This study aimed to clarify the association between the percentage of follicle number by size over antral follicle count (AFC) and subsequent reproductive performance. A total of 306 Japanese Black cattle underwent timed artificial insemination (TAI) 41-62 days postpartum; the AFC and numbers of small, medium, and large follicles were recorded 10 days before TAI. The cross-sectional and blood flow areas of the dominant follicle (DF) on the day of TAI and the corpus luteum (CL) six days after TAI were recorded. The total number of follicles ≥ 2 mm was defined as the AFC, and the percentages of follicle number by each size defined as small (S-AFC%; 2-2.9 mm), medium (M-AFC%; 3-8.4 mm), and large (L-AFC%; ≥ 8.5 mm) follicles. The AFC and S-, M-, and L-AFC% were further grouped into low, medium, and high tertiles, and the subsequent reproductive performance compared among the groups. Plasma anti-Müllerian hormone (AMH) levels were quantified on the day of AFC measurement. No differences were observed in reproductive performance between the AFC and L-AFC% groups. The high-S-AFC% group showed a 20.6% lower conception rate, 0.58 more AI numbers, and 21.9 longer days open than those of the low-S-AFC% group (P < 0.05). The low-M-AFC% group showed an 18.0% lower conception rate after TAI and 0.54 more AI numbers than those of the high-M-AFC% group (P < 0.05). DF and CL parameters did not differ among the AFC, S-, M-, and L-AFC% groups. Plasma AMH levels in the low-AFC group were the lowest in the tertile. In conclusion, the percentage of follicles by size could be used to estimate subsequent reproductive performance.
Assisted reproductive technologies (ART) to generate developmentally competent oocytes necessitates light exposure due to the use of microscopes. Previous studies in several species have reported that the wavelength of light during the light exposure period is a critical factor in embryo development. However, the effects of different light wavelengths on embryo development in pigs remain unexplored. This study aimed to identify the optimal light conditions to enhance oocyte maturation, parthenogenetic activation of mature oocytes, and pre-implantation development of parthenogenetic embryos in pigs. Conducted irradiation experiments during in vitro maturation (IVM), parthenogenesis (PG), and in vitro culture (IVC) using visible (390-750 nm), blue (445-500 nm), green (500-575 nm), yellow (575-585 nm), and red (620-750 nm) light. Variation in light wavelengths during IVM, PG, or IVC did not significantly influence oocyte maturation of cumulus-oocyte complexes (COCs) derived from median antral follicles (MAFs), developmental competence of in vitro-culture COCs after PG, and the production of blastocysts. However, continuous irradiation with green light throughout the entire process (IVM, PG, and IVC) significantly enhanced in vitro blastocyst production, and the resulting blastocysts showed significantly elevated HSP70 expression and a numerical increase in PCNA expression. We recommend conducting all in vitro procedures (IVM, PG, and IVC) for producing blastocysts from MAFs in porcine models under green light. This study will contribute to yielding higher success rates of porcine ART and reduce artificial stress to oocytes and embryos caused by in vitro manipulation under strong light exposure.
During mouse preimplantation development, zygotic genome activation (ZGA), which synthesizes new transcripts in the embryo, occurs during the 1-cell to 2-cell stage. Embryos at the 1- and 2-cell stages are totipotent, and as embryonic development progresses, their differentiation potential decreases, and the embryos become pluripotent. However, the roles of genes expressed during ZGA in mouse embryonic differentiation remain incompletely understood. Here, we show that periodic tryptophan protein 1 (Pwp1), a WD-repeat protein, is expressed from the ZGA and controls embryonic differentiation at later stages. Developmental potential was reduced when siRNAs or antisense oligonucleotides targeting Pwp1 were introduced into 1-cell stage mouse embryos. Further, Pwp1 knockdown resulted in irregular localization of YAP1 at the morula stage, upregulation of the inner cell mass marker Nanog, and downregulation of the trophectoderm marker Cdx2 at the blastocyst stage. Transcriptome analysis showed that Pwp1 knockdown upregulated ZGA gene expression at the morula stage. Because Pwp1 contributes to H4K20me3 histone modification, these results suggest that Pwp1 is required for mouse preimplantation development to control differentiation-associated genes via H4K20me3 modification. Elucidating the role of Pwp1 in embryonic differentiation is expected to contribute toward the advancement of assisted reproductive technologies.
The enzymatic isolation of preantral follicles (PAFs) is considered the most efficient method for retrieving a large number of intact follicles, offering significant advantages in terms of yield and processing time. However, the low success rate of enzymatically isolated follicles in long-term culture raises concerns regarding their impact on oocyte quality and developmental potential. This study addresses a critical gap in understanding how enzymatic retrieval of PAFs affects the oocyte-granulosa cell connection and its relationship with high mortality and culture failure observed during in vitro growth (IVG). By systematically comparing crude collagenases (IA and IV) and purified collagenases (Liberase TM and DH) with a mechanical isolation protocol, we identified the optimal enzyme concentrations that maximize follicle yield while minimizing cellular damage. Our results reveal that the enzymatic retrieval of PAFs corresponds to the loss of transzonal projections (TZPs) post-isolation, as well as premature oocyte extrusion and follicle deformities during IVG. Our findings also highlight the differential apoptotic responses in oocytes and granulosa cells. Although these enzymes sustain follicle cell integrity, they compromise oocyte viability during isolation. Notably, crude collagenases impair oocyte growth during prolonged culture, whereas purified collagenases preserve the developmental potential of oocytes. This study also provides the first evidence that enzymatic isolation of PAFs adversely affects TZPs. Overall, our study highlights the importance of selecting an appropriate method, enzyme type, and concentration for preserving the integrity of oocytes, follicles, and their connections, thereby supporting successful in vitro culture. Additionally, our results suggest that mechanical protocols and high-purity enzymes are preferred for maintaining oocyte competence.
Exposure of cumulus-oocyte complexes (COCs), mature oocytes, and preimplantation embryos to subphysiological temperatures during in vitro maturation (IVM), parthenogenesis (PG), and in vitro culture (IVC) has been shown to impair oocyte maturation, embryogenesis, and developmental competence. However, most previous studies have focused primarily on defining optimal embryo culture temperatures. Therefore, the present study aimed to establish optimal temperature conditions for in vitro manipulation, to minimize impairment of oocyte maturation, embryogenesis, and preimplantation development. All in vitro manipulation steps during the IVM, PG, and IVC processes were conducted using manipulation media maintained at defined temperatures (39°C, 38°C, 37°C, 35°C, 34°C, and 33°C) through the combined use of microscope warming plates and infrared irradiation. Oocyte maturation and the preimplantation developmental competence of embryos following parthenogenetic activation were subsequently evaluated. The use of infrared irradiation to control manipulation medium temperature precisely did not adversely affect oocyte maturation or embryonic developmental competence. Among the subphysiological temperature conditions examined (33°C-38°C), maintaining the in vitro manipulation media at 35°C throughout the IVM, PG, and IVC processes resulted in significantly reduced perivitelline space size, increased intra-ooplasmic glutathione levels, improved mitochondrial distribution normality, and enhanced preimplantation development compared with manipulation at 39°C, the physiological body temperature of pigs. Based on these results, we suggest that in vitro manipulation of immature COCs, oocytes, and embryos in pigs should be performed using manipulation media maintained at 35°C.
Improving and sustaining the health and productivity of dairy cows require integrated strategies that combine management and appropriate nutrition. This review adopts two complementary perspectives: optimizing periparturient management to support high productivity without compromising animal health and improving gestational nutrition to produce healthy replacement heifers. Several studies have examined how maternal nutritional and metabolic status before and after calving influence postpartum reproduction, productivity, placental development, and offspring health. Prepartum energy status is a key determinant of early postpartum ovarian function, with a timely first ovulation associated with adequate insulin-like growth factor-I exposure during follicular development. Insufficient β-carotene intake in energy-prioritized feeding systems adversely affects ovarian resumption. Uterine recovery is also nutrition-sensitive; cows with endometritis exhibit signs of impaired liver function, whereas delayed uterine involution is associated with inefficient ruminal nitrogen utilization. Supplementation with lysine, the first limiting amino acid in corn-based diets, further promotes uterine recovery. From a developmental perspective, maternal insulin resistance is associated with low birth weight and altered endocrine profiles in calves, which are indicative of long-term metabolic vulnerability. In contrast, lysine supplementation during the dry period supports neonatal protein synthesis and growth. Furthermore, in growing primiparous cows, greater milk yield during early pregnancy was associated with reduced anti-Müllerian hormone concentrations in female calves, suggesting a lower ovarian reserve. Collectively, these findings highlight the importance of gestational and peripartum nutritional management not only for improving dairy cow fertility and productivity but also for optimizing the health and reproductive potential of the next generation.
Freeze-dried (FD) somatic cells serve as a novel method for preserving animal genetic resources; however, the efficiency of cloned mice production from FD somatic cells remains low, owing to severe nuclear damage induced by the freeze-drying. In this study, we aimed to mitigate FD-induced damage by evaluating protectants that have been reported to exert protective effects during freeze-drying of microorganisms, and to identify those most effective for somatic cells. Results showed, that using Tris-EGTA as the basal medium for freeze-drying, together with monosodium glutamate (MSG) as a protective agent, significantly reduced DNA damage in FD somatic cells after injection into oocytes. Subsequent optimization of MSG concentration revealed that the addition of 3% MSG markedly increased the formation rate of premature chromosome condensation from 50.0% to 80.4% compared with the non-MSG condition. Moreover, the rate of normal chromosome segregation at the two-cell stage of cloned embryos increased from 0% to 20.0%. Furthermore, although no blastocysts were obtained in the absence of MSG, the addition of 3% MSG enabled the formation of morphologically good-quality blastocysts, albeit at a low frequency. These findings indicated that the addition of 3% MSG reduces DNA damage in FD somatic cells and improves the developmental competence of somatic cell nuclear transfer embryos, representing the first step toward the practical application of FD somatic cells as genetic resources.
Malonic acid (MA) is a dicarboxylic acid that inhibits the mitochondrial electron transport chain. Cumulus cells surrounding human oocytes that achieve conception after in vitro fertilization (IVF) contain higher levels of MA than those that do not. Although MA has no effect on embryo developmental culture, the effects of MA on cumulus cells and oocyte quality remain unclear. In this study, the addition of 10 μM MA to the in vitro maturation (IVM) medium cultured cumulus cell-oocyte complexes improved embryo production efficiency, meeting multiple conception-related criteria after IVF (P < 0.05). MA did not affect the nuclear maturation rate of oocytes after IVM; however, it increased lipid content and normal alignment ratio of cortical granules (P < 0.05), both of which are indicators of cytoplasmic maturation of oocytes. MA also increased the adenosine triphosphate (ATP) content in oocytes only when they were present with cumulus cells (P < 0.05). MA decreased mitochondrial membrane potential activity in cumulus cells but increased the ATP content and enhanced glycolytic activity (P < 0.05). The use of a glycolysis inhibitor resulted in a decrease in the ATP content of both cumulus cells and oocytes and reduced the amount of lipids in oocytes (P < 0.05). These results indicate that MA shifts ATP production in cumulus cells from the mitochondrial to the glycolytic pathway, resulting in improved ATP synthesis. Furthermore, an increased ATP supply from cumulus cells to oocytes promotes oocyte cytoplasmic maturation, leading to improved quality of IVF embryos.
Advanced paternal age affects embryonic development and offspring phenotypes. We previously demonstrated that resveratrol prevents age-associated declines in the mitochondrial DNA copy number (mt-cn) and telomere length (TL) in embryos. The present study was performed to investigate the effects of paternal resveratrol intake on the mt-cn and TL in pups and embryos produced either via in vitro fertilization (IVF) or IVF using sperm preincubated in epididymal fluid (EF). C57BL/6N male mice were administered drinking water containing either the vehicle (ethanol, 1/2500) or 0.1 mM resveratrol. When these males were mated with young ICR females, paternal resveratrol treatment did not affect the mt-cn or TL in offspring derived from young fathers (16-25 weeks of age). In contrast, in offspring of aged fathers (41-51 weeks of age), the mt-cn and TL in heart tissue were altered in a sex-dependent manner. Moreover, continuous resveratrol administration to males until an advanced paternal age resulted in sperm TL elongation. However, resveratrol treatment did not affect the TL in embryos but significantly increased the mt-cn and reduced the lipid content in blastocysts produced via IVF using oocytes from young females (4 weeks of age). RNA- sequencing revealed that resveratrol treatment affected metabolic pathways, including lipid metabolism. The preincubation of sperm from young untreated males (11 weeks of age) in EF derived from resveratrol-treated males did not affect the mt-cn or TL in blastocysts but reduced the lipid content. In conclusion, paternal resveratrol intake modulates embryonic and offspring phenotypes, potentially through alterations in sperm TL, sperm epigenetic modifications, and bioactive components in EF. Resveratrol intake may exert long-term effects on offspring.
Although it is well-known that sperm tyrosine-phosphorylated proteins increase in a cyclic AMP (cAMP)-dependent manner during capacitation, it remains unclear whether or not this reaction is involved directly in sperm fertilization-related events. This study aimed to examine the effects of pharmacological suppression of cAMP-dependent increases in tyrosine-phosphorylated proteins on the induction of full-type hyperactivation and penetration into eggs in boar spermatozoa. Ejaculated spermatozoa were treated with a cAMP analog (cBiMPS) and subsequently used for Western blotting, flagellar beating assays, and egg penetration assays. Marked increases in tyrosine-phosphorylated proteins and full-type hyperactivation were observed in spermatozoa treated with cBiMPS in the presence of CaCl2. These spermatozoa effectively penetrated eggs. A spleen tyrosine kinase (SYK) inhibitor OXSI2 suppressed large cBiMPS-dependent increases in sperm tyrosine-phosphorylated proteins, in contrast to the ineffectiveness of SU6656 (a SRC family kinase inhibitor) and dasatinib (a c-ABL inhibitor). However, this suppression was not associated with changes in the state of full-type hyperactivation or the outcomes of egg penetration assays conducted in insemination medium without OXSI2. Conversely, adding OXSI2 to both the cBiMPS treatment medium and the sperm insemination medium suppressed increases in sperm tyrosine-phosphorylated proteins during insemination and significantly decreased the average number of spermatozoa that penetrated eggs. These results suggest that high SYK activation and large cAMP-dependent increases in tyrosine-phosphorylated proteins are not absolutely necessary for inducing full-type hyperactivation and penetration into eggs in boar spermatozoa in vitro, but may influence sperm functions associated with the outcomes of egg penetration assays.
The 10th outbreak of Ebola Virus Disease (EVD) in the Democratic Republic of the Congo (DRC) in 2018-2020 was the largest in DRC's history and the second largest worldwide. Different strategic response plans (SRPs) were implemented, and the outbreak was eventually stopped after a large scale-up of operations with the SRP 4, which benefited from all public health measures deployed during SRPs 1-3, upon which it developed a more holistic approach including community engagement, logistics and security. We used modelling to characterise EVD transmission and assess the epidemiological impact of the two main response strategies (SRPs 1-3 vs SRP 4). We simulated potential future epidemics with different intervention scenarios, combined with a costing model to evaluate the incremental cost-effectiveness of different strategies. We estimated a mean effective reproduction number R of 1.19 (credible interval (95% CrI) = (1.13 ; 1.25)). The spatial spread was moderate with an average 4.4% (95% CrI = (3.5%; 5.4%)) of transmissions moving to different health zones. The scale-up of operations in SRP 4 coincided with a threefold reduction in transmission, and 30% faster control of EVD waves. In simulations, SRP 4 appears cost-saving, although most simulated outbreaks remain small even with SRPs 1-3. Most EVD outbreaks are expected to be small and can be contained with SRPs 1-3. In outbreaks with increased transmissibility or in the presence of insecurity, rapid scale-up to SRP 4 is likely to save lives and be cost-effective.
The cauda epididymis protects and stores mature sperm in mammals. Recently, comprehensive transcriptomic and proteomic analyses have been conducted to understand its molecular functions; however, fundamental information on its metabolites has not been reported. In this study, we optimized a system for the comprehensive metabolic analysis of the cauda epididymis in mature and juvenile mice. This system identified 116 and 92 metabolites in mature and juvenile mice, respectively. Comparative analysis revealed that 44 and 13 metabolites were upregulated and downregulated, respectively, in the cauda epididymis of mature and juvenile mice. Based on the identified metabolites, 34 metabolic and unique pathways (mature: four pathways and juvenile: one pathway) were determined. In conclusion, the levels of certain metabolites in the cauda epididymis differed between mature and juvenile mice. These results contribute to understanding of the unique functions of the cauda epididymis based on dynamic changes in metabolites.
Reduced glutathione (GSH), a thiol compound, plays an important role in protecting sperm from excessive levels of reactive oxygen species. Our previous study demonstrated that GSH supplementation was associated with improved in vitro fertilization (IVF) outcomes. However, GSH supplementation also increased the proportion of DNA-damaged sperm detected in the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, indicating involvement of its thiol components. In this study, we investigated the effects of several thiol compounds, including l-cysteine (Cys), N-acetyl-l-cysteine (NAC), and dithiothreitol (DTT), on bovine sperm DNA integrity. The addition of Cys or NAC (0.5, 1, or 5 mM) to the medium caused an increase in DNA damage, which was observed either throughout the sperm head or specifically in the post-acrosome (PA) region. DTT treatment increased DNA damage in both the PA and acrosomal regions, and it induced progressive sperm head lysis from the acrosome to the entire head. These treatments caused sperm DNA-damage rates that exceeded the normal range, suggesting that thiol groups enhance the detection of DNA damage. The effects of these thiols on IVF outcomes were further examined. Short-term exposure to 1 mM Cys before insemination improved cleavage rates, whereas prolonged exposure at concentrations ≥ 1 mM during insemination reduced cleavage rates. NAC supplementation had no significant effects. A mild negative correlation was observed between TUNEL-positive sperm rates and both cleavage and blastocyst formation rates following short-term treatment with 0.5 mM Cys. These findings suggest that thiol supplementation, when optimally dosed and timed, may enhance the sensitivity of DNA-damage detection rather than induce additional damage.
A thin endometrium can lead to low clinical pregnancy rates, low live birth rates, high spontaneous abortion rates, and low birth weight. However, current methods of treating thin endometria do not achieve ideal results. This study explored the effect of Indian Hedgehog (IHH) on thin endometrium and its mechanism of action. A thin endometrial rat model was established by infusion of 95% ethanol. IHH was overexpressed in model rats using adeno-associated viruses. The endometrial thickness and number of glands and vessels were determined using H&E staining. Endometrial fibrosis was detected using Masson's trichrome staining. Immunohistochemistry was performed to detect α-SMA, MUC-1, and CK19. After modeling, the rats were mated, and the number of gestational sacs was counted for fertility assessment. Western blotting was used to detect the angiogenesis markers vWF, PCNA, and vim and Hedgehog signaling-related proteins SMO, GLI1, and GLI3. IHH overexpression reduced ethanol-induced edema and bruising, repaired the appearance of damaged tissue, increased endometrial thickness, promoted glandular and vascular regeneration, and alleviated endometrial fibrosis. IHH overexpression inhibited the expression of fibroblast marker α-SMA while promoting the expression of vWF, PCNA, vim, CK19, and MUC-1. It also increased the number of gestational sacs and promoted the expression of SMO, GLI1, and GLI3. In conclusion, IHH ameliorates ethanol-induced thin endometrium and improves fertility by activating the Hedgehog signaling pathway.
Genomic selection using genomic estimated breeding values (GEBVs) based on single nucleotide polymorphism (SNP) genotyping has been widely adopted for efficient breeding of cattle. Recent advances in DNA analysis technologies have enabled comprehensive SNP genotyping using a small number of cells. Implementation of a preimplantation genomic selection (PGS) system, wherein GEBVs are calculated from a limited number of embryonic cells, can further enhance breeding efficiency through the selective production of genetically superior animals by embryo transfer. In addition, the use of bovine embryos produced by in vitro fertilization (IVF) has increased worldwide in recent years. However, the implantation potential of bovine IVF embryos is lower than that of in vivo-derived (IVD) embryos, and aberrant epigenetic modifications and gene expression are considered major contributing factors to the reduced developmental competence of bovine IVF embryos. Therefore, the differences in the gene expression characteristics of bovine IVD and IVF embryos need to be better understood for improved bovine in vitro production (IVP) systems. This review outlines the development and applications of PGS systems using bovine blastocysts, elongating conceptuses, and demi-embryos derived from blastomere separation. It also discusses the utility of gene expression profiling as a method for evaluating the quality of bovine preimplantation embryos, which may contribute to improvements in bovine IVP systems.
To efficiently produce high-quality bovine calves by transferring embryos obtained from in vitro fertilization (IVF), it is important to evaluate their ability to conceive and their ability to develop into normal litters. In this study, we aimed to clarify the effects of timing and morphology of blastomere cleavage on the gene expression status of bovine IVF embryos. Bovine IVF embryos were classified in four categories, which were divided according to the time of the first blastomere cleavage and the presence or absence of direct cleavage. In addition, the gene expression profiles of these embryos were examined. The timing and morphology of the blastomere cleavage was involved in pre-implantation development and gene expression status of bovine IVF embryos. Our results indicate the possibility of multiple evaluations for bovine IVF embryos and the selection of the most suitable embryos for embryo transfer.
Safe, stable, and nurturing relationships (SSNRs) are an essential component of healthy child development. However, there is a scintilla of data on the measurement of SSNRs and whether these conceptualizations have different associations with peer-violence. We examined two ways of measuring SSNRs (domains and latent class analysis (LCA)) and their relationship with peer violence victimization and perpetration among adolescents across three countries. Three longitudinal datasets (Wave 1-Wave 3) were used from Democratic Republic of Congo (DRC), China, and Indonesia (n = 6068). Adolescents were purposefully selected if they were 10-14 years old and living in urban poor communities during Wave 1. We examined associations between both SSNR conceptualizations (domains and LCA) and peer-violence experiences cross-sectionally and longitudinally using multivariable logistic regressions for each site. We found similarities and differences across sites. The class structure (through LCA) differed across sites. All sites had a 'high SSNR' class. In Indonesia and Shanghai there was a 'low SSNR' class, 'high parental factor' class, and 'high SSNR except social cohesion' class. In Indonesia and Kinshasa, associations with peer-violence were found for both conceptualizations (domain and LCA). In Shanghai, only the latent classes showed association with peer-violence experiences. In general, SSNRs were protective against peer-violence experiences during Wave 1. LCA provides a more subtle understanding of SSNRs. In particular, positive family relationships play a critical role in reducing peer violence experiences. Developing programs require cross-sectoral collaborations that involve families and communities to prevent peer-violence.
Luteal blood flow (LBF) is essential for progesterone (P4) biosynthesis in the corpus luteum (CL) and affects bovine fertility. However, the mechanism by which LBF affects fertility remains unclear. This study was conducted to investigate the effects of LBF on endometrial P4 concentrations and gene expression. Endometrial biopsies and blood samples were collected from 13 Japanese Black cows after ultrasound examination on Day 7 (Day 0 = day of estrus). Based on LBF, the cows were divided into low- (LV; n = 5), medium- (MV; n = 2), and high-vascularity (HV; n = 6) groups. Plasma and endometrial P4 concentrations were measured using enzyme immunoassays. RNA sequencing was performed to compare the endometrial gene expression profiles from three cows in each of the LV and HV groups. Reverse transcription-quantitative PCR was performed for genes selected from the differentially expressed genes (DEGs), P4 receptors (PGR, PGRMC1, and PGRMC2), and P4-regulated genes (ANPEP, DGAT2, DKK1, and LTF). No differences were observed in plasma or endometrial P4 concentrations between the HV and LV groups. CCN3 was identified as a DEG between the HV and LV groups and was upregulated in the HV group. Compared to those of the LV group, the HV group exhibited higher CCN3 and PGR mRNA expression levels and lower ANPEP, DGAT2, and DKK1 mRNA expression levels. In conclusion, LBF affects endometrial gene expression without changing plasma or endometrial P4 concentrations on Day 7.
Ethanolamine plasmalogens (EPls) and choline plasmalogens (CPls), unique glycerophospholipids may play important roles in milk production and reproduction in postpartum dairy cows. While CPls are more abundant in bovine blood, EPls are predominant in the brain. Brain EPls are the only recognized ligands of G protein-coupled receptor 61 (GPR61), a receptor that co-localizes with GnRH receptors on gonadotrophs. We hypothesized that chemosynthetic CPls stimulate gonadotropin secretion from bovine gonadotrophs, similar to the reported effects of chemosynthetic EPls. Anterior pituitary cells from healthy, post-pubertal heifers, were cultured for 3.5 days and then treated with increasing concentrations (0, 0.7, 7, 70, or 700 pM) of EPl with vinyl-ether-bonded stearic acid and ester-bonded oleic acid (C18:0-C18:1EPl) as a positive control, or CPls with vinyl-ether-bonded stearic acid and ester-bonded oleic acid (C18:0-C18:1CPl), arachidonic acid (C18:0-C20:4CPl), or docosahexaenoic acid (C18:0-C22:6CPl). After 2 h, the medium samples were harvested for FSH and LH assays. C18:0-C18:1EPl (7-700 pM) stimulated basal FSH and LH secretion (P < 0.01). None of the tested CPl concentrations stimulated LH secretion. Only 700 pM of C18:0-C18:1CPl, but not lower concentrations, stimulated FSH secretion (P < 0.05), an effect that was inhibited by a SMAD pathway inhibitor. However, both C18:0-C18:1CPl and C18:0-C20:4CPl synergized with GnRH to stimulate FSH secretion. In silico molecular-docking simulations using the deep-learning algorithm ColabFold revealed that CPls bind to the three-dimensional structural model of GPR61. In conclusion, C18:0-C20:4CPl stimulated FSH secretion exclusively in the presence of GnRH, whereas C18:0-C18:1CPl weakly stimulated FSH secretion and showed potential interaction with the GnRH signaling pathways.
Gonadotropin-releasing hormone (GnRH) tightly regulates the synthesis and the release of gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), while the intracellular molecular mechanisms following GnRH signal for the regulation of transcription remain incompletely understood. In this study, we used primary culture of rat anterior pituitary cells to investigate the role of NR4A transcription factors (NR4A1, NR4A2, and NR4A3) in GnRH regulation of gonadotropin secretion. GnRH agonist stimulation rapidly and transiently increased Nr4a1, Nr4a2, and Nr4a3 expression within one hour, accompanied by a time-dependent increase in Fshb mRNA levels and the secretion of both FSH and LH. The knockdown of each Nr4a gene using siRNA significantly reduced Fshb expression under GnRH stimulation. Nr4a1 knockdown caused the most pronounced decrease in FSH secretion. Although Lhb and Cga mRNA levels were largely unaffected, LH secretion was consistently reduced following NR4A knockdown. These findings suggest that NR4A transcription factors act downstream of GnRH signaling to promote Fshb transcription and facilitate gonadotropin secretion, thereby modulating GnRH-dependent control of FSH and LH secretion.