A Step Forward in Understanding the Expression of Classical Aquaporins in the Male Reproductive Tract: Study Findings in Cattle (Bos taurus).

Aquaporins (AQPs), also known as water channels, appear to be particularly promising in maintaining male reproductive potential. Therefore, this study aimed to determine the presence of classical AQPs in the bovine (Bos taurus) reproductive system and analyze changes in their expression with age using immunohistochemistry and Western blotting. Of the six classical AQPs, AQP0, AQP1, AQP4, AQP5 and AQP6 were detected, while AQP2 was absent. In the testis, AQP0 was visible in Leydig cells in selected animals, while AQP1 was found in myoid cells surrounding the seminiferous tubules of mature individuals. This characteristic expression patterns of AQP0, limited only to certain bulls, is difficult to explain unequivocally. It is possible that AQP0 expression in cattle is subject to individual variability or changes in response to specific physiological conditions. In the caput and corpus epididymis, AQP0 showed weak expression in epithelial cells of immature animals and stronger expression in basal and principal cells of reproductive bulls. In all animals, AQP1 was present on the apical surface of epithelial cells in the initial segment of the caput epididymis. AQP4, AQP5 and AQP6 were identified in principal and basal cells along the entire epididymis of reproductive bulls. The abundance of AQP4 and AQP6 increased from the caput to the cauda epididymis with the growth and development of the animals. In all males, AQP4, AQP5 and AQP6 were observed in epithelial cells of the vas deferens, and their expression in this section increased with age. In conclusion, the abundance and distribution of the classical AQPs in various cell types and parts of the male reproductive system indicate their crucial role in maintaining water homeostasis, which is essential for normal reproductive function in cattle.

Rhox8 homeobox gene ablation leads to rete testis abnormality and male subfertility in mice†.

The reproductive homeobox X-linked (Rhox) genes encode transcription factors that are expressed selectively in reproductive tissues including the testis, epididymis, ovary, and placenta. While many Rhox genes are expressed in germ cells in the mouse testis, only Rhox8 is expressed exclusively in the Sertoli cells during embryonic and postnatal development, suggesting a possible role of Rhox8 in embryonic gonad development. Previously, Sertoli cell-specific knockdown of RHOX8 resulted in male subfertility due to germ cell defects. However, this knockdown model was limited in examining the functions of Rhox8 as RHOX8 knockdown occurred only postnatally, and there was still residual RHOX8 in the testis. In this study, we generated new Rhox8 knockout (KO) mice using the CRISPR/Cas9 system. Sex determination and fetal testis development were apparently normal in mutant mice. Fertility analysis showed a low fecundity in Rhox8 KO adult males, with disrupted spermatogenic cycles, increased germ cell apoptosis, and reduced sperm count and motility. Interestingly, Rhox8 KO testes showed an increase in testis size with dilated seminiferous tubules and rete testis, which might be affected by efferent duct (ED) Rhox8 ablation dysregulating the expression of metabolism and transport genes in the EDs. Taken together, the data presented in this study suggest that Rhox8 in the Sertoli cells is not essential for sex determination and embryonic testis differentiation but has an important role in complete spermatogenesis and optimal male fertility.

Defects in efferent duct multiciliogenesis underlie male infertility in GEMC1-, MCIDAS- or CCNO-deficient mice.

GEMC1 and MCIDAS are geminin family proteins that transcriptionally activate E2F4/5-target genes during multiciliogenesis, including Foxj1 and Ccno Male mice that lacked Gemc1, Mcidas or Ccno were found to be infertile, but the origin of this defect has remained unclear. Here, we show that all three genes are necessary for the generation of functional multiciliated cells in the efferent ducts that are required for spermatozoa to enter the epididymis. In mice that are mutant for Gemc1, Mcidas or Ccno, we observed a similar spectrum of phenotypes, including thinning of the seminiferous tubule epithelia, dilation of the rete testes, sperm agglutinations in the efferent ducts and lack of spermatozoa in the epididymis (azoospermia). These data suggest that defective efferent duct development is the dominant cause of male infertility in these mouse models, and this likely extends to individuals with the ciliopathy reduced generation of multiple motile cilia with mutations in MCIDAS and CCNO.

Role of nuclear and membrane estrogen signaling pathways in the male and female reproductive tract.

Estrogen signaling through the main estrogen receptor, estrogen receptor 1 (ESR1; also known as ERα), is essential for normal female and male reproductive function. Historically, studies of estrogen action have focused on the classical genomic pathway. Although this is clearly the major pathway for steroid hormone actions, these hormones also signal through rapid non-classical effects involving cell membrane actions. Reports of rapid effects of estrogens extend for more than half a century, but recent results have expanded understanding of the identity, structure, function and overall importance of membrane receptors in estrogen responses. Key findings in this field were the immunohistochemical detection of ESR1 in cell membranes and demonstration that a portion of newly synthesized ESR1 is routed to the membrane by palmitoylation. These receptors in the membrane can then signal through protein kinases and other mechanisms following ligand binding to alter cell function. Another crucial advance in the field was development of transgenic mice expressing normal amounts of functional nuclear ESR1 (nESR1) but lacking membrane ESR1 (mESR1). Both male and female transgenic mice lacking mESR1 were infertile as adults, and both sexes had extensive reproductive abnormalities. Transgenic mice lacking mESR1 were highly protected from deleterious effects of neonatal estrogen administration, and estrogen effects on the histone methyltransferase Enhancer of Zeste homolog 2 that are mediated through mESR1 could have significant effects on epigenetic imprinting. In summary, signaling through mESR1 is essential for normal male and female reproductive function and fertility, and is a critical enabler of normal estrogen responses in vivo. Although the precise role of mESR1 in estrogen responses remains to be established, future research in this area should clarify its mechanism of action and lead to a better understanding of how mESR1 signaling works with classical genomic signaling through nESR1 to promote full estrogenic responses.

Motility of efferent duct cilia aids passage of sperm cells through the male reproductive system.

Motile cilia line the efferent ducts of the mammalian male reproductive tract. Several recent mouse studies have demonstrated that a reduced generation of multiple motile cilia in efferent ducts is associated with obstructive oligozoospermia and fertility issues. However, the sole impact of efferent duct cilia dysmotility on male infertility has not been studied so far either in mice or human. Using video microscopy, histological- and ultrastructural analyses, we examined male reproductive tracts of mice deficient for the axonemal motor protein DNAH5: this defect exclusively disrupts the outer dynein arm (ODA) composition of motile cilia but not the ODA composition and motility of sperm flagella. These mice have immotile efferent duct cilia that lack ODAs, which are essential for ciliary beat generation. Furthermore, they show accumulation of sperm in the efferent duct. Notably, the ultrastructure and motility of sperm from these males are unaffected. Likewise, human individuals with loss-of-function DNAH5 mutations present with reduced sperm count in the ejaculate (oligozoospermia) and dilatations of the epididymal head but normal sperm motility, similar to DNAH5 deficient mice. The findings of this translational study demonstrate, in both mice and men, that efferent duct ciliary motility is important for male reproductive fitness and uncovers a novel pathomechanism distinct from primary defects of sperm motility (asthenozoospermia). If future work can identify environmental factors or defects in genes other than DNAH5 that cause efferent duct cilia dysmotility, this will help unravel other causes of oligozoospermia and may influence future practices in genetic and fertility counseling as well as ART.

Bi-allelic mutations in MCIDAS and CCNO cause human infertility associated with abnormal gamete transport.

Reduced generation of multiple motile cilia (RGMC) and the consequent primary ciliary dyskinesia (PCD) cause infertility due to a substantial reduction in the number of multiciliated cells (MCCs) in the efferent ducts (EDs)/oviducts. MCIDAS acts upstream of CCNO to regulate the biogenesis of basal bodies (BBs); therefore, both genes play a vital role in the multiciliogenesis of the reproductive tract epithelium. In this study, whole-exome sequencing was performed to identify the causative genes in 10 unrelated infertile patients with PCD: seven males and three females. Notably, homozygous frameshift mutations in MCIDAS (c.186dupT, p.Pro63Serfs*22) and CCNO (c.262_263insGGCCC, p.Gln88Argfs*8) were identified in one male and one female participant from two unrelated consanguineous families. Haematoxylin-eosin staining/scanning electron microscopy revealed abnormal MCCs in the mutated EDs/oviducts. Furthermore, transmission electron microscopy revealed significantly reduced BBs. Immunofluorescence staining showed the absence of MCIDAS and CCNO signals in the affected tissues and confirmed that MCIDAS acts upstream of CCNO in the context of multiciliogenesis in the reproductive tract epithelium. In vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) was successful, with a positive pregnancy outcome in both MCIDAS- and CCNO-mutated patients. Our results support the use of IVF/ICSI interventions to treat infertility due to RGMC in couples.

Three-dimensional structure of efferent and epididymal ducts in mice.

The aim of the present study was to clarify the detailed morphology of efferent and epididymal ducts in adult mice using three-dimensional (3D) analysis. We reconstructed efferent and epididymal ducts in three adult mice using serial paraffin sections and high-performance 3D reconstruction software to draw the core lines of all ducts. By comparing the 3D core lines with the histological features in serial sections, we obtained detailed information on the gross characteristics of the ducts and identified the duct divisions accurately. The intra-testicular rete testis penetrated the tunica albuginea at one place and turned into the extra-testicular rete testis, which branched once or twice to give rise to four efferent ducts within 0.5 mm from the tunica albuginea. As these ducts approached the epididymis, they converged into one again and changed abruptly into the initial segment (IS) of the epididymis. The average length from the tunica albuginea to the IS was 19.7 ± 3.1 mm. In one mouse, we found four additional efferent ducts diverging from the common region with blind ends. The epididymal duct was a single highly convoluted duct with no branch and an average length of 767 ± 26 mm. By dividing the epididymal duct into five regions based on its cytological features and periodic acid-Schiff stainability, we calculated the length and diameter of individual regions accurately. Furthermore, we clearly showed locations of the connective tissue septa that divide the head epididymis into several segments. The epididymal duct followed a complicated, winding path within each segment while drawing a large spiral overall along the circumference of the epididymis. Sometimes the direction of this spiral reversed between adjacent segments. The present study revealed the detailed 3D structures of efferent and epididymal ducts in adult mice.

Three-dimensional analysis of junctions between efferent and epididymal ducts in the human caput epididymis.

BACKGROUND: Due to the scarcity of studies using human tissues, the limited information is currently available on the gross structure of the caput epididymis in humans, at which efferent ducts connect to the epididymal duct. OBJECTIVE: The present study investigated the three-dimensional structures of efferent and caput epididymal ducts in humans, with a focus on junctions between the former and the latter. MATERIALS AND METHODS: We examined three sets of human efferent and caput epididymal ducts in specimens obtained from prostatic carcinoma patients. They were reconstructed from serial paraffin sections using a segmentation model created by a deep learning protocol and high-performance three-dimensional reconstruction software. RESULTS: Serial sections and three-dimensional images of human efferent and caput epididymal ducts were combined to obtain the detailed anatomical information. When a single efferent duct was defined as a duct connecting to both the extra-testicular rete testis and epididymal duct, there were 14.7 efferent ducts with a total length of 3.0 m per specimen on average. The cranial portion of the efferent ducts joined to a single duct and terminated at the end of the epididymal duct, whereas other efferent ducts terminated independently on the side of the epididymal duct. These two types of junctions between the efferent and epididymal ducts differed in the patterns of the epithelial-type switch. The epididymal duct consisted of multiple segments, which were separated by a minimal amount of connective tissue septa or even without them. Efferent ducts occupied most of the volume of the caput epididymis. DISCUSSION AND CONCLUSIONS: By utilizing deep learning, we reconstructed human efferent and caput epididymal ducts and revealed their precise three-dimensional structures, which differed from those of rodents in several aspects. The present results may be useful for analyzing anatomical abnormalities related to some types of male infertility.

Selective inhibition of PDE4 in Wistar rats can lead to dilatation in testis, efferent ducts, and epididymis and subsequent formation of sperm granulomas.

Testicular tubular dilatation and degeneration and epididymal sperm granulomas were frequently seen in 4-week toxicity studies using different phosphodiesterase-4 (PDE4) inhibitors in Wistar rats, including the prototypic PDE4 inhibitor BYK169171. To investigate the pathogenesis of testicular and epididymal lesions, a time course study with BYK169171 was conducted with sequential necropsies after 7, 14, 21, and 28 days of treatment. After 7 days, a dilatation of efferent ducts and of the initial segment of the epididymis and a subacute interstitial inflammation were seen followed by a diffuse dilatation of seminiferous tubules in the testis. Dilatation and inflammation were most pronounced after 14 days. Single animals also exhibited vascular necrosis in the inflamed interstitium. Although dilatation decreased later in the study, the incidence and severity of tubular degeneration increased from 14 days onward. Sperm granulomas developed in efferent ducts and in the caput and cauda of the epididymis after 14 days. Our results demonstrate a clear time course of PDE4 inhibition-induced lesions, with dilatation preceding sperm granuloma formation. We conclude that the most likely mechanism of toxicity is a disturbance of fluid homeostasis in efferent and epididymal ducts resulting in abnormal luminal fluid and sperm contents, epithelial damage at specific sites of the excurrent duct system, sperm leakage, and granuloma formation.

Functional genomics of the human epididymis: Further characterization of efferent ducts and model systems by single-cell RNA sequencing analysis.

BACKGROUND: The human epididymis is poorly studied due to the lack of availability of tissue samples. Our understanding of its structure and function depends upon anatomical and histological observations of archived material. OBJECTIVES: Here we used single-cell RNA sequencing (scRNA-seq) technologies to elucidate the identity of cells within the human efferent ducts (EDs) and compared them to caput epididymis cells. We also compared the cellularity of primary tissues with those of 2D and 3D (organoid) culture models used for functional studies. MATERIALS AND METHODS: Human epididymis tissue was dissected to separate different anatomical regions and digested to release single cells for processing on the 10X Genomics Chromium platform. Primary human epididymis epithelial (HEE) cells and HEE organoids were grown as described previously and subjected to scRNA-seq. scRNA-seq data were processed by standard bioinformatics pipelines and used for comparative analysis. RESULTS: We define the cell types in the EDs which include specialized epithelial cells, connective tissue stromal cells, vascular endothelial cells, smooth muscle cells, and immune cells, but lack basal cells that are seen in the caput epididymis. Furthermore, we identify a sub-population of epithelial cells which have marker genes found in the bladder and urothelium. Comparative genomics analysis of the 2D and 3D culture models shows cellular identities adapted to the culture environment while still maintaining similarity to the primary tissue. DISCUSSION: Our data suggest that EDs are lined with a transitional epithelium, which like the urothelium is able to stretch and contract depending on luminal volume. This is consistent with its primary role in seminal fluid resorption and sperm concentration. Moreover, we describe the cellularity of models to study the human epididymis epithelium in vitro. CONCLUSION: Single-cell RNA-seq data from the human epididymis make a valuable contribution to our understanding of this highly specialized organ.

Essential Roles of Efferent Duct Multicilia in Male Fertility.

Cilia are microtubule-based hair-like organelles on the cell surface. Cilia have been implicated in various biological processes ranging from mechanosensation to fluid movement. Ciliary dysfunction leads to a plethora of human diseases, known as ciliopathies. Although non-motile primary cilia are ubiquitous, motile multicilia are found in restricted locations of the body, such as the respiratory tract, the oviduct, the efferent duct, and the brain ventricles. Multicilia beat in a whip-like motion to generate fluid flow over the apical surface of an epithelium. The concerted ciliary motion provides the driving force critical for clearing airway mucus and debris, transporting ova from the ovary to the uterus, maintaining sperm in suspension, and circulating cerebrospinal fluid in the brain. In the male reproductive tract, multiciliated cells (MCCs) were first described in the mid-1800s, but their importance in male fertility remained elusive until recently. MCCs exist in the efferent ducts, which are small, highly convoluted tubules that connect the testis to the epididymis and play an essential role in male fertility. In this review, we will introduce multiciliogenesis, discuss mouse models of male infertility with defective multicilia, and summarize our current knowledge on the biological function of multicilia in the male reproductive tract.

Morphology and immunolocalization of aquaporins 1 and 9 in the agouti (Dasyprocta azarae) testis excurrent ducts.

This study investigated the morphology and immunoexpression of aquaporins (AQPs) 1 and 9 in the rete testis, efferent ducts, epididymis, and vas deferens in the Azara's agouti (Dasyprocta azarae). For this purpose, ten adult sexually mature animals were used in histologic and immunohistochemical analyses. The Azara's agouti rete testis was labyrinthine and lined with simple cubic epithelium. Ciliated and non-ciliated cells were observed in the epithelium of the efferent ducts. The epididymal cellular population was composed of principal, basal, apical, clear, narrow, and halo cells. The epithelium lining of vas deferens was composed of the principal and basal cells. AQPs 1 and 9 were not expressed in the rete testis. Positive reaction to AQP1 was observed at the luminal border of non-ciliated cells of the efferent ducts, and in the peritubular stroma and blood vessels in the epididymis, and vas deferens. AQP9 was immunolocalized in the epithelial cells in the efferent ducts, epididymis and vas deferens. The morphology of Azara's agouti testis excurrent ducts is similar to that reported for other rodents such as Cuniculus paca. The immunolocalization results of the AQPs suggest that the expression of AQPs is species-specific due to differences in localization and expression when compared to studies in other mammals species. The knowledge about the expression of AQPs in Azara's agouti testis excurrent ducts is essential to support future reproductive studies on this animal, since previous studies show that AQPs may be biomarkers of male fertility and infertility.

The Therapeutic Potential of Amniotic Fluid-Derived Stem Cells on Busulfan-Induced Azoospermia in Adult Rats.

BACKGROUND: Busulfan is an alkylating chemotherapeutic agent that is routinely prescribed for leukemic patients to induce myelo-ablation. However, it also results in azoospermia and infertility in cancer survivors. This research was constructed to explore the possible therapeutic role of amniotic fluid-derived stem cells (AFSCs) in improving busulfan-induced azoospermia in adult rats. METHODS: Forty two adult male albino rats were randomized into: (1) control group, (2) azoospermia group, (3) spontaneous recovery group, and (4) AFSCs-treated group, in which AFSCs were transplanted through their injection into the testicular efferent ducts. The assessment included a histo-pathological examination of the seminiferous tubules by the light and transmission electron microscopes. Additionally, the confocal laser scanning microscope was used for confirmation of homing of the implanted cells. Moreover, we conducted an immuno-fluorescence study for detection of the proliferating cell nuclear antigen (PCNA) in the spermatogenic cells, epididymal sperm count, and a histo-morphometric study. RESULTS: AFSCs successfully homed over the basement membrane of the injured seminiferous tubules. They greatly attenuated busulfan-induced degenerative and oxidative changes. They also caused a re-expression of PCNA in the germ cells, leading to resumption of spermatogenesis and re-appearance of spermatozoa. CONCLUSION: AFSCs could be a promising treatment modality for male infertility induced by chemotherapy, as they possess prominent regenerative, anti-apoptotic, and anti-inflammatory potentials.

A possible highway system for the rapid delivery of sperm from the testis to the penis in the naked mole-rat, Heterocephalus glaber.

Gametogenesis is suppressed in most members of the eusocial naked mole-rat (NMR) colony, while the queen selects mainly one breeding male during her life span. Recently, it was reported that the NMR testicular organization seems to produce spermatozoa on demand after suppression of spermatogenesis during most of gestation. A Sertoli cell "pump" is then used to flush the spermatozoa into short tubuli recti and simplified rete testis to reach the excurrent duct system. We hypothesize that the components of this duct system are adapted for rapid delivery of spermatozoa to the penis and for numerous copulations with the queen. Therefore, the aim was to study the ultrastructure of the male NMR reproductive duct system using light microscopy and transmission electron microscopy. The NMR rete testis gives rise to six to eight efferent tubules joining the caput epididymis. The caput epididymis resembles that of other rodents but with less distinction in terms of histological zoning. The remainder of the epididymis is considerably reduced in length compared to other rodents. In contrast, the vas deferens epithelium is highly specialized in that a vast range of vesicles, often closely associated with the spermatozoa, were visible. The large ampulla is a factory for merocrine and apocrine secretions, producing even more diverse vesicles. The transitional epithelial cells of the bladder appear to secrete abundant mucous and the penis as well as its baculum is relatively small. We speculate that these modifications strongly suggest that the excurrent duct system has been simplified and adjusted to compensate for the absence of long maturation and storage of spermatozoa. We propose that these adaptations to the NMR reproductive tract are associated with a state of degenerative orthogenesis that was selected for due to the absence of sperm competition and apparently rapid delivery of spermatozoa from the testis.

G protein-coupled oestrogen receptor 1, oestrogen receptors and androgen receptor in the sand rat (Psammomys obesus) efferent ducts.

BACKGROUND: The efferent ducts are mainly involved in the reabsorption of the seminiferous tubular fluid. Testosterone and oestrogens regulate efferent ducts functions via their receptors. MATERIALS AND METHODS: This paper presents an experimental investigation on the location of the P450 aromatase, the 17-b oestradiol (E2), the androgen receptor (AR), the oestrogen receptor 1 (ESR1), the oestrogen receptor 2 (ESR2) and the G protein-coupled oestrogen receptor 1 (GPER1) in the efferent ducts using Psammomys obesus as an animal model to highlight the effect of the season on the histology and the distribution of these receptors. RESULTS: We observed a proliferation of the connective tissue, decreasing in the height of the epithelium during the resting season compared to the breeding season. Ciliated cells expressed P450 aromatase, AR, E2, ESR1, ESR2 and GPER1 during both seasons. Basal cells showed a positive staining for the ESR1 and the GPER1 during both season, the AR and E2 during the breeding season and ESR2 during the resting season. CONCLUSIONS: Our result shows that the expression of androgen receptor and oestrogen receptors in the efferent ducts vary by season witch suggest that they are largely involved in the regulation of the efferent ducts functions.

Revisiting structure/functions of the human epididymis.

BACKGROUND: The epididymis is the hallmark of all vertebrate species practicing internal fertilization. While the functions of the epididymis are well documented in laboratory rodents and some domestic animals, the structure and functions of the epididymis in humans remain poorly documented. OBJECTIVES: Using human tissues obtained with the collaboration of our local organ transplantation program, the histology, cell types, and three-dimensional organization of the excurrent duct were investigated. Microarrays were performed to determine the gene expression pattern along the human epididymis. MATERIALS AND METHODS: The histology of longitudinal sections of the proximal epididymis was described, and immunohistochemistry using specific antibodies was used to characterize cell types of the efferent duct and caput epididymis epithelia. The epididymis was divided into eight segments permitting gene profiling by microarray and gene ontology analysis. RESULTS: The proximal region of the human epididymis is formed exclusively by efferent ducts. These ducts form a complex histological structure particularly at the junction of the efferent ducts and caput epididymis. The efferent ducts exhibit a specific cellular signature when compared with the adjacent epididymis tubule. Efferent duct gene expression is not segmented and is dedicated to cilium differentiation and movement. The gene expression pattern of the caput segment is homogeneous and specialized in defense and immune responses and fertilization. DISCUSSION: In murine species, the epididymis is segmented into the initial segment, caput, corpus, and cauda regions, whereas in humans, the proximal region is formed by efferent ducts. The caput tubules have their own histological organization with a well-defined gene expression pattern. The distal corpus and cauda epididymis are distinct by a limited number of differentially expressed genes. CONCLUSIONS: Knowledge of epididymis functions and structure obtained using laboratory species should be extrapolated to humans with caution.

We, the developing rete testis, efferent ducts, and Wolffian duct, all hereby agree that we need to connect.

BACKGROUND: The mechanisms by which the rete testis joins the efferent ducts, which joins the Wolffian duct during development, are not known. Mouse and chick models have been helpful in identifying genes that are important for the development of each part, but genes have not been identified as to those that play a role in the joining of each part. Clinical implications of the failure of the male reproductive tract to form a fully functional conduit for spermatozoa are not trivial. Epididymal disjunction, the failure of the efferent ducts to join the testis, is one of several epididymal anomalies that have been observed in some boys who were cryptorchid at birth. OBJECTIVE: A systematic review of studies focusing on the morphogenesis of the mesonephric duct and mesonephric tubules in different species, and identification of clinical issues should there be failure of these tissues to develop. DESIGN: PubMed and GUDMAP databases, and review of books on kidney development were searched for studies reporting on the mechanisms of morphogenesis of the kidney and epididymis. MAIN OUTCOMES MEASURE(S): Gaps in our knowledge were identified, and hypotheses coupled with suggestions for future experiments were presented. RESULTS: A total of 64 papers were identified as relevant, of which 53 were original research articles and 11 were book chapters and reviews covering morphogenesis and clinical issues. Investigators utilized multiple species including, human, mouse, chick, Xenopus, bovine, and sheep. CONCLUSION: Fundamental understanding of the morphogenesis of the male reproductive tract is limited, especially the morphogenesis of the rete testis and efferent ducts. Therefore, it is not surprising that we do not understand how each part unites to form a whole. Only one mechanism of joining of one part of the tract to another was identified: the joining of the Wolffian duct to the cloaca via controlled apoptosis.

E2f4 and E2f5 are essential for the development of the male reproductive system.

The E2F transcription factors are primarily implicated in the regulation of entry and exit from the cell cycle. However, in vivo studies have established additional roles for E2Fs during organ development and homeostasis. With the goal of addressing the intestinal requirements of E2f4 and E2f5, we crossed mice carrying Vil-cre, E2f4 conditional and E2f5 germline alleles. E2f4 deletion had no detectable effect on intestinal development. However, E2f4f/f;E2f5+/-;Vil-cre males, but not E2f4f/f;Vil-cre littermates, were unexpectedly sterile. This defect was not due to defective spermatogenesis. Instead, the seminiferous tubules and rete testes showed significant dilation, and spermatozoa accumulated aberrantly in the rete testis and efferent ducts. Our data show that these problems result from defective efferent ducts, a tissue whose primary function is to concentrate sperm through fluid absorption. First, Vil-cre expression, and consequent E2F4 loss, was specific to the efferent ducts and not other reproductive tract tissues. Second, the E2f4f/f;E2f5+/-;Vil-cre efferent ducts had completely lost multiciliated cells and greatly reduced levels of critical absorptive cell proteins: aquaporin1, a water channel protein, and clusterin, an endocytic marker. Collectively, the observed testis phenotypes suggest a fluid flux defect. Remarkably, we observed rete testis dilation prior to the normal time of seminiferous fluid production, arguing that the efferent duct defects promote excessive secretory activity within the reproductive tract. Finally, we also detect key aspects of these testis defects in E2f5-/- mice. Thus, we conclude that E2f4 and E2f5 display overlapping roles in controlling the normal development of the male reproductive system.