α-Ketoglutarate (α-KG) is an intermediary metabolite in the tricarboxylic acid (TCA) cycle and functions to inhibit ATPase and maintain the pluripotency of embryonic stem cells (ESCs); however, little is known regarding the effects of α-KG on the development of preimplantation embryos. Herein, we report that α-KG (150 μM) treatment significantly promoted the blastocyst rate, the number of inner cell mass (ICM) cells and foetal growth after embryo transfer. Mechanistic studies revealed two important pathways involved in the α-KG effects on embryo development. First, α-KG modulates mitochondria function by inducing relatively low ATP production without modification of mitochondrial copy number. The relatively low energy metabolism preserves the pluripotency and competence of the ICM. Second, α-KG modifies epigenetics in embryos cultured in vitro by affecting the activity of the DNA demethylation enzyme TET and the DNA methylation gene Dnmt3a to increase the ratio of 5hmC/5mC ratio. Elevation of the 5hmC/5mC ratio not only promotes the pluripotency of the ICM but also leads to a methylation level in an in vitro embryo close to that in an in vivo embryo. All these functions of α-KG collectively contribute to an increase in the number of ICM cells, leading to greater adaptation of cultured embryos to in vitro conditions and promoting foetal growth after embryo transfer. Our findings provide basic knowledge regarding the mechanisms by which α-KG affects embryo development and cell differentiation.
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Zhenzhen Zhang, Changjiu He, Lu Zhang, Tianqi Zhu, Dongying Lv, Guangdong Li, Yukun Song, Jing Wang, Hao Wu, Pengyun Ji and Guoshi Liu
Lutz Konrad, Raimund Dietze, Pradeep Kumar Kudipudi, Fabian Horné and Ivo Meinhold-Heerlein
A diagnosis of endometriosis is based upon the histological identification of endometrial tissue at ectopic sites which are commonly located on the pelvic organs, the peritoneum and ovary. In rare cases, ectopic lesions can be found in other organs, such as kidney, bladder, lung or brain. Diagnosis is achieved by laparoscopic intervention followed by histological confirmation of endometriotic tissue. Prevalence is estimated at approximately 10% in the general female population with many patients experiencing pain and/or infertility. Currently, the implantation hypothesis by Sampson is the most accepted hypothesis about the pathogenesis of endometriosis. However, the occurrence of endometriosis in patients with Mayer–Rokitansky–Küster–Hauser (MRKH) syndrome who sometimes lack a uterus or endometrium seems to suggest metaplasia as a cause of endometriosis. A critical reevaluation of the literature about MRKH does not reveal conclusive evidence of an association of uterus/endometrium agenesis and endometriosis. Most often only MRI diagnoses of uterus/endometrium agenesis and only very rarely conclusive histological evidence of the endometriotic lesions are presented. In contrast, whenever biopsies were performed endometriosis always appeared together with uterus/endometrium remnants. Taken together, we suggest that MRKH patients only develop endometriosis if a uterus/endometrium is present which underscores and not contradicts the implantation hypothesis of Sampson.
Irene Mytilinaiou, Kiki Sarganaki, Eirini Sachouli, Sofia Tsagdi, Katerina Vardaki, Anna Vassiliadis and Irene Athanassakis
Mate choice has been postulated to be MHC-dependent, ensuring the maintenance of polymorphism for species survival. At the molecular level, MHC polymorphism is represented by class-I (MHCI), class-II (MHCII) antigens and their T cell receptors (TCRs). In order to evaluate the presence such immune molecules during male/female interaction, vaginal fluid, vaginal cells, urine, sperm, seminal fluid, cumulus cells, tubal fluid and epithelium were isolated from BALB/c mice and examined for the presence of membrane or soluble MHCI, MHCII, TCRαβ and TCRγδ, using immunofluorescence and ELISA techniques, respectively. These molecules were expressed on sperm and seminal fluid in a sperm quality-dependent manner and in vagina, fallopian tube, cumulus cells and urine in an estrus cycle-dependent manner. Vaginal cells showed increased expression of all molecules tested during estrus, while vaginal fluid showed an increase of TCRγδ and decrease of MHCI and MHCII levels, during estrus. Urine showed only increased concentrations of TCRαβ during estrus. Cumulus cells expressed MHCI, MHCII, TRCγδ but not TCRαβ, while sperm mainly expressed TCRαβ and TRCγδ. All molecules were detected in tubal fluids mostly during estrus, while they were almost undetectable during pregnancy. The vaginal environment was shown to affect sperm motility according to the estrus-cycle, whereas sperm motility was affected by antibodies against these molecules. In conclusion, the presence of complementary immune molecules in the male/female interactive environment, except for revealing novel markers for unexplained infertility, provides for the first time evidence for immune-mediated recognition of the two counterparts, enlightening thus a molecular basis for mate choice.
Neil A Youngson, G Mezbah Uddin, Abhirup Das, Carl Martinez, Haley S Connaughton, Sara Whiting, Josephine Yu, David A Sinclair, R John Aitken and Margaret J Morris
Male fertility and sperm quality are negatively impacted by obesity. Furthermore, recent evidence has shown that male offspring from obese rat mothers also have reduced sperm quality and fertility. Here, we extend work in this area by comparing the effects of both maternal obesity and offspring post-weaning diet-induced obesity, as well as their combination, on sperm quality in mice. We additionally tested whether administration of the NAD+-booster nicotinamide mononucleotide (NMN) can ameliorate the negative effects of obesity and maternal obesity on sperm quality. We previously showed that intraperitoneal (i.p.) injection of NMN can reduce the metabolic deficits induced by maternal obesity or post-weaning dietary obesity in mice. In this study, female mice were fed a high-fat diet (HFD) for 6 weeks until they were 18% heavier than a control diet group. Thereafter, HFD and control female mice were mated with control diet males, and male offspring were weaned into groups receiving control or HFD. At 30 weeks of age, mice received 500 mg/kg body weight NMN or vehicle PBS i.p. for 21 days. As expected, adiposity was increased by both maternal and post-weaning HFD but reduced by NMN supplementation. Post-weaning HFD reduced sperm count and motility, while maternal HFD increased offspring sperm DNA fragmentation and levels of aberrant sperm chromatin. There was no evidence that the combination of post-weaning and maternal HFD exacerbated the impacts in sperm quality suggesting that they impact spermatogenesis through different mechanisms. Surprisingly NMN reduced sperm count, vitality and increased sperm oxidative DNA damage, which was associated with increased NAD+ in testes. A subsequent experiment using oral NMN at 400 mg/kg body weight was not associated with reduced sperm viability, oxidative stress, mitochondrial dysfunction or increased NAD+ in testes, suggesting that the negative impacts on sperm could be dependent on dose or mode of administration.
Katrine Harpelunde Poulsen and Anne Jørgensen
Testicular development from the initially bipotential gonad is a tightly regulated process involving a complex signalling cascade to ensure proper sequential expression of signalling factors and secretion of steroid hormones. Initially, Sertoli cell specification facilitates differentiation of the steroidogenic fetal Leydig cells and establishment of the somatic niche, which is critical in supporting the germ cell population. Impairment of the somatic niche during fetal life may lead to development of male reproductive disorders, including arrest of gonocyte differentiation, which is considered the first step in the testicular cancer pathogenesis. In this review, we will outline the signalling pathways involved in fetal testis development focusing on the Nodal pathway, which has recently been implicated in several aspects of testicular differentiation in both mouse and human studies. Nodal signalling plays important roles in germ cell development, including regulation of pluripotency factor expression, proliferation and survival. Moreover, the Nodal pathway is involved in establishment of the somatic niche, including formation of seminiferous cords, steroidogenesis and Sertoli cell function. In our outline of fetal testis development, important differences between human and mouse models will be highlighted to emphasise that information obtained from mouse studies cannot always be directly translated to humans. Finally, the implications of dysregulated Nodal signalling in development of the testicular cancer precursor, germ cell neoplasia in situ, and testicular dysgenesis will be discussed – none of which arise in rodents, emphasising the importance of human models in the effort to increase our understanding of origin and early development of these disorders.
Rafal P Piprek, Michal Kolasa, Dagmara Podkowa, Malgorzata Kloc and Jacek Z Kubiak
The normal course of gonad development is critical for the sexual development and reproductive capacity of the individual. During development, an incipient bipotential gonad which consists of unorganized aggregate of cells, must differentiate into highly structured testis or ovary. Cell adhesion molecules (CAMs) are a group of proteins crucial for segregation and aggregation of different cell types to form different tissues. E-cadherin (Cdh1) is one of the CAMs expressed in the developing gonads. We used tissue-specific knockout of Cdh1 gene in OCT4+ germ cells and, separately, in SF1+ somatic cells of developing gonads. The knockout of E-cadherin in somatic cells caused decrease in the number of germ cells, while the knockout in the germ cells caused their almost complete loss. Thus, the presence of E-cadherin in both the germ and somatic cells is necessary for the survival of germ cells. Although the lack of E-cadherin did not impair cell proliferation, it enhanced apoptosis, which was a possible cause of germ cell loss. However, the somatic cells of the gonad differentiated normally into Sertoli cells in the testis cords, and into follicular cells in the ovaries. The testis and ovigerous cords maintained their integrity; they were covered by continuous basement membranes. The testicular interstitium with steroidogenic fetal Leydig cells did not show any noticeable changes. However, in the female gonads, because of the lack of germ cells, the ovarian follicles were absent. The sex determination and sexual differentiation of the gonad were not impaired. These results underscore an important role of E-cadherin in germ cell survival and gonad development.
Michele Boiani, Ellen Casser, Georg Fuellen and Elisabeth S Christians
The mammalian zygote is a totipotent cell that generates all the cells of a new organism through embryonic development. However, if one asks about the totipotency of blastomeres after one or two zygotic divisions, opinions differ. As it is impossible to determine the individual developmental potency of early blastomeres in an intact embryo, experiments of blastomere isolation were conducted in various species, showing that two-cell blastomeres could give rise to a new organism when sister cells were separated. A mainstream interpretation was that each of the sister mammalian blastomeres was equally totipotent. However, reevaluation of those experiments raised some doubts about the real prevalence of cases in which this interpretation could truly be validated. We compiled experiments that tested the individual developmental potency of early mammalian blastomeres in a cell-autonomous way (i.e. excluding nuclear transfer and chimera production). We then confronted the developmental abilities with reported molecular differences between sister blastomeres. The reevaluated observations were at odds with the mainstream view: A viable two-cell embryo can already include one non-totipotent blastomere. We were, thus, led to propose a revised model for totipotency continuity based on the construction of the zygote as a mosaic, which accounts for differential inheritance of totipotency-relevant components between sister blastomeres. This takes place with no preordained mechanisms that would ensure a reproducible partition. This model, which is compatible with the body of data on regulative properties of mammalian early embryos, aims at tempering the rigid interpretation that discounted maternal constraints on totipotency.
Patrycja Kurowska, Ewa Mlyczyńska, Alix Barbe, Christophe Staub, Ewa Gregoraszczuk, Joëlle Dupont and Agnieszka Rak
Vaspin, also known as visceral adipose tissue-derived serine protease inhibitor, is a member of the serine protease inhibitor family. Its expression is associated with obesity, insulin resistance and type 2 diabetes, and elevated concentration is observed in polycystic ovary syndrome. However, vaspin has never been studied in the ovary. Here, we identified vaspin in two prolific breeds of pigs: fat Meishan (MS) and lean Large White (LW). We then investigated the molecular mechanism involved in the regulation of its expression in response to gonadotropins, insulin, insulin-like growth factor type 1 (IGF-1) and steroids (progesterone, testosterone and oestradiol) in ovarian follicles cells. Using real-time PCR and Western blot, we found higher vaspin mRNA and protein expression in the ovarian follicles and adipose tissue at 10–12 days of the oestrous cycle in MS compared to LW. Moreover, vaspin expression, as well as its concentration in plasma and follicular fluid, decreased in ovarian follicles of LW during days of the oestrous cycle, while the opposite results were noted in MS. Immunohistochemistry showed vaspin in granulosa, theca, cumulus cells and oocytes as well as in adipocytes. Vaspin level in the ovary increased by gonadotropin, insulin, IGF-1 and steroids stimulation through kinases JAK/Stat, ERK1/2, PI3K and AMPK, as well as factor NF-κB. These findings all show vaspin expression and regulation in the pig ovary, indicating vaspin as a new regulator in female reproduction. Future studies will be necessary for understanding the role of vaspin on ovarian physiology providing new insights into the pathology of ovaries.
S Fair, K G Meade, K Reynaud, X Druart and S P de Graaf
In species where semen is deposited in the vagina, the cervix has the unique function of facilitating progress of spermatozoa towards the site of fertilisation while also preventing the ascending influx of pathogens from the vagina. For the majority of species, advances in assisted reproduction techniques facilitate the bypassing of the cervix and therefore its effect on the transit of processed spermatozoa has been largely overlooked. The exception is in sheep, as it is currently not possible to traverse the ovine cervix with an inseminating catheter due to its complex anatomy, and semen must be deposited at the external cervical os. This results in unacceptably low pregnancy rates when frozen-thawed or liquid stored (>24 h) semen is inseminated. The objective of this review is to discuss the biological mechanisms which regulate cervical sperm selection. We assess the effects of endogenous and exogenous hormones on cervical mucus composition and discuss how increased mucus production and flow during oestrus stimulates sperm rheotaxis along the crypts and folds of the cervix. Emerging results shedding light on the sperm-cervical mucus interaction as well as the dialogue between spermatozoa and the innate immune system are outlined. Finally, ewe breed differences in cervical function and the impact of semen processing on the success of fertilisation, as well as the most fruitful avenues of further investigation in this area are proposed.
Martín Ricardo Ventureira, Cristian Sobarzo, Felipe Argandoña, Wilder A Palomino, Claudio Barbeito and Elisa Cebral
Perigestational alcohol consumption up to early organogenesis can produce abnormal maternal vascularization via altered decidual VEGF/receptor expression. CF-1 female mice were administered with 10% ethanol in drinking water for 17 days prior to and up to day 10 of gestation. Control females received water without ethanol. Treated females had reduced frequency of implantation sites with expanded vascular lumen (P < 0.05), α-SMA-immunoreactive spiral arteries in proximal mesometrial decidua, reduced PCNA-positive endothelial cells (P < 0.01) and diminished uterine NK cell numbers (P < 0.05) in proximal decidua compared to controls. The VEGF expression (laser capture microscopy, RT-PCR, western blot and immunohistochemistry) was reduced in decidual tissue after perigestational alcohol consumption (P < 0.05). The uNK-DBA+ cells of treated females had reduced VEGF immunoexpression compared to controls (P < 0.01). Very low decidual and endothelial cell KDR immunoreactivity and reduced decidual gene and protein KDR expression was found in treated females compared to controls (P < 0.001). Instead, strong FLT-1 immunoexpression was detected in decidual and uNK cells (P < 0.05) in the proximal decidua from treated females compared to controls. In conclusion, perigestational alcohol ingestion induces the reduction of lumen expansion of spiral arteries, concomitant with reduced endothelial cell proliferation and uNK cell population, and uncompleted remodeling of the artery smooth muscle. These effects were supported by low decidual VEGF and KDR gene and protein expression and increased FLT-1 expression, suggesting that VEGF and KDR reduction may contribute, in part, to mechanisms involved in deficient decidual angiogenesis after perigestational alcohol consumption in mouse.