Biology of Reproduction最新文献

This study aimed to understand the physiological mechanisms regulating parturition and to identify potential biomarkers to predict onset of birth. Additionally, we compared hormone profiles between cows with shorter and longer gestation lengths. Twenty-eight days before due date until 3d postpartum, cows (n = 18) were blood-sampled daily. Circulating concentrations were measured for progesterone and estradiol by RIA, testosterone, prostaglandin F2α metabolite, cortisol, pregnancy-specific protein B by enzyme-linked immunosorbent assay, and lactate concentrations by colorimetric assay. At end of gestation, progesterone decreased from d-14 to d-4 (from 3.6 to 1.4 ng/mL), most likely from rapid loss of placental progesterone production (64% of decline in 24 h). A second rapid decrease in progesterone to undetectable concentrations was observed from d-2 to parturition (from 1.4 to 0.1 ng/ml; most likely luteal origin) corresponding to increase in prostaglandin F2α metabolite from d-2 to parturition (249.7 to 2868.4 pg/mL). E2 and pregnancy-specific protein B increased ~8-fold from ~13d before parturition with acute rise in E2 but not pregnancy-specific protein B (45% vs. 13% in first 24 h). Testosterone decreased slightly during the same period. Cortisol and lactate increased only at calving. Comparison of cows with shorter vs. longer gestation, when data were normalized to parturition day, a difference was detected in circulating E2 and prostaglandin F2α metabolite patterns, but not progesterone and pregnancy-specific protein B. Thus, the first significant hormonal changes associated with parturition begin at d-14 with E2 and pregnancy-specific protein B as two clear biomarkers of impending parturition. Cows with shorter and longer gestation had hormonal differences indicative of identifiable earlier placental maturation.

Although meiosis plays an essential role for the survival of species in natural selection, the genetic diversity resulting from sexual reproduction impedes human-driven strategies to transmit the most suitable genomes for genetic improvement, forcing breeders to select diploid genomes generated after fertilization, that is, after the encounter of sperm and oocytes carrying unknown genomes. To determine whether genomic assessment could be used before fertilization, some androgenetic haploid morula-stage bovine embryos derived from individual sperm were biopsied for genomic evaluation and others used to reconstruct "semi-cloned" (SC) diploid zygotes by the intracytoplasmic injection into parthenogenetically activated oocytes, and the resulting embryos were transferred to surrogate females to obtain gestations. Compared to controls, in vitro development to the blastocyst stage was lower and fewer surrogates became pregnant from the transfer of SC embryos. However, fetometric measurements of organs and placental membranes of all SC conceptuses were similar to controls, suggesting a normal post-implantation development. Moreover, transcript amounts of imprinted genes IGF2, IGF2R, PHLDA2, SNRPN, and KCNQ1OT1 and methylation pattern of the KCNQ1 DMR were unaltered in SC conceptuses. Overall, this study shows that sperm can be replaced by genotyped haploid embryonic-derived cells to produce bovine embryos carrying a predetermined paternal genome and viable first trimester fetuses after transfer to female recipients.

Summary sentence: Haploid morula-stage embryonic cells derived from a single sperm can be genotyped and injected into activated oocytes to reconstruct diploid zygotes that develop both in vitro into blastocysts and in vivo into viable post-implantation bovine conceptuses with predetermined paternal genomes.

Dcaf17, also known as DDB1- and CUL4-associated factor 17, is a member of the DCAF family and acts as the receptor for the CRL4 ubiquitin E3 ligase complex. Several previous studies have reported that mutations in Dcaf17 cause Woodhouse-Sakati syndrome, which results in oligoasthenoteratozoospermia and male infertility. As a model to explore the role of Dcaf17 in the male reproductive system, we created Dcaf17-deficient male golden hamsters using CRISPR-Cas9 technology; the results of which demonstrate that deletion of Dcaf17 led to abnormal spermatogenesis and infertility. To uncover the underlying molecular mechanisms involved, we conducted single cell Ribonucleic Acid sequencing analysis to evaluate the effect of Dcaf17 deficiency on transcriptional levels in spermatogenic cells during various stages of spermatogenesis. These data emphasize the significant regulatory role played by Dcaf17 in early spermatogenic cells, with many biological processes being affected, including spermatogenesis and protein degradation. Dysregulation of genes associated with these functions ultimately leads to abnormalities. In summary, our findings highlight the critical function of Dcaf17 in spermatogenesis and clarify the specific stage at which Dcaf17 exerts its effects, while simultaneously providing a novel animal model for the study of Dcaf17.

Endocrine-disrupting chemicals are natural and synthetic compounds found ubiquitously in the environment that interfere with the hormonal-immune axis, potentially impacting human health and reproduction. Exposure to endocrine-disrupting chemicals has been associated with numerous health risks, such as neurodevelopmental disorders, metabolic syndrome, thyroid dysfunction, infertility, and cancers. Nevertheless, the current approach to establishing causality between these substances and disease outcomes has limitations. Epidemiological and experimental research on endocrine-disrupting chemicals faces challenges in accurately assessing chemical exposure and interpreting non-monotonic dose response curves. In addition, most studies have focused on single chemicals or simple mixtures, overlooking complex real-life exposures and mechanistic insights, in particular regarding endocrine-disrupting chemicals' impact on the immune system. The ENDOMIX project, funded by the EU's Horizon Health Program, addresses these challenges by integrating epidemiological, risk assessment, and immunotoxicology methodologies. This systemic approach comprises the triangulation of human cohort, in vitro, and in vivo data to determine the combined effects of chemical mixtures. The present review presents and discusses current literature regarding human reproduction in the context of immunotolerance and chemical disruption mode of action. It further underscores the ENDOMIX perspective to elucidate the impact of endocrine-disrupting chemicals on immune-reproductive health.

Environmental hypoxia adversely impacts the reproduction of humans and animals. Previously, we showed that fetal hypoxia exposure led to granulosa cell (GC) autophagic cell death via the Foxo1/Pi3k/Akt pathway. However, the upstream regulatory mechanisms underlying GC dysfunction remain largely unexplored. Here, we tested the hypothesis that fetal hypoxia exposure altered gene expression programs in adult GCs and impaired ovarian function. We established a fetal hypoxia model in which pregnant mice were maintained in a high-plateau hypoxic environment from gestation day (E) 0-16.5 to study the impact of hypoxia exposure on the ovarian development and subsequent fertility of offspring. Compared with the unexposed control, fetal hypoxia impaired fertility by disordering ovarian function. Specifically, fetal hypoxia caused mitochondrial dysfunction, oxidant stress, and autophagy in GCs in the adult ovary. RNA sequencing analysis revealed that 437 genes were differentially expressed in the adult GCs of exposed animals. Western blotting results also revealed that fetal exposure induced high levels of hypoxia-inducible factor 1-alpha (Hif1a) expression in adult GCs. We then treated granulosa cells isolated from exposed mice with PX-478, a specific pharmacological inhibitor of Hif1a, and found that autophagy and apoptosis were effectively alleviated. Finally, by using a human ovarian granulosa-like tumor cell line (KGN) to simulate hypoxia in vitro, we showed that Hif1a regulated autophagic cell death in GCs through the Pi3k/Akt pathway. Together, these findings suggest that fetal hypoxia exposure induced persistent Hif1a expression, which impaired mitochondrial function and led to autophagic cell death in the GCs of the adult ovary.

Uterine infections cause ovarian dysfunction and infertility. The bacterial endotoxin, lipopolysaccharide (LPS), accumulates in the follicular fluid of dominant follicles of cows with uterine infections. Granulosa cells produce an innate inflammatory response to LPS, altering the follicular microenvironment of the oocyte. We hypothesized that developmental competence and embryo quality would be reduced when oocytes are matured in an inflammatory environment. Bovine mural granulosa cells were exposed to either 1 μg/mL of LPS or medium alone for 24 h to produce conditioned medium. Inflammatory responses of mural granulosa cells were confirmed by increased expression of CXCL8, IL1B, IL6 and TNF. Bovine cumulus-oocyte complexes were matured for 22 ± 1 h in medium supplemented with either 1 μg/mL of LPS, 10% v/v conditioned medium of granulosa cells treated with either LPS (LCM) or medium alone (CCM), or no supplementation (CON). In addition, polymyxin B (20 μg/mL) was added to maturation medium to sequester LPS. Following maturation, cumulus-oocyte complexes were fertilized and cultured for 7.5 days with no further treatment. Oocyte maturation using LPS or LCM impaired development to the blastocysts stage, reduced the number of total and CDX2 negative blastomeres and increased TUNEL positive cells in blastocysts. Polymyxin B could rescue these effects in the LPS group but not in the LCM group, indicating factors produced by granulosa cells and not LPS alone compromised oocyte development. These findings suggest that the inflammatory milieu produced by granulosa cells in response to LPS impairs oocyte competence and quality of resultant blastocyst-stage embryos.

The accurate diagnosis of non-obstructive azoospermia and obstructive azoospermia is crucial for selecting appropriate clinical treatments. This study aimed to investigate the pivotal role of microRNAs in circulating plasma extracellular vesicles in distinguishing between non-obstructive azoospermia and obstructive azoospermia, as well as uncovering the signaling pathways involved in azoospermia pathogenesis. In this study, differential expression of extracellular vesicle miR-513c-5p and miR-202-5p was observed between non-obstructive azoospermia and obstructive azoospermia patients, while the selenocompound metabolism pathway could be affected in azoospermia through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. The predictive power of these microRNAs was evaluated using receiver characteristic operator-area under the curve analysis, demonstrating promising sensitivity, specificity, and area under the curve values. A binomial regression equation incorporating circulating plasma levels of extracellular vesicles miR-202-5p and miR-513c-5p along with follicle-stimulating hormone was calculated to provide a clinically applicable method for diagnosing non-obstructive azoospermia and obstructive azoospermia. This study presents a potentially non-invasive testing approach for distinguishing between non-obstructive azoospermia and obstructive azoospermia, offering a possibly valuable tool for clinical practice.

In cattle, oviductal function is controlled by the ovarian sex-steroids estradiol and progesterone. Here, we tested the hypothesis that the exposure to contrasting sex-steroid milieus differentially impacts the oviductal fluid composition. Estrous cycles of non-lactating, multiparous Nelore cows were pre-synchronized and then synchronized with a protocol designed two induce ovulation of large or small follciles. Larger preovulatory follicle (day 0) and corpora lutea (day 4) and greater estradiol (day 0) and progesterone (day 4) concentrations were observed in the large follciles group. Four days after induced ovulation, oviductal fluid was collected post-mortem. Quantitative mass spectrometry was used to determine the concentration of amino acids, biogenic amines, acylcarnitines, phosphatidylcholines, lysophosphatidylcholines, sphingomyelins, hexoses, prostaglandins, and related compounds. Multivariate analyses (orthogonal projections to latent structures discriminant analysis) were conducted to compare the metabolomic signatures of oviductal fluids. Correlation network analysis was conducted to measure the strength and hierarchy of associations among metabolites. Of the 205 metabolites quantified, 171 were detected in at least 50% of the samples and were included in further data analysis. After orthogonal projections to latent structures discriminant analysis analysis, samples of the large follciles and small follciles were divided clearly into two non-overlapping clusters. Twenty metabolites had different or tended to have different concentrations in the oviductal fluid when comparing groups. Seven of these 20 analytes had greater concentrations in large follciles cows. Moreover, total sum of biogenic amines, phosphatidylcholines, and prostaglandins were higher in the small follciles group. The correlation network showed that the large follciles group metabolites' concentrations were highly intercorrelated, which was not observed in the small follciles group. We concluded that the periovulatory endocrine milieu regulates the composition of the oviductal fluid.

An increase in global infertility has coincided with the accumulation of microplastics (MPs) in the environment. This trend is particularly troubling because only 10% of male infertility cases can be attributed to identifiable causes, leaving a knowledge gap in our understanding of their underlying factors. To bridge this, it is important to explore the connection between the accumulation of MPs and the observed decline in male fertility. We assessed the presence of microplastics in epididymal sperm from bulls and used it as baseline concentrations for sperm exposure. MPs were detected in all epidydimal sperm (ES) samples, with a mean concentration of 0.37 μg mL-1. Next, to investigate the effects of MPs on fertility, bovine sperm was exposed to three different concentrations of a mixture of 1.1, 0.5, and 0.3 μm polystyrene (PS) beads: (1) 0.7 μg mL-1, blood concentration of PS in cows (bPS); (2) 0.37 μg mL-1, based on the concentration of total MPs found in ES (esMP); and (3) 0.026 μg mL-1, based on the concentration of PS found in ES (esPS). All sperm samples incubated with PS exhibited reduced motility compared with the control at 0.5 h. However, PS exposure did not affect acrosome integrity or induced oxidative stress. Embryos produced from sperm exposed to PS had reduced blastocyst rates, in addition to increased ROS formation and apoptosis. By employing physiological exposure, this research provided evidence of MPs in bovine epididymal sperm and demonstrated the detrimental effect of PS on sperm functionality.

Isthmus is the region of the oviduct considered a reservoir for spermatozoa, where they are retained and released synchronously with ovulation. Integrins mediate this interaction, and it is suggested that they regulate the viability and capacitation of spermatozoa. Spermatozoa retained in the oviductal epithelial cells show specific characteristics: normal morphology, intact acrosome and plasma membrane, no DNA fragmentation, and low levels of intracellular Ca2+, and protein phosphorylation at Tyr. This work aimed to define spermatozoa's ability to adhere to an immobilized fibronectin matrix and its effects on their viability and capacitation. We found that guinea pig spermatozoa showed a high affinity for adhering to an immobilized fibronectin matrix but not to those made up of type 1 collagen or laminin. This interaction was mediated by integrins that recognize the RGD domain. Spermatozoa adhered to an immobilized fibronectin matrix were maintained in a state of low capacitation: low levels of intracellular concentration of Ca2+, protein phosphorylation in Tyr, and F-actin. Also, spermatozoa kept their plasma membrane and acrosome intact, flagellum beating and showed low activation of caspases 3/7. The spermatozoa adhered to the immobilized fibronectin matrix, gradually detached, forming rosettes and did not undergo a spontaneous acrosomal reaction but were capable of experiencing a progesterone-induced acrosomal reaction. In conclusion, the adhesion of spermatozoa to an immobilized fibronectin matrix alters the physiology of the spermatozoa, keeping them in a steady state of capacitation, increasing their viability in a similar way to what was reported for spermatozoa adhered to oviductal epithelial cells.