Epididymal mRNA expression profiles for the protein disulfide isomerase gene family: Modulation by development and androgens.

BACKGROUND: The endoplasmic reticulum (ER) is the central hub for protein quality control, where the protein disulfide isomerases (PDIs), encoded by at least 21 genes, play a pivotal role. These multifunctional proteins contribute to disulfide bond formation, proper folding, and protein modifications, and may act as hormone-binding proteins (e.g., steroids), influencing hormone biology. The interplay between ER proteostasis, PDIs, and epididymis-a crucial site for sperm maturation-remains largely understudied. OBJECTIVES: This study characterizes transcriptional signatures of Pdi genes in the epididymis. MATERIAL AND METHODS: Transcriptional profiles of selected Pdi genes were assessed in adult Wistar rat tissues, and epididymis under different experimental conditions (developmental stages, surgical castration, and efferent ductules ligation [EDL]). In silico bioinformatic analyses identified expression trends of this gene family in human epididymal segments. RESULTS: P4hb, Pdia3, Pdia5, Pdia6, Erp44, Erp29, and Casq1 transcripts were detected in both reproductive and non-reproductive tissues, while Casq2 exhibited higher abundance in vas deferens, prostate, and heart. Pdilt, highly expressed in testis, and Pdia2, highly expressed in heart, showed minimal mRNA levels in the epididymis. In the mesonephric duct, epididymal embryonic precursor, P4hb, Pdia3, Pdia5, Pdia6, and Erp29 mRNAs were found at gestational day (GD) 17.5. Except for Erp29, which remained stable, these Pdi transcript levels increased from GD17.5 to GD20.5, when epididymal morphogenesis occurs, and were maintained to varying degrees in the epididymis during postnatal development. Surgical castration downregulated P4hb, Pdia3, Pdia5, Pdia6, Pdilt and Erp29 transcripts, an effect reversed by testosterone replacement. Conversely, transcript levels remained unaffected by EDL, except P4hb, which was reduced in caput epididymis. All 21 PDI genes exhibited diverse transcriptional profiles across the human epididymis. DISCUSSION AND CONCLUSION: The findings lay the foundations to explore Pdi genes in epididymal biology. As a considerable proportion of male infertility cases are idiopathic, targeting hormonal regulation of protein quality control in epididymis represents a route to address male infertility and advance therapeutic interventions in this domain.

Generation of a homozygous (MCRIi031-A-3) WT1 knockout human iPSC line.

The transcription factor WT1 plays a critical role in several embryonic developmental processes such as gonadogenesis, nephrogenesis, and cardiac development. We generated a homozygous (MCRIi031-A-3) WT1 knockout induced pluripotent stem cell (iPSC) line from human fibroblasts using a one-step protocol for CRISPR/Cas9 gene-editing and episomal-based reprogramming. The cells exhibit a normal karyotype and morphology, express pluripotency markers, and have the capacity to differentiate into the three embryonic germ layers. These cell lines will allow us to further explore the role of WT1 in critical developmental processes.

COUP-TFII regulates early bipotential gonad signaling and commitment to ovarian progenitors.

BACKGROUND: The absence of expression of the Y-chromosome linked testis-determining gene SRY in early supporting gonadal cells (ESGC) leads bipotential gonads into ovarian development. However, genetic variants in NR2F2, encoding three isoforms of the transcription factor COUP-TFII, represent a novel cause of SRY-negative 46,XX testicular/ovotesticular differences of sex development (T/OT-DSD). Thus, we hypothesized that COUP-TFII is part of the ovarian developmental network. COUP-TFII is known to be expressed in interstitial/mesenchymal cells giving rise to steroidogenic cells in fetal gonads, however its expression and function in ESGCs have yet to be explored. RESULTS: By differentiating induced pluripotent stem cells into bipotential gonad-like cells in vitro and by analyzing single cell RNA-sequencing datasets of human fetal gonads, we identified that NR2F2 expression is highly upregulated during bipotential gonad development along with markers of bipotential state. NR2F2 expression was detected in early cell populations that precede the steroidogenic cell emergence and that retain a multipotent state in the undifferentiated gonad. The ESGCs differentiating into fetal Sertoli cells lost NR2F2 expression, whereas pre-granulosa cells remained NR2F2-positive. When examining the NR2F2 transcript variants individually, we demonstrated that the canonical isoform A, disrupted by frameshift variants previously reported in 46,XX T/OT-DSD patients, is nearly 1000-fold more highly expressed than other isoforms in bipotential gonad-like cells. To investigate the genetic network under COUP-TFII regulation in human gonadal cell context, we generated a NR2F2 knockout (KO) in the human granulosa-like cell line COV434 and studied NR2F2-KO COV434 cell transcriptome. NR2F2 ablation downregulated markers of ESGC and pre-granulosa cells. NR2F2-KO COV434 cells lost the enrichment for female-supporting gonadal progenitor and acquired gene signatures more similar to gonadal interstitial cells. CONCLUSIONS: Our findings suggest that COUP-TFII has a role in maintaining a multipotent state necessary for commitment to the ovarian development. We propose that COUP-TFII regulates cell fate during gonad development and impairment of its function may disrupt the transcriptional plasticity of ESGCs. During early gonad development, disruption of ESGC plasticity may drive them into commitment to the testicular pathway, as observed in 46,XX OT-DSD patients with NR2F2 haploinsufficiency.

Generation of heterozygous (MCRIi030-A-1) and homozygous (MCRIi030-A-2) NR2F2/COUP-TFII knockout human iPSC lines.

The NR2F2 gene encodes the transcription factor COUP-TFII, which is upregulated in embryonic mesoderm. Heterozygous variants in NR2F2 cause a spectrum of congenital anomalies including cardiac and gonadal phenotypes. We generated heterozygous (MCRIi030-A-1) and homozygous (MCRIi030-A-2) NR2F2-knockout induced pluripotent stem cell (iPSC) lines from human fibroblasts using a one-step protocol for CRISPR/Cas9 gene-editing and episomal-based reprogramming. Both iPSC lines exhibited a normal karyotype, typical pluripotent cell morphology, pluripotency marker expression, and the capacity to differentiate into the three embryonic germ layers. These lines will allow us to explore the role of NR2F2 during development and disease.

Epididymal embryonic development harbors TLR4/NFKB signaling pathway as a morphogenetic player.

The Wolffian duct (WD) is an embryonic tissue that undergoes androgen-induced morphological changes to become the epididymis. Toll-like receptor 4 (TLR4)- and nuclear factor kB (NFKB)-induced effectors are expressed in the adult epididymis and represent important players in epididymal innate immune responses. TLR4/NFKB signaling pathway is evolutionarily conserved and plays a critical morphogenetic role in several species; however, its function during WD morphogenesis is unknown. We hypothesized that TLR4/NFKB pathway plays a role during WD development. Here we examined TLR4 expression and regulation of TLR4-target genes during rat WD morphogenesis between embryonic days (e) 17.5-20.5. The functionality of TLR4/NFKB signaling was examined using WD organotypic cultures treated with lipopolysaccharide (LPS) from E. coli (TLR4 agonist) and PDTC (NFKB inhibitor). TLR4 was detected at mRNA level in e17.5 (uncoiled duct) and e20.5 (coiled duct) WDs, and spatio-temporal changes in TLR4 immunoreactivity were observed between these two time points. Expression level analysis of a subset of TLR4-regulated genes showed that TLR4/NFKB pathway was activated after exposure of cultured WD to LPS (4 h), an event that was abrogated by PDTC. Long-term exposure of cultured WDs to LPS (96 h) resulted in dysregulations of morphogenetic events and LAMA1 immunodistribution changes, suggesting the extracellular matrix at the intersection between WD morphogenesis and balance of innate immune components. Our results unveil the epididymal morphogenesis as an event equipped with TLR4/NFKB signaling components that may serve developmental functions, and eventually transition to host defense function when the fetus is exposed to an infectious or noninfectious threat.

Intelligence Quotient Variability in Klinefelter Syndrome Is Associated With GTPBP6 Expression Under Regulation of X-Chromosome Inactivation Pattern.

Klinefelter syndrome (KS) displays a broad dysmorphological, endocrinological, and neuropsychological clinical spectrum. We hypothesized that the neurocognitive dysfunction present in KS relies on an imbalance in X-chromosome gene expression. Thus, the X-chromosome inactivation (XCI) pattern and neurocognitive X-linked gene expression were tested and correlated with intelligence quotient (IQ) scores. We evaluated 11 KS patients by (a) IQ assessment, (b) analyzing the XCI patterns using both HUMARA and ZDHHC15 gene assays, and (c) blood RT-qPCR to investigate seven X-linked genes related to neurocognitive development (GTPBP6, EIF2S3, ITM2A, HUWE1, KDM5C, GDI1, and VAMP7) and XIST in comparison with 14 (male and female) controls. Considering IQ 80 as the standard minimum reference, we verified that the variability in IQ scores in KS patients seemed to be associated with the XCI pattern. Seven individuals in the KS group presented a random X-inactivation (RXI) and lower average IQ than the four individuals who presented a skewed X-inactivation (SXI) pattern. The evaluation of gene expression showed higher GTPBP6 expression in KS patients with RXI than in controls (p = 0.0059). Interestingly, the expression of GTPBP6 in KS patients with SXI did not differ from that observed in controls. Therefore, our data suggest for the first time that GTPBP6 expression is negatively associated with full-scale IQ under the regulation of the type of XCI pattern. The SXI pattern may regulate GTPBP6 expression, thereby dampening the impairment in cognitive performance and playing a role in intelligence variability in individuals with KS, which warrants further mechanistic investigations.

Novel androgen-induced activity of an antimicrobial ß-defensin: Regulation of Wolffian duct morphogenesis.

The Wolffian duct (WD) undergoes morphological changes induced by androgens to form the epididymis, which is an organ essential for sperm maturation. Androgen action in WD epithelium involves paracrine factors of mesenchymal origin that function by still poorly understood mechanisms. Here we studied the antimicrobial ß-defensin SPAG11C as a new player in duct morphogenesis, localized prenatally in the WD mesenchyme. Organotypic culture of rat WDs and tissues from Androgen Receptor (AR) knockout mice (ARKO) were used. Our results show that androgen/AR signaling differentially regulated SPAG11C expression at mRNA and protein levels in the developing WD. WDs incubated with recombinant human SPAG11C were shorter and less coiled as a result of reduced epithelial cell proliferation, but not increased apoptosis. Our results suggested ß-defensin SPAG11C as an androgen-target required for WD morphogenesis. This highlights the multifunctional repertoire of the ß-defensin protein family and their potential contribution to the in utero environment that determines male reproductive success.

Generation and phenotypic characterization of a galanin overexpressing mouse.

In most parts of the peripheral nervous system galanin is expressed at very low levels. To further understand the functional role of galanin, a mouse overexpressing galanin under the platelet-derived growth factor-B was generated, and high levels of galanin expression were observed in several peripheral tissues and spinal cord. Thus, a large proportion of neurons in autonomic and sensory ganglia were galanin-positive, as were most spinal motor neurons. Strong galanin-like immunoreactivity was also seen in nerve terminals in the corresponding target tissues, including skin, blood vessels, sweat and salivary glands, motor end-plates and the gray matter of the spinal cord. In transgenic superior cervical ganglia around half of all neuron profiles expressed galanin mRNA but axotomy did not cause a further increase, even if mRNA levels were increased in individual neurons. In transgenic dorsal root ganglia galanin mRNA was detected in around two thirds of all neuron profiles, including large ones, and after axotomy the percentage of galanin neuron profiles was similar in overexpressing and wild type mice. Axotomy reduced the total number of DRG neurons less in overexpressing than in wild type mice, indicating a modest rescue effect. Aging by itself increased galanin expression in the superior cervical ganglion in wild type and transgenic mice, and in the latter also in preganglionic cholinergic neurons projecting to the superior cervical ganglion. Galanin overexpressing mice showed an attenuated plasma extravasation, an increased pain response in the formalin test, and changes in muscle physiology, but did not differ from wild type mice in sudomotor function. These findings suggest that overexpressed galanin in some tissues of these mice can be released and via a receptor-mediated action influence pathophysiological processes.

Relaxation of human temporal artery by endothelin ETB receptors.

Endothelin receptors have been characterized in human temporal artery by molecular biological methods and in vitro pharmacology. Reverse transcriptase-polymerase chain reaction was used to detect mRNA encoding ETA and ETB receptors in normal and endothelium-denuded arteries. Vasomotor response experiments with a specific ETA antagonist (FR 139317) suggested the presence of ETA subtypes. Marked ETB-mediated relaxation was obtained with ET-3 when ETA activity was blocked in precontracted arteries. Relaxation was significantly reduced by bosentan, indomethacin, and a nitric oxide synthase inhibitor. It may be speculated that the relaxant activity is mediated through ETB1 receptors.

On the role of cholecystokinin in the mediation of spinal reflex excitability in intact and axotomized rats.

The effects of intrathecal (i.t.) administration of cholecystokinin (CCK) on the hindlimb flexor withdrawal reflex were compared in decerebrate, spinalized, unanaesthetized rats with intact sciatic nerves or 14-26 days after unilateral transection of the sciatic nerve. The effect of CI-988, a CCK-B receptor antagonist, on the facilitation of the flexor reflex induced by conditioning stimulation (CS) of C-afferents was also examined. Intrathecal CCK induced facilitation of the flexor reflex over the dose range of 10 ng-1 micrograms in rats with intact and sectioned sciatic nerves. However, the facilitation induced by CCK in axotomized rats was significantly reduced compared with nerve intact animals. CI-988 did not block the facilitation of the flexor reflex induced by the CS and the increases in reflex magnitude during the CS train (wind-up) in rats with intact and sectioned sciatic nerves. The present results indicate that the excitatory effect of CCK on the spinal cord is reduced after axotomy, suggesting that nerve section may induce transsynaptic changes in the spinal cord leading to hyposensitivity to CCK. Furthermore, it is suggested that CCK may not play a significant role in the phenomenon of wind-up and central sensitization after repetitive C-fiber input under normal conditions or after axotomy, when expression of CCK and CCK-B receptors are increased in sensory neurons.

Extracellular cholecystokinin levels in the rat spinal cord following chronic morphine exposure: an in vivo microdialysis study.

Conflicting results concerning the issue of whether or not chronic morphine exposure induces an increase in CCK biosynthesis have been found in many CNS sites, including the spinal cord, where CCK activity may contribute to the facilitation of the development of opiate tolerance. The present study was undertaken in order to monitor the extracellular level of CCK under spontaneous and stimulus-evoked release in the spinal cord dorsal horn of drug naive and morphine tolerant rats. Tolerance was induced by implantation of two morphine pellets (2x75 mg) which induced a stable morphine plasma concentration after 48 h post-implantation. The tail-flick test and naloxone precipitated withdrawal were used as indexes of tolerance and dependence to morphine. The effect of morphine-pellet implantation on basal and K+-induced release of CCK-like immunoreactivity (CCK-LI) in the rat dorsal horn were monitored with in vivo microdialysis 96 h after implantation of morphine or placebo pellets, when rats showed tolerance and dependence. Basal CCK levels were below the detection limit of the assay (0.6 pM) in both tolerant and normal animals. K+ (100 mM) in the perfusion medium induced a more than 3-fold increase of the extracellular level of CCK-LI in control animals, and a more than 4-fold increase on CCK-LI in morphine-pellet implanted animals. However, this difference was not significant. In addition, naloxone (2 mg/kg; i.v.), did not induce any change in the extracellular level of CCK in either group. The present study suggests that the modulatory interaction between CCK and opioids in the development of tolerance in the spinal cord may occur without necessarily increasing the extracellular level of CCK. Another possible explanation of the finding is that the microdialysis technique is not sensitive enough to detect differences in unstimulated CCK levels in normal and tolerant animals.

Negative anticipatory contrast and preference conditioning: flavor cues support preference conditioning, and environmental cues support contrast.

In 2 experiments, access to a 0.15% saccharin solution was followed on alternating days by access to a 32% sucrose solution and the same saccharin solution. In Experiment 1, rats increased both intake of and preference for a flavored saccharin solution that predicted sucrose, but neither effect was found using a predictive odor cue alone. Experiment 2 replicated the predictive flavor results but showed suppression of saccharin intake when environmental cues predicted sucrose. When both flavor and environment predicted sucrose, saccharin intake did not change, but preference for the predictive flavor increased. Discriminative taste cues appear to facilitate the development of preference conditioning, but environmental cues favor negative anticipatory contrast effects. Also, preference conditioning and contrast may develop concurrently and compete for expression.

Time horizons in rats foraging for food in temporally separated patches.

An important tenet of optimal foraging theory is that foragers compare prey densities in alternative patches to determine an optimal distribution of foraging behavior over time. A critical question is over what time period (time horizon) this integration of information and behavior occurs. Recent research has indicated that rats do not compare food density in a depleting patch with that in a rich patch delayed by an hour or more (Timberlake, 1984). In the present research we attempted to specify over what time period a future rich patch would affect current foraging. The effect of future food was measured by early entry into the rich patch (anticipation) and by a decrease in food obtained in the depleting patch (suppression). The rats showed anticipation of a rich patch up to an hour distant, but suppressed current feeding only if the rich patch was 16 min distant or less. The suppression effect appeared mediated by competition for expression between anticipatory entries into the rich patch and continued foraging in the depleting patch. These results suggest that optimal foraging is based on a variety of specific mechanisms rather than a general optimizing algorithm with a single time horizon.

System-specific differences in behavior regulation: overrunning and underdrinking in molar nondepriving schedules.

In two experiments we tested the molar regulation prediction that animals adjust schedule performance to reduce deviations from baseline response totals. Both experiments constrained the baseline drink-burst length under molar nondepriving schedules but allowed rats to continue running without drinking. In Experiment 1, rats were required to run in order to drink. In Experiment 2, water was delivered independently of running by fixed-time (FT) schedules. Under the run-to-drink contingency, rats exceeded their baseline amounts of running (overrunning) but failed to maintain their baseline water intake (underdrinking). The total amount of running that did not lead to drinking approximated baseline running. Under the FT schedules, rats again underdrank, but total running approximated baseline. These results do not support previous studies that have shown molar equilibrium effects under nondepriving reciprocal schedules. We conclude that (a) contingent running may not substitute for independent running; (b) intermittent access to water reduces the total instigation for drinking; (c) molar regulation differs under reciprocal and nonreciprocal schedules; and (d) more dynamic, system-specific regulatory models need to be developed.

Schedule constraint on the average drink burst and the regulation of wheel running and drinking in rats.

Two experiments compared predictions of a molar-pattern model and a general molar behavior regulation model by requiring rats to wheel run for access to water. In both experiments schedule parameters constrained the baseline average burst length of drinking without constraining total drinking. Five levels of schedule constraint were imposed on time spent per drinking burst (Experiment 1) or the number of drinks per burst (Experiment 2). The results of both experiments supported the general molar behavior regulation view but not the molar-pattern model by showing no increase in total wheel running and no decrease in total drinking under schedule constraint. However, both experiments also showed local effects of drink burst constraint, including a direct relation between the degree of constraint and the local rate of drinking, and an approximation of the temporal distribution of baseline drinking under all degrees of schedule constraint. Most local changes support the view that rats defend the baseline temporal distribution of responding under schedule constraint, though some changes appear related to disruption of local response pattern characteristics.

Anticipation of future food: suppression and facilitation of saccharin intake depending on the delay and type of future food.

A series of studies examined the (Sprague-Dawley) rat's tendency to suppress intake of .15% saccharin when it was followed by a second food after 4-, 16-, or 32-min delays. The second foods examined were 32% sucrose, 64% sucrose, lab chow, a Nutrasweet solution, skim milk, and chocolate milk. Saccharin intake was influenced by both the delay and the specific food available. Subsequent analysis showed that saccharin intake before the 4-min delay was an inverse function of the caloric value of the second food. However, saccharin intake before the 16-min delay was better predicted as an inverse function of the hedonic value of the second food. The results suggest that the caloric and hedonic values of a food may influence food selection across different time courses, and that the effective time horizon for the sequential comparison of foods depends on the specific foods that are compared.

Interpellet delay and meal patterns in the rat.

Rats typically eat in short discrete meal periods separated by long intermeal intervals. The present study measured meal patterns and total intake of food and water when the rate of access to food pellets within the meal was reduced by delaying the time between pellet deliveries. In contrast to studies that reduce the caloric density of food, simple interpellet delay produced a marked reduction in meal size at the shorter (32 sec or less) interpellet delays. However, longer delays (up to 128 sec) produced no further change in average meal size. The results suggest that meal size is determined (1) by a positive feedback component with a relatively short decay time probably based on gustatory stimulation from feeding, and (2) by a negative feedback component capable of integrating total intake across delays of up to 1 hour. Increasing the delay between pellets appeared to interfere with the positive feedback component, but not with the negative feedback component.

Learning and meal-associated drinking: meal-related deficits produce adjustments in postprandial drinking.

Two experiments examined the effect of meal-related water deficits on the distribution of meal-associated drinking. In the first procedure free-feeding rats received 10-, 20-, or 30-min delays between the end of a meal and the subsequent availability of postprandial water. Each delay condition remained in effect for 10 consecutive days. The primary effect of the delay was to postpone the intake of postprandial drinking. None of the delay conditions produced an increase in preprandial drinking. However, when the rats were returned to baseline following the delay conditions a pronounced rebound effect was obtained in the proportion of postprandial drinking. The second experiment followed the same general procedure except that 5-min access to water was always presented after each meal and before the postprandial water restrictions. Under this procedure the rats increased their water intake during the 5-min period when the postprandial restrictions were imposed. These findings show that meal-related water deficits can affect the timing and proportion of postprandial drinking, but provide no evidence that meal-related deficits increase preprandial drinking. The results suggest that the anticipation of meal-related water deficits may play a role in shaping the rat's postprandial drinking pattern.

Anticipatory drinking in rats: compensatory adjustments in the local rate of intake.

The current experiment examined whether dehydrated rats could learn to anticipate disruption of access to water. Previous research showed that rats running in a wheel in order to drink compensated for local disruptions on intake by increasing their rate of intake after, but not prior to the initial disruption. The present experiment exposed rats to either a fixed-ratio (FR) schedule that required rats to run in order to drink or a matched, fixed-time (FT) schedule that allowed the animals intermittent access to water without running. Access to water was disrupted by restricting the quantity of water available per drink bout. Rats increased their local rate of water intake prior to the disruption under the FT schedule but not under the FR schedule. The results suggest that rats can learn to increase their local rate of intake in anticipation of local restrictions on water under response-independent (FT) schedules. It was hypothesized that these anticipatory increases in the local rate of intake resulted from a priming effect due to the motivating effects of stimuli associated with water and/or frustration resulting from attempts to drink prior to water availability.

The basis of superstitious behavior: chance contingency, stimulus substitution, or appetitive behavior?

This research examined three explanations for the "superstitious" behavior of pigeons under frequent fixed-time delivery of food: accidental response-reward contingency, stimulus substitution, and elicited species-typical appetitive behavior. The behavior observed in these studies consisted of occasional postfood locomotion away from the food hopper, and a predominant pattern of activity directed toward the hopper wall (wall-directed behavior), including approaching, stepping side to side, scratching with the feet, bumping with the breast, pendulum movements of the extended neck, and head bobbing, though not pecking. The consistency of these behavior patterns argued against explanation by accidental response contingencies, and the complexity of behavior was incompatible with the classic stimulus-substitution account. These studies also showed that: (1) response contingencies and prior stimulus experience can modify wall-directed behavior, but within definable limits; (2) pecking sometimes can be obtained in birds of specific strains, and by providing extended training; (3) placing the hopper in the floor at the center of a large chamber replaces wall-directed behavior with circling in a manner that resembles ground foraging for food. We conclude that superstitious behavior under periodic delivery of food probably develops from components of species-typical patterns of appetitive behavior related to feeding. These patterns are elicited by a combination of frequent food presentations and the supporting stimuli present in the environment.