Molecular Human Reproduction

Ovarian disorders, including anovulation, primary ovarian insufficiency (POI), and polyendocrine metabolic ovarian syndrome (PMOS), affect millions of reproductive-age women worldwide; however, mechanistic studies of ovarian biology and pathophysiology remain challenging because current experimental approaches often lack selectivity, tunability, or physiological relevance. Genetically modified animal models are labor-intensive and irreversible; small molecules frequently exhibit off-target effects; and conventional antibodies have limited tissue penetration and restricted temporal control. Designed ankyrin repeat proteins (DARPins) represent a highly modular protein engineering platform with advantages in specificity, size, stability, and extracellular targeting, but their utility in reproductive biology remains largely unexplored. Here, we used epidermal growth factor receptor (EGFR)-targeting DARPins as a proof-of-concept platform to interrogate ovarian signaling during ovulation. Screening of engineered anti-EGFR DARPins identified SX-006, a bispecific tetravalent construct with robust cross-species EGFR binding and potent biological activity. Using an ex vivo murine ovulation system, SX-006 inhibited follicle rupture in a dose-dependent manner with IC50 of 1.21 M without overt cytotoxicity. Lower concentrations of SX-006 preferentially perturbed follicle rupture while largely preserving oocyte meiotic maturation and luteinization, suggesting differential sensitivity of ovulatory processes to extracellular EGFR blockade. Comparative transcriptomic analyses further revealed that extracellular EGFR blockade and small molecule-based intracellular EGFR kinase inhibition produce overlapping but also distinct transcriptional responses, supporting biologically distinct modes of ovulatory signaling pathway perturbation. Together, these findings establish DARPins as a selective, tunable, and physiologically relevant platform for studying ovarian signaling and provide proof-of-concept for extracellular receptor targeting in ovarian biology, infertility research, and non-hormonal contraceptive development. Summary sentenceAn engineered EGFR-targeting DARPin selectively inhibits ovulation through extracellular receptor blockade and establishes a versatile platform for investigating ovarian signaling and reproductive disorders.

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.

Primary ovarian insufficiency (POI) and related infertility, early menopause, and endocrine disorders due to hormonal deficiency are major side effects in young female cancer patients undergoing cancer therapy. Current strategies preserving the fertility and hormonal functions of the ovary remain imperfect due to concerns of feasibility, efficacy, or safety. Herein, we identified c-Jun N-terminal kinase (JNK) as a pivotal regulator of the DNA damage response (DDR) signaling in oocytes of primordial follicles in response to DNA-damaging cancer therapy. Using pharmacological JNK inhibition and a genetically modified mouse model with oocyte-specific JNK deletion, together with histological, bioinformatic, and molecular approaches, we demonstrated that JNK inhibition prevented chemotherapy-induced oocyte apoptosis and POI, and preserved long-term reproductive cycles and fertility. Mechanistically, JNK was activated in response to chemotherapy-induced DNA damage in oocytes of primordial follicles, causing activation of transcription factor TAp63 and subsequent oocyte apoptosis, ultimately resulting in diminished ovarian reserve and POI. A more clinically relevant breast cancer-bearing mouse model revealed that JNK inhibition preserved the ovarian reserve without compromising anti-cancer efficacy of chemotherapy. Together, our study identifies oocyte-intrinsic JNK as a promising target for developing ovarian protectants and safeguarding reproductive health and fertility in young female cancer survivors.

Embryo culture, a required step during in vitro fertilization (IVF), exposes developing embryos to altered environmental conditions not normally experienced in vivo, including altered oxygen (O2) tension. Importantly, O2 influences gene expression, metabolism, and the activity of enzymes that sculpt the epigenetic landscape. The lowest O2 tension currently used in clinics during embryo culture is 5%, despite evidence that sections of the mammalian female reproductive tract have O2 levels as low at 2%. Lower O2 may therefore better mimic the in vivo environment and thus lead to improved pre- and postnatal outcomes in IVF-conceived offspring. Using our validated IVF mouse model, we show embryo culture at 2% O2 compared to culture under 5% O2 significantly improves embryo cell number, the chromatin landscape in preimplantation embryos, fetal and placental development during gestation, and metabolic function in adulthood. We further uncover mechanisms by which culture under ultra-low O2 mediates these improvements. Overall, these results suggest embryo culture with 2% O2 ameliorates adverse outcomes after IVF and provide evidence that IVF could be further improved by adjusting culture conditions to model the in vivo environment.

Ovarian tissue cryopreservation enables fertility preservation in females undergoing gonadotoxic therapies, restoring fertility in adults. Although offered even before puberty, the childhood ovary and its vulnerability to therapy remain poorly characterized. Here, ovarian tissue from 16 patients undergoing fertility preservation (aged 1-16 years) and 11 adult controls (aged 22-32 years) was analyzed using single-cell RNA sequencing, spatial transcriptomics, and multiplex immunostaining. In chemotherapy-naive samples, 13 somatic cell populations underwent extracellular matrix remodeling, vascular, neural, and stromal maturation during puberty, whereas changes in germline related to chromatin remodeling. Spatial transcriptomics resolved 23 clusters across, revealing distinct tissue organization and follicular niche composition between children and adults. Chemotherapy exposure depleted perifollicular and vascular cells, suppressed intercellular signaling, and dysregulated over half of puberty-associated genes, converging on stress responses and extracellular matrix remodeling, with SEPTIN7 as a potential biomarker. These findings uncover critical developmental vulnerabilities of the pediatric ovary relevant to fertility preservation.

To facilitate the investigation of signaling by the luteinizing hormone receptor (LHR), we created a mouse line called Lhr-COIN. This line allows for the conditional replacement of the Lhr coding sequence with enhanced green fluorescent protein (eGFP), resulting in both a conditional knockout line and a reporter line. By breeding these mice with mice expressing Cre recombinase, we generated mice in which either one or both Lhr alleles were replaced with eGFP. Notably, mice in which one Lhr allele in the granulosa cells was replaced with eGFP exhibited normal LH responsiveness. This enabled live imaging of LH-induced migration of LH-receptor-expressing granulosa cells within preovulatory ovarian follicles. The Lhr-COIN mouse line holds significant potential for future research on LHR function and localization in the ovary and other tissues.

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

Ovarian stimulation is widely used in assisted reproductive technologies, yet its effects on uterine architecture and embryo implantation remain poorly understood. Using a mouse model, we show that ovarian stimulation or superovulation disrupts pre-implantation luminal epithelial folding and induces aberrant smooth muscle structure and contractile function. These structural defects result in embryo trapping within aberrant longitudinal folds, impaired implantation chamber formation, misalignment of the embryo-uterine axis, and subsequent embryo loss. These ovarian stimulation effects were reversible after rest and restoration of normal estrus cycling. Transcriptomic analysis suggests widespread disruption in the stroma and immune compartments of the stimulated uteri. Pathway analysis revealed significant disruption of stromal extracellular matrix and enhanced probability of stroma-immune communication via collagen signaling. Platelet derived growth factor receptor A (PDGFRA) expression was elevated in both the stroma and smooth muscle of the stimulated uteri. Short-term pharmacological inhibition of PDGFRA in the stimulated uteri prior to implantation fully restored epithelial fold transition and implantation chamber formation and partially restored smooth muscle architecture and contractility. Importantly, PDGFRA protein was also elevated in endometrial biopsies from women undergoing ovarian stimulation when compared to natural cycle biopsies. Together, this study establishes muscle contractions and stromal and smooth muscle PDGFRA signaling as novel non-cell autonomous regulators of uterine epithelial architecture critical for embryo implantation.

Study questionHow does cryptorchidism affect germ cell development and UTF-1-mediated pluripotency potential at the time of orchiopexy? Summary answerCryptorchidism was associated with the following germ cell abnormalities: germ cell clustering with many cords/tubules lacking germ cells and reduced UTF-1 expression, suggesting limited germ cell differentiation into spermatogonia What is known alreadyCryptorchidism, affecting 1.6-9% of male newborns, is known to increase the risk of infertility and testicular cancer due to abnormal germ cell development. Germ cells and pluripotent stem cell gene, UTF-1, play critical roles in spermatogonia differentiation, self-renewal, and spermatogenesis. No prior study has evaluated the testicular development by immunohistochemically mapping of these cell populations, Study design, size, durationA cross-sectional study of 31 postnatal cryptorchid testis and 5 age-matched scrotal testicular biopsies obtained from UCSFs pathology department performed between 1993-2023. Participants/materials, setting, methodsSpecimens were grouped by age at surgery (6-18 months, 19 months-7 years, 8-12 years, and [≥]13 years) and testis location (palpable vs. non-palpable). Scrotal prepubertal testis biopsies were sourced through the Pedi-LIFE program, a fertility preservation research biobank, with at least one control per age group. Immunohistochemistry was performed to stain specimens for germ cell (DDX4, OCT4, TSPY), pluripotent cell marker (UTF-1), as well as other key testis cell markers (A-actin, AR, P450, Sox-9), with staining graded based on control expression levels. The number of germ cells per seminiferous tubule was quantified and compared across anatomical locations using appropriate statistical analyses. Main results and the role of chanceThis study included 36 specimens, comprising 31 cryptorchid testes (86%) and 5 scrotal control testes (16%). The cryptorchid group exhibited testicular dysgenesis and reduced germ cell expression, correlated with increased age and testis location. Qualitative assessment revealed reduced germ cell expression across all ages in cryptorchid testes. The number of germ cells per tubule was markedly reduced in cryptorchid compared with scrotal testes after 19 months of age for DDX4, TSPY, and UTF-1. Germ cell clusters were identified in 15 out of 31 cryptorchid specimens (48%) stained for DDX4 and TSPY. UTF-1 expression was lower in cryptorchid testes across all age groups. No significant differences were noted in other testicular cell markers. Large scale dataNA Limitations, reasons for cautionFirst, the power and generalizability of the study are limited by the availability of specimens within each age group, particularly for scrotal testes, as biopsies of these tissues are not routinely performed. Second, a cross-sectional study design limits a longitudinal comparison to evaluate changes in marker expression, delayed maturation, or irreversible germ cell loss. Third, immunohistochemistry data is semi-quantitative, and protein detection is affected by antibody sensitivity and tissue preservation and influenced by antibody sensitivity. Lastly, scrotal testis used as controls were obtained from cryopreserved tissue from patients with other unrelated pathology, which may influence histological profiles. Wider implications of the findingsCollectively, our findings support a model in which cryptorchidism involves both germ cell depletion and disrupted SSC lineage formation, with UTF-1 downregulation and germ cell clustering as early signatures of testicular dysgenesis. These features may help identify high-risk patients for worsening gonadal dysgenesis and infertility and can provide a rationale for earlier orchiopexy or SSC-preserving strategies. Study funding/competing interest(s)The authors declare no conflicts of interest and received no funding for this study. Data Availability StatementThe data underlying this article cannot be shared publicly due to ethical and legal restrictions related to the use of human tissue specimens, which may compromise donor privacy and confidentiality. Data are available from the corresponding author upon reasonable request and subject to institutional and ethical approvals.

Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder characterized by hyperandrogenism, ovulatory dysfunction, and polycystic ovaries, with granulosa cell dysfunction being a key pathological feature. This study aimed to investigate the role of microRNA-6818-5p in PCOS pathogenesis. Quantitative PCR revealed a significant upregulation of circulating miR-6818-5p in PCOS patients compared to healthy controls. In vitro, functional assays in the human granulosa cell line KGN demonstrated that miR-6818-5p overexpression markedly inhibited cell proliferation (assessed by CCK-8 assay) and promoted apoptosis (measured by Annexin V/PI flow cytometry). Mechanistically, dual-luciferase reporter assay and Western blotting identified HSD17B2 as a direct target of miR-6818-5p, with miR-6818-5p mimics significantly suppressing HSD17B2 protein expression. In conclusion, our findings reveal that elevated miR-6818-5p in PCOS may contribute to follicular development dysfunction by targeting HSD17B2 to disrupt granulosa cell proliferation and apoptosis balance, offering novel insights into PCOS pathology and highlighting miR-6818-5p as a potential diagnostic biomarker and therapeutic target.

Proper establishment of the primitive placenta and subsequent tissue homeostasis in the mature placenta are critical for successful pregnancy in humans. Placental insufficiency is associated with adverse pregnancy outcomes, including fetal growth restriction, preeclampsia, and pregnancy loss. Moreover, emerging evidence suggests that placental defects are associated with long-term health challenges that manifest well into adulthood; yet the etiologies of such diseases are largely unknown. Defining the mechanistic basis for placental deficiencies, therefore, has important implications for improving both reproductive health and the lifelong well-being of affected children. Down syndrome is characterized by placental defects of unknown mechanistic origin, and notably, individuals with Down syndrome are at increased risk of developing diseases commonly associated with placental insufficiency later in life. Using induced pluripotent stem cells from Down syndrome patients, we found that stem cell-based embryo models (i.e., blastoids) and directed differentiation systems recapitulate trophoblast cell fate defects observed in placentas affected by Down syndrome. Furthermore, we demonstrate that attenuated estrogen signaling contributes to placental syncytialization defects and identify NRIP1, a transcriptional corepressor of estrogen receptor that is located on chromosome 21, as a key driver of trophoblast cell fate defects. Increased gene dosage of NRIP1 in an otherwise diploid cell line phenocopies cell fate defects observed in trophoblasts affected by Down syndrome. Our study suggests that estrogen signaling is a crucial regulator of trophoblast development and may serve as a potential target for therapeutic intervention. Highlights and eTOC blurbO_LIEstrogen signaling mediates syncytiotrophoblast fusion C_LIO_LIHuman iPS cells provide a tractable model for trophoblast defects in Down syndrome C_LIO_LITrophoblast differentiation and estrogen signaling are disrupted in Down syndrome C_LIO_LIIncreased NRIP1 expression is sufficient to induce trophoblast defects C_LI Logsdon and colleagues apply patient-derived induced pluripotent stem cells to recapitulate placentation defects observed in Down syndrome. The authors demonstrate that attenuated estrogen signaling disrupts trophoblast differentiation and identify NRIP1, a gene found on chromosome 21 that dampens estrogen signaling, as a regulator of trophoblast maturation. NRIP1 and estrogen signaling may represent important therapeutic targets for infertility and Down syndrome.

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.

Chronically stressing male rodents can induce stress-specific epigenetic changes in sperm that contribute to altered offspring phenotypes. Whether similar phenomena occur in men is unclear. This study addresses this knowledge gap by analyzing sperm microRNAs (miRNAs) from 51 men exposed to various levels of adult trauma including crime, disaster, and physical or sexual violence, quantified by the Trauma History Questionnaire (THQ), a measure of risk for Post-Traumatic Stress Disorder (PTSD). Four sperm miRNAs, miR-532-3p, 491-5p, 375-3p and 361-3p correlated positively with mens THQ scores, showing 4X to 130X over expression in sperm from the most highly traumatized men. These changes were independent of mens adverse childhood experiences (ACEs), which we previously linked to decreased miR-34/449 in their sperm; and sperm miR-34/449 levels were not associated with THQ scores. Injecting these 4 miRNAs into fertilized mouse oocytes at levels comparable to those found in men reporting high THQ scores yielded offspring with elevated anxiety-and depression-like phenotypes. This finding differs from the stress related phenotypes we observed in offspring of mice fertilized by sperm with reduced levels of miR-34/449. Consistent with only a small subset of men with high THQ scores developing PTSD, we observed no statistically significant increase in overall anxiety or depression among this highly traumatized group, however there were indications of increased sleeplessness, appetite and concentration difficulties and negative self-concept among this group. Nevertheless, almost all men reporting high THQ scores had elevated levels of all 4 of these miRNAs in their sperm, suggesting these trauma-induced epigenetic changes may raise mental health risks in the offspring of men with only subtle mental health problems. Since [~]20 % of men report either THQ or ACE scores in the ranges linked here and in our earlier study to changes in sperm miRNAs that in mice lead to elevated levels of stress-related behaviors, a large human population with an elevated risk of transmitting stress-related traits to their offspring likely exists.

Actin-related protein T2 (ACTRT2) localizes to the perinuclear theca (PT) of male germ cells, yet its functional significance remains unclear. ACTRT2 is evolutionarily conserved and exhibits significant sequence similarity to other testis-specific actin-related proteins, with the highest conservation observed within the canonical actin core domain. We generated Actrt2-deficient mice which displayed male subfertility with pronounced acrosomal malformations originating during the Cap phase of acrosome biogenesis. Actrt2-deficient male mice showed reduced fertilization rate and poor blastocysts quality. Co-immunoprecipitation identified ACTRT2 interactions with PT proteins ACTRT1, ACTRT3, ACTL7A, ACTL9, PFN3, SPEM2 and CCIN while the interaction with CYLC1 was not detected. ACTRT2 overexpression in HEK293T cells altered cell morphology and F-actin distribution. Further, cytoskeletal regulator CFL1 was enriched in testis from Actrt2-deficient mice. We propose that ACTRT2 is a structural component of the PT stabilizing the acroplaxome during spermiogenesis and acrosome biogenesis by modulating actin dynamics. Finally, the high degree of sequence conservation and similarity with ACTRT1 and ACTRT3 together with their similar phenotypes when deleted, indicate that ACTRT2 shares a partial functional redundancy and compensatory capacity with other Arp proteins in testis. Taken together, these findings establish ACTRT2 as a structural regulator of sperm head architecture and male fertility in mice.

The endometrium is the innermost compartment of the uterus and undergoes cyclical remodeling throughout the human menstrual cycle and the rodent estrous cycle. The endometrium must thicken appropriately for embryonic implantation to occur; thus, it is crucial to understand the molecular mechanisms downstream of steroid hormone action that regulate endometrial thickness. Hedgehog (Hh) signaling is required for endometrial remodeling in both mice and humans, but the role of downstream Hh transcriptional effectors in endometrial remodeling is unknown. Here, we discover a role for the Hh transcriptional repressor, Gli3, in endometrial homeostasis: conditional knockout of Gli3 resulted in a constitutively thick endometrium throughout the estrous cycle. In our model, a constitutively thick endometrium could support pregnancy. Bulk RNA-sequencing data revealed that loss of Gli3 also resulted in dysregulated stromal-epithelial crosstalk, while immunofluorescent staining showed larger uterine glands and increased gland proliferation. These data deepen our understanding of molecular mechanisms controlling endometrial thickness, offering novel pathways to investigate endometrial factors in infertility.

Development of the female reproductive tract in mice occurs in early postnatal life. The current model identifies Trp63 as the master regulator that initiates differentiation of simple columnar Keratin 8+ epithelium in the cervix and vagina into a stratified squamous epithelium. Thereafter Trp63+ basal progenitors maintain cervicovaginal epithelial cell homeostasis and in the adult serve as the progenitor for hormone-regulated shifts in stratified squamous and secretory luminal cells. This model differs from the human in which two progenitors, one columnar and the other basal gives rise to secretory cells in the endocervix and stratified squamous epithelia in the ectocervix and vagina respectively. In the current study, we identify a population of Krt8+, Tp63- epithelial cells that are retained in the cervicovaginal epithelium during the postnatal developmental period and into adulthood. Single cell datasets from the cervices of adult mice, identify Olfactomedin 4 (Olfm4), as a unique marker of the Krt8+Trp63- population. Adult lineage tracing and reassessment of gene markers during postnatal development support a revised model in which two progenitors are delineated in the mouse cervix and vagina by PND15. Olfactomedin 4+ progenitors give rise to specialized secretory goblet cells, while Trp63+ basal progenitors give rise to stratified squamous luminal cells in the cervix and vagina of nonpregnant and pregnant mice. Consistent with the expansion of goblet cells in pregnancy, the Olfm4+ progenitor is highly proliferative in early pregnancy and progesterone regulates increased goblet cell differentiation. These findings reveal a previously unrecognized species similarity between mice and humans in which goblet cell and squamous keratinized cell subtypes are derived from two progenitor populations respectively. HIGHLIGHTSO_LITwo epithelial progenitors (Trp63 and Olfm4) populations are delineated in the cervix and vagina within the first two weeks of postnatal life. C_LIO_LIThe Trp63+ progenitor gives rise to keratinized epithelial cells, whereas the Olfm4+ progenitor cells give rise to secretory goblet cells. C_LIO_LIlfm4 is not required for maintenance of the luminal progenitor or differentiation of goblet cells in the cervix and vagina during adulthood and pregnancy. C_LIO_LIIn adults, progesterone promotes differentiation of Olfm4+ progenitors into goblet cells. C_LI

In mammals, a small population of spermatogonial stem cells (SSCs) is established shortly after birth. These cells self-renew and produce sperm for the entirety of a males reproductive lifespan, passing the genome on to the next generation. Thus, establishment of a population of SSCs with high genomic integrity is essential. SSCs are derived from a much larger precursor population of male germ cells (MGCs) that differentiate during fetal life. During the last third of gestation, MGCs undergo a prolonged period of G0 cell cycle arrest during which they sustain high levels of transcription and acquire epigenetic programming for SSC fate. Although these differentiation steps can cause cellular and genomic damage, it has been unclear whether selection for germ cell quality occurs during G0 arrest since no classic markers of cell death have been detected. In this study, we utilize a mouse model to characterize a population of MGCs that begin to accumulate markers if cell death, such as AnnexinV (AnV) and propidium iodide (PI), at E16.5. The AnV- and PI-positive MGC population is characterized by low expression of the RNA-binding protein, Dead End 1 (DND1), and exhibit dsDNA breaks and mitochondrial dysfunction. Interestingly, we do not see evidence of an active cell death cascade until the time of birth, where we see phosphorylation of MLKL, a hallmark of a necroptotic cell death mechanism. Based on these findings, we propose that variable cellular health is an important basis for selection of the SSC precursors. Significance StatementSpermatogonial stem cells (SSCs) are essential for reproductive fitness, yet how their precursors are selected during development is not known. Utilizing a mouse model, this study describes high levels of cellular damage within a subset of male germ cells (MGCs) during G0 arrest. The damaged MGC population was marked by low expression of the RNA-binding protein, DND1, and was strongly associated with mitochondrial dysfunction and dsDNA breaks. We observed signs of non-apoptotic cell death by embryonic day (E)16.5 and the appearance of necroptotic markers in MGCs at the time of birth. This study uncovers previously unknown heterogeneity in the MGC pool and points to MGC health as an important source of selection during G0 arrest.

Maternal transcripts are stored in the oocyte cytoplasm during oogenesis and play a pivotal role in early embryonic development after fertilization. However, specific maternal transcripts that reflect the developmental potential of embryos have not been systematically identified, and the use of maternal transcript levels as an indicator of successful development has not been explored. Here, we link the maternal transcriptome to the zygotes developmental potential by examining transcripts in a single polar body. The transcriptome of a zygote or an oocyte was highly similar to that of its accompanying polar body in mouse, cow, and human. We have identified a set of maternal transcripts whose expression levels fluctuate between poor- and good-quality zygotes. Specifically, Sipa1and Zmym6 were identified as marker transcripts that accurately reflect the developmental potential of zygotes. Using these marker genes, combined with machine learning, the development of zygotes to the blastocyst stage was successfully predicted with more than 80% specificity as early as 12 hours after fertilization. Furthermore, our prediction platform significantly improved implantation rates and live births to term. Thus, we have demonstrated a minimally invasive method for identifying maternal transcripts associated with zygote developmental potential. Our developed prediction system provides a generalizable conceptual framework for human infertility treatment to reduce the risk of implantation failure by excluding embryos with low developmental potential, especially when early embryos are transferred, and for livestock propagation to assess selected expressed maternal trait-associated variants before embryo transfer.

Impaired seminal redox balance is a main factor that contributes to male fertility disorders and reduced sperm survival during storage. Although several methods are available to measure antioxidant and reactive oxygen species (ROS) levels, their cost and complexity limit their use in routine sperm analysis. Recently, assessment of oxidation-reduction potential (ORP) has emerged as a convenient and comprehensive method for evaluating seminal redox status. While the implications of seminal ORP in humans have been extensively explored, its use in other species is limited. In this study, we explored the relationship between boar seminal ORP and sperm quality and its dynamics during liquid preservation. We found that the ORP of the porcine ejaculate was lower than that of the seminal plasma, while both parameters were correlated with the total antioxidant capacity (TAC) of seminal plasma. Sperm concentration and seminal pH influenced the seminal ORP, with lower values observed in ejaculates with higher sperm concentration and pH. Notably, a more oxidative seminal environment (characterized by high ORP or low TAC) was correlated with high mitochondrial activity and sperm velocity in fresh samples, which might be explained by increased ROS production by sperm mitochondria. Our results also show that seminal ORP increased during three days of liquid storage, while the ORP of the extender did not change significantly during the same period. Our findings advance our understanding of the implications of redox status in porcine sperm biology and pave the way for the broader application of ORP measurement in animal andrology. HighlightsO_LIThe ejaculates oxidation-reduction potential is lower than that of seminal plasma C_LIO_LISeminal oxidation-reduction potential is correlated with total antioxidant capacity C_LIO_LISeminal redox status is influenced by sperm concentration and semen pH C_LIO_LIAn oxidative seminal environment is correlated with high sperm metabolism C_LIO_LISeminal oxidation-reduction potential increases during 3 days of liquid storage C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/726780v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@18fd914org.highwire.dtl.DTLVardef@f4f179org.highwire.dtl.DTLVardef@1195e32org.highwire.dtl.DTLVardef@7760ad_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cryopreservation of ram semen induces structural and functional alterations that compromise sperm fertility. Since seminal plasma contributes to the regulation and preservation of sperm function, increasing attention has been directed toward seminal plasma extracellular vesicles (EVs) that are involved in sperm physiology. EVs act as carriers of proteins that are involved in sperm membrane organization and capacitation, suggesting that they may contribute to the maintenance of sperm stability during cryopreservation.. Thus, the aim of this study was to evaluate the effect of seminal plasma-derived EVs on post-thaw functional parameters of ram sperm. Semen was cryopreserved in the presence or absence of EVs isolated by ultracentrifugation that have been characterized by nanoparticle tracking analysis (NTA) and Western blotting (WB). Post-thaw sperm quality was assessed by evaluating viability, membrane lipid disorder, reactive oxygen species production, protein phosphorylation, acrosome status, intracellular calcium levels, and sperm motility. Sperm cryopreserved with an extender containing EVs showed a significant reduction in membrane lipid disorder and lower intracellular calcium levels compared to control samples (p < 0.05). CASA analysis revealed that EV supplementation did not affect total or progressive motility but modified sperm kinematic patterns, with increased linearity and straightness, indicating improved trajectory efficiency without induction of hyperactivated motility. No differences were detected in viability, ROS content, phosphorylation of proteins in residuous tyrosine (pY) or PKA or acrosome status. These results provide the first evidence that seminal plasma derived extracellular vesicles exert a protective effect during ram semen cryopreservation, preserving membrane organization and calcium homeostasis and improving sperm functional quality after thawing. Highlights- Seminal EVs protect ram sperm during cryopreservation. - EVs reduce membrane lipid disorder and intracellular Ca2+ levels. - EVs modify kinematics, increasing linearity and straightness. - No effects on viability, ROS, phosphorylation or acrosome status. - EVs improve post-thaw sperm functional quality and stability. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/732841v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@d1f8a9org.highwire.dtl.DTLVardef@11c3d6aorg.highwire.dtl.DTLVardef@104124forg.highwire.dtl.DTLVardef@4e355f_HPS_FORMAT_FIGEXP M_FIG C_FIG