Ovarian follicular atresia is mediated by heterophagy,autophagy, and apoptosis in Prochilodus argenteus and Leporinus taeniatus (Teleostei: Characiformes)

We investigated apoptosis, cell proliferation antigen (PCNA), and heat shock protein (HSP70) during ovarian follicular atresia in two freshwater teleost species from the São Francisco River basin, Brazil: curimatã-pacu, Prochilodus argenteus and piau-jejo, Leporinus taeniatus. Fishes were maintained in captivity after the reproductive period and ovarian regression was assessed by gonadosomatic index for three stages: early, advanced, and late regression. Follicular atresia was analysed by light and transmission electron microscopy, as well as by TUNEL and immunohistochemistry for HSP70 and PCNA. During early regression, atretic follicles exhibited zona pellucida breakdown, yolk degeneration, and hypertrophied follicular cells (e.g. granulosa in mammals). Intense heterophagy to engulf the yolk, and autophagy were detected in the follicular cells during advanced and late atresia. The TUNEL assay detected DNA fragmentation, mainly in late follicular atresia. The apoptosis rate of the follicular cells increased up to 10% during follicular atresia in both species and was negatively correlated with follicular area. Immunohistochemistry reaction for HSP70 stained the follicular cells strongly during advanced atresia, when they are intensively involved in yolk engulfment, whereas the reaction for PCNA labelled theca cells. We inferred that heterophagy, autophagy, and apoptosis contributed to follicular atresia in teleost ovaries, thereby achieving a more efficient removal of the degenerating oocyte and dying follicular cells. Additionally, HSP70 may protect the follicular cells before apoptosis when they are involved in yolk engulfment, and cell proliferation in the theca contributed to ovarian remodelling.     

Ovarian follicular atresia is mediated by heterophagy, autophagy, and apoptosis in Prochilodus argenteus and Leporinus taeniatus (Teleostei: Characiformes)

We investigated apoptosis, cell proliferation antigen (PCNA), and heat shock protein (HSP70) during ovarian follicular atresia in two freshwater teleost species from the São Francisco River basin, Brazil: curimatã-pacu, Prochilodus argenteus and piau-jejo, Leporinus taeniatus. Fishes were maintained in captivity after the reproductive period and ovarian regression was assessed by gonadosomatic index for three stages: early, advanced, and late regression. Follicular atresia was analysed by light and transmission electron microscopy, as well as by TUNEL and immunohistochemistry for HSP70 and PCNA. During early regression, atretic follicles exhibited zona pellucida breakdown, yolk degeneration, and hypertrophied follicular cells (e.g. granulosa in mammals). Intense heterophagy to engulf the yolk, and autophagy were detected in the follicular cells during advanced and late atresia. The TUNEL assay detected DNA fragmentation, mainly in late follicular atresia. The apoptosis rate of the follicular cells increased up to 10% during follicular atresia in both species and was negatively correlated with follicular area. Immunohistochemistry reaction for HSP70 stained the follicular cells strongly during advanced atresia, when they are intensively involved in yolk engulfment, whereas the reaction for PCNA labelled theca cells. We inferred that heterophagy, autophagy, and apoptosis contributed to follicular atresia in teleost ovaries, thereby achieving a more efficient removal of the degenerating oocyte and dying follicular cells. Additionally, HSP70 may protect the follicular cells before apoptosis when they are involved in yolk engulfment, and cell proliferation in the theca contributed to ovarian remodelling.     

Dual roles for autophagy during follicular atresia in fish ovary

Autophagy, a highly conserved catabolic program for degrading proteins and organelles, is essential for cell and tissue homeostasis. Primarily, this process has a cytoprotective role under nutrient deprivation, but several stress stimuli can induce autophagy and, thus, distinct programmed cell death (PCD) pathways can be actived when stress is not abolished. Fish ovaries are a suitable experimental model system for studying the mechanisms of PCD due to the presence of postovulatory and atretic (i.e., non-ovulated) follicles, which follow different routes after spawning. Apoptosis of the follicular cells is the major mechanism responsible for the rapid resorption of the postovulatory follicles. Recently, we investigated the contribution of PCD during follicular atresia in two species of freshwater fish. In contrast to mammals, this study revealed that follicular apoptosis is not a major process for deletion of follicular cells in atretic follicles. Furthermore, we detected autophagic vacuoles containing degenerating organelles increasing through follicular atresia in both species. In this addendum, we propose a hypothesis for follicular cell removal during ovarian regression in oviparous fish. In this model, autophagy could have dual roles in follicular atresia. Thus, fish ovaries after breeding are suitable models for studying the interactions among the different cell death pathways.     

Monoamine- and peptide-containing elements in the body wall and nervous trunks of nemerteans

Localization and morphological peculiarities of cateholamine-containing (CA-C), serotonin-(5HTIR), neurotensin-(NT-IR) and FMRFamide-immunoreactive (FMRFa-Ir) cells and processes in the body wall and lateral nervous trunks of nemerteans were investigated. The following nemertean species from three orders occurring in the White Sea were studied by fluorescent histochemistry and by immunohistochemistry: Lineus viridis, Lineus ruber and Cerebratulus sp. (Heteronemertini), Cephalothrix linearis (Palaeonemertini), and Amphiporus lactifloreus (Hoplonemertini). The similarity and characteristics of morphological types of monoamine-and peptide-containing elements, their distribution and possible participation in regulation of some functions in different nemertean species and in other invertebrates are discussed. We suggest that 5HT-IR and CA-C bi-and multipolar intra-and subepidermal open-type cells prevailing in the body wall perform a receptive function, while the FMRFa-Ir and NT-IR neurons of the nerve trunks and body wall are responsible for efferent innervation of musculature and connections within the nervous system.     

Relationship among follicular apoptosis,integrin β<Subscript>1</Subscript> and collagen type IV during early ovarian regression in the teleost <Emphasis Type="Italic">Prochilodus argenteus</Emphasis> after induced spawning

Early ovarian regression was analyzed in the neotropical freshwater teleost, curimatã-pacu (Prochilodus argenteus), in order to evaluate follicular apoptosis, basement membrane morphology, and integrin β₁ and collagen type IV immunostainning in postovulatory follicles. Mature females were induced to spawn by using carp pituitary extract for study of ovarian regression up to 5 days post-spawning. Morphometric analyses showed that the postovulatory follicle area decreased progressively after spawning and was coupled to the gonadosomatic index (r=0.92). During ovarian regression, follicular cells detached from the neighboring cells and basement membrane and then died by apoptosis. The follicular basement membrane became thicker and diffuse and was breached during regression of the postovulatory follicles. Follicular apoptosis was detected by TUNEL, histology, and electron microscopy. The ladder pattern of apoptotic DNA was revealed by agarose gel electrophoresis. The apoptotic index for the follicular cells increased until 3 days post-spawning and decreased thereafter. Immunohistochemistry reactions detected caspase 3, integrin β₁, and collagen type IV in the follicular layer of the postovulatory follicles. Labeling for integrin β₁ and collagen type IV decreased significantly, whereas a peak in cell death occurred 3 days post-spawning. At 4-5 days post-spawning, the connective theca was more thickened and vascularized. Simultaneously, granulocytes migrated toward the follicular lumen. Thus, follicular apoptosis contributes to early ovarian regression in P. argenteus. Additionally, our findings suggest integrin β₁ and collagen type IV as possible survival factors for follicular cells in teleost ovary.     

Unlike in Drosophila Meroistic Ovaries,Hippo Represses Notch in Blattella germanica Panoistic Ovaries,Triggering the Mitosis-Endocycle Switch in the Follicular Cells

During insect oogenesis, the follicular epithelium undergoes both cell proliferation and apoptosis, thus modulating ovarian follicle growth. The Hippo pathway is key in these processes, and has been thoroughly studied in the meroistic ovaries of Drosophila melanogaster. However, nothing is known about the role of the Hippo pathway in primitive panoistic ovaries. This work examines the mRNA expression levels of the main components of the Hippo pathway in the panoistic ovary of the basal insect species Blattella germanica, and demonstrates the function of Hippo through RNAi. In Hippo-depleted specimens, the follicular cells of the basal ovarian follicles proliferate without arresting cytokinesis; the epithelium therefore becomes bilayered, impairing ovarian follicle growth. This phenotype is accompanied by long stalks between the ovarian follicles. In D. melanogaster loss of function of Notch determines that the stalk is not developed. With this in mind, we tested whether Hippo and Notch pathways are related in B. germanica. In Notch (only)-depleted females, no stalks were formed between the ovarian follicles. Simultaneous depletion of Hippo and Notch rescued partially the stalk to wild-type. Unlike in the meroistic ovaries of D. melanogaster, in panoistic ovaries the Hippo pathway appears to regulate follicular cell proliferation by acting as a repressor of Notch, triggering the switch from mitosis to the endocycle in the follicular cells. The phylogenetically basal position of B. germanica suggests that this might be the ancestral function of Hippo in insect ovaries.     

Synthesis and Membrane Transport of Serotonin in the Developing Ovarian Follicle of Mouse

Using RT-PCR, we showed the presence of Tph1 mRNA in follicular cells and Tph2 mRNA in oocytes isolated from primary multilayer ovarian follicles of mouse and the absence of Ddc expression, which indicates that serotonin cannot be synthesized in a developing ovarian follicle. The membrane serotonin transporter Sert is expressed in follicular cells and oocytes. Experiments on the cultivation of follicles in vitro confirmed the absence of serotonin synthesis from 5-hydroxytryptophan and the presence of membrane transport activity in the oocyte.     

Autophagy and apoptosis interplay during follicular atresia in fish ovary: a morphological and immunocytochemical study

Follicular atresia in fish ovary provides an interesting model for studying autophagy and apoptosis. In order to improve knowledge of the mechanisms regulating ovarian regression, we investigated the immunolocalisation of various proteins involved in the complex network of autophagy and apoptosis. Females of three species of freshwater fish maintained in captivity were sampled after the reproductive period and the main events of follicular atresia were assessed by histology: splits in the zona radiata, yolk degradation and reabsorption, hypertrophy of the follicular cells, accumulation of autophagic vacuoles, closing of the follicular lumen and thickening of the theca. The interplay of apoptosis and autophagy was analysed by TUNEL in situ and by immunocytochemistry for caspase-3, bax, bcl-2, beclin-1 and cathepsin-D. During early and advanced stages of follicular regression, the actin cytoskeleton was well developed and labelling for bcl-2 and cathepsin-D were pronounced in the follicular cells at a stage when they were intensively involved in yolk phagocytosis. Immunofluorescence for beclin-1 was prevalent in the follicular cells, punctate labelling often surrounding autophagic vacuoles during the advanced stage of follicular regression, a critical step towards cell death. TUNEL-positive reaction and immunostaining for bax and caspase-3 demonstrated the participation of apoptosis in late follicular regression. Overall, this study provides evidence that autophagic and apoptotic proteins are activated in a coordinated fashion depending on the stage of follicular regression, with interplay between autophagy and apoptosis being essential in determining the fate of the cell during follicular atresia in fish ovary.     

Plant functional types and their ecological responses to salinization in saline grasslands,Northeastern China

Photosynthetic pathways (C₃, C₄, and CAM) and morphological functional types (e.g. shrubs, high perennial grasses, short perennial graminaceous plants, annual grasses, annual forbs, perennial forbs, halophytes, and hydrophytes) were identified for the species from salinity grasslands in Northeastern China, using the data from both stable carbon isotope ratios (δ¹³C) and from the references published between 1993 and 2002. 150 species, in 99 genera and 37 families, were found with C₃ photosynthesis, and most of these species are dominants [e.g. Leymus chinensis (Trin.) Tzvel., Calamagrostis epigeios (L.), Suaeda corniculata (C.A. Mey.) Bunge]. 40 species in 25 genera and 8 families were identified with C₄ photosynthesis [e.g. Chloris virgata Sw., Aeluropus littoralis (Gouan) Parlat] and 1 species with CAM photosynthesis. Gramineae is the leading family with C₄ photosynthesis (27 species), Chenopodiaceae ranks the second (5 species). The significant increase of C₄ proportions with intense salinity suggested this type plant is remarkable response to the grassland salinization in the region. 191 species were classified into eight morphological functional types and the changes of most of these types (e.g. PEF, HAL, and HPG) were consistent with habitats and vegetation dynamics in the saline grassland. My findings suggest that the photosynthetic pathways, combined with morphological functional types, are efficient means for studying the linkage between species and ecosystems in this type of saline grassland in Northeastern China.     

Study on properties and regulation peculiarities of glycogenolysis enzymes in fish skeletal muscles depending on peculiarities of the species motor activity

The activity values, properties and peculiarities of activation of glycogen phosphorylase (GP, EC 2.4.1.1) and glycogen phosphorylase kinase (GPK, EC 2.7.138) were studied in the white skeletal muscle of fishes differing in motor behavior. No differences in the GP and GPK activity levels were revealed in porgy Diplodus annularis (L.), horse mackerel Trachurus mediterraneus ponticus, trout Salmo trutta morphario, scorpionfish Scorpaena porcus, flounder Scophtalnus maeoticus maeoticus, and carp Cyprinus carpio; however, properties of the isolated enzymes and peculiarities of formation of their activated forms during swimming in a hydrodynamic tube are determined by functional peculiarities of the muscle tissue and are associated with the motor activity character of the species. The more rapid ion regulation prevails in fishes capable for the burst swimming type (scorpionfish, trout). The glycogenolysis hormonal regulation leading to a change of the GPK activity index has been found in other species.     

Distribution of laminin β2, collagen type IV, fibronectin and MMP-9 in ovaries of the teleost fish

Extracellular matrix in the ovarian follicle has been characterised for several mammalian species but there are no reports that describe the immunolocalisation of the extracellular matrix elements, matrix metalloproteinases, and its relation to plasma 17β estradiol levels and follicular apoptosis during the teleost’s reproductive cycle. The present study used immunohistochemistry to characterise the distribution of laminin β2, collagen type IV, fibronectin and matrix metalloproteinases-9 (MMP-9). The TUNEL in situ technique was used to quantify apoptosis and indirect immunofluorimetric to determine plasma 17β estradiol levels. The TUNEL-positive reaction associated with morphological features exhibited follicular apoptosis. During postovulatory follicle involution, the drop in plasma 17β estradiol levels after spawning contributed to the intense apoptosis observed. By immunohistochemical analysis, laminin β2 and collagen type IV were identified as the major constituents of the basement membrane. The loss of integrity of the basement membrane occurred due to lyses of the major constituents, and coincides with increased follicular apoptosis. The integrity of the basement membrane is important for the survival of follicular cells. Furthermore, the MMP-9 results suggest that this enzyme is involved in final oocyte maturation and regression of postovulatory follicles. Fibronectin was observed on the surface of follicular cells of the postovulatory follicle in P. argenteus, this being important for maintaining normal cell adhesion to extracellular matrix. In conclusion, our results suggest that the structure and composition of the extracellular matrix, and plasma 17β estradiol levels related to apoptosis, play an important role during the follicular development and post-spawning involution in teleost fishes.     

Distribution and Y397 phosphorylation of focal adhesion kinase on follicular development in the mouse ovary

Several protein tyrosine kinases (PTKs) are identified as follicle survival factors that suppress apoptosis in granulosa cells. Focal adhesion kinase (FAK/PTK2) interacts with numerous signaling partners and is important for cell adhesion, survival and other vital processes in which FAK autophosphorylation at Y397 (pY397 FAK) is critical for activating signaling pathways. Despite its important roles in apoptosis, the expression and function of FAK in the ovaries remain unknown. Here, we describe FAK expression, including pY397 FAK, in normal healthy mouse ovaries and its association with follicular development and/or atresia. Normal healthy mouse ovaries were used for western blot (n > 60) and immunohistochemical (n > 180) analyses. Western blot results in immature and mature mice revealed that total FAK and pY397 FAK were highly expressed in the ovary and immunohistochemistry results in 3-week-old mice showed they were localized to granulosa cells of ovarian follicles, especially preantral follicles. In 3-week-old mice treated with 5 IU pregnant mare serum gonadotropin (for obtaining homogenous populations of growing or atretic follicles), western blotting revealed that follicular atresia progression involved decreased phosphorylation of Y397 at 72 and 96 h after treatment, particularly in granulosa cells of atretic follicles, as shown by immunohistochemistry results at 72 h after treatment. Moreover, immunostaining patterns of FAK and cleaved caspase-3 were negatively correlated in serial sections of 3-week-old mouse ovaries. These results suggest that FAK is most active in ovarian follicle granulosa cells and that its phosphorylation at Y397 is histologically meaningful in follicular development in normal healthy ovaries.     

An ant species of the genus <Emphasis Type="Italic">Pyramica</Emphasis> (Hymenoptera,Formicidae) new for the fauna of Russia and the Northern Caucasus

Data on the location of the ant Pyramica argiola (Emery 1869) in Russia, in the territory of the Northern Caucasus (Nalchik), are presented for the first time. The species was previously known from Transcaucasia. The species chorology and ecology and morphological peculiarities of individuals from the regional population are considered.     

Follicular atresia in the prepubertal spiny mouse (<Emphasis Type="Italic">Acomys cahirinus</Emphasis>) ovary

This study was designed to determine follicular atresia in the newborn and the prepubertal spiny mouse. We analyzed the processes of follicle loss using classical markers of apoptosis (TUNEL reaction, active caspase-3) and autophagy (Lamp1). Numerous small clear vacuoles and autophagosomes as well as strong Lamp1 staining were observed in dying oocytes of all follicle types, especially of the primordial and primary ones. Active caspase 3 and the TUNEL reaction were detected only in the granulosa cells of large secondary and antral follicles. The expression of apoptosis and autophagy markers was also changing during the prepubertal period. Western blot analysis indicated that at the moment of birth, females undergo an increased rate of follicular atresia mediated by autophagy, while apoptosis is the dominant form of ovarian atresia in consecutive postnatal days. On the basis of these observations, we concluded that apoptosis and autophagy are involved in follicular atresia and these processes are cell and developmental stage-specific.     

EDS1-mediated activation of autophagy regulates <Emphasis Type="Italic">Pst</Emphasis> DC3000 (<Emphasis Type="Italic">AvrRps4</Emphasis>)-induced programmed cell death in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Autophagy is a conserved intracellular process through which cytoplasmic components are degraded and recycled under stress conditions. In the innate immunity of higher plants, autophagy has either pro-survival or pro-death functions in pathogen-induced programmed cell death (PCD). In aged leaves, autophagy negatively regulates PCD by eliminating redundant salicylic acid. However, in young leaves, the specific pro-death mechanisms of autophagy and signaling pathways related to the autophagic process have not been elucidated. Here, we demonstrate that enhanced disease susceptibility 1 (EDS1) mediated the activation of autophagy and played a key role in the pro-death mechanism of autophagy during avirulent Pst DC3000 (AvrRps4) infection. The path through which autophagosomes enter the vacuole was blocked. Additionally, formation of the ATG12–ATG5 complex and the level of enzymatic activity associated with ATG8 cleavage decreased in eds1 mutants. The expression of EDS1 in atg5 mutants was also much lower than that in wild-type plants during pathogen-triggered PCD. These findings implied that EDS1 may regulate autophagy by affecting the activities of the two ubiquitin-like protein-conjugating pathways. Moreover, autophagy may regulate immunity-related PCD by affecting the expression of EDS1 in young plants. Our results provide important insights into the mechanisms of EDS1 in autophagy during infection with avirulent Pst DC3000 (AvrRps4) in Arabidopsis.     

Assessment of Early Postpartum Reproductive Performance in Two High Producing Estonian Dairy Herds

Early postpartum (6 weeks) ovarian activity, hormonal profiles, uterine involution, uterine infections, serum electrolytes, glucose, milk acetoacetate and blood urea nitrogen (BUN) levels were studied in 2 Estonian high producing dairy herd with annual milk production of 7688 (Farm A) and 9425 (Farm B). From each farm 10 cows, with normal calving performance were used. Blood samples for the hormonal (PGF_2α-metabolite, progesterone) analyses were withdrawn. On day 25 PP blood serum samples were taken for the evaluation of metabolic/electrolyte status. On the same day estimation of milk acetoacetate values was done. The ultrasound (US) was started on day 7 PP and was performed every 3^rd day until the end of experiment. Uterine content, follicular activity and sizes of the largest follicle and corpus luteum were monitored and measured. Vaginal discharge and uterine tone were recorded during the rectal palpation. Each animal in the study was sampled for bacteriological examination using endometrial biopsies once a week. Two types of PGF_2α-metabolite patterns were detected: elevated levels during 14 days PP, then decline to the basal level and then a second small elevation at the time of final elimination of the bacteria from the uterus; or elevated levels during first 7 days PP, then decline to the basal level and a second small elevation before the final elimination of bacteria. Endometritis was diagnosed in 5 cows in farm A and in 3 cows in farm B respectively. In farm A, 5 cows out of 10 ovulated during experimental period and in 1 cow cystic ovaries were found. In farm B, 3 cows out of 10 ovulated. In 3 cows cystic ovaries were found. Altogether 40% of cows had their first ovulation during the experimental period. Three cows in farm A and 5 cows in farm B were totally bacteria negative during the experimental period. The most frequent bacteria found were A. pyogenes, Streptococcus spp., E. coli., F. necrophorum and Bacteroides spp. The highest incidence of bacteriological species was found during the first 3 weeks in both farms. All animals were free from bacteria after 5^th week PP in farm A and after 4^th week in farm B respectively. Serum electrolytes and glucose levels were found to be within the reference limits for the cows in both farms. No significant difference was found between farms (p > 0.05). Low phosphorus levels were found in both farms. Significant difference (p < 0.05) was found in BUN levels between farms. In both farms milk acetoacetate values were staying within the reference range given for the used test (<100 μmol/l). The uterine involution and bacterial elimination in the investigated cows could consider as normal but more profound metabolic studies could be needed to find reasons for later resumption of ovarian activity. Some recommendations to changing feeding regimes and strategies should also be given.     

Comparative Morphological Analysis of Ovaries in Cow and Reindeer

A comparative morphological analysis of ovaries of cow and reindeer is performed. The following peculiar features of structure of the reindeer ovary have been revealed: a marked twisting of the ovarian artery and less pronounced functional asymmetry of ovaries. Corpus luteum in both ruminant species in pregnancy has a greater weight than corpus luteum during the cycle. Physiological significance of the revealed peculiarities of the structure of ovaries as well as their possible.     

Transplantation of UC-MSCs on collagen scaffold activates follicles in dormant ovaries of POF patients with long history of infertility

Premature ovarian failure (POF) is a refractory disease for clinical treatment with the goal of restoring fertility. In this study, umbilical cord mesenchymal stem cells on a collagen scaffold (collagen/UC-MSCs) can activate primordial follicles in vitro via phosphorylation of FOXO3a and FOXO1. Transplantation of collagen/UC-MSCs to the ovaries of POF patients rescued overall ovarian function, evidenced by elevated estradiol concentrations, improved follicular development, and increased number of antral follicles. Successful clinical pregnancy was achieved in women with POF after transplantation of collagen/UC-MSCs or UC-MSCs. In summary, collagen/UC-MSC transplantation may provide an effective treatment for POF.