Human efferent ductules and epididymis display unique cell lineages with motile and primary cilia.

BACKGROUND: Previous research has illustrated the role of cilia as mechanical and sensory antennae in various organs within the mammalian male reproductive system across different developmental stages. Despite their significance in both organ development and homeostasis, primary cilia in the human male reproductive excurrent duct have been overlooked due to limited access to human specimens. OBJECTIVE: This study aimed to characterize the unique cellular composition of human efferent and epididymal ducts, with a focus on their association with primary cilia. MATERIALS AND METHODS: Human efferent ductules/epididymides from five donors aged 32-47 years, were obtained through our local organ transplant program. Cell lineage specificity and primary cilia features were examined by immunofluorescent staining and confocal microscopy in the efferent ductules and the distinct segments of the epididymis. RESULTS: The epithelium of the human efferent duct exhibited estrogen receptor-positive cells with primary cilia, FoxJ1-positive multiciliated cells with numerous motile cilia, and non-ciliated intraepithelial immune cells. Notably, intraluminal macrophages, identified by CD163/CD68 positivity, were observed to engage in sperm phagocytosis. In all three segments of the human epididymis, primary cilia were found on the surface of principal and basal cells. DISCUSSION AND CONCLUSIONS: Our research indicates that the human efferent ductules create a distinct environment, characterized by the presence of two types of ciliated cells that are in contact with immune cells. The discovery of sensory primary cilia exposed on the surface of reabsorptive cells in the efferent ductules, as well as on basal and principal cells in the epididymis, lays the foundation for complementary functional studies. This research uncovers novel characteristics exclusive to human efferent ductules and epididymides, providing a basis for exploring innovative approaches to male contraception and infertility treatment.

Cryptorchidism and testicular cancer in the dog: unresolved questions and challenges in translating insights from human studies†.

Cryptorchidism, the failure of one or both testes to descend into the scrotum, and testicular cancer show a strong correlation in both dogs and humans. Yet, long-standing medical debates persist about whether the location of undescended testes directly causes testicular cancer in humans or if both conditions stem from a common origin. Although testicular cancer is a prevalent disease in dogs, even less is known about its cause and correlation with testicular descent in this species. This review investigates the relation between these two disorders in dogs, drawing insights from human studies, and examines key biomarkers identified thus far. In addition, it explores potential causal links, including the impact of temperature on maturing testicular cells and a potential shared genetic origin. Notably, this literature review reveals significant differences between men and dogs in reproductive development, histological and molecular features of testicular tumors, and the prevalence of specific tumor types, such as Sertoli cell tumors in cryptorchid dogs and germ cell tumors in humans. These disparities caution against using dogs as models for human testicular cancer research and underscore the limitations when drawing comparisons between species. The paper concludes by suggesting specific research initiatives to enhance our understanding of the complex interplay between cryptorchidism and testicular cancer in dogs.

Role for Nongenomic Estrogen Signaling in Male Fertility.

Estrogen actions are mediated by both nuclear (n) and membrane (m) localized estrogen receptor 1 (ESR1). Male Esr1 knockout (Esr1KO) mice lacking functional Esr1 are infertile, with reproductive tract abnormalities. Male mice expressing nESR1 but lacking mESR1 (nuclear-only estrogen receptor 1 mice) are progressively infertile due to testicular, rete testis, and efferent ductule abnormalities similar to Esr1KO males, indicating a role for mESR1 in male reproduction. The H2NES mouse expresses only mESR1 but lacks nESR1. The goal of this study was to identify the functions of mESR1 alone in mice where nESR1 was absent. Breeding trials showed that H2NES males are fertile, with decreased litter numbers but normal pup numbers/litter. In contrast to Esr1KO mice, H2NES testicular, and epididymal weights were not reduced, and seminiferous tubule abnormalities were less pronounced. However, Esr1KO and H2NES males both had decreased sperm motility and a high incidence of abnormal sperm morphology. Seminiferous tubule and rete testis dilation and decreased efferent ductule epithelial height characteristic of Esr1KO males were reduced in H2NES. Consistent with this, expression of genes involved in fluid transport and ion movement that were reduced in Esr1KO (Aqp1, Car2, Car14, Cftr) were partially or fully restored to wild-type levels in H2NES. In summary, in contrast to Esr1KO males, H2NES males are fertile and have reduced phenotypic and functional abnormalities in the testis and efferent ductules. Thus, mESR1 alone, in the absence of nESR1, can partially regulate male reproductive tract structure and function, emphasizing its importance for overall estrogen action.

Single neonatal estrogen implant sterilizes female animals by decreasing hypothalamic KISS1 expression.

Reproductive sterilization by surgical gonadectomy is strongly advocated to help manage animal populations, especially domesticated pets, and to prevent reproductive behaviors and diseases. This study explored the use of a single-injection method to induce sterility in female animals as an alternative to surgical ovariohysterectomy. The idea was based on our recent finding that repetitive daily injection of estrogen into neonatal rats disrupted hypothalamic expression of Kisspeptin (KISS1), the neuropeptide that triggers and regulates pulsatile secretion of GnRH. Neonatal female rats were dosed with estradiol benzoate (EB) either by daily injections for 11 days or by subcutaneous implantation of an EB-containing silicone capsule designed to release EB over 2-3 weeks. Rats treated by either method did not exhibit estrous cyclicity, were anovulatory, and became infertile. The EB-treated rats had fewer hypothalamic Kisspeptin neurons, but the GnRH-LH axis remained responsive to Kisspeptin stimulation. Because it would be desirable to use a biodegradable carrier that is also easier to handle, an injectable EB carrier was developed from PLGA microspheres to provide pharmacokinetics comparable to the EB-containing silicone capsule. A single neonatal injection of EB-microspheres at an equivalent dosage resulted in sterility in the female rat. In neonatal female Beagle dogs, implantation of an EB-containing silicone capsule also reduced ovarian follicle development and significantly inhibited KISS1 expression in the hypothalamus. None of the treatments produced any concerning health effects, other than infertility. Therefore, further development of this technology for sterilization in domestic female animals, such as dogs and cats is worthy of investigation.

Expression patterns of sex steroid receptors in developing mesonephros of the male mouse: three-dimensional analysis.

The androgen pathway via androgen receptor (AR) has received the most attention for development of male reproductive tracts. The estrogen pathway through estrogen receptor (ESR1) is also a major contributor to rete testis and efferent duct formation, but the role of progesterone via progesterone receptor (PGR) has largely been overlooked. Expression patterns of these receptors in the mesonephric tubules (MTs) and Wolffian duct (WD), which differentiate into the efferent ductules and epididymis, respectively, remain unclear because of the difficulty in distinguishing each region of the tracts. This study investigated AR, ESR1, and PGR expressions in the murine mesonephros using three-dimensional (3-D) reconstruction. The receptors were localized in serial paraffin sections of the mouse testis and mesonephros by immunohistochemistry on embryonic days (E) 12.5, 15.5, and 18.5. Specific regions of the developing MTs and WD were determined by 3-D reconstruction using Amira software. AR was found first in the specific portion of the MTs near the MT-rete junction at E12.5, and the epithelial expression showed increasing strength from cranial to the caudal regions. Epithelial expression of ESR1 was found in the cranial WD and MTs near the WD first at E15.5. PGR was weakly positive only in the MTs and cranial WD starting on E15.5. This 3-D analysis suggests that gonadal androgen acts first on the MTs near the MT-rete junction but that estrogen is the first to influence MTs near the WD, while potential PGR activity is delayed and limited to the epithelium.

CEP164 is essential for efferent duct multiciliogenesis and male fertility.

Cilia are evolutionarily conserved microtubule-based structures that perform diverse biological functions. Cilia are assembled on basal bodies and anchored to the plasma membrane via distal appendages. In the male reproductive tract, multicilia in efferent ducts (EDs) move in a whip-like motion to prevent sperm agglutination. Previously, we demonstrated that the distal appendage protein CEP164 recruits Chibby1 (Cby1) to basal bodies to facilitate basal body docking and ciliogenesis. Mice lacking CEP164 in multiciliated cells (MCCs) (FoxJ1-Cre;CEP164fl/fl) show a significant loss of multicilia in the trachea, oviduct, and ependyma. In addition, we observed male sterility; however, the precise role of CEP164 in male fertility remained unknown. Here, we report that the seminiferous tubules and rete testis of FoxJ1-Cre;CEP164fl/fl mice exhibit substantial dilation, indicative of dysfunctional multicilia in the EDs. We found that multicilia were hardly detectable in the EDs of FoxJ1-Cre;CEP164fl/fl mice although FoxJ1-positive immature cells were present. Sperm aggregation and agglutination were commonly noticeable in the lumen of the seminiferous tubules and EDs of FoxJ1-Cre;CEP164fl/fl mice. In FoxJ1-Cre;CEP164fl/fl mice, the apical localization of Cby1 and the transition zone marker NPHP1 was severely diminished, suggesting basal body docking defects. TEM analysis of EDs further confirmed basal body accumulation in the cytoplasm of MCCs. Collectively, we conclude that male infertility in FoxJ1-Cre;CEP164fl/fl mice is caused by sperm agglutination and obstruction of EDs due to loss of multicilia. Our study, therefore, unravels an essential role of the distal appendage protein CEP164 in male fertility.

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.

The protein YWHAE (14-3-3 epsilon) in spermatozoa is essential for male fertility.

BACKGROUND: Spermatogenesis is a complex biological process highlighted by synthesis and activation of proteins that regulate meiosis and cellular differentiation occur during spermatogenesis. 14-3-3 proteins are adaptor proteins that play critical roles in kinase signaling, especially for regulation of cell cycle and apoptosis in eukaryotic cells. There are seven isoforms of the 14-3-3 family proteins encoded by seven genes (ß, ε, γ, η, θ/τ, ζ and σ). 14-3-3 isoforms have been shown to have many interacting partners in several tissues including testis. OBJECTIVE: While it is known that 14-3-3 proteins are expressed in the functions of testis and spermatozoon, the role for each of the seven isoforms is not known. In this study, we investigated the roles of 14-3-3η and 14-3-3ε isoforms in spermatogenesis. MATERIALS AND METHODS: To study the in vivo function of 14-3-3η and 14-3-3ε in spermatogenesis, we generated testis-specific and global knockout mice for each of 14-3-3η and 14-3-3ε isoforms (CKO and GKO, respectively). Computer-assisted semen analysis was used to assess sperm motility, while immunohistochemical studies were conducted to check spermatogenesis. RESULTS: Although both 14-3-3η and 14-3-3ε isoforms were present in mouse testis, only the expression of 14-3-3ε, but not 14-3-3η, was detected in spermatozoa. Mice lacking 14-3-3η were normal and fertile while 14-3-3ε CKO and GKO males showed infertility. Low sperm count with higher abnormal spermatozoa was seen in 14-3-3ε CKO mice. The motility of 14-3-3ε knockout spermatozoa was lower than that of the control. A reduction in the phosphorylation of both glycogen synthase kinase 3 and PP1γ2 was also seen in spermatozoa from 14-3-3ε CKO mice, suggesting a specific role of 14-3-3ε in spermatogenesis, sperm motility, and fertility. DISCUSSION AND CONCLUSION: This is the first demonstration that of the seven 14-3-3 isoforms, 14-3-3ε is essential for normal sperm function and male fertility.

Tumor-Associated Macrophages (TAM) are recruited to the aging prostate epithelial lesions and become intermingled with basal cells.

BACKGROUND: Prostate cancer remains one of the most common cancers in men. Macrophages are thought to be important regulators in cancers, and their potential involvement in prostate cancer should not be overlooked. Therefore, the association between macrophages and the pre-tumorous changes in prostate epithelium during aging deserves further investigation. OBJECTIVES: We sought to investigate whether macrophages would be recruited into the prostate epithelium that display pathological lesions commonly found during aging. MATERIALS AND METHODS: Prostates of aging rats, with and without treatment with a combination of testosterone and estradiol, were examined for premalignant and malignant epithelial lesions. For comparison, prostates of castrated rats were also investigated. RESULTS: Intraepithelial macrophages were found restricted to areas of premalignant and malignant lesions. An unprecedented interaction between macrophages and basal cells was observed in the aging pathological lesions. The intraepithelial macrophages were associated with autophagy, in contrast to those found after castration. In prostate lesions, the intraepithelial macrophages had TAM phenotype (CD68+/iNOS+/CD206+/ARG+), denoting a possible involvement in cancer progression. However, M2 macrophages (CD68+/CD163+) were recruited into the epithelium after castration, possibly to phagocytize cells undergoing apoptosis. DISCUSSION AND CONCLUSION: In conclusion, macrophages were recruited into the prostate epithelium and presented diverse phenotypes and morphology, consistent with changes reflected in the hormonal environment. Macrophages with the TAM phenotype were found restricted to areas of premalignant and malignant lesions in aging prostates, denoting a possible involvement in cancer progression. In contrast, M2 macrophages were found in the regressed epithelium after castration.

Rare mutations in the complement regulatory gene CSMD1 are associated with male and female infertility.

Infertility in men and women is a complex genetic trait with shared biological bases between the sexes. Here, we perform a series of rare variant analyses across 73,185 women and men to identify genes that contribute to primary gonadal dysfunction. We report CSMD1, a complement regulatory protein on chromosome 8p23, as a strong candidate locus in both sexes. We show that CSMD1 is enriched at the germ-cell/somatic-cell interface in both male and female gonads. Csmd1-knockout males show increased rates of infertility with significantly increased complement C3 protein deposition in the testes, accompanied by severe histological degeneration. Knockout females show significant reduction in ovarian quality and breeding success, as well as mammary branching impairment. Double knockout of Csmd1 and C3 causes non-additive reduction in breeding success, suggesting that CSMD1 and the complement pathway play an important role in the normal postnatal development of the gonads in both sexes.

Motile cilia of the male reproductive system require miR-34/miR-449 for development and function to generate luminal turbulence.

Cilia are cell-surface, microtubule-based organelles that project into extracellular space. Motile cilia are conserved throughout eukaryotes, and their beat induces the flow of fluid, relative to cell surfaces. In mammals, the coordinated beat of motile cilia provides highly specialized functions associated with the movement of luminal contents, as seen with metachronal waves transporting mucus in the respiratory tract. Motile cilia are also present in the male and female reproductive tracts. In the female, wave-like motions of oviductal cilia transport oocytes and embryos toward the uterus. A similar function has been assumed for motile cilia in efferent ductules of the male-i.e., to transport immotile sperm from rete testis into the epididymis. However, we report here that efferent ductal cilia in the male do not display a uniform wave-like beat to transport sperm solely in one direction, but rather exert a centripetal force on luminal fluids through whip-like beating with continual changes in direction, generating turbulence, which maintains immotile spermatozoa in suspension within the lumen. Genetic ablation of two miRNA clusters (miR-34b/c and -449a/b/c) led to failure in multiciliogenesis in murine efferent ductules due to dysregulation of numerous genes, and this mouse model allowed us to demonstrate that loss of efferent duct motile cilia causes sperm aggregation and agglutination, luminal obstruction, and sperm granulomas, which, in turn, induce back-pressure atrophy of the testis and ultimately male infertility.

Seasonal variation of cell proliferation and apoptosis in the efferent ductules and epididymis of the Neotropical bat Artibeus lituratus (Chiroptera, Phyllostomidae).

The balance between cell proliferation and apoptosis is important for maintenance of male fertility, being influenced by a variety of stimuli including androgens and estrogens. However, studies concerning regulation of these processes along the male reproductive tract under physiological conditions are scarce. Therefore, in this study, we investigated the profile of cell proliferation and apoptosis in the efferent ductules and epididymis of the Neotropical bat Artibeus lituratus, a seasonal breeder that presents natural variation in components of the androgen and estrogen responsive systems along the circannual cycle. Low rates of cell proliferation and apoptosis were found in the efferent ductules and epididymis of A. lituratus during the reproductive period, as few epithelial cells were positive for MCM7 (proliferation marker) and cleaved caspase-3 or TUNEL (apoptosis markers). In contrast, during the regressive period, the rate of both proliferating and apoptotic cells was significantly higher in the epithelium lining the efferent ductules as well as throughout the epididymis. The increased proliferative activity at this phase was positively correlated with the expression of estrogen receptor alpha (ERα), whereas the variation in apoptosis appears to be unrelated to the local expression of androgen and estrogen receptors. Together, these data suggest that cell proliferation and apoptosis are differentially modulated in the efferent ductules and epididymis of A. lituratus during the annual reproductive cycle, and support the hypothesis that ERα may be important in preparing the male reproductive tract for sexual recrudescence.

Seminal fluid metabolome and epididymal changes after antibiotic treatment in mice.

Paternal environment can induce detrimental developmental origins of health and disease (DOHaD) effects in resulting offspring and even future descendants. Such paternal-induced DOHaD effects might originate from alterations in a possible seminal fluid microbiome (SFM) and composite metabolome. Seminal vesicles secrete a slightly basic product enriched with fructose and other carbohydrates, providing an ideal habitat for microorganisms. Past studies confirm the existence of a SFM that is influenced by genetic and nutritional status. Herein, we sought to determine whether treatment of male mice with a combination of antibiotics designed to target SFM induces metabolic alterations in seminal vesicle gland secretions (seminal fluid) and histopathological changes in testes and epididymides. Adult (10- to 12-week-old) National Institutes of Health (NIH) Swiss males (n = 10 per group) were treated with Clindamycin 0.06 mg/kg day, Unasyn (ampicillin/sulbactam) 40 mg/kg day and Baytril (enrofloxacin) 50 mg/kg day designed to target the primary bacteria within the SFM or saline vehicle alone. Fourteen-day antibiotic treatment of males induced metabolomic changes in seminal vesicles with inosine, xanthine and l-glutamic acid decreased but d-fructose increased in glandular secretions. While spermatogenesis was not affected in treated males, increased number of epididymal tubules showed cribriform growth in this group (7 antibiotic-treated males: 3 saline control males; P = 0.01). Antibiotic-treated males showed more severe cribriform cysts. Current findings suggest antibiotic treatment of male mice results in seminal fluid metabolome and epididymal histopathological alterations. It remains to be determined whether such changes compromise male reproductive function or lead to DOHaD effects in resulting offspring.

Estrogen in the male: a historical perspective.

Estrogens have traditionally been considered female hormones. Nevertheless, the presence of estrogen in males has been known for over 90 years. Initial studies suggested that estrogen was deleterious to male reproduction because exogenous treatments induced developmental abnormalities. However, demonstrations of estrogen synthesis in the testis and high concentrations of 17ß-estradiol in rete testis fluid suggested that the female hormone might have a function in normal male reproduction. Identification of estrogen receptors and development of biological radioisotope methods to assess estradiol binding revealed that the male reproductive tract expresses estrogen receptor extensively from the neonatal period to adulthood. This indicated a role for estrogens in normal development, especially in efferent ductules, whose epithelium is the first in the male reproductive tract to express estrogen receptor during development and a site of exceedingly high expression. In the 1990s, a paradigm shift occurred in our understanding of estrogen function in the male, ushered in by knockout mouse models where estrogen production or expression of its receptors was not present. These knockout animals revealed that estrogen's main receptor (estrogen receptor 1 [ESR1]) is essential for male fertility and development of efferent ductules, epididymis, and prostate, and that loss of only the membrane fraction of ESR1 was sufficient to induce extensive male reproductive abnormalities and infertility. This review provides perspectives on the major discoveries and developments that led to our current knowledge of estrogen's importance in the male reproductive tract and shaped our evolving concept of estrogen's physiological role in the male.

Basal Cells Show Increased Expression of Aromatase and Estrogen Receptor α in Prostate Epithelial Lesions of Male Aging Rats.

Besides androgens, estrogen signaling plays a key role in normal development and pathologies of the prostate. Irreversible synthesis of estrogens from androgens is catalyzed by aromatase. Interestingly, animals lacking aromatase do not develop cancer or prostatitis, whereas those with overexpression of aromatase and, consequently, high estrogen levels develop prostatitis and squamous metaplasia via estrogen receptor 1 (ERα). Even with this evidence, the aromatase expression in the prostate is controversial. Moreover, little is known about the occurrence of age-dependent variation of aromatase and its association with histopathological changes commonly found in advanced age, a knowledge gap that is addressed herein. For this purpose, the immunoexpression of aromatase was evaluated in the prostatic complex of young adult to senile Wistar rats. ERα was also investigated, to extend our understanding of estrogen responsiveness in the prostate. Moderate cytoplasmic immunoreactivity for aromatase was detected in the glandular epithelium. Eventually, some basal cells showed intense staining for aromatase. The expression pattern for aromatase appeared similar in the normal epithelium when young and senile rats were compared; this result was corroborated by Western blotting. Conversely, in senile rats, there was an increase in the frequency of basal cells intensely stained for aromatase, which appeared concentrated in areas of intraepithelial proliferation and prostatitis. These punctual areas also presented increased ERα positivity. Together, these findings suggest a plausible source for hormonal imbalance favoring estrogen production, which, by acting through ERα, may favor the development of prostatic lesions commonly found in advanced age.

Estrogens in Male Physiology.

Estrogens have historically been associated with female reproduction, but work over the last two decades established that estrogens and their main nuclear receptors (ESR1 and ESR2) and G protein-coupled estrogen receptor (GPER) also regulate male reproductive and nonreproductive organs. 17ß-Estradiol (E2) is measureable in blood of men and males of other species, but in rete testis fluids, E2 reaches concentrations normally found only in females and in some species nanomolar concentrations of estrone sulfate are found in semen. Aromatase, which converts androgens to estrogens, is expressed in Leydig cells, seminiferous epithelium, and other male organs. Early studies showed E2 binding in numerous male tissues, and ESR1 and ESR2 each show unique distributions and actions in males. Exogenous estrogen treatment produced male reproductive pathologies in laboratory animals and men, especially during development, and studies with transgenic mice with compromised estrogen signaling demonstrated an E2 role in normal male physiology. Efferent ductules and epididymal functions are dependent on estrogen signaling through ESR1, whose loss impaired ion transport and water reabsorption, resulting in abnormal sperm. Loss of ESR1 or aromatase also produces effects on nonreproductive targets such as brain, adipose, skeletal muscle, bone, cardiovascular, and immune tissues. Expression of GPER is extensive in male tracts, suggesting a possible role for E2 signaling through this receptor in male reproduction. Recent evidence also indicates that membrane ESR1 has critical roles in male reproduction. Thus estrogens are important physiological regulators in males, and future studies may reveal additional roles for estrogen signaling in various target tissues.

Membrane estrogen receptor 1 is required for normal reproduction in male and female mice.

Steroid hormones, acting through their cognate nuclear receptors, are critical for many reproductive and non-reproductive functions. Over the past two decades, it has become increasingly clear that in addition to cytoplasmic/nuclear steroid receptors that alter gene transcription when liganded, a small fraction of cellular steroid receptors are localized to the cell membranes, where they mediate rapid steroid hormone effects. 17ß-Estradiol (E2), a key steroid hormone for both male and female reproduction, acts predominately through its main receptor, estrogen receptor 1 (ESR1). Most ESR1 is nuclear; however, 5-10% of ESR1 is localized to the cell membrane after being palmitoylated at cysteine 451 in mice. This review discusses reproductive phenotypes of a newly-developed mouse model with a C451A point mutation that precludes membrane targeting of ESR1. This transgenic mouse, termed the nuclear-only ESR1 (NOER) mouse, shows extensive male and female reproductive abnormalities and infertility despite normally functional nuclear ESR1 (nESR1). These results provide the first in vivo evidence that membrane-initiated E2/ESR1 signaling is required for normal male and female reproductive functions and fertility. Signaling mechanisms for membrane ESR1 (mESR1), as well as how mESR1 works with nESR1 to mediate estrogen effects, are still being established. We discuss some possible mechanisms by which mESR1 might facilitate nESR1 signaling, as well as the emerging evidence that mESR1 might be a major mediator of epigenetic effects of estrogens, which are potentially linked to various adult-onset pathologies.

Membrane-Localized Estrogen Receptor 1 Is Required for Normal Male Reproductive Development and Function in Mice.

Estrogen receptor 1 (ESR1) mediates major reproductive functions of 17ß-estradiol (E2). Male Esr1 knockout (Esr1KO) mice are infertile due to efferent ductule and epididymal abnormalities. The majority of ESR1 is nuclear/cytoplasmic; however, a small fraction is palmitoylated at cysteine 451 in mice and localized to cell membranes, in which it mediates rapid E2 actions. This study used an Esr1 knock-in mouse containing an altered palmitoylation site (C451A) in ESR1 that prevented cell membrane localization, although nuclear ESR1 was expressed. These nuclear-only estrogen receptor 1 (NOER) mice were used to determine the roles of membrane ESR1 in males. Epididymal sperm motility was reduced 85% in 8-month-old NOER mice compared with wild-type controls. The NOER mice had decreased epididymal sperm viability and greater than 95% of sperm had abnormalities, including coiled midpieces and tails, absent heads, and folded tails; this was comparable to 4-month Esr1KO males. At 8 months, daily sperm production in NOER males was reduced 62% compared with controls. The NOER mice had histological changes in the rete testes, efferent ductules, and seminiferous tubules that were comparable with those previously observed in Esr1KO males. Serum T was increased in NOER males, but FSH, LH, and E2 were unchanged. Critically, NOER males were initially subfertile, becoming infertile with advancing age. These findings identify a previously unknown role for membrane ESR1 in the development of normal sperm and providing an adequate environment for spermatogenesis.

Transcription Factor RFX2 Is a Key Regulator of Mouse Spermiogenesis.

The regulatory factor X (RFX) family of transcription factors is crucial for ciliogenesis throughout evolution. In mice, Rfx1-4 are highly expressed in the testis where flagellated sperm are produced, but the functions of these factors in spermatogenesis remain unknown. Here, we report the production and characterization of the Rfx2 knockout mice. The male knockout mice were sterile due to the arrest of spermatogenesis at an early round spermatid step. The Rfx2-null round spermatids detached from the seminiferous tubules, forming large multinucleated giant cells that underwent apoptosis. In the mutants, formation of the flagellum was inhibited at its earliest stage. RNA-seq analysis identified a large number of cilia-related genes and testis-specific genes that were regulated by RFX2. Many of these genes were direct targets of RFX2, as revealed by chromatin immunoprecipitation-PCR assays. These findings indicate that RFX2 is a key regulator of the post-meiotic development of mouse spermatogenic cells.

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.