REVERBA couples the circadian clock to Leydig cell steroidogenesis.

The involvement of the molecular clock in regulating cell physiological processes on a specific time scale is a recognized concept, yet its specific impact on optimizing androgen production in Leydig cells has been unclear. This study aimed to confirm the role of the REVERBA (NR1D1) gene in controlling the transcription of key genes related to Leydig cell steroid production. We investigated daily variations by collecting Leydig cells from rats at various times within a 24-h period. Chromatin immunoprecipitation study showed a time-dependent pattern for genes linked to steroid production (Nur77, Star, Cyp11a1, and Cyp17a1), which closely matched the 24-h REVERBA levels in Leydig cells, peaking between zeitgeber time (ZT) 7-11. To understand the physiological significance of REVERBA's interaction with promoters of steroidogenesis-related genes, Leydig cells from rats at two different times (ZT7 and ZT16; chosen based on REVERBA expression levels), were treated with either an agonist (GSK4112) or an antagonist (SR8278). The results revealed that the REVERBA agonist stimulated gene transcription, while the antagonist inhibited it, but only when REVERBA was sufficiently present, indicating a reliance on REVERBA's circadian fluctuation. Moreover, this REVERBA-dependent stimulation had a clear impact on testosterone production in the culture medium, underscoring REVERBA's involvement in the circadian regulation of testosterone. This study indicates that REVERBA, in addition to being a core component of the cellular clock, plays a key role in regulating androgen production in Leydig cells by influencing the transcription of critical steroidogenesis-related genes.

Transcriptional Profiles of Mitochondrial Dynamics Markers Are Disturbed in Adrenal Glands of Stressed Adult Male Rats.

Mitochondrial dynamics plays a significant role in shaping the mitochondrial network and maintaining mitochondrial function. Imbalanced mitochondrial dynamics can cause mitochondrial dysfunction leading to a wide range of diseases/disorders. The aim of this study was to investigate the expression of mitochondrial dynamics markers and regulatory molecules in whole adrenal glands, cortices, and medullae obtained from adult male rats exposed to acute and repeated psychophysical stress, the most common stress in human society. The transcriptional profiles of most of the mitochondrial dynamics markers investigated here were altered: 81%-(17/21) in the whole adrenal gland, 76.2%-(16/21) in the adrenal cortex, and 85.7%-(18/21) in the adrenal medulla. Changes were evident in representatives of every process of mitochondrial dynamics. Markers of mitobiogenesis were changed up to 62.5%-(5/8) in the whole adrenal gland, 62.5%-(5/8) in the adrenal cortex, and 87.5%-(7/8) in the adrenal medulla. Markers of mitofusion were changed up to 100%-(3/3) in the whole adrenal gland, 66.7%-(5/8) in the adrenal cortex, and 87.5%-(7/8) in the adrenal medulla, while all markers of mitofission and mitophagy were changed in the adrenal glands. Moreover, almost all markers of mitochondrial functionality were changed: 83.3%-(5/6) in the whole adrenal, 83.3%-(5/6) in the cortex, 66.7%-(4/6) in the medulla. Accordingly, the study highlights the significant impact of acute and repeated stress on mitochondrial dynamics in the adrenal gland.

Circadian desynchrony disturbs the function of rat spermatozoa.

Decreased male fertility is a growing health problem that requires a better understanding of molecular events regulating reproductive competence. Here the effects of circadian desynchrony on the rat spermatozoa functionality were studied. Circadian desynchrony was induced in rats that lived for 2 months under disturbed light conditions designed to mimic shiftwork in humans (two days of constant light, two days of continual dark, and three days of 14:10 h light:dark schedule). Such a condition abolished circadian oscillations in the rats' voluntary activity, followed by a flattened transcriptional pattern of the pituitary gene encoding follicle stimulating hormone subunit (Fshb), and genes important for germ cell maturation (Tnp1 and Prm2) as well as the clock in seminiferous tubules. However, the number of spermatozoa isolated from the epididymis of the rats suffering from circadian desynchrony did not deviate from the controls. Nevertheless, spermatozoa functionality, estimated by motility and progesterone-induced acrosome reaction, was reduced compared to the control. These changes were associated with the altered level of main markers of mitochondrial biogenesis (Pprgc1a/PGC1A, Nrf1/NRF1, Tfam, Cytc), decreased mitochondrial DNA copy number, ATP content, and clock genes (Bmal1/BMAL1, Clock, Cry1/2, and Reverba). The principal-component-analysis (PCA) points to a positive association of the clock and mitochondrial biogenesis-related genes in spermatozoa from rats suffering circadian desynchrony. Altogether, the results show the harmful effect of circadian desynchrony on spermatozoa functionality, targeting energetic homeostasis.

The cost of the circadian desynchrony on the Leydig cell function.

The increased frequency of different lifestyles that disrupts circadian rhythms, together with a trend in the accretion of male idiopathic infertility, imposes the necessity to understand the contribution of circadian rhythms disruption to fertility regulation. In this study, the effects of circadian desynchrony (CD) on the steroidogenic capacity of adult Leydig cells were studied. Adult rats were housed under a disturbing light regime (2 days of constant light, 2 days of continual dark, and 3 days of 12:12 h light:dark schedule) designed to mimic shiftwork in humans. CD was characterized by changed and decreased rhythmic locomotor activity and reduced blood testosterone. In the Leydig cells changed transcription of the clock genes (Bmal1, Clock, Cry1 and Reverba/b increased while Per1/2 reversed phase) was detected. This was followed by reduced transcription of genes (Star, Cyp11a1, and Hsd3b1/2) primarily involved in mitosteroidogenesis. In parallel, mitochondrial membrane potential (Δψi) and ATP production declined losing their characteristic oscillatory pattern. Also, the main markers of mitochondrial biogenesis (Ppargc1a, Nrf1, Tfam, Cytc), fusion (Mfn2), and mitophagy (Pink1 and Tfeb) were disturbed. Collectively, CD targets mitochondria in Leydig cells by reducing mitosteroidogenesis, mitoenergetics, and disturbing mitochondrial dynamics. These changes contribute to testosterone decline compromising androgen-dependent functions, including reproduction.

Spermatozoa Develop Molecular Machinery to Recover From Acute Stress.

This study was designed to search for the possible mechanism(s) of male (in/sub)fertility by following the molecular response of spermatozoa on acute psychological stress (the most common stress in human society) and on a 20-h time-dependent recovery period. To mimic in vivo acute stress, the rats were exposed to immobilization once every 3 h. The recovery periods were as follows: 0 (immediately after stress and 3 h after the light is on-ZT3), 8 (ZT11), 14 (ZT17), and 20 (ZT23) h after stress. Results showed that acute stress provoked effects evident 20 h after the end of the stress period. Numbers of spermatozoa declined at ZT17 and ZT23, while functionality decreased at ZT3 and ZT11, but recovered at ZT17 and ZT23. Transcriptional profiles of 91% (20/22) of tracked mitochondrial dynamics and functionality markers and 91% (20/22) of signaling molecules regulating both mitochondrial dynamics and spermatozoa number/functionality were disturbed after acute stress and during the recovery period. Most of the changes presented as increased transcription or protein expression at ZT23. The results of the principal component analysis (PCA) showed the clear separation of acute stress recovery effects during active/dark and inactive/light phases. The physiological relevance of these results is the recovered positive-acrosome-reaction, suggesting that molecular events are an adaptive mechanism, regulated by acute stress response signaling. The results of the PCA confirmed the separation of the effects of acute stress recovery on gene expression related to mitochondrial dynamics, cAMP, and MAPK signaling. The transcriptional patterns were different during the active and inactive phases. Most of the transcripts were highly expressed during the active phase, which is expected given that stress occurred at the beginning of the inactive phase. To the best of our knowledge, our results provide a completely new view and the first presentation of the markers of mitochondrial dynamics network in spermatozoa and their correlation with signaling molecules regulating both mitochondrial dynamics and spermatozoa number and functionality during recovery from acute stress. Moreover, the interactions between the proteins important for spermatozoa homeostasis and functionality (MFN2 and PRKA catalytic subunit, MFN2 and p38MAPK) are shown for the first time. Since the existing literature suggests the importance of semen quality and male fertility not only as the fundamental marker of reproductive health but also as the fundamental biomarkers of overall health and harbingers for the development of comorbidity and mortality, we anticipate our result to be a starting point for more investigations considering the mitochondrial dynamics markers or their transcriptional profiles as possible predictors of (in/sub)fertility.

Stress alters the transcriptional activity of Leydig cells dependently on the diurnal time.

The increasing amount of data points to the circadian timing system as an essential part of processes regulating androgen homeostasis. However, the relationship between stress response, timekeeping-, and steroidogenesis-related systems is unexplored. Here, the stress-response of the testosterone-producing rat Leydig cells depending on the time of stressful events was studied. The study analyzes the effects of 3-h immobilization (IMO) applied at different periods during the day. The IMO performed once [1 time immobilization stress (1×IMO)] or repeated in 10 consecutive days [10 time repeated immobilization stress (10×IMO)]. Both types of IMO increased corticosterone and decreased testosterone blood level. However, the effect of 10×IMO occurring in the active phase on blood testosterone was less pronounced. This is related to different sensitivity to IMO-events depending on the diurnal time. Most steroidogenesis-related genes [gene encoding luteinizing hormone/choriogonadotropin receptor (Lhcgr), gene encoding cytochrome P450, family 11, subfamily a, polypeptide 1 (Cyp11a1), gene encoding hydroxy-δ-5-steroid dehydrogenase, 3 ß- and steroid δ-isomerase 1 (Hsd3b1/2), and gene encoding cytochrome P450, family 17, subfamily a, polypeptide 1 (Cyp17a1)] were downregulated in the inactive phase but unchanged or even upregulated in the active phase of the day. Both types of IMO stimulated the expression of clock elements gene encoding aryl hydrocarbon receptor nuclear translocator-like (Bmal1)/aryl hydrocarbon receptor nuclear translocator-like (BMAL1), gene encoding period circadian regulator 1 (Per1)/period circadian regulator 1 (PER1) regardless of the day's stage and reduced gene encoding nuclear receptor subfamily 1, group D, member 1 (Rev-erba) in the inactive phase. The principal component analysis (PCA) confirmed a major shift, for both IMO-types, in the transcription of the genes across the passive/active stage. Further, 10×IMO changed a diurnal pattern of the glucocorticoid receptor [gene encoding nuclear receptor subfamily 3, group C, member 1 (Nr3c1)/nuclear receptor subfamily 3, group C, member 1 (GR)] expression, whereas the observed time-dependent IMO-response of the Leydig cells correlated with different corticosterone engagements. Altogether, the Leydig cell's stress response depends on the daytime of the stressful event, emphasizing the importance of the circadian system in supporting androgen homeostasis and male fertility.

Spermatozoal Mitochondrial Dynamics Markers and Other Functionality-Related Signaling Molecules Exert Circadian-like Response to Repeated Stress of Whole Organism.

In the search for the possible role of the mitochondrial dynamics markers in spermatozoa adaptation, an in vivo approach was designed to mimic situations in which human populations are exposed to 3 h of repeated psychological stress (the most common stress in human society) at different time points during the day (24 h). The hormones (stress hormone corticosterone and testosterone), the number and the functionality of spermatozoa (response to acrosome-reaction-inducer progesterone), as well as the transcriptional profiles of 22 mitochondrial dynamics and function markers and 22 signaling molecules regulating both mitochondrial dynamics and spermatozoa number and functionality were followed at three time points (ZT3, ZT11, and ZT23). The results show that repeated stress significantly decreased the number and functionality of spermatozoa at all time points. In the same samples, the transcriptional profiles of 91% (20/22) of mitochondrial dynamics and functionality markers and 86% (19/22) of signaling molecules were disturbed after repeated stress. It is important to point out that similar molecular changes in transcriptional profiles were observed at ZT3 and ZT23, but the opposite was observed at ZT11, suggesting the circadian nature of the adaptive response. The results of PCA analysis show the significant separation of repeated stress effects during the inactive/light and active/dark phases of the day, suggesting the circadian timing of molecular adaptations.

Stress-induced glucocorticoids alter the Leydig cells' timing and steroidogenesis-related systems.

The study aimed to analyze the time-dependent consequences of stress on gene expression responsible for diurnal endocrine Leydig cell function connecting them to the glucocorticoid-signaling. In the first 24h after the stress event, a daily variation of blood corticosterone increased, and testosterone decreased; the testosterone/corticosterone were lowest at the end of the stress session overlapping with inhibition of Leydig cells' steroidogenesis-related genes (Nr3c1/GR, Hsd3b1/2, Star, Cyp17a1) and changed circadian activity of the clock genes (the increased Bmal1/BMAL1 and Per1/2/PER1 and decreased Cry1 and Rev-erba). The glucocorticoid-treated rats showed a similar response. The principal-component-analysis (PCA) displayed an absence of significant differences between treatments especially on Per1 and Rev-erba, the findings confirmed by the in vivo blockade of the testicular glucocorticoid receptor (GR) during stress and ex vivo treatment of the Leydig cells with hydrocortisone and GR-blocker. In summary, stressful stimuli can entrain the clock in the Leydig cells through glucocorticoid-mediated communication.

Mitochondrial Dynamics Markers and Related Signaling Molecules Are Important Regulators of Spermatozoa Number and Functionality.

Here, we study possible mechanisms of (in/sub)fertility related to the acute or repeated psychological stresses (the most common stresses in human society) by following the transcriptional profile of 22 mitochondrial dynamics/function markers and 22 signaling molecules regulating both mitochondrial dynamics and spermatozoa number/functionality. An in vivo study mimicking acute (once for 3 h) and repeated (3 h for 10 consecutive days) psychophysical stress was performed on adult rats. The analysis of hormones, the number/functionality of spermatozoa, and 44 transcriptional markers were performed on individual samples from up to 12 animals per group. Results showed that both types of stress reduced spermatozoa functionality (acute by 4.4-fold, repeated by 3.3-fold) and ATP production (acute by 2.3-fold, repeated by 14.5-fold), while only repeated stress reduces the number of spermatozoa (1.9-fold). Stress significantly disturbed transcription of 34-out-of-44 markers (77%). Mitochondrial dynamics and functionality markers: 18-out-of-22 =>82% (mitochondrial-biogenesis-markers ->6-out-of-8 =>75%; mitochondrial-fusion-markers ->3-out-of-3 =>100%; mitochondrial-fission-markers ->1-out-of-2 =>50%; mitochondrial-autophagy-markers ->3-out-of-3 =>100%; mitochondrial-functionality-markers ->5-out-of-6 =>83%). Markers of signaling pathways regulating both mitochondrial dynamics/functionality and spermatozoa number/functionality important for male (in/sub)fertility ->16-out-of-22 =>73% (cAMP-signaling-markers ->8-out-of-12 =>67%; MAPK-signaling-markers ->8-out-of-10 =>80%). Accordingly, stress-triggered changes of transcriptional profile of mitochondrial dynamics/functionality markers as well as signaling molecules regulating both mitochondrial dynamics and spermatozoa number and functionality represent adaptive mechanisms.

Growing Up Under Constant Light: A Challenge to the Endocrine Function of the Leydig Cells.

The factors influencing Leydig cell maturity and the acquisition of functional capacity are incompletely defined. Here we analyzed the constant light (LL) influence on Leydig cells' endocrine function during reproductive maturation. Rats were exposed to LL from P21 to P90. Data were collected at juvenile (P35), peri/pubertal (P42, P49), and adult (P90) stages of life. The results proved the effect of LL on rats' physiology by changing of bimodal voluntary activity pattern into free-running. Additionally, the peripheral clock in Leydig cells changed in LL condition, indicating disturbed rhythm: the positive element (Bmal1) increased in pre-/pubertal but decreased in the adult period, while negative elements (Per2 and Reverba) were increased. The effects of LL were most prominent in puberty: pituitary genes encoding gonadotropic hormones (Cga, Lhb, Fshb) decreased; serum corticosterone increased, while serum androgens and mass of testicular and sex accessory organs reduced; markers of Leydig cells maturity/differentiation (Insl3, Lhcgr) and steroidogenesis-related genes (Scarb1, Star, Cyp11a1, Cyp17a1) decreased; the steroidogenic and energetic capacity of the Leydig cell mitochondria decreased; the mtDNA copy number reduced, and mitochondrial dynamics markers changed: fusion decreased (Opa1 and Mfn2), and mitophagy increased (Pink1). In adults, the negative effect of LL on mitochondrial function and steroidogenic capacity persists in adult Leydig cells while other parameters reached control values. Altogether, the results indicate that LL slows down Leydig cells' maturation by reducing the endocrine and energy capacity of cells leading to the delay of reproductive development.

Aging-Related Increase of cGMP Disrupts Mitochondrial Homeostasis in Leydig Cells.

Since mitochondria play an essential role in the testosterone biosynthesis, serve as power centers and are a source of oxidative stress, a possible mitochondrial dysfunction could be connected with decreased activity of Leydig cells and lowered testosterone production during aging. Here we chronologically analyzed age-related alterations of mitochondrial function in Leydig cells correlated by the progressive rise of cGMP signaling and with respect to testosterone synthesis. To target cGMP signaling in Leydig cells, acute or long-term in vivo or ex vivo treatments with sildenafil (phosphodiesterase 5 [PDE5] inhibitor) were performed. Aging-related accumulation of cGMP in the Leydig cells is associated with mitochondrial dysfunction illustrated by reduced ATP and steroid production, lowered O2 consumption, increased mitochondrial abundance and mtDNA copies number, decreased expression of genes that regulate mitochondrial biogenesis (Ppargc1a/PGC1a-Tfam-Nrf1/NRF1), mitophagy (Pink1), fusion (Mfn1, Opa1), and increased Nrf2/NRF2. Acute in vivo PDE5 inhibition overaccumulated cGMP and stimulated testosterone but reduced ATP production in Leydig cells from adult, middle-aged, and old rats. The increased ATP/O ratio observed in cells from old compared to adult rats was diminished after stimulation of cGMP signaling. Opposite, long-term PDE5 inhibition decreased cGMP signaling and improved mitochondrial function/dynamics in Leydig cells from old rats. Mitochondrial abundance in Leydig cells decreased while ATP levels increased. Chronic treatment elevated Tfam, Nrf1, Nrf2, Opa1, Mfn1, Drp1, and normalized Pink1 expression. Altogether, long-term PDE5 inhibition prevented age-related NO and cGMP elevation, improved mitochondrial dynamics/function, and testosterone production. The results pointed on cGMP signaling in Leydig cells as a target for pharmacological manipulation of aging-associated changes in mitochondrial function and testosterone production.

Deficiency in insulin-like growth factors signalling in mouse Leydig cells increase conversion of testosterone to estradiol because of feminization.

AIM: A growing body of evidence pointed correlation between insulin-resistance, testosterone level and infertility, but there is scarce information about mechanisms. The aim of this study was to identify the possible mechanism linking the insulin-resistance with testosterone-producing-Leydig-cells functionality. METHODS: We applied in vivo and in vitro approaches. The in vivo model of functional genomics is represented by INSR/IGF1R-deficient-testosterone-producing Leydig cells obtained from the prepubertal (P21) and adult (P80) male mice with insulin + IGF1-receptors deletion in steroidogenic cells (Insr/Igf1r-DKO). The in vitro model of INSR/IGF1R-deficient-cell was mimicked by blockade of insulin/IGF1-receptors on the primary culture of P21 and P80 Leydig cells. RESULTS: Leydig-cell-specific-insulin-resistance induce the development of estrogenic characteristics of progenitor Leydig cells in prepubertal mice and mature Leydig cells in adult mice, followed with a dramatic reduction of androgen phenotype. Level of androgens in serum, testes and Leydig cells decrease as a consequence of the dramatic reduction of steroidogenic capacity and activity as well as all functional markers of Leydig cell. Oppositely, the markers for female-steroidogenic-cell differentiation and function increase. The physiological significances are the higher level of testosterone-to-estradiol-conversion in double-knock-out-mice of both ages and few spermatozoa in adults. Intriguingly, the transcription of pro-male sexual differentiation markers Sry/Sox9 increased in P21-Leydig-cells, questioning the current view about the antagonistic genetic programs underlying gonadal sex determination. CONCLUSION: The results provide new molecular mechanisms leading to the development of the female phenotype in Leydig cells from Insr/Igf1r-DKO mice and could help to better understand the correlation between insulin resistance, testosterone and male (in)fertility.

Reduced spermatozoa functionality during stress is the consequence of adrenergic-mediated disturbance of mitochondrial dynamics markers.

Here we investigate the stress-signaling responsible for the effects of acute/repeated psychological stresses (the most common stresses in human society) on spermatozoa number and functionality, as well as the transcriptional profile of mitochondrial dynamics markers by using the in vivo and ex vivo approaches. Acute and repeated stress inhibit spermatozoa functionality (acute -> 3.2-fold, repeated -> 2.5-fold), while only repeated stress reduces the spermatozoa number (1.7-fold). Stress hormones mimic these effects and decrease the spermatozoa functionality (adrenaline: 10 µM -> 2.4-fold, 100 µM - > 2.8-fold; hydrocortisone: 50 pM -> 2.7-fold, 500 pM -> 8.5-fold). They also significantly disturb the transcriptional profile of all main mitochondrial dynamics markers in spermatozoa. Ex vivo manipulation of stress signaling in spermatozoa reveals that most of these effects are mediated through ɑ1-and/or-ß-adrenergic receptors. The transcription of these receptors and their kinases in the same samples is under the significant influence of adrenergic signaling. Our results are the first to show the importance of mitochondrial dynamics markers in spermatozoa since the transcriptional profiles of sixteen-out-of-ninteen are disturbed by manipulation of stress-hormones-signaling. This is a completely new molecular approach to assess spermatozoa functionality and it is important for a better understanding of the correlations between stress, environmental-life-style and other factors, and male (in)fertility.

Dependence of Leydig Cell's Mitochondrial Physiology on Luteinizing Hormone Signaling.

Knowledge about the relationship between steroidogenesis and the regulation of the mitochondrial bioenergetics and dynamics, in steroidogenic cells, is not completely elucidated. Here we employed in vivo and ex vivo experimental models to analyze mitochondrial physiology in Leydig cells depending on the different LH-cAMP environments. Activation of LH-receptor in rat Leydig cells ex and in vivo triggered cAMP, increased oxygen consumption, mitoenergetic and steroidogenic activities. Increased mitoenergetic activity i.e., ATP production is achieved through augmented glycolytic ATP production and a small part of oxidative phosphorylation (OXPHOS). Transcription of major genes responsible for mitochondrial dynamics was upregulated for Ppargc1a (regulator of mitogenesis and function) and downregulated for Drp1 (main fission marker), Prkn, Pink1 and Tfeb (mitophagy markers). Leydig cells from gonadotropin-treated rats show increased mitogenesis confirmed by increased mitochondrial mass, increased mtDNA, more frequent mitochondria observed by a transmission electron microscope and increased expression of subunits of respiratory proteins Cytc/CYTC and COX4. Opposite, Leydig cells from hypogonadotropic-hypogonadal rats characterized by low LH-cAMP, testosterone, and ATP production, reduced markers of mitogenesis and mitofusion (Mfn1/2, Opa1) associated with reduced mtDNA content. Altogether results underline LH-cAMP signaling as an important regulator of mitochondrial physiology arranging mitochondrial dynamics, bioenergetic and steroidogenic function in Leydig cells.

Luteinizing hormone signaling is involved in synchronization of Leydig cell's clock and is crucial for rhythm robustness of testosterone production†.

In mammals, circadian clock regulates concentration of many reproductive hormones including testosterone. Previously, we characterized pattern of circadian transcription of core clock genes in testosterone-producing Leydig cells. Here, the potential role of luteinizing hormone receptor (LHR)-cAMP signaling in synchronization of Leydig cell's circadian clock and rhythmic testosterone production were examined. Results showed that activation of LHR-cAMP signaling in primary rat Leydig cell culture increased Star/STAR and changed expression of many clock genes (upregulated Per1/PER1, Dec1/2, and Rorb, and downregulated Bmal1 and Rev-erba/b). Inhibition of protein kinase A prevented LHR-triggered increase in transcription of Per1 and Dec1. Effect of stimulated LHR-cAMP signaling on Leydig cell's clock transcription was also confirmed in vivo, using rats treated with single hCG injection. To analyze in vivo effect of low LH-cAMP activity on rhythmical Leydig cell function, rats with experimental hypogonadotropic hypogonadism were used. Characteristics of hypogonadal rats were decreased LH and testosterone secretion without circadian fluctuation; in Leydig cells decreased arrhythmic cAMP and transcription of steroidogenic genes (Cyp11a1 and Cyp17a1) were observed, while decreased Star/STAR expression retains circadian pattern. However, expression of clock genes, despite changes in transcription levels (increased Bmal1, Per2, Cry1, Cry2, Rora, Rorb, Rev-erba/b/REV-ERBB, Dec1, Csnk1e, and decreased Npas2 and PER1) kept circadian patterns observed in control groups. Altogether, the results strengthened the hypothesis about role of LH-cAMP signaling as synchronizer of Leydig cell's clock. However, clock in Leydig cells is not sufficient to sustain rhythmicity of testosterone production in absence of rhythmic activity of LH-cAMP signaling.

Insulin/IGF1 signaling regulates the mitochondrial biogenesis markers in steroidogenic cells of prepubertal testis, but not ovary.

Controlled changes in mitochondrial biogenesis and morphology are required for cell survival and homeostasis, but the molecular mechanisms are largely unknown. Here, male and female prepubertal mice (P21) with insulin and IGF1 receptors deletions in steroidogenic tissues (Insr/Igf1r-DKO) were used to investigate transcription of the key regulators of mitochondrial biogenesis (Ppargc1a, Ppargc1b, Pparg, Nrf1, Tfam) and architecture in Leydig cells, ovaries, and adrenals. Results showed that the expression of PGC1, a master regulator of mitochondrial biogenesis and integrator of environmental signals, and its downstream target Tfam, significantly decreased in androgen-producing Leydig cells. This is followed by reduction of Mtnd1, a mitochondrial DNA encoded transcript whose core subunit belongs to the minimal assembly required for catalysis. The same markers remained unchanged in ovaries. In contrast, in adrenals, the pattern of transcripts for mitochondrial biogenesis markers was the same in both sexes, but opposite from that observed in Leydig cells. The level of transcripts for markers of mitochondrial architecture (Mfn1, Mfn2) significantly increased in Leydig cells from Insr/Igf1r-DKO, but not in ovaries. This was followed by mitochondrial morphology disturbance, suggesting that the mitochondrial phase of steroidogenesis could be affected. Indeed, basal and pregnenolone stimulated progesterone productions in the mitochondria of Leydig cells from Insr/Igf1r-DKO decreased more than androgen production, and were barely detectable. Our results are the first to show that INSR/IGF1R are important for mitochondrial biogenesis in gonadal steroidogenic cells of prepubertal males, but not females and they serve as important regulators of mitochondrial architecture and biogenesis markers in Leydig cells.

Long-term inhibition of PDE5 ameliorates aging-induced changes in rat testis.

NO-cGMP signaling pathway has been implicated in reduction of testicular steroidogenesis during aging. Here we analyzed the effect of PDE5 inhibition on old testicular phenotype formation. The old phenotype exhibited low testosterone and increased nitrite levels in circulation, increased cGMP accumulation in testicular interstitial fluid (TIF), progressive atrophy of testicular seminiferous tubules and enlargement of interstitial area followed by rise in blood vessel density and slight increase in the number of Leydig cells and macrophages. Leydig cells have reduced steroidogenic capacity, increased MAP kinases expression (MEK, ERK1/2, JNK) and antiapoptotic PRKG1 and AKT, suggesting increased proliferation/survival and accumulation of senescent Leydig cells in testis. In 12 month-old rats, a long-term treatment with sildenafil (PDE5 inhibitor) normalized testosterone/nitrite levels in circulation and cGMP accumulation in TIF; improved Leydig cell steroidogenic capacity; decreased MEK, ERK1/2 and PRKG1 expression; prevented an increase in the Leydig cells number and atrophy of seminiferous tubules leading to histological appearance of young rat testes. In 18 month-old rats, long-term PDE5 inhibition partially recovered testosterone and nitrite levels in serum; normalized PRKG1 expression without effect on MEK and ERK1/2; and slowed down Leydig cell and macrophage accumulation and regressive tubular changes. Culturing of primary Leydig cells from aged rats in presence of PDE5-inhibitor stimulated steroidogenic and MAPK gene expression. Taking together, results indicate that cGMP targeting alter both steroidogenesis and signaling pathways associated with cell proliferation/survival. The long-term PDE5 inhibition improves testicular steroidogenesis and slows-down regressive changes in testes during aging.

Insulin and IGF1 receptors are essential for the development and steroidogenic function of adult Leydig cells.

The insulin family of growth factors (insulin, IGF1, and IGF2) are critical in sex determination, adrenal differentiation, and testicular function. Notably, the IGF system has been reported to mediate the proliferation of steroidogenic cells. However, the precise role and contribution of the membrane receptors mediating those effects, namely, insulin receptor (INSR) and type-I insulin-like growth factor receptor (IGF1R), have not, to our knowledge, been investigated. We show here that specific deletion of both Insr and Igf1r in steroidogenic cells in mice leads to severe alterations of adrenocortical and testicular development. Double-mutant mice display drastic size reduction of both adrenocortex and testes, with impaired corticosterone, testosterone, and sperm production. Detailed developmental analysis of the testes revealed that fetal Leydig cell (LC) function is normal, but there is a failure of adult LC maturation and steroidogenic function associated with accumulation of progenitor LCs (PLCs). Cell-lineage tracing revealed PLC enrichment is secondary to Insr and Igf1r deletion in differentiated adult LCs, suggesting a feedback mechanism between cells at different steps of differentiation. Taken together, these data reveal the cell-autonomous and nonautonomous roles of the IGF system for proper development and maintenance of steroidogenic lineages.-Neirijnck, Y., Calvel, P., Kilcoyne, K. R., Kühne, F., Stévant, I., Griffeth, R. J., Pitetti, J.-L., Andric, S. A., Hu, M.-C., Pralong, F., Smith, L. B., Nef, S. Insulin and IGF1 receptors are essential for the development and steroidogenic function of adult Leydig cells.

Teaching Animal Physiology: a 12-year experience transitioning from a classical to interactive approach with continual assessment and computer alternatives.

In response to the Bologna Declaration and contemporary trends in Animal Physiology education, the Animal Physiology course at the Faculty of Sciences, University of Novi Sad, Serbia, has evolved over a 12-yr period (2001-2012): from a classical two-semester course toward a one-semester course utilizing computer simulations of animal experiments, continual assessment, lectures, and an optional oral exam. This paper presents an overview of student achievement, the impact of reforms on learning outcomes, and lessons that we as educators learned during this process. The reforms had a positive impact on the percentage of students who completed the course within the same academic year. In addition, the percentage of students who completed the practical exam increased from 54% to >95% following the transition to a Bologna-based approach. However, average final grades declined from 8.0 to 6.8 over the same period. Students also appear reluctant to take the optional oral exam, and 82-91% of students were satisfied with the lower final grade obtained from only assessments and tests administered during the semester. In our endeavor to achieve learning outcomes set during the pre-Bologna period, while adopting contemporary teaching approaches, we sought to increase students' motivation to strive toward better performance, while ensuring that the increased quantity of students who complete the course is coupled with increased quality of education and a more in-depth understanding of animal physiology.