Functional Importance of 1α,25(OH)2-Vitamin D3 and the Identification of Its Nongenomic and Genomic Signaling Pathways in the Testis

The 1α,25(OH) 2 -vitamin D 3 (1,25-D 3 ) is known by its classic effects on Cametabolism and regulation of cellular proliferation and differentiation. The hormone 1,25-D 3 acts in the testis through nongenomic and genomic events being implicated in the success of spermatogenesis in rats and in human being. The aim of this review was to highlight the effect and intracellular pathways of 1,25-D 3 to modulate the spermatogenesis. The pivotal role of 1,25-D 3 in male reproduction is reinforced by the presence of VDR and 1α-hydroxylase in reproductive tract. Also, the marked expression of VDR and the VD metabolizing enzymes in human testis, ejaculatory tract, andmature spermatozoa implicates the 1,25-D 3 in spermatogenesis andmaturation of human spermatozoa. Among genomic events, 1,25-D 3 influences the expression of calcium binding protein and stimulates aromatase gene expression through a nongenomic activation of the membrane-bound VDR receptor involving the PKA pathway in the testis. Also, 1,25D 3 stimulates amino acid transport and exocytosis in testis by nongenomic events coupled to ionic currents triggered at plasma membrane. All together, the demonstration that 1,25-D 3 regulates both Sertoli cell and sperm function may be useful for the study and development of new therapeutic strategies for the male reproductive disorders.


General Aspects
The first scientific report associated with vitamin D deficiency and bone disease rickets was around 1645.However, the recognition of the rickets in patients with no sunshine exposition was only in the 20th century.In 1924, an additional key for vitamin D present in skin was the discovery that a precursor of vitamin D could be converted into vitamin D by exposure to sunlight or ultraviolet [1].The hallmark era of vitamin D began in 1965-1970 since the chemical characterization of an active metabolite of vitamin D, 1,25(OH) 2vitamin D 3 (1,25-D 3 ), and its nuclear receptor (VDR) was reported [1,2].
It is known that vitamin D 3 does not have any intrinsic biological activity [3].After the knowledge that the precursor of vitamin D can be converted into vitamin D by exposure to ultraviolet light (UVB), it is now comprehensive that vitamin D is correctly named as vitamin only when sunlight exposure does not exist [4].Nowadays, the biologically active form of vitamin D is known to be a steroid hormone and endocrine system of vitamin D is well accepted.The chemical characterization, the production of an active metabolite of 1,25-D 3 , and its nuclear receptor occurred between 1965-1970 [5].
The interesting thing about vitamin D is that it undergoes obligatory metabolism prior to generation of its biological response [6].The endogenous synthesis of 1,25-D 3 in the kidney represents the main source of 1,25-D 3 in the body.The UVB radiation starts the conversion of 7-dehydrocholesterol to cholecalciferol (provitamin D 3 ).So vitamin D 3 also can be endogenously produced by the animals or humans.However, in an inadequate sunlight access, the animal (or human) might largely depend on this vitamin from the diet or by individual oral intake of vitamin D 3 supplements [3,7,8].

Advances in Andrology
After the cholecalciferol formation (inactive vitamin D 3 ), it undergoes two hidroxylations to form the compound 1,25-D 3 [2,9].In the liver, the production of 25(OH)D 3 , the major form of vitamin D circulating in the blood compartment, is mainly mediated by the enzyme CYP2R1 encoded by the gene CYP27A1.So 25(OH)D 3 , which enters in blood circulation and subsequently in the kidney (that functions as an endocrine gland), is hydroxylated by 1-hydroxylase encoded by the gene CYP27B1 generating the hormone 1,25-D 3 and the candidate hormone 24R,25(OH) 2 D 3 , as well as 38 other vitamin D 3 metabolites [2,3,10].So, both these dihydroxylates metabolites can be transported to distal organs by the plasma vitamin D binding protein (DBP), a serum glycoprotein-globulin produced by the liver [11].The pool of DBP-bound vitamin D metabolites can be readily used or kept as a reservoir to be used during the reduced intake or production [12][13][14].In target tissues, the active hormone 1,25-D 3 can bind and activate the vitamin D receptor (VDRnuc or VDRmem) and after that can be inactivated by CYP24A1 [2,10,15].
The effects of 1,25-D 3 are mediated through the 1,25-D 3 receptor (VDR) and VDR is expressed in over 38 tissues, which include brain and endocrine organs such as pancreas, pituitary, muscle, kidney, ovary, prostate, and testis [7,16,17].As much in human as in animals models, it was demonstrated that 1,25-D 3 is a key regulatory factor of calcium homeostasis in both male and female.Although several studies suggest that 1,25-D 3 has a role in reproductive function, the association between hypospermatogenesis and a disturbance in vitamin D endocrine system is not clearly understood [18][19][20].In this review, the physiological role and genomic and nongenomic effects of 1,25-D 3 on the testis and on spermatogenic process will be discussed.

1,25-D 3 Receptor Distribution in the Testis of Rodents
Concerning the putative role of 1,25-D 3 in male reproduction, little information is available.The 1,25-D 3 classical receptor (VDR) is a member of the superfamily of nuclear receptors.It was discovered and identified in the intestine of chickens vitamin D-deficient, a chromatin-associated protein that binds the active hormone 1,25-D 3 [21].It is distributed in more than 37 tissues able to generate genomic and/or rapid responses [22].VDR is a DNA-binding transcription factor with a molecular weight of about 50 kDa [22].The heterodimer of the 1,25(OH) 2 D 3 -liganded VDR and unoccupied retinoid X receptor (VDR-RXR) recognize vitamin D responsive elements (VDREs) in the DNA sequence of vitamin Dregulated genes [23,24].Among 38 cell types and tissues, where the presence of VDR was detected, the testis of rodent and human beings is included.Several reports show the nuclear 1,25-D 3 receptors distribution in the testis of rodents [25][26][27].Using whole testis, seminiferous tubules, and isolated testicular cells, a wide distribution of VDR mainly in Sertoli and in seminiferous tubules was characterized which are strongly related to cell proliferation and differentiation.The role of 1,25-D 3 in male reproduction was reinforced since a significant binding of the hormone was detected in certain processes during spermatogenesis and spermiogenesis, on sperma maturation, on epididymal fluid resorption, and on secretion and transport of spermatozoa, in rats [17,28].In addition, the spermatogenesis of 1,25-D 3 -depleted rat is incomplete and exhibits impaired development, and degenerative changes in the seminiferous tubules are observed [27].These are in agreement with a gonadal insufficiency that occurs in VDR null mutant mice which showed a decreased sperm count and motility and significant histological abnormalities in the testis [18].
Recently, the pivotal role of 1,25-D 3 in male reproduction was described using concomitant evaluation of VDR and 1-hydroxylase expression in all organs of male mice reproductive tract.Epithelial cells of epididymis, seminal vesicle, coagulating gland, ductus deferens, preputial gland, and prostate were the prominent cell types that concomitantly expressed VDR and 1-hydroxylase.And an interesting fact was that specific band of approximately 54 kDa was observed in all male mice reproductive organs but not in the testis.Instead, in this organ 3 distinct bands of about 45, 57, and 63 kDa were seen in all mice examined and 45 kDa band was also observed in prostate tissue.Furthermore, testis and prostate express a higher level of 1-hydroxylase.In addition, for VDR, Sertoli cells were the prominent cell in testis that expressed very high levels of nuclear VDR.All other intratubular cells including primary spermatocytes and spermatogonia expressed varying degree of VDR in their nucleus and cytoplasm.Also Leydig cells showed strong nuclear staining [29].From these finds, increased evidence provides a central role of 1,25-D 3 in an active and full spermatogenesis.However, the aim is to understand the mechanism of action 1,25-D 3 in the testis to coordinate the complex spermatogenesis during the sexual development in order to warrant the male reproductive function.

1,25-D 3 Receptor Distribution in the Testis of Human Beings
In the last few years, studies on the effect and mechanism of action of 1,25-D 3 through VDR have focused on human male reproduction.In human testis, the specific high affinity and low binding capacity of VDR for 1,25-D 3 were demonstrated [16].Studies from Nangia et al. [30] revealed six forms of the VDR in the nuclear matrix of human testis.And in human testis was demonstrated a protein of 57 and 52 kDa molecular weight compared with 57 and 37 kDa in the rat testis.However, if the difference in molecular weight proteins with the anti-VDR antibody within tissues or species may represent different isoforms, proteolytic cleavage of a larger VDR, or even a posttranslational modification, it is not solved yet.
The advances in human germ cells increased the comprehension about the role of 1,25-D 3 in male fertility.Corbett et al. [31], through a prospective study of sperm collected from ten fertile men, showed for the first time the VDR on human sperm located predominantly on the head/nucleus of the sperm and midpiece.Thus, in an attempt to understand the gonadal insufficiencies with 1,25-D 3 deficiency and VDR, Aquila et al. [32] studied the localization of human VDR in normozoospermic samples and the role of 1,25-D 3 /VDR in sperm survival and capacitation.Although the action of 1,25-D 3 depends on its concentration, the authors suggested that 1,25-D 3 /VDR may have an important role in sperm survival and in the acquisition of fertilizing ability.Also it was shown that the human sperm expresses the VDR and that 1,25-D 3 is locally produced since 25(OH)D 3 -1,hydroxylase was detected in sperm [33].In addition, the effect of 1,25-D 3 through VDR increased intracellular Ca 2+ levels, motility, and acrosin activity revealing an unexpected significance of this hormone in the acquisition of fertilizing ability.So taking in mind that the cellular response for 1,25-D 3 is complex (since it depends on VDR expression, the cellular uptake of circulating 1,25-D 3 , and also presence and activity of VD metabolizing enzymes), BlombergJensen et al. [34] performed a comprehensive analysis of the expression of VDR, VD activating (CYP2R1, CYP27A1, and CYP27B1), and VD-inactivating (CYP24A1) enzymes in the testis, epididymis, seminal vesicle, prostate, and spermatozoa.From those results and based on the marked expression of VDR and the VD metabolizing enzymes in human testis, ejaculatory tract, and mature spermatozoa, the authors suggested that 1,25-D 3 is important for spermatogenesis and maturation of human spermatozoa.
Interesting results concerning the association of testicular failure with bone mineral density were recently published [19].The authors investigated CYP2R1 expression in pathological testis samples and relate this to vitamin D metabolism in young testiculopathic patients.Surprisingly, in all testiculopathic patients, 25-hydroxyvitamin D levels were significantly lower and parathyroid hormone levels higher compared to controls groups and the patients showed osteopenia and osteoporosis despite normal testosterone levels.
Another interesting point in human spermatozoa was about the use of VD-inactivating enzyme as a marker of the quality of the semen.It was described that the VDinactivating enzyme CYP24A1, titrating the cellular responsiveness to VD, is transcriptionally regulated by VD and has a distinct expression at the sperm annulus.So the CYP24A1 expression was investigated and pointed as a marker for VD metabolism in human spermatozoa.In addition, the CYP24A1 expression correlated positively with total count, the concentration, motility, and morphology of the sperm was associated with semen quality.Also, based on functional studies, 1,25-D 3 increased intracellular calcium and sperm motility in young healthy men but was unable to increase motility in subfertile patients.Then, the authors suggested that CYP24A1 expression at the annulus may serve as a novel marker of semen quality [35].

Genomic Effects of 1,25-D 3 in the Testis
It is well known that vitamin D undergoes obligatory metabolism in order to generate its biological response.
Briefly, an enzymatic insertion of a 25-hydroxyl group gives 25-hydroxylvitamin D (25-OH-D) that occurs in the liver.And a second step of metabolite modification occurs in the kidney where 25-OH-D may be converted into either 1,25-D 3 or 24R,25-dihydroxyvitamin D (24,25-D 3 ) [15,36,37].One of the most known genomic effects for the active steroid metabolite of vitamin D, 1,25-D 3 , is the induction of de novo synthesis of a vitamin D-dependent calcium-binding protein (CaBP) in intestinal mucosa [38].Regarding testicular genomic events, vitamin D 3 via its active metabolite 1,25-D 3 influences cell proliferation and cell differentiation and expresses calcium binding protein mediated by VDR in rat testis and in TM4 Sertoli cell line [25,[39][40][41][42].In addition, 1,25-D 3 nuclear receptors have been demonstrated in Sertoli cells and this hormone is also critical for the maintenance of normal reproduction in male rats since vitamin D-deficient rats have reduced fertility compared with normal vitamin D-replete rats [17,27,28].These finds were confirmed since that vitamin D receptor null mutant mice showed gonadal insufficiencies and decreased motility and sperm count with histological abnormality of the testis [18].
It is well-known that 1,25-D 3 regulates aromatase (CYP19A1) in humans in a tissue specific way [43][44][45] and it is implicated in estrogen and androgen production and metabolism.Also the association with serum testosterone and 25-hydroxyvitamin D serum levels or vitamin D supplementation and testosterone levels in men was well characterized [46,47].
Aromatase converts irreversibly androgens into estrogens and is present in the endoplasmic reticulum of various tissues including the mammalian testis [48,49].The aromatase enzyme complex comprises two proteins: a specific cytochrome P450 (P450arom) encoded by the CYP19 gene and a ubiquitous NADPH cytochrome P450 reductase [50,51].In mouse, the Cyp19 gene is localized on chromosome 9, and three promoters that control specifically the aromatase gene expression were also described [52].In rat, the Cyp19 gene is situated on chromosome 8 and up to now three promoters were described: the promoter PI.f in brain [53] and in testis [54], the promoter PII [55], which is the main one directing aromatase gene expression in gonads [56,57], and the promoter PI.tronly used in testis [54].Zanatta et al. [58] described that VDR transcripts were present in immature Sertoli cells, in adult testicular germ cells, and in somatic cells as much as 1,25-D 3 increased the amount of aromatase transcript, mainly in 30-day-old rats.The physiological role of 1,25-D 3 in Sertoli cells was corroborated due to the stimulation of aromatase gene expression by the agonist 1,25(OH) 2 lumisterol 3 and the suppression of the 1,25-D 3 effect by the antagonists 1,25(OH) 2 vitamin D 3 and (23S)-25-dehydro-1 (OH)-vitamin D 3 -26,23-lactone.In a whole, these data suggested that besides a genomic effect of 1,25-D 3 , the existence of nongenomic activation of the membranebound VDR receptor involves the PKA pathway.

Nongenomic Effects of 1,25-D 3 on Testis
The secosteroid hormone, 1,25-D 3 , mediates classic transcriptional events [59] and also acts through a VDRmem for the rapid stimulation of calcium uptake in isolated rat intestinal epithelial cells, as well as calcium transport in perfused chick duodenum [60,61].Herein, some studies were summarized concerning nongenomic evidence for 1,25-D 3 in the testis able to modify testicular behavior and spermatogenesis as well.
The neutral nonmetabolizable amino acid transport model previously introduced [62], thereby distinguishing protein synthesis from alternative amino acid accumulation pathways, has been useful for identification of substances at plasma membrane and/or nuclear effect.The transport of neutral amino acids involves three major systems.Among them, the alanine-preferring (A) systems, specific for Nmethylaminoisobutyric acid, are sodium, pH, and energy dependent.The hormonal effects on amino acid uptake appear to be restricted to system "A" [63].This system is characterized to be composed of an inorganic component (electrochemical gradient) and an organic component (plasma membrane carriers) which are essential for cell survival [64,65].In the testis, the neutral amino acid transport, "A" system, was identified to be regulated by folliclestimulating hormone, retinol, triiodothyronine, 1,25-D 3 , and thyroxine [65][66][67][68][69][70][71][72][73].So using this electrochemical amino acid "A" system as a tool, we provided evidence for rapid responses of 1,25-D 3 in immature rat testis involving voltage-dependent calcium channels (VDCC) and Ca 2+ -dependent K + channels activities at plasma membrane to regulate neutral amino acid accumulation [65].However, the involvement of VDRmem in amino acid uptake in the testis remains obscure and further computational studies on Sertoli cells are needed to better understand the role of this putative plasma membrane receptor for 1,25-D 3 .
The involvement of voltage-gated chloride channels activity as an electric shunt that couples to H + -ATPase-driven loading of secretory vesicles that are crucial for the onset of exocytosis is now well known.The steroid, 1,25-D 3 , activates chloride channels and it seems to be needed for exocytosis in bone cells [74].In particular, the secretory activities of Sertoli cells are critical to spermatogenesis and this cell expresses a variety of ionic channels involved in cellular secretion [75][76][77][78][79]. Furthermore, the hormonal regulation of fluid secretion by Sertoli cells is important for the development of spermatogenesis wave and involves the modulation of ionic channel activities [65,73,[80][81][82].Taking in mind that the secretory activities are highly dependent on ionic channel functions and critical for the spermatogenesis success [75], the role of 1,25-D 3 on exocytosis was investigated.Surprisingly, the rapid response to 1,25-D 3 in the mouse immature Sertoli cell line TM4 was mediated by chloride channel activation and culminated in cell secretion.Additionally, chloride currents were potentiated by the nongenomic VDR agonist 1,25(OH) 2 lumisterol D 3 (JN), while 1,25-D 3 potentiation of channels was suppressed by nongenomic VDR antagonist 1,25(OH) 2vitamin D 3 (HL).In particular, the expression of outwardly rectifying ClC-3 channels in TM4 cells, which is sensitive to the specific blocker 4,4  -diisothiocyanatostilbene-2,2 disulfonic acid (DIDS) and has outward rectifying characteristics, was detected at relatively high levels, among the ClC-7 members of the ClCn gene family investigated.Additionally, the agonists/antagonists of PKC and PKA increased and abolished the chloride currents in TM4 cells, respectively [83].So we demonstrated for the first time that nongenomic 1,25-D 3 potentiation of chloride currents is coupled to exocytosis in TM4 Sertoli cells and pointed a functional role for 1,25-D 3 in male fertility via stimulation of secretory activities in the testis.
The plasma membrane effects and rapid responses describe for 1,25-D 3 include the opening of voltagedependent calcium [84] and chloride channels [85] as also demonstrated for the testis [65,83].Zanatta et al. [86] reported that 1,25-D 3 induced calcium uptake in rat testis through a nongenomic mechanism of action mediated, at least in part, by PKA, PKC, and MEK.The ionic influence on the stimulatory effect of 1,25-D 3 in calcium uptake was characterized by ATP-and Ca 2+ -dependent K + channels and Ca 2+ -dependent chloride channels participation in the hormone mechanism of action.Beyond the calcium from the stocks involved, the effect of 1,25-D 3 on calcium uptake may also result from Na + /K + -ATPase pump inhibition.Also, in this study, the action of 1,25-D 3 in an enzyme associated with the plasma membrane of Sertoli cells was demonstrated for the first time.Gamma-glutamyl transpeptidase (GGTP; EC 2.3.2.2) has been detected in several tissues and also in the testis [87].GGTP located at plasma membrane in high concentrations in cells involved in secretory activity [88] and in the testis participates in transferrin secretion from Sertoli cells [89].This enzyme catalyzes the transfer of gamma-glutamyl peptides either to other peptides or to L-amino acids and may play a role in the synthesis of specific proteins known to be secreted by Sertoli cells [89,90].
Particularly, in Sertoli cells, the exposition of 1,25-D 3 or the agonist of VDRmem (JN) produced similar stimulatory effect on calcium uptake suggesting that 1,25-D 3 action occurs via a putative membrane receptor.From these data, the participation of VDCC, PKA, PKC, and ERK activation as well as the influence of the Na + /K + -ATPase inhibition on Na + /Ca 2+ exchanger activation in reverse mode and consequently induction on calcium uptake into the cells were triggered in Sertoli cells after the treatment with 1,25-D 3 , reinforcing once more the hormonal effect at plasma membrane [91,92].
A recent report correlates the mechanism of action of 1,25-D 3 in whole testis and in Sertoli cells from immature rats on calcium uptake.It was characterized that the rapid responses to 1,25-D 3 on calcium uptake in rat Sertoli cells are mediated by VDCCs, PKC, ERK1/2, and p38 MAPK pathways.In addition, the VDCC activities are mandatory for a full stimulatory effect of 1,25-D 3 as much in whole testis as in Sertoli cells.Potassium and chloride channels also are strongly involved in this rapid response coordinated by 1,25-D 3 .Some interesting finds were about the participation of PKC and ERK1/2 upstream activity in p38 MAPK activation that strongly pointed to a possible intracellular cross-talk between rapid calcium uptake and genomic events.Additionally, in Sertoli cells, the comparative effect of colchicine and ClC-3 channel blocker on calcium uptake provides evidence for a secretory activity triggered by 1,25-D 3 .These data highlight that 1,25-D 3 activates p38 MAPK and reorganizes microtubules, involving calcium, PKC, and ERK1/2 as upstream regulators and that extracellular calcium has a central role to rapidly start hormone-induced gene transcription and/or the secretory activity of immature Sertoli cell [93].
Although the calcium increase mediated by 1,25-D 3 in human male gamete has been reported, the role of this ion is unclear [33].1,25-D 3 increases intracellular calcium and sperm motility and induces the acrosome reaction in mature spermatozoa and the serum levels of the hormone are associated with sperm motility.In addition, 1,25-D 3 increases intracellular calcium through VDR and sperm motility in young healthy men but was unable to increase motility in subfertile patients [35,94].
The role of calcium on mechanism of action of 1,25-D 3 in the testis has increased evidences that this secosteroid triggers rapid responses in testicular cells in some significant physiological events.Vimentin is an abundant protein in the intermediate filaments in the cytoplasm of Sertoli cells from immature and adult rat testis [95].Vimentin is a target for the action of FSH, testosterone, thyroid hormones, and also 1,25-D 3 [96][97][98][99].In the testis, the increase of vimentin phosphorylation is mediated by the activation of PKA, PKC, and MAPK but is independent of protein synthesis.Furthermore, the intra-and extracellular calcium are involved in rapid responses of 1,25-D 3 .In addition, there is growing evidence that 1,25-D 3 serves as a primal regulator of ionic channels and also for PKA, PKC, and MAPK as well as aromatase expression in testis and Sertoli cells [73,82,92].However, whether the action triggered by hormone at plasma membrane is independent to mediate rapid responses (exocytosis, enzymes activities) or cross-talk pathways can also culminate in genomic activities remains unclear.
First, using chick duodenal caveolae-enriched membrane fraction (CMF) isolated without the use of detergents to study 1,25-D 3 binding at plasma membrane evidenced the action of 1,25-D 3 on a putative VDRmem [14].Also, as 1,25-D 3 is a secosteroid, the analogues are useful tools to be assayed in ex vivo and in vitro and also lately it has helped to understand the mechanism of action of 1,25-D 3 by in silico approach, since there is the possibility of the hormone binding at VDRmem or at VDRnuc to induce the alteration of cellular biological activity.

Structure/Function and Computational Analysis of 1,25-D 3 Binding in VDR-Genomic Pocket or Alternative Pocket
The flexibility of the secosteroid 1,25-D 3 can occur in three regions of the molecule: in the side chain with 360 ∘ rotation around the 5 carbon-carbon single bonds, in the broken Bring with a 360 ∘ rotation around the 6,7 carbon-carbon single bond, and in the A-ring where a cyclohexane-like chair ↔ chair interconversion occurs which changes the orientation of 1-hydroxyl and 3-hydroxyl between the equatorial and axial orientations [14,15].The rotation around 6,7 single carbon bond permits generation of potential ligand shapes extending from the 6-s-cis (steroid like) to the 6-s-trans (extended).Several evidences reinforce that the preferred shape for VDRmem that triggers rapid responses is that represented by the 6-s-cis conformation and the preferred shape for VDRnuc is the 6-s-trans [13,22,100].So in order to study the preferred shape of the ligands for nongenomic and genomic responses, a number of conformationally restricted analogs of 1,25-D 3 locked in the 6-s-cis or 6-s-trans conformation have been developed [100].
The effectiveness of these analogs to stimulate transcaltachia (a rapid response) or to induce osteocalcin expression in human osteoblast MB-63 cell (a genomic response) in comparison to 1,25-D 3 was measured [2,100,101].The structure-function studies showed that the 6-s-cis analog, 1,25-dihydroxylumisterol3 (JN), can efficiently induce transcaltachia in intestinal epithelium and also stimulate Ca 2+ uptake in osteosarcoma cell line via VDRmem.However, this analog did not activate the genomic action, and it possessed only weak ability to bind to VDRnuc [100].On the other hand, the 1,25-dihydroxyvitamin D3 (HL) blocked rapid responses induced by 1,25-D 3 or JN and was recognized as a specific antagonist of the nongenomic action [102,103].Also the 1,25-D 3 genomic response was blocked by coincubation with the analog (23S)-25-dehydro-1(OH)vitamin D 3 -26,23-lactone (MK), of which antagonistic action is caused by the inhibition of heterodimer formation between VDR and RXR and of VDR interaction with coactivator, steroid receptor coactivator 1 (SRC-1) [104].Taking it in whole, 1,25-D 3 analog effects have demonstrated that the comparison of 6-s-cis-and 6-s-trans-locked analogs of 1,25-D 3 indicates that the 6-s-cis conformation is preferred for rapid nongenomic biological responses and that neither 6-scis-nor 6-s-trans-locked analogs are preferred for genomic biological responses [22,100].The interesting properties of some selected analogs of 1,25-D 3 , after in-depth evaluation of biological activity based on their affinity for the VDRnuc and/or VDRmem, have supported to be very good tool to investigate a genomic pocket (GP) and alternative pocket (AP) that mediate regulation of gene transcription and rapid responses for the hormone, in in silico studies.
Also in the testis, the action of 1,25-D 3 occurs by interaction with both a well characterized VDRnuc to regulate gene transcription and with an as yet uncharacterized membraneassociated protein/receptor (VDRmem) to induce a variety of rapid, nongenotropic responses [58,65,83].The studies of 1,25-D 3 at plasma membrane mediated by VDRmem started in whole testes, TM4 Sertoli cells line, primary culture of Sertoli cells, and germ cells carried out in in vitro studies [92].
Recently, the structure/function and computational analysis suggest that the VDRnuc or an isoform thereof can function as the membrane receptor propagating the rapid effects of 1,25-D 3 in Sertoli cells [105].Following some data about 1,25-D 3 /JN-induced and HL-antagonized, nongenomic responses as chloride channel opening coupled to exocytosis in TM4 Sertoli cells and TM4 Sertoli cells [72], 1,25-D 3 /JN-induced and HL-antagonized, aromatase expression in testicular cells, mediated by rapid responses [58], and also because JN and HL are equipotent with 1,25-D 3 in stimulating (JN) or antagonizing (HL) rapid responses but have 100-fold lower affinities for VDR, Menegaz et al. [105] hypothesized that a novel membrane receptor must exist and serve as the mediator of vitamin D sterol rapid responses.Furthermore, it is in accordance with previous studies that showed a nuclear VDR present in the caveolae-enriched membrane fraction from human, rodent, and chick tissues [14,106].So the studies carried out by Menegaz et al. [105] postulated that the VDR contains two overlapping ligand binding sites, a GP and an AP, that mediate regulation of gene transcription and rapid responses, respectively.The authors showed that the flexible VDR ligand docking calculations predict that the major blood metabolite, 25D 3 , and curcumin (CM) bind more selectively to the VDR-AP when compared with the 1,25-D 3 .In VDR wild-type-transfected COS-1 cells and TM4 Sertoli cells, 1,25-D 3 , 25D 3 , and CM each trigger voltage-gated outwardly rectifying chloride channel (ORCC) currents that can be blocked by the VDR antagonist 1,25(OH) 2 -vitamin D 3 and the chloride channel antagonist (4,4  -diisothiocyanatostilbene-2,2  -disulfonic acid).In addition, VDR mutational analysis in transfected COS-1 cells demonstrates that the DNA-binding domain is not required, but the ligand binding and hinge domains of the VDR are required, for 1,25-D 3 and 25D 3 to activate the ORCC.Through dose-response curves it was demonstrated that 25D 3 and 1,25-D 3 are approximately equipotent in stimulating ORCC rapid responses, whereas 1,25-D 3 was 1000-fold more potent than 25D 3 and CM in stimulating gene expression.The VDR-AP agonist effects of 1,25-D 3 , 25D 3 , and low-dose CM are lost after pretreatment of TM4 cells with VDR small interfering RNA.Collectively, these results are consistent with an essential role for the VDR-AP in initiating the signalling required for rapid opening of ORCC.The fact that 25D 3 is equipotent to 1,25-D 3 in opening ORCC suggests that reconsideration of the ability of 25D 3 to generate biological responses in vivo may be in order.The demonstration that 1,25-D 3 regulates both Sertoli cell and sperm function may be useful for the study and development of new therapeutic strategies to the treatment of male reproductive disorders.

Conclusion
The spermatogenesis and steroidogenesis are the main functions of the testis and both are tightly regulated by endogenous and exogenous factors that can result in success of spermatogenesis or in male infertility.In contrast to steroids, the secosteroid 1,25-D 3 is able to achieve an interconversion to the 6-s-cis conformation referred to as steroidlike conformation with preferred binding at VDRmem and also display mobility to 6-s-trans conformation referred to as extended conformation with preferred binding at VDRnuc.The hormonally active form of vitamin D, 1,25-D 3 , is a pivotal hormone to maintain basic cellular processes through transcriptional events mediated by VDRnuc or rapid responses triggered by a putative plasma membrane receptor designated as VDRmem.A multitude of reports describe the testis as a target for 1,25-D 3 since both VDR and enzymes that metabolize vitamin D are present in rodents and in human.However, the upcoming studies should clarify the physiological relevance of autocrine and paracrine action of 1,25-D 3 and also the cross-talking between VDRnuc and VDRmem pathways to complete or keep an active ongoing spermatogenic wave and male fertility.Based on the existence of a VDR AP and VDR GP that mediate regulation of gene transcription (by 6-s-trans 1,25-D 3 form) and rapid responses (by 6-s-cis 1,25-D 3 form) on signalling cascades stimulated by 1,25-D 3 , the in silico model may provide a useful platform for drug development through docking studies of new or known therapeutic drugs for the reproductive disorders based on the endocrine system of vitamin D. In addition, we have used electrophysiology modeling, molecular biology, and biochemistry to characterize strategic sites of action for drug target for further therapy in male infertility.Also, it is worthwhile to pay attention that some proteins known in the vitamin D metabolism or in the signalling cascade mediated by VDRmem or VDRnuc may be of clinical interest even for the male infertility diagnosis.