Oocyte and embryo culture under oil profoundly alters effective concentrations of small molecule inhibitors.

Culture of oocytes and embryos in media under oil is a cornerstone of fertility treatment, and extensively employed in experimental investigation of early mammalian development. It has been noted anecdotally by some that certain small molecule inhibitors might lose activity in oil-covered culture systems, presumably by drug partitioning into the oil. Here we took a pseudo-pharmacological approach to appraise this formally using mouse oocytes and embryos. Using different culture dish designs with defined media:oil volume ratios, we show that the EC50 of the widely employed microtubule poison nocodazole shifts as a function of the media:oil ratio, such that nocodazole concentrations that prevent cell division in oil-free culture fail to in oil-covered media drops. Relatively subtle changes in culture dish design lead to measurable changes in EC50. This effect is not specific to one type of culture oil, and can be readily observed both in oocyte and embryo culture experiments. We subsequently applied a similar approach to a small panel of widely employed cell cycle-related inhibitors, finding that most lose activity in standard oil-covered oocyte/embryo culture systems. Our data suggest that loss of small molecule activity in oil-covered oocyte and embryo culture is a widespread phenomenon with potentially far-reaching implications for data reproducibility, and we recommend avoiding oil-covered culture for experiments employing inhibitors/drugs wherever possible.

Spindle assembly checkpoint insensitivity allows meiosis-II despite chromosomal defects in aged eggs.

Chromosome segregation errors in mammalian oocyte meiosis lead to developmentally compromised aneuploid embryos and become more common with advancing maternal age. Known contributors include age-related chromosome cohesion loss and spindle assembly checkpoint (SAC) fallibility in meiosis-I. But how effective the SAC is in meiosis-II and how this might contribute to age-related aneuploidy is unknown. Here, we developed genetic and pharmacological approaches to directly address the function of the SAC in meiosis-II. We show that the SAC is insensitive in meiosis-II oocytes and that as a result misaligned chromosomes are randomly segregated. Whilst SAC ineffectiveness in meiosis-II is not age-related, it becomes most prejudicial in oocytes from older females because chromosomes that prematurely separate by age-related cohesion loss become misaligned in meiosis-II. We show that in the absence of a robust SAC in meiosis-II these age-related misaligned chromatids are missegregated and lead to aneuploidy. Our data demonstrate that the SAC fails to prevent cell division in the presence of misaligned chromosomes in oocyte meiosis-II, which explains how age-related cohesion loss can give rise to aneuploid embryos.

Kinetochore deterioration concommitant with centromere weakening during aging in mouse oocyte meiosis-I.

Age-related oocyte aneuploidy occurs as a result of chromosome segregation errors in female meiosis-I and meiosis-II, and is caused by a progressive age-related deterioration of the chromosome segregation machinery. Here, we assess the impact of age upon the kinetochore, the multi-protein structure that forms the link between the chromosome and spindle microtubules. We find that in meiosis-I the outer kinetochore assembles at germinal vesicle breakdown, but that a substantially smaller outer kinetochore is assembled in oocytes from aged mice. We show this correlates with a weaker centromere in aged oocytes and, using nuclear transfer approaches to generate young-aged hybrid oocytes, we show that outer kinetochore assembly always mirrors the status of the centromere, regardless of cytoplasmic age. Finally, we show that weaker kinetochores in aged oocytes are associated with thinner microtubule bundles, that are more likely to be mis-attached. We conclude that progressive loss of the centromere with advancing maternal age underpins a loss of the outer kinetochore in meiosis-I, which likely contributes to chromosome segregation fallibility in oocytes from older females.

Distinct classes of lagging chromosome underpin age-related oocyte aneuploidy in mouse.

Chromosome segregation errors that cause oocyte aneuploidy increase in frequency with maternal age and are considered a major contributing factor of age-related fertility decline in females. Lagging anaphase chromosomes are a common age-associated phenomenon in oocytes, but whether anaphase laggards actually missegregate and cause aneuploidy is unclear. Here, we show that lagging chromosomes in mouse oocytes comprise two mechanistically distinct classes of chromosome motion that we refer to as "class-I" and "class-II" laggards. We use imaging approaches and mechanistic interventions to dissociate the two classes and find that whereas class-II laggards are largely benign, class-I laggards frequently directly lead to aneuploidy. Most notably, a controlled prolongation of meiosis I specifically lessens class-I lagging to prevent aneuploidy. Our data thus reveal lagging chromosomes to be a cause of age-related aneuploidy in mouse oocytes and suggest that manipulating the cell cycle could increase the yield of useful oocytes in some contexts.

Human oocytes harboring damaged DNA can complete meiosis I.

OBJECTIVE: To determine whether human oocytes possess a checkpoint to prevent completion of meiosis I when DNA is damaged. DESIGN: DNA damage is considered a major threat to the establishment of healthy eggs and embryos. Recent studies found that mouse oocytes with damaged DNA can resume meiosis and undergo germinal vesicle breakdown (GVBD), but then arrest in metaphase of meiosis I in a process involving spindle assembly checkpoint (SAC) signaling. Such a mechanism could help prevent the generation of metaphase II (MII) eggs with damaged DNA. Here, we compared the impact of DNA-damaging agents with nondamaged control samples in mouse and human oocytes. SETTING: University-affiliated clinic and research center. PATIENT(S): Patients undergoing ICSI cycles donated GV-stage oocytes after informed consent; 149 human oocytes were collected over 2 years (from 50 patients aged 27-44 years). INTERVENTIONS(S): Mice and human oocytes were treated with DNA-damaging drugs. MAIN OUTCOME MEASURE(S): Oocytes were monitored to evaluate GVBD and polar body extrusion (PBE), in addition to DNA damage assessment with the use of γH2AX antibodies and confocal microscopy. RESULT(S): Whereas DNA damage in mouse oocytes delays or prevents oocyte maturation, most human oocytes harboring experimentally induced DNA damage progress through meiosis I and subsequently form an MII egg, revealing the absence of a DNA damage-induced SAC response. Analysis of the resulting MII eggs revealed damaged DNA and chaotic spindle apparatus, despite the oocyte appearing morphologically normal. CONCLUSION(S): Our data indicate that experimentally induced DNA damage does not prevent PBE in human oocytes and can persist in morphologically normal looking MII eggs.

Segregating Chromosomes in the Mammalian Oocyte.

Chromosome segregation errors in human oocytes lead to aneuploid embryos that cause infertility and birth defects. Here we provide an overview of the chromosome-segregation process in the mammalian oocyte, highlighting mechanistic differences between oocytes and somatic cells that render oocytes so prone to segregation error. These differences include the extremely large size of the oocyte cytoplasm, the unique geometry of meiosis-I chromosomes, idiosyncratic function of the spindle assembly checkpoint, and dramatically altered oocyte cell-cycle control and spindle assembly, as compared to typical somatic cells. We summarise recent work suggesting that aging leads to a further deterioration in fidelity of chromosome segregation by impacting multiple components of the chromosome-segregation machinery. In addition, we compare and contrast recent results from mouse and human oocytes, which exhibit overlapping defects to differing extents. We conclude that the striking propensity of the oocyte to mis-segregate chromosomes reflects the unique challenges faced by the spindle in a highly unusual cellular environment.

Meiosis: SUMO Keeps a Tight Grip on Sister Chromatids.

Maintaining cohesion between sister chromatids during the first meiotic cell division is crucial for preventing oocyte aneuploidy. In a new paper in Current Biology, Yi and colleagues present evidence that the Small Ubiquitin-related Modifier (SUMO) pathway protects centromeric sister cohesion during the meiosis I-II transition in mouse oocytes.

The first cell-fate decision of mouse preimplantation embryo development: integrating cell position and polarity.

During the first cell-fate decision of mouse preimplantation embryo development, a population of outer-residing polar cells is segregated from a second population of inner apolar cells to form two distinct cell lineages: the trophectoderm and the inner cell mass (ICM), respectively. Historically, two models have been proposed to explain how the initial differences between these two cell populations originate and ultimately define them as the two stated early blastocyst stage cell lineages. The 'positional' model proposes that cells acquire distinct fates based on differences in their relative position within the developing embryo, while the 'polarity' model proposes that the differences driving the lineage segregation arise as a consequence of the differential inheritance of factors, which exhibit polarized subcellular localizations, upon asymmetric cell divisions. Although these two models have traditionally been considered separately, a growing body of evidence, collected over recent years, suggests the existence of a large degree of compatibility. Accordingly, the main aim of this review is to summarize the major historical and more contemporarily identified events that define the first cell-fate decision and to place them in the context of both the originally proposed positional and polarity models, thus highlighting their functional complementarity in describing distinct aspects of the developmental programme underpinning the first cell-fate decision in mouse embryogenesis.

Rho-associated protein kinase regulates subcellular localisation of Angiomotin and Hippo-signalling during preimplantation mouse embryo development.

The differential activity of the Hippo-signalling pathway between the outer- and inner-cell populations of the developing preimplantation mouse embryo directs appropriate formation of trophectoderm and inner cell mass (ICM) lineages. Such distinct signalling activity is under control of intracellular polarization, whereby Hippo-signalling is either supressed in polarized outer cells or activated in apolar inner cells. The central role of apical-basolateral polarization to such differential Hippo-signalling regulation prompted us to reinvestigate the role of potential upstream molecular regulators affecting apical-basolateral polarity. This study reports that the chemical inhibition of Rho-associated kinase (Rock) is associated with failure to form morphologically distinct blastocysts, indicative of compromised trophectoderm differentiation, and defects in the localization of both apical and basolateral polarity factors associated with malformation of tight junctions. Moreover, Rock-inhibition mediates mislocalization of the Hippo-signalling activator Angiomotin (Amot), to the basolateral regions of outer cells and is concomitant with aberrant activation of the pathway. The Rock-inhibition phenotype is mediated by Amot, as RNAi-based Amot knockdown totally rescues the normal suppression of Hippo-signalling in outer cells. In conclusion, Rock, via regulating appropriate apical-basolateral polarization in outer cells, regulates the appropriate activity of the Hippo-signalling pathway, by ensuring correct subcellular localization of Amot protein in outer cells.

The first two cell-fate decisions of preimplantation mouse embryo development are not functionally independent.

During mouse preimplantation embryo development, three distinct cell lineages are formed, represented by the differentiating trophectoderm (TE), primitive endoderm (PrE) and the pluripotent epiblast (EPI). Classically, lineage derivation has been presented as a two-step process whereby outer TE cells are first segregated from inner-cell mass (ICM), followed by ICM refinement into either the PrE or EPI. As ICM founders can be produced following the fourth or fifth cleavage divisions, their potential to equally contribute to EPI and PrE is contested. Thus, modelling the early sequestration of ICM founders from TE-differentiation after the fourth cleavage division, we examined ICM lineage contribution of varying sized cell clones unable to initiate TE-differentiation. Such TE-inhibited ICM cells do not equally contribute to EPI and PrE and are significantly biased to form EPI. This bias is not caused by enhanced expression of the EPI marker Nanog, nor correlated with reduced apical polarity but associated with reduced expression of PrE-related gene transcripts (Dab2 and Lrp2) and down-regulation of plasma membrane associated Fgfr2. Our results favour a unifying model were the three cell lineages are guided in an integrated, yet flexible, fate decision centred on relative exposure of founder cells to TE-differentiative cues.

Intratesticular alpha1-adrenergic receptors mediate stress-disturbed transcription of steroidogenic stimulator NUR77 as well as steroidogenic repressors DAX1 and ARR19 in Leydig cells of adult rats.

The aim of the present study was to define the role of testicular α1-adrenergic receptors (α1-ADRs) in stress-triggered adaptation of testosterone-producing Leydig cells of adult rats. Results showed that in vivo blockade of testicular α1-ADRs prevented partial recovery of circulating androgen levels registered after 10× repeated immobilization stress (10 × IMO). Moreover, α1-ADR-blockade diminished 10 × IMO-triggered recovery of Leydig cell androgen production, and abolished mitochondrial membrane potential recovery. In the same cells, 10 × IMO-induced increase in Star transcript was abolished, Lhcgr transcript decreased, while transcription of other steroidogenic proteins was not changed. α1-ADR-blockade recovered stress-induced decrease of Nur77, one of the main steroidogenic stimulator, while significantly reduced 10 × IMO-increased in the transcription of the main steroidogenic repressors, Arr19 and Dax1. In vitro experiments revealed an adrenaline-induced α1-ADR-mediated decrease in Nur77 transcription in Leydig cells. Adrenaline-induced increase of repressor Dax1 also involves ADRs in Leydig cells. Accordingly, α1-ADRs participate in some of the stress-triggered effects on the steroidogenic machinery of Leydig cells.

Molecular adaptations of testosterone-producing Leydig cells during systemic in vivo blockade of the androgen receptor.

This study systematically evaluates the effects of androgen receptor (AR) blockade on molecular events in Leydig cells. Results showed that intramuscular administration of testosterone-enanthate, at clinically relevant dose, decreased testosterone in interstitial fluid and Leydig cells from adult rats. AR-blocker (Androcur) prevented this effect and testosterone-reduced Leydig cells steroidogenic capacity/activity. Testosterone-reduced expression of some steroidogenic enzymes/proteins (Tspo,StAR,Hsd3b1/2) and transcription factors (Nur77,Gata4,Dax1) was completely abrogated, while decreased expression of Star,Cyp11a1,Cyp17a1,Hsd17b4,Creb1a was partially prevented. In the same cells, increased expression of Hsd3b5/HSD3B and Ar/AR was abolished. Androcur-treatment abolished testosterone-reduced cAMP, coupled with a changed expressional milieu of cAMP signaling elements. Results from in vitro experiments suggest that some of these effects are testosterone-AR dependent, while others could be due to disturbed LH and/or other signals. Presented data provide new molecular insight into Leydig cells function and are important in terms of human reproductive health and the wide-spread use of Androcur as well as use/abuse of testosterone-enanthate.

In vivo blockade of α1-adrenergic receptors mitigates stress-disturbed cAMP and cGMP signaling in Leydig cells.

The molecular mechanism of stress-associated reproductive dysfunction is complex and largely unknown. This study was designed to systematically analyze molecular effects of systemic in vivo blockade of α1-adrenergic receptors (α1-ADRs) on stress-induced disturbance of cAMP/cGMP signaling in testosterone-producing Leydig cells using the following parameters (i) level of circulating stress hormones, LH and testosterone; (ii) level of main molecular markers of Leydig cell functionality (testosterone, Insl3, cAMP); (iii) expression of cAMP signaling (cAMP 'producers'/'effectors'/'removers') and (iv) expression of NO-cGMP signaling (NO-cGMP 'producers'/'effectors'/'removers'). The results showed that oral administration of α1-ADR blocker before stress increased cGMP and diminished stress-reduced cAMP production in Leydig cells. In the same cells, stress-induced effects on cAMP/cGMP signaling pathways elements were changed. Sustained in vivo α1-ADR blockade completely abolished stress-increased transcription of most abundantly expressed phosphodiesterase that remove cAMP (Pde4b) and potentiated stress-increased expression of PRKA, the main stimulator of Leydig cell steroidogenesis. In the same Leydig cells, stress-decreased NOS3 expression was abolished, while stress-increased GUCY1 (cGMP 'producer') and PRKG1 (cGMP 'effector') were potentiated. It is possible that all molecules mentioned could contribute, at least in part, in recovery of Leydig cell testosterone production. Presented data provide new role of α1-ADRs in stress-triggered disturbance of cAMP/cGMP signaling, and new molecular insights into the relationship between stress and mammalian reproduction. Regardless of whether the effects of α1-blocker + stress are direct or indirect, the results are important in terms of human reproductive health and the wide use of α1-ADR antagonists, alone or in combination, to treat post-traumatic stress disorders, hypertension, benign prostatic hyperplasia symptoms and potential drugs for prostate cancer prevention/treatment.

Sustained in vivo blockade of α1-adrenergic receptors prevented some of stress-triggered effects on steroidogenic machinery in Leydig cells.

This study was designed to systematically analyze and evaluate the effects of in vivo blockade of α1-adrenergic receptors (α1-ADRs) on the stress-induced disturbance of steroidogenic machinery in Leydig cells. Parameters followed 1) steroidogenic enzymes/proteins, transcription factors, and cAMP/testosterone production; 2) the main hallmarks of stress (epinephrine, glucocorticoids); and 3) transcription profiles of ADRs and oxidases with high affinity to inactivate glucocorticoids. Results showed that sustained blockade of α1-ADRs prevented stress-induced 1) decrease of the transcripts/proteins for main steroidogenic CYPs (CYP11A1, CYP17A1); 2) decrease of Scarb1 and Hsd3b1 transcripts; 3) decrease of transcript for Nur77, one of the main activator of the steroidogenic expression; and 4) increase of Dax1 and Arr19, the main steroidogenic repressors in Leydig cells. In the same cells, the expression of steroidogenic stimulatory factor Creb1, StAR, and androgen receptor increased. In this signaling scenario, stress-induced stimulation of Adra1a/Adra1b/Adrbk1 and Hsd11b2 (the unidirectional oxidase with high affinity to inactivate glucocorticoids) was not changed. Blockade additionally stimulated stress-increased transcription of the most abundantly expressed ADRs Adra1d/Adrb1/Adrb2 in Leydig cells. In the same cells, stress-decreased testosterone production, the main marker of Leydig cells functionality, was completely prevented, while reduction of cAMP, the main regulator of androgenesis, was partially prevented. Accordingly, the presented data provide a new molecular/transcriptional base for "fight/adaptation" of steroidogenic cells and new molecular insights into the role of α1-ADRs in stress-impaired Leydig cell steroidogenesis. The results are important in term of wide use of α1-ADR selective antagonists, alone/in combination, to treat high blood pressure, nightmares associated with posttraumatic stress disorder, and disrupted sexual health.

The opposite roles of glucocorticoid and α1-adrenergic receptors in stress triggered apoptosis of rat Leydig cells.

The stress-induced initiation of proapoptotic signaling in Leydig cells is relatively well defined, but the duration of this signaling and the mechanism(s) involved in opposing the stress responses have not been addressed. In this study, immobilization stress (IMO) was applied for 2 h daily, and animals were euthanized immediately after the first (IMO1), second (IMO2), and 10th (IMO10) sessions. In IMO1 and IMO2 rats, serum corticosterone and adrenaline were elevated, whereas serum androgens and mRNA transcription of insulin-like factor-3 in Leydig cells were inhibited. Reduced oxygen consumption and the mitochondrial membrane potential coupled with a leak of cytochrome c from mitochondria and increased caspase-9 expression, caspase-3 activity, and number of apoptotic Leydig cells was also observed. Corticosterone and adrenaline were also elevated in IMO10 rats but were accompanied with a partial recovery of androgen secretion and normalization of insulin-like factor-3 transcription coupled with increased cytochrome c expression, abolition of proapoptotic signaling, and normalization of the apoptotic events. Blockade of intratesticular glucocorticoid receptors diminished proapoptotic effects without affecting antiapoptotic effects, whereas blockade of intratesticular α(1)-adrenergic receptors diminished the antiapoptotic effects without affecting proapoptotic effects. These results confirmed a critical role of glucocorticoids in mitochondria-dependent apoptosis and showed for the first time the relevance of stress-induced upregulation of α(1)-adrenergic receptor expression in cell apoptotic resistance to repetitive IMOs. The opposite role of two hormones in control of the apoptotic rate in Leydig cells also provides a rationale for a partial recovery of androgen production in chronically stressed animals.

Repeated immobilization stress disturbed steroidogenic machinery and stimulated the expression of cAMP signaling elements and adrenergic receptors in Leydig cells.

This study was designed to evaluate the effect of acute (2 h daily) and repeated (2 h daily for 2 or 10 consecutive days) immobilization stress (IMO) on: 1) the steroidogenic machinery homeostasis; 2) cAMP signaling; and the expression of receptors for main markers of 3) adrenergic and 4) glucocorticoid signaling in Leydig cells of adult rats. The results showed that acute IMO inhibited steroidogenic machinery in Leydig cells by downregulation of Scarb1 (scavenger receptor class B), Cyp11a1 (cholesterol side-chain cleavage enzyme), Cyp17a1 (17α-hydroxylase/17,20 lyase), and Hsd17b3 (17ß-hydroxysteroid dehydrogenase) expression. In addition to acute IMO effects, repeated IMO increased transcription of Star (steroidogenic acute regulatory protein) and Arr19 (androgen receptor corepressor 19 kDa) in Leydig cells. In the same cells, the transcription of adenylyl cyclases (Adcy7, Adcy9, Adcy10) and cAMP-specific phosphodiesterases (Pde4a, Pde4b, Pde4d, Pde7a, Pde8a) was stimulated, whereas the expression of the genes encoding protein kinase A subunits were unaffected. Ten times repeated IMO increased the levels of all adrenergic receptors and ß-adrenergic receptor kinase (Adrbk1) in Leydig cells. The transcription analysis was supported by cAMP/testosterone production. In this signaling scenario, partial recovery of testosterone production in medium/content was detected. The physiological significance of the present results was proven by ex vivo application of epinephrine, which increased cAMP/testosterone production by Leydig cells from control rats in greater fashion than from stressed. IMO did not affect the expression of transcripts for Crhr1/Crhr2 (corticotropin releasing hormone receptors), Acthr (adrenocorticotropin releasing hormone receptor), Gr (glucocorticoid receptor), and Hsd11b1 [hydroxysteroid (11-ß) dehydrogenase 1], while all types of IMO stimulated the expression of Hsd11b2, the unidirectional oxidase with high affinity to inactivate glucocorticoids. Thus, presented data provide new molecular/transcriptional base for "fight/adaptation" of Leydig cells and new insights into the role of cAMP, epinephrine, and glucocorticoid signaling in recovery of stress-impaired Leydig cell steroidogenesis.

Transient rise of serum testosterone level after single sildenafil treatment of adult male rats.

INTRODUCTION: Phosphodiesterase type 5 (PDE5) inhibitors have been established in therapy for a variety of physiological disorders including erectile dysfunction. Despite its popularity and wide usage in erectile dysfunction treatment, the short-term effect of PDE5 inhibition on Leydig cell functionality and testosterone dynamics is missing. AIM: This study was designed to assess the acute in vivo effects of sildenafil citrate (Viagra) treatment on testosterone production. METHODS: Male adult rats were given sildenafil (1.25 mg/kg BW) per os, and testosterone production were analyzed 30, 60, 120, and 180 minutes after treatment. Additionally, in vitro effect of sildenafil extract on Leydig cell steroidogenesis was estimated. MAIN OUTCOME MEASURES: The formation of testicular interstitial fluid (TIF), and testosterone, cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP) content was followed. Occurrence and phosphorylation of mature steroidogenic acute regulatory protein (StAR) and interaction with protein kinase G 1 (PRKG1) were assessed by immunoprecipitation and Western blot. RESULTS: Serum testosterone was increased 60 and 120 minutes after sildenafil treatment. In 60 minutes, TIF volume was doubled and stayed increased till the end of the experimental period. cGMP and testosterone content in TIF were increased 30 minutes after treatment, and cAMP decreased in 60 minutes. Further, sildenafil-induced stimulation of testosterone production was abolished by ex vivo addition of PRKG1 inhibitor but not by protein kinase A inhibitor. Sildenafil treatment increased the level of phosphorylated and total StAR protein. Moreover, co-immunoprecipitation of StAR and PRKG1 was increased following sildenafil treatment suggesting the active role of this kinase in initiation of testosterone synthesis. Additionally, sildenafil extract applied in vitro on primary Leydig cell culture increased cGMP accumulation and testosterone production in time- and dose-dependent manner without effect on cAMP level. CONCLUSION: Acute sildenafil treatment enlarged TIF volume but also stimulated testosterone production which may be significant considering the positive testosterone effect in regulation of sexual activity.

Pharmacological doses of testosterone upregulated androgen receptor and 3-Beta-hydroxysteroid dehydrogenase/delta-5-delta-4 isomerase and impaired leydig cells steroidogenesis in adult rats.

Anabolic androgenic steroids (AAS) are testosterone derivatives originally designed to enhance muscular mass and used for the treatment of many clinical conditions as well as in contraception. Despite popular interest and abuse, we still lack a broad understanding of effects of AAS on synthesis of steroid hormones on the molecular level. This study was designed to systematically analyze the effects of pharmacological/high doses of testosterone on steroidogenic machinery in Leydig cells. Two different experimental approaches were used: (1) In vivo experiment on groups of adult male rats treated with testosterone for 1 day, 2 weeks, and 2 months; (2) Direct in vitro testosterone treatment of Leydig cells isolated from intact rats. Result showed that prolonged in vivo treatment with testosterone decreased the expression of Scarb1 (scavenger receptor class B type 1), Tspo (translocator protein), Star (steroidogenic acute regulatory protein), Cyp11a1 (cholesterol side-chain cleavage enzyme), and Cyp17a1 (17α-hydroxylase/17, 20 lyase) in Leydig cells. Oppositely, the expression of Hsd3b (3-beta-hydroxysteroid dehydrogenase/delta-5-delta-4 isomerase), Ar (androgen receptor), and Pde4a/b (cyclic adenosine monophosphate-dependent phosphodiesterases) was increased. Androgenization for 2 weeks inhibited Cyp19 (aromatase) transcription, whereas 2-month exposure caused the opposite effect. Direct in vitro testosterone treatment also decreased the expression of Cyp11a1, Cyp17a1, and Cyp19a1, whereas Hsd3b was upregulated. The results of expression analysis were supported by declined steroidogenic capacity and activity of Leydig cells, although conversion of pregnenolone to progesterone was stimulated. The upregulation of AR and 3ßHSD in testosterone-impaired Leydig cells steroidogenesis could be the possible mechanism that maintain and prevent loss of steroidogenic function.