Cell polarity opposes Jak/STAT-mediated Escargot activation that drives intratumor heterogeneity in a Drosophila tumor model.

In proliferating neoplasms, microenvironment-derived selective pressures promote tumor heterogeneity by imparting diverse capacities for growth, differentiation, and invasion. However, what makes a tumor cell respond to signaling cues differently from a normal cell is not well understood. In the Drosophila ovarian follicle cells, apicobasal-polarity loss induces heterogeneous epithelial multilayering. When exacerbated by oncogenic-Notch expression, this multilayer displays an increased consistency in the occurrence of morphologically distinguishable cells adjacent to the polar follicle cells. Polar cells release the Jak/STAT ligand Unpaired (Upd), in response to which neighboring polarity-deficient cells exhibit a precursor-like transcriptomic state. Among the several regulons active in these cells, we could detect and further validate the expression of Snail family transcription factor Escargot (Esg). We also ascertain a similar relationship between Upd and Esg in normally developing ovaries, where establishment of polarity determines early follicular differentiation. Overall, our results indicate that epithelial-cell polarity acts as a gatekeeper against microenvironmental selective pressures that drive heterogeneity.

Follicle cell contact maintains main body axis polarity in the Drosophila melanogaster oocyte.

In Drosophila melanogaster, the anterior-posterior body axis is maternally established and governed by differential localization of partitioning defective (Par) proteins within the oocyte. At mid-oogenesis, Par-1 accumulates at the oocyte posterior end, while Par-3/Bazooka is excluded there but maintains its localization along the remaining oocyte cortex. Past studies have proposed the need for somatic cells at the posterior end to initiate oocyte polarization by providing a trigger signal. To date, neither the molecular identity nor the nature of the signal is known. Here, we provide evidence that mechanical contact of posterior follicle cells (PFCs) with the oocyte cortex causes the posterior exclusion of Bazooka and maintains oocyte polarity. We show that Bazooka prematurely accumulates exclusively where posterior follicle cells have been mechanically detached or ablated. Furthermore, we provide evidence that PFC contact maintains Par-1 and oskar mRNA localization and microtubule cytoskeleton polarity in the oocyte. Our observations suggest that cell-cell contact mechanics modulates Par protein binding sites at the oocyte cortex.

Knockdown of E1- and E2-ubiquitin enzymes triggers defective chorion biogenesis and modulation of autophagy-related genes in the follicle cells of the vector Rhodnius prolixus.

In insects, the last stage of oogenesis is the process where the chorion layers (eggshell) are synthesized and deposited on the surface of the oocytes by the follicle cells. Protein homeostasis is determined by the fine-tuning of translation and degradation pathways, and the ubiquitin-proteasome system is one of the major degradative routes in eukaryotic cells. The conjugation of ubiquitin to targeted substrates is mediated by the ordered action of E1-activating, E2-conjugating, and E3-ligase enzymes, which covalently link ubiquitin to degradation-targeted proteins delivering them to the proteolytic complex proteasome. Here, we found that the mRNAs encoding polyubiquitin (pUbq), E1, and E2 enzymes are highly expressed in the ovaries of the insect vector of Chagas Disease Rhodnius prolixus. RNAi silencing of pUbq was lethal whereas the silencing of E1 and E2 enzymes resulted in drastic decreases in oviposition and embryo viability. Eggs produced by the E1- and E2-silenced insects presented particular phenotypes of altered chorion ultrastructure observed by high-resolution scanning electron microscopy as well as readings for dityrosine cross-linking and X-ray elemental microanalysis, suggesting a disruption in the secretory routes responsible for the chorion biogenesis. In addition, the ovaries from silenced insects presented altered levels of autophagy-related genes as well as a tendency of upregulation in ER chaperones, indicating a disturbance in the general biosynthetic-secretory pathway. Altogether, we found that E1 and E2 enzymes are essential for chorion biogenesis and that their silencing triggers the modulation of autophagy genes suggesting a coordinated function of both pathways for the progression of choriogenesis.

Evaluation of ImageJ software in ultrasonic image analysis: Follicular and luteal morphological characteristics of cattle.

This study was conducted to compare the effectiveness of two methodologies in evaluating B- and Doppler-mode ultrasonic images: analysis using ultrasonic software and utilizing a computer with ImageJ software. To determine if ImageJ software utilization is an efficacious alternative to ultrasonic software device- analysis, there were comparisons of values when using the two methods for morphological and vascular characteristics of follicular dynamics and luteal function in 18 crossbred cattle. From day 8 of an ovarian dynamics synchronization treatment regimen period until the time of ovulation (Day 10), B-mode and power-flow ultrasonic cineloops were obtained every 12 h to assess follicular diameter, wall area, and wall blood perfusion area. On Day 14 after ovulation, US cineloops of ovaries were obtained in B mode and power flow to evaluate various morphological and vascular characteristics of the corpus luteum (CL), including luteal diameter, luteal area, and CL blood perfusion area. Cineloops were evaluated and analyzed using ultrasonic software, and in a computer with ImageJ software. To evaluate consistency in results between the two methods, there was evaluation utilizing paired t-test, Pearson correlation coefficient, Bland-Altman plot, and Linear Regression Test procedures to calculate proportion of bias between values for measurements of variables evaluated. Results indicated none of the values for variables before and after ovulation differed (P > 0.05). It, therefore, was concluded that utilization of ImageJ software is an efficacious biomedical technique to analyze ultrasonic images of morphological and vascular characteristics before and after ovulation in cattle.

Expression of steroidogenic enzymes and TGFß superfamily members in follicular cells of prepubertal gilts with distinct endocrine profiles.

Regulation of the transforming growth factor beta (TGFß) superfamily by gonadotrophins in swine follicular cells is not fully understood. This study evaluated the expression of steroidogenic enzymes and members of the TGFß superfamily in prepubertal gilts allocated to three treatments: 1200 IU eCG at D -3 (eCG); 1200 IU eCG at D -6 plus 500 IU hCG at D -3 (eCG + hCG); and the control, composed of untreated gilts. Blood samples and ovaries were collected at slaughter (D0) and follicular cells were recovered thereafter. Relative gene expression was determined by real-time PCR. Serum progesterone levels were greater in the eCG + hCG group compared with the other groups (P < 0.01). No differences were observed in the expression of BMP15, BMPR1A, BMPR2, FSHR, GDF9, LHCGR and TGFBR1 (P > 0.05). Gilts from the eCG group presented numerically greater mean expression of CYP11A1 mRNA than in the control group that approached statistical significance (P = 0.08) and greater expression of CYP19A1 than in both the eCG and the control groups (P < 0.05). Expression of BMPR1B was lower in the eCG + hCG treatment group compared with the control (P < 0.05). In conclusion, eCG treatment increased the relative expression of steroidogenic enzymes, whereas treatment with eCG + hCG increased serum progesterone levels. Although most of the evaluated TGFß members were not regulated after gonadotrophin treatment, the downregulation of BMPR1B observed after treatment with eCG + hCG and suggests a role in luteinization regulation.

Molecular characterization, expression profile and transcriptional regulation of the CYP19 gene in goose ovarian follicles.

Cytochrome P450 Family 19 (CYP19) is a crucial enzyme to catalyze the conversion of androgens to estrogens. However, the regulatory mechanism of goose CYP19 gene remains poorly understood. The present study attempted to obtain the full-length coding sequence (CDS) and 5'-flanking sequence of CYP19 gene, to investigate its expression and distribution profiles in different sized follicles, and to analyze the transcriptional regulatory mechanism of CYP19 gene in goose. Results showed that its CDS consisted of 1512 nucleotides and the encoded amino acid sequence contained a classical P450 structural domain. Homology analysis showed that there were high homologies of nucleotide and amino acid sequences between goose and other avian species. Its promoter sequence spanned from -1925 bp to the transcription start site (ATG) and several transcriptional factors were predicted in this region. Further analysis from luciferase assay showed that the luciferase activity was the highest spanning from -118 to -1 bp by constructing deletion promoter reporter vector. In addition, result from quantitative real-time polymerase chain reaction indicated that the mRNA level of CYP19 gene were highly expressed in theca layer of the fifth largest follicle, and the cellular location was in the theca externa cells by immunohistochemistry. Taken together, it could be concluded that the transcription activity of CYP19 gene was activated by transcriptional factors in its proximal region of promoter to promote the synthesis of estrogens, regulating the selection of pre-hierarchical into hierarchical follicle in goose.

El consumo semicrónico moderado de alcohol altera la foliculogénesis y la calidad núcleo-citoplasmática oocitaria, en el ratón / Moderate semi-chronic alcohol consumption alters folliculogenesis and oocyte nuclear-cytoplasmic quality in mice

Resumen El consumo crónico de alcohol es un problema de salud mundial que afecta particularmente a la población femenina. Sin embargo, los efectos de la ingesta semicrónica en cantidades moderadas a bajas en el ovario y el oocito son poco conocidos. En un modelo murino, se administró etanol al 10% en agua de bebida (hembras tratadas) o agua (hembras control) por 15 días, y luego de la superovulación o no (ovulación espontánea), se analizó el ciclo estral y la calidad ovárico-gamética. En las hembras tratadas, la frecuencia y duración del diestro aumentó, y las frecuencias de folículos y cuerpos lúteos disminuyeron vs hembras controles, valores que se restauraron luego de la superovulación. Sin embargo, en las hembras tratadas, la tasa de proliferación celular folicular y el desbalance de la expresión ovárica de VEGF (factor de crecimiento endotelial) persistieron luego de la superovulación. El número de ovocitos ovulados con metafase II anormal, fragmentados y activados partenogenéticamente fue mayor en las hembras tratadas respecto las controles. En conclusión, el consumo semicrónico moderado de alcohol produce anestro, ciclo estral irregular, foliculogénesis deficiente y anomalías núcleo-citoplasmáticas en los oocitos ovulados. Estas alteraciones podrían constituirse en un factor etiológico de pérdida gestacional temprana y desarrollo embrionario anormal luego del consumo de alcohol.

Abstract Chronic alcohol consumption is a global health problem that particularly affects the female population. However, the ef-fects of semi-chronic ethanol intake in low-moderate amounts on the ovary and oocyte are poorly understood. In a mouse model, 10% ethanol was administered in drinking water (treated females) or water (control females) for 15 days, and after superovulation or not (spontaneous ovulation), the estrous cycle and ovarian-gametic quality were analyzed. In treated females, the frequency and duration of the diestrus increased, and the frequencies of follicles and corpus luteum decreased vs control females, values that restored after superovulation. However, in treated females, the follicular cell proliferation rate and the imbalance in ovarian expression of VEGF (endothelial growth factor) persisted after superovulation. The number of ovulated oocytes with abnormal metaphase II, fragmented and parthenogenetically activated was higher in treated females than in control ones. In conclusion, moderate semi-chronic alcohol consumption produces anestrum, irregular estrous cycle, poor folliculogenesis, and nuclear-cytoplasmic abnormalities in ovulated oocytes. These alterations could constitute an etiological factor of early gestational loss and abnormal embryonic development after alcohol consumption.

Single-Cell Transcriptomics Analysis of Human Small Antral Follicles.

Human ovarian folliculogenesis is a highly regulated and complex process. Characterization of follicular cell signatures during this dynamic process is important to understand follicle fate (to grow, become dominant, or undergo atresia). The transcriptional signature of human oocytes and granulosa cells (GCs) in early-growing and ovulatory follicles have been previously described; however, that of oocytes with surrounding GCs in small antral follicles have not been studied yet. Here, we have generated a unique dataset of single-cell transcriptomics (SmartSeq2) consisting of the oocyte with surrounding GCs from several individual (non-dominant) small antral follicles isolated from adult human ovaries. We have identified two main types of (healthy) follicles, with a distinct oocyte and GC signature. Using the CellphoneDB algorithm, we then investigated the bi-directional ligand-receptor interactions regarding the transforming growth factor-ß (TGFß)/bone morphogenetic protein (BMP), wingless-type (MMTV)-integration site (WNT), NOTCH, and receptor tyrosine kinases (RTK) signaling pathways between oocyte and GCs within each antral follicle type. Our work not only revealed the diversity of small antral follicles, but also contributes to fill the gap in mapping the molecular landscape of human folliculogenesis and oogenesis.

Maintenance of quiescent oocytes by noradrenergic signals.

All females adopt an evolutionary conserved reproduction strategy; under unfavorable conditions such as scarcity of food or mates, oocytes remain quiescent. However, the signals to maintain oocyte quiescence are largely unknown. Here, we report that in four different species - Caenorhabditis elegans, Caenorhabditis remanei, Drosophila melanogaster, and Danio rerio - octopamine and norepinephrine play an essential role in maintaining oocyte quiescence. In the absence of mates, the oocytes of Caenorhabditis mutants lacking octopamine signaling fail to remain quiescent, but continue to divide and become polyploid. Upon starvation, the egg chambers of D. melanogaster mutants lacking octopamine signaling fail to remain at the previtellogenic stage, but grow to full-grown egg chambers. Upon starvation, D. rerio lacking norepinephrine fails to maintain a quiescent primordial follicle and activates an excessive number of primordial follicles. Our study reveals an evolutionarily conserved function of the noradrenergic signal in maintaining quiescent oocytes.

Equine Chorionic Gonadotropin as an Effective FSH Replacement for In Vitro Ovine Follicle and Oocyte Development.

The use of assisted reproductive technologies (ART) still requires strategies through which to maximize individual fertility chances. In vitro folliculogenesis (ivF) may represent a valid option to convey the large source of immature oocytes in ART. Several efforts have been made to set up ivF cultural protocols in medium-sized mammals, starting with the identification of the most suitable gonadotropic stimulus. In this study, Equine Chorionic Gonadotropin (eCG) is proposed as an alternative to Follicle Stimulating Hormone (FSH) based on its long superovulation use, trans-species validation, long half-life, and low costs. The use of 3D ivF on single-ovine preantral (PA) follicles allowed us to compare the hormonal effects and to validate their influence under two different cultural conditions. The use of eCG helped to stimulate the in vitro growth of ovine PA follicles by maximizing its influence under FBS-free medium. Higher performance of follicular growth, antrum formation, steroidogenic activity and gap junction marker expression were recorded. In addition, eCG, promoted a positive effect on the germinal compartment, leading to a higher incidence of meiotic competent oocytes. These findings should help to widen the use of eCG to ivF as a valid and largely available hormonal support enabling a synchronized in vitro follicle and oocyte development.

Hsa-mir-548 family expression in human reproductive tissues.

BACKGROUND: Hsa-miR-548ba expressed in ovarian granulosa cells targets PTEN and LIFR, which are essential for ovarian follicle activation and growth. The expression pattern of hsa-miR-548ba correlates with its host gene follicle-stimulating hormone receptor (FSHR), and FSH has a positive influence on hsa-miR-548ba expression. However, hsa-miR-548ba is a member of a large hsa-mir-548 family with potentially overlapping targets. The current study aims to investigate the co-expression of hsa-mir-548 family members in FSHR-positive reproductive tissues and to explore the potential co-regulation of pathways. RESULTS: For the above-described analysis, small RNA sequencing data from public data repositories were used. Sequencing results revealed that hsa-miR-548ba was expressed at the highest level in the ovarian granulosa cells and uterine myometrial samples together with another twelve and one hsa-miR-548 family members, respectively. Pathway enrichment analysis of microRNA targets in the ovarian samples revealed the hsa-miR-548ba and hsa-miR-548b-5p co-regulation of RAB geranylgeranylation in mural granulosa cells. Moreover, other hsa-mir-548 family members co-regulate pathways essential for ovarian functions (PIP3 activates AKT signalling and signalling by ERBB4). In addition to hsa-miR-548ba, hsa-miR-548o-3p is expressed in the myometrium, which separately targets the peroxisome proliferator-activated receptor alpha (PPARA) pathway. CONCLUSION: This study reveals that hsa-mir-548 family members are expressed in variable combinations in the reproductive tract, where they potentially fulfil different regulatory roles. The results provide a reference for further studies of the hsa-mir-548 family role in the reproductive tract.

Ovarian Folliculogenesis and Uterine Endometrial Receptivity after Intermittent Vaginal Injection of Recombinant Human Follicle-Stimulating Hormone in Infertile Women Receiving In Vitro Fertilization and in Immature Female Rats.

The uterine first-pass effect occurs when drugs are delivered vaginally. However, the effect of vaginally administered recombinant human follicle-stimulating hormone (rhFSH) on ovarian folliculogenesis and endometrial receptivity is not well established. We aimed to compare the efficacy of rhFSH administered vaginally and abdominally in clinical in vitro fertilization (IVF) treatment, pharmacokinetic study, and animal study. In IVF treatment, the number of oocytes retrieved, endometrial thickness and uterine artery blood perfusion were not different between women who received the rhFSH either vaginally or abdominally. For serum pharmacokinetic parameters, significantly lower Tmax, clearance, and higher AUC and T1/2_elimination of rhFSH were observed in women who received rhFSH vaginally, but urine parameters were not different. Immature female rats that received daily abdominal or vaginal injections (1 IU twice daily for 4 days) or intermittent vaginal injections (4 IU every other day for two doses) of rhFSH had more total follicles than the control group. In addition, the serum progesterone and progesterone receptors in the local endometrium were significantly higher in the groups treated with intermittent abdominal or vaginal injection of rhFSH, compared with those who recieved daily injection. In summary, vaginal administration of rhFSH may provide an alternative treatment regimen in women receiving IVF.

Upregulation of Low-Density Lipoprotein Receptor of the Steroidogenesis Pathway in the Cumulus Cells Is Associated with the Maturation of Oocytes and Achievement of Pregnancy.

The maturation of the oocyte is influenced by cumulus cells (CCs) and associated with pregnancy rate, whereas the influencing factors have not been completely elucidated in the CCs. In this study, we identified new regulators of CCs for high-quality oocytes and successful pregnancies during assisted reproductive techniques. CCs were collected from cumulus-oocyte complexes (COCs) in young (≤33 years old) and old (≥40 years old) women undergoing intracytoplasmic sperm injection (ICSI) procedures. We screened for factors differentially expressed between young vs. old CCs and pregnancy vs. non-pregnancy using whole mRNA-seq-next-generation sequencing (NGS). We characterized the transcriptome of the CCs to identify factors critical for achieving pregnancy in IVF cycles. Women in the young and old pregnancy groups exhibited the up- and downregulation of multiple genes compared with the non-pregnancy groups, revealing the differential regulation of several specific genes involved in ovarian steroidogenesis in CCs. It was shown that the low-density lipoprotein (LDL) receptor to the steroidogenesis pathway was upregulated in CCs with higher maturity rates of oocytes in the pregnancy group. In conclusion, a higher pregnancy rate is related to the signaling pathway of steroidogenesis by the LDL receptor in infertile women undergoing IVF procedures.

Orexin-A Regulates Follicular Growth, Proliferation, Cell Cycle and Apoptosis in Mouse Primary Granulosa Cells via the AKT/ERK Signaling Pathway.

Granulosa cells (GCs) are essential for follicular growth, development, and atresia. The orexin-A (OXA) neuropeptide is widely involved in the regulation of various biological functions. OXA selectively binds to orexin receptor type 1 (OX1R) and mediates all its biological actions via OX1R. This study aimed to explore the expression of OXA and OX1R and their regulatory role in GCs proliferation, cell cycle progression, apoptosis, oocyte maturation, and underlying molecular mechanisms of these processes and elucidate its novel signaling pathway. Western blotting and RT-qPCR showed that OXA and OX1R were expressed during different developmental stages of GCs, and siRNA transfection successfully inhibited the expression of OX1R at the translational and transcriptional levels. Flow cytometry revealed that OX1R knockdown upregulated GCs apoptosis and triggered S-phase arrest in cell cycle progression. RT-qPCR and Western blotting showed significantly reduced expression of Bcl-2 and elevated expression of Bax, caspase-3, TNF-α, and P21 in OX1R-silenced GCs. Furthermore, the CCK-8 assay showed that knockdown of OX1R suppressed GCs proliferation by downregulating the expression of PCNA, a proliferation marker gene, at the translational and transcriptional levels. Western blotting revealed that knockdown of OX1R resulted in a considerable decrease of the phosphorylation level of the AKT and ERK1/2 proteins, indicating that the AKT/ERK1/2 pathway is involved in regulating GCs proliferation and apoptosis. In addition, OX1R silencing enhanced the mRNA expression of GDF9 and suppressed the mRNA expression of BMP15 in mouse GCs. Collectively, these results reveal a novel regulatory role of OXA in the development of GCs and folliculogenesis by regulating proliferation, apoptosis, and cell cycle progression. Therefore, OXA can be a promising therapeutic agent for female infertility.

The Drosophila ribosome protein S5 paralog RpS5b promotes germ cell and follicle cell differentiation during oogenesis.

Emerging evidence suggests that ribosome heterogeneity may have important functional consequences in the translation of specific mRNAs within different cell types and under various conditions. Ribosome heterogeneity comes in many forms, including post-translational modification of ribosome proteins (RPs), absence of specific RPs and inclusion of different RP paralogs. The Drosophila genome encodes two RpS5 paralogs: RpS5a and RpS5b. While RpS5a is ubiquitously expressed, RpS5b exhibits enriched expression in the reproductive system. Deletion of RpS5b results in female sterility marked by developmental arrest of egg chambers at stages 7-8, disruption of vitellogenesis and posterior follicle cell (PFC) hyperplasia. While transgenic rescue experiments suggest functional redundancy between RpS5a and RpS5b, molecular, biochemical and ribo-seq experiments indicate that RpS5b mutants display increased rRNA transcription and RP production, accompanied by increased protein synthesis. Loss of RpS5b results in microtubule-based defects and in mislocalization of Delta and Mindbomb1, leading to failure of Notch pathway activation in PFCs. Together, our results indicate that germ cell-specific expression of RpS5b promotes proper egg chamber development by ensuring the homeostasis of functional ribosomes.

Single-cell transcriptome and cell-specific network analysis reveal the reparative effect of neurotrophin-4 in preantral follicles grown in vitro.

BACKGROUND: In-vitro-grow (IVG) of preantral follicles is essential for female fertility preservation, while practical approach for improvement is far from being explored. Studies have indicated that neurotrophin-4 (NT-4) is preferentially expressed in human preantral follicles and may be crucial to preantral follicle growth. METHODS: We observed the location and expression of Tropomyosin-related kinase B (TRKB) in human and mouse ovaries with immunofluorescence and Western blot, and the relation between oocyte maturation and NT-4 level in follicular fluid (FF). Mice model was applied to investigate the effect of NT-4 on preantral follicle IVG. Single-cell RNA sequencing of oocyte combined with cell-specific network analysis was conducted to uncover the underlying mechanism of effect. RESULTS: We reported the dynamic location of TRKB in human and mouse ovaries, and a positive relationship between human oocyte maturation and NT-4 level in FF. Improving effect of NT-4 was observed on mice preantral follicle IVG, including follicle development and oocyte maturation. Transcriptome analysis showed that the reparative effect of NT-4 on oocyte maturation might be mediated by regulation of PI3K-Akt signaling and subsequent organization of F-actin. Suppression of advanced stimulated complement system in granulosa cells might contribute to the improvement. Cell-specific network analysis revealed NT-4 may recover the inflammation damage induced by abnormal lipid metabolism in IVG. CONCLUSIONS: Our data suggest that NT-4 is involved in ovarian physiology and may improve the efficiency of preantral follicle IVG for fertility preservation.

Transcriptome profile of goat folliculogenesis reveals the interaction of oocyte and granulosa cell in correlation with different fertility population.

To understand the molecular and genetic mechanisms related to the litter size in one species of two different populations (high litter size and low litter size), we performed RNA-seq for the oocytes and granulosa cells (GCs) at different developmental stages of follicle, and identified the interaction of genes from both sides of follicle (oocyte and GCs) and the ligand-receptor pairs from these two sides. Our data were very comprehensive to uncover the difference between these two populations regarding the folliculogenesis. First, we identified a set of potential genes in oocyte and GCs as the marker genes which can be used to determine the goat fertility capability and ovarian reserve ability. The data showed that GRHPR, GPR84, CYB5A and ERAL1 were highly expressed in oocyte while JUNB, SCN2A, MEGE8, ZEB2, EGR1and PRRC2A were highly expressed in GCs. We found more functional genes were expressed in oocytes and GCs in high fertility group (HL) than that in low fertility group (LL). We uncovered that ligand-receptor pairs in Notch signaling pathway and transforming growth factor-ß (TGF-ß) superfamily pathways played important roles in goat folliculogenesis for the different fertility population. Moreover, we discovered that the correlations of the gene expression in oocytes and GCs at different stages in the two populations HL and LL were different, too. All the data reflected the gene expression landscape in oocytes and GCs which was correlated well with the fertility capability.

Effects of Er:YAG laser treatment on re-vascularization and follicle survival in frozen/thawed human ovarian cortex transplanted to immunodeficient mice.

PURPOSE: The huge loss of ovarian follicles after transplantation of frozen/thawed ovarian tissue is considered a major drawback on the efficacy of the procedure. Here we investigate whether Er:YAG laser treatment prior to xenotransplantation can improve re-vascularization and subsequently follicle survival in human ovarian tissue. METHODS: A total of 99 frozen/thawed human ovarian cortex pieces were included of which 72 pieces from 12 woman were transplanted to immunodeficient mice. Tissues from each woman were included in both an 8-day and an 8-week duration study and treated with either full-beam laser (L1) or fractionated laser (L2), or served as untreated controls. Vascularization of the ovarian xenografts were evaluated after 8 days by qPCR and murine Cd31 immunohistochemical analysis. Follicle densities were evaluated histologically 8 weeks after xenografting. RESULTS: Gene expression of Vegf/VEGF was upregulated after L1 treatment (p=0.002, p=0.07, respectively), whereas Angpt1, Angpt2, Tnf-α, and Il1-ß were significantly downregulated. No change in gene expression was found in Cd31/CD31, ANGPT1, ANGPT2, ANGTPL4, XBP1, or LRG1 after any of the laser treatments. The fraction of Cd31 positive cells were significantly reduced after L1 and L2 treatment (p<0.0001; p=0.0003, respectively), compared to controls. An overall negative effect of laser treatment was detected on follicle density (p=0.03). CONCLUSIONS: Er:YAG laser treatment did not improve re-vascularization or follicle survival in human ovarian xenografts after 8 days and 8 weeks grafting, respectively. However, further studies are needed to fully explore the potential angiogenic effects of controlled tissue damage using different intensities or lasers.

Generation of ovarian follicles from mouse pluripotent stem cells.

Oocytes mature in a specialized fluid-filled sac, the ovarian follicle, which provides signals needed for meiosis and germ cell growth. Methods have been developed to generate functional oocytes from pluripotent stem cell-derived primordial germ cell-like cells (PGCLCs) when placed in culture with embryonic ovarian somatic cells. In this study, we developed culture conditions to recreate the stepwise differentiation process from pluripotent cells to fetal ovarian somatic cell-like cells (FOSLCs). When FOSLCs were aggregated with PGCLCs derived from mouse embryonic stem cells, the PGCLCs entered meiosis to generate functional oocytes capable of fertilization and development to live offspring. Generating functional mouse oocytes in a reconstituted ovarian environment provides a method for in vitro oocyte production and follicle generation for a better understanding of mammalian reproduction.