In recent years considerable effort has been devoted to understanding the epigenetic control of sperm development, leading to an increased appreciation of the importance of RNA interference pathways, and in particular miRNAs, as key regulators of spermatogenesis and epididymal maturation. It has also been shown that sperm are endowed with an impressive array of miRNA that have been implicated in various aspects of fertilization and embryo development. However, to date there have been no reports on whether the sperm miRNA signature is static or whether it is influenced by their prolonged maturation within the male reproductive tract. To investigate this phenomenon, we employed next-generation sequencing to systematically profile the miRNA signature of maturing mouse spermatozoa. In so doing we have provided the first evidence for the posttesticular modification of the sperm miRNA profile under normal physiological conditions. Such modifications include the apparent loss and acquisition of an impressive cohort of some 113 and 115 miRNAs, respectively, between the proximal and distal epididymal segments. Interestingly, the majority of these changes occur late in maturation and include the uptake of novel miRNA species in addition to a significant increase in many miRNAs natively expressed in immature sperm. Because sperm are not capable of de novo transcription, these findings identify the epididymis as an important site in establishing the sperm epigenome with the potential to influence the peri-conceptual environment of the female reproductive tract, contribute to the inheritance of acquired characteristics, and/or alter the developmental trajectory of the resulting offspring.
Spermatogonial stem cells (SSCs) are a subset of undifferentiated spermatogonia responsible for ongoing spermatogenesis in mammalian testes. Spermatogonial stem cells arise from morphologically homogeneous prospermatogonia, but growing evidence suggests that only a subset of prospermatogonia develops into the foundational SSC pool. This predicts that subtypes of undifferentiated spermatogonia with discrete mRNA and protein signatures should be distinguishable in neonatal testes. We used single-cell quantitative RT-PCR to examine mRNA levels of 172 genes in individual spermatogonia from 6-day postnatal (P6) mouse testes. Cells enriched from P6 testes using the StaPut or THY1(+) magnetic cell sorting methods exhibited considerable heterogeneity in the abundance of specific germ cell and stem cell mRNAs, segregating into one somatic and three distinct spermatogonial clusters. However, P6 Id4-eGFP(+) transgenic spermatogonia, which are known to be enriched for SSCs, were more homogeneous in their mRNA levels, exhibiting uniform levels for the majority of genes examined (122 of 172). Interestingly, these cells displayed nonuniform (50 of 172) expression of a smaller cohort of these genes, suggesting there is substantial heterogeneity even within the Id4-eGFP(+) population. Further, although immunofluorescence staining largely demonstrated conformity between mRNA and protein levels, some proteins were observed in patterns that were disparate from those detected for the corresponding mRNAs in Id4-eGFP(+) spermatogonia (e.g., Kit, Sohlh2, Stra8), suggesting additional heterogeneity is introduced at the post-transcriptional level. Taken together, these data demonstrate the existence of multiple spermatogonial subtypes in P6 mouse testes and raise the intriguing possibility that these subpopulations may correlate with the development of functionally distinct spermatogenic cell types.
The asynchronous cyclic nature of spermatogenesis is essential for continual sperm production and is one of the hallmarks of mammalian male fertility. While various mRNA and protein localization studies have indirectly implicated changing retinoid levels along testis tubules, no quantitative evidence for these changes across the cycle of the seminiferous epithelium currently exists. This study utilized a unique mouse model of induced synchronous spermatogenesis, localization of the retinoid-signaling marker STRA8, and sensitive quantification of retinoic acid concentrations to determine whether there are fluctuations in retinoid levels at each of the individual stages of germ cell differentiation and maturation to sperm. These data show that processive pulses of retinoic acid are generated during spermatogonial differentiation and are the likely trigger for cyclic spermatogenesis and allow us, for the first time, to understand how the cycle of the seminiferous epithelium is generated and maintained. In addition, this study represents the first direct quantification of a retinoid gradient controlling cellular differentiation in a postnatal tissue.
This study examined the effect of the presence of single or multiple embryos on the transcriptome of the bovine oviduct. In experiment 1, cyclic (nonbred, n = 6) and pregnant (artificially inseminated, n = 11) heifers were slaughtered on Day 3 after estrus, and the ampulla and isthmic regions of the oviduct ipsilateral to the corpus luteum were separately flushed. Oviductal epithelial cells from the isthmus region, in which all oocytes/embryos were located, were snap-frozen for microarray analysis. In experiment 2, heifers were divided into cyclic (nonbred, n = 6) or pregnant (multiple embryo transfer, n = 10) groups. In vitro-produced presumptive zygotes were transferred endoscopically to the ipsilateral oviduct on Day 1.5 postestrus (n = 50 zygotes/heifer). Heifers were slaughtered on Day 3, and oviductal isthmus epithelial cells were recovered for RNA sequencing. Microarray analysis in experiment 1 failed to detect any difference in the transcriptome of the oviductal isthmus induced by the presence of a single embryo. In experiment 2, following multiple embryo transfer, RNA sequencing revealed 278 differentially expressed genes, of which 123 were up-regulated and 155 were down-regulated in pregnant heifers. Most of the down-regulated genes were related to immune function. In conclusion, the presence of multiple embryos in the oviduct resulted in the detection of differentially expressed genes in the oviductal isthmus; failure to detect changes in the oviduct transcriptome in the presence of a single embryo may be due to the effect being local and undetectable under the conditions of this study.
Polycystic ovary syndrome (PCOS) pathophysiology is poorly understood, due partly to lack of PCOS animal models fully recapitulating this complex disorder. Recently, a PCOS rat model using letrozole (LET), a nonsteroidal aromatase inhibitor, mimicked multiple PCOS phenotypes, including metabolic features absent in other models. Given the advantages of using genetic and transgenic mouse models, we investigated whether LET produces a similar PCOS phenotype in mice. Pubertal female C57BL/6N mice were treated for 5 wk with LET, which resulted in increased serum testosterone and normal diestrus levels of estradiol, similar to the hyperandrogenemia and follicular phase estrogen levels of PCOS women. As in PCOS, ovaries from LET mice were larger, polycystic, and lacked corpora lutea versus controls. Most LET females were acyclic, and all were infertile. LET females displayed elevated serum LH levels and higher Lhb mRNA in the pituitary. In contrast, serum FSH and Fshb were significantly reduced in LET females, demonstrating differential effects on gonadotropins, as in PCOS. Within the ovary, LET females had higher Cyp17, Cyp19, and Fsh receptor mRNA expression. In the hypothalamus, LET females had higher kisspeptin receptor mRNA expression but lower progesterone receptor mRNA levels. LET females also gained more weight than controls, had increased abdominal adiposity and adipocyte size, elevated adipose inflammatory mRNA levels, and impaired glucose tolerance, mirroring the metabolic phenotype in PCOS women. This is the first report of a LET paradigm in mice that recapitulates both reproductive and metabolic PCOS phenotypes and will be useful to genetically probe the PCOS condition.
To begin embryonic development, the zygote must resume the cell cycle correctly after stimulation by sperm-borne oocyte-activating factors (SOAFs). The postacrosomal WW domain-binding protein (PAWP) is one of the strongest SOAF candidates and is widely conserved among eutherian mammals. It has been reported that the microinjection of recombinant PAWP protein can trigger not only Ca2+ oscillations in mammalian eggs but also intracellular Ca2+ release in amphibian eggs. It was also suggested that PAWP is involved in the formation of high-quality spermatozoa. On the other hand, negligible SOAF activity for PAWP cRNA has also been reported. In this study, we generated PAWP null mice and examined the fertilizing ability of male mice. Electron microscopy showed no aberrant morphology in spermatogenesis. Intracytoplasmic injection of a single spermatozoon from the null mouse line showed that depletion of PAWP elicited no quantitative differences in Ca2+ oscillations or in subsequent development of the embryos. We conclude that PAWP does not play an essential role in mouse fertilization.
Expansion of the cumulus complex surrounding the oocyte is critical for ovulation of a fertilizable egg. The ovulation-inducing surge of luteinizing hormone leads to an increased expression of genes such as prostaglandin-endoperoxide synthase 2 (Ptgs2), pentraxin-related protein 3 (Ptx3), and tumor necrosis factor alpha-induced protein 6 (Tnfaip6) that support cumulus expansion. Factors released by mural granulosa and cumulus granulosa cells into the follicular fluid induce paracrine signaling within the follicular compartment. The follicular fluid that separates these distinct granulosa cell types is an enriched fluid containing numerous proteins, nucleic acids, and other macromolecules. Extracellular vesicles (EVs) are also present; however, no physiologically relevant functions of follicular EVs have yet been demonstrated. In our study, the effect of follicular EVs on cumulus-oocyte complex (COC) expansion and relevant gene expression was assayed. Follicular EVs were isolated using ultracentrifugation from follicular fluid of small (3-5 mm) and large (>9 mm) antral bovine follicles, then characterized by nanoparticle tracking analysis, electron microscopy, and Western blot analysis. To test for bioactivity, mouse and bovine COCs were cultured with follicular EVs. Cumulus expansion and Ptgs2, Ptx3, and Tnfaip6 gene expression were measured following COC maturation culture. The results demonstrated that follicular EVs can support both measurable cumulus expansion and increased gene expression.
Mobilization of fatty acids from adipose tissue during metabolic stress increases the amount of free fatty acids in blood and follicular fluid and is associated with impaired female fertility. In a previous report we described the effects of the three predominant fatty acids in follicular fluid (saturated palmitate and stearate; unsaturated oleate) on oocyte maturation and quality. In the current study the effects of elevated fatty acid levels on cumulus cells were investigated. The three fatty acids dose-dependently induced lipid storage in cumulus cells accompanied by an enhanced immune labeling of perilipin-2, a marker for lipid droplets. Lipidomic analysis confirmed incorporation of the administered fatty acids into triglyceride, resulting in a 3-6 fold increase of triglyceride content. In addition, palmitate selectively induced ceramide formation, which has been implicated in apoptosis. Indeed, of three fatty acids tested, palmitate induced reactive oxygen species formation, caspase 3 activation, and mitochondria deterioration, leading to degeneration of the cumulus cell layers. This effect could be mimicked by addition of ceramide C2 analog and could be inhibited by the ceramide synthase inhibitor fumonisin B1. Interfering with the intactness of the cumulus cell layers, either by mechanical force or by palmitate treatment, resulted in enhanced uptake of lipids in the oocyte and increased radical formation. Our results show that cumulus cells act as a barrier, protecting oocytes from in vitro induced lipotoxic effects. We suggest that this protective function of the cumulus cell layers is important for the developmental competence of the oocyte. The relevance of our findings for assisted reproduction technologies is discussed.
In mammals, follicular atresia can be partially triggered by granulosa cell apoptosis. However, very little is known about the functions of miRNAs in granulosa cell apoptosis. We previously reported that hsa-mir-23a (miR-23a) and hsa-mir-27a (miR-27a) were highly expressed in the plasma of patients with premature ovarian failure, but the action of these two miRNAs in follicular development was unclear. In this study, we explored the roles of miR-23a and miR-27a in the granulosa cells of women undergoing in vitro fertilization/embryo transfer. Using Hoechst staining, we found that miR-23a and miR-27a promoted apoptosis in human granulosa cells. In addition, the Western blotting results suggested that the miR-23a/miR-27a-mediated apoptosis occurred via the FasL-Fas pathway. Based on the results of a luciferase-reporter assay and quantitative RT-PCR and Western blotting analyses, we found that SMAD5 is a target gene of both miR-23a and miR-27a. Furthermore, knocking down SMAD5 expression increased the rate of apoptosis, as well as the levels of Fas, FasL, cleaved caspase-8, and cleaved caspase-3 protein. Taken together, these data suggest that miR-23a and miR-27a target SMAD5 and regulate apoptosis in human granulosa cells via the FasL-Fas pathway. These findings provide an improved understanding of the mechanisms underlying granulosa cell apoptosis, which could potentially be used for future clinical applications.
Humans are ubiquitously exposed to di(2-ethylhexyl) phthalate (DEHP), which is an environmental toxicant present in common consumer products. DEHP potentially targets the ovary through its metabolite mono(2-ethylhexyl) phthalate (MEHP). However, the direct effects of MEHP on ovarian folliculogenesis and steroidogenesis, two processes essential for reproductive and nonreproductive health, are unknown. The present study tested the hypotheses that MEHP directly accelerates early folliculogenesis via overactivation of phosphatidylinositol 3-kinase (PI3K) signaling, a pathway that regulates primordial follicle quiescence and activation, and inhibits the synthesis of steroid hormones by decreasing steroidogenic enzyme levels. Neonatal ovaries from CD-1 mice were cultured for 6 days with vehicle control, DEHP, or MEHP (0.2-20 mu g/ml) to assess the direct effects on folliculogenesis and PI3K signaling. Further, antral follicles from adult CD-1 mice were cultured with vehicle control or MEHP (0.1-10 mu g/ml) for 24-96 h to establish the temporal effects of MEHP on steroid hormones and steroidogenic enzymes. In the neonatal ovaries, MEHP, but not DEHP, decreased phosphatase and tensin homolog levels and increased phosphorylated protein kinase B levels, leading to a decrease in the percentage of germ cells and an increase in the percentage of primary follicles. In the antral follicles, MEHP decreased the mRNA levels of 17alpha-hydroxylase-17, 20-desmolase, 17beta-hydroxysteroid dehydrogenase, and aromatase leading to a decrease in testosterone, estrone, and estradiol levels. Collectively, MEHP mediates the effect of DEHP on accelerated folliculogenesis via overactivating PI3K signaling and inhibits steroidogenesis by decreasing steroidogenic enzyme levels.
Seminal fluid interacts with epithelial cells lining the female reproductive tract to induce expression of proinflammatory cytokines and chemokines, initiating immune tolerance mechanisms to facilitate pregnancy. TGFB cytokines are key signaling agents in seminal plasma but do not fully account for the female response to seminal fluid. We hypothesized that additional molecular pathways are utilized in seminal fluid signaling. Affymetrix microarray was employed to compare gene expression in the endometrium of mice 8 h after mating with either intact males or seminal fluid deficient (SVX/VAS) males. Bioinformatics analysis revealed TLR4 signaling as a strongly predicted upstream regulator activated by the differentially expressed genes and implicated TGFB signaling as a second key pathway. Quantitative PCR and microbead data confirmed that seminal fluid induces endometrial synthesis of several TLR4-regulated cytokines and chemokines, including CSF3, CXCL1, CXCL2, IL1A, IL6, LIF, and TNF. In primary uterine epithelial cells, CSF3, CXCL1, and CXCL2 were strongly induced by the TLR4 ligand LPS but suppressed by TGFB, while IL1A, TNF, and CSF2 were induced by both ligands. TLR4 was confirmed as essential for the full endometrial cytokine response using mice with a null mutation in Tlr4, where seminal fluid failed to induce endometrial Csf3, Cxcl2, Il6, and Tnf expression. This study provides evidence that TLR4 contributes to seminal fluid modulation of the periconception immune environment. Activation of TLR4 signaling by microbial or endogenous components of seminal fluid is thus implicated as a key element of the female tract response to seminal fluid at the outset of pregnancy in mice.
Xenobiotic estrogens, such as bisphenol A (BPA), disrupt a wide variety of genomic estrogen actions, but their nongenomic estrogen actions remain poorly understood. We investigated nongenomic estrogenic effects of low concentrations of BPA and three related alkylphenols on the inhibition of zebrafish oocye maturation (OM) mediated through a G protein-coupled estrogen receptor 1 (Gper)-dependent epidermal growth factor receptor (Egfr) pathway. BPA (10-100 nM) treatment for 3 h mimicked the effects of estradiol-17beta (E2) and EGF, decreasing spontaneous maturation of defolliculated zebrafish oocytes, an effect not blocked by coincubation with actinomycin D, but blocked by coincubation with a Gper antibody. BPA displayed relatively high binding affinity (15.8% that of E2) for recombinant zebrafish Gper. The inhibitory effects of BPA were attenuated by inhibition of upstream regulators of Egfr, intracellular tyrosine kinase (Src) with PP2, and matrix metalloproteinase with ilomastat. Treatment with an inhibitor of Egfr transactivation, AG1478, and an inhibitor of the mitogen-activated protein kinase (MAPK) 3/1 pathway, U0126, increased spontaneous OM and blocked the inhibitory effects of BPA, E2, and the selective GPER agonist, G-1. Western blot analysis showed that BPA (10-200 nM) mimicked the stimulatory effects of E2 and EGF on Mapk3/1 phosphorylation. Tetrabromobisphenol A, 4-nonylphenol, and tetrachlorobisphenol A (5-100 nM) also inhibited OM, an effect blocked by cotreatment with AG1478, as well as with the GPER antagonist, G-15, and displayed similar binding affinities as BPA to zebrafish Gper. The results suggest that BPA and related alkylphenols disrupt zebrafish OM by a novel nongenomic estrogenic mechanism involving activation of the Gper/Egfr/Mapk3/1 pathway.
ABSTRACT In mammals, follicular atresia can be partially triggered by granulosa cell apoptosis. However, very little is known about the functions of miRNAs in granulosa cell apoptosis. We previously reported that hsa-mir-23a (miR-23a) and hsa-mir-27a (miR-27a) were highly expressed in the plasma of patients with premature ovarian failure, but the action of these two miRNAs in follicular development was unclear. In this study, we explored the roles of miR-23a and miR-27a in the granulosa cells of women undergoing in vitro fertilization/embryo transfer. Using Hoechst staining, we found that miR-23a and miR-27a promoted apoptosis in human granulosa cells. In addition, the Western blotting results suggested that the miR-23a/miR-27a-mediated apoptosis occurred via the FasL-Fas pathway. Based on the results of a luciferase-reporter assay and quantitative RT-PCR and Western blotting analyses, we found that SMAD5 is a target gene of both miR-23a and miR-27a. Furthermore, knocking down SMAD5 expression increased t...
This study aimed to characterize the endometrial transcriptome and functional pathways overrepresented in the endometrium of cows treated to ovulate larger (>= 13 mm) versus smaller (<= 12 mm) follicles. Nelore cows were presynchronized prior to receiving cloprostenol (large follicle [LF] group) or not (small follicle [SF] group), along with a progesterone (P4) device on Day (D) - 10. Devices were withdrawn and cloprostenol administered 42-60 h (LF) or 30-36 h (SF) before GnRH agonist treatment (D0). Tissues were collected on D4 (experiment [Exp.] 1; n = 24) or D7 (Exp. 2; n = 60). Endometrial transcriptome was obtained by RNA-Seq, whereas proliferation and apoptosis were assessed by immunohistochemistry. Overall, LF cows developed larger follicles and corpora lutea, and produced greater amounts of estradiol (D - 1, Exp. 1, SF: 0.7 +/- 0.2; LF: 2.4 +/- 0.2 pg/ml; D - 1, Exp. 2, SF: 0.5 +/- 0.1; LF: 2.3 +/- 0.6 pg/ml) and P4 (D4, Exp. 1, SF: 0.8 +/- 0.1; LF: 1.4 +/- 0.2 ng/ml; D7, Exp. 2, SF: 2.5 +/- 0.4; LF: 3.7 +/- 0.4 ng/ml). Functional enrichment indicated that biosynthetic and metabolic processes were enriched in LF endometrium, whereas SF endometrium transcriptome was biased toward cell proliferation. Data also suggested reorganization of the extracellular matrix toward a proliferation-permissive phenotype in SF endometrium. LF endometrium showed an earlier onset of proliferative activity, whereas SF endometrium expressed a delayed increase in glandular epithelium proliferation. In conclusion, the periovulatory endocrine milieu regulates bovine endometrial transcriptome and seems to determine the transition from a proliferation-permissive to a biosynthetic and metabolically active endometrial phenotype, which may be associated with the preparation of an optimally receptive uterine environment.
The aim of this study was to identify conceptus-derived proteins, in addition to IFNT, that may facilitate pregnancy recognition in cattle. Analysis of the protein content of the uterine luminal fluid (ULF) from cyclic heifers on Day 16 by nano liquid chromatography tandem mass spectrometry identified 334 proteins. Comparison of these data with 299 proteins identified in the ULF of pregnant heifers on Day 16 identified 85 proteins only present in the ULF of pregnant heifers. Analysis of Day 16 conceptus-conditioned culture medium revealed the presence of 1005 proteins of which 30 proteins were unique to ULF from Day 16 pregnant heifers. Of these 30 proteins, 12 had mRNA expression values at least 2-fold higher in abundance (P < 0.05) in the conceptus compared to the endometrium (ARPC5L, CAPG, CKMT1, CSTB, HSPA8, HSPE1, LGALS3, MSN, NUTF2, P4HB, PRKAR2A, TKT) as determined by RNA sequencing. In addition, genes that have a significant biological interaction with the proteins (ACO2, CKMT1, CSTB, EEF2, GDI1, GLB1, GPLD1, HNRNPA1, HNRNPA2B1, HNRNPF, HSPA8, HSPE1, IDH2, KRT75, LGALS3, MSN, NUTF2, P4HB, PRKAR2A, PSMA4, PSMB5, PSMC4, SERPINA3, TKT) were differentially expressed in the endometrium of pregnant compared to cyclic heifers during the pregnancy recognition period (Days 16-18). These results indicate that 30 proteins unique to ULF from pregnant heifers and produced by short-term in vitro cultured Day 16 conceptuses could potentially be involved in facilitating the interactions between the conceptus and the endometrium during the pregnancy recognition period.
The antiviral activity of interferon (IFN) increases in uterine vein serum (UVS) during early pregnancy in sheep. This antiviral activity in UVS collected on Day 15 of pregnancy is blocked by anti-IFN-tau (anti-IFNT) antibodies. Conceptus-derived IFNT was hypothesized to induce IFN-stimulated gene (ISG) expression in endometrium and extrauterine tissues during pregnancy. To test this hypothesis, blood was collected from ewes on Days 12-16 of the estrous cycle or pregnancy. Serum progesterone was >1.7 ng/ml in pregnant (P) and nonpregnant (NP) ewes until Day 13, then declined to <0.6 ng/ml by Day 15 in NP ewes. A validated IFNT radioimmunoassay detected IFNT in uterine flushings (UFs) on Days 13-16 and in UVS on Days 15-16 of pregnancy. IFNT detection in UF correlated with paracrine induction of ISGs in the endometrium and occurred prior to the inhibition of estrogen receptor 1 and oxytocin receptor expression in uterine epithelia on Day 14 of pregnancy. Induction of ISG mRNAs in corpus luteum (CL) and liver tissue occurred by Day 14 and in peripheral blood mononuclear cells by Day 15 in P ewes. Expression of mRNAs for IFN signal transducers and ISGs were greater in the CL of P than that of NP ewes on Day 14. It is concluded that: 1) paracrine actions of IFNT coincide with detection of IFNT in UF; 2) endocrine action of IFNT ensues through induction of ISGs in peripheral tissues; and 3) IFNT can be detected in UVS, but not until Days 15-16 of pregnancy, which may be limited by the sensitivity of the IFNT radioimmunoassay.
During fertilization, spermatozoa make essential contributions to embryo development by providing oocyte activating factors, centrosomal components, and paternal chromosomes. Protamines are essential for proper packaging of sperm DNA; however, in contrast to the studies of oocyte-related female infertility, the influence of sperm chromatin structure on male infertility has not been evaluated extensively. The objective of this study was to determine the sperm chromatin content of bull spermatozoa by evaluating DNA fragmentation, chromatin maturity/protamination, PRM1 protein status, and nuclear shape in spermatozoa from bulls with different fertility. Relationships between protamine 1 (PRM1) and the chromatin integrity were ascertained in spermatozoa from Holstein bulls with varied (high vs. low) but acceptable fertility. Sperm DNA fragmentation and chromatin maturity (protamination) were tested using Halomax assay and toluidine blue staining, respectively. The PRM1 content was assayed using Western blotting and in-gel densitometry, flow cytometry, and immunocytochemistry. Fragmentation of DNA was increased and chromatin maturity significantly reduced in spermatozoa from low-fertility bulls compared to those from high-fertility bulls. Field fertility scores of the bulls were negatively correlated with the percentage of spermatozoa displaying reduced protamination and fragmented DNA using toluidine blue and Halomax, respectively. Bull fertility was also positively correlated with PRM1 content by Western blotting and flow cytometry. However, detection of PRM1 content by Western blotting alone was not predictive of bull fertility. In immunocytochemistry, abnormal spermatozoa showed either a lack of PRM1 or scattered localization in the apical/acrosomal region of the nuclei. The nuclear shape was distorted in spermatozoa from low-fertility bulls. In conclusion, we showed that inadequate amount and localization of PRM1 were associated with defects in sperm chromatin structure, coinciding with reduced fertility in bulls. These findings are highly significant because they reveal molecular and morphological phenotypes of mammalian spermatozoa that influence fertility.
Sex-reversal cases in humans and genetic models in mice have revealed that the fate of the bipotential gonad hinges upon the balance between pro-testis SOX9 and pro-ovary beta-catenin pathways. Our central query was: if SOX9 and beta-catenin define the gonad's identity, then what do the gonads become when both factors are absent? To answer this question, we developed mouse models that lack either Sox9, beta-catenin, or both in the somatic cells of the fetal gonads and examined the morphological outcomes and transcriptome profiles. In the absence of Sox9 and beta-catenin, both XX and XY gonads progressively lean toward the testis fate, indicating that expression of certain pro-testis genes requires the repression of the beta-catenin pathway, rather than a direct activation by SOX9. We also observed that XY double knockout gonads were more masculinized than their XX counterpart. To identify the genes responsible for the initial events of masculinization and to determine how the genetic context (XX vs. XY) affects this process, we compared the transcriptomes of Sox9/beta-catenin mutant gonads and found that early molecular changes underlying the XY-specific masculinization involve the expression of Sry and 21 SRY direct target genes, such as Sox8 and Cyp26b1. These results imply that when both Sox9 and beta-catenin are absent, Sry is capable of activating other pro-testis genes and drive testis differentiation. Our findings not only provide insight into the mechanism of sex determination, but also identify candidate genes that are potentially involved in disorders of sex development.
We have previously shown that the carboxyl terminus (cT) of human follicle-stimulating hormone (FSH, follitropin) receptor (FSHR) is clipped before insertion into the plasma membrane. Surprisingly, several different constructs of FSHR fluorescent fusion proteins (FSHR-FPs) failed to traffic to the plasma membrane. Subsequently, we discovered that substituting the extreme cT of luteinizing hormone (LH) receptor (LHR) to create an FSHR-LHRcT chimera has no effect on FSHR functionality. Therefore, we used this approach to create an FSHR-LHRcT-FP fusion. We found this chimeric FSHR-LHRcT-FP was expressed in HEK293 cells at levels similar to reported values for FSHR in human granulosa cells, bound FSH with high affinity, and transduced FSH binding to produce cAMP. Quantitative fluorescence resonance energy transfer (FRET) analysis of FSHR-LHRcT-YFP/FSHR-LHRcT-mCherry pairs revealed an average FRET efficiency of 12.9 +/- 5.7. Advanced methods in single-molecule analyses were applied in order to ascertain the oligomerization state of the FSHR-LHRcT. Fluorescence correlation spectroscopy coupled with photon-counting histogram analyses demonstrated that the FSHR-LHRcTFP fusion protein exists as a freely diffusing homodimer in the plasma membrane. A central question is whether LHR could oligomerize with FSHR, because both receptors are coexpressed in differentiated granulosa cells. Indeed, FRET analysis revealed an average FRET efficiency of 14.4 +/- 7.5 when the FSHR-LHR cT-mCherry was coexpressed with LHR-YFP. In contrast, coexpression of a 5-HT2cVSV-YFP with FSHR-LHR cT-mCherry showed only 5.6 +/- 3.2 average FRET efficiency, a value indistinguishable from the detection limit using intensity-based FRET methods. These data demonstrate that coexpression of FSHR and LHR can lead to heterodimerization, and we hypothesize that it is possible for this to occur during granulosa cell differentiation.
Lipid peroxidation products such as the naturally occurring aldehyde 4-hydroxynonenal (4-HNE) are known to be cytotoxic toward different cell types, including spermatozoa. In order to understand this at the molecular level, we have employed a proteomic approach to characterize direct 4-HNE adducts on human spermatozoa. Several proteins were identified to be of particular interest, including aldehyde labeling of histone methyltransferase and dynein heavy chain. In addition, we found that 4-HNE bound to part of the activation segment, cysteine residue 199, of protein kinase A (PKA). Interestingly, at low levels, addition of 4-HNE had a stimulatory effect on PKA. However, this did not correlate to increased phosphotyrosine levels during capacitation. This data explains the link between reactive oxygen species and sperm toxicity. Given that epigenetic regulation is likely affected in oxidative-stressed spermatozoa, this data show that spermatozoa appear to shut down under these conditions before reaching the egg.