Reproduction, fertility, and development最新文献

Context: Melatonin influences female reproduction, but expression of the melatonin system has not been characterised in the ovine uterus.

Aims: We aimed to determine whether synthesising enzymes (arylalkylamine N-acetyltransferase (AANAT) and N-acetylserotonin-O-methyltransferase (ASMT)), melatonin receptors 1 and 2 (MT1 and MT2), and catabolising enzymes (myeloperoxidase (MPO) and indoleamine 2,3-dioxygenase 1 and 2 (IDO1 and 2)), are expressed in the ovine uterus, and if they are influenced by the oestrous cycle (Experiment 1) or by undernutrition (Experiment 2).

Methods: In Experiment 1, gene and protein expression was determined in sheep endometrium samples collected on days 0 (oestrus), 5, 10 and 14 of the oestrous cycle. In Experiment 2, we studied uterine samples from ewes fed either 1.5 or 0.5times their maintenance requirements.

Key results: We have demonstrated the expression of AANAT and ASMT in the endometrium of sheep. AANAT and ASMT transcripts, and AANAT protein were more elevated at day 10, then decreased to day 14. A similar pattern was observed for MT2 , IDO1 , and MPO mRNA, which suggests that the endometrial melatonin system might be influenced by ovarian steroid hormones. Undernutrition increased AANAT mRNA expression, but seemed to decrease its protein expression, and increased MT2 and IDO2 transcripts, whereas ASMT expression was unaffected.

Conclusions: The melatonin system is expressed in the ovine uterus and is affected by oestrous cycle and undernutrition.

Implications: The results help explain the adverse effects of undernutrition on reproduction in sheep, and the success of exogenous melatonin treatments in improving reproductive outcomes.

Context: Arachidonic acid (AA) is the precursor of prostaglandins, which may play autocrine roles during early embryo development.

Aims: To test the developmental effects of addition of AA to pre- and post-hatching culture media on in vitro -produced bovine embryos.

Methods: Pre-hatching effects of AA were tested by culturing bovine zygotes in synthetic oviductal fluid (SOF) supplemented with 100 or 333μM AA. Post-hatching effects of AA were tested by culturing Day 7 blastocysts in N2B27 supplemented with 5, 10, 20 or 100μM AA up to Day 12.

Key results: Pre-hatching development to blastocyst was completely abrogated at 333μM AA, whereas blastocyst rates and cell numbers were not altered at 100μM AA. Impaired post-hatching development was observed at 100μM AA, whereas no effect on survival rates was noted at 5, 10 and 20μM AA. However, a significant reduction in Day 12 embryo size was observed at 10 and 20μM AA. Hypoblast migration, epiblast survival and formation of embryonic-disc-like structures were unaffected at 5-10μM AA. AA exposure downregulated the genes PTGIS , PPARG , LDHA and SCD in Day 12 embryos.

Conclusions: Pre-hatching embryos are mostly irresponsive to AA, whereas AA was observed to have negative effects during early post-hatching development.

Implications: AA does not improve in vitro bovine embryo development and is not required up to early post-hatching stages.

Caveolae are invaginations in the plasma membrane of most cell types and are present in the cells of normal prostate tissue. Caveolins are a family of highly conserved integral membrane proteins that oligomerise to form caveolae and interact with signalling molecules by providing a scaffold that sequesters signal transduction receptors in close proximity to each other. Signal transduction G proteins and G-protein-coupled receptors (GPCR), including oxytocin receptor (OTR), are localised within caveolae. Only one OTR has been identified, and yet, this single receptor both inhibits and stimulates cell proliferation. As caveolae sequester lipid-modified signalling molecules, these differing effects may be due to a change in location. The cavin1 necessary for caveolae formation is lost in prostate cancer progression. With the loss of caveolae, the OTR moves out onto the cell membrane influencing the proliferation and survival of prostate cancer cells. Caveolin-1 (cav-1) is reportedly overexpressed in prostate cancer cells and is associated with disease progression. This review focuses on the position of OTRs within caveolae, and their movement out onto the cell membrane. It explores whether movement of the OTR is related to changes in the activation of the associated cell signalling pathways that may increase cell proliferation and analyse whether caveolin and particularly cavin1 might be a target for future therapeutic stratagies.

Context: Proliferation, differentiation, migration and apoptosis of trophoblastic cells are influenced by hypoxia, as well as adequate modulation of oxidative stress and the unfolded protein response (UPR) pathway.

Aims: We aimed to evaluate the expression profile of redox and UPR mediators in the placenta of rats throughout pregnancy.

Methods: Placental expression of hypoxia-inducible factor 1α (HIF1α), 8-Hydroxy-2'-deoxyguanosine (8-OHdG), superoxide dismutase 1 (SOD1), glutathione peroxidase (GPX), catalase (Cat), activating transcription factor 6 (ATF6), protein kinase RNA-like endoplasmic reticulum kinase (PERK), 78 kD glucose-regulated protein (GRP78) and C/EBP-homologous protein (CHOP), as well as reactive oxygen species (ROS) and peroxynitrite production, were evaluated in Wistar rats on the 10th, 12th, 14th, 16th and 18th day of pregnancy (DP).

Key results: Increased immunostaining of HIF1α was observed on the 16th and 18th DP, while 8-OHdG and ROS production were greater on the 14th DP. SOD1 and Cat had increased immunostaining on the 14th and 18th DP, while staining of GPX1/2, GRP78 and CHOP was greater on the 18th DP. With regard to gene expression, Hif1α and Sod1 showed increased mRNA expression on the 12th and 16th DP, while Gpx1 had increased expression on the 10th and 16th DP. Cat , Perk and Grp78 gene expression was greater on the 14th DP, unlike Atf6 , which showed greater expression on the 12th DP. In contrast, Chop maintained increased expression from the 12th to the 18th DP.

Conclusions: The placental expression of redox and UPR mediators in rats is influenced by gestational age, with greater expression in periods of greater HIF1α and 8-OHdG expression and at the end of the pregnancy.

Implications: This study provides data on the physiological modulation of redox and UPR mediators during placental development in rats.

Context: Telomerase reverse transcriptase is a key factor responsible for structural and cellular alterations in aged oocytes and changes in the structure of the zona pellucida and mitochondria. Telomerase expression is reduced in aged cumulus oocyte complexes, and its activation or enhanced expression would be beneficial for in vitro oocyte maturation and in vitro embryo development.

Aims: This study aimed to investigate telomerase activation by cycloastragenol and its effect on bovine oocyte in vitro maturation, fertilisation, and early embryo development.

Methods: We used qPCR, Western blot, immunofluorescence, reactive oxygen species (ROS) assay,TUNEL assay, JC-1 assay, and invasion assay to analyse the affect of cycloastragenol (CAG) on bovine oocyte maturation, embryo development, embryo quality and implantation potential.

Key results: Cycloastragenol treatment of oocytes in in vitro maturation (IVM) media significantly (P <0.05) improved oocyte IVM (90.87%), embryo cleavage (90.78%), blastocyst hatching (27.04%), and embryo implantation potential. Telomerase also interacts with mitochondria, and JC-1 staining results showed significantly (P <0.05) higher mitochondrial membrane potential (ΔΨ m) in the CAG-treated group. Furthermore, the inner cell mass (OCT4 and SOX2) and trophoblasts (CDX2) of the control and CAG groups were examined. Moreover, CAG treatment to primary cultured bovine cumulus cells substantially enhanced telomerase activity.

Conclusions: Telomerase activation via cycloastragenol is beneficial for bovine oocyte IVM and for the production of high-quality bovine embryos.

Implications: Cycloastragenol is a natural telomerase activator, and could be useful as a permanent component of oocyte maturation media.

Context: Sphingosine-1-phosphate (S1P) is synthesised by follicle granulosa cells under the influence of follicle-stimulating hormone and seems to be necessary for the biological effects of this gonadotrophin.

Aims: To determine if luteinising hormone (LH) increases S1P production and if this sphingolipid, either induced by LH or added to culture media, regulates steroidogenesis and cell viability in bovine theca cells.

Methods: We used bovine theca cell cultures treated with: S1P (0, 0.1, 1 and 10μM; Experiment 1), LH (0, 0.02, 0.2 and 2ngmL-1 ; Experiment 2) and LH (0.02ngmL-1 ) plus a sphingosine kinase inhibitor (SKI-178; 0, 5 and 10μM; Experiment 3).

Key results: Treatment with S1P did not affect (P >0.05) theca cell viability or their ability to produce progesterone and testosterone. LH (0.02ngmL-1 ) increased (P <0.05) S1P production, and stimulated the expression of phosphorylated sphingosine kinase-1 (pSPHK1). However, the inhibition of SPHK1, by a specific SPHK1 inhibitor (SKI-178), reduced (P <0.05) cell viability and progesterone secretion. Additionally, the use of SKI-178 increased theca cell testosterone production (P<0.05).

Conclusions: S1P added to culture media did not affect cell viability or steroid synthesis. However, LH stimulated the production of S1P, by increasing phosphorylation of SPHK1 in theca cells. This intracellular S1P was inhibitory on testosterone production but augmented progesterone and viable cell number.

Implications: These results suggest a novel signalling pathway for LH in theca cells and underline the importance of S1P in the regulation of steroid synthesis.

Context: Heat shock protein 70 (HSP70) and glutathione peroxidase 5 (GPX5) are biomarkers of oxidative stress and stress in temperate, tropical environments, which are crucial for male reproduction. Their expression and distribution patterns in the testis and epididymis of Bactrian camels are still unknown.

Aims: This study aims to investigate the HSP70 and GPX5 expression and localisation in 3- and 6-year-old Bactrian camel testis and epididymis.

Methods: Reverse transcription quantitative polymerase chain reaction (qRT-PCR), Western blot and immunohistochemistry were used to detect HSP70 in the testis and epididymis (caput, corpus and cauda) and GPX5 in the epididymis at two developmental stages (3-year-old puberty group and 6-year-old adult group).

Key results: HSP70 was upregulated in the testis. Immunohistochemistry results indicated the HSP70 protein was mainly detected in spermatids and Leydig cells of testicular tissue. In the epididymis, HSP70 was located at the luminal spermatozoa, the epithelium lining the epididymal and the epididymal interstitium. GPX5 expression was significantly higher in the caput epididymis than in the corpus and cauda epididymis. GPX5 protein was observed in the epithelium lining the epididymal, interstitium and luminal spermatozoa in the epididymis by immunohistochemistry.

Conclusions: Bactrian camel HSP70 and GPX5 exhibited spatiotemporal expression specificity.

Implications: HSP70 and GPX5 may be essential for germ cell development and reproductive success after sexual maturation in Sonid Bactrian camels.

Context: Implantation of fertilised eggs and survival of a semi-allogenic embryo rely on the interactions between the cells and molecules preparing the uterus. We investigated the effect of regulatory T cell (Treg) therapy on the mechanism of local immune tolerance of mice prone to spontaneous abortion.

Methods: Naive T cells were stimulated in vitro with 17β-oestradiol (E2), progesterone (P4) and TGF-β1 for 96h to generate induced Tregs (iTreg). The iTregs were injected into DBA/2-mated pregnant CBA/J female mice (abortion prone model). On day 14 of pregnancy, mice were killed and decidual and placental tissues were collected for cellular composition analysis.

Results: Abortion prone mice (PBS treated) showed significantly lower survival rates (P <0.0001), increased CD3+ CD8+ (P <0.05), lower IDO+ (P <0.05) and increased natural killer cells (uNK) cell numbers (P <0.001) in the uterus, as well increased NK cells in the placenta (P <0.05) than in normal pregnant mice (CBA/J×BALB/c). Adoptive transfer of iTregs increased fetal survival in abortion-prone mice (P <0.01) and histopathological evaluation revealed a significantly decreased number of uNK cells in the uterus of TGF-β1-, E2- and P4-iTregs (P<0.05, P<0.0001 and P<0.05, respectively) than in the PBS treated group. In the placenta, we found significantly lower numbers of uNK cells from TGF-β1-, E2- and P4-iTregs than in the PBS treated group (P <0.05, P <0.05 and P <0.01, respectively).

Conclusions: We propose that modulation of uterine NK cell activity through immunotherapy using Treg cells should be given more attention as an immunological strategy in the treatment of recurrent miscarriage.

Despite its important role in numerous physiological functions, including regulation of appetite and body weight, immune function and normal sexual maturation, raised leptin levels could result in significant damaging effects on sperm. The adverse effects of leptin on the male reproductive system result from its direct actions on the reproductive organs and cells instead of the hypothalamus-pituitary-gonadal axis. Binding of leptin to the receptors in the seminiferous tubular cells of the testes increases free radical production and decreases the gene expression and activity of endogenous enzymatic antioxidants. These effects are mediated via the PI3K pathway. The resultant oxidative stress causes significant damage to the seminiferous tubular cells, germ cells and sperm DNA leading to apoptosis, increased sperm DNA fragmentation, decreased sperm count, increased fraction of sperm with abnormal morphology, and decreased seminiferous tubular height and diameter. This review summarises the evidence in the literature on the adverse effects of leptin on sperm, which could underlie the often-reported sperm abnormalities in obese hyperleptinaemic infertile males. Although leptin is necessary for normal reproductive function, its raised levels could be pathologic. There is, therefore, a need to identify the cut-off level in the serum and seminal fluid above which leptin becomes pathological for better management of leptin associated adverse effects on male reproductive function.

Context: Sulfasalazine (SAS) is a drug prescribed for pregnant and breastfeeding women with chronic inflammatory bowel diseases. SAS treatment induces transitory infertility in both adult men and male rats. Although SAS crosses the placenta and passes into maternal milk, the consequences of maternal SAS exposure on the reproductive development of male offspring needs further study.

Aims: The current study evaluated whether maternal SAS exposure interferes with the reproductive development of male rat offspring in the neonatal, infant, pubertal and adulthood periods.

Methods: Pregnant Wistar rats (n =10/group) received 300mg/kg/day of SAS dissolved in carboxymethyl cellulose (CMC), by gavage, from gestational day 0 to lactation day 21, and 3mg/kg/day of folic acid during gestation. The control group received CMC.

Key results: During puberty, maternal SAS exposure increased the total length of seminiferous tubules, and round cells were observed in the lumen of caput and cauda epididymis. Moreover, SAS induced oxidative stress-related alterations in the testes of infant and adolescent rats.

Conclusions: Although maternal SAS treatment caused reproductive alterations in infant and adolescent male rats, in adulthood, there were no impairments in sperm parameters that could compromise fertility.

Implications: This study investigated the consequences of maternal exposure to SAS on the reproductive development of male rat offspring from birth to adulthood, employing a human-relevant dose. Thus, this study provides information for better understanding of SAS treatment during critical periods of development.