Biology of Reproduction最新文献

The creatine (Cr) biosynthesis pathway buffers ATP in metabolically active tissues. We investigated whether sex of fetus and day of gestation influence Cr in endometrial and conceptus tissues from gilts on Days 60 and Day 90 (n = 6 gilts/day) of gestation. Uterine and conceptus tissues associated with one male and one female fetus from each gilt were analyzed for creatine, mRNAs, and proteins for Cr biosynthesis. Total Cr decreased in amniotic fluid but increased in allantoic fluid between Days 60 and 90 of gestation for male (P < 0.05), but not for female fetuses (P > 0.05). Endometrial expression of CKM, CKMT1, and SLC6A8 mRNAs increased (P < 0.05) between Days 60 and 90 only for female fetuses. On Day 60, expression of CKB and CKMT1 mRNAs was greater (P < 0.05) for placentae of female than male fetuses. Livers of male fetuses had greater expression of AGAT and CKB than for females on Day 60, while kidneys of female fetuses had greater expression of GAMT than male fetuses on Day 90 (P < 0.05). Localization of GAMT, CKB, CKMT1 and SLC6A8 proteins to uterine and chorionic epithelium was not influenced by gestational age or fetal sex. AGAT localized to fetal kidneys and appeared greater on Day 90 than Day 60 in both sexes. Thus, expression of the creatine-creatine kinase-phosphocreatine (Cr-CK-PCr) system at the uterine-conceptus interface is affected by gestational age and fetal sex to influence energy homeostasis in pigs.

The mammalian target of rapamycin (mTOR) signaling pathway is activated by luteinizing hormone in preovulatory follicle. However, its impact on ovulation remains inadequately explored. Utilizing in vivo studies and in vitro fertilization, we demonstrated that the negative effect of inhibition of mTOR signaling by rapamycin on oocyte quality during the ovulatory phase, with a notable decrease in the total cell count of blastocysts, a reduction in gastrula size, and fetal degeneration on the 16th day of gestation while not affecting ovulated oocyte count or granulosa cell luteinization. Mechanistically, our study elucidated that in the ovulatory phase, mTOR signaling inhibition enhances lipid consumption, mitochondrial membrane potential of oocytes, and ATP generation. As a result, embryos derived from these oocytes exhibit higher levels of reactive oxygen species, insufficient energy supply, and lower developmental potency. Furthermore, the impact of mTOR signaling on oocytes remains consistent across various species, and its inhibition has been demonstrated to enhance energy metabolism during the in vitro maturation process of bovine oocytes. These findings demonstrate the critical role of mTOR signaling during the ovulatory phase in balancing oocyte energy metabolism, enriching our understanding of the role of mTOR on ovulation regulation.

Economic losses in cattle farms are frequently associated with failed pregnancies. Some studies found that the transcriptomic profiles of blood and endometrial tissues in cattle with varying pregnancy outcomes display discrepancies even before artificial insemination (AI) or embryo transfer (ET). In the study, 330 samples from seven distinct sources and two tissue types were integrated and divided into two groups based on the ability to establish and maintain pregnancy after AI or ET: P (pregnant) and NP (nonpregnant). By analyzing gene co-variation and employing machine learning algorithms, the objective was to identify genes that could predict pregnancy outcomes in cattle. Initially, within each tissue type, the top 100 differentially co-expressed genes (DCEG) were identified based on the analysis of changes in correlation coefficients and network topological structure. Subsequently, these genes were used in models trained by seven different machine learning algorithms. Overall, models trained on DCEGs exhibited superior predictive accuracy compared to those trained on an equivalent number of differential expression genes (DEGs). Among them, the deep learning models based on differential co-expression genes in blood and endometrial tissue achieved prediction accuracies of 91.7% and 82.6%, respectively. Finally, the importance of DCEGs was ranked using SHapley Additive exPlanations (SHAP) and enrichment analysis, identifying key signaling pathways that influence pregnancy. In summary, this study identified a set of genes potentially affecting pregnancy by analyzing the overall co-variation of gene connections between multiple sources. These key genes facilitated the development of interpretable machine learning models that accurately predict pregnancy outcomes in cattle.

Reproductive success requires accurately timed remodeling of the cervix to orchestrate the maintenance of pregnancy, the process of labor, and birth. Prior work in mice established that a combination of continuous turnover of fibrillar collagen and reduced formation of collagen cross-links allows for the gradual increase in tissue compliance and delivery of the fetus during labor. However, the mechanism for continuous collagen degradation to ensure turnover during cervical remodeling is still unknown. This study demonstrates the functional role of extracellular and intracellular collagen degradative pathways in two different settings of cervical remodeling: physiological term remodeling and inflammation-mediated premature remodeling. Extracellular collagen degradation is achieved by the activity of fibroblast-derived matrix metalloproteases MMP14, MMP2, and fibroblast activation protein (FAP). In parallel, we demonstrate the function of an intracellular collagen degradative pathway in fibroblast cells mediated by the collagen endocytic mannose receptor type-2 (MRC2). These pathways appear to be functionally redundant as loss of MRC2 does not obstruct collagen turnover or cervical function in pregnancy. While both extracellular and intracellular pathways are also utilized in inflammation-mediated premature cervical remodeling, the extracellular collagen degradation pathway uniquely employs fibroblast and immune-cell derived proteases. In sum, these findings identify the dual utilization of two distinct degradative pathways as a failsafe mechanism to achieve continuous collagen turnover in the cervix, thereby allowing dynamic shifts in cervical tissue mechanics and function.

The adult mammalian testis is filled with seminiferous tubules, which contain somatic Sertoli cells along with germ cells undergoing all phases of spermatogenesis. During spermatogenesis in postnatal mice, male germ cells undergo at least 17 different nomenclature changes as they proceed through mitosis as spermatogonia (=8), meiosis as spermatocytes (=6), and spermiogenesis as spermatids (=3) [1-6]. Adding to this complexity, combinations of germ cells at each of these stages of development are clumped together along the length of the seminiferous tubules. Due to this, considerable expertise is required for investigators to accurately analyze changes in spermatogenesis in animals that have spontaneous mutations, been genetically modified (transgenic or knockout/knockin (KO/KI)), or been treated with pharmacologic agents. Here, we leverage our laboratory's expertise in spermatogenesis to optimize the open source "Quantitative Pathology & Bioimage Analysis" (QuPath) software platform for automated analyses of germ and somatic cell populations in both the developing and adult mammalian testis.

Unlike differentiated somatic cells, which possess elongated mitochondria, undifferentiated cells, such as those of preimplantation embryos, possess round, immature mitochondria. Mitochondrial morphology changes dynamically during cell differentiation in a process called mitochondrial maturation. The significance of the alignment between cell differentiation and mitochondrial maturity in preimplantation development remains unclear. In this study, we analyzed mouse embryos into which liver-derived somatic mitochondria were introduced (SM-embryos). Most SM-embryos were arrested at the two-cell stage. Some of the introduced somatic mitochondria became round, while others remained elongated and large. RNA-sequencing revealed a disruption of both minor and major zygotic gene activation (ZGA) in SM-embryos. Minor ZGA did not terminate before major ZGA, and the onset of major ZGA was inhibited, as shown by histone modification analyses of histone H3 lysine 4 trimethylation and histone H3 lysine 27 acetylation. Further analysis of metabolites involved in histone modification regulation in SM-embryos showed a significantly lower NAD+/NADH ratio in SM-embryos than in control embryos. Additionally, the mitochondrial membrane potential, an indicator of mitochondrial function, was lower in SM-embryos than in control embryos. Our results demonstrated that introducing somatic mitochondria into an embryo induces mitochondrial dysfunction, thereby disrupting metabolite production, leading to a disruption in ZGA and inducing developmental arrest. Our findings reveal that the alignment between cell differentiation and mitochondrial maturity is essential for early embryonic development.

The first interactions among the embryo, endometrium, and corpus luteum are essential for pregnancy success. Small extracellular vesicles (sEVs) are part of these interactions. We previously demonstrated that small extracellular vesicles from in vivo- or in vitro-produced bovine embryos contain different miRNA cargos. Herein we show: (1) the presence and origin (in vivo or in vitro) of the blastocyst differentially reprograms endometrial transcriptional profiles; (2) the endometrial explant (EE) cultured with in vivo or in vitro embryos release small extracellular vesicles with different miRNA contents, and (3) the luteal explant (CLE) exposed to these small extracellular vesicles have distinct mRNA and miRNA profiles. To elucidate this, the endometrial explant were cultured in the presence or absence of a single Day-7 in vivo (EE-artificial insemination; EE-AI) or in vitro (EE-in vitro fertilization; EE-IVF) embryo. After of culture we found, in the endometrial explant, 45 and 211 differentially expressed genes associated with embryo presence and origin, respectively. Small extracellular vesicles were recovered from the conditioned media (CM) in which endometrial explant and embryos were co-cultured. Four miRNAs were differentially expressed between small extracellular vesicles from CC-EE-AI and CC-EE-IVF. Luteal explants exposed in culture to these small extracellular vesicles showed 1360 transcripts and 15 miRNAs differentially expressed. The differentially expressed genes associated with embryo presence and origin, modulating cells' proliferation, and survival. These results demonstrate that in vivo- or in vitro-produced bovine embryos induce molecular alterations in the endometrium; and that the embryo and endometrium release small extracellular vesicles capable of modifying the messenger RNA (mRNA) and miRNA profile in the corpus luteum. Therefore, the small extracellular vesicles-mediated embryo-endometrium-corpus luteum interactions possibly regulate the corpus luteum viability to ensure pregnancy success.

The blastocyst of the European roe deer (Capreolus capreolus) undergoes a period of decelerated growth and limited metabolism. During this period known as embryonic diapause, it floats freely in the uterus encircled by the histotroph. Prior to implantation, reactivation is marked by rapid embryonic growth and conceptus elongation. We hypothesized that the uterine fluid, which is known to undergo changes in its composition to support early embryonic development, contributes to controlling embryonic growth during diapause and elongation. We therefore characterized the pre-implantation uterine fluid metabolome during diapause and at elongation by mass spectrometry and particularly assessed nonpolar lipids, polar metabolites, acylcarnitines, and polyamines. Our results show that triglycerides and diglycerides levels decreased at elongation, likely serving as a source for membrane synthesis rather than for energy production. A functional analysis identified glycolysis as a key pathway during elongation, which may compensate for the energy requirements during this phase. We also observed an increase of sphingomyelin; prostaglandin precursors; and the amino acids asparagine, glutamine, and methionine upon elongation. The sphingolipid and glycerophospholipid metabolism pathways were implicated during elongation. Particularly, spermidine, and to some extent spermine but not putrescine-levels significantly increased in the uterine fluid during elongation, indicating their significance for reactivation and/or proliferation at embryo elongation. We conclude that the roe deer uterine fluid sustained dynamic compositional changes necessary to support the energy- and resource-intensive conceptus elongation. However, it remains to be determined whether these changes are the cause or a consequence of embryo elongation. Studying the metabolic changes and molecular interactions in the roe deer during diapause and elongation not only reveals insights into aspects of its reproductive strategy, but also deepens our knowledge of embryo metabolic demands and developmental velocities across species.

Antral follicle count (AFC) and anti-Müllerian hormone (AMH) concentrations are reflective for ovarian reserve and have been associated with improved reproductive performance in cattle. Key events for regulation of uterine receptivity are orchestrated by progesterone. As progesterone concentrations are greater in animals with high than low AFC, we tested the hypothesis, if the resulting improved uterine environment will lead to improved conceptus elongation and endometrial response to interferon tau. For four years, 10 heifers with lowest and highest AFC, respectively, were selected from 120 heifers. Reproductive tracts and blood samples for progesterone and AMH analysis were collected after synchronization and insemination. For a recovered conceptus, length was determined, and interferon tau (IFNT) transcript abundance was analyzed. Endometrial transcript abundance of interferon-stimulated gene 15 (ISG15) and oxytocin receptor (OXTR) were analyzed. Progesterone concentrations did not differ between low and high AFC groups (P = 0.1). A difference in conceptus length was not observed. Endometrial abundance of ISG15 did not differ between pregnant low and high AFC heifers. Abundance of OXTR was greater in open low AFC than open high AFC heifers (P < 0.01). Interaction of AMH and pregnancy status was determined, with greater AMH in pregnant than open high AFC heifers (P < 0.05). Improved uterine environment in high vs. low AFC heifers did not result in longer conceptuses or improved endometrial response. As the increase in OXTR transcript abundance was only detected in low AFC heifers, reported differences in reproductive performance might be associated with earlier initiation of luteolysis.

Since females grow faster in penaeid shrimp, all-female aquaculture was proposed. Environmental conditions in the Pacific white shrimp were not found to affect genetic sex determination (ZZ/ZW system). The androgenic gland-secreting insulin-like androgenic gland hormone is a key controlling factor in crustacean male differentiation. However, functional sex reversal (neo-male) in penaeid shrimp has not yet been achieved by manipulating the insulin-like androgenic gland hormone-sexual switch. Therefore, understanding the molecular mechanisms of gonadal differentiation may help build appropriate tools to generate neo-male for all-female breeding. This study describes the potential role of the novel penaeid-specific testicular zinc finger protein (pTZFP) in the gonads of Pacific white shrimp. First, pTZFP transcripts show a male-bias expression pattern in undifferentiated gonads, which is then exclusively expressed in the testis and absent or slightly expressed in the ovary and other tissues. Besides, the knockdown of pTZFP in undifferentiated males results in smaller testes but no sex reversal. Immunohistochemical staining of proliferating cell nuclear antigen further confirmed that the smaller testes in pTZFP-deficient males are due to the lower proliferating activity of spermatogonia. These data reveal that pTZFP may be involved in testicular development but have fewer effects on gonadal differentiation. Moreover, testicular pTZFP transcription levels were not reduced with estradiol-17β (E2) administration or AG excision. Therefore, our data suggest that pTZFP may regulate testicular development through downstream genes regulating spermatogonia proliferation. Moreover, our data provide an appropriate molecular marker for identifying the sex of undifferentiated gonads.