Compared to ovarian antral follicle development, the mechanism underlying preantral follicle growth has not been well documented. Although C-type natriuretic peptide (CNP) involvement in preantral folliculogenesis has been explored, its detailed role has not been fully defined. Here, we used mouse preantral follicles and granulosa cells (GCs) as a model for investigating the dynamic expression of CNP and natriuretic peptide receptor 2 (NPR2) during preantral folliculogenesis, the regulatory role of oocyte-derived growth factors (ODGFs) in natriuretic peptide type C (Nppc) and Npr2 expression, and the effect of CNP on preantral GC viability. Both mRNA and protein levels of Nppc and Npr2 were gradually activated during preantral folliculogenesis. CNP supplementation in culture medium significantly promoted the growth of in vitro-cultured preantral follicles and enhanced the viability of cultured GCs in a follicle-stimulating hormone (FSH)-independent manner. Using adult and prepubertal mice as an in vivo model, CNP pre-treatment via intraperitoneal injection before conventional superovulation also had a beneficial effect on promoting the ovulation rate. Furthermore, ODGFs enhanced Nppc and Npr2 expression in the in vitro-cultured preantral follicles and GCs. Mechanistic study demonstrated that the regulation of WNT signaling and estrogen synthesis may be implicated in the promoting role of CNP in preantral folliculogenesis. This study not only proves that CNP is a critical regulator of preantral follicle growth, but also provides new insight in understanding the crosstalk between oocytes and somatic cells during early folliculogenesis.
Guangyin Xi, Wenjing Wang, Sarfaraz A Fazlani, Fusheng Yao, Mingyao Yang, Jing Hao, Lei An and Jianhui Tian
Michelle L Johnson, M Jill Saffrey and Victoria J Taylor
Pregnancy and lactation increase maternal appetite and adiposity, which in humans can lead to long-term body mass retention. Previous rat reproduction studies suggest that appetite-inhibiting gut hormone, peptide-YY (PYY), is elevated, despite hyperphagia also that gastrointestinal size increases. The present study characterised changes in orexigenic (appetite-stimulating) ghrelin and anorexigenic (appetite-inhibiting) PYY and glucagon-like peptide-1 (GLP-1), and gastrointestinal architecture during pregnancy and lactation, in matched fed and fasted plasma and gut tissue samples taken during the dark phase. Enteroendocrine cells were immunolabelled, and gut masses and lengths were measured. Fasted plasma ghrelin reduced during pregnancy: it was lowest by day 18, recovered to control values at parturition, then increased by the end of lactation. Ghrelin-immunoreactive stomach cells and stomach ghrelin concentrations were highest at birth, prior to the onset of lactation-associated hyperphagia. Plasma fed GLP-1 concentrations were elevated during pregnancy, and together with higher colon concentrations of PYY and GLP-1 during early lactation, they were associated with gastrointestinal tissue expansion, not satiety. Body mass increased during lactation, whereas white adipose tissue depots depleted. Extensive gut remodelling coincided with elevated colon concentrations of PYY and GLP-1. Modifications included stomach and caecum expansion, and duodenal, ascending and descending colon circumference increases, all peaking by day 10 of lactation; increased intestinal masses and lengths peaking at lactation day 10 for small intestine and lactation day 25 for large intestine. If these physical tissue increases persist post-partum, they could accelerate future nutrient assimilation and storage in dams, and may contribute to increased obesity risk.
Dongmei He, Hong Zeng, Jingfei Chen, Lan Xiao, Yuhao Zhao and Nenghui Liu
Integrin β3 (ITGB3), which is the target gene of the miRNA let-7 that can be antagonized by long noncoding RNA (lncRNA) H19, is well known to have a critical role in endometrium receptivity. However, the regulation of ITGB3 in cell–cell or cell–extracellular matrix adhesion and invasion for the maintenance of early pregnancy remains unknown. This study aimed to explore the role of the H19/let-7/ITGB3 axis in regulating trophoblastic spheroid adhesion and in vitro invasion ability using the HTR-8/SVneo cell line and to investigate the expression levels of lncRNA H19 and ITGB3 in human products of conception. The in vitro knockdown of H19 resulted in decreased expression of ITGB3 at the mRNA and protein levels and reduced the adhesion and invasion ability. In the embryonic chorion tissue of spontaneous abortion (SA), the expressions of H19 and ITGB3 at both the mRNA and protein levels decreased. The results of quantitative RT-PCR, Western blot analysis, dual-luciferase report gene and functional miRNA let-7 rescue experiments, adhesion assay and in vitro transwell invasion assay confirmed that H19 regulated trophoblastic spheroid adhesion with endometrial stromal cells through the H19/let-7/ITGB3 axis, thereby providing an improved understanding of the molecular mechanism of SA.
Alexandra J Harvey
Mitochondria, originally of bacterial origin, are highly dynamic organelles that have evolved a symbiotic relationship within eukaryotic cells. Mitochondria undergo dynamic, stage-specific restructuring and redistribution during oocyte maturation and preimplantation embryo development, necessary to support key developmental events. Mitochondria also fulfil a wide range of functions beyond ATP synthesis, including the production of intracellular reactive oxygen species and calcium regulation, and are active participants in the regulation of signal transduction pathways. Communication between not only mitochondria and the nucleus, but also with other organelles, is emerging as a critical function which regulates preimplantation development. Significantly, perturbations and deficits in mitochondrial function manifest not only as reduced quality and/or poor oocyte and embryo development but contribute to post-implantation failure, long-term cell function and adult disease. A growing body of evidence indicates that altered availability of metabolic co-factors modulate the activity of epigenetic modifiers, such that oocyte and embryo mitochondrial activity and dynamics have the capacity to establish long-lasting alterations to the epigenetic landscape. It is proposed that preimplantation embryo development may represent a sensitive window during which epigenetic regulation by mitochondria is likely to have significant short- and long-term effects on embryo, and offspring, health. Hence, mitochondrial integrity, communication and metabolism are critical links between the environment, the epigenome and the regulation of embryo development.
Marcelo H Ratto, Marco Berland, Mauricio E Silva and Gregg P Adams
The type of stimuli triggering GnRH secretion has been used to classify mammalian species into two categories: spontaneous or induced ovulators. In the former, ovarian steroids produced by a mature follicle elicit the release of GnRH from the hypothalamus, but in the latter, GnRH secretion requires coital stimulation. However, the mechanism responsible for eliciting the preovulatory LH surge in induced ovulators is still not well understood and seems to vary among species. The main goal of this review is to offer new information regarding the mechanism that regulates coitus-induced ovulation. Analysis of several studies documenting the discovery of β-NGF in seminal plasma and its role in the control of ovulation in the llama and rabbit will be described. We also propose a working hypothesis regarding the sites of action of β-NGF in the llama hypothalamus. Finally, we described the presence of β-NGF in the semen of species categorized as spontaneous ovulators, mainly cattle, and its potential role in ovarian function. The discovery of this seminal molecule and its ovulatory effect in induced ovulators challenges previous concepts about the neuroendocrinology of reflex ovulation and has provided a new opportunity to examine the mechanism(s) involved in the cascade of events leading to ovulation. The presence of the factor in the semen of induced as well as spontaneous ovulators highlights the importance of understanding its signaling pathways and mechanism of action and may have broad implications in mammalian fertility.
Alan J Conley, Erin L Legacki, C Jo Corbin, Scott Stanley, Carl R Dahlen and Lawrence P Reynolds
Dexamethasone (DEX) initiates parturition by inducing progesterone withdrawal and affecting placental steroidogenesis, but the effects of DEX in fetal and maternal tissue steroid synthetic capacity remains poorly investigated. Blood was collected from cows at 270 days of gestation before DEX or saline (SAL) treatment, and blood and tissues were collected at slaughter 38 h later. Steroid concentrations were determined by liquid chromatography tandem mass spectrometry to detect multiple steroids including 5α-reduced pregnane metabolites of progesterone. The activities of 3β-hydroxysteroid dehydrogenase (3βHSD) in cotyledonary and luteal microsomes and mitochondria and cotyledonary microsomal 5α-reductase were assessed. Quantitative PCR was used to further assess transcripts encoding enzymes and factors supporting steroidogenesis in cotyledonary and luteal tissues. Serum progesterone, pregnenolone, 5α-dihydroprogesterone (DHP) and allopregnanolone (3αDHP) concentrations (all <5 ng/mL before treatment) decreased in cows after DEX. However, the 20α-hydroxylated metabolite of DHP, 20αDHP, was higher before treatment (≈100 ng/mL) than at slaughter but not affected by DEX. Serum, cotyledonary and luteal progesterone was lower in DEX- than SAL-treated cows. Progesterone was >100-fold higher in luteal than cotyledonary tissues, and serum and luteal concentrations were highly correlated in DEX-treated cows. 3βHSD activity was >5-fold higher in luteal than cotyledonary tissue, microsomes had more 3βHSD than mitochondria in luteal tissue but equal in cotyledonary sub-cellular fractions. DEX did not affect either luteal or cotyledonary 3βHSD activity but luteal steroidogenic enzyme transcripts were lower in DEX-treated cows. DEX induced functional luteal regression and progesterone withdrawal before any changes in placental pregnene/pregnane synthesis and/or metabolism were detectable.
Monika Fluks, Katarzyna Szczepanska, Takao Ishikawa and Anna Ajduk
In fully grown ovarian follicles both transcriptionally active (NSN) and inactive (SN) oocytes are present. NSN oocytes have been shown to display lower developmental potential. It is possible that oocytes that have not completed transcription before meiosis resumption accumulate less RNA and proteins required for their further development, including those responsible for regulation of Ca2+ homeostasis. Oscillations of the cytoplasmic concentration of free Ca2+ ions ([Ca2+]i) are triggered in oocytes by a fertilizing spermatozoon and are crucial for inducing and regulating further embryonic development. We showed that NSN-derived oocytes express less inositol 1,4,5-triphosphate receptor type 1 (IP3R1), store less Ca2+ ions and generate weaker spontaneous [Ca2+]i oscillations during maturation than SN oocytes. Consequently, NSN oocytes display aberrant [Ca2+]i oscillations at fertilization. We speculate that this defective regulation of Ca2+ homeostasis might be one of the factors responsible for the lower developmental potential of NSN oocytes.
Bart Leemans, Tom A E Stout, Catharina De Schauwer, Sonia Heras, Hilde Nelis, Maarten Hoogewijs, Ann Van Soom and Bart M Gadella
In contrast to various other mammalian species, conventional in vitro fertilization (IVF) with horse gametes is not reliably successful. In particular, stallion spermatozoa fails to penetrate the zona pellucida, most likely due to incomplete activation of stallion spermatozoa (capacitation) under in vitro conditions. In other mammalian species, specific capacitation triggers have been described; unfortunately, none of these is able to induce full capacitation in stallion spermatozoa. Nevertheless, knowledge of capacitation pathways and their molecular triggers might improve our understanding of capacitation-related events observed in stallion sperm. When sperm cells are exposed to appropriate capacitation triggers, several molecular and biochemical changes should be induced in the sperm plasma membrane and cytoplasm. At the level of the sperm plasma membrane, (1) an increase in membrane fluidity, (2) cholesterol depletion and (3) lipid raft aggregation should occur consecutively; the cytoplasmic changes consist of protein tyrosine phosphorylation and elevated pH, cAMP and Ca2+ concentrations. These capacitation-related events enable the switch from progressive to hyperactivated motility of the sperm cells, and the induction of the acrosome reaction. These final capacitation triggers are indispensable for sperm cells to migrate through the viscous oviductal environment, penetrate the cumulus cells and zona pellucida and, finally, fuse with the oolemma. This review will focus on molecular aspects of sperm capacitation and known triggers in various mammalian species. Similarities and differences with the horse will be highlighted to improve our understanding of equine sperm capacitation/fertilizing events.
L K Akison, K M Moritz and N Reid
Fetal alcohol exposure results in well-characterised neurobehavioural deficits in offspring, which form the basis for diagnosing fetal alcohol spectrum disorder. However, there is increasing interest in the full range of health complications that can arise in children and adults with this disorder. We used a systematic review approach to locate all clinical and preclinical studies across a broad range of health outcomes in offspring exposed to prenatal alcohol. Our search encompassed four databases (PubMed, CINAHL, EMBASE and Web of Science) and titles/abstracts from retrieved studies were screened against strict inclusion/exclusion criteria. This review specifically evaluated studies reporting on reproductive outcomes in both males and females. A total of 23 studies were included, 5 clinical and 18 preclinical. Although there was a wide range in the quality of reporting across both clinical and preclinical studies, and variable results, trends emerged amongst the reproductive measures that were investigated. In females, most studies focussed on age at first menarche/puberty onset, with evidence for a significant delay in alcohol-exposed offspring. In males, offspring exposed to prenatal alcohol had altered testosterone levels, reduced testes and accessory gland weights and reduced sperm concentration and semen volume. However, further studies are required due to the paucity of clinical studies, the narrow scope of female reproductive outcomes examined and inconsistencies in outcomes across preclinical studies. We recommend that adolescents and individuals of reproductive age diagnosed with f-etal alcohol spectrum disorder be assessed for reproductive dysfunction to allow appropriate management of their reproductive health and fertility.
Claire Stenhouse, Charis O Hogg and Cheryl J Ashworth
Integrins regulate adhesion at the foeto-maternal interface by interacting with secreted phosphoprotein 1 (SPP1) and fibronectin (FN). It is hypothesised that impaired foetal growth of ‘runt’ piglets is linked to altered integrin signalling at the foeto-maternal interface. Placental and endometrial samples associated with the lightest and closest to mean litter weight (CTMLW) (gestational day (GD18, 30, 45, 60 and 90), of both sex (GD30, 45, 60 and 90) (n = 5–8 litters/GD), Large White × Landrace conceptuses or foetuses were obtained. The mRNA expression of the integrin subunits (ITG) ITGA2, ITGAV, ITGB1, ITGB3, ITGB5, ITGB6, ITGB8, SPP1 and FN was quantified by qPCR. Temporal changes in mRNA expression were observed, with different profiles in the two tissues. Endometrial ITGB1 (P ≤ 0.05, GD45) and SPP1 (P ≤ 0.05, all GD combined and GD60) expression was decreased in samples supplying the lightest compared to the CTMLW foetuses. Placentas supplying female foetuses had decreased expression of ITGB6 (GD45, P ≤ 0.05) and FN (GD90, P ≤ 0.05) compared to those supplying male foetuses. Endometrial samples supplying females had increased ITGB3 (P ≤ 0.05, GD60) and FN (P ≤ 0.05, GD30) expression and decreased SPP1 (P ≤ 0.05, GD60) expression compared to male foetuses. Correlations between mean within-gilt mRNA expression and percentage prenatal survival, number of live foetuses or conceptuses and percentage male foetuses were observed. This study has highlighted novel and dynamic associations between foetal size, sex and integrin subunit mRNA expression at the porcine foeto-maternal interface. Further studies should be performed to improve the understanding of the mechanisms behind these novel findings.