis. MST analysis further provided target-engagement evidence supporting a direct interaction between crocin and HSP90AA1. In TM4 Sertoli cells, crocin improved cell viability, enhanced Ki-67 expression, and protected against lipopolysaccharide/nigericin-induced pyroptosis, as evidenced by reduced lactate dehydrogenase release, decreased PI-positive cells, and downregulated expression of IL-1β, IL-18, NLRP3, Caspase-1, and GSDMD. Moreover, under HSP90AA1-overexpressing conditions, crocin partially reduced pyroptosis-related injury and inflammatory mediator expression, supporting the involvement of HSP90AA1 in crocin-mediated regulation of Sertoli cell pyroptosis. Collectively, these findings indicate that crocin enhances Sertoli cell proliferation and mitigates inflammasome-driven pyroptosis through an HSP90AA1-associated mechanism. These findings should be interpreted as preliminary in vitro evidence for crocin-mediated regulation of Sertoli cell proliferation and pyroptosis, and further validation in animal models and clinical samples is required.

Early embryonic mortality is a major concern to the beef industry, as nearly 32.3% of multiparous cows will not maintain pregnancy past the first sixteen days of gestation. During this period, the uterine environment provides nutrients to support embryonic growth and development. Therefore, our objective was to (1) modulate the luminal metabolome at the onset of conceptus elongation in pregnant beef cows and (2) identify changes in the endometrial epithelial transcriptome during the peri-elongation period. Cows received either TARG: 100 g of a rumen-inert mono- and polyunsaturated fatty acid source plus 60 g of a rumen-protected choline source (n = 50) or CON: 114 g of a saturated fatty acid source (n = 50) daily from 30 days before (d -30) to 30 days after breeding (d 30). Uterine cytobrush was collected from pregnant cows on day 14 for metabolomics (CON = 17, TARG = 15) and on Day 16 for transcriptomics analysis (CON = 6, TARG = 7). On Day 14, the concentration of 23 metabolites (p < 0.1) differed; the concentration of fatty acids, triacylglycerols, hexosylceramides, cholesterol esters, phospholipids, biogenic amines, carboxylic acids, methyl donors, and amino acids was greater in TARG. On Day 16, 675 genes were differentially expressed (FDR ≤ 0.1). Among these genes, 506 genes were upregulated in TARG, involving important genes in pathways such as interferon signaling, fatty acid transport, synthesis, metabolism, immune response, and cell-to-cell adhesion. In conclusion, the dietary supplementation provided herein modulated the uterine environment during the period of conceptus elongation.

Nearly 200 years ago, scientists began examining reproductive cells in calcareous sponges (Calcarea, Porifera). Since then, 238 studies have been published, encompassing descriptions of gametes and embryos (including ultrastructural studies), life history, asexual reproduction, regeneration, and molecular aspects of development. Here, we present a comprehensive synthesis of two centuries of research on the development of Calcarea and review the different steps of the reproductive processes within this class. We analyzed temporal trends, thematic foci, authors, and geographic distribution of the studied species, and the reproductive characteristics of both subclasses (Calcinea and Calcaronea). Bibliographic productivity increased in gradual waves, from classical histology through ultrastructural and experimental embryology to advanced live imaging and molecular approaches. The past half-century has shown acceleration, yet with a persistent geographic bias toward the Mediterranean and North Atlantic species. Molecular datasets are beginning to link phylogenetic context and gene expression during development, but remain fragmentary and lack standardization within the class. We outline priorities for the coming years and identify critical unanswered questions. While significant challenges remain, confronting them actively advances research on Calcarea reproduction and development and firmly establishes it as a distinct field within sponge reproductive science.

Establishing a successful pregnancy relies on effective communication between the mother and fetus. Small extracellular vesicles (sEVs) are pivotal in facilitating intercellular communication, transferring messenger RNA (mRNA), noncoding RNA (ncRNA), and proteins. Considering the prevalence of in vitro fertilization (IVF) in bovine production, despite its lower birth rate, we aimed to evaluate whether there are differences in transcript content in sEVs between pregnancies from artificial insemination fixed-time (FTAI) and in vitro fertilization-embryo transfer (IVF-ET). Uterine fluid was collected on Days 18 and 32 from AI- and IVF-derived pregnancies in beef cattle. sEVs were isolated, and total RNA was extracted for transcriptome and microtranscriptome analysis. Differences in transcript abundance were greatest when comparing Days 18 and 32 within the IVF-ET, suggesting that temporal changes in transcripts are highly influenced by reproductive technique. Gene set enrichment analysis showed 22 gene sets in IVF-ET18 x FTAI18, with mRNAs involved in embryonic pre-adhesion that may impair embryo fixation. In IVF-ET32 x IVF-ET18, target genes of differentially expressed (DE) microRNAs (miRNAs) were enriched in endocytosis pathway, while target genes of DE lncRNAs were enriched in complement and coagulation cascades pathway. In summary, sEVs derived from FTAI and IVF-ET pregnancies have different coding and noncoding transcript content, suggesting failures in maternal-fetal communication post-IVF.

Oocyte and granulosa cells undergo dramatic molecular alterations prior to ovulation, which are critical for acquisition of oocyte developmental competence. These events are orchestrated by a complex bidirectional communication system mediated by the surrounding follicular cells, with follicular fluid reflecting the metabolic output of this system. The composition of follicular fluid is highly dynamic during folliculogenesis, especially following an ovulation-inducing surge of luteinizing hormone, as granulosa cells and theca cells respond and synthesize new metabolites. In the present study, untargeted metabolomics was performed on follicular fluid collected from synchronized Bos taurus cattle at 0, 6, and 22 h after gonadotropin releasing hormone-induced ovulation. High-performance liquid chromatography-mass spectrometry revealed a temporal shift in metabolite abundance across the periovulatory window. Partial least squares discriminant analysis demonstrated clear separation between 0- and 22-h samples, while 6-h samples displayed an intermediate metabolite profile. A total of 489 features were retained after variance-based filtering (interquartile range threshold of 25%), and a covariate-adjusted linear model identified 327 features as significantly altered across time points (p ≤ 0.05). Lipid-associated metabolites, including polyunsaturated fatty acids, oxylipins, and steroid-derived metabolites were enriched prior to induced ovulation while amino acids and related metabolites were progressively enriched from 0 to 22 h. Functional analysis of features that were progressively enriched across the periovulatory window revealed nine significantly affected pathways (false discovery rate < 0.1) associated with amino acid metabolism and biosynthesis, as well as galactose metabolism. These results enhance our understanding of the periovulatory follicular environment and have potential applications for improving in vitro oocyte maturation protocols.

LIM and SH3 protein 1 (LASP1) is a widely expressed scaffold protein associated with actin-rich structures. In Lasp1-knockout mice, reproductive defects such as longer intervals between litters and reduced pup numbers were observed. Consistently, data from the Human Protein Atlas show strong LASP1 expression in spermatids, suggesting a reproductive role. Mammalian fertilization involves sequential processes of sperm activation, capacitation, and acrosome reaction, driven by bicarbonate-mediated PKA activation, CNP-regulated PKG stimulation, F-actin polymerization, Ca[2+]-signaling, and MAPK signaling pathways - culminating in membrane fusion and CD44 cluster formation for interaction with the egg. In this context, we observed differential isoform expression of PKG in human and mice. Our data provide the first evidence of LASP1 presence in the head, midpiece, and tail of human and mouse spermatozoa. During capacitation, LASP1 undergoes dynamic changes, co-localizes with F-actin and stabilizes the equatorial ring segment. In mice, loss of LASP1 did not affect sperm morphology, motility or chemotaxis, but led to changes in protein phosphorylation, incomplete capacitation, and reduced hyaluronan receptor surface expression, revealing a novel role of LASP1 in sperm fertilization.

In this study, we evaluated the effect of a previous exposure of in vitro produced blastocysts to hypothermia (33°C) on their further cryotolerance. In Experiment 1, embryos were exposed to hypothermia for either 6 or 12 h on day 5 or 6 of development, and the blastocyst rates and kinetics were evaluated. In Experiment 2, the transcriptome of embryos exposed to hypothermia on either day 5 or 6 for 12 h was assessed. In Experiment 3, embryos were exposed to hypothermia for 12 h on day 6, vitrified-warmed, and their re-expansion, apoptotic index, and total cell number were assessed. Results showed that hypothermia had no effect on blastocyst rate but reduced the percentage of hatched blastocysts when applied for 12 h. Moreover, hypothermia was associated with changes in the transcriptional profile, particularly in embryos exposed on day 6, including increased expression of the cold-shock-related transcripts RBM3 and CIRBP. Finally, hypothermia on day 6 did not improve embryo re-expansion after cryopreservation but did reduce apoptosis in the surviving embryos. Our results provide insight into transcriptomic responses associated with mild hypothermia in bovine embryos and support the hypothesis that hormetic-like responses may contribute to improved embryo quality after cryopreservation.

Animal cloning, while promising for livestock breeding and genetic improvement, is hampered by low efficiency largely due to placental abnormalities. This study investigated this issue in cloned pigs at the 35th day of gestation, where a 69.8% developmental abnormality rate was observed. Morphological analyses revealed structural defects in placentas from failed pregnancies, including delayed fold development and abnormal areolae formation, coupled with reduced endometrial glands and maternal-side blood vessels. Immunohistochemistry demonstrated impaired placental function, evidenced by aberrant E-cadherin expression compromising trophoblast adhesion, and significantly reduced glycogen/polysaccharide secretion. Furthermore, Plet1 expression was disrupted. Transcriptomic profiling indicated that upregulated genes were enriched in cell-cell tight junctions, while downregulated genes were associated with oxygen binding, oxidative stress, stromal differentiation, and vascular development. Cross-species comparison with cloned mice suggested conserved genetic mechanisms underlying placental defects. These findings provide a crucial theoretical foundation for elucidating the mechanisms of placental failure in animal cloning and for developing strategies to enhance its efficiency.

Understanding the molecular mechanisms that guided early animal evolution is a central challenge of evolutionary developmental biology. Sponges (Porifera), as one of the earliest-branching metazoan lineages, are crucial for reconstructing the ancestral genetic toolkit for development. Here, we present the first transcriptomic analysis of embryogenesis in the demosponge Halisarca dujardinii, characterizing gene expression across three key stages: cleavage, blastula, and prelarva. We identified significant transcriptional shifts, including upregulation of cadherins during blastula formation, suggesting the establishment of adherens junctions, and a wave of kinase expression in the prelarva. Furthermore, we phylogenetically classified and analyzed the developmental expression of 186 transcription factors from the zinc finger, homeobox, Forkhead, and T-box families. Strikingly, spatial expression analysis revealed that Wnt ligands and T-box genes are expressed in complementary domains along the embryonic anterior-posterior axis, with HduWntL specifically marking the future posterior pole from the blastula stage. Our results demonstrate that despite its morphological simplicity, H. dujardinii employs a complex repertoire of developmental transcription factors and signaling molecules, and provides evidence for the early specification of the primary body axis, offering new insights into the evolution of developmental mechanisms in metazoans.

Flow cytometric sorting can be used to select sperm subpopulations with defined physiological or molecular traits; however, the impact of sorting on sperm function and subsequent embryo development in the porcine model remains unresolved. The objective of this study was to evaluate the effect of fluorescence-activated cell sorting (FACS) on boar sperm motility and developmental competence following in vitro fertilization (IVF). Pooled semen from boars was divided into sorted and unsorted (control) treatments and analyzed for motility and kinematic parameters using computer-aided sperm analysis (CASA) with both population-level (conventional, population-averaged) metrics and single-cell track data collected before and after sorting. Oocytes were fertilized with either treatment, and cleavage (≥ 2-cell stage) and blastocyst development were evaluated. Cleavage and blastocyst percentages were reduced in the sorted (48.8% ± 9.9% and 8.5% ± 4.2%) compared with control (92.2% ± 1.5% and 28.4% ± 2.8%, respectively; p < 0.001) group. Population-level CASA revealed statistically significant but biologically modest changes in motility and kinematic parameters following sperm sorting. In contrast, single-cell CASA coupled with k-means clustering identified four distinct motility subpopulations and demonstrated that sorting redistributed existing motility states, characterized by depletion of fast, progressive sperm and enrichment of less energetically demanding populations. These findings suggest that FACS alters the composition of sperm motility subpopulations, alongside a marked reduction in post-fertilization developmental competence in porcine IVF.

Sex determination in Porifera remains one of the least understood aspects of early metazoan biology despite the group's key phylogenetic position. Sponges display exceptional diversity in sexual systems-ranging from stable gonochorism to sequential hermaphroditism and sex reversal-yet lack morphological dimorphism and any discrete gonadal structures, blurring the boundary between sex determination and gametogenesis. Here we synthesize current knowledge on sexual systems, environmental and genetic influences on sexual fate, and the evolutionary origins of sexual systems in sponges. Evidence for environmental modulation is substantial, particularly temperature-driven shifts in sex ratios and reproductive timing, but environmental sex determination remains unsupported by causal evidence yet. Conversely, conserved molecular components of metazoan sex-determining pathways, such as fem-1 and Dmrt genes, are present in several sponge lineages, though their functional roles remain unresolved. We also evaluate the hypothesis that microbial symbionts contribute to sexual allocation by buffering the energetic costs of gametogenesis. Overall, sponges exhibit an evolutionarily labile system in which sexual fate may emerge from the interaction of conserved genetic modules, environmental cues, and physiological constraints. Clarifying these mechanisms will be essential for understanding the origins of sex determination in Metazoa and for predicting reproductive resilience under environmental change.

Premature ovarian insufficiency (POI) patients usually have a few residual dormant primordial follicles that are difficult to activate. We investigated the effects of branched-chain amino acids (BCAAs) on mouse primordial follicle activation. Leucine, but not isoleucine and valine, promoted primordial follicle activation in the cultured neonatal mouse ovaries. Furthermore, leucine significantly elevated the levels of phosphorylated protein kinase B (p-Akt) and phosphorylated forkhead Box O3a (p-FOXO3a), and increased the number of oocytes exhibiting nuclear export of FOXO3a in mouse ovaries. LY294002 and PI3K-IN-1, the inhibitors of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, reversed the promoting effects of leucine on mouse primordial follicle activation. Importantly, intraperitoneal injection of leucine in 3-day-old neonatal mice and oral administration of leucine in 21-day-old mice with or without cyclophosphamide treatment significantly increased the number of growing follicles. Thus, leucine activates mouse primordial follicles through the PI3K/Akt pathway. Leucine may be a potential oral supplement for treating infertility in POI patients with residual dormant primordial follicles.