Venlafaxine increases aromatization, reduces apical V-ATPase in clear cells and induces increased number of mast cells and smooth muscle cells death in rat cauda epididymis.

Depressive disorders (DD) have affected millions of people worldwide. Venlafaxine, antidepressant of the class of serotonin and norepinephrine reuptake inhibitors, has been prescribed for the treatment of DD. In rat testes, venlafaxine induces testosterone (T) aromatization and increases estrogen levels. Aromatase is a key enzyme for the formation of estrogen in the epididymis, an essential organ for male fertility. We investigated the impact of serotonergic/noradrenergic venlafaxine effect on the epididymal cauda region, focusing on aromatase, V-ATPase and EGF epithelial immunoexpression, smooth muscle (SM) integrity and mast cells number (MCN). Male rats were distributed into control (CG; n = 10) and venlafaxine (VFG, n = 10) groups. VFG received 30 mg/kg b.w. of venlafaxine for 35 days. The epididymal cauda was processed for light and transmission electron microscopy (TEM). The expression of connexin 43 (Cx43) and estrogen alpha (Esr1), adrenergic (Adra1a) and serotonergic (Htr1b) receptors were analyzed. Clear cells (CCs) area, SM thickness, viable spermatozoa (VS) and MCN were evaluated. Apoptosis was confirmed by TUNEL and TEM. The following immunoreactions were performed: T, aromatase, T/aromatase co-localization, V-ATPase, EGF, Cx43 and PCNA. The increased Adra1a and reduced Htr1b expressions confirmed the noradrenergic and serotonergic venlafaxine effects, respectively, corroborating the increased MCN, apoptosis and atrophy of SM. In VFG, the epithelial EGF increased, explaining Cx43 overexpression and basal cells mitotic activity. T aromatization and Esr1 downregulation indicate high estrogen levels, explaining CCs hypertrophy and changes in the V-ATPase localization, corroborating VS reduction. Thus, in addition to serotonergic/noradrenergic effects, T/estrogen imbalance, induced by venlafaxine, impairs epididymal structure and function.

Estrogens and development of the rete testis, efferent ductules, epididymis and vas deferens.

Estrogen has always been considered the female hormone and testosterone the male hormone. However, estrogen's presence in the testis and deleterious effects of estrogen treatment during development have been known for nearly 90 years, long before estrogen receptors (ESRs) were discovered. Eventually it was learned that testes actually synthesize high levels of estradiol (E2) and sequester high concentrations in the reproductive tract lumen, which seems contradictory to the overwhelming number of studies showing reproductive pathology following exogenous estrogen exposures. For too long, the developmental pathology of estrogen has dominated our thinking, even resulting in the "estrogen hypothesis" as related to the testicular dysgenesis syndrome. However, these early studies and the development of an Esr1 knockout mouse led to a deluge of research into estrogen's potential role in and disruption of development and function of the male reproductive system. What is new is that estrogen action in the male cannot be divorced from that of androgen. This paper presents what is known about components of the estrogen pathway, including its synthesis and target receptors, and the need to achieve a balance between androgen- and estrogen-action in male reproductive tract differentiation and adult functions. The review focuses on what is known regarding development of the male reproductive tract, from the rete testis to the vas deferens, and examines the expression of estrogen receptors and presence of aromatase in the male reproductive system, traces the evidence provided by estrogen-associated knockout and transgenic animal models and discusses the effects of fetal and postnatal exposures to estrogens. Hopefully, there will be enough here to stimulate discussions and new investigations of the androgen:estrogen balance that seems to be essential for development of the male reproductive tract.

Hippo kinases MST1 and MST2 control the differentiation of the epididymal initial segment via the MEK-ERK pathway.

Although the roles of the Hippo pathway in organogenesis and tumorigenesis have been well studied in multiple organs, its role in sperm maturation and male fertility has not been investigated. The initial segment (IS) of the epididymis plays a critical role in sperm maturation. IS differentiation is governed by ERK1/2, but the mechanisms of ERK1/2 activation in IS are not fully understood. Here we show that double knockout (dKO) of mammalian sterile 20-like kinases 1 and 2 (Mst1 and Mst2), homologs of Hippo in Drosophila, in the epididymal epithelium led to male infertility in mice. Sperm in the cauda epididymides of mutant mice were immotile with flagellar angulation and severely disorganized structures. Loss of Mst1/2 activated YAP and increased proliferation and cell death in all the segments of epididymis. The mutant mice showed substantially suppressed MEK/ERK signaling in the IS and failed IS differentiation. Deletion of Yap restored the reduced MEK/ERK signaling, and partially rescued the defective IS differentiation and fertility in Mst1/2 dKO mice. Our results demonstrate that YAP inhibits the MEK/ERK pathway in IS epithelial cells, and MST1/2 control IS differentiation and fertility at least partially by repressing YAP. Taken together, the Hippo pathway is essential for sperm maturation and male fertility.

Maternal Low-Protein Diet Impairs Prostate Growth in Young Rat Offspring and Induces Prostate Carcinogenesis With Aging.

Carcinogenesis is frequently linked to genetic background, however, exposure to environmental risk factors has gained attention as the etiologic agent for several types of cancer, including prostate. The intrauterine microenvironment has been described as a preponderant factor for offspring health; and maternal exposure to insult has been linked to chronic disease in older offspring. Using a model of maternal exposure to low-protein diet (LPD; 6% protein), we demonstrated that impairment of offspring rat prostatic growth on postnatal day (PND) 21 was associated with prostate carcinogenesis in older offspring (PND 540). One explanation is that maternal LPD consumption exposed offspring to an estrogenic intrauterine microenvironment, which potentially sensitized prostate cells early during glandular morphogenesis, increasing cellular response to estrogen in older rats. The onset of accelerated prostatic growth, observed on PND 21, associated with an unbalanced estrogen/testosterone ratio and increased circulating IGF-1 in older offspring appears to contribute to the development of prostate carcinoma in groups on gestational low protein and gestational and lactational low protein diets (33 and 50%, respectively). Our study strongly indicated maternal exposure to LPD as a potential risk factor for induction of slow-growing prostate carcinogenesis in rat offspring later in life.

Reduced levels of stromal sex hormone-binding globulin and androgen receptor dysfunction in the sperm storage region of the rat epididymis.

The cauda epididymidis is the major sperm storage region whose androgenic supply, essential for the sperm viability, is provided by the vasculature and is dependent upon testosterone diffusion through the stromal tissue to reach the epithelial cells. We have focused our efforts on examining the regulation of this important epididymal region by evaluating the impact of the androgen disrupter cimetidine on the epithelial-stromal androgenic microenvironment. Male rats received 100 mg/kg cimetidine (CMTG) or saline (CG) for 50 days, serum testosterone levels were measured and the epididymal cauda region was processed for light and transmission electron microscopy. In the proximal cauda region, the duct diameter was measured and birefringent collagen in the stroma was quantified. TUNEL-labeled epithelial cells were quantified, and androgen receptor (AR), karyopherin alpha (KPNA) and sex hormone-binding globulin (SHBG) levels were analyzed by immunofluorescence and Western blot. CMTG showed reduced duct diameter and high number of apoptotic epithelial cells. In the epithelium, the total AR concentration and the KPNA immunoreactivity were reduced, and a weak/absent AR nuclear immunofluorescence was observed in contrast to the enhanced AR immunolabeling observed in the cytoplasm of the epithelial cells. A significant reduction of collagen and SHBG levels in the stroma was also observed. Cimetidine treatment impairs AR nuclear import in the epithelium, causing androgenic dysfunction and subsequent epithelial cell apoptosis and duct atrophy. The connective tissue atrophy and reduction of SHBG stromal levels associated with epithelial androgenic dysfunction indicate a possible role of stromal SHBG in the androgenic supply of the sperm storage region of the epididymis.

Alteration of transporter activities in the epididymides of infertile initial segment-specific Pten knockout mice.

A fully functional initial segment, the most proximal region of the epididymis, is important for male fertility. Our previous study generated a mouse model to investigate the importance of initial segment function in male fertility. In that model, phosphatase and tensin homolog (Pten) was conditionally removed from the initial segment epithelium, which resulted in epithelial de-differentiation. When spermatozoa progressed through the de-differentiated epithelial duct, they developed angled flagella, suggesting compromised sperm maturation, which eventually resulted in male infertility. To understand the molecular mechanisms, by which PTEN regulates epididymal sperm maturation, we compared the transcriptome profile of the initial segment between controls and initial segment-specific Pten knockouts and revealed that water, ion, and organic solute transporter activities were one of the top molecular and cellular functions altered following loss of Pten. Alteration in protein levels and localization of several transporters following loss of Pten were also observed by immunofluorescence analysis. Epithelial cells of the initial segment from knockouts were more permeable to fluorescein isothiocyanate-dextran (4000 Da) compared to controls. Interestingly, conditional deletion of Pten from other organs also resulted in changes in transporter activity, suggesting a common role of PTEN in regulation of transporter activity. Taken together, our data support the hypothesis that loss of Pten from the initial segment epithelium results in changes in the transporting and permeability characteristics of the epithelium, which in turn altered the luminal fluid microenvironment that is so important for sperm maturation and male fertility.

Protein tyrosine kinase 7 regulates extracellular matrix integrity and mesenchymal intracellular RAC1 and myosin II activities during Wolffian duct morphogenesis.

Wolffian duct morphogenesis must be highly coordinated with its specialized function of providing an optimal microenvironment for sperm maturation. Without normal Wolffian duct morphogenesis, male infertility will result. Our previous study showed that mediolateral and radial intercalation of epithelial and mesenchymal cells respectively, were major drivers of ductal elongation and were regulated by protein tyrosine kinase 7 (PTK7), a member of the planar cell polarity (PCP) non-canonical Wnt pathway. To understand the mechanism by which PTK7 regulates cell rearrangement/intercalation, we investigated the integrity of the extracellular matrix (ECM) and the activity of intracellular cytoskeleton mediators following loss of Ptk7. Abnormal assembly of nephronectin, laminin, and collagen IV at the basement membrane and fibrosis-like deposition of fibrilla collagen in the interstitium were observed in Ptk7 knockout Wolffian ducts. Further, the activity levels of RAC1 and myosin II, two cytoskeleton mediators, decreased in the Ptk7 knockout mesenchyme compared to controls. In addition, in-vitro experiments suggested that alterations of ECM and cytoskeleton mediators resulted in changes in Wolffian duct morphogenesis. When in-vitro-cultured Wolffian ducts were treated with collagenase IV, the degree of cross-linked fibrilla collagen was reduced, Wolffian duct elongation and coiling were significantly reduced, and an expanded cyst-like duct was observed. When Wolffian ducts were treated with RAC1 inhibitor NSC23766, mesenchymal fibrilla collagen was disassembled, and Wolffian duct elongation was significantly reduced. Our findings provide evidence that PTK7 regulates ECM integrity and the activity levels of RAC1 and myosin II, which in turn regulates Wolffian duct morphogenesis and therefore, epididymal function.

Initial Segment Differentiation Begins During a Critical Window and Is Dependent upon Lumicrine Factors and SRC Proto-Oncogene (SRC) in the Mouse.

Without a fully developed and functioning initial segment, the most proximal region of the epididymis, male infertility results. Therefore, it is important to understand the development of the initial segment. During postnatal development of the epididymis, many cellular processes of the initial segment are regulated by lumicrine factors, which are produced by the testis and enter the epididymis with testicular luminal fluid. In this report, we showed that prior to Postnatal Day 15 (P15), the initial segment was lumicrine factor independent in the mouse. However, from P19 onward, lumicrine factors were essential for the proliferation and survival of initial segment epithelial cells. Therefore, P15 to P19 was a critical window that established the dependency of lumicrine factors in the initial segment epithelium. The initial segment-specific kinase activity profile, a marker of initial segment differentiation, was also established during this window. The SFK (SRC proto-oncogene family kinases), ERK pathway (known as the RAF/MEK/ERK pathway) components, and AMPK (AMP-activated protein kinases) pathway components had increased activities from P15 to P19, suggesting that lumicrine factors regulated SFK/ERK/AMPK signaling to initiate differentiation of the initial segment from P15 to P19. Compared with litter mate controls, juvenile Src null mice displayed lower levels of MAPK3/1 (mitogen-activated protein kinase 3/1) activity and a reduced level of differentiation in the initial segment epithelium, a similar phenotype resulting from inhibition of SRC activity within the window of P15 to P19. Therefore, lumicrine factor-dependent SRC activity signaling through MAPK3/1 is important for the initiation of initial segment differentiation during a critical window of development.

Protein tyrosine kinase 7 is essential for tubular morphogenesis of the Wolffian duct.

The Wolffian duct, the proximal end of the mesonephric duct, undergoes non-branching morphogenesis to achieve an optimal length and size for sperm maturation. It is important to examine the mechanisms by which the developing mouse Wolffian duct elongates and coils for without proper morphogenesis, male infertility will result. Here we show that highly proliferative epithelial cells divide in a random orientation relative to the elongation axis in the developing Wolffian duct. Convergent extension (CE)-like of cell rearrangements is required for elongating the duct while maintaining a relatively unchanged duct diameter. The Wolffian duct epithelium is planar polarized, which is characterized by oriented cell elongation, oriented cell rearrangements, and polarized activity of regulatory light chain of myosin II. Conditional deletion of protein tyrosine kinase 7 (PTK7), a regulator of planar cell polarity (PCP), from mesoderm results in loss of the PCP characteristics in the Wolffian duct epithelium. Although loss of Ptk7 does not alter cell proliferation or division orientation, it affects CE and leads to the duct with significantly shortened length, increased diameter, and reduced coiling, which eventually results in loss of sperm motility, a key component of sperm maturation. In vitro experiments utilizing inhibitors of myosin II results in reduced elongation and coiling, similar to the phenotype of Ptk7 knockout. This data suggest that PTK7 signaling through myosin II regulates PCP, which in turn ensures CE-like of cell rearrangements to drive elongation and coiling of the Wolffian duct. Therefore, PTK7 is essential for Wolffian duct morphogenesis and male fertility.

PTEN signaling through RAF1 proto-oncogene serine/threonine kinase (RAF1)/ERK in the epididymis is essential for male fertility.

Without a fully developed initial segment, the most proximal region of the epididymis, male infertility results. Therefore, it is important to understand the development and regulation of this crucial region. In addition to distinctively high activity levels of the components of the ERK pathway, which are essential for initial-segment differentiation, the initial segment exhibits high protein and activity levels of phosphatase and tensin homolog (PTEN). To understand the role of PTEN in the regulation of the initial segment, we generated a mouse model with a conditional deletion of Pten from the epithelial cells of the proximal epididymis from postnatal day 17 (P17) onward. Shortly after Pten deletion, hypertrophy of the proximal epididymis became evident. Loss of Pten resulted in activation of the AKT (protein kinase B) pathway components from P28 onward, which in turn gradually suppressed RAF1 proto-oncogene serine/threonine kinase (RAF1)/ERK signaling through the interaction between AKT and RAF1. Consistent with progressive changes in RAF1/ERK signaling, loss of Pten progressively altered cell shape, size, organization, proliferation, and survival in the initial-segment epithelium and resulted in dedifferentiation and extensive epithelial folding. Most importantly, knockout males progressively lost fertility and became infertile from 6 to 12 mo. Spermatozoa from older knockout mice showed a lower percentage of motility and a higher percentage of flagellar angulation compared with controls, suggesting compromised sperm maturation. Therefore, under normal physiological conditions, PTEN suppresses AKT activity to maintain activation of the RAF1/ERK signaling pathway, which in turn maintains normal function of the initial segment and therefore, normal sperm maturation.

The Role of fibroblast growth factor receptor substrate 2 (FRS2) in the regulation of two activity levels of the components of the extracellular signal-regulated kinase (ERK) pathway in the mouse epididymis.

The components of the extracellular signal-regulated kinase (ERK) pathway are involved in the regulation of epididymal cellular processes. Interestingly, our previous studies showed that there are two different activity levels of the ERK pathway components in the epididymal epithelium: a basal level in most regions and a higher level in the differentiated initial segment (IS). In this study we analyzed the role of fibroblast growth factor receptor substrate 2 (FRS2) in the regulation of these two levels. Two mouse models were generated. In the first model, Frs2 was deleted from epithelial cells of most epididymal regions except for the IS from the embryonic period onward. Loss of Frs2 dampened the basal activity level of the ERK pathway components, which resulted in an increase in apoptosis along the epididymal duct. This was observed during the period when FRS2 expression level was highest in wild-type epididymides. In the second model, Frs2 was deleted from the proximal epididymal epithelium from Postnatal Day 17 onward. Most of the epididymides in this model exhibited normal morphology. Loss of Frs2 in these epididymides did not affect the high activity level of the ERK pathway components in the IS. However, a subgroup of epididymides in the second model showed increased apoptosis which resulted in an abnormally shaped proximal region or development of granulomas. Therefore, data from these two models showed that FRS2 played different roles in the regulation of two activity levels of the ERK pathway components in the epididymis.

p-MAPK1/3 and DUSP6 regulate epididymal cell proliferation and survival in a region-specific manner in mice.

A fully developed, functional epididymis is important for male fertility. In particular, it is apparent that without the most proximal region, the initial segment (IS), infertility results. Therefore, it is important to understand the development and regulation of this crucial epididymal region. We have previously shown that many functions of the IS are regulated by luminal fluid factors/lumicrine factors from the testis. This study provides evidence that lumicrine factors activated the ERK pathway only in epithelial cells of the IS from Postnatal Day (P) 14 to P19 and sustained this activation into adulthood. The activated ERK pathway promoted cell proliferation and differentiation in the developing IS, although in the adult, its role was switched to maintain cell survival. To understand further the regulation of cell proliferation in the IS, we examined the role of DUSP6, an MAPK1/3 (ERK1/2) preferred phosphatase that is also regulated by lumicrine factors in the IS. Utilizing Dusp6(-/-) mice, our studies, surprisingly, revealed that Dusp6 was a major regulator of cell proliferation in the caput and corpus regions, whereas components of the ERK pathway, together with PTEN and SRC, were the major regulators of cell proliferation in the IS. We hypothesize that region-specific regulation of cell proliferation is caused by differences in the balance of activities between pro- and antiproliferation signaling pathway components for each epididymal region. An understanding of the mechanisms of cell proliferation may provide clues as to why the epididymis rarely succumbs to cancer.

Cellular signaling by fibroblast growth factors (FGFs) and their receptors (FGFRs) in male reproduction.

The major function of the reproductive system is to ensure the survival of the species by passing on hereditary traits from one generation to the next. This is accomplished through the production of gametes and the generation of hormones that function in the maturation and regulation of the reproductive system. It is well established that normal development and function of the male reproductive system is mediated by endocrine and paracrine signaling pathways. Fibroblast growth factors (FGFs), their receptors (FGFRs), and signaling cascades have been implicated in a diverse range of cellular processes including: proliferation, apoptosis, cell survival, chemotaxis, cell adhesion, motility, and differentiation. The maintenance and regulation of correct FGF signaling is evident from human and mouse genetic studies which demonstrate that mutations leading to disruption of FGF signaling cause a variety of developmental disorders including dominant skeletal diseases, infertility, and cancer. Over the course of this review, we will provide evidence for differential expression of FGFs/FGFRs in the testis, male germ cells, the epididymis, the seminal vesicle, and the prostate. We will show that this signaling cascade has an important role in sperm development and maturation. Furthermore, we will demonstrate that FGF/FGFR signaling is essential for normal epididymal function and prostate development. To this end, we will provide evidence for the involvement of the FGF signaling system in the regulation and maintenance of the male reproductive system.

Putative regulation of expression of members of the Ets variant 4 transcription factor family and their downstream targets in the rat epididymis.

Several genes expressed in the initial segment of the epididymis depend on factors from the testis that reach the epididymis via the luminal system. These include gamma-glutamyl transpeptidase mRNA IV (Ggt_pr4), steroid 5 alpha reductase (Srd5a1), glutathione peroxidase 5 (Gpx5), and cystatin-related epididymal spermatogenic (Cst8) genes. Promoter analyses indicated that these genes contain several ETS DNA-binding sites. Members of the polyomavirus enhancer activator 3 (ETV4) family bind to ETS sites on the promoter of target genes to regulate transcription. In this study, the role of ETV4 family members (ETV4, ETV5, ETV1) in the transcription of initial segment specific genes was evaluated. All three ETV4 family mRNAs are expressed in the principal cells of the initial segment and depend upon the presence of testicular luminal fluid factors. ETV4 protein was localized to principal cell nuclei and displayed the highest expression in the most proximal region of the initial segment. In addition, ETV4 protein levels were diminished after loss of testicular luminal fluid factors. A dominant-negative construct of ETV5 was in vivo electroporated into the initial segment to determine if ETV4 family members can regulate the transcription of testicular luminal fluid factor-regulated genes. Quantitative PCR indicated that 1 day postelectroporation, all three ETV4 family member mRNAs were significantly decreased. In addition, Ggt_pr4, Srd5a1, and Gpx5 mRNA levels were also significantly decreased. The data suggest that ETV4 family members regulate their own expression, and that they regulate transcription of a subset of genes that are dependent upon testicular luminal fluid factors.

Characterization of epididymal epithelial cell-specific gene promoters by in vivo electroporation.

The mammalian epididymis plays a critical role in sperm maturation, a function dependent on testicular androgens. However, the function of the initial segment, the most proximal part of the epididymis, is also dependent on luminal factors of testicular origin. Efferent duct ligation (EDL), which prevents luminal testicular fluid from reaching the epididymis, results in changes in gene expression within this region. Cystatin-related epididymal spermatogenic (cres) gene and gamma-glutamyl transpeptidase (GGT) mRNA IV are highly expressed in the initial segment and are regulated by luminal testicular factors. EDL results in decreased expression of both genes. To evaluate these promoters in the context of their native physiological state, an in vivo electroporation procedure was used. Significant differences were observed in vivo compared to previous in vitro results. Whereas two C/EBP sites were necessary for transcriptional activity from a 135-base-pair (bp) cres promoter in vitro, only the 5' site displayed functional activity in the in vivo system. A 135-bp GGT promoter IV construct was sufficient for reporter gene expression in vitro. However, in vivo, substantial expression was not observed until the construct was extended to 530 bp. Three polyoma enhancer activator 3 (PEA3) sites were found to be necessary for in vivo reporter gene expression from this construct. A cis-acting negative regulatory element between -530 and -681 bp was also identified that was not previously recognized in the in vitro studies. These studies demonstrate the utility of in vivo electroporation for elucidating promoter elements that may not be identified when traditional in vitro methods are used.

5' hox genes and meis 1, a hox-DNA binding cofactor, are expressed in the adult mouse epididymis.

Hox genes determine the formation of segmented structures during development. The epididymis shows a segmented organization in its structure and function beyond embryogenesis. This study examined the adult mouse epididymis and vas deferens for expression of 5' hox genes and a hox-DNA binding cofactor. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed the expression of hoxa-9, hoxa-10, hoxa-11, hoxd-9, and hoxd-10 in all regions including the vas deferens. Semiquantitative RT-PCR revealed highest mRNA levels for hoxa-11 in the distal part of the epididymis and vas deferens, and this was confirmed by Northern blot analysis. To determine protein presence an antibody raised against a peptide N-terminal to the homeodomain of hoxa-11 was produced in rabbits. The antibody recognized a band of approximately 37-39 kDa in Western blot analysis. Immunohistochemistry indicated the presence of hoxa-11 in the nuclei of the epithelial cells with some staining in the cytoplasm. Staining was also detected in nuclei of interstitial cells throughout the entire organ and the vas deferens. A DNA binding cofactor for hoxa-11, Meis 1, was investigated for its presence in the epididymis. Semiquantitative RT-PCR identified both transcripts for Meis 1 (Meis 1a and Meis 1b) in all regions. Protein presence was confirmed by Western blot analysis, and this detected one band of approximately 53-55 kDa. Immunohistochemistry localized Meis 1 in the nuclei of interstitial cells throughout the entire organ and the vas deferens. Our study provides preliminary data from which we suggest the involvement of homeodomain transcription factors in the maintenance of segmental function of the adult epididymis and vas deferens.