Spatial Proteomics for Further Exploration of Missing Proteins: A Case Study of the Ovary.

In the quest for "missing proteins" (MPs), the proteins encoded by the human genome still lacking evidence of existence at the protein level, novel approaches are needed to detect this challenging group of proteins. The current count stands at 1,343 MPs, and it is likely that many of these proteins are expressed at low levels, in rare cell or tissue types, or the cells in which they are expressed may only represent a small minority of the tissue. Here, we used an integrated omics approach to identify and explore MPs in human ovaries. By taking advantage of publicly available transcriptomics and antibody-based proteomics data in the Human Protein Atlas (HPA), we selected 18 candidates for further immunohistochemical analysis using an exclusive collection of ovarian tissues from women and patients of reproductive age. The results were compared with data from single-cell mRNA sequencing, and seven proteins (CTXN1, MRO, RERGL, TTLL3, TRIM61, TRIM73, and ZNF793) could be validated at the single-cell type level with both methods. We present for the first time the cell type-specific spatial localization of 18 MPs in human ovarian follicles, thereby showcasing the utility of the HPA database as an important resource for identification of MPs suitable for exploration in specialized tissue samples. The results constitute a starting point for further quantitative and qualitative analysis of the human ovaries, and the novel data for the seven proteins that were validated with both methods should be considered as evidence of existence of these proteins in human ovary.

Proteome-wide and matrisome-specific atlas of the human ovary computes fertility biomarker candidates and open the way for precision oncofertility.

Our modern era is witnessing an increasing infertility rate worldwide. Although some of the causes can be attributed to our modern lifestyle (e.g., persistent organic pollutants, late pregnancy), our knowledge of the human ovarian tissue has remained limited and insufficient to reverse the infertility statistics. Indeed, all efforts have been focused on the endocrine and cellular function in support of the cell theory that dates back to the 18th century, while the human ovarian matrisome is still under-described. Hereby, we unveil the extracellular side of the story during different periods of the ovary life, demonstrating that follicle survival and development, and ultimately fertility, would not be possible without its involvement. We examined the human ovarian matrisome and described its remodeling from prepuberty until menopause, creating the first ovarian proteomic codex. Here, we confidently identified and quantified 98 matrisome proteins present in the three ovary groups. Among them, 26 were expressed differently among age groups, delineating a peculiar matrisomal fingerprint at each stage. Such proteins could be potential biomarkers phenotyping ovarian ECM at each age phase of female reproductive life. Beyond proteomics, our study presents a unique approach to understanding the data and depicting the spatiotemporal ECM-intracellular signaling networks and remodeling with age through imaging, advanced text-mining based on natural language processing technology, machine learning, and data sonification. Our findings provide essential context for healthy ovarian physiology, identifying and characterizing disease states, and recapitulating physiological tissues or development in vitro. This comprehensive proteomics analysis represents the ovarian proteomic codex and contributes to an improved understanding of the critical roles that ECM plays throughout the ovarian life span.

Elucidation of the changes occurring at the proteome level in ovaries of high-fat diet-induced obese rats.

High-fat diet-induced obesity adversely affects the female reproductive system. The metabolic changes that the high-fat diet causes on the ovaries have not been elucidated. Herein, to understand the molecular and cellular mechanisms underlying the effects of long-term high-fat diet-fed, the changes in the global proteomic profile of the rat ovaries were investigated. The female rats were randomly divided into two groups based on their diets: the ones that were fed with the high-fat diet and the other ones that were fed with the control diet for 18 weeks. To identify differentially expressed proteins, the changes in ovary proteomes were investigated by two-dimensional electrophoresis coupled to matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight and label-free quantification with nano-high performance liquid chromatography to tandem mass spectrometry (nHPLC-MS/MS). A total of 80 proteins were differentially regulated. The upregulated proteins were involved in responses to chemical and organic substances, cytokines, external stimuli, and lipids. These proteins were particularly associated with vesicles, microbodies, and cell surface proteins. The downregulated proteins were involved in biological processes associated with cellular respiration. Those proteins created a network consisting of proteins involved in aerobic respiration and energy generation. Our results demonstrated that the mechanisms related to energy production in the ovary tissue were particularly affected by the high-fat diet.

Developmental changes in gene expression and gonad morphology during sex differentiation in Atlantic salmon (Salmo salar).

The Atlantic salmon (Salmo salar) is a globally important species for its value in fisheries and aquaculture, and as a research model. In order to characterise aspects of sex differentiation at the morphological and mRNA level in this species, the present study examined developmental changes in gonad morphology and gene expression in males and females between 0 and 79 days post hatch (dph). Morphological differentiation of the ovary (indicated by the formation of germ cell cysts) became apparent from 52 dph. By 79 dph, ovarian phenotype was evident in 100% of genotypic females. Testes remained in an undifferentiated-like state throughout the experiment, containing germ cells dispersed singularly within the gonadal region distal to the mesentery. There were no significant sex-related differences in gonad cross-section size, germ cell number or germ cell diameter during the experiment. The expression of genes involved in teleost sex differentiation (anti-müllerian hormone (amh), cytochrome P450, family 19, subfamily A, polypeptide 1a (cyp19a1a), forkhead box L2a (foxl2a), gonadal soma-derived factor (gsdf), r-spondin 1 (rspo1), sexually dimorphic on the Y chromosome (sdY)), retinoic acid-signalling (aldehyde dehydrogenase 1a2 (aldh1a2), cytochrome P450 family 26 a1 (cyp26a1), cytochrome P450 family 26 b1 (cyp26b1), t-box transcription factor 1 (tbx1a)) and neuroestrogen production (cytochrome P450, family 19, subfamily A, polypeptide 1b (cyp19a1b)) was investigated. Significant sex-related differences were observed only for the expression of amh, cyp19a1a, gsdf and sdY. In males, amh, gsdf and sdY were upregulated from 34, 59 and 44 dph respectively. In females, cyp19a1a was upregulated from 66 dph. Independent of sex, foxl2a expression was highest at 0 dph and had reduced âˆ¼ 47-fold by the time of morphological sex differentiation at 52 dph. This study provides new insights into the timing and sequence of some physiological changes associated with sex differentiation in Atlantic salmon. These findings also reveal that some aspects of the mRNA sex differentiation pathways in Atlantic salmon are unique compared to other teleost fishes, including other salmonids.

Parental diuron exposure causes lower hatchability and abnormal ovarian development in offspring of medaka (Oryzias melastigma).

Diuron is one of the most widely used herbicides worldwide. It has been widely detected in various aquatic environments, especially in marine ecosystems. Although direct effects of diuron exposure on various organisms have been reported, little is known about its effects on marine fishes including multigenerational effects. Herein, the filial generation (F1) of diuron-exposed marine medaka (Oryzias melastigma) (F0) was raised in clean seawater from fertilized eggs to adulthood and used as a marine fish model to study the potential multigenerational effects of diuron. We found that the successful hatching of F1 larvae was significantly reduced and that ovarian development in F1 females was retarded. A significant increase in the percentage of previtellogenic oocytes, along with a visual decrease in the percentage of vitellogenic and mature oocytes in the F1 ovary, were observed. The hormone levels of the hypothalamus-pituitary-gonad-liver axis and vitellogenin-related transcription were downregulated. In addition, the mRNA levels of DNA methyltransferase in the brain, ovary and liver of F1 adult fish exhibited significant upregulation, suggesting that the probable underlying multigenerational mechanism might be associated with epigenetic modifications. Taken together, these results demonstrated that chronic environmental diuron exposure in F0 marine medaka can inhibit F1 ovary development and suggested that diuron may affect marine fish thriving in the ocean.

Concentrations of Tetrodotoxin (TTX) and Its Analogue 4,9-Anhydro TTX in Different Tissues of the Silver-Cheeked Pufferfish (Lagocephalus sceleratus, Gmelin, 1789) Caught in the South-Eastern Mediterranean Sea, Lebanon.

Pufferfishes are among the best-known marine organisms that accumulate marine biotoxins such as Tetrodotoxin (TTX). In the Mediterranean Sea, the silver-cheeked toadfish Lagocephalus sceleratus is the most reported TTX-bearer, causing many fatal and non-fatal cases. In Lebanon, no previous studies have measured TTX levels although the possibility of TTX-poisoning is high since L. sceleratus is caught in different sizes and can be mistaken with other small fishes. Hence, this study reports TTX and its analogue 4,9-anhydro TTX in L. sceleratus collected from Lebanese waters in the Eastern Mediterranean Sea. The results show that TTX concentrations in fish tissues varied between 0.10 and 252.97 µg/g, while those of 4,9-anhydro TTX oscillated between 0.01 and 43.01 µg/g. Internal organs of L. sceleratus were the most toxic parts of its body, with the highest TTX levels found in gonads (mainly ovaries) and liver, followed by the muscles and skin with concentrations always exceeding the safety level. Toxicity fluctuations of L. sceleratus, its expansion, ecological and economic effects were also elucidated. Based on the present findings, it has been confirmed that L. sceleratus constitutes a health, ecological and economic risks, and therefore its trade in seafood markets should be banned to avoid any potential intoxication.

Three classes of epigenomic regulators converge to hyperactivate the essential maternal gene deadhead within a heterochromatin mini-domain.

The formation of a diploid zygote is a highly complex cellular process that is entirely controlled by maternal gene products stored in the egg cytoplasm. This highly specialized transcriptional program is tightly controlled at the chromatin level in the female germline. As an extreme case in point, the massive and specific ovarian expression of the essential thioredoxin Deadhead (DHD) is critically regulated in Drosophila by the histone demethylase Lid and its partner, the histone deacetylase complex Sin3A/Rpd3, via yet unknown mechanisms. Here, we identified Snr1 and Mod(mdg4) as essential for dhd expression and investigated how these epigenomic effectors act with Lid and Sin3A to hyperactivate dhd. Using Cut&Run chromatin profiling with a dedicated data analysis procedure, we found that dhd is intriguingly embedded in an H3K27me3/H3K9me3-enriched mini-domain flanked by DNA regulatory elements, including a dhd promoter-proximal element essential for its expression. Surprisingly, Lid, Sin3a, Snr1 and Mod(mdg4) impact H3K27me3 and this regulatory element in distinct manners. However, we show that these effectors activate dhd independently of H3K27me3/H3K9me3, and that dhd remains silent in the absence of these marks. Together, our study demonstrates an atypical and critical role for chromatin regulators Lid, Sin3A, Snr1 and Mod(mdg4) to trigger tissue-specific hyperactivation within a unique heterochromatin mini-domain.

Mitochondrial Localization of CB1 in Progesterone-producing Cells of Ovarian Interstitial Glands of Adult Mice.

Localization of cannabinoid receptor type 1 (CB1) immunoreactivity on mitochondrial membranes, at least their outer membranes distinctly, was detected in progesterone-producing cells characterized by mitochondria having tubular cristae and aggregations of lipid droplets in ovarian interstitial glands in situ of adult mice. Both immunoreactive and immunonegative mitochondria were contained in one and the same cell. Considering that the synthesis of progesterone is processed in mitochondria, the mitochondrial localization of CB1 in the interstitial gland cells suggests the possibility that endocannabinoids modulate the synthetic process of progesterone in the cells through CB1.

The GnRH Antagonist Degarelix Suppresses Gonadotropin Secretion and Pituitary Sensitivity in Midgestation Sheep Fetuses.

The specific role of gonadotropin-releasing hormone (GnRH) on brain sexual differentiation remains unclear. To investigate whether gonadotropin and, in turn, testosterone (T) secretion is regulated by GnRH during the critical period for brain differentiation in sheep fetuses, we attempted to selectively suppress pituitary-testicular activation during midgestation with the long-acting GnRH antagonist degarelix. Fetuses received subcutaneous injections of the antagonist or vehicle on day 62 of gestation. After 2 to 3 weeks we examined consequences of the intervention on baseline and GnRH-stimulated plasma luteinizing hormone (LH) and T levels. In addition, we measured the effect of degarelix-treatment on messenger RNA (mRNA) expression for the pituitary gonadotropins and key gonadal steroidogenic enzymes. Baseline and GnRH-stimulated plasma LH levels were significantly suppressed in degarelix-treated male and female fetuses compared to control values. Similarly, T concentrations were suppressed in degarelix-treated males. The percentage of LHß-immunoreactive cells colocalizing c-fos was significantly reduced by degarelix treatment indicating that pituitary sensitivity was inhibited. Degarelix treatment also led to the significant suppression of mRNA expression coding for the pituitary gonadotropin subunits and for the gonadal enzymes involved in androgen synthesis. These findings demonstrate that pharmacologic inhibition of GnRH early in gestation results in suppression of LH secretion and deficits in the plasma T levels of male lamb fetuses. We conclude that GnRH signaling plays a pivotal role for regulating T exposure during the critical period of sheep gestation when the brain is masculinized. Thus, disturbance to gonadotropin secretion during this phase of gestation could have long-term consequence on adult sexual behaviors and fertility.

Transcriptomic Analysis of Laying Hens Revealed the Role of Aging-Related Genes during Forced Molting.

Molting in birds provides us with an ideal genetic model for understanding aging and rejuvenation since birds present younger characteristics for reproduction and appearance after molting. Forced molting (FM) by fasting in chickens causes aging of their reproductive system and then promotes cell redevelopment by providing water and feed again. To reveal the genetic mechanism of rejuvenation, we detected blood hormone indexes and gene expression levels in the hypothalamus and ovary of hens from five different periods during FM. Three hormones were identified as participating in FM. Furthermore, the variation trends of gene expression levels in the hypothalamus and ovary at five different stages were found to be basically similar using transcriptome analysis. Among them, 45 genes were found to regulate cell aging during fasting stress and 12 genes were found to promote cell development during the recovery period in the hypothalamus. In addition, five hub genes (INO80D, HELZ, AGO4, ROCK2, and RFX7) were identified by WGCNA. FM can restart the reproductive function of aged hens by regulating expression levels of genes associated with aging and development. Our study not only enriches the theoretical basis of FM but also provides insights for the study of antiaging in humans and the conception mechanism in elderly women.

Cathepsin D knockdown regulates biological behaviors of granulosa cells and affects litter size traits in goats.

Cathepsin D (CTSD), the major lysosomal aspartic protease that is widely expressed in different tissues, potentially regulates the biological behaviors of various cells. Follicular granulosa cells are responsive to the increase of ovulation number, hence indirectly influencing litter size. However, the mechanism underlying the effect of CTSD on the behaviors of goat granulosa cells has not been fully elucidated. This study used immunohistochemistry to analyze CTSD localization in goat ovarian tissues. Moreover, western blotting was applied to examine the differential expression of CTSD in the ovarian tissues of monotocous and polytocous goats. Subsequently, the effects of CTSD knockdown on cell proliferation, apoptosis, cell cycle, and the expression of candidate genes of the prolific traits, including bone morphogenetic protein receptor IB (BMPR-IB), follicle-stimulating hormone (FSHR), and inhibin α (INHA), were determined in granulosa cells. Results showed that CTSD was expressed in corpus luteum, follicle, and granulosa cells. Notably, CTSD expression in the monotocous group was significantly higher than that in the polytocous group. In addition, CTSD knockdown could improve granulosa cell proliferation, inhibit cell apoptosis, and significantly elevate the expression of proliferating cell nuclear antigen (PCNA) and B cell lymphoma 2 (Bcl-2), but it lowered the expression of Bcl-2-associated X (Bax) and caspase-3. Furthermore, CTSD knockdown significantly reduced the ratios of cells in the G0/G1 and G2/M phases but substantially increased the ratio of cells in the S phase. The expression levels of cyclin D2 and cyclin E were elevated followed by the obvious decline of cyclin A1 expression. However, the expression levels of BMPR-IB, FSHR, and INHA clearly increased as a result of CTSD knockdown. Hence, our findings demonstrate that CTSD is an important factor affecting the litter size trait in goats by regulating the granulosa cell proliferation, apoptosis, cell cycle, and the expression of candidate genes of the prolific trait.

Expression profiling of ovarian BMP antagonists reveals the potential interaction between TWSG1 and the chordin subfamily in the ovary.

The TGF-ß superfamily members and their antagonists comprise an indispensable system that controls mammalian ovarian development in a sophisticated manner. In contrast to a plethora of studies on the ovary-expressed TGF-ß superfamily members, knowledge regarding their antagonists, including their expression profiles and antagonism preferences, is still lacking. Using quantitative PCR in rats and transcriptomic dataset comparisons in mice and humans, we set out to characterize the relative expression levels of most antagonists in the mammalian ovary. We found that Twsg1 and Nbl1 are the most abundant BMP antagonists expressed in the rodent and human ovaries, respectively. TWSG1 has been reported to have synergistic action with the chordin subfamily, including CHRD and CHRDL1, the genes of which also showed moderate expression in the mammalian ovary. Therefore, their ovarian expression profiles and antagonisms against the ovary-expressed TGF-ß superfamily members were further characterized. Bioactivity tests indicated that TWSG1 alone can directly inhibit the signaling of BMP6 or BMP7. In addition, it can further enhance the antagonizing ability of CHRD towards BMP2, BMP4, BMP7 and GDF5, or CHRDL1's antagonism towards BMP2, BMP4, GDF5 and activin A. In combination with their distinct transcript profiles in ovarian compartments, our findings suggest that TWSG1 may work coordinately with CHRD within theca/interstitial shells and also with CHRDL1 in developing granulosa cells; these interactions would modulate the intraovarian functions of the TGF-ß superfamily members, such as the control of progesterone production.

Comparison of age-related anti Müllerian hormone, inhibin-b levels and follicle reserve in rat ovarium / Comparación de la hormona anti-mülleriana relacionada con la edad, los niveles de inhibina-b y la reserva folicular en el ovario de rata

SUMMARY: Anti-Müllerian hormone (AMH) and Inhibin B (INHB) in the glycoprotein structure are members of the transforming growth factor β family and expressed by granulosa cells from puberty. AMH is a factor that increases the life span of small developing follicles. For this reason, it is widely used to determine the ovarian reserve and age. Inhibin-B secreted from granulosa cells plays a role in regulation of the Follicle Stimulating Factor (FSH) and determination of the follicle diameter. There are few studies on the effect of these two age-related hormones on ovarian histology in rats. In this study, AMH and INHB expression in ovarian tissues of female rats of different age groups, their relationship with ovarian structure and folliculogenesis were examined histologically and biochemically. Wistar Albino rats were used in the study and a total of 3 groups were formed. The ovaries of rats in the pre-oestrous period were collected, and follicle count was performed on tissue sections in batches. Expression of AMH in the follicles was identified immunohistochemically. In serum, AMH and INHB levels were assessed by ELISA method and their significance was evaluated statistically. Results from light microscopic examination determined that AMH was expressed from the granulosa cells of developing follicles. INHB expression during the prepubertal period and AMH had a protective effect on the ovarian reserve and the number of developing follicles, respectively.

RESUMEN: La hormona antimülleriana (AMH) y la inhibina B (INHB) en la estructura de la glicoproteína son miembros de la familia del factor de crecimiento transformante β y se expresan en las células de la granulosa desde la pubertad. La AMH es un factor que aumenta la vida útil de los pequeños folículos en desarrollo. Por este motivo, se utiliza frecuentemente para determinar la reserva ovárica y la edad. La inhibina B secretada por las células de la granulosa tiene un rol en la regulación del factor estimulante de (FSH) y en la determinación del diámetro del folículo. Hay pocos estudios sobre el efecto de estas dos hormonas relacionadas con la edad en la histología ovárica en ratas. Se examinaron histológica y bioquímicamente la expresión de AMH e INHB en tejidos ováricos de ratas hembras de diferentes grupos de edad, su relación con la estructura ovárica y la foliculogénesis. Se utilizaron ratas Wistar Albino en el estudio y se formaron 3 grupos. En los ovarios de ratas en el período preestro se realizó el recuento de folículos en secciones de tejido. La expresión de AMH en los folículos se identificó inmunohistoquímicamente. En suero, los niveles de AMH e INHB se evaluaron mediante el método ELISA y su importancia se evaluó estadísticamente. Los resultados del examen con microscopio óptico determinaron que la AMH se expresaba a partir de las células de la granulosa de los folículos en desarrollo. La expresión de INHB durante el período prepuberal y AMH tuvo un efecto protector sobre la reserva ovárica y el número de folículos en desarrollo, respectivamente.

MRPL15 is a novel prognostic biomarker and therapeutic target for epithelial ovarian cancer.

PURPOSE: To analyze the role of six human epididymis protein 4 (HE4)-related mitochondrial ribosomal proteins (MRPs) in ovarian cancer and selected MRPL15, which is most closely related to the tumorigenesis and prognosis of ovarian cancer, for further analyses. METHODS: Using STRING database and MCODE plugin in Cytoscape, six MRPs were identified among genes that are upregulated in response to HE4 overexpression in epithelial ovarian cancer cells. The Cancer Genome Atlas (TCGA) ovarian cancer, GTEX, Oncomine, and TISIDB were used to analyze the expression of the six MRPs. The prognostic impact and genetic variation of these six MRPs in ovarian cancer were evaluated using Kaplan-Meier Plotter and cBioPortal, respectively. MRPL15 was selected for immunohistochemistry and GEO verification. TCGA ovarian cancer data, gene set enrichment analysis, and Enrichr were used to explore the mechanism of MRPL15 in ovarian cancer. Finally, the relationship between MRPL15 expression and immune subtype, tumor-infiltrating lymphocytes, and immune regulatory factors was analyzed using TCGA ovarian cancer data and TISIDB. RESULTS: Six MRPs (MRPL10, MRPL15, MRPL36, MRPL39, MRPS16, and MRPS31) related to HE4 in ovarian cancer were selected. MRPL15 was highly expressed and amplified in ovarian cancer and was related to the poor prognosis of patients. Mechanism analysis indicated that MRPL15 plays a role in ovarian cancer through pathways such as the cell cycle, DNA repair, and mTOR 1 signaling. High expression of MRPL15 in ovarian cancer may be associated with its amplification and hypomethylation. Additionally, MRPL15 showed the lowest expression in C3 ovarian cancer and was correlated with proliferation of CD8+ T cells and dendritic cells as well as TGFßR1 and IDO1 expression. CONCLUSION: MRPL15 may be a prognostic indicator and therapeutic target for ovarian cancer. Because of its close correlation with HE4, this study provides insights into the mechanism of HE4 in ovarian cancer.

The Osa-Containing SWI/SNF Chromatin-Remodeling Complex Is Required in the Germline Differentiation Niche for Germline Stem Cell Progeny Differentiation.

The Drosophila ovary is recognized as a powerful model to study stem cell self-renewal and differentiation. Decapentaplegic (Dpp) is secreted from the germline stem cell (GSC) niche to activate Bone Morphogenic Protein (BMP) signaling in GSCs for their self-renewal and is restricted in the differentiation niche for daughter cell differentiation. Here, we report that Switch/sucrose non-fermentable (SWI/SNF) component Osa depletion in escort cells (ECs) results in a blockage of GSC progeny differentiation. Further molecular and genetic analyses suggest that the defective germline differentiation is partially attributed to the elevated dpp transcription in ECs. Moreover, ectopic Engrailed (En) expression in osa-depleted ECs partially contributes to upregulated dpp transcription. Furthermore, we show that Osa regulates germline differentiation in a Brahma (Brm)-associated protein (BAP)-complex-dependent manner. Additionally, the loss of EC long cellular processes upon osa depletion may also partly contribute to the germline differentiation defect. Taken together, these data suggest that the epigenetic factor Osa plays an important role in controlling EC characteristics and germline lineage differentiation.

Pressurized intra-peritoneal aerosol chemotherapy (PIPAC): increased intraperitoneal pressure does not affect distribution patterns but leads to deeper penetration depth of doxorubicin in a sheep model.

BACKGROUND: Pressurized Intra-Peritoneal Aerosol Chemotherapy (PIPAC) is an innovative treatment against peritoneal carcinomatosis. Doxorubicin is a common intra-venous chemotherapy used for peritoneal carcinomatosis and for PIPAC. This study evaluated the impact of increased PIPAC intraperitoneal pressure on the distribution and cell penetration of doxorubicin in a sheep model. METHODS: Doxorubicin was aerosolized using PIPAC into the peritoneal cavity of 6 ewes (pre-alpes breed): N = 3 with 12 mmHg intraperitoneal pressure ("group 12") and N = 3 with 20 mmHg ("group 20"). Samples from peritoneum (N = 6), ovarian (N = 1), omentum (N = 1) and caecum (N = 1) were collected for each ewe. The number of doxorubicin positive cells was determined using the ratio between doxorubicine fluorescence-positive cell nuclei (DOXO+) over total number of DAPI positive cell nuclei (DAPI+). Penetration depth (µm) was defined as the distance between the luminal surface and the location of the deepest DOXO+ nuclei over the total number of cell nuclei that were stained with DAPI. Penetration depth (µm) was defined as the distance between the luminal surface and the location of the deepest DOXO+ nuclei. RESULTS: DOXO+ nuclei were identified in 87% of samples. All omental samples, directly localized in front of the nebulizer head, had 100% DOXO+ nuclei whereas very few nuclei were DOXO+ for caecum. Distribution patterns were not different between the two groups but penetration depth in ovary and caecum samples was significantly deeper in group 20. CONCLUSIONS: This study showed that applying a higher intra-peritoneal pressure during PIPAC treatment leads to a deeper penetration of doxorubicin in ovarian and caecum but does not affect distribution patterns.

Conjugation of (-)-epigallocatechin-3-gallate and protein isolate from large yellow croaker (Pseudosciaena crocea) roe: improvement of antioxidant activity and structural characteristics.

BACKGROUND: Large yellow croaker (Pseudosciaena crocea) roe is the main by-product in the processing of large yellow croaker. Previous studies have found that its protein isolates are composed of vitellogenin, as well as vitellogenin B and C, having good functional properties. (-)-Epigallocatechin-3-gallate (EGCG) is a natural antioxidant component that can be combined with protein to improve antioxidant activity and structural characteristics of protein. RESULTS: EGCG was bound with the P. crocea roe protein isolate (pcRPI) by the free radical method to prepare the conjugate. The formation of pcRPI-EGCG conjugates was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography, which showed that the calculated weight-average molar masses of native-pcRPI and pcRPI-EGCG conjugates were 86.9 and 215.3 kDa, respectively. The results of fluorescence, ultraviolet, circular and infrared spectra indicated that the conjugation of EGCG with native-pcRPI changed the secondary and tertiary structure of native-pcRPI. The pcRPI-EGCG conjugates exhibited higher thermal stability than native-pcRPI. The radical scavenging and reducing power of native-pcRPI were increased by 2.0-2.5- and 1.4-fold, respectively, after the EGCG-grafting reaction. CONCLUSION: These results indicate that the binding of pcRPI and EGCG effectively improved the antioxidant properties and structural characteristics of the pcRPI. © 2021 Society of Chemical Industry.

Brenner tumor associated with rete ovarii: a histologic and immunohistochemical analysis of six cases exploring the relationship between these entities.

The association of Brenner tumor (BT) with rete ovarii (RO) has been rarely alluded to in the literature. Both entities have debatable histogenesis. In this study of six cases of BT associated with RO, we describe the morphologic features and performed immunohistochemical staining for markers of Mullerian, Wolffian, mesothelial, and sex cord stromal derivation to explore the relationship between these entities. Histologically, all BTs were benign, microscopic, and incidental. RO was prominent and hyperplastic with gradual or abrupt transition to BT. In addition, focal areas of rete entrapped between BT nests were seen. All BTs were positive for GATA-3 and negative for PAX-8. Conversely, the RO in all cases was negative for GATA-3 and positive for PAX-8. WT-1 was positive in both entities. Sex cord stromal and mesothelial markers (other than WT-1) were negative in BT and RO. Although morphologically, BTs seem to arise from RO in these cases, they have a distinct immunophenotype. It is possible that at least some BTs arise from metaplastic changes in RO epithelium.

Discovery of the Dmrt gene family members based on transcriptome analysis in mud crab Scylla paramamosain.

Doublesex and mab-3 related transcription factors (Dmrts) play crucial roles in sex determination/differentiation and gonad development. The information on Dmrts and their functions are still scarce in mud crab Scylla paramamosain. In this study, 12 published transcriptome data of S. paramamosain were retrieved, pooled, and assembled. From the assembly, 7 Dmrt gene family members were identified and consisted of Spdmrt-like, Spdmrt-1a, Spdmrt-3, Spdmrt-11E, Spidmrt-1, Spdoublesex (Spdsx), and Spidmrt-2. These dmrt genes were predicted to encode 224 aa, 465 aa, 435 aa, 276 aa, 520 aa, 552 aa, and 266 aa protein precursors, respectively. The expression patterns of the dmrt genes were characterized by semi-quantitative PCR. The Spdmrt-like and Spdmrt-1a were exclusively detected in gonads, of which both expression levels in the testis were higher than that in the ovary. The Spdmrt-3, Spdmrt-11E, Spidmrt-1, Spdsx, and Spidmrt-2 were observed in various tissues; all these genes were sexually dimorphic except for dmrt-11E. Specifically, the expression level of Spdmrt-3 and Spidmrt-2 were higher in the testis than that in the ovary. On the contrary, the Spdsx and Spidmrt-1 expression level were higher in ovary than that in testis. The present study's findings provided a fundamental understanding of Dmrt gene family members involving sex determination/differentiation and gonad development in the S. paramamosain.

The protective effects of resveratrol pretreatment in cyclophosphamide-induced rat ovarian injury: an vivo study.

OBJECTIVES: To explore whether resveratrol (Res) pretreatment could exert a protective effect on cyclophosphamide (Cy) induced ovarian toxicity in a rat model. METHODS: Twenty-four female 7-week old Sprague-Dawley rats were randomly divided into four groups: Con, administered with vehicle solutions; Cy, treated with Cy; Res + Cy, treated with Cy + Res combined; Res, treated with Res. After 21 d of treatments, the rats were euthanized and blood samples were collected to evaluate the levels of anti-Müllerian hormone (AMH). The Ovaries were processed for immunohistochemical and western blotting. RESULTS: Cy-treat caused the decrease of body weights and ovarian weight. AMH was lower in Cy group, whereas AMH levels were similar among other groups. Histomorphology showed a large number of primordial follicles were activated in Cy groups, whereas the primordial follicles were inhibited in the Res and Res + Cy groups. The expressions of Sirt1, Foxo3a were up-regulated and p53, Caspase-3, and Bax were down-regulated in Res + Cy and Res groups (p < .05). CONCLUSIONS: Res can prevent the primordial follicle activation and decrease apoptosis induced by Cy. Res may be an effective protection for ovarian function during chemotherapy, which means a new nonsurgical application for protection of ovarian reserve.