Semen evaluation in human and animal reproduction relies on sperm motility and morphology; however, these often fail to predict fertility. The domestic boar (Sus scrofa) serves as a biomedical model for male reproduction due to similarities in sperm size, capacitation dynamics, and acrosomal structure to humans in comparison to traditional rodent models. This study evaluated sperm capacitation biomarkers, particularly zinc signatures, to predict cleavage success after in vitro fertilization (IVF). Semen from 20 boars (3 replicates each) was analyzed at 0, 1 and 4 h post-in vitro capacitation (IVC) using image-based flow cytometry to assess 4 zinc signatures, plasma membrane integrity, and acrosomal remodeling. Capacitation kinetics were quantified between timepoints. Motility was measured by computer-assisted semen analysis, and IVF cleavage percentages were determined. Zinc signature 1 at 4 h post-IVC negatively correlated with cleavage percentage (r = -0.366), indicating higher noncapacitated sperm proportions reduce fertilization potential. The delta of zinc signature 3 from 1 to 0 h also negatively correlated (r = -0.441), suggesting excessively rapid capacitation impairs fertilization. Models combining capacitation biomarkers, motility, kinetics, and morphology parameters had higher predictive power (R2 = 0.469) than motility models alone. Zinc signatures may serve as mechanistic fertility biomarkers in a translational boar model applicable to animal breeding and human-assisted reproduction.
Exposure to low temperature prior to insemination increases the likelihood of polyspermy in Lytechinus variegatus eggs. Electrophysiological recordings reveal that cold shock partially disrupts the egg's action potential, producing a subthreshold depolarization during the fertilization response. This attenuated shift in membrane potential appears insufficient to activate the fast block to polyspermy, allowing entry of multiple sperm. These findings identify a temperature-sensitive vulnerability in the electrical barrier to polyspermy in sea urchin eggs.
Sponges (phylum Porifera) are an early-branching lineage of Metazoa. The long independent evolution of sponges makes them an essential group for comparative studies of the emergence and early evolution of various aspects of metazoan biology, including asexual reproduction. This review provides a current critical overview of the modes of asexual reproduction in sponges with an emphasis on the morphogeneses accompanying it. Asexual reproduction occurs in all poriferan clades and has three modes: fragmentation, budding, and gemmulation. Fragmentation seems to be a universal, but unspecialized and passive form of asexual reproduction; it relies on the pronounced regeneration capabilities of sponges. Budding and gemmulation are processes that are triggered by endogenous factors and are an integral part of the life cycle in many species. Budding seems to occur in all poriferan classes but differs in its mechanisms between classes: buds in Demospongiae are formed through mesenchymal morphogeneses, while in Homoscleromorpha and Calcarea-through epithelial ones. In contrast to other modes of asexual reproduction, gemmulation is restricted to freshwater demosponges and a few brackish-water marine demosponges. Gemmules represent compact groups of dormant cells, thesocytes, coated by a thick protective coat; in favorable conditions, these cells give rise to a new individual. Gemmulation represents not only a reproduction mechanism but also a mechanism for enduring adverse environmental conditions becoming a very important alternative reproduction strategy for sponges living in discontinuous-fragmented and/or unstable environments.
In vitro oocyte maturation and embryo culture are critical for preserving and expanding genetic lines, and are necessary to produce genetically engineered pigs for biomedical and agricultural purposes. However, suboptimal in vitro conditions compromise oocyte and embryo viability. Recent improvements to in vitro maturation medium, including the addition of FGF2, LIF, and IGF1 (FLI), have increased the number of oocytes that reached the metaphase II stage, doubled the number of oocytes that reached the blastocyst stage, and quadrupled the number of piglets born after embryo transfer. Despite these benefits, the underlying cellular mechanisms remain not fully understood. Given the essential role of cumulus cells (CCs) in oocyte maturation, we investigated how FLI affects CCs gene expression. Cumulus-oocyte complexes were matured for 24 h in control or FLI media, and CCs from oocytes that reached the blastocyst stage underwent RNA sequencing. FLI altered 1257 transcripts (423 upregulated, 834 downregulated; adj-p < 0.05), with enrichment of junctional communication genes and downregulation of extracellular matrix organization. Immunofluorescence confirmed increased TJP1, TJP2, and GJA4 in FLI-treated complexes. Additionally, cortical granule localization suggested enhanced cytoplasmic maturation with FLI supplementation. These findings indicate that FLI promotes CC communication and supports improved oocyte competence in porcine in vitro maturation.
7,12-Dimethylbenz(a)anthracene (DMBA) is a polycyclic aromatic hydrocarbon formed by the combustion of organic substances and has ovotoxic and carcinogenic effects on ovarian follicular development in rodents. The Hippo signaling pathway contributes to the understanding of various molecular mechanisms, including cell proliferation, differentiation, apoptosis, organ size regulation, and tumorigenesis, as an evolutionarily conserved pathway. In this study, we hypothesized that the Hippo signaling pathway may play a role in the mechanism of DMBA-induced ovotoxicity. We aimed to identify Hippo signaling pathway proteins in DMBA-treated ovaries via immunohistochemistry and quantitative real-time PCR (qRT-PCR). Twenty-eight-day-old 18 BalbC female mice were used and divided into three groups. The control group received no treatment, the vehicle group was injected daily with sesame oil, and the DMBA group was injected intraperitoneally with 1 mg/kg/day DMBA (dissolved in sesame oil) for 14 consecutive days. The sections were subjected to periodic acid-Schiff (PAS) staining to demonstrate the morphological differences between the DMBA and control groups of mouse ovaries. Anti-Mullerian hormone (AMH) serum levels were analyzed via ELISA, and follicles were counted to evaluate the follicle reserve. Immunohistochemistry was performed to determine the localization of the Hippo signaling pathway proteins MST1/2, LATS1/2, YAP1, and TEAD4 and to assess oxidative stress with a nitrotyrosine (NTY) antibody. The mRNA levels of Hippo signaling components were detected via qRT-PCR. The Hippo signaling pathway may play a role in the mechanisms underlying the rapid depletion of follicles through increased oxidative stress, an increased number of atretic follicles, and a decreased number of corpus luteum in DMBA-induced ovotoxicity. We demonstrated that in the DMBA-induced ovotoxicity model, the Hippo signaling pathway is inactivated by YAP/TAZ translocation to the nucleus, and the increase in YAP1 and TEAD4 expression is at the translational level. This study provides the first evidence of the relationship between ovotoxicity and the Hippo signaling pathway in DMBA-induced ovotoxicity in mice. Therefore, our findings suggest that the Hippo pathway could be pharmacologically targeted to regulate DMBA-induced ovotoxicity.
Gynogenesis has wide application in genetic improvement program for the generation of inbred line, mapping population, monosex population, and conservation. In the present study, embryonic development in gynogenesis of Pengba (Osteobrama belangeri [O. belangeri]) was analyzed to evaluate the efficacy of using conspecific and heterospecific sperm. For the selection of suitable sperm donor, three heterospecific males (Tilapia, Rohu, and Java barb) were experimented with no UV irradiation and thermal shock. The result showed high fertilization rate in case of Rohu and Java barb thus, they were omitted from further gynogenesis, in view of possibilities of producing hybrid offspring. For gynogenesis study, three experiments were conducted; first, conspecific as well as heterospecific sperm (Tilapia) were used to fertilize Pengba eggs (UV-irradiated sperm of Pengba and thermal shock to maintain diploidy) and the fertilization, hatching and survivability rates were recorded as 18%-20%, 5%-6%, and 2%-3%, respectively. Fertilization rate in Tilapia sperm without UV irradiation and thermal shock and UV-irradiated sperm of tilapia and thermal shock to maintain diploidy experiments was recorded as 4%-5%. However, hatching did not occur in either of the cases. Our results form the first report of gynogenesis in O. belangeri using conspecific and heterospecific sperm, which will be helpful in its genetic stock improvement programme and ensure conservation of native stock as well as better production in aquaculture.
Recurrent spontaneous abortions (RSA) are defined as a loss of two or more consecutive clinically recognized pregnancies before the 20th week of gestation. In RSA, several causative factors are known, but still, 50% of the cases remain unexplained. Two large clusters of microRNAs (miRNAs), namely C14MC (Chromosome 14 microRNA cluster) and C19MC (Chromosome 19 microRNA cluster), are imprinted and expressed in the placenta. Hence, we studied the expression of miRNAs coded by these clusters and their mRNA targets in RSA cases in order to elucidate the involvement of these two clusters in the pathogenesis of RSA. Upon miRNA sequencing, we found a total of seven miRNAs to be differentially expressed in RSA group. Further, using bioinformatics tools such as TargetScan and DAVID pathway analysis, we found that the mRNA targets were involved in the functionally relevant processes of the placenta such as cell migration, cell-cell adhesion, and angiogenesis. Upon validation of sequencing data, we found differential expression of five miRNAs from C14MC and C19MC clusters and differential expression of genes regulating processes such as cell migration, cell adhesion, and angiogenesis in RSA cases which are targeted by these miRNAs.
During recent decades a myriad of approaches have emerged for generating surrogate gametes or achieving in vitro gametogenesis (IVG). The most recent approaches to IVG include the development of increasingly complex culture systems, use of companion cells combined with germ line cells in two- or three-dimensional configurations, derivation of germ line-like cells from pluripotent stem cells, and genetic and chromosomal manipulations of pluripotent stem cells to enable the production of bi-maternal and bi-paternal offspring. This review summarizes the major approaches in IVG, the challenges remaining, and considers the significance of these technologies within the context of departure from exclusively dioecious sexual reproduction for the human species.
The search for male-biased genes that may drive sexual differentiation in sturgeon gonadal tissue has remained largely fruitless until now. This study explored the presence, expression, and sexual steroid regulation of gonadal soma-derived factor (Gsdf) in Siberian sturgeon (Acipenser baerii). Three sequences compatible with the protein-coding gene gsdf were identified in a Siberian sturgeon gonadal transcriptome database. The phylogenetic analysis shows the presence of two paralogues gsdfa and gsdfb, and two clearly identifiable gsdfa isoforms (gsdfa1 and gsdfa2). gsdfa was expressed in sex-undifferentiated gonads, brain, hypophysis, heart, interrenal, intestine, liver, stomach, embryos and larvae, while gsdfb was specific to the gonads. gsdfb showed significantly higher expression in genetically sexed males during late stage of sex differentiation. In contrast, gsdfa1 and gsdfa2 showed similar gonadal expression levels in both sexes. In terms of sensitivity to sexual steroids, only gsdfb was repressed significantly and in a dose-dependent manner by estrogens. Taken together, these results indicate both that gsdfb is part of the male gonadal differentiation pathway in sturgeon and that its repression by estrogens is fundamental for female development.
Diabetes-associated spermatogenic dysfunction remains mechanistically unclear despite its clinical significance. We aimed to elucidate the effect and molecular mechanism of sperm damage induced by type 2 diabetes mellitus (T2DM). In a mouse model of T2DM, transcriptome sequencing identified Art1 as a differentially expressed gene. T2DM induced structural damage to the seminiferous tubules, deregulated sperm parameters, and suppressed Art1 expression in mice and caused reproductive damage in their offspring. Danshensu administration rescued these pathologies. In vitro, high-glucose/high-lipid exposure induced GC-2 cell damage, while Art1 overexpression reduced oxidative stress and apoptosis. Art1-knockout mice exhibited impaired sperm motility and tubule structural defects, confirming Art1's critical role in spermatogenesis maintenance. This study demonstrated that T2DM triggers testicular oxidative stress, apoptosis, and Art1 suppression, leading to transgenerational reproductive dysfunction. Experiments have shown that the overexpression of Danshensu or Art1 can rescue these pathological damages. The results from the Art1-knockout mice confirm the indispensable regulatory role of Art1 in spermatogenesis maintenance. We speculate that T2DM may impair male reproductive ability by inhibiting the expression of Art1, which will provide a new molecular target and theoretical basis for further investigation into the interaction mechanism between T2DM and the reproductive system.
In ungulates, the development of the trophoblast lineage follows a peculiar process, as implantation is preceded by a relatively long period of trophoblast elongation, the molecular control of which is still not well known. Here, we have established novel bovine trophoblast stem cells (b2iTSCs) growing in a completely defined medium containing inhibitors of GSK3b and MEK, and WNT3 during establishment. These cells can grow either as adherent, or as vesicles in suspension. These fluid-filled vesicles show high proliferative capacity and apical polarity. Transcriptome analysis reveals that b2iTSCs are distinct from bovine embryonic fibroblasts and exhibit gene expression profiles related to lipid metabolism, steroid hormone synthesis, and nutrient absorption. The cells also express different Pregnancy Associated Glycoproteins, such as PAG11. Comparative transcriptome analysis shows that 2iTSCs have a signature intermediate between early trophectoderm and elongating trophoblast, expressing canonical trophoblast markers but not IFNt. Additionally, b2iTSCs can differentiate into binucleate cells in vitro and participate in the formation of bovine blastoïds when cocultured with bovine embryonic stem cells, demonstrating their potential for studying trophoblast development and function.
The synthesis and degradation of Cyclins are critical for cell cycle progression. Cyclin K (CCNK), a member of the Cyclin family, is known to regulate transcriptional elongation and the MAPK signaling pathway by binding to CDK12/13. While CCNK has been extensively studied in cancers, its roles in oocyte maturation remains unclear. In this study, we found that overexpression of CCNK accelerates the resumption of meiosis in oocytes. This effect is primarily attributed to the early nuclear entry of Cyclin B1 (CCNB1) and the premature activation of maturation promoting factor. These findings suggest that CCNK plays a vital role in the process of oocyte maturation, and its dysregulation could disrupt the normal progression of meiosis.
Yellowish myotis presented four reproductive stages and long-term sperm storage in cauda epididymis. Aiming to investigate the morphofunctional aspects of the epididymis and spermatozoa of yellowish myotis, 48 adult male bats were captured in Santuário do Caraça, Minas Gerais, Brazil, for histomorphometric evaluations of the epididymis and sperm analysis. Yellowish myotis spermatozoa were observed in the caput and corpus epididymis only during the Mature and Regressed stages, coinciding with increased epithelial height in these stages, indicating their possible role in sperm maturation. In contrast, spermatozoa were found in the cauda epididymis during all reproductive stages, with the epithelium maintaining a consistent height, which suggests that this region is adapted for long-term sperm storage. The spermatozoa remained alive and motile for up to 25 days during the Regressed and Early Rest stages. Mating period in yellowish myotis occurs during the Rest stage, when spermatozoa in the cauda epididymis exhibit reduced DNA fragmentation and minimal chromatin abnormalities. During this period, sperm with wider heads and longer midpieces dominate, reflecting selection for traits that would enhance fertilization success. These results reinforced the importance of cauda epididymis physiology for sperm survival and future events of capacitation and fertilization.
FOXR1 belongs to the large conserved F-box family of DNA binding transcription factors that are involved in various developmental processes and diseases. The gene is important for embryogenesis in a fish model, brain development in mammals, and human FOXR1 was shown to possess oncogenic properties when it is abnormally expressed as a fusion gene. Earlier work on Foxr1 expression in mouse and human suggested roles for the protein in embryogenesis and sexual reproduction. We generated a mouse gene deletion model that revealed an embryonic lethal phenotype with partial penetrance and normal fertility in persistent homozygous male mutants. The results suggest that Foxr1 is functionally redundant in adult male gonads but needed for normal early embryo development and post-natal viability. We discuss our results in the context of publicly available human and rodent genomic and genetic data.
Cumulus-oocyte complexes (COCs) used for in vitro production (IVP) of bovine embryos originate from antral follicles of different sizes, leading to variations in developmental competence. To address this, pre-in vitro maturation (pre-IVM) allows oocytes with additional time to acquire developmental competence. Given the role of follicular fluid-derived extracellular vesicles (EVs) in ovarian follicle communication, which has been shown to vary in content and function across folliculogenesis, we investigated whether EVs from early versus late antral follicles influence COCs during pre-IVM. EV supplementation significantly altered gene expression in cumulus cells and oocytes. In cumulus cells, affected pathways included MAPK signaling, Gap junctions, Cytokine-cytokine receptor interaction, Axon guidance, cAMP, and Cushing syndrome. In oocytes, fewer genes were altered, with effects on Inositol phosphate metabolism, p53 signaling and Cholesterol metabolism. Despite these changes, no significant effects of the EV treatment were noted on oocyte chromatin configuration and developmental competence, except for a significant increase of mitochondrial membrane potential (Δψm) in blastocysts. In conclusion, EV supplementation during pre-IVM significantly altered the transcriptional profile of COCs, with EVs from early follicles modulating the expression of genes regulating cumulus cell proliferation and gap junctions, while EVs from late follicles impacted pathways associated with meiotic resumption, cumulus cell expansion, and apoptosis. Along with improved Δψm in blastocysts, these results support a positive effect of EVs on bovine COCs, but further research is needed to better characterize the functional consequences, mainly in terms of the effects of early versus late follicle-derived EVs on oocyte developmental potential.
This study aimed to assess differences in the immunostaining intensity of follicle-stimulating hormone receptor (FSHr) and leutenizing hormone receptor (LHr) receptors in the ovarian follicles of Bos indicus cows with high or low antral follicle counts (AFCs). Ovaries from cyclic Nelore cows (N = 20) were obtained from a local slaughterhouse and classified based on AFC (≥ 3 mm) into high- (≥ 30 follicles, N = 10) and low- (≤ 15 follicles, N = 10) AFC groups. Immunohistochemical studies were performed for FSHr and LHr. Immunostaining intensity was measured using ImageJ software with the IHC Profiler plugin, and pixel intensity was measured on a scale of 0 (darkest) to 255 (lightest). An interaction was observed between the AFC group and follicular developmental stage for FSHr immunostaining intensity, with preantral follicles from the low-AFC group showing highest immunostaining intensity (p < 0.0001). The FSHr immunostaining intensity of antral follicles from the low-AFC group was higher than that of the high-AFC group (p = 0.03). LHr immunostaining intensity also was higher in the low-AFC group than in the high-AFC group (p = 0.002). These findings suggest that ovarian follicle characteristics of low-AFC cows have distinct characteristics that could affect their response to reproductive treatments.
Embryo genome activation (EGA) is a crucial event implicated in proper embryonic development. Similarly, the DNA Damage Response (DDR) is essential for correcting or preventing occasional errors during embryonic cell division that could result in embryo development arrest or the propagation of genetic abnormalities. Although both EGA and DDR are regulated by epigenetic mechanisms, the role of microRNAs (miRNAs) in these processes has not been explored in pig embryos. Herein, we assessed the abundance of miRNAs linked to embryo development and DDR across four developmental stages: Day 2 (D2), Day 3 (D3), and Day 4 (D4), and at the blastocyst stage, as well as after DNA damage induction. mRNA levels of EGA-related genes confirmed our timepoints as representing EGA timeframe, while immunofluorescence for γH2AX validated DNA damage induction. Significant decrease in blastocyst rate and total cell number per blastocyst was detected in UV-exposed embryos. Analysis of miRNA abundance revealed increased levels of miR-200a-5p on D3, which were partially maintained on D4. Both miR-15a and miR-24-3p increased on D4, but their levels were downregulated in UV-exposed embryos at the same stage of development. In blastocysts, UV exposure upregulated miR-29a-3p and miR-344b-3p. Together, these findings provide the first characterization of miRNAs expression in porcine embryos during EGA and following DNA damage induction.
To identify the optimal in vitro maturation (IVM) duration for bovine oocytes with different nuclear maturation speeds (NMS), this study assessed how varying IVM durations (24, 28, and 32 h) affect developmental competence and embryo quality in oocytes with fast- or slow-predicted NMS classified via machine learning. Developmental competence was evaluated through cleavage rates, first cleavage timing and patterns, and blastocyst formation under individual culture. Embryo quality was assessed via differential staining of inner cell mass and trophectoderm and expression analysis of quality-related genes in formed blastocysts. For oocytes with slow-predicted NMS, extending IVM to 28 h increased cleavage rates and accelerated first cleavage timing (p < 0.01). The lower blastocyst formation rates of oocytes with slow-predicted NMS matured for 24 h improved when IVM reached 28 h, becoming comparable to fast-predicted NMS oocytes. However, extended IVM decreased expression of pluripotency-related genes (e.g., NANOG and OCT4; p < 0.01) regardless of predicted NMS. In conclusion, extending IVM duration to 28 h improved developmental competence of slow-predicted NMS oocytes, highlighting the importance of fertilization timing relative to nuclear maturation completion, though it reduced expression of key pluripotency genes. Individualized IVM protocols based on predicted NMS can enhance bovine embryo production efficiency.
Dairy cows often experience a period of negative energy balance (NEB) during the post-calving period, which can significantly impact economic outcomes due to extended calving-to-conception intervals and overall reduced fertility. This reduction is due, in part, to the impact on uterine biology by high nonesterified fatty acids (NEFA) and beta-hydroxybutyrate concentration. The uterine fluid (UF) contains small extracellular vesicles (UF-EVs) that, through their cargo, including microRNAs (miRNAs), respond to metabolic stress, affecting the uterine environment. This study aimed to assess the long-term impact of NEB intensity on the uterine environment of dairy cows. Post-partum dairy cows were classified based on NEFA concentrations in their blood during the 3 weeks post-calving as having either Low or High NEB. At 30 and 60 DPC, the synchronization protocol was started, and UF samples were collected (corresponding to ~15 days after initiation of the synchronization protocol) to isolate UF-EVs and uterine epithelial cells for miRNA and transcriptome profiling. We also investigated whether UF-EVs could modulate epithelial uterine naïve cells. Our results indicate that the uterine environment of dairy cows experiencing a High NEB post-calving is unfavorable for embryo development at 60-day post-calving. Importantly, we show that UF-EVs can reproduce this phenotype in epithelial uterine naïve cells, suggesting that UF-EVs may act as modulators of the uterine response to metabolic challenges.
