single-particle-interferometric-reflectance-imaging-characterization

Single-Particle Interferometric Reflectance Imaging Characterization of Individual Extracellular Vesicles and Population Dynamics

Extracellular vesicles (EVs) are nanometer-sized vesicles with a lipid bilayer that are secreted by most cells. EVs carry a multitude of different ...

University of Kansas Medical Center

Estrogens are essential hormones for the regulation of fertility. Cellular responses to estrogens are mediated by estrogen receptor α (ESR1) and estrogen receptor β (ESR2). In mouse and rat models, disruption of Esr1 causes infertility in both males and females. However, the role of ESR2 in reproductive function remains undecided because of a wide variation in phenotypic observations among Esr2-mutant mouse strains. Regulatory pathways independent of ESR2 binding to its cognate DNA response element have also been implicated in ESR2 signaling. To clarify the regulatory roles of ESR2, we generated two mutant rat models: one with a null mutation (exon 3 deletion, Esr2ΔE3) and the other with an inframe deletion selectively disrupting the DNA binding domain (exon 4 deletion, Esr2ΔE4). In both models, we observed that ESR2-mutant males were fertile. ESR2-mutant females exhibited regular estrous cycles and could be inseminated by wild-type (WT) males but did not become pregnant or pseudopregnant. Esr2-mutant ovaries were small and differed from WT ovaries by their absence of corpora lutea, despite the presence of follicles at various stages of development. Esr2ΔE3- and Esr2ΔE4-mutant females exhibited attenuated preovulatory gonadotropin surges and did not ovulate in response to a gonadotropin regimen effective in WT rats. Similarities of reproductive deficits in Esr2ΔE3 and Esr2ΔE4 mutants suggest that DNA binding-dependent transcriptional function of ESR2 is critical for preovulatory follicle maturation and ovulation. Overall, the findings indicate that neuroendocrine and ovarian deficits are linked to infertility observed in Esr2-mutant rats.

Defining the Role of Estrogen Receptor β in the Regulation of Female Fertility.

Estrogen receptor 2 (ESR2) plays a critical role in folliculogenesis and ovulation. Disruption of ESR2-function in the rats results in female infertility due to failure of ovulation. Ovulation failure occurred in two distinct rat models, a null mutant and a DNA binding domain (DBD) mutant of ESR2, indicating that transcriptional regulation by ESR2 is indispensable for ovulation. To define the regulatory role of ESR2 in preovulatory follicular maturation and ovulation, we investigated ovarian responsiveness to exogenous gonadotropins in prepubertal females. Granulosa cells (GCs) play a vital role in follicle maturation and ovulation, and ESR2-dependent estrogen signaling is predominant in GCs, therefore, we examined the differential expression of gonadotropin-induced genes in GCs. Of 32,623 genes detected by RNA-sequencing, 1696 were differentially expressed in Esr2-mutant rats (789 downregulated, and 907 upregulated, absolute fold change 2, FDR p < 0.05). Molecular pathway analyses indicated that these differentially expressed genes are involved in steroidogenesis, follicle maturation, and ovulation. Many of these genes are known regulators of ovarian function and a subset were also disrupted in Esr2-mutant mice. Interestingly, Kiss1 was identified as one of the differentially expressed genes implicating a potential role within the follicle and its regulation by ESR2. Our findings indicate that ESR2 regulates key genes in GCs that are essential for follicle maturation and ovulation in the rat.

ESR2 regulates granulosa cell genes essential for follicle maturation and ovulation.

RNA seq analyses were performed in granulosa cells (GCs) collected from gonadotropin treated ESR2 mutant rats. Data obtained from a null mutant with  exon 3 deletion (∆3) and another DNA binding domain (DBD) mutant with exon 4 deletion (∆4) were compared to that of wildtype (WT) rats. The raw data were analyzed using CLC genomics workbench. High quality RNA-sequencing reads were aligned to the  genome. Differentially expressed genes in ∆3 or ∆4 -mutant GCs were identified based on the following criteria: FDR p-Value ≤0.05 and an absolute fold change of 2. Fewer differentially expressed genes were identified in ∆3 compared to the ∆4 mutant group. As both mutant groups demonstrated a common phenotype of ovulation failure, differentially expressed genes common to both in ∆3 and ∆4 mutant rats were emphasized and further analyzed in the companion article "ESR2 regulates granulosa cell genes essential for follicle maturation and ovulation" [1].

Differentially regulated genes in -mutant rat granulosa cells.

Estrogens are traditionally considered to be female sex steroid hormones and most of the studies examining estrogen regulation of metabolic function in the liver have been conducted in females. However, the liver expresses high levels of estrogen receptor alpha (ESR1) in both males and females, which mediates the hepatic response to estrogens. In this data article, we investigated whether metabolic disorders in  knockout () male rats were linked with loss of transcriptional regulation by ESR1 in liver. To identify the ESR1 regulated genes in the mutant liver, RNA-sequencing was performed on liver RNAs purified from young male rats. The raw data were analyzed using the CLC Genomics Workbench and high-quality RNA-sequencing reads were aligned to the  genome. Transcriptome data obtained from  liver RNAs were compared to that of wild type rats. Based on an absolute fold change of 2 with a -value ≤ 0.05, a total of 618 differentially expressed genes were identified in the  male liver. Pathway analyses demonstrated that the majority of differentially expressed genes are regulators of carbohydrate and lipid metabolism in the liver. These differentially expressed genes and their potential roles were further examined in a companion manuscript, "Disruption of ESR1 alters the expression of genes regulating hepatic lipid and carbohydrate metabolism in male rats" (Khristi et al., 2018).

Liver transcriptome data of  knockout male rats reveals altered expression of genes involved in carbohydrate and lipid metabolism.

The liver helps maintain energy homeostasis by synthesizing and storing glucose and lipids. Gonadal steroids, particularly estrogens, play an important role in regulating metabolism. As estrogens are considered female hormones, metabolic disorders related to the disruption of estrogen signaling have mostly been studied in females. Estrogen receptor alpha (ESR1) is the predominant receptor in both the male and female liver, and it mediates the hepatic response to estrogens. Loss of ESR1 increases weight gain and obesity in female rats, while reducing the normal growth in males. Although Esr1-/- male rats have a reduced body weight, they exhibit increased adipose deposition and impaired glucose tolerance. We further investigated whether these metabolic disorders in Esr1-/- male rats were linked with the loss of transcriptional regulation by ESR1 in the liver. To identify the ESR-regulated genes, RNA-sequencing was performed on liver mRNAs from wildtype and Esr1-/- male rats. Based on an absolute fold change of ≥2 with a p-value ≤ 0.05, a total of 706 differentially expressed genes were identified in the Esr1-/- male liver: 478 downregulated, and 228 upregulated. Pathway analyses demonstrate that the differentially expressed genes include transcriptional regulators (Cry1, Nr1d1, Nr0b2), transporters (Slc1a2), and regulators of biosynthesis (Cyp7b1, Cyp8b1), and hormone metabolism (Hsd17b2, Sult1e1). Many of these genes are also integral parts of the lipid and carbohydrate metabolism pathways in the liver. Interestingly, certain critical regulators of the metabolic pathways displayed a sexual dimorphism in expression, which may explain the divergent weight gain in Esr1-/- male and female rats despite common metabolic dysfunctions.

Disruption of ESR1 alters the expression of genes regulating hepatic lipid and carbohydrate metabolism in male rats.

SATB homeobox 1 (SATB1) and its heterodimeric partner SATB2 play an important regulatory role in maintaining proliferation of trophoblast stem (TS) cells and in inhibiting trophoblast differentiation. To identify the SATB-regulated genes in TS cells, we studied the transcriptome changes in a 'loss of function' model of Rcho-1 rat TS cell line.  gene expression was silenced by lentiviral delivery of shRNAs targeted to exon 9 and exon 12. An  shRNA was used as a non-targeted control. Total RNA was purified from shRNA-transduced Rcho-1 cells, and whole transcriptome was assessed by RNA-sequencing on an Illumina HiSeq X platform. Differentially expressed genes in  shRNA-transduced cells were identified by analyses of the RNA-sequencing data using CLC Genomics Workbench. Differentially expressed genes with each of the two different shRNAs were compared to identify SATB1-target genes and to eliminate the potential off-targets of the shRNAs. These datasets can be used to identify the SATB-regulated genes in TS cells and to understand the molecular mechanisms that regulate trophoblast proliferation and inhibit differentiation.

SATB1-regulated transcriptome datasets of Rcho-1 rat trophoblast stem cells.

Follicle development beyond the preantral stage is dependent on gonadotropins. FSH signaling is crucial for the advancement of preantral follicles to the antral stage, and LH signaling is essential for further maturation of preovulatory follicles. Estrogen is intricately tied to gonadotropin signaling during the advanced stages of folliculogenesis. We observed that Erβ ovarian follicles fail to develop beyond the antral stage, even after exogenous gonadotropin stimulation. As ERβ is primarily expressed in the granulosa cells (GCs), we explored the gonadotropin-regulated GC genes that induce maturation of antral follicles. Synchronized follicle development was induced by administration of exogenous gonadotropins to wildtype 4-wk-old female rats. The GC transcriptome was analyzed via RNA-sequencing before and after gonadotropin stimulation. An Erβ mutant model that fails to show follicle maturation was also included in order to identify the ERβ-regulated genes involved at this step. We observed that specific groups of genes were differentially expressed in response to PMSG or hCG administration in wildtype rats. While some of the PMSG or hCG-induced genes showed a similar expression pattern in Erβ GCs, a subset of PMSG- or hCG-induced genes showed a differential expression pattern in Erβ GCs. These latter ERβ-regulated genes included previously known FSH or LH target genes including Lhcgr, Cyp11a1, Cyp19a1, Pgr, Runx2, Egfr, Kiss1, and Ptgs2, which are involved in follicle development, oocyte maturation, and ovulation. We also identified novel ERβ-regulated genes including Jaml, Galnt6, Znf750, Dusp9, Wnt16, and Mageb16 that failed to respond to gonadotropin stimulation in Erβ GCs. Our findings indicate that the gonadotropin-induced spatiotemporal pattern of gene expression is essential for ovarian follicle maturation beyond the antral stage. However, expression of a subset of those gonadotropin-induced genes is dependent on transcriptional regulation by ERβ.

Granulosa cell genes that regulate ovarian follicle development beyond the antral stage: The role of estrogen receptor β.

Ovarian steroids dramatically impact normal homeostatic and metabolic processes of most tissues within the body, including muscle, bone, neural, immune, cardiovascular, and reproductive systems. Determining the effects of spaceflight on the ovary and estrous cycle is, therefore, critical to our understanding of all spaceflight experiments using female mice. Adult female mice (n = 10) were exposed to and sacrificed on-orbit after 37 days of spaceflight in microgravity. Contemporary control (preflight baseline, vivarium, and habitat; n = 10/group) groups were maintained at the Kennedy Space Center, prior to sacrifice and similar tissue collection at the NASA Ames Research Center. Ovarian tissues were collected and processed for RNA and steroid analyses at initial carcass thaw. Vaginal wall tissue collected from twice frozen/thawed carcasses was fixed for estrous cycle stage determinations. The proportion of animals in each phase of the estrous cycle (i.e., proestrus, estrus, metestrus, and diestrus) did not appreciably differ between baseline, vivarium, and flight mice, while habitat control mice exhibited greater numbers in diestrus. Ovarian tissue steroid concentrations indicated no differences in estradiol across groups, while progesterone levels were lower (p < 0.05) in habitat and flight compared to baseline females. Genes involved in ovarian steroidogenic function were not differentially expressed across groups. As ovarian estrogen can dramatically impact multiple non-reproductive tissues, these data support vaginal wall estrous cycle classification of all female mice flown in space. Additionally, since females exposed to long-term spaceflight were observed at different estrous cycle stages, this indicates females are likely undergoing ovarian cyclicity and may yet be fertile.

Anamika Ratri

Department of Molecular and Integrative Physiology

University of Kansas Medical Center

Single-Particle Interferometric Reflectance Imaging Characterization of Individual Extracellular Vesicles and Population Dynamics