Reproduction

Conventional semen cryopreservation involves equilibration at 4{degrees}C and optimum freezing rates. We hypothesized that a cholesterol-based semen extender obviates the need for equilibration, minimizing total processing time for semen cryopreservation. Experiments were conducted to determine the effects of semen extender (egg yolk- or cholesterol-based) and freezing method (routine or fast) on post-thaw sperm characteristics and fertility of beef and bison semen. In Experiment 1, beef semen diluted in tris-egg yolk-glycerol (TEYG) or cholesterol-cyclodextrin tris-glycerol (CCTG) extender underwent routine or fast freezing method. Cholesterol from animal and plant origins were compared. The routine method included 90-min equilibration at 4{degrees}C and routine freezing (RE-RF, total time 97 min) whereas the fast method included no equilibration and fast freezing (NE-FF, total time 14 min). Post-thaw sperm quality was assessed by CASA, and in vitro fertilization. Post-thaw sperm motility was not affected by the origin of cholesterol (animal or plant), but was lowest in the TEYG NE-FF group (24% vs 43-51%, P < 0.05). In vitro cleavage and blastocyst development rates did not differ between RE-RF and NE-FF groups. In Experiment 2, bison semen was diluted in TEYG or plant-CCTG extender and frozen as in Experiment 1. Post-thaw sperm motility was lowest in the TEYG NE-FF group (10% vs 39-51%, P < 0.05). In Experiment 3, beef semen diluted in TEYG or plant-CCTG extender underwent either a routine (RE-RF) or modified freezing (NE-RF, total time 25 min) method. Post-thaw sperm characteristics did not differ between extenders but were greater using routine freezing (RE-RF) compared to the modified method of freezing (NE-RF). Pregnancy rates were similar between extenders (TEYG vs plant-CCTG) using the modified freezing method without equilibration and insemination at 72 h after progesterone device removal. In conclusion, beef and bison semen diluted in cholesterol-based extender may be cryopreserved without equilibration.

In the terminal segment of the seminiferous tubules, SOX17 expression in the rete testis (RT) epithelium plays a crucial role in the formation of the Sertoli valve (SV), as revealed by phenotypic analyses of RT-specific Sox17 conditional knockout (cKO) mouse testes. In these RT-specific Sox17 cKO testes, SV disruption leads to the backflow of RT fluid into the seminiferous tubules, resulting in defective spermiogenesis and male infertility. Although valve deformation in the Sox17 cKO testes is likely caused indirectly by impaired downstream actions of Sox17 in the RT, the mechanisms by which SOX17 in RT influences SV formation in the seminiferous tubules remain unclear. To address this, we generated a novel AMH-Sox17 transgenic (Tg) mouse line carrying a human AMH promoter-driven Sox17 cDNA cassette. We analyzed the phenotypes of the Sertoli valve and spermatogenesis in AMH-Sox17 Tg mice, as well as in RT-specific Sox17 cKO; AMH-Sox17 Tg double mutant mice. Ectopic SOX17 (SOX17+) expression in Sertoli cells resulted in excessive Sertoli valve structures with acetylated tubulin bundles in the terminal segment of the AMH-Sox17 Tg testes, along with enhanced WNT4/RSPO1 signaling, suggesting the enhanced valve formation of ectopic SOX17+ Sertoli cells by themselves. Moreover, the AMH-Sox17 Tg could partially rescue the SV deformation and infertility in RT-specific Sox17 cKO mice, leading to proper SV formation, normal spermiogenesis and a partial recovery of male fertility in AMH-Sox17 Tg; RT-specific Sox17 cKO double mutant mice. These findings genetically demonstrate that ectopic SOX17+ Sertoli cells can compensate for SOX17 paracrine signaling in the RT, underscoring a key shared downstream pathway between RT and SV. Summary statementThe paracrine actions downstream of ectopic SOX17 expression in the Sertoli cells not only promote the valve formation, but also partially rescue the defective spermiogenesis of the rete testis-specific Sox17-null mice.

Study questionDoes the human placenta utilize the creatine phosphagen system for energy homeostasis during development? Summary answerComponents of the creatine (Cr)-creatine kinase (CK)-phosphocreatine (PCr) system are dynamically expressed by the trophoblast and mesenchymal compartments throughout gestation wherein creatine kinase is required for cellular ATP metabolism, cell cycle, and proliferation of trophoblast cells. What is known alreadyThe Cr-CK-PCr system maintains ATP homeostasis in tissues with high energy demand and is required for proliferation, migration, and invasion of tumor cells. The term human placenta can synthesize and transport creatine locally. Early placental development involves trophoblast proliferation, an event requiring ATP, but the role of the creatine phosphagen system during early placental development remains unknown. Study design, size, durationWe performed immunohistochemistry (IHC) and immunofluorescence (IF) for different components (biosynthesis, transport, utilization) of the Cr-Ck-PCr system in human placentae (n=3/group) across gestation including first trimester, second trimester, and term. Using primary human trophoblast stem cells (hTSCs) and trophoblast organoids (TO), we determined the role of the creatine phosphagen system in trophoblast growth by functional inhibition of creatine kinase. Participants/materials, setting, methodsIHC/IF were performed in human placentae across gestation for proteins involved in biosynthesis (AGAT and GAMT), transport (SLC6A8, SLC22A15, and SLC6A13) and utilization (CKB and CKMT1) of creatine to determine the presence of the creatine phosphagen system locally in the placenta. For delineating the functional importance of this system in placental development, cyclocreatine (cCr), a creatine analogue, was used for functional inhibition of CK. Primary hTSCs were culture in medium containing 0 (control), 1, 10, 20 mM cCr for 48 hours followed by analysis of cell growth (cell count), cell cycle (EdU incorporation assay), apoptosis (Annexin V/PI flow cytometry), energy metabolism (Sea horse mito-stress and glycolytic stress tests), and gene expression (qPCR). Primary TO were also treated with 20mM cCr for 6 days in vitro to determine the role of Cr-CK-PCr system in placental development. Main results and the role of chanceAGAT localized to the fetal villous mesenchyme, while GAMT was broadly expressed in the trophoblast and fetal mesenchyme compartments across gestation. CKB localized primarily to fetal mesenchyme with strongest expression at term. CKMT1 was broadly expressed in all trophoblast subtypes. SLC6A8 was abundant in early syncytiotrophoblast but absent at term, where its expression shifted to fetal blood vessels. SLC22A15 was expressed in the endothelial cells of fetal capillaries across gestation. In primary hTSCs, cyclocreatine (20mM) treatment reduced proliferation (P<0.001), decreased expression of trophoblast epithelial marker EGFR (P<0.05), induced G0/G1 and G2/M arrests (P<0.0001), enhanced early and late apoptosis (P<0.0001), and downregulated GPX8 expression (P<0.05). Seahorse analysis revealed marked reductions (P<0.01) in mitochondrial (basal, maximal, and ATP-linked) and glycolytic (rate, capacity, and reserve) function compared to controls. In primary human TO, cyclocreatine treatment reduced the growth of organoids (P<0.05) as well the expression of EGFR (P<0.05). Large scale dataN/A Limitations, reasons for cautionFurther experiments assessing apoptosis, cellular stress and redox imbalance may provide more mechanistic role of the creatine phosphagen system in trophoblast metabolism and function. Since the functional role of the Cr-CK-PCr system was investigated in vitro, findings of this study should be taken with caution for implications of in vivo placental development. Nevertheless, reproducible results of reduced growth of trophoblast cells using both 2D and 3D cultures is highly suggestive of the importance of the creatine phosphagen system in early placental development. Wider implications of the findingsThis study provides foundational knowledge that the placenta contains the creatine phosphagen system, known for ATP homeostasis, and that this system ensures proper cell division, survival and placental development. Dysregulation of components of Cr-CK-PCr system in placenta has been observed in pregnancy disorders such as preeclampsia and fetal growth restriction warranting continued investigation into mechanisms and potential remediation using creatine supplementation. Stem cells share similar metabolic features so findings of this study can be implicated in other stem cells models as well. Study funding/competing interest(s)This work was supported by CIRM EDUC4-12804 Interdisciplinary Stem Cell Training Grant and a Lalor Foundation Postdoctoral Fellowship awarded to NS, and by the California Institute for Regenerative Medicine (DISC0-13757) and the National Institute of Child Health and Human Development (R01-HD096260) award to FS. The authors have no competing interest to declare.

In eutherian mammals, blastocyst implantation is often associated with a quasi-inflammatory reaction in the endometrium, which is resolved with the establishment of the definitive placenta. This is understandable in the case of invasive placentation, since implantation entails a nidatory injury to the maternal tissue due to the invading blastocyst. Quasi-inflammatory processes have also been documented in pregnant pigs, even though the blastocyst only attaches to, rather than invades into, the endometrium of the uterus. In this study, we asked what processes in early porcine pregnancy lead to the resolution of attachment-associated inflammation. In generic wound healing the transition from a pro- to an anti-inflammatory state is caused by a corresponding transition from M1 to M2 polarized macrophages following efferocytosis by macrophages of apoptotic neutrophils. In order to determine whether this scenario applies to the pregnancy-related resolution of inflammation in the porcine uterus, we produced a series of bulk transcriptome samples spanning days (D) 13 to 25 of gestation. This time span corresponds to the transition from pre- to post-attachment stages of pregnancy. We found slower changes in the transcriptome between D20 and D25 than prior to D20, suggesting a turning point in pregnancy-related reprogramming. The turning point at D20 corresponds to the time of firm attachment of trophectoderm to uterine luminal epithelium and the cessation of IFNG signaling from the blastocyst. This transition coincides with increased expression of RNAs of genes implicated in resolution of inflammation and M2 polarization such as ARG1, MRC1/CD206, CD86, TGFb1 and IL10, as well as a significant increase in expression of HGPD, the enzyme that metabolizes prostaglandins. While immunoreactivity for ARG1 was found in putative macrophages in the sub-epithelial stratum compactum, other markers of M2 polarized macrophages were localized to non-immune cells: MRC1 was found on fibroblast-like stromal cells, CD86 on trophoblast cells, and IL10 in luminal and glandular epithelia. These results suggest that intrauterine immune regulation is decoupled from that of the rest of the body by engaging non-immune cell types as anti-inflammatory mediators during the peri-attachment period of pregnancy.

Vitamin D signaling has recognized roles in female reproductive physiology, but its effects at the chromatin level in endometrial stromal cells are still unclear. Here, we investigated how the active form of vitamin D, 1,25-dihydroxyvitamin D3, or calcitriol, influences the accessible chromatin landscape of human endometrial stromal cells. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) was performed on T-HESCs treated with either a vehicle or 1,25(OH)2D3. Ligand treatment increased overall chromatin accessibility, shown by higher ATAC-seq signal intensity, while causing only minor changes in the total number of called peaks. Peak annotation revealed that accessible regions were spread across both promoter-proximal and distal genomic areas. Integrating this data with CUT&RUN and RNA sequencing showed that most vitamin D-responsive cistromic modifications and transcripts were linked to nearby open chromatin, though fewer were associated with regions that were significantly differentially accessible. These results suggest that 1,25(OH)2D3-dependent transcription mainly occurs within a permissive, pre-accessible chromatin environment. This study offers new evidence that active vitamin D influences the epigenomic landscape of human endometrial stromal cells, establishing the chromatin-based molecular response to a chemically-defined VDR ligand, 1,25(OH)2D3, relevant to stromal differentiation and preparation for decidualization. HighlightsO_LIFirst evidence suggesting the direct impact of active vitamin D, 1,25-dihydroxyvitamin D3, 1,25(OH)2D3, enhanced the signal intensity of chromatin accessibility in human endometrial stromal cells C_LIO_LIMost accessible chromatin regions were shared between vehicle and ligand-treated human endometrial stromal cells C_LIO_LI1,25(OH)2D3-responsive transcription occurs largely within pre-accessible chromatin in human endometrial stromal cells C_LIO_LIAssay for transposase-accessible chromatin sequencing (ATAC-seq) defines a chromatin-level pharmacologic response to a chemically defined VDR ligand in human endometrial stromal cells C_LI

Development and use of assisted reproductive technologies (ARTs) in non-domestic species provides novel tools for species conservation. As a first step towards in vitro embryo production, we developed an OPU technique for two antelope species, scimitar horned oryx (Oryx dammah) and roan antelope (Hippotragus equinus) utilizing a custom-made needle guide and existing OPU equipment utilized by livestock and human practitioners. Females were anesthetized and placed in sternal recumbency for transvaginal OPUs. Prior to OPUs (36 - 45 hours), SHO and roan were either hormonally stimulated with follicle stimulating hormone (FSH, 140 or 250IU) as a single injection or not. A total of 32 and 26 OPUs were completed in SHO (n=10) and roan (n=7), respectively, representing one to four OPUs per animal at monthly intervals. A total of 141 oocytes were recovered from 215 follicles in SHO and 31 oocytes from 58 follicles in roan. FSH dose (250IU) increased (P<0.05) the number of follicles aspirated and the number of oocytes recovered in SHO. No effects of FSH were observed in roan (P>0.05). Good quality oocytes were recovered from all females and procedures were conducted in four consecutive months with no evidence of scar tissue buildup or reduced capacity to recover quality oocytes. These ARTs can be used to develop in vitro embryo production tools for population management and the preservation of female genetics; bolstering genetic diversity and guarding against extinction.

Current evaluation of male fertility is largely based on indirect sperm parameters such as viability, concentration, morphology, and motility; however, each of these parameters, alone or combined, has been shown to have limited predictive value for successful fertilization. To address this problem, we introduce hSPICER (human SPerm-Induced CEll-cell fusion Requiring JUNO), an assay that evaluates sperm function based on their ability to induce fusion of somatic cells expressing human JUNO (hJUNO), the egg-specific sperm receptor. Similarly to our previous discovery in mice, we found that human sperm can fuse with somatic cells expressing hJUNO on their surface (pseudo-eggs) and promote content mixing between cells in culture, as measured using a split GFP system. The assay is sensitive, specific, and species-dependent, requiring hJUNO for optimal signal. We generated a stable cell line expressing hJUNO, enhancing reproducibility and sensitivity. We also show that hSPICER is compatible with cryopreserved sperm and consistent over different days. Importantly, hSPICER values correlate with fertilization outcomes of patients during fertility treatments, indicating its potential as a functional diagnostic tool. Beyond diagnostic uses, hSPICER establishes a platform to explore sperm fusion mechanisms and to screen for therapeutic compounds and interventions to treat low fertility, enhance fertilization, and develop non-hormonal contraceptives for males and females, as well as quality assessment of semen samples in fertility clinics and sperm banks.

Pregnancy leads to many adaptations in the maternal body, most of which are reversible. However, reproductive experience can also result in permanent effects. Here, we investigated how pregnancy influences the somatotrophic system and the lasting effects of reproductive experience on the maternal organism. Reproductive experience induced a pronounced increase in lean body mass and longitudinal growth in both wild-type and growth hormone (GH)-deficient mice compared with age-matched virgins. Body growth was primarily observed during the first pregnancy, whereas a second gestation was mostly associated with increased adiposity. Data from a cohort of women with isolated GH deficiency (IGHD) caused by a loss-of-function mutation in the GHRHR gene revealed that nulliparous women were 7 cm shorter than those with one or more pregnancies. Increased GH secretion was observed in pregnant wild-type mice but not in pregnant GHRHR-deficient mice. Pregnancy-induced body growth is preserved despite disruption of GH-, ghrelin-, and estrogen-related signaling pathways. In conclusion, reproductive experience induces permanent changes in the maternal organism, promoting body growth in models that allow this response. Pregnancy-induced body growth appears to be independent of GH action. These findings underscore the need for further studies to investigate the long-lasting consequences of reproductive experience in females.

During early development of the placenta, a subset of murine trophectoderm stem cells (TSCs) undergo endoreplication, an unusual form of cell division cycle that decouples DNA synthesis from cytokinesis, resulting in physiological polyploidy. Oscillations in CDK2 activity are essential for the orderly progression of the cell cycle to ensure replicated DNA is accurately partitioned into two daughter cells. However, it remains underexplored how the dynamics of CDK2 activity regulate endoreplication in the context of TSCs differentiation. To address this question, we leveraged the variability in cell fate decisions in an established in vitro system of TSCs differentiation that relies on removal of a growth factor, FGF4, to induce endoreplication. Using quantitative single-cell live confocal microscopy of a precise CDK2 biosensor, DHB-Venus, we identified at least three different outcomes upon FG4 removal: self-renewal, endoreplication, and migration. Our quantitative analyses showed high levels of Cdk2 activity in self-renewing cells whereas intermediate DHB-Venus turnover is linked to increased nuclear and cell size, indicating a shift to endoreplication. Importantly, we also characterize a third class of differentiating TSCs with migratory characteristics that correlate with low levels Cdk2 activity without a change in nuclear size. In sum, our results demonstrated a correlation between different fate outcomes and specific thresholds of CDK2 activity. Our findings show that TSCs can distinguish between different outcomes through modulating the central kinase of the cell cycle, CDK2, positioning it as a key regulator of early trophoblast differentiation. Summary StatementThis study investigates the oscillatory behavior of CDK2 activity during murine trophectoderm differentiation and its potential role in guiding cell fate decisions.

Plac1 is an X-linked gene essential for placental and embryonic development. A knockout (KO) mouse model was used to identify Plac1-regulated gene expression at E16.5 and E18.5 using gene expression microarray. Genes exhibiting at least 1.5-fold change in expression and FDR < .05 were considered significant. At E16.5, 717 genes were downregulated and 798 were upregulated in male KO placentas versus wild type (WT), whereas at E18.5, 1122 genes were downregulated and 1149 were upregulated. GO, KEGG, and IPA analyses revealed downregulated genes were enriched for Rho GTPase-mediated and actin-cytoskeleton based processes that transmit extracellular cues through canonical signaling pathways, including Integrin, GPCR, Wnt, Notch, VEGF, BMP and TGF-beta, documented to impact trophoblast development, vasculogenesis, vascular tone, branching morphogenesis, and immunomodulation. Furthermore, a preeclampsia-associated transcriptomic signature was induced that strengthened over time. By contrast, upregulated genes reflected immune activation and adaptations to oxidative stress resulting from impaired placental function. These findings indicate that Plac1 supports signaling required to maintain placental structure and regulatory function. Its absence disrupts essential regulatory processes and triggers cellular stress and immune activation, contributing to fetal growth restriction, increased risk for embryopathy and preeclampsia, consistent with the Developmental Origins of Health and Disease (DOHaD) framework.

Early pregnancy requires a tightly regulated pro-inflammatory environment shared between the primitive placenta and decidua. While immune balance supports successful implantation and placental invasion, disruptions in immune signaling during this period can impair implantation and lead to embryo loss. In this study, we investigated the molecular mechanisms underlying immune imbalance during implantation using a trophoblast stem cell (TSC) model. TSCs were cultured in either stem cell or syncytiotrophoblast (STB) differentiation medium and treated with either lipopolysaccharides (LPS) or interferon beta (IFNB). RT-qPCR and Western blotting revealed that LPS failed to induce a pro-inflammatory cytokine response in TSCs or STBs. In contrast, IFNB triggered a strong antiviral response in both TSCs and STBs. RNA-sequencing of IFNB-treated TSC and STB 3D spheroids revealed subtle differences between the TSCs and STB responses to interferons. Both TSC and STB IFNB-treated spheroids mount an interferon-mediated antiviral response; however, STB spheroid genes associated with the type I interferon response, viral RNA/DNA sensing, and antigen processing were upregulated. We also compared the interferon response between the CT27 (female) and CT29 (male) TSCs and STBs. While STBs showed minimal differences, the CT29 TSCs exhibited a markedly stronger interferon response than the CT27 TSCs. Collectively, these findings suggest that the primitive placenta is selectively responsive to interferon signaling rather than direct pathogen-associated stimuli. This implies that maternal immune activation, rather than microbial invasion, likely drives that placental immune response and embryo success at this stage. Understanding these dynamics underscores the importance of the maternal immune balance in early pregnancy success.

Estrogen is an essential hormone that critically impacts bodily and brain functions, supporting learning, memory, and motor activities. A decrease in estrogen levels is associated with cognitive decline and motor dysfunction, such as muscle weakness. While conventional hormone replacement treatments (HRT) exist, those have limitations and potentially severe side effects. NAP (davunetide) is the smallest neuroprotective peptide site of activity-dependent neuroprotective protein (ADNP), a master regulator of cognition, essential for brain formation. It is known that NAP restores ADNP activity in cases of deficiency and it has already shown potential in preventing cognitive impairment, protecting against tauopathy, and improving motor function in various animal models and in clinical trials. Based on the dynamic regulation of ADNP by the estrous cycle and its involvement in steroidogenic pathways, we hypothesize that NAP may restore ADNP activity and thus serve as an alternative to conventional hormonal treatments. To test this, 3-month-old female ICR mice underwent bilateral ovariectomy (OVX) or Sham surgery and received daily intranasal administration of NAP, estrogen, or vehicle. Results showed a significant reduction in weight-normalized forelimb grip strength in the OVX model. Daily administration of NAP or estrogen resulted in intermediate grip strength levels that did not statistically differ from either the Sham control or untreated OVX groups. Interestingly, grip strength was the only test that yielded significant results, and no significant differences were observed in the Novel Object Recognition (NOR) test or computed tomography (CT) scans. These findings suggest that NAP may effectively prevent the loss of physical force production typically seen following ovarian hormone depletion, presenting a viable, non-hormonal candidate strategy for managing musculoskeletal symptoms. We hypothesize that the lack of significance in other parameters was due to soy-derived phytoestrogens in the diet, which may have exerted a systemic estrogenic effect that masked the expected physiological phenotypes typically observed in OVX models. Future replication using phytoestrogen-deficient food is required to isolate the specific neuroprotective and musculoskeletal effects of NAP from dietary influence and clarify the broader therapeutic benefits of NAP.

Bioengineers strive to recreate in vivo microenvironments in vitro to reduce our use of animal models and provide insights into human biology. While liver models show promise, sex differences in liver biology remain largely neglected in preclinical studies. Despite the 2014 EU mandate for the inclusion of women in clinical trials, decoupling of research data by sex is historically rare, with only 11% of papers disaggregating data by sex. This gap contributes to women being more susceptible to drug-induced liver injury (DILI) and being underserved in drug development, as well as to costly drug attrition levels. Here we present a novel approach to modelling sex differences in vitro. Human induced pluripotent stem cells (iPSCs) from both male (XY) and female (XX) donors, were differentiated into hepatocyte liver spheroids and exposed to in vivo-mimicking levels of testosterone, progesterone, and oestrogen in high-throughput microwell format. We successfully recapitulated sex-specific metabolic profiles and demonstrated significant differences in CYP1A2 and CYP3A4 drug metabolism and gene expression patterns consistent with reported in vivo observations, without compromising cell viability. These findings validate the utility of sex-differentiated microenvironments in early-stage research, offering a pathway to refine animal and clinical trials and improve therapeutic outcomes for all sexes.

Phthalates are pervasive endocrine-disrupting chemicals widely used in consumer products. The wide use of many phthalates results in chronic human exposure to complex mixtures rather than single compounds. Despite extensive studies on individual compounds, the combined effects of phthalate metabolites on oogenesis remain poorly understood. Here, we developed a precise microinjection-based single-oocyte toxicological assay to examine the impact of a defined phthalate metabolite mixture on meiotic progression. Phthalate mixture exposure markedly impaired oocyte maturation, as most oocytes failed to extrude the first polar body. Mechanistic analyses revealed severe meiotic defects, including disrupted spindle morphology, chromosome misalignment, disorganized actin cytoskeleton, and impaired mitochondrial function, accompanied by excessive reactive oxygen species (ROS) accumulation and DNA damage. Single-cell transcriptomic profiling further identified differentially expressed genes enriched in biological processes related to exocytosis, secretory pathway regulation, and cytoskeletal organization, as well as in MAPK, JAK-STAT, cGMP-PKG, and GnRH signaling pathways that are essential for follicular development and oocyte maturation. Together, these findings demonstrate that combined phthalate exposure directly compromises female gamete quality and underscore the importance of evaluating mixture effects when assessing risks to womens reproductive health.

Animal tissues have diverse architectures and cell behaviors across the epithelial-mesenchymal spectrum. Cell adhesion mediated by classical cadherins is foundational. Cadherins nucleate complexes of dozens of proteins connecting junctions to the cytoskeleton and signaling downstream. Many junctional proteins are well-studied in epithelia, but less is known about roles during mesenchymal migration. The nascent myotubes of the pupal Drosophila testis provide an excellent model for N-cadherin mediated mesenchymal migration. We combined a proximity proteomics dataset of adherens junction proteins in mammalian epithelial cells with genome-wide shRNA libraries knocking down Drosophila genes to begin to define the subset of junctional proteins important in mesenchymal migration. While N-cadherin is predominant, E-cadherin plays a supporting role. Surprisingly, several proteins with key roles in epithelial morphogenesis, including Afadins homolog Canoe, ZO-1s homolog Polychaetoid, and Par3s homolog Bazooka play at most modest roles. Twenty-two genes with diverse cell biological roles had strong to moderate defects in testis morphogenesis. These will provide a community resource. We followed up two. The kinase Par-1 is important for migration and gap closure, with knockdown phenotypes paralleling those of myosin. The Rab GAP RN-tre does not have roles until after migration and works in parallel with N-cadherin during testis spiralization.

Pluripotent stem cells derived from livestock species represent valuable systems for studying early mammalian development and for establishing renewable, well-defined cell sources; however, direct comparative characterization of distinct pluripotent stem cell platforms in sheep remains limited. In this study, we established and evaluated two ovine pluripotent stem cell types: reprogrammed induced pluripotent stem cells (siPSCs) and embryonic disc-derived stem cells (sEDSCs). Both siPSCs and sEDSCs exhibited core features of pluripotency, including compact colony morphology, alkaline phosphatase activity, expression of key pluripotency-associated markers, and maintenance of a normal ovine karyotype. Flow cytometry and quantitative RT-PCR analyses revealed broadly overlapping yet distinguishable pluripotency marker expression profiles between the two cell types. Functional pluripotency was confirmed by embryoid body formation and in vitro differentiation into derivatives of all three germ layers. To further assess lineage-specific differentiation competence and compare functional outputs relevant to mesodermal differentiation, both pluripotent stem cell types were directed towards the adipogenic lineage. While siPSCs and sEDSCs were each capable of adipogenic differentiation, differences in differentiation efficiency and marker expression were observed. Together, these findings demonstrate that ovine siPSCs and sEDSCs share core pluripotency characteristics while retaining distinct molecular and functional properties, providing a robust comparative framework for studies of ovine pluripotency, lineage specification, and stem cell biology.

Zinc is an essential trace element involved in numerous biological processes, including cellular signalling, development, and reproduction. Zinc homeostasis is regulated by zinc transporters, yet the physiological roles of many transporters remain poorly understood in vivo. Here, we investigated the function of the zinc transporter ZIP9 (SLC39A9) using a zebrafish (Danio rerio) knockout model. Elemental imaging using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) revealed altered zinc distribution in zip9-deficient larvae. Synchrotron-based X-ray fluorescence (XRF) imaging further showed reduced zinc levels in the brain region of mutant zebrafish. Consistent with these observations, loss of zip9 was associated with altered expression of key neuroendocrine genes within the hypothalamic-pituitary-gonadal (HPG) axis. Zip9 mutant females exhibited disrupted ovarian follicle development, reduced spawning rates, and decreased egg production. In addition, embryos derived from zip9 mutant parents displayed reduced size, impaired early development, and decreased survival. Together, these findings identify ZIP9 as a regulator of zinc distribution in vivo and suggest that ZIP9-mediated zinc signalling contributes to reproductive regulation in zebrafish.

Emerging evidence indicates that the maternal in utero environment has enduring effects on offspring neurodevelopment. The obesity epidemic in the United States affects nearly one-third of women before pregnancy, potentially predisposing offspring to harmful developmental conditions. Glucose, the primary energy source for the brain, is highly regulated by facilitative diffusion glucose transporters (GLUTs). However, our understanding of how maternal obesity influences perinatal cerebral glucose metabolism remains limited. We hypothesized that maternal obesity is associated with altered expression of key GLUTs and dysregulated energy-sensing mechanisms in the fetal brain. Female C57BL/6J mice were randomly assigned to either a control diet (CON) or an obesogenic diet (DIO) (60% kcal from fat, 17.5% kcal from sucrose) for 10 weeks, time-mated with control males, and fed their respective diets throughout gestation. At 18.5 days post coitum, fetal brain tissue was collected for protein analysis. DIO diet did not affect litter size, offspring body weight, or brain weight when compared to CON. Whole brain GLUT1 expression was elevated only in female DIO offspring, while GLUT3 and GLUT4 expression was increased in all DIO offspring without modification by sex. However, maternal diet was not associated with differences in the activation of energy regulatory pathways adenosine monophosphate-activated protein kinase (AMPK) or the nutrient-sensing pathway mechanistic target of rapamycin (mTOR) in the fetal brain. These findings suggest that maternal obesogenic diet alters glucose transporter expression in the fetal brain, indicating a potential disruption in cerebral glucose metabolism during critical periods of perinatal development.

Perinatal opioid exposure is a prevalent clinical concern linked to respiratory instability and adverse infant outcomes. The opioid buprenorphine is prescribed as a medication for opioid use disorder during pregnancy and used to treat neonatal opioid withdrawal syndrome, yet its direct effects on neonatal control of breathing have not been examined. Here, we asked how acute buprenorphine exposure affects breathing at rest, and during chemoreceptor stimulation. Using dual-chamber head-out plethysmography, we measured pulmonary ventilation rate ([V]I) and metabolic rate in awake male and female Sprague-Dawley neonatal rats on postnatal days 4-5 (P4-5) during eupnea and a hypoxic-hypercapnic (HH) challenge. The effects of buprenorphine and two opioid receptor antagonists, naloxone hydrochloride, or peripherally restricted naloxone methiodide, were assessed using a repeated measures design. [V]I during eupnea and HH were markedly depressed following buprenorphine administration. Buprenorphine reduced [V]O2 and [V]CO2 and produced ventilatory equivalents for O2 and CO2 consistent with frank hypoventilation, driven by reduced breathing frequency and tidal volume (VT). When administered after buprenorphine, neither naloxone hydrochloride nor naloxone methiodide could rescue the buprenorphine-mediated hypoventilation in eupnea or during HH. In contrast, pre-treatment with either naloxone hydrochloride or naloxone methiodide attenuated buprenorphine-induced hypoventilation by preserving VT. These findings demonstrate that neonatal protective chemoreceptor reflexes are depressed by buprenorphine and suggest that pre-treatment with a peripheral opioid receptor antagonist could mitigate buprenorphine-induced hypoventilation without inducing opioid withdrawal. Key PointsO_LIAcute buprenorphine exposure significantly depressed pulmonary ventilation rate ([V]I) during eupnea and hypoxic hypercapnia (HH) in awake neonatal rats. C_LIO_LIBuprenorphine-induced hypoventilation was driven by reduced tidal volume (VT) and breathing frequency. C_LIO_LIBuprenorphine also reduced oxygen consumption ([V]O2) and carbon dioxide production ([V]CO2). C_LIO_LINaloxone given after buprenorphine failed to reverse hypoventilation. C_LIO_LIIn contrast, pre-treatment with either naloxone hydrochloride or peripherally restricted naloxone methiodide mitigated buprenorphine-induced hypoventilation by preserving VT. C_LI

Transcriptional bursts regulate gene expression by altering burst size or burst frequency. Here, we present a protocol that integrates fixed-cell smFISH and live-cell single-molecule imaging to analyze estrogen-responsive transcriptional bursting of the TFF1 gene in human breast cancer cell lines. This workflow enables measurement of burst size, burst initiation, and active allele frequency to determine how endocrine disruptor chemicals modulate transcriptional bursting dynamics. For complete details on the use and execution of this protocol, please refer to Day, Yasar et al.1